CA2688795C - Systems and methods for monitoring health and delivering drugs transdermally - Google Patents

Abstract

The present invention pertains to a system and method for transdermal sampling, comprising: at least one sampler for retrieving and transferring at least one analyte obtained transdermally from the skin of a subject; at least one detector system for identifying and quantifying said at least one analyte; and at least one logic module for: (i) receiving and storing input data from said at least one detector, (ii) relating the input data to other data obtained from the subject, (iii) displaying output information, (iv) transmitting the output information to another system, and (v) controlling the operation of said at least one sampler and at least one detector.

Description

SYSTEMS AND METHODS FOR MONITORING
HEALTH AND DELIVERING DRUGS
TRANSDERMALLY
BACKGROUND OF THE INVENTION
Field of the Invention;
The present invention relates generally to portable biomedical monitoring. More specifically, this invention relates to non-invasive and minimally invasive molecular monitoring and optionally the implementation of protective feedback measures and remote monitoring through telemetry.
Description of the Related Art Non-invasive transdermal sampling of body fluids has long been a goal in medical research. The notion that valuable diagnostic information comprising the concentrations of key analytes within the bloodstream could be obtained without breaching the skin has spurred many lines of research. With such technology, long-term convenient health monitoring and screening without needles or outpatient care would become a reality: diabetics could monitor blood glucose without drawing blood; markers for microbial', fungal or viral infections could be monitored; and environmental exposure to toxins could be assessed non-invasively.

Biomarkers have been utilized effectively to dentrrneasere, Ina menu exposure levels to envitomnental chemicals deemed hazardous and toxic to human life. The sensitivity of biomarken allows them to act as early wanting indicators to subtle alterations in the environment. Their specificity can be used to establish the nature of the imposing chemical agent, determine exposure level and define a suitable comae of action. Environmentally induced diseases affect everyone to one degree or another, however individual susceptibilities can predispose the dome of tcodc reaction of one group over another. It is worthwhile noting that in 1996, there were 86,912 cases of pesticide exposures to reported to American Association of Poison Centers, of which 26 were fatalities. In particular, individuals in their developmental stages, ranging from the embryonic phase to adolescence, are particularly susceptible to such environmental stresses since key body functions have not maimed to a level where they can tolerate, process and hanille such exposures The use of 15 biomarkers for determination of children's environmental health will allow for the early detection of toxins, prevention of impairmemt in their physical condition, and determine a course of treatment for children who have; been exposed to a toxic environment Especially important in the field of pediatrics is the use of health 20 evaluation tools that are minimally intrusive.
Many transdermal sampling techniques have been reported, but all to date suffer from one or uxce serious drawbacks. Conventional techniques have disadvantages of being grossly invasive (and potentially injuricms) and sweat or interstitial fluid dependent, except for the: passive, non-sweat dependent 25 transdermal analyte collection and detection techniques.

2 One approach to transdermal sampling has einployed thecollecdbil ol4 sweat. For example, M. Philips and M.H. McAloon. Alcohol Clin Exp. Res. 4 391 (1980) disclose an adsorbent patch which is a salt-impregnated, cellulose pad under an occlusive, adhesive cover. However, such a method of transdermal sampling is dependent upon the sweat rate, requires sweat extraction by centrifbgation, and calls for external chemical analysis. S.
Balabanova and E. Schneider. Beitr. Oerichd. Med 48, 45 (1990) disclose Pilocarpine-induced sweat secretion, but the system requires Iontclphoresis-induced inflation of pilocarpine and analyte dilution. U.S. Patent No.
5,203,327, issued to Schoendorfer et id., discloses an absorbent pad under a water vapor-penneable, occlusive, adhesive cover, but the system is sweat rate dependent and requires chemical extraction and external rbrodral analysis. F.P. Smith and DA. Kidwell, Foram* Sci. Int. 83, 179 (1996) discloses a cotton sweat wipe, but this system is sweat volume-dependent and requires extraction and external is chemical analysis. G.L. Henderson and B.K. Wilson,. Res. Commun. Chem.
Pathol. Pharmacol. 5, 1 (1973) discloses the collection of liquid sweat following exercise, but the system requires vigorous exercise, is sweat volume-dependent, and requires extraction and external chemical analysis.
C.C. Peck, D.P. Conner, et al. Skin Pharmacol 1, 14(1988) discloses a gel with an analyte binding reservoir under an occlusive adhesive cover.
However, this reference requires extraction and external chemical analysis.
US. Patent No. 4,909,256, issued to Peck discloses a chy binding reservoir under an occlusive adhesive cover. However, this rah:mace requires extraction and external chemical analysis.

3 U.S. Patent No. 4,821,733, issued to Peck discloses a collection and detection system under an occlusive adhesive cover. However, this reference requires highly sensitive detection components.
U.S. Patent No. 4,775,361, issued to Jacques discloses emhanced migration of analyte to a skin surface. However, this reference requires introduction of light energy into the body.
U.S. Patent No. 5,362,307, issued to Guy discloses iontophoretic enhanced smalyte collection mon skin. However, this reference requires the introduction of electrical energy into the body.
to U.S. Patent No. 5,722,397, issued to Eppstein discloses ultrasound enhanced analyte collection across skin. However, this reference requires the introduction of sonic energy and chemicals into the body.
U.S. Patent No. 5,885,211, issued to Eppstein, discloses micropore formation using heated water vapor, physical lancet, sonic energy, high pressure jet of fluid, or electricity. However, this reference requires puncture of the skin using heat, SOO* or electrical energy, phytdcal or hydraulic fbrce.
These is, therefore, a need within the trunsdermal sampling field for a mmally invasive sampling technique and apparatus suitable for rapid, inexpensive, unobtrusive, and pain-free monitoring of important biomedical markers and envhonmental toxin exposure. These properties and advantages of

4 the resent invention will become apparent to those ofnklleak open reading the following disclosure.
BRIEF SUMMARY OF THE INVENTION
l'he esent invention pertains to a tnmsdermal sampling system, = comprising: at least one sampler for retrieving and transferring at least one analyte obtained Candi:m*11y from the skin of a subject; at least one detector system for idemtifying and quantifying said at least one analyte; and at least one logic module for (i) receiving and storing input data from said at least one detector, (h) relathig the input data to other data obtained from the subject, (iii) displaying output information, (iv) transmitting the output information to another system, and (v) controlling the operation of said at least one sampler and at least one detector.
= The present invention also pertains to a miaofabricated device for Is allowing remote monitoring of a subject, comprising: at least one sampler unit body for retrieving and transferring at least one acolyte obtained transdemially from the skin of a subject at least one detector system connected to said at least one sampler unit body for identifying and quantifying at least one analyte obtained from a subject; and a transmitter/receiver for tninsmitting data relating to at least one analyte detected by said detection system to a logic module for processing thereby, and for allowing control of the microfabricated device by a logic nlodule The present invention also pertains to a microfabricated device for ' sampling analytes from the skin of a subject, compisinc a detection chamber for receiving analytes retrieved from the skin of a subject; a photonk detection system, comprising a photonics source located attached to said microfabricated device in association with said detection chamber, and detectors associated with said detection chamber for detecting analytes received in said detection chamber.
The present invention also pertains to a microfabricated device for sampling analytes from the slcin of a subject, comprising: a detection chamber for receiving analytes retrieved from the skin of a subject; a patch which changes color when contacted by predetermined analytes, located attached to said mierofabricated device in association with said detection chamber; and detectors associated with said detection chamber, for detecting a change of color of the patch indicating the presence of a predetermined analyte.
The present invention also pertains to a microfabricated device for sampling and detecting analytes retrieved from the skin of a subject, comprising: at least one conduit for retrieving and transmitting an analyte from the skin of a subject to a detector, and means for enhancing permeability of the skin ata subject for retrieving said at least one analyte therefrom.
It is an aspect of the present invention to provide a transdermal sampling VACS, It is another aspect of the present invention to provide an integrated detection system using patch type detector.
It is still another aspect of the present invention to provide an integrated detection system using integrated photonics.
It is a further aspect of the present invention to provide a microfluidic perfusion system for enhancing transdermal transfer of biological molecules.

=
It is yet another aspect of the present invention to provide a thermal ablation meebttnism by resistive: heating for removal of the stratum comm.
It is still =Why aspect of the present invention to provide a laser ablation mtschaniant for removal of stratum cometmt It is a fiuther aspect of the present invention to provide a microfluidic tumbled Salami:02W MINTY action.
It is another aspect of the present invention to provide an adhesive for holding trimsdemnd tantpling system on skin It is anotimr aspect of dm present invention to provide a chemi,cal modification of channel surfaces with antibodies containing fluorescently labeled antigens that are expelled from the =face and detected down stream by competitive binding.
A greater understanding of the present invention and its concomitant advantages will be obtained by referring to the following figures and detailed description provided below.
BRIEF DESCRIPTION OF THE MURES
Figure I is a schematic illustration of the overall architecture of the rnicrosystem of the present invention.
Figure 2 illustrates in cross-section a single reservoir capillary pair.
Figure 3 shows test results obtained for a back of the band colorimetric test for blood alcohol.
Figure 4 shows the seal structure as viewed (a) from the bottom, and (b) in cross-section.

Figure 5 is a causs-sectka of a device of the ;Resent invention illustrating the non-invasive sampling sequence.
Figure 6 is a cross-sectional view illustrating the sequence of operation of the Bio-Fluidic Integrable Tamsdennal (B.M) microsystem.
Figure 7 schematkally illustrates the basic fakication stqm far the three main components of the system, shown in cross-section.
Figure 8 is a schematic illustration of a detection scheme using fluorescently kbeled proteins or metabolite&
Figure 9 illustrates in transverse section an alternative waveguide and sample to chamber configuration.
Figure 10 illustrates a Et-FTT mktrosystem.
Figure 11 illustrates a cross-sectional view of type C bed ilk:stating the detection scheme.
Figure 12 illustrates an overview of the ELM microsystem infotmational component.
Figure 13 illustrates a cross-sedional view of type CI showing the microfluidic interconnect, Coupling the external tubing with the silicon capillaty.
Figure 14 alushrides a cross-sectional view of an alternative which uses a silicon sleeve mould the DR113 =palmy hole, showing tbe silicon sleeve microfhidic interconnect, coupling the amoral tubing with the silicon capillary fabricated as wafer through-holes, sad external tubing connected to silicon capillary.
Figure 15 illustrates a aoss-section view of a third bed structure incorporating a collection chamber for the smalyte, which has flowed up through DRIB capillary through-wafer hole by capillary action.

Figure 16 illustrates a cross-sectional view of a fourth bed, designated type CIC, incorporating collection chamber and fluidic interconnect.
Figure 17 ilhanates the general fabrication process for the type CI array, showing, (a) photoresist (PR) patterning for silicon sleeve, (b) (=de patterning of s sleeve, (c) re-application of PR, (d) pattern for DRIB of bore hole, (a) remove PR and DIM sleeve, and (f) remove oxide.
Figure 18 illustrates a cross-section showing the double sided processing necessary to fabricate the type CC (and type CIC) device.
Figure 19 illustrates a magnified view of anchored spiropynms in a silicon io capillary.
Figure 20 illustrates a single reservoir capillary pair.
Figure 21 illustrates a single reservoir =pills' ry pair.
Figure 22 illustrates fabrication steps for wafer #2.
Figure 23 illustrates retention volumes at varying concentrations of (3H}-EB.
IS Figure 24 illustrates retention volumes at varying pH and ionic strengths.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides an enhanced system and method for monitoring the health ofan individual and delivering drugs to an individual 20 transdamally. Specifically, the present invention provides an integrated, cost-effective, rapid and =obtrusive assessment of a subject' medical condition.
The invention further provides means for transdanud delivery of drugs in response to the aforementioned assessment of a subject's medical condition.
Embodiments include, for example, monitoring a subject for pesticide exposure, 25 monitoring the Sten status of a war-fighter; phenotyping using the enzyme N-acetyl transferase to indicate an infected or diseased state; monitoring external exposure and internal contamination of a person with either organophosphate nerve agents (tabun, sark, soman) or organophosphate insecticides (parathion and metabolites thereof); monkeying inflammatory sequeli in response to microbial infection (interleukin-1, interleukin-6, tumor necrosis factor); monitoring microbial toxins (anthrax, botulimms, endotoxin);

