WO2007077218A1

WO2007077218A1 - An optical system; an optical chip for an optical system and a method of using an optical chip for an analytical operation

Info

Publication number: WO2007077218A1
Application number: PCT/EP2007/000029
Authority: WO
Inventors: Sergey I Bozhevolnyi; Mads Hoy Sorensen; Peter Theilade Thomsen; Michael Hansen; Kristjan Leosson; David Edward Williams
Original assignee: Inverness Medical Switzerland Gmbh
Priority date: 2006-01-03
Filing date: 2007-01-03
Publication date: 2007-07-12

Abstract

The present invention provides an optical system for use in analytical operations. The system comprises an optical chip with a chamber for a sample with a lower refractive index than the material of at least a part of the optical chip defining a transparent window into the chamber. The system also comprises at least one lens, a light source and a detector. The light source is arranged to direct a light beam into the chip- sample interface to generate an evanescent field, and the detector is arranged to collect light (such as fluorescence or scattered light) from objects in the sample induced by the evanescent field via the lens, which is either incorporated into the optical chip or is placed between the optical chip and the detector to collect and direct light to the detector. Also provided are a method of using the optical system and an optical chip.

Description

AN OPTICAL SYSTEM; AN OPTICAL CHIP FOR AN OPTICAL SYSTEM AND A METHOD OF USING AN OPTICAL CHIP FOR AN ANALYTICAL

OPERATION

This invention relates to an optical system allowing light incoupling (i.e., incoming light) in the total internal reflection (TIR) geometry to a sensing surface and light outcoupling (i.e., outgoing light) suitable for imaging/sensing the sensing surface onto a detector unit. The invention also relates to an optical chip design with added functionality for use in biological research, drug discovery and diagnostics, specifically in studies of cellular events, surface binding events, blood, antibodies, protein/DNA detection. Furthermore the invention relates to a method of using an optical chip for an analytical operation that employs optical detection.

Molecular diagnostics is of critical importance to public health. This area of medical technology will facilitate the detection and characterization of disease, the monitoring of drug response, and the identification of genetic modifiers and disease susceptibility. Molecular diagnostics will also be used to identify food, water, and environmental contamination and the possible presence of biological warfare agents. Our method also supports nucleic acid-based methods to support DNA detection and analysis and to pinpoint changes in gene expression. This is done with high accuracy and reproducibility of the tests and literally no training required by users.

Sensitivity and Markers

Today many diagnostic tests are hampered by the lack of sufficiently sensitive detection platforms. Also the ease of use may be hampered by the platform because washing steps and reaction steps are time consuming. Higher sensitivities of the platform may result in the availability of new markers and new targets for our diagnostic platform which can be used for a variety of fluids such as blood or urine in the attempt to detect proteins, antibodies, DNA and other components. Nucleic Acid Testing at the Point of Care

POC (point-of-care) nucleic acid methodologies could play a pivotal role in the early diagnosis of diseases, facilitating their prompt treatment. The tests provide a powerful tool for identifying myriad microorganisms known to be associated with sexually transmitted diseases, infections of the immunocompromised host, and many other diseases. Further, these tests aid in the critical evaluation of disease predisposition.

The test is completely self-contained, eliminating any risk of cross-contamination. Our system may be included in or combined with gene chips, DNA probe technology, micro fluidics, and tandem mass spectrometry, among others. This will add high sensitivity to speed in probe-based tests.

Nucleic acid amplification techniques are a main focus of interest because of the general need for high sensitivity. This involves selective amplification of the target region of the molecule (which may also be used to label the product intended for detection), and specific detection of the amplified product. PCR is the dominant amplification technology considered but other methods may challenge this in various areas. Genomics-based assays represent an application too in relation to genomic tests such as Factor V Leiden and cystic fibrosis panels. Larger molecular infectious- disease opportunities such as viral load testing, viral genotyping, and detection of sexually transmitted diseases are considered too.

