CA1332221C

CA1332221C - Enzymatically amplified piezoelectric specific binding assay

Info

Publication number: CA1332221C
Application number: CA000602569A
Authority: CA
Inventors: Michael David Ward; Richard Calvin Ebersole
Original assignee: EI Du Pont de Nemours and Co
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1988-04-06
Filing date: 1989-03-31
Publication date: 1994-10-04
Anticipated expiration: 2011-10-04
Also published as: JPH03503567A; DE68916423T2; DE68916423D1; WO1989009937A1; US4999284A; EP0408578B1; EP0408578A1

Abstract

ABSTRACT
A quartz crystal microbalance assay in which the binding of analyte to a surface on or near a quartz crystal microbalance (QCM) is detected by a conjugate which comprises an enzyme capable of catalyzing the conversion of a substrate to a product capable of accummulating on or reacting with a surface of the QCM leading to a mass change and, hence, a change in resonant frequency.

Description

~ 33~2 ~

~ITLE CR-8402 ENZYMATICALLY AMPL~FIED PIEZOELECTRIC
SPECIFIC BINDING ASSAY

Field of the Invention The present invention relates to an enzymatically amplified piezoelectric specific binding assay in which analyte suspected of being present in a liquid sample is bound either on or in the proximity of a quartz crystal microbalance by means of a capture reagent, and the bound analyte is reacted with an anti-analyte reagent/enzyme conjugate. The conjugate is reacted with a substrate specific for the enzyme to form a product that is capable of either reacting with and/or accumulating on the surface of the quartz crystal microbalance.
The mass change on the surface of the quartz crystal microbalance resulting from these reactions results in a ehange in the resonant frequency of the quartz crystal microbalance, which can be used to determine the analyte concentration in the 6ample.
3ackground of the I~vçntion The use of quartz crystal microbalances (also known as piezoelectric occillators) in immunoassays has been described previously. These devices consist of sinqle crystal w~r~ sandwiched ~etween tvo electrodes. The electrodes ~re provided wit~ means for connecting these devic~s to an external ~scillator circuit that drives the gu~rtz crystal at its resonant frequency. Th~G frequency dependent on the mas~ o~ the cryfit~ s well a~ the ~ass of ~ny layers confined to the el~ctrode areas of the cryst~l. Thus, the frequency is altered by changes in mass on the surface of the electrodes or in any layers on those electrodes. In gener~l, t~e ~ 1 ~
, ' 1 J3222i .

change in resonant frequency of these devices can be correlated to the amount of mass change; if the ~, quartz crystal microbalance and any attached layer~
s obey rigid-layer behavior, the mass change can be determined from the frequency change by the Sauerbrey relationship ~ f 210~m ~,`.s AJ~
.`..~1 , where ~ f is the measured freguency shift, fo the parent frequency of the quartz crystal, ~ m the mass change, A the piezoelectrically active area, pg the density of quartz (2.648 g cm~3) ~nd uq the shear modulus (2.947 x loll dynes cm~2 for AT-cut quartz). ~-~
Shons et al. describe a piezoelectric quartz crystal microbalance which has been ~odified for the determination of antibody activity in solution. A guartz crystal, precoated w$th antigen, is exposed to antisera of varying concentration and specificity. Antisera ~pecific for the antigen coating will form an ~ddition~l prote$n layer on the crystal. The t~ickness of the ~ntibody layer, measured by the frequency ~hift of the dry cry~tal, is proportion~l to the concentrstion of ~pecific ~nt$body in ~olution. ~J. B$~med. Mater. Res., Vol.
6, pp. 565-570 (1972)~
U. S . Patent No. 4,235,983, $~sued to Rice on December 2, 1980, discloses ~ ~ethod for tbe . . . .
determ$nation of ~ part$cular 6ubc1~6s of ant$body.
The ~ethod ut$1$zes ~ p$ezoelectr$c oscillator hav$ng bound to $ts ~urface an ant$gen ~pec$f$c for the nt$body to be determined. The ~nt$gen-cQated ~ '. ~ . `
~:' . ~