monitoring spore metabolites arising from human catabolism via lymphatic or hepatic pathways; monitoring stimulants such as caffeine, antilisbunines (deacomethorphan, caffeine); monitoring stress through alterations in blood glucose concentration or altered metabolism of insulin/glucose.
An overall architecture of a prefened embodiment of the present invention is shown in Figure 1. The disposable B-FIT 100 is adapted to detect analytes of interest and is mounted in a receptacle 101 to provide mechanical support and electrical connections, including electrical connections to the thermal heaters of the B-FIT. The connection receptacle 101 also accurately ' aligns the B-FIT with respect to a switchable photonic bwicplane. The connection receptacle also preferably contains a power source 102; logic control 103; and electronic circuits for power management, electronic storage of results, electronic circuits for processing biochemical analysis data, electronic circuits for timing events, and metms for coromunicating results 104 either directly or via telemehy. Optical components are providedõ preferably located within B-.
FIT 100 or ME2v1S physiochip 106. In one embodiment, fluorescence measurements are made sequentially upon each of a plurality of analysis chambers contained in the B-F1T. Drug delivery chips 105 are also optionally provided by the present invention and are used to deliver potent drugs transdemmily, for example, drugs used to counteract nerve gas mew be 'delivers& In addition, a physiochip 106 is optiorudly provided that gathas continuous basic vital information, including blood pressure and pulse rate.
The mundenoal subsystem% located widdn the B-FIT and drug delivery chip, functions to contact the eldn with a physiologically compatible solution or a physiologically compatible soludon containing a drug. The B-Ft is organized into a dense army of sometvhat independent single reservoir capillary pairs.
The capillary pairs each comprise a reservoir capillary 211 for retaining a physiologically compatible solution, the reservoir having a breakable seal 215 (illustrated in a ruptured state), and an adjacent transport capillary 212 for transporting phyaiologically compatible solution. which has contacted skin. to an analyte measuring site. An adhesive Mier 216 is provided upon the lower surface of the B-FIT. In use, the adhesive layer is interposed between the lower surface ofthe B-FIT and the skin, and attaches the 8-FIT to the akin.
In a prefened embodiment, a thennal perforation subsystem functions to ablate a microscopic portion of the stratum comeum, the topmost layer ce skin, so that the interstitium can be exposed. The thermal perforation subsystem is preferably comprised of a miao-heater in close proximity to the skin surface, together with electrical components that control current to the micro-heaters.
A capillary away subsystem is preferably provided microfabricated into silicon wafers that comprise the B-FIT. The invention preferably provides a plurality of capillary-array subsystems, each Of which comprises a fluid delivay chamber or reservoir chamber 201 to deliver a fluid to the skin surface, a capillary channel 202 to recover fluid from the skin surface, and at least one transverse capillary channel in which the analyte or analytes we detected. The B-FIT 200 is prefect* comprised of a nrultilayered assembly of micromschined silicon wafers: a first wafer 204, a second wafer 206, and a detection layer 203. The detection layer preferably comprises a platonics system for visible or fluorescence messuremene, or a layer that comprises calorimetric reagents that develop a color change in the presence of an analyte, or other means for detection of an analyte. The capillary subsystem thus preferably comprises =pinnies for storage, passage and analysis of phydological fluids. The diameter and surface coatings (Idle capillaries are preferably optimized for conttoffing flow of the fluid and to prevent non-specific adsorition of fluid components onto the capillary walls.
An optional integrated photonics system is provided by the preset invention to determine, either qualitatively or quantitatively, the presence of one or more analytes. The Integrated photanica system comprises waveguides, ' lenses, mirtors, light sources, and light detectors. Preferably, the integrated photonics system is housed within connection receptacle 101, which is attached to a surfs= of B-FIT 100 that faces away nun the skin. In some embodiments, the integrated photonics subsystem is replaced by a colorimetric analyte sensitive region, wherein a color change, perceived directly by an observer, indicates the presence of an analyte.
Each of these subsystems and the interactions between the subsystems is described in getter detail below.
The B-FIT preferably contains an array of somewhat independent analyte sensing devices, termed "single reservoir capillary pairs" 200. As used herein, the teem "physiological fluid" represents a fluid that is biologically compatible with living tissue, and is, therefore, isotonically and otherwise physiologically (for example, pH) suitable as a medium for contacting for example, viable epidamid cells IN cells of the stratum comeum. Aa example of a physiological solution within the current meaning is physiological saline solution. Each single reservoir capillary pair preferably contains a reservoir capillary 201 that stores and releases a physiological fluid to irrigate the skin surface or a small region of the stratum comeum and recover analytes. A
breakable seal 205 is preferably provided to control the timing of the release of the fluid to irrigate the skin. The fluid is preferably recovered into a capillary channel 202 that caries the flukl to an analysis location, for example a detection patch 203. The transdermal subsystem preferably utilizes single reservoir capillary pairs to ensure that the analyte of interest, if presort, is accessible to the fluid.
As the tam is used in the present application, "transdamal dosimetry"
refete to the collection and detection of trace quantities of analytes that reach the surface Of the skin by passive diffiision from interstitial fluid underlying the outermost layer of skin, the snatum caneum. It will be appreciated that, in one embodiment of the present invention, the interstitial fluid is sampled for the presence of snalytes of intaesit. It will further be appreciated that, in another embodiment of the present invention, ablation of a microscopic portion of stratum comeum enables the physiologic solution from the reservoir to come into contact with the upper region of the underlying viable epidermis, enabling analytes in interstitial fluid to migrate into the physiologic solution via passive diffusion for analysis.
"Non-invasive tnundamal detection," as the term is used in the present application, maims detection of substation below the skin that is achieved without physical or chemical modifIcadons ofthe normal skin border. Small molecular weight snalyns dad exhibit both water and lipid solubilities can be sdapled try non-invasive tedmiques.
For examples, sweat can be sampled from the surface of the sldn and analyzed far alcohol content by a colorimetdc test indicative ofblood alcohol concentration, as illustrated in Figure 3. In this exempla of non-invasive detection, alcohol is detected in sweat *Wined from the backs of the hands of seven male 'subjects who have ingested 0-4 alcoholic drinks prior to the test Alcohol contains' d in dte sweat reacts with reagent, contained within a reactive to layer, resulting in a quantitadve mean= of alcohol content of the blood However, non-invasive techniques are not pracdcal where the analyte has a high molecular weight (for example, ptetein), is highly polar (for =maple, glucose), or is poorly soluble. The outward flux dutch molecules across the skin can be greatly enhanced by ablation of the stratum corneum. Ablation is peribrmed to a typical depth of 30-60 pm, exposing the underlying viable epidennis, from which fluid can be collected and analyzed for analytes that only poorly penetrate =ablated stratum comm. This technique is herein tamed "minimally invasive" because only the stratum come= is ablated while the underlying viable epidermis is not breached. In one preferred embodiment of the present invention, minimally invasive tnmsdermal detection is achieved by microscopic heat ablation of the stmum comeum layer. In anodmprefinred embodiment of the present invention, minimally invasive transdermal detecdon is achieved by laser ablation of the.stratum comma layer.
An adhesive layer preferably provides an interface between the device of the present invention and the skin. The adhesive layer is affixed to the lower surface of the B-FIT assembly and fimctions to attach the B-PIT assembly to a suitable pordon of skin surface, thereby minimizing motion of the B-PIT
assembly relative to the skin for efficient sampling. Gaps in the adhesive layer are provided over each capillary pair to permit the physiologic.al sohnion to contact the skin. The adhesive layer prevents leakage of fluid laterally, and is preferably comprised da Band-Aid-type adhesive that is relatively water impermeable.
It will be appreciated that that portion of the B-FIT that interfaces with the dermis preferably Notions to firmly and occhnively place the B-FIT
to system in direct contact the external surface of skin (stratum comeum) or uppermost region of the viable epidermis. Occlusive contact are preferably such that prevent lateral or vertical movement of the B-FIT from its initial position on the skin, that limit release of B-FIT materials externally, and preclude entry of external materials. Movement preventive properties include preferably an adhesive element located peripherally on the lowermost surface of the B-F1T and/or covering the entire B-FIT and adjacent skin surface.
Additionally, the lowermost surface of the B-FIT can be adhered to the demds to prevent sheer forces that would displace the B-F1T from its initial position.
The occlusive nature of the attachment of the B-FIT to the skin serves to confine all substances migrating from the body or skin within the B-Fff, including water vapor. This captured water wpor facilitates tranadamal permeation by hydrating the stratum comeum, naderhrg it more permeable to a wide variety of and,ytes or therapeutic drugs.
In one prefened embodiment of the present invention, minimally invasive heat ablation of the stratum comeum is employed to achieve significant enhancement of the efflux of certain analytes. In preferred embodiments, thermal ablation is used to remove the stratum comeum over a microscopic region of the skin through a mechanism of resistive heating. A miao-aNation %mit containing a micro-heater is preferably fabricated upon the surface of the B-PIT adjacent to each capillary pair, and provides a conductive heat path to the stratum coniam. The micro-heater preferably comprises a pair of electrodes competed by a conductive pathway that is arranged, either by the use of a resistive material or by a serpentine conductive pathway, to provide sufficient resistance to the flow of electricity such that an effective amount of heat is produced so as to locally ablate an appropriate portion of the stratum comeum.
Electrical connections are also provided to each of the two electrodes to connect the micro-heating unit to a controller that controls the application of an electrical current source to the electrodes. In preferred embodiments, it is advantageous that the micro-heater protrude finni the surface of the silicon substrate of the B-FIT to provide improved heat transfer to the stratum conteum and reduce the power consumption of the micro-heater. In one embodiment, a heat-sink material iiincorporated on top of the micro-heater to direct the thenn.al flow towards the skin barrier rather than through the bulk silicon material. In another embodiment, the micro-heater is fabricated onto a silicon mesa that protrudes from the main silicon substrate of the B-FIT. Such an embodiment may preferably require non-planar fabrication of electrical connections to ;amide analucting pathways from the silicon mesa to the contiguous bulk silicon substrate. Such non-planar fabrication techniques are known to those of slcill in the art, as illustrated in Paranjape et al., Technical Digest, 1997 hitemational Conference on Solid-State Sensors and Actuators, Chicago, Minois, Vol. 1, pp. 397 (1997).
The tbsnnal ablation micro-heater is pulsed with a suitable alternating or direct current to provide local ablation. Control of the duration and intensity of the heating pulse is preferably carried out to effect ablation of the correct area and depth. The micro-ablation preferably occurs in a confined volume of the stratum corneum of approximately 50 gm x 50 gm x 30 pm.
Figures 4(a) and 4(b) illustrate the seal structure as viewed (a) from the bottom and (b) in cross-section.
A physiological compatible solution that may or may not contain one or more dmgs is retained within the reservoir capillary 401 by a breakable seal 405 =
prior to use. The seal preferably provides an electronically addressable means for opening the reservoir capillary and contacting the skin surface or exposed stratum corneum to the physiological solution. The seal comprises a closure at the bottom end of the reservoir capillary and a means for opening the reservoir capillary. In a preferred embodiment, the seal comprises a thin membrane 400 that is preferably a dielectric bilayer that ruptures at elevated temperatures and a metal conducting path. Preferabli, any thin, non-toxic, membraneous material that is sufficiently tough not to tear prior to intended use, is not electrically conducting, and ruptures at elevated temperatures is a suitable material for use as the seal closure. A preferred material is low-stress nitride. Control of the film stresses of the membrane is required during fabrication. Kinard et al:, ME Trans. on Inst. Meas., 46(2), 347 (1998).
To fabricate the seal, a metal conducting path 402 is surface deposited upon a low-stress silicon dielectdc 400. Preferred metals for microheating elements include evanohm. Since the heat used to nipture the seal is optionally also used to ablate die skin in certain embodiments, a careful balance of film stresses, thickness and resistance is preferably achieved so as to provide both the desired heating and rupture properties. Deposition of the metal upon the fihn also requires deposition of metal Upon an irregular topography.
Such techniques are known to those of skill in the art. Geist et al., MST
Journal of Research 95(6), 631 (1990).
The conductive path preferably terminates at two electrical contact pads 403, 404, to facilitate passage of electricity through conductive pathway 402. In a preferred mode of operation, an electrical current passing through the thin conductive pathway heats the metal of conductive pathway 402 and causes the rupturing of the underlying dielectric layer, thus, opening the reservoir capillary. It should be noted that an advantage of this preferred embodimait of the present invention and this preferred sad, in particular, is that mechanical moving parts are absent, thereby enhancing reliability.
In certain preferred embodiments, the seal seals both the reservoir capillary and the capillary channel, and both are thereby opened simultaneously.
Figure 5 illustrates a B-FIT device cross-section, showing details on the non-invasivehninhnally invasive sampling sequence. An exemplary unused capillary pair 502 has an intact seal wherein the physiological solution is retained within the reservoir capillary; upon application of a suitable electric current, the seal 501 is ruptured and the physiological compatible solution first contacts the skin and is then recovered into the transport capillary; and filially a used capillary with a ruptured seal 500 is illustrated. In this preferred embodim' ent, each capillary pair functions as a single-use unit so as to utilize the seal and physiological solution.
Similarly, Figure 6 illustrates the sequence of operations of a minimally invasive embodiment of a B-FIT system to determine blood glucose concentration. A micro-heater 603 preferably operates to ablate a portion of the stratum corneum located below a gap in the adhesive layer 604 at the same time, or immediately prior to, rupture of the seal 601. Such a device provides an "on-demand" analysis. A physiological solution is preferably expelled onto the exposed viable epidermis; and recovered into the tnmsport capillary. The to transport iv Tillery preferably conducts the solution to a detection patch where the glucose is detected in a colorimanc reaction that produces a blue reaction.
Note that in this preferred embodiment, the current pulses delivered to the micro heater and the seal may be the same or different the heater and seal may, therefore, be electrically connected either in series or parallel.
Capillaries within the silicon body of the B-FIT device can preferably be fabricated by several techniques, for example by micro-machining, or by etching in place using deep resistive ion etching (DRIll) techniques.
Referring now to Figure 7, construction of a preferred embodiment of the B-FIT is illustrated. The device comprises three main parts: the main body 700 which is preferably made of sill= 702, and contains sevaal serpentine capillary channels 706, each with its own reservoir clumnel 707; a bottom capping section 701 that forms the lower part of the serpentine structure ad contains the micro-heating elements 703; and a top capping section 704, which foams the upper part of the serpentine channel 706, and which optionally contains electrodes for assisting the flow of physiological fluids using electro-osmotic pumping through the horizontal segments Of the serpentine channel. The top-capping section 704 is, in some embodiments, bonded to the main body: an advantage of such an arrangement is good coupling of light into the capillary that is thereby achieved. The main body is preferably made of silicon. T'he main = body 700 and the bottom capping section 701 are preferably permanently affixed to each other to comprise a sensor 705, that can, in certain embodiments, be detached from the top capping section 704 after use and replaced with a fresh way.
The reservoir and capillary channels are preferably fabricated within a =
standard silicon wafer. The dimensions of the capillaries are selected to facilitate the transport of sweat, interstitial fluid, or other physiological fluid, out of the open end of the reservoir under the force of gravity, and into a capillary channel through capillary action. In a preferred embodiment, the capillary channels are 25 pm in diameter and are approximately 500 pm in length, and the reservoir channels are 50 pm in diameter but are etched slightly shorter than 500 pm in length to provide a back wall. A lateral portion of the serpentine capillary channel 708 is provided, which provides for a region of fluid flow that is parallel and adjacent to the upper surface of the main body of the device for optical detection of analyte. The lower inside surface of the lateral portion Is optionally provided with a reflective surface, such as a reflective metal coating, to facilitate optical detection. The lateral portion of the serpentine capillary is, in a preferred embodiment, completed by a surface of the top capping section. In use, the transport òf physiological fluids and the recovery of analyte is enhanced by rinsing the skin with fluid previously maintained within the reservoir clumnel and then recovering the stung into the corresponding capillary charmel.
The surface of the capillazy sway system is preferably functionalized to improve the properties of the surface, for example to reeved adsorption of protein, andke to attach biomolecules such as antibodies to the surface.
Molecules that bind specific amdytes are used to immobilize analytes for subsequent detection and. quantitative analysis. Suitable biomolecules include, but are not limited to, antibodiea, antibody fragments, artificial antibodies, lectins, hybridizable nucleic acids, nucleic acid binding proteins, proteins that to bind nucleic acids, proteins that bind other proteins, proteins that.
bind cofactors, cofactors (for example, flavins, pterins, thiamine, pyridoxals, quinone), and other reagents that specifically bind biological tmalytes.
Capillsry tubes are preferably modified by either chemical or plasma treatment This step aids surface cleaning of organic contaminants and 13 introduces surface hydroxyl groups on the capillary surface, which are preferably reacted with a aflame such as aminopropyl trimethoxysilane (APTS) to provide a fme amine stoup as an anchor for coupling reagents such as antibodies. In a preferred embodiment, polyethykne glycol (PEG) silane derivalives are used to provide a surface coating that prevents adsorption of 20 protein.
In one embodiment, a solution containing antibodies directed to an analyte of interest is exposed to mildly crxidizing conditions known to those of skill in the art, which ptovides aldehyde groups upon the surface of the antibodies. The aldehyde functionality is then coupled to a free amine on the capillary tube surface via a Schiff base reaction, thus unmointszusg me antfblidp to the capillary tube surface.
In a preferred embodiment, detection of the analyte of interest is by done by means of fluorescence. A substance that is capable of specifically bindh*
an analyte (for example, an antibody) 802 is covalently attached to the surface of the capillary, as descrthed previously. The binding sites of the immobilized substance 802 are filled with fluorescently labeled analyte 801, prior to use of the invention. When analyte, 800, is present, it competes for the specific binding sites, displacing a portion of the labeled analyte molecules into the solution. The to degree of displacement of labeled analyte depends upon the concentration of analyte in the solution. Therefore, measurement of the amount of fluorescence displaced into the solution, when suitably calibrated, provides a quantitative measure of the concentration of analyte 800.
By preferably immobilizing a plurality of antibodies of different binding .
15 specificity, the binding sites of which are separately filled with their respective analytes tagged with fiuorophores with distinct emission and excitation spectra, multiple analyte determinations; can pnsferably be made within a shsgle capillary pair. The use of spectral filters and/or alternative light sources is used in a preferred embodiment to photoacite and detect fluorescence from the different 20 fluorophores, and thereby; determine the contribution of each fluorophore to the total fluorescent properties of the sample.
Preferred fiuorophores for the present invention include rhodamines, fluoresceins, Texas red, Oregon green, Bidipy dyes, and aminonaphthalenea In one embodiment, N-acetyl transfers" isozyme 2, (NAT-2) activity is 25 measured as a marker of adverse drug effects, toxicity and predisposition to disease. The NAT-2 phenotype can detected, for example, by detecting the redo of two metabolites of caffeine produced by NAT-2,

5-acetylamino-6-formylamino-3-methyl uracil (AMU) and 1-methybcanthine (IX). U1li2i3g the ratio of AFIAU to 1X, the activity of NAT-2 can be determined. Polyclonsd antibodies can be raised to these two metabolites and then purified. These antibodies can alai be used to detect AMU and 1X in urine samples by ELEA.
In a preferred embodiment &the present invention, the reservoir capillary is provided with a micro-heating element located at the opposite end of the capillary to thaseal. The micro-heater is activated to provide local heating of the physiological fluid so as to produce a bubble, thereby forcibly expelling the physiological solution from the capillary once the seal is ruptured. Note that the micro-heater fimctions as a pump means, but that the pumping is achieved without mechanically moving parts, thereby assuring increased reliability.
IS The micro-heating elements are preferably comprised of a resistive conducting pathway deposited by conventional deposition methods upon the surface of the silicon. Unlace the breakable seal, the heating elements are designed to withstand elevated temperatures without destruction of the conductive pathway. The conductive pathway is, in one preferred embodiment a serpentine pathway, in which a high-resistance pathway and localized heat generation sue achieved through the use of a serpentine pathway comprised of thin conductive pathways densely arranged upon within a small' surface area.
Another profaned aspect of the present invention is an integrated photonics analysis subsystem. The inWgration of photopics components into the B-FIT system permits increased density of assays, reduced size, lower power consumption, and decreased cost. In a preferred embodiment, the photonics components are housed within a plastic housing that comprises the top capping section of the device. Note that other detection methods are envisaged in the present invention and are discussed below.
In such a an integrated photonica analysis subsystem, photonics sources, for example LED's or lasers, are combined with detectors, waveguides, couplers, and minors, to provide a fully-integrated optical detection system for detecdng analytes in the present invention. The photonics components are preferably located upon, and attached to, the top surface of the main body of the B-FIT device in a top cappingsection.
Polymer waveguides with couplers for source and detector arrays are fabricated as integrated 'Ilex circuits* for mounting. Fully integrated waveguide structures= are fabricated by mama known to those of skill in the art, such as monolithic fabrication of the waveguide by dry resist processes. -Low n waveguide material < u. 1.33) is preferred.
Figure 9 illustrates a preferred embodiment of a waveguide and sample chamber. In a preferred embodiment, capillary fluorescence is used to detect the analyte within the capillary. LED sources emitting green, blue, yellow, or red light, can be used to excite fluorophores. The choice of exciting wavelength is dictated primarily by the excitation spectrum of each fluorophore. In other embodiments, laser sources can be used to provide specific excitation wavelengths, although the cost, size, and power consumption of lasers is generally higher than for LED's.
In a preferred embodiment, the upper inner surface of the lateral portion of the serpentine capillary is completed by a surface of the top capping section 900. Optical detection is preferably performed within the lateral portkm.
Light is conducted to and from the lateral portion by an integral waveguide fabricated within the, preferably plastic, top capping section. The orientation of the waveguide nms parallel to the silicon surface.
In another embodiment, the transverse capillary interrupts the path of the waveguide 903, so that the light conchmted by the waveguide passes directly through a pordon of the solution contained in transverse capably 900. This embodiment has the advantage of simplicity: lenses and mirrors are not required to divert and collate the lightbeam. Fluorescence OT absorbance measurements are preferably made within the portion of the transverse conduit that interrupts the waveguide. A preceding conduit portion 902 preferably contains the binding reagents that give rise to the displacement into the solution of fluorophore when analyte is present. Subsequent conduit 901 pref.:ably conducts the soludon out of the light Path-In an alternative embodiment of the present invention, light measurements are made within a capllkny that is constructed of a material =
having an Wet of refraction lower than that of water. This embodiment also eliminates the need for lenses and minors and offers superior signal to noise Properties.
Figure 10 illustrates a preferred embodiment of the B-Fit system platform. To facilitate the coupling of light from the waveguide 1008 into the latent portion, and from the lateral portion into the waveguide, a micro-minor 1005 is pr.:lib:ably provided. The minor is integrated as a pressed component in the top capping section, or is a separate component placed within the plastic housing by injection motdin& or is fabricated by any other appropriate means.