Infectious Diseases Infectious-disease testing demands high sensitivity and specificity with rapid turnaround, all of which are offered by our chip platform. This expands to testing for pathogen identification, strain verification, viral load monitoring, and drug-resistant strains, pharmacogenomics will be used in concert. The capabilities of these tests will allow the physician not only to identify the illness-causing pathogen, but also to determine its resistance or susceptibility to a drug. Clinical Genetic Testing

Only a portion of non-infectious-disease testing can be considered genomic. Most molecular cancer-related testing is aimed at detecting tumor-associated changes in gene expression: translocations, loss of heterozygosity, or other abnormal patterns of expression.

Our chip may be used in current strategies for prenatal testing which rely primarily on biochemical assays and imaging methods for whole-chromosome analysis. Also genomic testing associated with preimplantation genetic diagnosis is considered. Paternity testing is based mostly on measuring the size of selected short tandem repeats, although some believe that this field will in time adopt testing for single- nucleotide polymorphisms (SNPs) as a more cost-effective approach. Many genetic diseases are detected through assays directed at proteins or metabolites rather than by characterizing DNA sequences. We keep a main focus on assays for thrombotic risk, cystic fibrosis, and certain other heritable genetic disorders, for human leukocyte antigen typing, and for predictive testing in selected populations.

Theranostics and Pharmacogenomics

A main focus in genomic testing is kept on theranostics, a shorthand term for therapy- specific diagnostics. This approach differs from traditional medical practice in which therapeutic choices follow a diagnosis that may be based on clinical signs alone or may also take into account results of an in vivo or in vitro diagnostic test. In traditional practice, neither the effectiveness of the prescribed drug therapy nor the likelihood of side effects can be predicted for individual patients in many cases. The primary current application of theranostic assays is to select patients for drug treatments that are likely to benefit them and unlikely to produce adverse effects. In addition, theranostics will provide an early indication of treatment efficacy for a particular patient. Personalized diagnostic tests thus will provide healthcare professionals with information useful for individualizing and optimizing the therapeutic regimen of each patient. Also pharmacogenomic profiling may be able to be applied to existing drugs or may even be used to resurrect old drugs that were recalled owing to adverse effects in identifiable subgroups. General TIR geometry:

When using standard and fluorescence microscope techniques, the investigations of biological phenomena are often hampered by a low signal and a high noise level. Therefore, there has been a focus on developing more advanced techniques to overcome these problems. In particular, microscopy based on illuminating a sample by total internal reflection has found applications in the biological sciences due to its unique capabilities for studying surface events.

Refractive index differences at an interface regulate how light is refracted or reflected as function of incident angle. Total internal reflection occurs when light is striking a medium with a lower refractive index at an incident angle above the critical angle, where the critical angle is described by Snell's law (0c=ArcSin(n2/nl), where nl and n2 is respectively the higher and lower refractive index). Light incident at any angles higher than the critical angle is totally reflected at the interface and generates a thin electromagnetic field near the surface of the lower refractive index media. This field is called the evanescent field and its intensity decays exponentially with distance from the surface, usually over 200 run. This surface confined illumination principle allows selective monitoring of surface and binding events without introducing background signal as known from conventional illumination techniques, e.g. conventional epi- illumination. The intensity of the evanescent field as function of the distance from the interface can be determined via solving standard boundary conditions obtained from Maxwells equations, in this document we refer to the results obtained by Nikolas Chronis, Luke P Lee, Lab Chip, 2004, 4, 125-130.