~; 1 33222 1 oscillator is exposed to a solution containing an ~` unknown amount of the antibody. After the antibody i in the solution is attached to the antigen on the -`~ oscillator, the oscillator is exposed to a so-called -1 sandwiching substance which selectively binds to a specific subclass of the antibody being determined.
The frequency of the oscillator is measured in the j dry state before and after exposure to the !
sandwiching substance. The change in frequency is related to the amount of the subclass of antibody bound to the oscillator, and the amount of the subclass of antibody in the solution can be determined by reference to a standard curve.
Roederer et al. disclose an in-situ immunoassay using piezoelectric guartz crystals, specifically, surface acoustic wave devices. Goat anti-human IgG was ~mmobilized on the guartz crystal surface with a coupling agent. The piezoelectric crystals were then placed in an electric oscillator circuit and tested for detec~ion of the antigen human -IgG. Detection was based upon the fact that 6urface mass changes by adsorption are reflected as shifts in the resonant freguencies of the crystals. The ~uthors concluded that the ~ethod suffers from both poor sensitivity and poor detection limits. The authors also concluded that the antigen to be detected must be of high molecular wèight: low olecular weight analytes cannot be directly detected by this ~ethodology. [Analytical Chemistry, Vol. 55, (1983)]
Ngeh-Ngwainbi et ~1. de~cribe the use of piezoelectric quartz crystals coated with antibodies against parathion which are used for the assay of parathion in the gas phase. When the coated ~nt~body binds with parathion by a direct reaction in the gas : ' .:

: :~ .: ~ - - ~ : : : : . . . - : : :

1 ~ 3 2 2 2 1 :i i phase, the resulting mass change on the crystal generates a frequency shift proportional to the concentration of the pesticide. [J. Mat. Chem. Soc., Vo~. 108, pp. 5444-5447 (1986)]
European patent application 0 215 669, published March 25, 1987, discloses an analytical devic~ and method for t~e in-situ analysis of biochemicals, microbes and cells. Again, the ~ethod is predicated on a resonant frequency change caused by a weig~ change on the surface of a piezoelectric crystal on which are immobilized receptor materials pecific for the analyte tv be detected.
Grabbe et al. describe a quartz crystal resonator, used in conjunction with cyclic voltammetry, to ctudy the binding of human IgG and anti-IgG at a silver electrode. tG. Electroanal.
Chem. Vol. 223, pp. 67-78 (1987)]
As discussed by Roederer et al., piezoelectric crystal-based immunoassays in which mass chan~e is attributable only to the immunological reaction between an antigen and an antibody can, under certain circumstances, suffer from poor sensitivity and poor detection limit. Conseguently there is a need in the art for a piezoelectric crystal-based specific binding assay in which the reaction between a binding agent and ~ts ligand can be amplified to provide a more ~ensitive and reliable assay.
Summary of the Invention This need i5 met by the present invention which, in one aspect, is a process ~or measuring an analyte utilizing a coniugate compri6ing an enzyme and either an anti-analyte reagent or the analyte.
The conjugate is capable of reacting with (or competing with) an analyte indirectly bound to a ' - . . . . , ~ ~

~ <3222~

quartz crystal microbalance by a capture reagent that is directly bound to a surface of the quartz crystal microbalance. The quartz crystal microbalance may have at least one of its 6urfaces modified by ~ny combination of chemically reactive, priming, coating or anti-analyte layers prior to exposure to the analyte. æuch a modified guartz crystal microbalance is referre~ to herein as ~ biologically modified quartz crystal microbalanceO or BMQCM. Once the conjugate is bound to the BMQCM-bound analyte, a substrate ~pecific for the enzyme is added to the system. The enzyme catalyzes a reaction in which the substrate is converted to a product which either tl) accumulates on the surface of the BMQCM; (2) reacts with and is subsequently incorporated into the BMQCM, either electrostatically, covalently or by simple adsorption; or (3) reacts with the BMQCM, but results in incorporation of a species other than the enzymatic reaction product. The resulting mass changes produce corresponding changes in the resonant freguency of the quartz crystal, as measured by an external oscillator circuit and freguency meter.
In another aspect, the present invention is a process for measuring an analyte utilizing a reaction chamber in which a quartz crystal microbalance is placed opposite and in close proximity to a surface havinq capture reagent adsorbed thereon. Upon exposure to the ~ample, analyte is bound by the capture resgent. The resulting bound complex i~ then reacted with a conjugate comprising an enzyme ~nd either an anti-analyte reagent or the analyte. A substrate is ~-~
then introduced. The enzymç catalyzes a reaction in which the substrate is convered into ~ product wh~ch accumulates on the surface of the quartz crystal .