Preferably the micro-miner 1003 is rioted at approximately 45* relative to the silicon surface of the lateral portion of the capillary, and is positioned directly above the lateral section. A highly reflective surface coating, such as a metal coating, is preferably deposited upon the surface of the mirror to reflect light from the horizontal waveguide downwards into the lateral portion of the capillary. A lens is preferably provided to collate the fluorescence excitation and emission light beams. Micro-lens 1012 is, in one embodiment, convex to provide divergence of the light beam entering the lateral section flora the waveguide, and convergent with respect to light leaving the lateral portion and entering the waveguide 1008. In embodiments in which fluorescence detection is used, light from spectral region capable of exciting the fluorophme is conducted along the waveguide, stiles the divergent mirror and enters liquid ' contained within the lateral conduit. A fluorophore within the lateral conduit is preferably excited and emits light of a longer wavelength. The emitted light strikes the mirror, which converges the light, and re-enters the waveguide.
Bandpass or notch filters may preferably be interposed in the light path to optimize the signal-to-noise ratio of the detected fluorescence, depending on the bandwidth sensitivity of photodetector embodiment . Light sources for the integrated photonics analysis subsystem include LED's, which have recently become available in light-emission cokes from blue to green, thus essentially covering at least a portion of the excitation spectra of most commonly used fluorescent probes. See, for example, Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed. W.T. Mason, ed. Academic Press (1999). Alternatively, microelectronic lasers can preferably be used where specific wavelengths are required. Any light detection means can be used to detect the emitted fluorescent light. Photodiodes, phot=3transistors, Darlington pair phototransistors, or photoresistors can be fabricated onto the silicon surface of the main body, or can be provided as separate components.
Standard low power CMOS fabrication is preferably used to power the microsystem, to provide sequential logic control, and to permit storage of data in memory and its manipulation.
It should be noted that, despite the foregoing disclosure of fluorescence detection of analytes, the present invention is not restricted to fluorescence to measurements. Other detection methods that are advantageously used in the present invention include, but are not limited to, Raman, UV-VIS, and FUR
spectroscopy, including two-dimensional techniques, and fluorescence correlation spectroscopy. Furthermore, radiation senors and magnetic field sensors are also useful as the basis of detection in certain embodiments. For 15 monitoring radiation workers and the like, a preferred sensor embodiment is an optical random access memory (ORAM) material. These materials are composed of a photochromic molecule such as spirobenzopyram embedded in a poly(methyl methacxylate) matrix. The measurement approach is based upon measurement of radiation-induced tracks in optical memory media.
20 An optical deflection magic field sensor is preferably utilized where magnetic field monitoring is desired. The microsensor comprises an ahaninum beam that is suspended above a micromachined silicon substrate using four alumimmi support arms. These arms hold the beam at its nodal points, which are points of zero displace:mat when the beam vibrates at the fundamental resonant 25 frequency. A sinusoidal current is forced to flow through one support arm, through the length of the beam, and out thrones the other support am. The frequency of the sinusoidal current is essentially identical to that of the mechanical resonant frequency of the beam. In the absence of a maroctic field, the beam is unaffected. However, in the presence of a magnetic field oriented 3 peependiadar to the beam, a magnetic force causes deflection of curia%
which in turn cantos the beam to vibrate at its resonant frequency. The amplitude of the vibration is directly proportional to the magnetic field strength, which am be measured using a laser.
Figure 10 illustrates the operation of an embodiment of the II-FIT
systaa with respect to analyte detection. The physiological soludon preferably contacts the exposed viable epidermis following operation of microheaters 1006 to ablate a portion of the stratum comeum, rupture the seal, and expel the physiological solution front the reservoir channel 1002. Solution containing analyte recovered from the interstitial fluid bahhig the viable epidermis ts preferably enters the capillary channe11004. Within the capillary channel, analyte displaces fluorescently labeled analyte from analyte binding molecules affixed to the capillary walls. Displaced fluorescently labeled analyte is preferably carried to the lateral portion where it is excited by light conducted by the waveguide 1008, micto-mirror 1005, and micro-lens 1012. Light of a longer wavelength that is emitted by the fluarophore is, in one preferred embodiment, conducted back into the waveguide 1000 by the reversed optical pathway, and propagates to a detector.
The integration aspect of the present invention also preferably includes the aspect that real-time monitoring of a subject permits the use of adaptive 23 contml algorithms to optimize the conditiout Mr example, heating pulse charactedstica sampling rate, among others), and drug delivery regimen, in response to data obtained. In this preferred embodiment of the invention, data machine-learning tecimiques are preferably employed to derive or learn some function that relates one measure of the health of a subject to analyte = measurements, thereby possibly acquiring the ability to predict the health measure from subsequent analyte measurements. Adaptive control algorithms utilized in the present invention embody the steps of learniitg, adaptation, feedback, and decision-making. Since the body is a dynamic system, these steps occur simultaneously and continuously throughout the life of the device of the present invention.
Figure 12 illustrates an overview of the ELISA microsystan informational component. A preferred aspect of the present invention is the large number of individual measurements that are possible over an extended time period,. With extended periods of measurement, baseline drift must be accounted for so that significant deviations are accunstely detected. The present invention preferably provides computational means for accounting for baseline drift, and for thereby detecting deviations from a current baseline. This means is illustrated for an embodiment directed to monitoring health in a subject. With improved monitoring techniques, day-to-day variations in metabolism are preferably established in the healthy individual, and limits set to detect early stages of infection, disease progression, and exposure to toxins.
The metabolism of exogenous compounds such as drugs is mediated by a series of enzymes. The type and amount of these enzymes in each individual is reflected in the person's genotype and, based upon the genetic information, individuals can be classified as more efficient metabolizers (FAST) and others as less efficient metabolizers (SLOW). In healthy individuals, the relationship' between genetic makeup (genotype) and its expression (phenotype) is conserved, i.e. FAST genotypes produce FAST phenotypes, while SLOW
genotypes produce SLOW phenotypes. However, a discs* state of the, individual can alter this relationship, as can diet, smoking, alcohol, environmental chemicals, and biological or chemical warfare agents, among other factors. The determination of a person's NAT-2 genotype and the monitoring of that individual's NAT-2 phenotype can be used as a direct and sensitive probe of heath and clinical status.
In this approach, polyclonal antibodies are preferably developed against the caffeine metabolites AFMU and 1X, and are used to determine NAT-2 phenotypes in an embodiment of the present invention. Blood glucose levels, cytokine levels, and dextromethorphan metabolite levels, can also be monitored.
Machine-leaming algorithms are preferably used to acquire a metabolic baseline and to indicate when an individuars body begins to enter a state of distress or disease. The Winnow and Weighted-Majority Algorithms (Littlestone & Watmuth, Information and Computations 108, 212, (1994) can preferably be used. These algorithms, with well-understood fonnal properties, are capable of learning and performing in non-stationary environments (i.e., in the presence of baseline drift).
The readings of the two caffeine metabolites, AFMU and 1X, are pmferably provided as inputs for computation, and the computation preferably proceeds in two alternating and cooperative modes: a learning mode and a performance mode. In the laming mode, the device preferably continually calibrates itself to the wearer's body chemishy using an adaptive algorithm, which adjusts a set of weights, With the aid of feedback. User hgeraction is =

necessary only if the body is stimulated in such a way that the levels of the metabolites are not imitative of normal body function (Le., the user svill provide feedback only thr false-negatives). In the performance mode, the device preferably takes the readinp of the caffehre metabolites and, using the =rent concept descripthms (Le, weights), mabm a decision about the body's state of health, which is then commimicated to the user. Since the body is a dynamic system, this process of leaning, adaptadon, feedback, and decision-making preferably occurs conthmously and throughout the life of the device.
The device also prefriably acquires a model of the weaves healthy state, and uses this model to predict states of health in the future.
Formally, maohine-learning methods dedve or teen some Ration fn from a set of lc, y pairs, such that ri f(tc). Naturally, fo is an approximation to the tme fuoction, which is unknown.
is However, when levels o say, troponin I begin to increase (suggesting an irmninent heart attack), than the device will preferably need to sample more frequently, as the rate of clumge from one measurement to the next will be increasing. In this situation, adaptive control algoridmut are preferably employed, powerful enough to properly control the sampling rate, but simple enough to be realized in micrO-hardware.
Thus, in a preferred embodiment, adapdve control algorithms can be used to task the tranadermal compcment to sample its wearer for the target substances, and machine-leandng algorithms can be used to acquire a model, which may change, of the wearer's healthy state.

=

Reng back, Figure 10 illustrates a preferred embocliment of a B-F1T
mierosystem. This total modular system preferably includes: (1) the fluid transport system including reservoir dnumel 1002, and capillary channel 1004;
(2) micro-heatee(s)1006, (3) the photonics system including waveguide 1008, micro-mirror 1005, and raicro-lens 1012, and (4) the chemistry for analysis of selected analytes. The iramatitial fluid containing molecules indicative of biomadcas are prefinably obtained using a minimally invasive tecimique employing controlled thermal micro-ablation of the stratum commun. The micm4eater(s)1006 used for this are preferably incoxporated directly into the ilicon-based subsystem antis part of the B-FIT microsystem. For opthnal hunsport of interstitial fluids or enables through the analysis capillary. a second reservoir capillary, containing a physiologlisdly impale fluid, is preferably used to drive all fluids towards the upper surface of the module. The driving force is preferably provided by microhester(s)1006 that prodece bubbles to lime the liquid to flow out of the memoir capillary and over the thermally ablated region of the skin. Once the interstitial and physiological appropriate liquids containing tagged and =tagged mole:ides mach the top helding-civity, analysis can begin. The top of this total tiensclermal detection platform can prefixebly be integrated with optical waveguides, comprising micro-minor(s)1005 and micto-lensea 1012 for propedy directin" g the light within the holding-chamber. The light that strikes the analysis region of the holding-cavity is used to excite the fiumeecenliy tagged molecules. The intensity of this fluorescence is preferably picked up through the mina path by the same optical wavegtdde.

The modular nature of the microsystem provides an excellent platform that can be easily adapted for many innovative applications by applying new cab:miseries for the detection of selected analytes. For example, one analyte or biomarker that is especially important to children exposed to pesticides is acetylcholine. Acetylcholine is located throughout the body and when it is released, it acts as am excitatory neurotransmitter to propagate nerve conduction in the periphaal and central nervous systems, or to initiate muscle contraction.
Exposing to organophosphorus pesticides causes inhibition of acetylcholinesterase activity resulting in an accumulation of acetylcholine.
This increase in acetylcholine concentration will act as a biomarkee, measured using the device by first establishing a baseline in an unexposed child. The MEMS-based patch is small and unobtrusive, permitting a child to live his/her daily life while being continuously monitored for impost= to pesticide) contamination and providing early warning diagnostics.
Thus, one embodiment of the portable biomedical monitoring device of the present invention is as a pediatric micro patch system (PuP). In providing such a Pt& device there are three tasks. Task 1 is the fabrication of silicon bed structures that fimction analogous to the B-FTT Microsystem. As described above, the bed fimctions to deliver fluids to the interior of the capillaries and to the collection chamber. In addition, the bed minors the B-FIT microsystem with regard to the integration of the chemistry. Task 2 is the chemistry to detect acetylcholine. Ibis task includes chemically modifying a flat sample of silicon, which enables a fbnctioning method for integrating chemistry to the bed. Task involves the testing and validation phase, where the chemistry protocol is adopted for the capillary bed. Detection limits ot acetytchohne are established and sample bodily interstitiai fluid is tested.
With regard to the B-FIT system, the fabrication of the capillary bed structures relies, in one embodiment, on ballt miciomachining of silicon, accomplished linough either deep reactive ion etching (DR1E) or wet chemical etching. The DRIE process preferably enables the fabrication of high aspect mtio through-wafer holes that fonn narrow micro-capillaries of varying diameters. Wafers with a nominal thickness of 500 pm are used; however, a preferred thickness can be established through surface modification testing.
to For the exemplary bed structure shown in Figure 10, (type C), an army of capillaries with varying diameters me preferably formed using lithographic patterning and DRIE. This type of structure enables selection for the optimal dimensions required for capillary action to allow liquids to be drawn up and inside the channel, because wet chemistries are involved in both capillary-wall 15 surface modifications and during fluorescence validation using a test solution contain' ing acetylcholine. Once the capillaries have been chemically modified, testing for fluorescence is preferably accomplished using a laser source at the top-side entrance port of the silicon capillary, and a detector located at the exit port on the bottom-side.
20 Returning to Figure 1 1 illustrating this preferred detection scheme, the spot size of the laser light path 1102 can preferably be adjusted to match the diameter of silicon capillary hole 1104 etched in silicon substmte 1106, while its excitation wavelength is preferably held at 430 run, to match the frequency required to excite the fluorophore causing it to emit fluorescent light 1108.
The 25 detector preferably includes photomultiplier 1110, and monocluomator 1112 is prekrably used to tune the detector to the fluorescence wavelength of 567 um.
In addition, notch filter 1114 is preksably used to greatly attenuate the unwanted laser light frequency from reaching the photomultiplier.
A second exemplary bed structure, termed type CI, is similar to the bask capillary array with a modification to the adatace port tint is fitted with a microfluidic interconnect. This design is prefbrably used as an alternative to type C, in a situation where capillary action perhaps does not &notion appropriately. In such circumstences, type CI provides an interconnect mechanism allowing for external tubing or syringe ports to be directly coupled to the silicon capillary for surface modification and tesdng purposes.
= Figure 13 illustrates a cross-sectional view of a type CI bed structure showing the microfluidic interconnect, coupling the external tubing with the silicon capillary. Typically, a hole produced by DRIB can preferably be made so that its inner and outer diameters match that of the interconnect tubing, which is inserted into the opening and held in place with adhesive 1301 Thus, DRS
microcapillaties fabdcated as wafer through holes 1304 are in silicon substrate 1306. The holes are preferably produced such that the inner and outer diameters match that of external tubing connected to silicon capillary 1308, wherein the tubing is held in place with adhesive 1310. However, care must be taken such that the adhesive used to hold the tubing does not seep into the capillaries blocking the flow.
Figure 14 illustrates across-sectional view of an alternadve embodiment which uses a silicon sleeve around the DRIB capillary hole, showing the silicon sleeve microfluidic internonnect, coupling the external tubing with the silicon capillary. The sleeve preferably provides enhanced mechanical integrity for the externs' flc coreponme, but also prevents adhesive 1402 torn seeping and plugging the capilley hole. Once the external tubing is attached to the silicon subetrate, chemicals and analytes can prehrahly be injected using either pressure gradients or syringe. The external tubing is than removed, having Milled its purpose of introducing fluids into the norm capillary channel. and the verification procedure to detect fluorescence can start, as for the type C

device. Figure 14 thus shows adhesive 1402, silicon substrate 1404, DRIB
mictocapillaries fabricated as wafer through-holes 1406, and external tubing connected to silicon cuplliary 1408.
Figure 15 illustates a. acus-stetional view of a third bed structure incorporating a collection dumber for thes;nalyte, which hes flowed up through DRIB capillary through-wafer hole 1502 by capillary action..nu this microstructure, called type CC, the silicon capillary is profitably fabricated using DRIB followed by an anisottopk wet silicon etch to create the collection = chamber on the fion-aide of silicon substrate 1504. vrith Ws bed, it is sufficient to chemically mortify only the surfice of this dumber. The analyte preferably flows up the capillary charmed and reacts with the immobilized chemistry in the chamber to produce florescence light path 1506.
Figure 15 also 'Mutates preferred excitation laser 1508 and fluorescence light path 1510 detection set-up. The excitation and detection method preferably mares use denied= leer 1508, photomultiplier 1512, and monochrometer 1514, respectively, as beton, however, now the setup is preferably only (nth* fandraide ofthe bed structure. The photomultiplier 1512 rod monochrome 1514 are preferably set directly above the collection zs reservoir to act as the fluorescence detector. In this sited." the impinging laser light can preferably be directed towards the collecdon chamber at an angle such that its reflection does not caddbute to idrotomidtiplier 1512 detection.

Nevertheless, notch-filter 1516 can preferably be used between the detection unit and the chamber to eliminate any stray light fix= excitation laser 1508.
Figure 16 illustrates a cross-sectional view of a fourth pretkaed bed, designated type ac, inccaporadng collection chamber 1602 and fitddic Interconnect. In addition to the silicon capillary chtumel and collection chantber of type CC, tide bed preferably also includes a fluidic interconnect mechanism on the beck-side of the wafer. As befbre, this design am prorate* save as a io fidlback mechanism if the capillary action does not provide enough capillary force to draw the fluid up to the collection chamber. The fluidic interconnect preferably mattes use of the silicon sleeve 1604, as described in the type CI
test-bed structure. DRD3 cspillay through-walk hole 1606 and silicon substrate 1606 are also shown.
is For each variation in bed structure, Mikados of the through-wafer capillary arrays is made on singlo-sided polished, <100>-type 4 inch silicon wafers. The army of capillary holes preferably consists of four diameter values (25 SAM, SO pm. 75 pm, and 100 pm) with a nominal length of 500 m, which corresponds to the waft thickness. For the type C design, the patterns for the 20 holes are preferably formed in a photonsist layer, which acts as an idad masking law to the DRIB process. A single (standard) photolithographic step prefendgy produces patterns on the fioat-side of the polished silicon seduce.
Although the DRM process renders ari anisotropically etched cavity, some undercutthig of the mask takes place. Thus, the pattern of the mask takes into 25 account this unavoidable lateral etch to achieve the desired diameters for the capillaries. Depending on the type of DRIE system used, the ratio of vertical-to-lateral etch is better than 5040-1. That is, for every 50 pm of etch depth, there is approximately I ism of under-etch beneath the masking hryer.
The maddng dimensions are therefore dependent on this etch parameter, which can be determined through prior testing. DRIB services can be obtained, for example,through one albs National Nanafabdcation Facilities, or through the MEM Exchange program.
Figures 17(a) to (f) illustrate a prefermd general fabrication process for the type CI array, showing. (a) photoresist (PR) patterning for silicon sleeve, (b) oxide inflaming of sleeve, (c) re-application of PR, (d) pattern for DRIE of bore hole, (e) remove PR and DRIE sleeve, and (i) remove oxide. The initial step, prior to lithography, is to grow a thin layer of thennal silicon dioxide over the entire silicon surface. Photormist is applied, and the oxide is patterned and etched to delineate the locations of the silicon sleeves that are located around each capillary hole providing for microfluidic interconnections. This step is followed by another application of photoresist, and the capillary locations are patterned into both photoresist and oxide. Thtough-wafer holes are again formed using DRIE, as with the type C device. The photoresist is subsequently removed, leaving the pre-patterned layer of thermal oxide on the front-side of the silicon surface. A much shorter DRIE step is performed, with the oxide layer acting as the masking layer, to create the silicon sleeve.
For both type CC and CIC the thbricalion process preferably requires the wafers to be double-sided polished since back to front alipment is required.
For the type CC device, the collection chamber is preferably bulk micromachined into the front-side of the wafer using an anisotropic wet chanical etches*