The TER. concept has been introduced in to microscope systems, e.g. Olympus, Nikon and Zeiss. The newest systems use microscope objectives with high numerical aperture to enable light to be guided through the objective and onto an interface above the critical angle. When an object flows through or settles near the interface at which total internal reflection occurs, scattered light or fluorescence is emitted that allows imaging of the object. These events can be imaged using the same microscope objective. DISCLOUSURE OF THE INVENTION

In a first aspect, the invention provides an optical system for use in analytical operations, comprising:

- an optical chip with a chamber for a sample, the sample having a lower refractive index than the material of at least the part of the optical chip defining a transparent window into the chamber, the interface between the transparent window and a sample in the chamber being defined as the chip- sample interface;

- at least one lens;

- a light source; and

- a detector, wherein the light source is arranged to direct a light beam into the chip-sample interface to generate an evanescent field, and the detector is arranged to collect light from objects in the sample induced by the evanescent field via the lens, which is either incorporated into the optical chip or is placed between the optical chip and the detector to collect and direct light to the detector.

The sample may be a fluid, such as a bodily fluid. The sample may be an aqueous sample.

In a second aspect, the invention provides an optical chip comprising:

- a chamber for a sample; - a transparent window into the chamber, the interface between the transparent window and the chamber being defined as the chip-chamber interface; and

- an optical element directing an incoming light beam towards the chip- chamber interface.

In a third aspect, the invention provides a method of using an optical chip for an analytical operation of a sample, comprising the steps of: - providing an optical chip with a chamber comprising a transparent window into the chamber, wherein the material of the window has a refractive index which is higher than the refractive index of the sample;

- directing a light beam towards the chip-sample interface between the transparent window and a sample with a light incident higher than the critical angle to generate an evanescent field;

- arranging a lens and a detector to collect light from objects in the sample induced by the evanescent field, the lens being arranged between the optical chip and the detector.

The method may also comprise one or more washing steps.

The optical chip of the second aspect may be used in the optical system of the first aspect and/or the method of the third aspect.

Preferred features of each aspect of the invention are set out in the dependent claims.

An optical system of the invention is as defined in the claims and comprises an optical chip, e.g. a microfluidic device with a chamber for a sample with a lower refractive index than the material of at least the part of the optical chip defining a transparent window into the chamber. Often the optical chip is composed of one material. Alternatively the optical chip may be composed of a combination of two or more materials. The interface between the transparent window and a sample in the chamber is defined as the chip-sample interface. The sample will most often be a liquid sample, e.g. comprising dispersed objects (objects in this application should be construed to includes biological compositions and components as well as fragments thereof, such as antibodies, proteins, nucleotides, cells, cell-agglomerations, bacteria's and similar components). The system also comprises at least one lens for collecting and focusing light. The system further comprises a light source, which in principle may be any type of light source which can emit light to generate an evanescent field at the chip-sample interface, e.g. a laser. Further, the system comprises a detector for detecting light (such as fluorescence or scattered light) from objects in the sample induced by the evanescent field.

The critical angle is measured in relation to the normal incidence of light.

The optical chip which is a part of the system may in principle be any kind of optical chip which has a chamber for a sample and a transparent window into the chamber. The optical chip of the optical system may e.g. be a micro fluidic device with or without incorporated optical devices. Useful optical chips are e.g. as disclosed in US 6100541, which with respect to its design, fabrication and materials are hereby incorporated by reference.

The optical system may e.g. be used for biological application, specifically for studies of cellular events and surface binding events. The system can be used for sensing/imaging biological samples, especially fluorescence or scattered light from metallic probes. The method does not require the sample to be optically transparent.

The optical system may in one embodiment be used for intensity measurement, measuring the light intensity from the sample. In another embodiment the optical system is used for imaging of the sensed area. A combination of the two measurements is also possible. The lens and the detector unit should preferably be selected to the desired measurement.

A person skilled in the art will be able to design the lens and detector unit according to the optical chip application. The lens can be designed using geometric optics as described in standard text books in optics. Also, commercially available software is available to design optical systems, e.g. Zemax, OSLO. The design criteria are related to the numerical aperture, depth of focus, wavelength, material, resolution, aperture stop and magnification of the optical system. Based on the design criteria ray tracing formulas are developed by applying the laws of refraction and ray propagation which the lens designer uses for adjusting the lens material, shape and position in attempt to optimize its performance. The detector unit is capable of detecting light (such as fluorescence or scattered light) from objects in the sample induced by the evanescent field. The detector can be any photosensitive device such as photodiodes and photomultipliers, intensified detectors, semiconductor sensors such as CCD and CMOS devices. The detector can be an imaging detector. For example a CCD, CMOS or intensified camera can be used to make measurements in viewing regions on the surface of the chip-sample interface.