~ ` 1332221 microbalance, thereby changing its mass and resonant frequency. The accumulation of product on the microbalance can be mediated by a reactive layer on the microbalance. The reactive layer can be chosen to mediate mass accumulation by, for example, physical adsorption, ion complexation or covalent attachment of the catalysis product. Alternatively, the reactive layer can be chosen so that the ~ catalysis product causes a change in the reactive q 10 layer that results in simple adsorption, ion exchange or covalent attachment of another reagent in the reaction medium.
Brief DescriPtion of the Drawing , The drawing consists of seven figures.
15 Figures 1 through 7 (excluding Figure 6 which does not form part of this specification) depict various modes of carrying out the present invention. Figure 8 depicts suitable circuitry for measuring the J resonant frequency of the BMQCM.
Detailed Description of the Invention The invention may be understood by reference .1 to the Drawing wherein like reference numerals are used to indicate like elements.
~j Referring now to Figure 1, there is seen a biologically modified quartz crystal microbalance (BMQCM) indicated generally by the reference numeral 10. The BMQCM comprises a quartz crystal wafer 12 , sandwiched between two electrodes 14, 16. Adsorbed to one surface 18 of the electrode 16 is capture i;` 30 reagent 20. -Upon exposure of the BMQCM having capture reagent bound thereto to a solution (not shown) containing analyte 22, the analyte 22 will be bound by the adsorbed capture reagent 20, thus forming a - 35 bound complex. After a suitable incubation period, unbound analyte is washed away.

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1 3 3 ~ 2 2 1 The QCM is then contacted with a conjugate 24 comprising anti-analyte reagent and an enzyme, designated generally by E. After a suitable incubation period, unbound conjugate is washed away.
The QCM, having the conjugate bound thereto, is then contacted with a 601ution containing a substrate, designated generally by S, which is specific f~r the enzyme E. The enzyme will then catalyze a reaction in which the substrate is converted to a produ~t P. The enzyme and substrate system is chosen such that the product P is insoluble and precipitable on the BMQCM surface. The product P
will accumulate on the surface 18, thereby leading to a change in mass and hence a change in resonant frequency, as measured by an external circuit 26.
Suitable anti-analyte reagents and capture reagents include those reagents which are capable of participating in complexation reaction with the analyte. Preferred reagents include antibodies, lectins, chelating agents, binding proteins, DNA and RNA polynucleic acid pro~es, and cell receptors. The choice of reagent will depend on the analyte to be ~easured. The anti-analyte reagent and capture reagent may be the same or different chemically.
Suitable analytes include proteins, hormones, enzymes, antibodies, drugs, carbohydrates, nucleic acids, etc.
Examples of enzyme/substrate ~ystems which are capable of producing an insoluble product which is capable of accumulating on the surface of the BMQCM include alkaline phosphatase ~nd 5-bromo-4- ~-chloro-3-indolylphosphate (BCIP). The enzymatically catalyzed hydrolysis of ~CIP produces an insoluble dimer which precipitates on the ~urface of the BMQC~
Other nnalogous substrates having the phosphate :
.
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1 3 3 ~ ~ 2 1 moiety replaced with such hydrolytically cleavable functionalities as galactose, glucose, fatty acids, fatty acid esters and amino acids can be used with their complementary enzymes. `-Other enzyme/substrate systems includeperoxidase enzymes, for example horseradish peroxidase (HRP) or myeloperoxidase, and one of the following: benzidene, benzidene dihydrochloride, diaminobenzidene, o-tolidene, o-dianisidine ~nd tetramethylbenzidene, carbazoles, particul~rly 3-amino-9-ethylcarbazole, all of which have been reported to form precipitates upon reaction with peroxidases. Also, oxidases suc~ as alphahydroxy acid oxidase, aldehyde oxid~se, glucose oxidase, L-amino acid oxidase and xanthine oxidase can be used with oxidizable substrate systems such as a phenazine methosulfate-nitriblue tetrazolium mixture.
Referring now to Figure 2, there is seen an alternative embodiment of the BMQCM shown in Figure 1. Specifically, the surface 18 has been modified by coating it with a layer 28. The layer 28 can 6erve as a ~priming~ layer, which enhances attachment of the capture reagent 20. The layer 28 can also ~erve to enhance mass accumulation on the BM~CM by (1) pecific reaction between product P and the layer 28, (2) ion exchange between P and t~e layer 28 or (3) simple absorption o~ P into the layer 28. `~
Illustrative surfaces 28 are polymer fil~s and silane reagents that serve to enhance the binding of the capture reagent durin~ equilibration by either hydrophobic interactions or covalent interactions. An example of a polymer film i6 polystyrene, which, itself, c~n be applied by conventional ~ethods, such as spin coating. Higher ~
surface area coatings for greater capture reagent ~;