Subsequently, a thin thermal oxide is grown only on thetrontartdbidiacrats passivation layer, while the back-side is coated with photoresist. The DUI
procedure for tha capillaries is perftemed from the back-side so the capillary hole aligns with one side of tbs collection chamber.
Figure 18 illustrates a cross-section showing the double sided processing = necessary to fabricate the type CC (and type CIC) device. The wafer is then inverted so that subsequent processing of the capillaries and silicon sleeve interconnects on the beck-side follows the same procedure as that for the type CI device.
With regard to a preferred surface modification aspect of the present invention, the technical approach to providing a surface bound fluorescent probe specific for the biomarlosr acetylcholine is preferably accomplished by modifying the method developed for liquid phase detection set faith in Inouye, M. et al., Nondestructive Detection of Acetyl Cholla. in Protic Media Artificial Signaling Acetylcholine Receptors, J. Am. Chem. Soc., 116, 5517 (1994). In a preferred embodiment, the method utilizes spiropyrans, which are inexpensive and readity available from commercial sources. They are known for their spectral properties and are very robust, especially compared with molecules used for standard ELISA detection methods. The spiropyrans are synthetically surface immobilized on the silicon bed, in either a collection chamber or in a capillary, using slime chemistry anistandard coupling chemistry.
Pio= 19 illustrates a magnified view of anchored spiropyrans in a silicon capillary. A spitopyran (for example, C-methylcalix[4]resorcinarene) is preferably modified to incorporate a carboxylic acid cross-linking group that can be coupled to a free amino-silane modified silicon surface. Stoichiometric addition of base to the spiropyran allows for the reaction of an ce-bromocarboxylic acid (for example, 5-bromopentanoic arid). The length of this molecule is rekded to its solubility and reaction efficiency. Longer carbon chain-lengths are more soluble, but harder to couple to tit* surface, while longer carbon chain-lengths are less soluble and more Moly to couple to the surface.
The synthesis can be followed by NMR spectroscopy as needed to examin' e and characterize reaction products.
The spiropyran or resorcinol/acetaldehyde tettamer preferably forms a tetraphenolate inakaline media that arranges in a bowl shaped cavity and can to complex alkylammonium cations. When complexed with a pyrene modified N-alkyl pyridinium cation (PPC), no fluorescence is observed. The PPC may be purchased or synthesized depending on the selected method. One preferred method of incorporation of PPC is by solution complexation with the spiropyran. After anchoring the spiropyran, PPC is introducectind the complex 15 is formed. The competitive binding of acetylcholine kicks off PPC and produces a fluorescent complex. This complex is detected using the laser/detector scheme described above in the microfabrication approach. PPC can also be incorporated by forming mixed monolayers of the spiropyran and the PPC. In this way, complexes are formed at the solution surface interface. Another Feigned way 20 of making PPC complex with the spiropyran is by synthetically attaching PPC
to the spiropyren as dossed* by Inouye et al., supra. This method allows for intramolecular quenching of the fluorescence as opposed to intermolecular quenching as described in the first two methods.
Upon completion of synthesis, silicon substrates are preferably 25 derivatized with silence such as 3-aminopropyltrimethoxysilane. The reaction provides a five amino group Oa the silicon mime that can be coupled using a water-soluble carboditaide, such u EDC, to the carboxylic acid Of the modified spiropyran. X-ray plmtoeleckon specttoecopy (cps) and contact Ingle measuzements can be employed to analyze the progress of varying surface attacimuut reactions. In a prekked embodiment, the highest mike coverage is achieved. In addliiem to surface coves" the fluorescence efficiencies is examined using a damascenes microscope. This aids in qualifying the activity of the stashed spiropyrans. A simple experiment monitoring the qualitative fhaxescence intensity bedbm addition of acetylcholine and after the addition of to acetylcholine provides a baseline. At this point, tbe method is tremsitioned into bed devices iix testing.
During the microfabrication task, DRIE and wet chemical etch rates are paten* deenstined using teit samples in order to produce the proper bed structures. In addition, preferably samples are cleaved and viewed through a ts scmming electron microscope to determine whether the proper aoss-sectional geometry ofthe through-we capillades ha been achieved. For the chemistry task, surface modification and chemical synthesis is palatably used to validate the immobilization premed on a fist sample of silicon. This determination requires the detection offinorescence after excitation on a relatively large 20 sample; thus, a fiumescence ndcroacope is used during dds testing procedures.
Once the chemical synthesis and swam modification tasks are completed through rigorous huge ample testing, the chemistry is then tested on the small-scale capffiaty bed structenes. For this phase, a photonics-based test set-up is prelim* en4goyed based on the excitation and emission of 25 fluorophores. Exdtadon is through direct absorption ftom an external laser source. Several 'referred sources are available for various testing strategies, including tunable continuous wave (CW) argon ion pumped dye laser, an air-cooled argon ion laser, a Nd:YAG nanosecond pulsed laser which pumps an optical parametric oscillator, and several smaller HeNe lasers with both red (632.8 am) and green (543 nm) wavelength outputs. The Ar ion pumped dye laser has outputs from the pump laser at wavelengths of 488 nm and 514 rim, with a maximum power of 9W. The maximum power from the CV/ dye laser is 3W, and is trmable in the ranges of 590 nm. to 600 nm and 610 nm to 630 nm, with an additional output at 577 nm. The air-cooled Ar ion laser has a single output at 514 nm and provides approximately 70 mW of power. The Nd:YAG
laser has a ftmdamental wavelength of 1064 nm, along with the doubled (532 nm) and tripled (3.55 mn) outputs achieved with internal harmonic generators (KDP crystals). The pulse width is S to 7 nanoseconds, with peak pulse powers of over 200 ml. However, since the fluorophore being immobilized on the silicon surface needs to be excited at 430 nm, the power from the tripled output Nd:YAG law can be used to pump an optical parametric oscillator (0P0), based on a beta Barium borate crystal (BBO). This is essentially a resonant optical cavity containing the nonlinear BBO crystal. The pump beam is converted to the so-called signal and idler beams, where the wavelengths following the relation:
+ramp beer+ +slew which arises from the photon energy conservation requirement The ratio between the two output wavelengths is governed by the angle of the Bl30 crystal with respect to the incident beam. Using this, the output wavelength can be tuned by changing the crystal angle. Either the signal or idler output can be elimbiated using a high- or low-pass optical filter at the output port of the OPO.
Tide output is tunable in different ranges ikom about 400 nm. to 2200 nm. The ranges are set by the resonimt cavity inirxrir properdes and the output filters.
Peak pulse energies in these roses me on the orderof I0 The output fluorescence is prefenbly detected either in the forward direction or at some imgle depending on the microstructure gecanehy. In either case, the incident laser light is preferably blocked using both a holographic notch filter and a monochromatot. 'The notch filter, commtndy used in Raman spectrometers, cuts all light at the wavelemgth ofthe excitation light, whether coining directly from the laser or from Rayleigh scattering. The monocbromator provides Author rejection dm/anted light, both ambient and num the laser.
Th.e monochromator also allows tuning to the maidminn of the emission spectrum, for optimization of the signal-to-noise ratio. Finally, detection is preferably accomplished using a photomultiplier and a boxce,brtegrator 13 detection scheme. with pdng from the laser electronics. Alteniately, the signal is detected with a silicon avalimche photodiode, providing higher detection efficiency.
With the type C bed, capillary action is preferably tested bi order to, first, modify the internal silicon surface wall of the capillary hole, and then, second, to draw up suety* 'which reacts with the immobilized sidewall chemistry. Lf the capillary fortes are not sufficient to draw up significant amounts of fhdd for die capillary dimensions being tested, then the type CI
test-bed is preferably used. This allows direct pb,ysical insertion of fluids within the capillary using a pressure gradient or a syrinp pump comiected to the micrufluidic interconnects. In either case, the photonic detection system b prefbrably used to test the feaslisility of performing in-capillary fluorescence measurement& In contrut, tbe type CC test-bed requires chemical modification only withhs the silicon surface of the collusion chamber. Therefore, tbis device can also be used to test capillaty action as well as the photonic detection system situated on the front-side of the wafer. Again, the device can preferably be tested physically by hued* the analyte into the capillary array, using bed type CIC.
Once a particular bed is selected, further testing preferably relates to determining the selectivity and sensidvity of the biomarker to the immobliked chemistry within the capillary army. Mk type oftesting is preferably conducted, for example, by introducing* solution containing acetylcholine at varying concentration levels. By determining the amount of fluorescence variation, using ihe output ikint the photomultiplier, with corresponding changes in acetylcholine concentration ieveb, a quantitative inclicadon of the lower limit of deiced= of acetykholine, and therefore device smnithity, is obtained. To determine selectivity, another series &tests are preferably performed to introduce other nenvotransmittes in venous concentrations within the anaiyte solution and to determine their rektive fluorescence with respect to that obtained fbr acetylcholine. The mbre common neurotransmitters that can be used in this testing phase, other than acetylcholine, include adrenaline, dopamine, suotonin, tryptamine, histamine, and Amine. Fat comprehensive testing of selectivky, the device is preferably tested thr other neurotransmitters such as noradretudine, tylemine, ghnsmic acid, asps* acid, tanzineõ and proline that could introduce an unwanted aoss-sensitivity fluorescence 23 responsa Finally, in vino testing of the device preferably using traditionally extracted interstitial fluid from human donors is conducted. Samples are taken from individuals exposed to high levels of pesticides, and a control set is taken from those who were not exposed to toxic environments.
A sample result of this fabrication and testing process is preferably a silicon-based capillary ansy bed (PO) with a chemical immobilization protocol for the detection of acetylcholine, which is a biomarker for ogannphosphate type pesticide exposure. In addition, a sensitivity profile is established.
The PAP
microstructure design allows it to be readily interfaced and integrated as a module to the B-FIT trimsdermal sampling platform. The transdermal sampling process is preferably initiated using a minimally invasive micro-thermal ablation heater to reach the stratum comeum/viable epidermis' interface, allowing for extraction of interstitial fluid. The B-FIT microsystem makes use of silicon fabricated capillary ansys to allow for interstitial fluid transport to a glucose-sensing patch situated on top of the array. The basic capillary array structure of the PpP microdevice can be incorporated into the B-FIT. The detection mechanism for the biomarker acetylcholine in PpP preferably consists of the synthesis of a fluorescent spyropyran that is surfer.. immobilized With the chemistry identified, surface modification of the bed is carried out, allowing for ease of manufacture of low-cost, mieireidty-intrusive chip scale detection.
The chemistry developed relies upon an alternate preferred embodiment of the B-FIT microsysasm device, incorporating a photonics component instead of a glucose patch. This system is preferably fitted with waveguide technology on the top of the amry, which is used to transmit and detect excitation and fluorescence light, respectively. Again, the PpP microdevice is ideally suited as a module to the B-FfT, in view of the fluoresamce detection of acelylcholine.

Testing of simulated body fluid and human serum am preferably be done on the test bed to determine the samidvity and the specificity of the chemistry.
The present invention allows for the fabrication and chendcal 'immobiliation of any number of biamarkas, thaeby creating a set of modules to be "plugged into" the B-FIT platform. Numerous examples, possibilities, and applications exist, rosins from avast number of molecmlar biomakers for health monitoring through enzyme end metabcdite detection, to hotmones. For pesticide detection some of the other key biomakas would bet acetylcholinestetase, acetic acid, and choline. It is also important to detect other to analytes, aside from orgenophosphates, made possible through the use of the PAP microdevice. These include smticholinesterese insecticides (phosphorothicmates), organoc.hlorine insecticides (DDT, Dieldrin, Lindane), pyrethroid insecticides (Nemeth** Fenvalerate), habicidea (IVDD, Pasqua), and rodenticides (Warfarin, Diphacinone, sodium fiuoroacetate, strychnine).
Other key biomarkers to trace would be the antidotes such as atropine and pralidoxime.
Processing steps and the respective equipment in the fatnication of the PAP preferably include the following: (I) lithography: a front-side mask aligner capable of 1 Am line resolution with UV and deep-UV photolithography; a fixture capable of two sided alignmen4 a photo-resist spinner, pre- and post-bake ovens and associated processing chemicals: (2) deposition: a magnetron sputtedng system capable of depositing metals (Al, W, Nl, 11, Pt, etc.), and magnetron reactive sputtering of oxides which can be provided in the fainicatiox an e-beam evaporator with three hearths for low energy deposition of metals; and deposition apparatus for PECVD oxides and nitrides for coating seams to ad*. t fir stresses and adhesion; (3) film treatment, to adjust the stresses and strengths of films and membranes using rapid thermal annealing capability; (4) photo-mask design and fabrication; (5) etching: deep reactive ion etcher (DRIE), RIB equipment and wet TMAH etching (6) diffusion and heat treatment: high tempuatme furnaces capable of wet and dry oxide growth and famace soak annealing which can be required for heaters comprised of dolled silicon; and (7) measurement a thin film stress tester and a Leitz thin film analyzer or a Nanometrics Automatic Film Thickness measuring apparatus for measuring film thickness. becroscopic examination is available with a high quality Leitz microscope and a Zeiss SEM with EDS.
The transdermal transfer system (1'rs) is preferably manufactured using various standard processing and fabrication technologies. The TTS microdevice fabrication also relies on several miaomadtin. ins steps, from simple bulk mioromachining to deep reactive ion etching (DIM) procedures.
The fabrication process steps of the ITS microdevice prefesably involve silicon processing of two wafers, as indicated in Figure 20. Wafer #1 preferably comprises the reservoir channel, capillary channel, micro-ablation unit, and bre:skill* seal. The micro-ablation unit contains the micro-heater along with a heat-sink to provide a highly conductive thermal path towards the stratum corneum. By incorporating a heat-sink on the micro-heater, the heat transfer is more favorably directed towards the stratum corneum. Wafer #2 preferably contains the reservoir micro-heater, which is preferably aligned to mate with the top of the reservoir channel.
Figure 21(a-e) providers cross-sectional fabrication diagrams of the wafer processing steps for wafer #1. Double-sided polished, 300 gm thick silicon walks me pinfembly used in the progenies steps because world g will be done on both the fmnt and back sides. Initially, in one embodiment, the microablation heater is formed by depositing and patterning a mekd layer onto a patterned silicon dielntric law to form a serpentine bating element dnough widch cusent is passed. The dielectric lspantbrably panned as square region where the heating element resides. A preferred bona menial can be selected dsough thermal simtdation. In addition to the heating element, a temperature sensor can preferably be integrated akesside to monitor the local temperatures generated by the curten through the beadng coil. This is indicated in Figure 2Is, whine processing occurs on the top-aide ofthe walls, but will eventually be inverted to become tie bottom. In tide version of the design, all bonding peds and traces are located in the plane of the heating element. The metallic traces and heating elements an profitably instdated and protected by depositing a layer of low-stress silicon nitride across the wafer. Although et:en-free nitride is not required fbs this passivation putpose, it can find an application in the subsequent step.
Refining back to Figure 4 (a-b), the second step is preferably to fabricate the breakable seal. The seal is preferably composed of a bilayer formed by the low-skess silicon nitride* layer, deposited in the earlier step, and a metal that can weaken at elevated temperatures. The area where the metal is deposited determines tie location of the reservoir capillary. Since the capillary dimenske is prole:ably on the order of 75 tun, the breakable seal must be situated in this 75 Au region in ceder to open the reservoir capillary. Firms shows the bottom view design of the Washable seal. It consists of two low-stress silicon nitride flaps brldpd by the seal metaL By passing large enough currents through this metal strip, the heat is AM* 00 weaken the metal seal thereby releasing the nitride daps. Although the nitride layer is low in stress, preferably there is some controllable tensile or compressive strain.
By adjusting the deposition conditions of the silicon nitride, it can be made in slight compression so that when the seal ruptures, the nitride lisps crudely act as unidirectional valves.
The preferred third major processing step is to form the heat-sink on top of the micro-ablation besting element. As discussed above, a hen-sink prefers* directs the hest towards the atm= =num instead of within the to bulk silicon material. 'Without the hest-stink the majority of the heat travels through the &Moon, because its thermal amductivity is higher than air. By deposithsg an alneinum hest-sink oe the her, the resulting beet fiow is approximately divided evenly betweas silicon and aluminum. This is because =
the dismal conductivities of both aeon and altintiltUM ere comparable, but by %pier:ring a metal with a higher thermal conductivity than that of saloon and aluminum, a more efficient hest tnestilar con be achieved. In a preferred embodiment, aluminum is used as the hest-eink material, however additional meterials can be applicable. In addition to increasing thermal flow towards the stratum COIlleU20. tbe placement of* heat-sink prehrably reduces the overall distance between the WW1= of heat and the skin banter, thereby reducing power amsumption. The ahmsimen I ptedembly patterned using a lift-off procedure.
However, since a thick metal layer may be needed, preferably either a thick photoresist is used or the aluminum is deposited over the entire wafer. This may cause problems with the metal wait therefore a din protective isolating layer is 23 preferably deposited prim tOthe aluminum. The ahuninum is then lithographically patterned after the fourth step, to remain only on the micro-ablation heater. The patterning is done at a later stage in older to keep the surface planar for subsequent processing steps.
The fourth preferred processing step involves inverting the double-sided polished wafer to reveal the as yet unprocessed side. A photoresist masking layer is preferably deposited in order to pattern the openings when both reservoir atel thin capillaries are formed simultaneously. Thews capillaries are both fitlxicated using deep reactive ion etching in ouler to obtain narrow, high aspect ratio through-v/2hr holes. The thin capillary is preferably designed to be 25 ttm in diameter while the reservoir capillary is about 75 *ruin diameter, both with nominal lengths of 300 pin. During the DRIB process, the silicon is anisotropically etched until holes are made through the wafer. However, for the reservoir capillary, the etching process terminates on the silicon-nitride that is already preset on the wafer backside. This is because the nitride acts as an etch-stop for the DRIB etch process. As all processing of wafer #1 is now complete, the alumiman heat-sink can be defined and the isolating layer can be removed 'The preferred pzocesaing steps for wafer #2 can also be outlined by a cross-sectional fabrication diagram, as shown in Figure 22. The sequence of steps is far less laborious, however some alignmar issues still exist. The first step in this preferred process is to fabricate the reservoir heating element on the front side of the silicon wafer. This is preferably done, as before, by depositing a heater material onto a silicon dielectric surface. The material is prefaably patterned in the forrn of a heating coil, and is subsequently covered by a protective silicon nitride passivation layer. The preferred next step is to deposit on the autos an etch-stop didectdc layer. Next, the wafer is inverted and patterned using DRIB to ism the connecting capillary opening. Once complete, the third step preferably involves depositing a layer of silicon dioxide onto the ids containing the reservoir hater. This allows for the Bad step, anodiadbr bonding wafer I with wafer O. Care and attendon preferably is taken to wane that the reservoir heaur mates with the reservoir capillary opening, and also to ensure dat the capillary from wafer 1 comets properly with the capillary formed in wafer it2..
Each capillary and reservoir pair is preferably addressed individtally so to as to expose only one such pair to the skin sauface in order to perfonn a single fluid analysis. Once employed, the open end ofthe capillary andinues to remain exposed to the skin. but is not addressed fOr any Author use.
A preened embodimen inclabs additional considerations regarding the dmthg fir signals to open seals and control beaten. The total amount of 15 energy imparted to the heaters that affect the ablation of the *atom comeum and the time over widch that a argy is imparted are also considerations. The system is designed and tested for abbtion energy. That is, the minimum enemy for the minimum duration is a signifiamt parameter for operation, resulting in minimal damage of the underlying viable epidamb, and 20 therefon minimizing the invasive nature of the process.
Other timing issues are also considerations, including the timing of the ablation incase (heaters) with relation to the opening eta capillary sat During the time the seal for a given capillary is being ruptured, the micro-ablation heater is preferably pubed with an appropriate alternadng Gwent to the:molly 23 ranove successive layers of the stratum comeum.

Another timin' g consideration is the heater pulses associated with the reservoir erriptying process. The timing of heater pulsing is a consideration to keep the reservoir flowing, and not taking up fluid from the stratum comeum.
The heater at the top of the fluid reservoir preferably forces out the liquid contents. Control of this heater permits control of the flow of liquid during the reservoir/capillary analysis lifetime.
Preferably tests of the various subsystems are done to establish the dermatmdcological and clinical pharmacological advantages. The test sequence is preferably sequential, starting with simple tests on various mataials, to progressing to in vitro tests on hunt= cadaver skin or animal skin, then to complete animal testing and finally clinical pharmacological testing with human subjects. One shot valves covering the capillary and reservoir are prefaably =
tested and optically examined for successful deployment. The liquid reservoir is preferably initially tested to prove that it can be emptied of liquid contents.
Initial tests are preferably also done on adsorbent surfaces. Further testing is preferably done on a nonabsorbent surfaces to prove flow of liquid up the capillary. Determination of the optimal flow rate for the reservoir and capillary combination is preferably determined based on glucose concentration at the patch detector.
The glucose detector patch material is preferably tested for sensitivity using standard in vitro wet chemistry methods, to assure that its cellulose platform-glucose detector =axial is capable of reflectance densitometric detection of at least 10 fg of glucose per Am2.
In vitro (using cadaver skin) and in Vi3/0 animal and human biomechanical tests of FDA approved biocompatible adhesives and adhesive membranes obtained from 3M, Inc and Adhesives Research, Inc, are preferably conducted to determine optimal adhesive components and skin preparation conditions for occlusive, fluid tight adhesion requirements of the B-FIT
device.
Upon completion of initial tests of the B-FTI' system, preclin' ical dermatcodcological testing begins. These tests preferably consist of a demonstration of the biophysics of the device, done in vitro using human cadaver skin or animal skin, and evaluation of local dermatological effects, done on live animals.
Dennatcodcological testing is preferably undertaken to demonstrate the to biophysical properties of the B-FIT device. Biophysical testing is preferably conducted on mimal or human cadaver skin. Full thickness human abdominal skin specimens can be obtained commercially from Vitron, Inc. (Phoenix, AZ) and other vendors. Animal tissue samples can preferably be used to establish a baseline and adtigate costs. The skin samples preferably serve as a platform to investigate and optimize die thermal ablation mechanism. The B-FIT system has several different ways to ablate skin. The goal of the heat/ablation step is to remove the stratum comeum with no damage to the viable epidermis. The first set of experiments preferably determines the optimal ablation conditions, for example, temperature peak, pulse duration, number of pulses, among others.
Tests to determine the optimum ablation conditions are preferably accomplished using optical and electron microscopy and surface profilometry using an atomic force microscope in order to view and measure (a) depth and volume of ablation hole, and (b) the epidermal cell structural integrity so as to provide sufficient ablation of the stratum comer= without penetrating the viable epidermis.