The invention also relates to a particular optical chip (also called a TIR optical chip) which in one embodiment may be a part of the optical system. The optical chip of the invention is as defined in the claims.

hi one embodiment the invention relates to a TIR-based optical chip with integrated optics that provides a system which makes it more simple to use, as the optical chip makes the system essentially alignment free.

In one embodiment the optical chip consists of a polymer material like Polystyrene, TOPAS, PMMA or glass. The chip may have chambers for biological samples. The samples are presumably a liquid solution like blood, other body liquid, or derivatives thereof.

Our device is based on a variety of substrate based technologies, such as lipid plates, chips or slides as well as solid beads or micro particles. A number of chemical surface modifiers can be added to substrates to attach the probes to the substrates (see US patent 6,905,816).

The principle of the optical chip structure and path of a light beam is illustrated in figure 1. The optical chip is designed to direct a light beam at an angle larger than the critical angle onto a chip-sample interface (e.g. a polymer-liquid interface). The evanescent field near the surface of the sample can induce scattered light or fluorescence from objects located in the evanescent field. The chip may consist furthermore of an optical readout/collection device. One or more lenses may in one embodiment be integrated in the chip to thereby collect and direct fluorescence or scattered light from objects in the sample (e.g. a liquid solution) onto a detector unit. In one embodiment the lens or lenses is/are not integrated into the optical chip, but is/are simply placed between the optical chip and the detector. The detector unit can be an array type to allow imaging of an area or just a single-unit to detect the light intensity of scattered light and/or fluorescence.

The design of the optical chip makes it possible to position a light source without the need to align the light beam into the chip with high accuracy. The light source can be integrated into the chip or be externally positioned. Light from the light source can be directed onto the chip-sample interface via a prism-like interface, grating interface or mirror-like interface to generate the evanescent field.

The optical chip of the invention comprises a chamber for a sample and a transparent window into the chamber, and an optical element for directing an incoming light beam towards the interface between the transparent window and the chamber to hit this interface, which is referred to as the chip-chamber interface if the chamber does not comprise a sample, and the chip-sample interface when the optical chip comprises a sample in its chamber.

The skilled person will by a calculation as specified by Snell's law be able to calculate a light incident angle which results in formation of TIR and the evanescent field.

In one embodiment the optical element of the optical chip of the invention is capable of providing a chip-chamber interface light incident at an angle above 50 degrees, such as above 55 degrees.

In one embodiment the optical element of the optical chip of the invention is capable of providing a chip-sample interface light incident with an angle above the critical angle when the chamber comprises a sample. In one embodiment the optical element of the optical chip of the invention is capable of providing a chip-sample interface light incident with an angle above the critical angle when the chamber comprises an aqueous sample.

hi one embodiment of the optical chip of the invention the optical element is a prism, e.g. as shown in figure 6.

hi one embodiment of the optical chip of the invention the optical element is a grating, e.g. as shown in figure 7.

hi one embodiment of the optical chip of the invention, the optical element is provided by arranging the chip-chamber interface with at least two sections including a side wall chip-chamber interface and a measuring area chip-chamber interface with an angle to each other, so that a light beam directed towards the side wall chip- chamber interface is reflected and redirected towards the measuring area chip- chamber interface in an angle generating an evanescent field. An example of such embodiment is shown in figure 8.

hi one embodiment the lens is integrated into the optical chip making the optical readout system robust and essentially alignment-free, hi another embodiment one or more lenses can be positioned externally. The lens can in general be any shape performing the desired function such as spherical, aspheric, Fresneltype or microlens array. The desired functions include magnifying, demagnifying, collimating, light delivery, light collection or focusing functions and the like. The shape of the surface determines the lens properties. The diameter of the lens, distance to the chip-sample interface plane and shape may be selected according to the optical chip application.