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-" I 33222 1 coverages can be achieved by fabrication of irregular and three dimensionally shaped surfaces, such as by aerosol application which deposits minute droplets of polymer. Suitable silanes include the generaI class of alkyl trichlorosilanes, which covalently bind to the metal and glass surfaces of the quartz cry6tal microbalance by M-O-Si and si-o-si linkages, respectively. The general class of aminosilanes, when attached to the QCM surface via ~-O-Si or si-o-si linkages, can be used to bind the capture reagent by covalent linkages between the nitrogen atom of the aminosilane and an ~ppropriate functional group on the capture reagent. Surfaces can also be treated with reactive films, for example, redox polymer films such as polyvinylferrocene, PV-Fc, which serve as hydrophobic layers to enhance binding of the capture reagent, as well as reactive layers that react with the enzymatic reaction product, P, leading to an increased mass and changed resonant frequency.
Examples of enzyme/substrate systems which result in the production of a product for which the BMQCM surface can ~ave a ~pecific affinity include -~
horseradish peroxidase and hydrogen peroxide/iodide ~ixtures. In this system, the substrate is catalytically converted by the enzyme to I2/I3- which oxidizes a PV-Fc film on the ~urface of the BMQCM.
After oxidation, I3- is ~pecifically bound by the ` ~
PV-Fc+ sites in the film. ~ -Referring now to Figure 3, there is seen yet another embodiment of the BMQCM. Specifically, a layer 28 is chosen to react with the product P to induce ~ chemical change in the layer 28 mak$ng it specifically reactive with a chemical species, 9 ~ ~

., ., ~......................... . . .. .

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~ 33222 1 :

designated generally by A, which is different from P
and is present in the reaction systsm.
Suitable layers 28 for the embodiments shown in Figures 2 and 3 could comprise organic thin films, redox polymers and conducting polymers which are capable of incorporating anions upon oxidation.
Illustrative ~rganic redox p~lymers and thin films are poly~inylferr~cene, polypyrrole, polythiophene, polyacetylene and phthalocyanines. Suitable anions for the emb~di~ent shown in Pigure 3 include halides, polyhalides, ferro/ferricyanide and nitrate.
Illustrative enzyme/substrate systems are peroxidase/H202/I~ and peroxidase/ferrocyanide.
Referring now to Figure 4, there is seen an alternat-ve embodiment of the method according to the present invention. In this embodiment, the capture reagent 20 is attached to a support surface 30 which is different from the QCM surface 18 or layer 28.
However, the support surface 30 must be in close proximity to the layer 28. In this embodiment, analyte 22 is bound by capture reagent 20. The bound analyte is then reacted with conjugate 24. The conjugate 24 is then reacted with substrate S to produce product P which diffuses to layer 28 where it accumulates to produce a mass change and, hence, a resonant frequency change. ~;
Figure 5 depicts substantially the same embodiment, except that the interaction of the product P with the layer 28 induces a chemical change in the layer 28, making it reactive with a species A
present in the reaction ~ystem. Suitable layers 28, enzyme/substrate ~ystems and ~peeies A have been discussed above in connection with Figure 3.
-, 10 ' :

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332~? 1 ll For the embodiments described in Figures 4and 5 suitable support surfaces include porous and nonporous polymeric thin films, cellulosic membranes and nitrocellulose membranes. The reactive films 28 may include organic thin films, polymers, redox polymers and conducting polymers which are capable of adsorbing the product P or reacting with P, followed by incorporation of P or a different species A. The polymers or organic thin films may include polyvinylferrocene, polypyrrole, polythiophene, polyacetylene and phthalocyanines. Illustrative anions are halides, polyhalides, ferro/ferricyanide and nitrate. An illustrative enzyme/substrate system is peroxidase/H2O2/I . In this enzyme/substrate system, peroxidase catalyzes the conversion of I to I2/I3 , which, in turn, oxidizes a polyvinylferrocene film. Subsequent incorporation of I3 in the film to maintain -charge balance results in a mass increase and a ~-measurable change in frequency of the QCM.
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-~ 33222 1 In the description relating to Figures 1 i through 5, above, the sample and reag~nts are de~cribed as being added sequentially. It should be understood, however, that the reagentC may be added simultaneously.
~, Figure 7 depicts another embodiment of the 3~ invention i~ ~hich the analyte 22 and a conjugate of analyte and enzy~e 24 compete for a limited nu~ber of capture reagent 20 sites on the surface of the QCM.
The sample can be added before the conjugate 24 or toget~er with the conjugate 24. After a suitable incubation period, the unbound conjugate 24 is washed away, and substrate added. The remainder of the assay is performed as described above. In this case, the response due to accumulation of mass on the BMQCM
- is inversely proportioned to the analyte concentration. It should be understood that this emb~di~ent may also be performed using a capture reagent surface which is in proximity to the surface of the QCM, analogous to the embodiments shown in Figures 4 and 5.
Figure 8 depicts circuitry which may be used to measure the resonant frequency of the BMQCM.
Such circuitry is conventional and well-known in ~he ` art.
In general, attachment of the capture reagent 20 is accomplished by incubation of the QCM
in a phosphate buffered saline (PBS) buffer ~olution of the capture reagent, in ooncentrations ranging from 50 to 100 ug/ml, depending on the capture reagent and the type of ~urface on the quartz crystal microbalance. Another ~uitable method involve immobilization of the capture reagent ~y chemical crosslinking, for example with glutaraldehyde. After :
. , .: 5~