To obtain a preclinical evaluation of safety, in vivo animal testing of the B-FT system is prefixal* odertaken, utilizing. for example, a hairless rat, guinea pig, or fuzzy rat specie. Clinical observations for puss evidence of skin irritation, ulcer formation, and hilammatory reactions are preferably made.
3 Skin biopsies, CX8Milled using light and electron microscopy provide a closer examinatice dile device biophysical effects. Serial clinical and microscopic observations fbllovibsg removal of the device enable assessment of the healing time for thathermal ablation ksions.
Clinical pharmacological testing is preferably undertaken to determine the analytical recision and accuracy ofmethods to determine glucose levels via transdermal sampling relative to previously validated plasma assays. A
preferred assay technology is based on glucose oxidase immobilization in micromachined capillaries. A validated plasma assay for glucose with acceptable limits of detection and quantification and with acceptable intra-and 15. inter-day coefficients of variation is used to compare with these assays in the clinical settings described in the two trials outlined in detail below. The disclosed trials are of identical design: the fust in normal volunteers, and the secoral in patients with Type 11 (Adult Onset) diabetes mellitus.
In order to validate the analytical sensitivity of transdermal sampling to zo measure glucose, ten healthy men and non-pregnant women who have signed an informed consent, fasted overnight, and have been screened to satisfy the inclusion and exclusion criteria of the study are enrolled in a clinical trial to measure glucose connotations in their plasma or interstitial fluid before and during a glucose toleamce test. The B-F1T system is attached to the dorsal 25 surface of the right hand Wag adhesive tape. An 18-gauge intravenous catheter is inserted in a forearm vein in the Mit arm. VOW= bloothialiPlei (approximately 5 co. sod not less than 4 cc) are taken at appmpriate hared*
for the determination of plasma glucose concentrations. Concentrations demote in the plasma are determined by a validated assay, routinely used in clinical settinp. These command= are compand to those determined in interstitial fluid using the B-FIT system. Plasma concentrations ate measured on 11 occasions at 15 mhsute intervals over two hours, while interstitial fluid co3Mentrations am measured for 0.5. 1, 2, 5, 10 and 15 minute periods over dm same 2 hours before the administration of 75 grams demon by mouth. These data are used to optimise the sampling that tbr the &FIT system Afia the adminbtration of glucose, plasma cementations and &FIT system-estimated concentrations are measured at 30 minute intavals fbr a father two hours. In healthy vohmteers, the glucose plasma concentrations should range from 90 to 140 medl under these conditions (Wallington Manua of Medical is Therapeutics, 28th edition., 1995.) ledusion criteria for the clinical trail are, as fbllows: Group 1: men and .
women who are ova the age of 21 years and under the age 475 yeas, Group 2: male and female volunteers who are over the age of 21 and under the age of 75, and who carry the diagnosis of adult-esset diabetes by a board-certified endocrinologist. Group 1: taking no Feseiption medications or natural products. Group 1: showing elhdadly normal laboratory 'ohms for complete blood counts, serum chanistries (Na, IC. CI, HCO3, BUN, glucose asd creatinine) and clinically normal liva enzyme Follies: SOOT, SOFT, alkaline phosphatess and bilirubin; ability to understand and carry out a signed informed cons= describing this Fotocol.

The following subjects are excluded from the trial: subjects who, in the opinion of the investigator, is noncompliant with the protocol requirements;
and women who are pregnant.
Once a subject has consented to participate in the study, the following procechnes are conducted. Screening procedures are conducted within 21 days of study initiation and include: medical history and physical examinadon;
review of inclusion and exclusion criteria; and blood and urine specimen collection. Subsequent to inclusion in the study, subjects undergo the following ;anecdote& (1) subjects arrive at the location of the clinical tried at approximately 9a.m. in the morning after an overnight fast. Vital signs (heart rate, respiratory rate, blood pressure and temperature) are recorded:
The B-FIT system is placed on the dorsal surface of the right hand and attached securely with tape. Recording mute via a 50 micron cauterized lesion in the skin made by a small needle on the underside of the monitor that is not ts visible. The monitor is checked to ensure that it is recording. Vital signs (heart rate, respinetety rate, blood pressure and temperature) are recorded once the device has been attached once more. Samples of venous blood (S cc or one teaspoon) are drawn from a catheter inserted in the a left forearm vein for the measurement of glucose according to the above schedule while the subject is supine. Additional blood samples are drawn four hours and eight hours after the first. The B-FIT system monitor tape and device is removed. Patients are discharged and allowed to return home.
With regard to the blood sampling schedule, five mL venous blood saMples are collected in vaeutainers in the manner described above. The total number of blood draws during the COMM of the study including the screening samples is 14, (12 study draws and 2 screwing draws for hematology, = chemistry, and liver enzymes, respectively.) The total volume of blood drawn should not exceed 100 mL. Vital signs (heart rate, respiratory rate, blood pressure and temperature) are taken before and alter placentent of the device and catheter and after the last blood draw has been taken and the device and catheter has been removed. Patients are encouraged to report any notable irritation on the ann where the device is placed. A physician is constantly available to subjects enrolled in the study fa concerns related to bruising or infection in the skin due to the intravenous catheter or multiple blood draws.
In addition, symptoms of polynria and polydypsia are carefully noted and paid attention to during the study with diabetic patients, and insulin is available for immediate injection by physicians and muses should the need arise. Statistical analysis include plasma glucose concentrations determined using the clinical plasma assay as compared with the values obtained using the B.FIT system. If the correlation coefficient is > 0.8 with a significance p < 0.05, the measteements are deemed valid.
The second clinical trial with diabetic patients is conducted using an identical study design. Patients are allowed to take oral hypoglycemic medications on the day before, but not on the morning of the study, and are asked not to inject insulin during the study period: Once the study pmiod is over, patients are allowed to eat and to resume their routine &ahem regune. In addition to the safety considerations described above, careful clinical monitoring and thc. availability ofinsulin is paid great attention to while these subjects are tmder study.

The El-FIT system preferably utilizes many different microfabrication technologies and strategies, ranging from simple bulk micromachining to the more complicated de, reactive ion etching (D1UE). Referring back to Figure cross-secdon of a pastured one channel system mirnodevice is shown, s pregerably comprising dthree maht components: (1) the main body containing several serpentine capillary chimes, each with its own reservoir charnel, to sample and analyze physiologically compatible fluid; (2) a bottom capping section to form the lower part of the serpentine structure and to contain micro-heating elements to thernnally porde the epidermal layer for substantial physiologically compatible fluid extractkac and (3) a top capping section which forms the upper part of tbe serpentine channel, end, if necessary, to contain electrodes for assisting the flow of physiologically compatible fluids using elect . osmotic pumpiSg through horizontal segments of the serpentine channel. The first and second components together form the disposable modules of the system. These hate:changeable 8-FIT elements we inserted into the main connection receptacle after all analysis capillaries have been used.
The vssermir and capillary chamois are predbrably flabrtcated in a standard silicon wafer using deep reactive ion etching in order to obtain narrow, high aspect ratio through-wafer holes. The capillary charnels are preferably designed to be 25 tun in diameter with a nominal length of SOO um, while the reservoir channels are 50 lam in diameter, but etched slightly less than SOO
pm.
The lateral pordon of the serpentine capillary chamiel is preferably formed by recessing the silicon surface by 25 lam. 'Ibis region also preferably has a highly reflective metal deposited on the surfitce to facilitate the mechanian for optical detection of the andyte. After bonding drti silicon with a top capping section, the serpentine structure becomes complete. With these dimensions, physiologically compatible fitddsõ such u perspiration 4N intastitial fluids, am be drawn into the open cads of the channels through capillary action.
Furthermore, the fluid widdn the reservoir is preferably used in conitmction with capillary anion, and washes over the dermal region being tested, thereby assisting in the transport of the physiologladly compadble fluids through the smaller channel. By activating the internal capillary chased =faces to sum&
a specific antibody immobilization, the fluids can be preferably analyzed by antibody-andgen complexation. A safes of such capillaries, each with its own to reservoir chinned, is contained within a single device element. Each analysis capillary and reservoir pair is preferably addressed individually to expose only one earl pair to the skin surface in order to peafowl a single fluid analysis.

Once employed, the open end ofthe capillary coininues to remain exposed to the skin.
15 Tee bottom capping unit is also preferably made using silicon and serves two other major *notions, 'mitts from the role of forming the lower structure of the serpentine channel. Micromachined heating demons imorporated within this section are preferably used to thermally prate the skin surface, allowing greater availability of interstithd and physiologically compatible fluids within 20 the channels.
Smetana:fully occulting during the stratum comeum potadon procedtse, the miao-systans are prefixably used to individually address each of the capillary-reserroir pars. InitWly, all open ends ofthe cbnmels in contact with the skin are covered by a sad that can be "Nowa" to reveal a single analysis capillary. ilia aadting procedure am be effectively controlled using 25 large thermal gradients in closeproximity to the seal, such as those afforded by silicon micro-resistors. The micro-heatus are preferably integrated in the silicon region !mounding each of** capillary-reservoir Ciannels. The connecting micro-capillaries in this section are preferably formed using DRD3, each being aligned with the vertical micro-capillaries from the main body.
Unitle the two previous components, the top capping layer is preferably made out of plastic and used to accomplish several tasks. Firsdy, it completes the upper structure, or lateral portion, *Me serpentine channel. This area, preferably, is the detection region of our microsystem. Secondly, by using = plastic, an imbedded waveguide can preferably be fabricated within the material, with its orientation fuming parallel to the silicon surface and forming the basis of the integrated phokmics analysis system. In addition, to couple the light from the waveguide into the detection region, a micro-minor is preferably integrated within the plastic. In addition, for more effective light coupling and better efficiency, a micro-lens is preferably integrated within the plastic located directly above the detection region. The micro-minor is preferably integrated as a pressed component within this top section by using a triangular fonn to indent the plastic. The resulting indentedon pnferably has a 45 angle with respect to the surface. Deposition of a higWy reflective material is made on the resulting beveled angle to render a micro-mirror that reflects the horizontally-directed light from the waveguide downward. The ink:gated microlenses can also be stamped directly into the pleads or can be incorporated as separate units placed within the plastic by using injection molding. In either case, the lens does not need to be of high quality, but should simply be able to diverge light originating = from the waveguide. The illumination produced by the light from the waveguide will cause the tagged amdytes to fluoresce. The light produced is than reflected from the bottom surface of the detection region back through the now converging lens.
Returning to Figure 6, the three possible (controllable) states of the individual microcaplary systems are shown. The leftmost micro-capilloy system (# I) shows an exhausted capillary pair that has already been used for an analysis procedure. This first pair shows the completed thermal ablation, microfluid flow and capture of glucose from exposed interstitial fluid, encounter with the glucose detection patch, and bluish color reaction evident at the upper surface of the chip. The middle micro-culinary system (#2) is performing an to on-demand analysis. 'The rightmost capillary system is ready for a future, on-demand analysis.
A purpose of the microdevice is to facilitate the transfer of molecules of glucose or other poorly permeable analyte(s) from interstitial fluid in the viable epidermis, located just beneath the inner surface of the stratum comeum, to the ts detection patch situated on top of the microdevice. The microdevice enables contact of the microfluidic sampling fluid directly with inteintitial fluid by thermal micro-ablalion of the stratum comeum. By direct interface with intostitid fluid, the miandevice enables sampling of not only the normally-inaccessihle polar molecules, but also impermeable larger molecules 20 such as proteins.
The first in a profaned programmed sequence of events is the flow of electrical current through, the reservoir honing element to create a minute hydraulic pressure in the sealed reservoir contain' ing physiologically compatible fluid. The second and third steps occur almost in unison, and comprise two 25 separate currents through both the breakable seal and the micro-ablation heater.

The seal preferably is a metal-dielectric bilayer that raptures at elevated temperatures. The metal seal is preferably surface deposited on a low-stress silicon dielectric element to reduce the chances of compromising the seal integrity prior to its operation. Once the seal is broken during an analysis 3 procedure, the physiologically compatible fluid preferably flows down from the reservoir and across the region that has been thermally ablated by the micro-heater. During the time the seal is being ruptured, the raicro-ablation heater is preferably being pulsed with alternating current to theanally remove successive layers of the stratum comeum, which is typically about 30-60 pm in to thickness. The micro-ablation preferably occurs in a highly confined volume of the stratum common, approximately 50 pm x 50 pm x 30 pm. The physiologically compatible fluid fkom the now-open reservoir interfaces with the interstitial fluid and, due to the dual actions of the reservoir heater and capffiary force, the mixture is transported towards the detection patch The bulk 15 of the physiological sampling fluid is preferably forced out of the reservoir, emptying over the skin surface region and into the absorbent detection patch.
In addition, a strong, Band-Aid-like adhesive film preferably keeps the microdevice in fluid-tight contact with the skin, preventing escape of interstitial and physiologically compatible fluids from the analysis region. The fluids are 20 preferably forced up the analysis capillary to the detection patch, directly above the capillaries on the microdevice and, for example, in one anbodiment, generate a color change to indicate the presence of glucose.
The miaosystem component of the present invention is preferably based on molecular scale manipulation using enhanced transdermal transfer of 25 metabolites from interstitial fluids, and resultant detection with enzyme immobilized chemistries. Samples me preferably collected using a minimally am-invasive transdelmal microdevice and trace quatities of andytes, which reach the skin auface by passive diet:lion from interstitial fluid tmdedying the , antennae layer of skin (the stratum comet* can be detected. Since these analytes originate from other parts of the body, transported to the interstitW
fluid via blood ckoalation, they DAM a variety ofpbydological processes including body exposure to envircannemtal chemicals or microbes, as well as internal metabolism. Micro-layen of tbe stratum conesten are gently removed enabling uptake ofinterstitial fluid from the viable epidemis, which lies jot to beneath the *Mtn comma.
A preferred detection scheme for determination of health and other important biological madosrs utilizes similar SIAM and biochenddry fbr each assay. Returning to Figun: 11, a prekned procedtue for the detection scheme is shown. A prefeered procedure is to covelently attach antibodies of the protein or IS metabolite of interest to a capillary wall that inamoratas a fluorescentlY tagged antigen. The tagged antigen is replaced by competitive binding with the protein or metabolite of interest from dm interstitial fluid amroled. The flumoscent antis= is kidced off into solution to be detected down stream in the collecting chamber by the photonics component. The fluorophom mrcitation and emission 20 characteristics are matched to the photonics and. villa versa for integrating the right VIIIVOkligth source, detector and fillers to propedy excite and determine emisdon in the photonics module.
As an ample, tbe following three protein' of various molecular weights can be used for monitoring health properties: troponin C-reactive 25 protein, and prealbtanin Anti-troponin I is cove's* attached to the capillary wall tbliowing a aliens =AM besbnemt olantinopropyttdmethoxyalane (APTS)h Troponkt I is fhmemendy tagged ash* *Wm fluorescein or rhodamins and bound to the antibody attached in the capillary. Using competitive binding of toponin I from samplin& the flume:catty tagged troponin I is replaced into solution and detected downstream. The above procedure can also used for both C-reactive protein and prettibumin, albeit modified to take into account differences between these putein.
The sure chemistry is characterized stepwise to ensure sufficient surface coating. The amotmt of bound antibody and competitive binding studies to is tested using a variety of diflbrent instruments such as XPS, fluorescent plate reader, fluorescing microscope, or separation techniques.
In another example, polycloml antibodies raised against the caffeine metabolites 5-acetylamino-6-foratyl-3-methyl urea (AFMU) and 1-methybrantidne (DQ are immobilized on the micromacbined capillaries. The ts capillary tubes are modified by chemical treatment in order to introduce hydroxyl moieties on the capillary suffice. The surface hydroxyl groups are then reacted with APT& produdng a molecular tether with fine amine moieties at the end of the dreamt= chain.
The supr residues in the Fc region of the antibodies raised against 20 AFMU and IX are oxidized using periodate to generate aldehydes. The antibodies are anchored to surface of the ndcromachined capillaries through Schiff base formation between the aldehydes on the antibodies and the amines on the molecular tether. In this mammy, the antibody binding regions is directed away from the surface of the micromachined channels.