An optical chip with an integrated lens can be designed by the skilled person, based on the present teaching, to collect and focus light from the sensing area (the area where the evanescent field is generated) onto a detector, e.g. a photodiode, for intensity measurements. Such a configuration is shown below. In another embodiment, an optical chip can be designed with an integrated imaging lens to image part of the sensing area onto an array-type detector, e.g. CCD or CMOS sensor. Such a configuration is shown below in figure 3. The imaging lens can be designed to produce a real image of very small objects in the sensing area onto a detector unit.

In a further embodiment one or more lenses are externally positioned and can function either as a focusing lens/lenses for intensity measurements or as an imaging lens/lenses for imaging measurements.

In another embodiment a chip with integrated lens can be combined with one or more lenses for intensity or imaging purposes. Such a configuration is illustrated in Figure 4.

The incident light can be similarly coupled out and detected to obtain a calibration reference for the incident light as illustrated in Figure 2.

An image sensor, CCD or CMOS or similar, can be used to image the sensing area. Below, an image (figure 5) shows a simulated sensor image provided by an optical system of the invention of an object density corresponding to 1 bead/100 image sensor pixels. The images can be processed to evaluate the number of objects in the image. The numbers of objects in the images can e.g. be related to the number of surface binding events.

In one embodiment of the optical chip of the invention the lens is arranged on the same side of the optical chip as the optical element capable of directing an incoming light. Thereby the scattered light or fluorescence from the object generated by the evanescent field need not travel through the whole sample in the chamber, but only the few ran layer of sample between the chip-sample interface and the object. Therefore the sample need not be optically transparent. In one embodiment of the optical chip of the invention the lens is arranged on the side of the optical chip opposite the optical element capable of directing an incoming light. In this embodiment it is desired that the sample is optically transparent because the scattered light or fluorescence from the object generated by the evanescent field, should be capable of traveling through the sample in the chamber.

One or more lenses can in one embodiment of the optical system be inserted between the chip and the detector unit to increase the imaging quality and/or relax the manufacturing and positioning tolerances.

The optical chip can be manufactured by any suitable method depending on which kind of material is used e.g. the method of producing a microfluidic device as disclosed in US 6100541. In one embodiment the optical chip is manufactured using injection moulding.

The optical chip may include fluidic or micro fluidic sample transport, such as it is generally known from prior art microfluidic devices.

The design can in one embodiment incorporate multiple detection sites on the same optical chip.

The optical chip can in one embodiment consist of one chamber. In another embodiment the optical chip can contain one or more chambers. The chambers may also serve mixing, reaction, detection or other functionalities.

In one embodiment it is desired that the light source has low divergence angle so that the light can easily be directed. Sources suitable for such purposes include primarily laser sources such as gas laser, solid state laser, diode laser, VCSEL and liquid laser. In addition to lasers suitable sources also include sources such as e.g. an amplified stimulated emission (ASE) source, edge emitting light emitting diodes (EE-LEDS) and LEDs. Finally suitable sources can involve white light sources such as e.g. Xe- lamp The invention also relates to a method of using an optical chip for an analytical operation of a sample. Several steps of the method have already been described above.

The method comprises the steps of

• providing an optical chip with a chamber comprising a transparent window into the chamber, wherein the material of the window has a refractive index which is higher than the refractive index of the sample; • directing a light beam towards the chip-sample interface between the transparent window and a sample with a light incident higher than the critical angle;

• arranging a lens and a detector to collect light (such as fluorescence or scattered light) from objects in the sample induced by the evanescent field, the lens being arranged between the optical chip and the detector.

The optical chip may preferably be an optical chip as described above.