~ t3222 1 equilibration, the BMQaM is washed with PBS buffer solution to remove any excess cap~ure reagent that is not strongly attached to the desired surface.
After formation of the BMQCM assembly, the BMQCM is exposed to 6Dlutions of the analyte 22 ~t room temperature for a period of time predetermined to be optimal for the capture reagent/analyte system under study. After this exposure, the surface is washed with TRIS test buffer to remove non-specifically adsorbea ~aterial. The conjugate 24 is then added in concentrations ranging from 0.05 to 20 ug/mL in TRIS test buffer to the BMQCM/analyte assembly at room temperature. Conjugate can be prepared, for example, according to the method of Imagawa [J. Biochem., 92, 1413 (1982)] or Avrameas et al. [Scand. J. Immunol., Vol. 8, Suppl. 7, 7-23 (1978)] and maintained at 4'C as a stock solution.
Conjugates of enzymes and 6ynthetic polynucleic acids -~
can be prepared according to the ~ethod of Ruth et al. ~DNA, 4, 93 (1985)]. This is then followed by washing with TRIS test buffer solution to remove nonspecifically adsorbed conjugate 24. The total amount of conjugate 24 should be added at a concentration to exceed the amount of specifically adsorbed analyte 22. A~ternatively, the analyte and conjugate may be added 6imultaneously.
In a so-called competitive mode, the ,.~.;
analyte and an ~nalyte~enzyme conjugate ~ay be reacted with the ~M~C~ The analyte and conjugate ~-- may be added sequentially or simultaneously.
The frequen~ of the guartz crystal microbalance i~ ~eas~rea in 50 MM TRIS wash ~uffer ~olution, and ~hen a ~t~ndard ~olution of the ~ substrate is directly aaded. The rate of fr~quency ;` change, as well a~ t~e total freguency change after a , .
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t 33222 1 i 14 i time considered to ~e the optim~m measurement j interval, are measured in solution and, since conjugate is present only when analyte is bound to the surface, are indicative of the amount of analyte expose~ to the BMQ~. The signal measured i~
amplifi~d ~y the large turnover numbers of the enzymatic reaction, which produces concentrations of produ~t far exceeding that of the analyte.
~ he present invention can be embodied in diagnostic ~it~s CD } ising crystals treated with the desired capture reagent and any modifying layers 28, and an oscillator circuit with direct readout of the resonant frequency of the quartz crystal microbal~nce. In typical use, the ~nalyte solution, -~ for example patient serum, would be added to a ~; compartment containing the BMQCM, followed by a wash with buffer solution, followed by addition of the appropriate enzyme/2nti-analyte reagent conjugate, followed ~q ~ ~sh. Ihe substrate would then be ~` added and the freguency change directly measured.
The preferred mode of operation would include the use of a refer~nc~ crystal which is exposed to the identical solutions, but which has not been modified so as to ha~e capt~re reagent on or in proximity to its surface. In thifi ranner, the difference in frequency between the sample and reference crystals can be ~easured and error~ due to changes in viscosity, ~YYçer~re ~nd non-~pecific binding minimiz~d. ~es~icn with only the 6ample crystal, however, is al80 feasible, because (1) the viscosity and temperat~re change6 during addition of the substrate and during ~easurement are not large, (2) interference frn~ nnnspecific ~dsorption i~ ized ` ~:

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by t~e washing step, and (3) the measurement step poses no risk of mass changes from processes other , than those induced by P.
;; The invention is further illustrated by the following nonlimiting examples.