The amount of antibody immobilization on thenifogs pAtlie microchannel is preferably determined by analysis of the protein content of the binding solution before and after exposure to the ochannels. The binding activity of the immobilized antibodies is detamin" ed using displacement of the fluorescent-Meted AFMU and IX probes and the observed activity is compared to the activity of equivalent concentrations of non-immobilized antibodies to yield binding affty per mg of immobilize' d antibody indices MAD.
For the caffeine metabolites, in vivo and in ;vitro testing is conducted to to assess the specificity of the antibodies AFMU and 1X. The ability to measure the ratio of these metabolites using the device of the present invention is assessed by comparing the redo obtained using the portable biomedical monitoring system with the ratio obtained employing conventional HPLC
methods. Devices modified with this assay are tested both in vivo and in vitro 15 for provide a preclinical evaluation. These data are utilized as a baseline and preliminary data for testing the algorithm.
Antibodies for prearbumin, CRP, troponin I are currently available on the market and are used to assess the specificity of these antibodies to their protein compliment. As SOMC commercial antibodies are not active or specific, 20 this prescreening test is preferred to determine activity and specificity for each protein of interest. Specificity is preferably tested for each antibody by adding other substances similar in structure, which should not cross-react. For example, in assessing prealbumin the proteins such as albumin and globulins, among others, are added. In assessing caffeine metabolites, xanthines and xanthine 25 metabolites are added. Assays using the antibody, the ligand and the fluonacentlylabeded lipid are pasficably developed ushqg techniques such as flow injection analysis (FIA) Each completed. assay is stressed for accuracy, reproducibility, linearity, rend maks me compered**. these of exist' log procedures currently used in the cliniad laboratry. A precludesl eralualion for in vivo and in vitro testing is done and the data is utilized in tbs algorftlen developed.
The portable biomedical monitoring system of the peewit invention is preferably based on molecular scale manipdation ming enhanced tranalennal tranahrofinetabolites and other body snalytee using trasidamal dosimetry immobilized anlihodies ln microchermels, capillary action kr fluidic mobility, and integrated photonics fbr detection. Thus, the micro-fluitlic chip intedhce technologies of the present invention provide controlled sample collection from host fluids, (circulatory and noncirculatory) and for the controlled delivery of fiukle (drup, chemicals) and target probes (*mtibodies, proteins, signal 13 molecules). in addition, sample collection platforms of the present invention can simidteneously employ an "outward Archie device canponemt for sampling air or liquid borne environmental target analytes and an %ward fleeing" component for detection of target analytes emanating from the skin airtime or acceedble body fluid.
The appends and process of portable biomedical mattering disclosed herein is adaptable to a wide variety ofchemistries. Fre a temple, Ihe portable biomedical umnitoring device can include chips which monitor health (Chip "A") and illness or infection (Chip "B*). Chip A can measure molecules like:
glucose to establish a baseline of the subject's health state in both nomial and 23 high stress situation" Changes from these baseline limits will signal a need for Chip B. Chip 13 is designed to determine the exact cause of illness For example, Chip Ef can contain antibody conjugates for parathion and its metabolites that emulate a chemical wedge agent The structtne of the microsystem is adaptable for many other types of chemistries based upon drag metabolism and/or "probe drugs".
Drug metabolism is the process by which drugs are converted, by enzyme-catalyzed Inactions, to products or metabolites which are readily excreted in the urine and bile. One pathway of drag metabolism are phase I
reactions, which involve the creation or modification of a functional group in the substrate molecule. The cytochrome P450 -dependent (CYP) microsomal mixed function oxidase system is a very important enzyme systmn for these reactions. A second major pathway involves phase 11 reactions, in which the drug or a phase I metabolite is conjugated with a water soluble endogenous substrate. Phase II reactions involve a diverse group of enzymes known collectively as transferases. This group includes UDP-glucuronyltransferase, UDPglycosyltransferase, glutathione-S-transfbrase, sulphotranderase, methyltrensferase, and N-acetyltransfemse. =
Drug metabolism is affected by dietary and environmental factors. For exempla, alcohol, certain ibod constituents and compounds in cigarette smoke have been observed to affect the biotransformation of many drugs, as have industrial pollutants and pesticides. Genetic factors also play an important part in the control of drug metabolism and it has been observed that there is much variation in drug effects between individuals. For some enzymes, discrete genetic subgroups are present in the human population. These genetic polymorphism* are generated by mutations in the genes coding for these enzymes which cause decreased, increased or absent enzyme expresaion or activity. Gene* polymorphisms of sevaal CYPs have been identified and their activity falls into two deftly defined and qualitatively different population' s:
individuals whose rate and extent of metabolism is poor (poce metabolizes, PMs) sod those who liave faster or more extsedve metabolism (extensive metabolizes, Ms). Genetic polymaphisms of some phase II enzymes also exist For example, N-acetyltransferase-2 (NAT-2) is ratted in this way and this acetYladon polymotphism nista* to the metabolism of a variety o drugs and carcinogens. Numerous alleles me associated with decreased ihnction of to this enzyme snd s bhnoild distribution is observed: 50.60 % of individuals are genotypically dow acetylators and the mat of the population are fast.
= In healthy individuals, the metabolic genotype normally ptedicts the metsbolio phenotype. Thm is, for a particular enzyme, genotypically extensive metabolizers are observed to efficiently metabolize drugs that are substrates for 13 that enzyme, and genotypically poor metabolimrs are deficient in that process.
However, drug intaections, infection, disease progression and malnutrition may produce changes in the relative levels and activities of metabolizing enzymes.

Thus, in healthy individuals the relationship between genotype and its expression (phenotype) is conserved; Le. FAST genotypes produce FAST
20 phenotypes, while SLOW genotypes produce SLOW phenotypes. However; a disease state of the individual maker this relationship, as can diet sznoldng, alcohol, environmental chemicals, and biological or chemical infirm agents.
For this reason, the determination of metabolic phenotype (the measure of actual enzyme activity) is fittest importsmce and can be used as a direct and as sensitive probe of health and clinical status. In a preferred embodimers, identifiattion and qmintification of specific metabolite patterns produced by innocuous tat compotmds or probe drugs can be utilized to determine the metabolic phenotype of a subject. For ample, caffeine is metabolized by several routes inchsding one involving NAT-2. Thus the urinary ratio of 2 metabolites, 5-acetylamino-6-formylaminoamethyluracil (AFMU) to 1-methylxanthine (IX) is an index of NAT-2 activity.
Examples of numerous embodiments follow. Each embodiment can be practiced alone or in cosiunction with other embodiments of the invention.
For example, as mentioned above, dispositional or metabolic markers of "areas can be monitored, including but not limited to, chemistries for die detection of diffenat chemical probes of human health, such as glucose, caffeine, ethanol, and dextromettorphan. "Stress" can manifest itself via detectable situations dimly internal metabolic pathways, such as in altered insulin-ghscose patterns or aberrant hepatic cataboliam of safe, commonly used stimulants (caffeine) or antihistamines (dextromethotphan).
The enzyme N-acetyl transfivase (NAT-2) metaboli2ns caffeine. This ammo is highly polymorphic. The activity of NAT-2 is known to be associated with adverse drug ants, diverse toxicitias and predis' position to disease.
Two major metabolic phenotypes have been identified: at and slow N-acetylatccs.
The expressed activity ofNAT-2 (phenotype) has been shown to be affected by acute and ChM& disease atates. For example, in HIV+ and AIDS patients, the presence of an acute illness reduces the egg:tressed activity of NAT-2, changing a patient with a fast NAT-2 phenotype into one with a slow NAT-2 phenotype.
When the illness is resolved and the patient is returned to the initial clinical state, the patient again expresses a fast NAT-2 phenotype. Thus, the determination of an individual's NAT-2 phenotype and the monitoring of changes in this phenotype cm be a direct and sensitive probe of that individual's health and clinical status Ma determinaion allows prediction of whether patients are FAST or SLOW metabolized prior to initiating drug regimens. This approach also allows ear the screening of all patients before drug augment is inithrtal so that appropriate dosage regimens are given at the outset of treatmag and drug overtreatment or undestreatment is avoided.
The NAT-2 phenotype can be determined by a number of probes. In the preferred embodiment, caffeine is used because of its wide (retribution and to relative safety. In studies using caffeine as the probe, the phenotype of the enzyme is deteradned by the ratio of two caffedne metabolites: AFMU to I X.
Based on the ratio of these metabolites, the activity of the enzyme can be determined. Polydonal antibodies are grown against the metabolites AFMU
and 1X and then purified. These antibodies are successfully used to determine Is NAT-2 phenotypes.
The preferred detection scheme consists of the anchoring of antibodies of a particular metabolite or chemical antigen to the surface of the capillary. The antibody is bound to a special rmtigen attached to a fluorescent tag such as rhodamine. As the smidgen flows into the chiumel it will release the fluorescent 20 tag which is detected downstream.
Thus, in an alternative embalming, phenotyping using NAT-2 is conducted to indicate an infiscted or diseased state. The enzyme NAT-2 is highly polymorphic. The activity ofNAT-2 bag been associated with adverse drag effects, diverse toxicities and predisposition of diSaus The monitoring of changes in this phenotype is a direct and sensitive pro* bf fhltitikilers WM
and infection sums.
In a thither embodiment,mganophosphate chemicals (nerve) agents are monitored using the imecticide surrogate model compound parathion. Since *nerve pa" type chemical weapons Ian Tabun, Sad% and Somas act by Inhibition of acetylcholinaterase, the oripmophophste insecticide, parathion (or ka metabolites) provkles an excellenesanopte enable to detect exposure A
parathion monitor, thus, has important industrial and civilian applications.
In a, anther embodiment, inflammatory segued to microbial toxins are =
monitored, including, for cramp* inter1euk1n-1 (IL I); interleu,k*-6 (IL 6) and honor necrosis Actor (TNFX moos 'others. Cheulating IL 1, 1L6, and TN?
present candidate analytes that can be collected and detected using permeation enhanced transtkrmal tedmiquea and advanced detectice system designs in accordance with the present inventkm.
13 In a Anther embodiment, microbial nudns are monitored, including, for example, anthrax, Wolin= toxin, endotoxin, among otbstu. Although microbial toxins are typically large molecules, their extremely high biological potency, coupled with enhanced outward migration using microscopic physical barrier modification techniques (for example, thermal microabladon) can pamit traredermel dosimetry employing detedion systems incorporating toxin responsive components. /a addition, antibody tap can be used fbr identification ofinfbcdng agents and determination ofbactedal and viral loads. Moreover, the determinadon of D- amino acids (from bacterial sources) am enable the monitorhe of the response* antibiotic thmapy.

In a further embodiment, spore metabolites can be monitored Circulating biochemical metabolites arising from human catabolism via lymphatic and hepatic pathways of microbial spores are collected and detected using the techniques and optimized detection systerct designs of the present =
invention.
In a further embodiment, specific proteins are monitored, such as those referred to in the table below.
Protein Concentration MoL Wt.
(WA) (IcD) Prealburcdn 70-390 54 C-reactive Protein 0.06-8.2 115-140 Troponin I < 0.0001 76 to Prealbumin (MW 54,000) is known as being an important marker for nutritional status. The reference range for 0-1 month old is 70-390 mg/L. Uses of this embodiment include but are not limited to screening inner city pediatric populations for nutritional status, as well as screening all patients for nutritional status, particularly prior to surgery.
15 C-reactive protein (MW 115,000-140,000) is an acute phase reactant and as such is elevated in many disease processes. The reference range in adults is 68-8200 pg/L. The measurement of this protein provides a good indication of health vs. disease. C-reactive protein is also an important prognosticator of heart disease and impending myocardial infarction. Thus, this assay could also be 20 used to screen for cardiovascular health.
Troponin I is recognized as a useful and specific marker for acute myocardial infarction. The reference range in adults is <0.1 pg/L. In myocardial infarction patients it is > 0.8 pg/L This assay provides a real time evaluation of troponin I in the emergency rooms of hospitals and provide the earliest recognition that a patient needs to be admitted to intensive care units.
In addition to monitoring, the biomedical monitoring system of the present invention can provide drug delivery with feedback control in bursts to maintain concentrations of a specific agent within the body at specific levels throughout the day, levels which can vary on a day to day basis and during the day. Examples of such agents include the hormones estrogen and testosterone.
The decrease that occurs in estrogen with age is intimately related to the increased risk of osteoporosis and cardiovascular disease in women. Moreover, to the replacement with phannacologic estrogen may improve mortality from cardiovascular disease, reduce the risk of osteoporotic fractures and may play an important role in protecting women agaihst Alzheimer's disease. These diseases have immense societal impact and financial cost, but their treatment with replacement estrogen is associated with a host of side effects including, not least the development of breast cancer and uterine cancer, but also a host of other effects including skin changes, weight changes and depression. Although no medicine has been shown to be as effective as estrogen itself a hugueffort has been expended to develop modified estrogens that have selective actions on bone, breast or other tissues (the development of specific estrogen receptor =Mann or SERMs). The approach of administering effective estrogen in a physiologic, controlled and monitored manna is attractive in that it remains the most effective medication and innovative therapeutic regimens utilizing it may prove of great benefit.
The delivery of testosterone in a controlled and monitored manner can also be useful. Serum concentrations of testosterone also decline with age, as they do in a number of pathological conditions, includibrifIVecistaterehli replacement strategies for the treatment of HIV and cancer wasting, male osteoporosis and chronic obstructive pulmonary disease are emerging and can also be useful for short term controlled administration post-operatively after major surgery to enhance the rate and the likelihood of successful recovery.
Feasibility of these embodiments is investigated through tramdermal detection of estnaliol (E2) in Rhesus monkeys and human females. A prototype solid phase E2 detection system crm can be incorporated in a transdemial patch that immobilizes antibody against E2 in the TED, and analyzed ex-situ using a radioimmtmoassay procedure. The 132 detection system is capable of detection less than 0.125 picograms. TEDs are first tested by emplacement for 24 hours on the chests of partially or fully castrated female Rhesus monkeys (n=3), treated with placebo or 20 ugilrg estradiol benzoate. The TED measurements distinguish between monlrzys that have high circulating E2 concentrations and those who have none. TEDs can also be affixed on the forearms of four . reproductive age human females who exhibit a large range of circulating E2 concentrations (48-382 pg/ml). E2 collected in TEDs range from 0.06 to 0.5 pg, and correlate roughly with circulating E2 =mutations. These data are consistent with an in vivo permeability coefficient of 4.3 +/- 0.5 x 105 cmlbr.
bi a further embodiment, the portable biomedical monitoring device of the present invention can be used for pain management, determining how best to deliver codeine and morphine, among others, to minimiw. cytotoxicity, while achieving pain control.
In a further embodiment of the present invention, a ME148-based physiochip can be used to non-invasively monitor fundamental physiological aspects related to human function under typical and atypical environmental conditions By carefully monitoring of relevant physiological data such as body temperature, pulse rate, blood pressure, and heart activity (electrocardiogram) an infinitesimal change or anomalous behavior can provide an early indicator of stress to the human system.
In a further embodiment, passive or non-invasive transdermal dosimetry is used without physical or c.hetnical modification of the normal skin barrier.
This embodiment is practical for small molecular weight analytes that exhibit both lipid and water solubilities.
The following description of experiments and clinical trials is provided so as to demonstrate how various embodiments of the present invention perform. Suitable analytes for demonstrating the operation of these embodiments of the present invention are provided below. However, it is to be recognized that the systems and methods of the present invention contemplate analysis of a much larger set of analytes in the various embodiments of the present invention.
Develqmpest of humobilizediticotinic acetylcholine receptor (nAChRal based-HPIX stationary phases and the application of these phases to the on-line determination of drus-receptor intesaction&
Preparation of nACIA-detergent solution. Rat whole forebrain or transfected cells are suspended in 50 mM Tris-HCI, pH 7.4, (buffer A), homogenized for 30 seconds with Brinkmamt Polytron, and centrifuged at 40,000 x g for 10 min at 4 C. The pellet is resuspended in ml of 2%
deoxycholate or 2% cholate in buffer A and stirred for 2 hours. The mixture is centrifuged at 35,000 x g for 30 minutes, and the supernatant containing nAChR-deoxycholate solution is collected.

kogyabilbstion of nAChRs on M/vfpartides or Superdex 200 gel beads.
Dried IAM particles are suspended in 4 ml of the obtained den:egad solutions containing nAChR subunits or subtypes. For the immobilization of one nAChR
subtype, the mixture of IAM-detagent-receptor is stirred for I hour at room temperature. The suspension is dialyzed against 2 x 1 L buffer A for 24 hours at 4 C. The IAM LC support with immobilized nAChits is then washed with buffer A, centrifuged and the solid collected.
A dried lipid mixture of 60 mg L-a-Lecitin (20% phosphatidylcholine), mg L-a-phosphatidythaine, and 20 mg cholesterol is solubilized with 4 ml of 10 obtained nACbR-detergent solution. The nAChR-lipid-cholate solution is mixed with 50 mg dry Superdex 200 beads. The suspension is dialyzed against buffer A for 24 houn at 4 "C. Non-immobilized liposomes are removed by centrifilgal washing with butrerA at 2,000 x g.
((311)-epibatidine ([311)-EB) binding assays for the suspensions of nAChR-IAMparticles and nAChR-Superdeac 200 beads: The nAChR-IAM
particles, IAlvt particles, nAChR-Superdex 200 gel beads and Superdex 200 gel beads, corresponding to 30 mg dry material, are each suspended in 1.25 ml buffer A. A 250 gl aliquot of each suspension is incubated with 250 gl of [3HJ-EB [1.5 nMifor 4h at 24 C in a fmal volume of 2.5 ml.
Experiments are carried out with and without added 100 al of 300 p.M
(-)-nicotine. Bound and free ligands are separated by vacumn filtration through Whitman GF/C filters treated with 0.5% polyethylenimine. The filter-retained radioactivity is determined by liquid scintillation counting. Specific binding is defined as the difference between total binding and nonspecific binding. The amount of protein is detaimined using BCA reagent (Pierce, Rockford, IL, USA) measured at 570 inn.
Cinematography based on nAChR-LAMcolumn or nAChR-liposome-Superdex 200 colunm: The nACIAWAM particles or nAChR-Superdex gel beads are packed in a IBM glass column and connected to a FIPLC pump. [311]-EB is used is a marker and an on-line flow scintllation detector (525 'TR) monitors the elution profile. All chromatographic experiments are performed at flow rate 0.4 ml/min at room temperature.
In zonal chromatographic experiments, a 100 pl-loop is used to apply the sample. The cinematographic data is summed up in 0.5-min intervals and smoothed using the Microsoft Excel program with a 5 point moving average.
In frontal chromatogram 50-ml sample superloop are used to apply a series of [3111-BB concentration through the nAChR-column to obtain elution profiles showing a front and plateau regions. The chromatographic data is IS sununed up in 1-min intervals and smoothed using the Microsoft Excel program .
with a 10 point moving average.
Results: Immobilization of nAChR subunits or subtypes. About 63 mg protein isolated from the membrane of transfected cells and 14 mg of protein prepared from the brain tissues are respectively immobilized on the per gram of IAM particles or Superdea 200 gel beads. Receptor binding assays using [3111-EB showed that the nAChR binding activities are retained after the immobilization procedure as shown in the table below. In parallel experiments, no specific binding of [311]-EB is detected on IAM particles and Superdex 200 gel beads.

Sample Spedfie Binding nAChR Density (A) (nmoVg protein) a443.2 nAChR-detergent solution 62 (L 1 4 44/02 nAChR-IAhor 49 0.81 c13434 nAChR-detergent so1ution3 100 8.57 e3414 nAChR-IAM2 97.8 5.09 a3/04 nAChR-liposome Superdex 2002 29.4 1.45 premed from rat forebrain with detergent deoxycholata 2 prepared fiom transfected cells with detergent cholate.
Frontal chromatognqty with 43434 nAChR-IAM stationary phase: The retention vohnnes of [311143B are 23 ml at the concentmlion of 60 pM. This retardation is primarily due to the specific binding to saturable sites of the receptors as indicated by a decrease in retention vohnne to 8 ml when the concentration of NIES is increased to 450 pM (Figure X, profile B). The binding of [3/11-BB to the 43/114 nAChR-IAM stationary phase could be reduced in competitive displacement experiments using known a3/fi4 nAChR ligands in the mobile phase. For example, the retention volume of 60 pM [311]-EB
decreased fiom 23 ml to 18 ml vihen a 60 nM concentration of the nAChR-ligand (-)-nicotine is added to the mobile phase and feil to 0.9 nil when the (-)-nicotine concentration is increased to 1000 nM. The decreases in retention volumes of [41]-EB relative to mobile phase concentrations of a displacer reflect the thnding affinity of the displacer for the receptor.
Using this technique, the relative affinities of nicotinic drugs for the a3/04 nAChR. are readily classified by determining the concentrations required to decrease the retention volumes of [11]-BB to a predetermined level.
To decrease the retention volumes of 60 pM (3}11-EB from 9.5 ml to 6 ral on an 43/134 nAChR colunm (0.5 x 1.25 cm), requires mobile phase concentrations of 0.12 nM of (+)-EB, 1.7 nM of A85380, 45 nM of (-)-nicotine, 1,200 riM of carbachol or 21,000 nM of atropine, respectively. The relative affinities of these drugs for the a31(34 nAChR determined by this method are therefore (*)-BB > A85380> (+nicotine > carbachol > atropine which is consistent with results finm ligand binding assays using membrane homogenates. The relative affinities can be classified by the association constants calculated from the resulting data in the table below.
Ligsnd 141 (n4) 1Cd2 OAP
(*)-Epibatidine 0.27 0.05 0.38 * 0.07 A85380 17.21Ø5 73.6*6.3 (-)-Nicotine 88 * 33 475 * 52 Carbachol 1,280 *t 30 3,839 276 Atropine 14,570 2600 -Ifinntal chromatography with a31(34-1AM stationary phase (0.5 x 1.3 cm).
2Binding assay using cell membrane homogenates.
These dissociation constants (ICd) values show the same rank order as those of the values measured with binding assays using membrane homogeoates. The low affinity of atropine (Kd: 17,200 aM) is also consistent with literature values.
Zpnal cbummagraby for determination of different specific binding activitiel of immobilized nAClits subtypes: Binding of (3/1]-EB is also measured in zonal format on the columns containing ta subunits only, 134 subunits only, a mixture of the two cell types, or a3/134 nAChRs. The retention of elfj-EB on o.3 nAChR-IAM (peak 1, Figure 23a),134 nAChR-IAM (peak 2, Figure 23A) and a3/04 nAChR-IAM (peak 3, Figure 23A) is Iow, and no significant change in the retention volumes is observed when a displacer, (-)-nicotine, is included in the mobile phase, [311]-13B is retained on the IAM
column containing the immobilized a3/114 nAChR-IAM (peak 4, Figure 23A).