The present invention will be described further with reference to the following non- limiting drawings and example, in which:

Figure 1 is a diagrammatic representation of one optical chip in accordance with the invention;

Figure 2 is a diagrammatic representation of another optical chip in accordance with the invention;

Figure 3 is a diagrammatic representation of a further optical chip in accordance with the invention;

Figure 4 is a diagrammatic representation of a yet further optical chip in accordance with the invention; Figure 5 is a simulated image of an optical system in accordance with the invention with imaging properties;

Figure 6 is a diagrammatic representation of a still further optical chip in accordance with the invention;

Figure 7 is a diagrammatic representation of a still further optical chip in accordance with the invention;

Figure 8 is a diagrammatic representation of a still further optical chip in accordance with the invention;

Figure 9 is a diagrammatic representation of a still further optical chip in accordance with the invention;

Figure 10 shows a graph of the intensity of the evanescent wave at the interface versus the incident angle;

Figure 11 shows a graph of the theoretical angular dependence of the penetration depth;

Figure 12 shows a graph of the theoretical wavelength dependence of the cross section for scattering of light from a gold sphere in a TIR geometry.

The principle of the optical chip structure and path of a light beam is illustrated in figure 1. An optical chip of the invention 1 is shown with a chamber with sample 4, a chip-sample interface 5, and an integrated lens 6. An external lens 7 and detector 8 are also shown. The path of light of the light incoupling 2 is shown entering the optical chip of the invention 1. An optical element 3 directs the light in a TIR configuration. The optical element shown in Figure 1 is a grating, though the element can also be a prism or mirror or combination thereof. The ray of light in the light outcoupling 9 is also shown. The example illustrates that the optical system can operate with multiple detection sites. The example illustrates a case where a lens is integrated into the optical chip and a case without an integrated lens.

Figure 2 shows a chip with integrated lens designed to collect and focus light from the TIR sensing area onto a single-unit detector, e.g. a photodiode, for intensity measurements. The example illustrates a case where a lens is integrated into the chip and a case where light is coupled out and detected to obtain a calibration reference for the incident light. 1. Indicates the optical chip. 2. The path of light of the light incoupling. 3. Optical element for directing the light in a TIR configuration, this example illustrates a grating, the element can also be a prism or mirror or combination thereof. 4. Chamber with sample. 5. Chip-sample interface. 6. Integrated lens. 7. Optical element for directing the light out of the chip, this example illustrates a grating, the element can also be a prism or mirror or combination thereof. 8. Detector. 9. Illustrating the ray of light in the light outcoupling. 10. Detector.

Figure 3 shows a chip with integrated imaging lens for imaging events from the TIR sensing area onto an array-type detector, e.g. a CCD or CMOS sensor. The example illustrates a case where the imaging lens is integrated into the chip and a case where light is coupled out and detected to obtain a calibration reference for the incident light. 1. Indicates the optical chip. 2. The path of light of the light incoupling. 3. Optical element for directing the light in a TIR configuration, this example illustrates a grating, the element can also be a prism or mirror or combination thereof. 4. Chamber with sample. 5. Chip-sample interface. 6. Integrated lens. 7. Optical element for directing the light out of the chip, this example illustrates a grating, the element can also be a prism or mirror or combination thereof. 8. Detector (CCD or CMOS). 9. Illustrating the ray of light in the light outcoupling. 10. Detector.

Figure 4 shows a chip with integrated and external imaging lenses for imaging events from the TIR sensing area onto an array-type detector, e.g. a CCD or CMOS sensor. 1. Indicates the optical chip. 2. The path of light of the light incoupling. 3. Optical element for directing the light in a TIR configuration, this example illustrates a grating, the element can also be a prism or mirror or combination thereof. 4. Chamber with sample. 5. Chip-sample interface. 6. Integrated lens. 7. External lens. 8. Detector (CCD or CMOS). 9. Illustrating the ray of light in the imaging.

Figure 5 shows a simulated image of the optical system with imaging properties. A bead density of 1 bead/ 100 image sensor pixels is simulated.