Example 1 .i :
I Assay of adenosine-5'-pbosphosulfate (APS) reductase using alkaline phosphatase/5-~romo-4-chloro-3-indolylphophate (BC~P) on unmodified quartz crystals.
:.
~ The procedure was performed accordiny to - the mode illustrated in Figure 1, with anti-APS
~^ reductase antibody as the capture reagent 20, APS
- reductase as the analyte 22, anti~APS reductase antibody with alkaline phosphatase enzyme as t~e conjugate 24, and 5-bromo-4-chloro-3-indolylphophate -~ (BCIP) as the substrate, S. ~he first step was adsorption of the anti-APS antibody on the gold/
~ guartz surfaces of a quartz crystal. Thi~ was ;~ performed by equilibration of the gold/quartz crystal with 2 mL of 100 ug/mL anti-APS reductase antibody in PBS buffer solution for 2 hours. The ` crystal was then washed once with PBS buffer `~ containing 0.1~ bovine serum ~lbumin (BSA) and then 3 times with PBS buffer to remove eYcess anti-APS
reductase anti~ody snd bloc~ any nonspecific binding sites. The crystal was then exposed to varied concentrations of APS reductase ranging from 0 to 400 ng in 1.5 mL of TRIS test buffer 601ution ~or 20 minutes; the dosage concentrations were varied to determine the response characteristics of the device.
, After being washed wit~ TRIS test buffer to remove ``,`' :' ' 1 33222i nonspecifically adsorbed ~PS reductase, the crystal was exposed for twenty minutes to 1.5 mL TRIS test buffer solution containing 30 mL conjugate comprised of anti-APS reductase antibody and alkaline phosphatase enzyme. The crystal was then washed again 2 times with test buffer and once with 50 MM
TRIS wash solution.
The detection step was performed by i~mersing the crystal in 0.5 mL of TRIS wash buffer in a cell holder, followed by addition o~ 0.5 mL of a standard solution of BCIP reagent solution (SIGNA).
A positive response for antigen was measured by a decrease in frequency, corresponding to the precipitation of the oxidized dimer of BCIP, an indigo dye analog. Precipitation results from the enzymatically catalyzed hydrolysis of the phosphate functionality of BCIP, which, in turn, only occurs if the alkaline phosphatase conjugate is present~ which, in turn, is only possible when the APS reductase is present~ Table 1 indicates the frequency response of the BMQCM to different dosage levels of APS
reductase. It is clear that the frequency change and the rate of change increase with larger dosage rates, as expected. The relative responses agreed with those determined by the spectroscopically measured optical density of the blue BCIP indigo dimer deposited on the surface 18 of the quartz crystal microbalance. Notably, these responses were observed for APS reductase levels in whi~h the direct binding of APS reductase ~ould not be observed. That is, the frequency change resulting from addition of APS
reductase to the BMQCM was not detectable.
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Table 1 APS reductase ~ frequency/sec a frequency concentration(Hz/sec)in 30 min.
(ng/mL) 200 0.24 406 ~; 75 0.17 250 O.lO 170 7.5 0.002 6.3 ., .
:. --Example 2 Assay of APS using alkaline phosphatase/5-bromo-4-chloro-3-indolylphophate (BCIP) on quartz crystals with polyvinylferrocene modifying layers.

The procedure was performed according to the mode illustrated in Figure 2, with a polyvinyl-ferrocene (PV-Fc) layer 28 on the quartz crystal, an anti-APS reductase antibody as the capture reagent 20, APS reductase as the analyte 22, anti-APS
reductase antibo~y with alkaline phosphatase enzyme as the conjugate 24, and 5-bromo-4-chloro-3-indolyl-phophate (BCIP) as the substrate, S. The PV-Fc layer serves as a hydrophobic layer, enhancing adsorption of the anti-APS reductase antibody.
The first ~tep was adsorption of the anti-APS reductase antibody onto a PV-Fc layer, applied by sipin coating from a lO~i o-chlorotoluene siolution onto the gold/guartz ~urface 18 of ~ quartz crystal. This was performed ~y oquilibration of the PV-Fc modified gold/quartz crystal with 1.5 ~L of ~: ~