The retention volume is decreased when the concentration of ['P]-BB is increased or when (-)-nicotine is included in the mobile phase, peak 4 (dash line) Figtre 23B.
Specific binding activities of immobilized nAChRs subtypes. The results of binding to immobilized receptors showed that [311]-EB and (-)-nicotine have higher binding affinities at nAChR 44/02 subtype than at a3/P4 -subtype and these results are consistent with the results determined from ligand binding assays using membrane homogenates as shown in the tabk below. 'The Kd values obtained from u4/132 nAChR-liposome-Superderc 200 column are similar as those determined using a4432 nAChR-IAM column.
Fonnats of nAChRs Kd of (*)-epibatidine (nM) Kd of (+nicotine (11M) a3/04 - nAChR-IAM 0.27 * 0.05 88 * 33 13 a3/P4 nAChR membrane 0.38 0.07 475 * 52 a4/02 -nAChR-IAMB 0.044* 0.005 1.0* 2.3 a4/02 -nAChR membrane 0.053 * 0.002 7.2 * 1.3 a4412-nAChR-liposome-Superclex 200 0.020 0.08 7.4 2 Bffects of ionic strewth and pH of the mobile phase on the binding of 13111-EB: The effect of mobile phase ionic strength and pH on the binding affinities of [4-BB are determined with a a3/114 nAChR-column. The retention volumes increased when the pH of mobile phase is increased from pH 4.0 to pH
7.0 and remained constant between pH 7.0 to 9.5. 'The retention volumes of [311]-E13 are higher at low ionic strength (5-mM ammonium acetate) and decrease as the ionic concentration of the mobile phase increases.
Stability and reproducibility of nAChR columns; One a3/04 nAChR-IAM coltmm is used continuously over a ten day period and then stored for 40 days at 4 C. The retention volumes for 60 pM [3 HI-EB are 9.5 * 0.05 ml (from day 1 to day 10) and 9.7 0.08 ml (day 50). The relative affinities of EB

and (-) nicotine obtained on three a3/(34 nAChR-IAM columns prepared from different batch of cell lines ate reproducible as shown in the table below, although the retention volumes of EB at the same concentration differed from column to column.
=
Column size Kd of ED 1C4 (AO-Nicotine Binding sites (ca) (nM) (uM) (pmoitati bed) 0.5x1.8 0.344.04 52110 7.5102 0.5x1.3 0.2710.05 88133 13.54.3 0.5 x 1.7 0.21 4.06 130145 15.010.4 Preparation of Immobilized OABAA and nicotinic acetylctoline receptors on an IAM support from rat whole brain: Rat whole brain (4 brains) is homogenized in 30 ml of TRIS-HC1 buffer [50 mM, pH 7.4] containing 5 mM
EDTA, 3 mM benzamidine and 0.2 mM PMSF (Phenyl methyl sulfonyl chloride) for 3 x 20 seconds using a Brinkman Polytron at setting 6. The mixture is kept in an ice bath for 20 seconds between each homogenization step to prevent excessive heating of the tissue. Homogenized brain tissue is centrifuged for 10 min/4 C at 21,000 rpm. Supernatant is removed using a Pasteur pipette and discarded. The pellets are suspended in 10 ml of Solubffization Haw containing 100 mM NaC1, 2 mM MgC12, 3 mm CaC12, 5 mM KCI, 2 % Na-cholate and 10 Itg/m1Leupeptin in TRIS-HC1 buffer [50 mM, pH 7.4]. The resulting mixture is stirred for 12h/4 C and centrifuged at 21,000 IP=
Supernatant (receptor-cholate suspension) is mixed with 200 mg of dried IAM-PC packing material and stirred gaudy for 1 h/25 C, transfetred into dialysis tubing and dialyzed for 48 h/4 C against 3 x 600 nil of Dialysis Buffer containing 5 mM EDTA, 100 mM NaC1, 0.1 mM Caa2 and 0.1 mM PMSF in TRIS-HCI buffer [50 mM, pH 7.4].
The receptor4AM-PC is centrifuged for 3 min/4 C at 2,000 rpm.
Supernatant is discarded. Pellets are washed with TRIS-Ha buffer [50 mM, pH
7.4] and centrifuged until the supernatant is clear. The resulting pellets are used to pack the column.
Determination of binding affinities to the immobilized QABAA receptx (OR) using frontal chromatography: The OR-IAM particles are packed in a HR5/2 glass column and connected to a HPLC pump. [311]-Flrmitrazepam ([311)-FTZ), a GABAA receptor ligarxi, is used as a marker and an on-line flow scintillation detector (525 TR) monitored the elution puffin Ail chromatographic experiments are perfumed at flow rate 0.4 ml/min at room temperature. In frontal cbroraatography, a 50-ml sample superloop is used to apply a series of [3 H]-FTZ concennations through the GR-column to obtain elution profiles showing a front and plateau regions. The chromatographic data is summed up in 1-min intervals and smoothed using the Microsoft Excel program with a 10 point moving average.
When the GABAA receptor ligand diazepam (DAZ) is added to the mobile phase, the retention volume of [311)-FTZ is reduced in proportion to the concentration of DAZ in the mobile phase. These results indicate that the retention of FTZ on the GR-IAM is due to specific interactions with the immobilized GABAA receptor. The dissociation constants (&) of FTZ and DAZ are determined on the '3R-IAM. The calculated Kti of FTZ and DAZ
obtained by frontal chromatography are consistent with those determined by classical binding assays, as shown in the table below.

IAPod Frontal Chromatography Binding Assays Flunitrazepen 1.3 1.7 Diazepam 1.0 1.3 /10411A19111111.111016i21.4.2111.aaa-The Estrogen Reactor (ER) is pad of the Nuclear Receptor Superhmily. It is made up of five different regions: A, B. D and B. The B region. also known as, the lipnd binding to domain (LBD) is where the agonise end antagonists bind. The ER-LBD
has been equaled in yeast and also in bacteria via a hake product between protein A and the LBD. The Production of recombinant Estrogen Receptor Protein is descdbed: The DNA sequence coding for the ligand binding domain of the human estrogen receptor a protein (amino acids 302.595) is obtained by 15 KR Using the NI length cDNA as the template. The product of the PCR
reaction is subcloned into dte pan plasmid in trine with a 6 histidine tag on the N-teetrdnal. end of the protein.' The His tag is used for the purification (XS*
protein front dm bacterial proteins. The *amid is transikroned into the 8L21 codon+ bacteds. The becteda are grown In standard LB Broth to an optiad 20 density at X 600 al¨ LS.
The bacteria are harvested by centrifugation and fro= at -80 C until Anther porificadon. The WOMB pollen am lysed in a ures/HEPBS lysis buffer by sonication and clarified by cent:Minion and filtradon. Tbe lysete is loaded onto a 5 ml Ni-TA nickd affinity column that is preegullibtated with the 25 urea/WIPES lysis buflit. The Ni-141TA column selectively binds proteins with the 6-Hl. tag. The nontagged proteins are washed off the column with the urea/HEPES buffer. The estrogen receptor is refolded on the column by gradually changing the buffer to a PBS (phosphate barred saline) buffer.

Finally, the asrogen receptor protein is eluted with a PBS buffer containing imidazole, which competes with the His tag fbr binding to the Ni-NTA column.
The fractions containing the estrogen receptor protein are determined by gel electropbresis and staining with Gelcode Blue and by western blot analysis using a antibody against the human estrogen receptor. The comentration of protein plated is determined via bicinchoninic acid (BAC) protein assay.
Bindles activity of the ISR-In. A binding assay is carried out to determine the activity of the Anion protein. lbe classical method using dextran-coated charcoal is initially used and gives the activity of the protein.
However, the method is improved with the use of Nicktil-NTA aprose beads to isolate the ftnion protein. RougWy 200 pmoles of protein is placed per tube.
For total binding, varying concentration of [3111-estradiol is added and for nonspecific binding a 200 fold excess of the cold estradiol is added prior to the addition of the auliolabeled estmdiol. The solutions are incubated at MOM
IS temperature fbr 2 hours. Following incubation, the Nickel-NTA is added. After one wash, the protein is displaced with imidazole. The Kdis determined to be approximately 3.4 nhi (an average Kd of several experiments). Although estradiol had a slightly stronger affinity for the native ER (02 nM), this is sufficient.
Inunnbilization of*, ER-LBD. The initial immobilization of the isolated fusion protein is coaled out using a silica based immobilized artificial membrane: IAM.PC. This membrane contains a silica core, which is attached to a hydrophobic spacer with a polar head group. The procedure for immobilization of the protein onto these membranes is known in the art.
23 Varying concentrations of IAM are used to determine the optimal conditions for immobilization. It is determined that 25 mg of LAM is optittarvntr343 3C
incorporation.
However, upon testing for activity it becomes apparent that [31.1]-esttadiol is not only binding to the protein but also to the hydrophobic 5= layer of the membrane. Increasing the ethanol concentration in solution does not significantly reduce the binding tethe membrane. Using a modified IAM
stationmy phase, the IAM-MO, that is more hydrophilic only slightly reduces the nonspecific binding.
The ER-LBD is then immobilized in a new column format containing a silica backbone and a hydrophobic spacer (C 10). The ER-LBD is immobilized and retained its binding activity but the nonspecific binding of elij-estradiol is still to great for effecdve use of the column. The Cl() spacer is replaced by a hydrophilic spacer and the nonspecific binding of (3Hkestradiol is elim' inated and the ER-LBD-SP column is synthesized.
The Ke of the estradiol marker ligsmd is then determined on-line using the ER-LBD-SP column. The ER-LBD-SP column is connected with on-line flow scintillation monitoring (kadiometric FLO-ONE Beta 500 TR. instrument, Packard Instrument Co., Meridian, CT) and run s1 room temperature for 97.5 minutes at a flow rate of 02 mlimin. The system setup is as described by Zhang, et al., Immobffized Nicotinic Receptor Stationary Phase For On-Line Liquid Cinematographic Determination of Drug-Receptor Affinities, Anal Biochem. 264., 22 (1998). 18 mL samples of 0.5 nM [314]-Estradio1 (13H)-E2) supplemented with a range of concentration of cold Estradiol (0-7 nM) are run by frontal c.hromatogsaphy. The elution volume data is used to calculate the dissociation constant of the ligand. The ICe value of estradiol is calculated by nonlinear rpgression with Prism (GraphPad Software) using one site binding equation: Y=Bmax [Ealtotal/(Ka + [E2]total). The Ka values of estradiol is calculated as previously described to be (0.189 e 0.06) nM. The radioactive signal is recorded every 6 seconds by an on-line flow scintillation detector.
lreparation of the ER-LSD: The recombinant ER-LBD is obtained and purified as described above.
Immobilization of the ER-LBIX The ER-LBD is then immobilized in the micromachined capillaries. The immobilization is accomplished through activation of the silanol groups on the silica chips using dicyclohexylcarbodilmide (DCC) and then coupling of the C2 spacer with a flee carboxyl group to the activated surface. The ER-LBD is then bound to the derivatized surface using the procedures developed in the previous studies with the liquid chromatographic stationary phase composed of silica gel beads. The amount of protein immobilized on the surface of the microchannels is determined by analysis of the protein content of the binding solution before and after exposure to the microchannels.
Ifthe initial experimental approach to the immobilizadon of ER-LBD is not successild the following procedures are investigated: I) if the problem exists at the during the activation of the Autol groups at the silica surface, DCC is replaced by dimedrylaminopyridine (DMAP); 2) if the problem arises frbm the C2 spacer, C3 to C4 spacers are examined; 3) if a problem exists with the immobilization to the new surface, an epoxide activated approach is explored by a method such as descdbed in J.B. Wheatley et al: Sah-induced immobilization of affinity ligands onto epoxide-activated supports, J. Chromatogr. A, 849, 1 (1999); D. Zhou, et d.: Membrane affmity chromatography for analysis mid purification of biopolymes. Chromatographis, 50, 27 (1999), or an approach utilizing streptavidin-biotinylation such as described by L.A. Paige, et al.:
Estrogen receptor (ER) modulators each induce distinct conformational changes in ERct and ERA Proc. Net. Acad. Sci., 96, 3999 (1999).
Bindimactivity of tbe immobilized ER-LBD: The binding activity of the.immobilized ER-LBD is determined using [3H]-estradiol (0.005 nM in phosphate buffer [0.1 M, pH 7.4) ([3H]-E2) supplemented with a range of concentration of cold estradiol to produce a range of from 0.001 to 0.050 nM
in Phosphate buffer [0.1 M, pH 7.4]). The solutions containing the [311X2 are applied to the microchtumels =Seining' the immobilized ER-LBD, microchannels containin' g the hmnobilized support (without the ER-LBD, a positive control) and bare microchannels (negative control). The solution containing microchannels is incubated at room temperature for 30 minutes. The c.hemels are then washed three times with phosphate buffer [0.1 M, pH 7.4], the washing is collected and assayed for ('H]-E2 content using a scintillation detector. The 114 value of E2 is calculated by nonlinear regression with Prism (GraphPad Software) using one site binding equation: Ir Bmax [P2]tota1/(114 [E2]total). The observed binding affinities and extent of binding is compared to the data from parallel binding studies carried out using an equivalent concentration of non-immobilized ER-LBD. 'These studies will yield binding affinity/mg immobilized ER-LBD indices (BA1) which is used to characterize the immobilized receptor.
thliiligAtigniatift/linliblithililLitlkilinabliinlii= The immobilization of the ERLBD is optimized through the investigation of the effect of ER-LBD concentration, reactio' n time, tempest= and chemistry used in the immobiliza' tion. Each of the variables is independently investigated in a step-wise optimization approach. The outcome of each iteration is assessed using the BAL Once an optinunn irnm.obilization procedure has been determined, the intra-day and inter-day reproducibility of the procedure is determined. A variance of no greater than 10% is deemed acceptable. ff -this cannot be achieved under the initially detamined "optimal" conditions, other previously determined conditions is investigated using the BIA as the selecting variable.
Determination of the limits of ouantikition and_ dee:alma the immobilized ER-LBD chip: The estradiol ligand is daivatized with fluorescein-5-maleimide to produce the fluaescent-ligand (132-FM) which is used in the clinical patch. ff this fluorescent-tag does not produce enough sensitivity, other agents are utilized. The immobilized ER-L13D chip is suspended over and then brought into surface contact with solutions containing ts E2. The concentrations of the E2 solutions are serially diluted from the initial concentration of 0.050 nM until displacement of E2-FM can no longer be observed. The measured optical density at lax at 488 Dm and lem 520 rim is plotted against the E2 concennutions of the test solutions to construct standard curves. Standard inter-day and infia-day validation studia are conducted to establish tite reproducibility of the measurements, the lower limits of quantitation and the lower limits of detection. Once this has been established, the chip is ready for clinical testing.
Preparation of the AR-LBD: The androgen receptor ligand binding domain (AR-LBD) fusion protein is produced and purified following known procedures. Once the protein is expressed and purified, the binding amity of 11$

the AR-LBD is determined by conventional methods following the procedure described for the BR-LBD.
Preparation of the immobilized AR-LBD chip and validation of its activity: The immobilization of the AR-LBD, determination of the bindin' g activity of the immobilized ERLBD, determination of the limits of quantitation and detection as well as the initial clinical validation is carried out based upon the results obtained with the ER-LBD.
The protein is immobilized in a similar fashion as the ER-LBD, and the Kd is determined by frontal chromatography. The stability of the column is also to determined.
The immobilization of these receptors allows for rapid screening and determination of presence of biologically activermactive compounds on the estrogen and/or androgen receptors.
The recombinant ER-LBD is obtained and purified as described above.
15 lmmobilization of the ER- : D: The AR-L/3D is then immobilized following the procedures described above for the ER-LBD.
The clinical high described herein determine the analytical precision and accuracy of methods to determine estrogen and testosterone via transdennal sampling in comparison with validated plasma assays. Validated plasma PPM
20 assays for estrogen and testosterone with acceptable limits of detection and quantification and with acceptable intra- and inter-day coefficients of variation are used to cerapare with these assays in the clirdcal settings described in the four trials outlined in detail below.
Clinical Triall : A Pilot Trial Correlating Estrogen Concentrations ig 25 plasma and Interstitialfluid in Pre and Post-Menopausal Women. In order to validate the analytical sensitivity of tranadermal sampling to measure estrogen, eight healthy, menstruating women and eight healthy post-menopausal women who are taking no estrogen-containing medications are followed for two months to measure the estrogen concentrations in their plasma or interstitial fluid.
Concentrations of estrogen in the plasma is determined by a validated can' ical EL1SA assay, routinely used in clinical settings. These concentrations are compared to those deteamined in interstitial fluid using estrogen receptor-based assays using the HW monitoring device. Plasma and interstitial fluid concentrations are measured daily during the period from the end of after to menstruation until 10 days afterward in menstruating women and over a ten day = period in post-menopausal women.
Two groups of women are recruited: a group of eight women who are pre-menopausal and who, by history, experience regular menstrual cycles; and a group of wornen who have passed through menopause Inclusion Criteria. Group 1: women who are over the age of 21 years and under the age of 40 years. Group 2: women who are over the age of 55 and under the age of 75. All women must conform with the following,: (1) taking no prescription medications or natural products intended to produce estrogen like effects (for example, ginseng or black kohosh); (2) with clinically normal laboratory values for complete blood counts, serum chemistries (Na, K, Cl, HCO3, BUN, glucose and creatinine) and clinically normal liver enzyme pinnies: SGOT, SOFT, alkaline phosphatase and bilintbin; (3) ability to understand and Gary out a signed informed consent describing this protocol.
Exclusion Criteria. The following individuals are excluded from the study: (1)smokers; (2) impaired liver or renal fanction as demonstrated by serum SOOT, SON' or bilirubin above the normal mange of laboratory values, or serum reediting greater than 1.5 ing/c1L; (3) positive urke drug screen;
(4) subjects who test positive for Human Immunodeficiency Virus or hepatitis; (5) subjects who have taken an investigational drug within 30 days of study start;