Figure 6 shows a schematic of an optical system where the TER is obtained in a prism like configuration. Light is incident at a measuring area at an angle greater than the critical angle so that TIR results. The system includes a lens 6 that allows sensing/imaging of the measuring area onto a detector unit. The angle of the tilted sidewall interface is defined to direct light onto a sensing/imaging surface above the critical angle. 1. Indicates the optical chip. 2. The path of light of the light incoupling. 3. Optical element for directing the light in a TIR configuration, this example illustrates a prism configuration. 4. Chamber with sample. 5. Chip-sample interface. 6. Integrated lens.

Figure 7 shows a schematic of an optical system where the TIR is obtained in a grating configuration. Light is incident at a grating that is diffracted onto measuring area at an angle greater than the critical angle so that TER. results. The system includes a lens that allows sensing/imaging of the measuring area onto a detector unit. The periodicity of the grating is defined to direct light onto a sensing/imaging surface above the critical angle. 1. Indicates the optical chip. 2. The path of light of the light incoupling. 3. Optical element for directing the light in a TIR configuration, this example illustrates a grating configuration. 4. Chamber with sample. 5. Chip- sample interface. 6. Integrated lens.

Figure 8 shows a schematic of an optical chip where the TIR is obtained in a TIR configuration. Light is incident at a side wall interface section where TIR occurs and light is reflected onto the measuring area interface at an angle greater than the critical angle so that TIR results. The system includes a lens that allows sensing/imaging of the measuring area onto a detector unit. The angle of the tilted side wall interface is defined to allow TIR at this side wall interface. In this way the tilted sidewall functions as a mirror that directs light onto a sensing/imaging surface. The sidewall can be coated to function as a mirror. 1. Indicates the optical chip. 2. The path of light of the light incoupling. 3. Optical element for directing the light in a TIR configuration, this example illustrates a mirror configuration. 4. Chamber with sample. 5. Chip-sample interface. 6. Integrated lens.

Figure 9 shows a schematic of an optical chip as described in figure 8. The chip furthermore contains: 7. Antibodies and colloids coupled to antibodies, that may cause surface binding events. In this example the surface binding events are detected from scattered light from the metal colloids entering the evanescent field.

Preferred features of each aspect of the invention are as for each of the other aspects, mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law.

EXAMPLE

Quantitative analysis

The chip platform may also be used for real time measurement and quantification of bound gold beads or other bead like structures or colloids. This can be done by counting the gold beads or measuring the overall intensity of a signal. The binding mechanism may be DNA or protein mediated in the form of antibody-specific antigens or subunits thereof or any other intermediating substances. The measurements may be mediated by imaging involving a detector unit, e.g. a camera platform, and other hardware as well as software including specific algorithms. The quantification may also be done using fluorescence and in the case of kinetic studies the binding may appear both linear and nonlinear. The fluids to be examined may be any body fluid and components thereof and in particular blood. A quantitative analysis of the properties of the evanescent field and configuration for the optical chip has been performed.

An optical chip as disclosed in figure 8 is used. This optical device is provided in polystyrene material with a refractive index of n=l .59. A liquid sample of blood (or derivatives thereof) is introduced into the chamber. Thereafter an antibody labeled colloid metal is dissolved in the chip system. The liquid reaches a reaction chamber where an antigen of interest can mediate the coupling of an Au-antibody to another Antibody which is immobilized into the reaction chamber. The excess blood is removed at the end of the chip. After this one or more washing steps may follow (see figure 9).

Using Snell's law the critical angle for total internal reflection is 60.2 degrees. A laser with wavelength 532 nm is used to provide a light beam with an incident between 62-75 degrees towards the chip-sample interface to generate an evanescent field. A CCD camera is used to make measurements in viewing regions on the surface of the chip-sample interface.