~i ~
~ ~ 33222 1 100 ug/mL anti-APS reductase antibody in PBS buffer solution for 2 hours. The crystal was then washed 3 times with PBS buffer to remove excess anti-APS
reductase antibody. The crystal was then exposed to 0 to 80 mL of APS reduc~ase (0.5 mg/mL) in 2 mL TRIS
test buffer solution for 30 minutes; the dosage rate was varied to determine the response characteristic~
of the device. After being washed with TRIS test buffer to remove nonspecifically adsorbed APS
reductase, the crystal was exposed for thirty minutes to l.S mL TRIS test buffer solution containing 30 mL
of conjugate comprised of anti-APS reductase antibody and alkaline phosphatase enzyme. The crystal was then washed again with TRIS test and TRIS wash buffers as described in Example 1.
The detecti~n step was performed by immersing the crystal in 0.5 mL of TRIS buffer in a cell holder, followed by addition of 0.5 mL of a standard detection solution containing S0% dilution of BCIP substrate reagent (SIGMA) in 50 MM TRIS
buffer (SIGMA). A positive response for antigen was .
measured by the decrease in frequency, corresponding to the precipitation of the oxidized dimer of BCIP, an indigo dye analog. Precipitation results from the enzymatically catalyzed hydrolysis of the phosphate functionality of BCIP, which, in turn, only occurs if the alkaline phosphatase conjugate is present, which, in turn, is only possible when the APS reductase is present. Table 2 indicates the frequency response of the ~MQCM to different dosage levels of APS
.
reductase. It is clear that the frequency change and the rate of change increase with larger dosage rates, as expected. The relative responseC agreed .
i` 18 ~.
l~j ~ ~ 1 33222 i : ::
19 - ~
with those deter~ined by the spectroscopically measured optical density of the blue BCIP indigo ~ dimer deposited on the ~urface 18 of the quartz i~,3 crystal microbalance.

:l Table 2 APS reductase ~ freguency/sec a frequency concentration (Hz/sec) in 30 min.
3 ( ng/mL) .,, ~

- 200 0.22 278 o.og 80 :~
0.05 69 7.5 0~025 22 .

~ Example 3 - Assay of human chorio~ic gonadotropin (hCG) using horseradish peroxidase/~ydrogen peroxide-iodide on ~- nylon membranes situated opp~site to a quartz crystal -~ microbalance modified with polyvinylferrocene `~ modifying layers.

;- The procedure was performed according to `~ the mode illustrated in Figures 4 ~nd 6, with a polyvinylferrocene (PV-Fc) layer 28 on the guartz J crystal situated opposite to a nylon membrane 36 who~e ~urface 30 was modified with capture re~gent : The 6pacing of the compartment was approximat~ly 1 - mm. In this example, anti-hCG antibody was the . --` ~ 3322~ i ` 20 capture reagent 20, hCG was the analyte 22, anti-hCG
antibody with horseradish peroxidase (HRP) enzyme was the conjugate 24, and a hydrogen peroxide-iodide mixture was the substrate, S. The nylon membrane served as a hydrophobic layer that adsorbed the anti-hCG antibody, and presented the enzymatic reaction product to the PV-Fc film. The enzymatically catalyzed formation of iodine/triiodide that occu~ed when the conjugate was present resulted in oxidation of the PY-Fc filM, followed by incorporation of triiodide into the film to maintain electroneutrality. This led to an increase in mass of the PV-Fc film and accordingly a larqe change in the resonant frequency of the BMQCM device.
The first step was adsorption of the anti-hCG antibody to the nylon surface 30 of membrane 36. This was performed by equilibration of the nylon membrane 30 wit~ 2 mL of 100 ug/mL anti-hCG antibody in PBS buffer solution for 2 hours. The membrane was then washed once with PBS buffer containing 0.1%
BSA and then 3 times with PBS buffer to remove excess anti-hCG antibody and block any nonspecific binding sites. The membrane was then exposed for thirty ` minutes to different concentrations of hCG in PBS~`
buffer containing 0.1% BSA buffer solution. After being washed with PBS buffer to remove -~-nonspecifically adsorbed hCG antigen, the membrane was exposed for twenty ~inutes to 2 ~L buffer 601ution containing 20 mL of ~tock conjugate comprised of anti-hCG antibody and HRP enzyme.
The mem~rane was then washed again with PBS/0.1% BSA
buffer ~olution.

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. ~

1 332~2 1 The detection step was performed by placing ~ the membrane opposite to the quartz crystal modified i with the PV-Fc film, with a 1 mm diameter separator between the two surfaces to form a reaction compartment. The compartment was filled with a pH=5.0 citrate/phosp~ate/iodide detection buffer.
After the frequency stabilized, 10 mL of a standard solution of 0.01% hydrogen peroxide was added. The -` enzymatically catalyzed reaction product iodine/triiodide, P, diffuses across the compartment to the PV-Fc film, resulting in oxidation of the PV-Fc film by one-half of an equivalent of P. This is subsequently followed by incorporation of an equivalent of triiodide into the oxidized PV-Fc film, resulting in an increase i~ mass of the film and a corresponding decrease in resonant frequency of the quartz crystal microbalance. Triiodide incorporation results from the enzymatically catalyzed conversion of iodide to iodine/triiodide, which, in turn, only ~- occurs if the HRP conjugate is present, which, in turn, is possible only when the hCG antigen is -~ present.