(6) subjects taking any enzyme inducing or inhibiting medications (for example, tifampin or phenytoin) for 30 days prior to dosing and (7) subjects who, in the opinion of the Investigator, is =compliant with the protocol requirements.
Retrictions kande that subjects are instructed to refrain from the to following (1) taking any prescription medications for two weeks prior to dosing C2) consuming' caffeke and/or xanthine containing products and alcohol from at least 48 bows pdor to Study Day 1 tmtil after the last blood sampk has beim collected; (3) smoking; (4) strenuous exercise during the entire study to avoid dislocation of the measuring device.
Once a subject has consented to participate in the study, the following procedures am conducted. Screening procedures are conducted within 21 days of study initiation and include: medical history and physical examination;
review of inclindon and exclusion criteria; blood and urine specimen collection;
analysis of blood sample for hanatolotg, serum chemistry, liver enzymes, HIV
and hepatitis B and C; and analysis of urine sample for screening of drugs of abuse.
&tbsequent to inclusion in the study, subject will undergoes the following procedures: subjects arrive at the testing facility at approximately 9am. in the morning. Whale the precise date is not important for post-menopausal women, menstruating mines are asked to report on the last day of their regular period. At this time estrogen levels are low and the detection limits attic assays is appropriately tested. Vital signs (heart rate, respiratory rate, blood pressure and temperature) are recorded. The portable biomedical monitoring device is placed on a forearm and attached securely with tape.
Recording occurs via a 50 micron cauterized lesion in the skin made by a small needle on the underside of the monitor that is not visible. The monitor is checked to ensure that it is recording. Vital signs (heart rate, respiratory rate, blood pressure and temperature) are recorded.
A single sample of venous blood (5 cc or one teaspoon) is drawn from a to forearm vein for the measurement of estrogen while the subject is supine. The device is instructed to measure estrogen for periods of 0.25, .5, 1, 1.5, 2, 3, 6, and 8 hours in order to test the optimal time required Additional blood samples are drawn 4 hours and eight hours after the first. The tape and device is removed. Patients are then discharged and allowed to return home before returning at 9 a.m. the next morning. This procedure is repeated on the following 9 days for a total of 10 days with each subject.
Blood Sampling Schedule. five mL venous blood samples are collected in vacutainen containing BDTA on days 1 through 10 in the rammer described above. The total number of blood draws during the course of the study including the screening and exit samples, is 35, (Thirty study draws and five screening draws for hematology, chemistry, liver enzymes, HIV and Hepatids and C
respectively.) and the total yob= of blood dravm does not exceed 200 mL.
Vital signs (heat rate, respiratory rate, blood pressure and ternperanne) are taken before and after placement of the device and before and after each 2$ blood draw on days 1 through 10.

Patients are encouraged to report any notable irritation on the arnt where the device is placed. A physician is constantly available to subjects enrolled in the study for concerns related to bruising or infection in the skin due to multiple blood draws.
Estrogen concentrations detennined using plasma ELBA is compared with the values obtained using the portable biomedical monitor. Utile correlation coefficient is > 0.8 with a significance p < 0.05 the measurements are deemed valid.
A Pik; Tdal contlating Testosterone Csocentratops in plasma and Interstitial Fluid in Men. Ten healthy men, chosen to represent a range of ages between 21 and 70 years old and who are taking no medications have plasma and interstitial fluid testosterone concentrations measured daily for 5 consecutive days both by validated plasma assay and by tnui.sdermal sampling using the portable biomedical monitoring device.
*Win Criteria. Men who are over the age of 21 and under the age of 75. All men must cod= with the following: (1) taking no prescription medications or natural products intended to produce testosterone like effects (for example, androstenedione or DHEA); (2) with clinically noimal laboratory values for complete blood counts, serum chemistries (Na, IC, CI, HCO3, BUN, glucose and creatinine) and clinically normal liver enzyme profiles: SOOT, SOPT, alkaline phosphatesc and bilimbin; and (3) ability to understand and carry out a signed infarmed consent describing this protocol.
Exclusion Criteria. The following individuals are excluded from the study: (1) smokers; (2) impaired liver or renal fimction as demonstrated by serum SOOT, SOPT or bilirubin above the nonnal range of laboratory values, or serum creatinine greater than 1.5 mgfdL; (3) positive urine dng screen; (4) subjects who test positive for Human Immtmodeficiency Virus or hepatitis.
Restrictions include subjects are instructed to refrain from the following:
(1) =noir* and (2) stramous exercise duting the entire study to avoid s dislocadon of the measuring device.
Once a subject has consented to participate in the study, the following procedures are conducted: Screening procedures are conducted within 21 days of study initiation and include: medical history and physical examination;
review of inclusion and exclusion criteria; blood and urine specimen collection;
analysis of blood sample fa hemeology, serum chemistry, liver ammo, HIV
and hepatitis B and C; and analysis of urine sample for screening of drugs of abuse.
Subsequent to inclusion in the study, subject will undergoes the =
following procedures: Subjects wive at the testing facility at approximately 9 a.m in the morntn' g. Vital signs (heart rate, respiratory rate, blood pressure and =
temperature) are recorded The portable biomedical monitoring device is placed on a &rearm and attached securely with ape. Recording occurs vial 50 micron cauterized lesion in the skin made by a small needle on the underside ofthe monitor that is not visible. The monitor is checked to ensure that it is recording.
Vital signslheart rate, respiratory rate, blood pressure and temperature) are recorded A single sample of venous blood (5 cc or one teaspoon) is drawn from a forearm vein for the measintmlent of testosterone addle the subject is supine.

The device is instructed to MU= estrogen for periods of 0.25, .5, I, 1.5, 2, 3, 6, and 8 hours in order to test the optimal time required. Additional blood samples are drawn 4 hours and eight hours after the first. The tape and device is removed. Patients are then discharged and allowed to return home before returning M 9 am. the next morning. This procedure is repeated on the following 4 days for a total of 5 days with each subject.
Five mL venous blood samples are collected in Vacutainen containing EDTA on Days 1 through 5 in the manna descdbed 11130Ve. The total number of blood draws during the course of the study including the screening and mit samples, is 20, (Fifteen study draws and five screening draws for hematology, chemistry, fiver enzymes, FIN and Hepadtis and C respectively.) and the total to volt= of blood drawn does not exceed 200 mL.
Vital signs (head rate, respiratory rate, blood pressure and temperature) are taken before and alter placeman of the device and before and after each blood draw on days 1 through 5.
Patients are encouraged to report any notable irritation on the arm where S the device is placed. A physician is constantly available to subjects enrolled in the study for CO0CO211S related to bruising or infection in the skin due to multiple blood draws.
Testosterone concentrations determined using plasma ELISA is compared with the values obtained using the portable biomedical monitor. ffthe 20 conelation coefficient is > 0.8 whit a significance p < 0.05 the measurements are deetmed The purpose of this third clinical trial is to administer appropriate concentrations of estrogen to postmenopausal women who are healthy volunteers and to meastire the resulting concentrations. Estrogen is administered 25 in microgram pubes over a period of 3 days after a 3-day nm-in for baseline measurement using the optimal machine settings for the monitor as defined above.
The purpose of this fourth clinical trial is to administer appropriate concentrations of testosterone to men between the ages of 55 and 75 years who are healthy volunteers and to measure the resulting concentrations. Testosterone ins administered in microgram pulses over a 5 day period after a 3 day run-in period that is used to determine baseline testosterone concentrations in these men before the administration of testosterone. The resulting concentrations is determined using the analytical specifications as defined above.

Claims (56)

Claims:

1. A transdermal sampling system, comprising: at least two samplers for transdermally retrieving and transferring at least one analyte from the skin of a subject, wherein each of the at least two samplers are included in a single microfabricated device comprising a microfluidic assembly; each of the at least two samplers including at least one microheater configured for ablating a portion of the stratum corneum of the skin of a subject on demand to facilitate access to interstitial fluid from the subject's underlying viable epidermis; at least one detector system for identifying and quantifying said at least one analyte separately for each of the at least two samplers; and at least one logic module for (i) receiving and storing input data from said at least one detector, (ii) relating the input data to other data obtained from the subject relating to the condition of the subject, (iii) displaying output information indicative of health and clinical state of the subject as determined from the relating of the input data to the other data, (iv) transmitting the output information to another system, and (v) controlling the operation of said at least one sampler and at least one detector.

2. The system of claim 1, wherein said at least one detector system comprises an optical detection system comprised of at least one light source effective to excite fluorophores, and at least one detector for detecting fluorescence from excited fluorophores.

3. The system of claim 2, wherein said light source comprises at least one LED or at least one laser.

4. The system of claim 1, further comprising means for holding the the microfabricated device in contact with the skin of the subject.

5. The transdermal sampling system according to claim 4, wherein the adhesive substantially prevents movement of the transdermal sampling system relative to the skin of the subject.

6. The transdermal sampling system according to claim 4, wherein the adhesive serves to prevent loss of a physiologically compatible fluid.

7. The transdermal sampling system according to claim 4, wherein the adhesive is water impermeable.

8. The system of claim 4, wherein the means for holding is an adhesive.

9. The system of claim 1, further comprising at least one substance located in each of said at least two samplers which is capable of binding an analyte of interest, and said substance being detectable by said at least one detector system.

10. The system of claim 1, further comprising means for monitoring predetermined physiological data as said other data, for relating to the input data by said at least one logic module.

11. The system of claim 1, further comprising means for monitoring environmental conditions data as said other data, for relating to the input data by said at least one logic module.

12. The system of claim 1, wherein said at least one detector system comprises a patch sensitive to at least one analyte for changing color in response to contact with said at least one analyte, and at least one detector for detecting a change in color of the patch.

13. The device of claim 1, wherein said at least one detector system comprises a colorimetric detection system comprised of a colorimetric analyte sensitive region, wherein a color change in the colorimetric analyte sensitive region by an observer indicates the presence of an analyte.

14. The transdermal sampling system according to claim 1, wherein the microfluidic assembly comprises at least one serpentine capillary channel.

15. The transdermal sampling system according to claim 1, wherein each of the at least two samplers further comprises: at least one reservoir channel: at least one bottom capping section: and at least one top capping section.

16. The transdermal sampling system according to claim 15, wherein the at least one reservoir channel further comprises at least one seal for retaining a physiologically compatible fluid within the at least one reservoir channel.

17. The transdermal sampling system according to claim 16, wherein the at least bottom capping section includes the at least one microheater.

18. The transdermal sampling system according to claim 15, wherein the at least one top capping section comprises two or more electrodes.

19. The transdermal sampling system according to claim 18, wherein the two or more electrodes serve to assist the flow of a physiologically compatible fluid through an at least one serpentine capillary channel.

20. The transdermal sampling system according to claim 15, further comprising a sensor for detecting the at least one analyte.

21. The transdermal sampling system according to claim 20, wherein the sensor for detecting the at least one analyte comprises a patch sensitive to the at least one analyte for changing color in response to contact with the at least one analyte, and at least one detector for detecting a change in color of the patch.

22. The transdermal sampling system according to claim 1, wherein the microfluidic assembly contains a physiologically compatible fluid that transfers at least one analyte obtained transdermally from the skin of a subject.

23. The transdermal sampling system according to claim 1, wherein at least one surface of the microfluidic assembly is modified in a manner to enhance sampling function.

24. The transdermal sampling system according to claim 23, wherein the modification of the at least one surface of the microfluidic assembly prevents the adsorption of protein to the at least one surface of the microfluidic assembly.

25. The transdermal sampling system according to claim 23, wherein the modification of the at least one surface of the microfluidic assembly attaches to the at least one surface of the microfluidic assembly at least one specific-binding molecule which specifically binds the at least one analyte.

26. The system of claim 1, further comprising at least one substance located in each of said at least two samplers which is capable of reacting with an analyte of interest, and said substance being detectable by said at least one detector system.

27. A microfabricated device for allowing remote monitoring of a subject, comprising: at least one sampler unit body for transdermally retrieving at least one analyte from interstitial fluid of a subject, wherein the sampler unit body is a microfabricated device including; at least two samplers; at least one microheater associated with each of the at least two samplers configured for ablating a portion of the stratum corneum of the skin of a subject on demand to facilitate access to interstitial fluid from the subject's underlying viable epidermis at least one detector system connected to said at least one sampler unit body for identifying and quantifying at least one analyte obtained from a subject separately for each of the at least two samplers; and a transmitter/receiver for transmitting data relating to at least one analyte detected by said detection system to a logic module for processing thereby, and for allowing control of the microfabricated device by a logic module.

28. The device of claim 27, wherein said at least one detector system comprises a plurality of detector systems, each of said plurality of detector systems connected to a corresponding one of said at least two samplers.

29. The device of claim 27, wherein the sampler unit body is configured to substantially adhere to the skin of a subject.

30. The device of claim 27, further comprising at least one substance located in said sampler unit body which is capable of binding an analyte of interest, and said substance being detectable by said at least one detector system.

31. The device of claim 27, wherein said at least one detector system comprises a patch sensitive to at least one analyte for changing color in response to contact with said at least one analyte, positioned for contact with said at least one analyte, and at least one detector positioned for detecting a change in color of the patch.

32. The device of claim 27, wherein each of said at least two samplers includes a capillary channel extending therethrough for sampling analyte from the skin of a subject through said capillary channel; and at least one detector chamber associated with said at least one detection system, and connected with said capillary channel for detecting analytes received within the detector chamber.

33. The device of claim 32, wherein each of said at least two samplers comprises at least one reservoir connected with said capillary channel, wherein a fluid retained in the at least one reservoir transfers from the reservoir to the subject's skin into said capillary channel.

34. The device of claim 33, wherein said sampler unit body further comprises a microheater disposed to be in close proximity to a subject's skin surface at a location proximate to where said fluid is pumped into contact with the skin of a subject, wherein the microheater ablates a portion of the stratum corneum of the skin of a subject.

35. The device of claim 32, wherein said sampler unit body is silicon.

36, The device of claim 27, wherein said at least one detector system comprises an optical detection system comprised of light sources effective to excite fluorophores, and at least one detector for detecting fluorescence from excited fluorophores.

37. The device of claim 36, wherein said light source comprises at least one LED or at least one laser.

38. The device of claim 27, further comprising at least one substance located in each of said at least two samplers which is capable of reacting with an analyte of interest, and said substance being detectable by said at least one detector system.

39. A microfabricated device for sampling analytes from the skin of a subject, comprising: at least two samplers for transdermally retrieving and transferring at least one analyte from the skin of a subject, wherein each of the at least two samplers are included in a single microfabricated device; each of the at least two samplers including at least one microheater configured for ablating a portion of the stratum corneum of the skin of a subject on demand to facilitate access to interstitial fluid from the subiect's underlying viable epidermis; a detection chamber for receiving analytes retrieved from the skin of a subject; a photonic detection system, comprising a photonics source located attached to said microfabricated device in association with said detection chamber for separately detecting analytes for each of the at least two samplers.

40. The device of claim 39, wherein said photonics source is at least one LED or at least one laser.

41. The device of claim 39, further comprising substances in said device which bind with selected analytes, and which fluoresce when said photonics source applies radiation thereto.

42. A microfabricated device for sampling analytes from the skin of a subject, comprising: at least two samplers for transdermally retrieving and transferring at least one analyte from the skin of a subject, wherein each of the at least two samplers are included in a single microfabricated device; each of the at least two samplers including at least one microheater configured for ablating a portion of the stratum corneum of the skin of a subject on demand to facilitate access to interstitial fluid from the subject's underlying viable epidermis; a detection chamber for receiving analytes retrieved from the skin of a subject; a patch which changes color when contacted by predetermined analytes, located attached to said microfabricated device in association with said detection chamber far separately detecting analytes for each of the at least two samplers.

43. A device for sampling and detecting analytes retrieved from the skin of a subject, comprising: at least two microfluidic channels for retrieving and transferring at least one analyte from the skin of a subject to a detector, wherein said detector identifies and quantifies said at least one analyte separately for each of the at least two microfluidic channels; and each of the at least two microfluidic channels including a microheater configured for ablating a portion of the stratum corneum of the skin of a subject on demand to facilitate access to interstitial fluid from the subject's underlying viable epidermis which may contain the at least one analyte.

44. A microfabricated device for allowing remote monitoring of a subject, comprising: at least one sampler unit body, wherein the sampler unit body is a microfabricated device comprising a microfluidic assembly, said microfluidic assembly including: at least one reservoir containing a fluid and at least one channel either directly or indirectly fluidly connected to the at least one reservoir arranged wherein the at least one channel receives the fluid from the at least one reservoir after the fluid retrieves the at least one analyte from the skin and transfers the at least one analyte through the at least one channel; and at least one detector in association with the at least one channel to detect the at least one analyte transferred through the at least one channel.

45. The device of claim 44, wherein the reservoir retains the fluid in the reservoir with a breakable seal, wherein the breaking of the breakable seal releases the fluid.

46. The system of claim 44, wherein a pump pumps the fluid into contact with the skin of a subject.

47. The system of claim 46, further comprising a microheater disposed to be in close proximity to a subject's skin surface at a location proximate to where said fluid is pumped into contact with the skin of a subject, wherein the microheater ablates a portion of the stratum corneum of the skin of a subject.

48. The system of claim 44 wherein the at least one channel is a capillary.

49. A transdermal sampling system, comprising: at least two samplers for transdermally retrieving at least one analyte from the skin of a subject, wherein each of the at least two samplers are included in a single microfabricated device; each of the at least two samplers including at least one microheater configured for ablating a portion of the stratum corneum of the skin of a subject on demand to facilitate access to interstitial fluid from the subject's underlying viable epidermis; at least one detector system for identifying and quantifying said at least one analyte separately for each of the at least two samplers; and at least one logic module for (i) receiving and storing input data from said at least one detector, (ii) relating the input data to other data (iii) displaying output information as determined from the relating of the input data to the other data, (iv) transmitting the output information to another system, and (v) controlling the operation of said at least one sampler and at least one detector.

50. The system of claim 49, wherein said at least one detector system comprises an optical detection system comprised of at least one light source effective to excite fluorophores, and at least one detector for detecting fluorescence from excited fluorophores.

51. The system of claim 50, wherein said light source comprises at least one LED or at least one laser.

52. The system of claim 49, further comprising means for holding the microfabricated device in contact with the skin of the subject.

53. The system of claim 49, further comprising at least one substance located in each of said at least two samplers which is capable of binding an analyte of interest, and said substance being detectable by said at least one detector system.

54. The system of claim 49, further comprising means for monitoring predetermined physiological data as said other data, for relating to the input data by said at least one logic module.

55. The system of claim 49, further comprising means for monitoring environmental conditions data as said other data, for relating to the input data by said at least one logic module.

56. The system of claim 49, wherein said at least one detector system comprises a patch sensitive to at least one analyte for changing color in response to contact with said at least one analyte, and at least one detector for detecting a change in color of the patch.