Table 3 .
:
hCG concentra~ion a frequency/sec ~ frequency ~ng/ml)(HZ/sec) in 10 min.

0 ~ 0.005 ~ 3 ~; 600 0.01 48 ~"

~ 21 - . ~ , ', 22 The invention is defined by the following claims, although it will be appreciated by those skilled in the art that various modifications can be made without departing from the spirit thereof.

' ~`

;q , ~ .
h`

r ' ., ., ' ~.

'~J~` . ` . . ::.':
"' ~: ' ' ' ` , ~ ` ' ' ' '

Claims

1. In a process for detecting an analyte suspected of being present in a liquid sample, which process comprises reacting the sample with a quartz crystal microbalance (QCM) having an analyte capture reagent bound to a surface thereof thereby binding the analyte to the QCM by the analyte capture reagent, the improvement comprising reacting the bound analyte with (1) a conjugate comprising an enzyme and either an anti-analyte reagent or the analyte, and (2) a substrate which is capable of being catalyzed by the enzyme to form a product which is capable of accumulating on or reacting with the QCM surface to induce a mass change, thereby leading to a resonant frequency change of the QCM.

2. The method of Claim 1 wherein the conjugate comprises an enzyme and an anti-analyte reagent.

3. The method of Claim 1 wherein the analyte capture reagent is an antibody, lectin, chelating agent, binding protein, polynucleic acid probe, or cell receptor.

4. The method of Claim 3 wherein the analyte capture reagent is an antibody or polynucleic acid probe.

5. The method of Claim 4 wherein the analyte capture reagent is an antibody.

6. The method of Claim 1 wherein the anti-analyte reagent is an antibody, lectin, chelating agent, binding protein, polynucleic acid probe, or cell receptor.

7. The method of Claim 3 wherein the analyte capture reagent is an antibody or polynucleic acid probe.

8. The method of Claim 4 wherein the analyte capture reagent is an antibody.

9. The method of Claim 1 wherein the enzyme is alkaline phosphatase and the substrate is 5-bromo-4-chloro-3-indolylphosphate.

10. The method of Claim 1 wherein the enzyme is horseradish peroxidase.

11. The method of Claim 1 wherein the surface is coated with a silane, polymer or organic thin film.

12. The method of Claim 11 wherein the polymer is polyvinylferrocene, polypyrrole, polythiophene, polyacetylene or phthalocyanine.

13. The method of Claim 12 wherein the enzyme is horseradish peroxidase and the substrate is hydrogen peroxide/iodide, urea peroxide/iodide or ferrocyanide.

14. The method of Claim 13 wherein the substrate is hydrogen peroxide/iodide.

15. A method for detecting an analyte suspected of being present in a liquid sample, comprising reacting the sample with a support surface having an analyte capture reagent bound thereon, said support surface being in close proximity to the surface of a quartz crystal microbalance, and reacting the support surface with a (1) conjugate comprising an enzyme and either an anti-analyte reagent or the analyte and (2) a substrate which is capable of being catalyzed by the enzyme to form a product which is capable of accummulating on or reacting with the QCM surface to induce a mass change, thereby leading to a resonant frequency change of the QCM.

16. The method of Claim 15 wherein the conjugate comprises an enzyme and an anti-analyte reagent.

17. The method of Claim 15 wherein the analyte capture reagent is an antibody, lectin, chelating agent, binding protein, polynucleic acid probe, or cell receptor.

18. The method of Claim 16 wherein the analyte capture reagent is an antibody or polynucleic acid probe.

19. The method of Claim 17 wherein the analyte capture reagent is an antibody.

20. The method of Claim 15 wherein the anti-analyte reagent is an antibody, lectin, chelating agent, binding protein, polynucleic acid probe, or cell receptor.

21. The method of Claim 17 wherein the analyte capture reagent is an antibody or polynucleic acid probe.

22. The method of Claim 18 wherein the analyte capture reagent is an antibody.

23. The method of Claim 15 wherein the enzyme is horseradish peroxidase.

24. The method of Claim 15 wherein the quartz crystal microbalance surface is coated with a silane, polymer or organic thin film.

25. The method of Claim 24 wherein the polymer is polyvinylferrocene, polypyrrole, polythiophene, polyacetylene or phthalocyanine.

26. The method of Claim 25 wherein the enzyme is horseradish peroxidase and the substrate is hydrogen peroxide/iodide, urea peroxide/iodide or ferrocyanide.

27. The method of Claim 26 wherein the substrate is hydrogen peroxide/iodide.

28. The method of Claim 15 wherein the support surface is nylon or nitrocellulose.