Figure 10 shows a graph of the intensity of the evanescent wave at the interface versus incident angle. Left: p-polarization. Right: s-polarisation. The theoretical analysis has used polystyrene, refractive index n=1.59, and a sample with refractive index of 1.38. The critical angle for TIR is just above 60 degrees. The horizontal axis displays the angle in degrees, the vertical axis displays the intensity in arbitrary units (normalized to incoming intensity).

Figure 10 depicts the theoretical angular dependence of the intensity of the evanescent field at the interface. For simplicity the s-polarized and p-polarized incident amplitudes at the interface are assumed to be unity. Large incident angles reduce the evanescent intensities reducing the scattering or fluorescence signal from an object in the field. However, the analysis illustrates that the proposed chip design at angle close to the critical angle can produce an evanescent field comparable to the incoming field. Figure 11 shows a graph of the angular dependence of the penetration depth. The horizontal axis displays the angle in degrees; the vertical axis displays the penetration depth in run (1/e). The theoretical analysis has used polystyrene, refractive index n=l .59, and a sample with refractive index of 1.38.

Figure 11 depicts the theoretical angular dependence of the penetration depth of the evanescent field. The figure illustrates that the normal penetration depths are approximately 100 nm. At a very narrow window just above the critical angle higher penetration depths are possible.

Figure 12 depicts the wavelength dependence of the cross section for scattering of light from a gold sphere in a TIR geometry. The calculations are based on scattering from gold colloids with 40 nm radius.

Figure 12 depicts the theoretical wavelength dependence of the cross section for scattering of light from a gold sphere in a TIR geometry. The analysis illustrates that a wavelength around 530 nm yields the highest scattering efficiency. Analysis has also indicated that s-polarization will give the highest scattering efficiency perpendicular to the surface from a gold sphere.

Claims

1. An optical system for use in analytical operations, comprising:

- an optical chip with a chamber for a sample, the sample having a lower refractive index than the material of at least the part of the optical chip defining a transparent window into the chamber, the interface between the transparent window and a sample in the chamber being defined as the chip-sample interface;

- at least one lens;

- a light source; and - a detector, wherein the light source is arranged to direct a light beam into the chip-sample interface to generate an evanescent field, and the detector is arranged to collect light from objects in the sample induced by the evanescent field via the lens, which is either incorporated into the optical chip or is placed between the optical chip and the detector to collect and direct light to the detector.

2. An optical system according to claim 1, wherein the light from objects in the sample induced by the evanescent field is fluorescence or scattered light.

3. An optical system according to claim 1 or claim 2, wherein the light source is a gas laser, a solid state laser, a diode laser, VCSEL, a liquid laser, an amplified stimulated emission (ASE) source, edge emitting light emitting diodes (EE-LEDS), light emitting diodes (LEDs) or a white light source.

4. An optical system according to claim 3, wherein the white light source is a Xe lamp.

5. An optical system according to any one of the preceding claims, wherein the light source is arranged to direct a light beam towards the chip-sample interface at an angle higher than the critical angle to thereby generate an evanescent field.

6. An optical system according to any one of the preceding claims, wherein the lens is placed closer to the optical chip than to the detector.

7. An optical system according to claim 6, wherein the lens is integrated in the optical chip.

8. An optical system according to any one of the preceding claims, wherein the optical chip comprises an optical element capable of directing the light beam towards the chip-sample interface.

8. An optical system according to claim 7, wherein the optical element is a reflector, a grating, a prism, or a combination thereof.

9. An optical system according to claim 8, wherein the reflector is a mirror or a TIR based reflector.

10. An optical chip comprising:

- a chamber for a sample;

- a transparent window into the chamber, the interface between the transparent window and the chamber being defined as the chip-chamber interface; and - an optical element directing an incoming light beam towards the chip- chamber interface.

11. An optical chip according to claim 10, wherein the optical element is a reflector, a grating, a prism, or a combination thereof.

12. An optical chip according to claim 11, wherein the reflector is a mirror or a TIR based reflector.

13. An optical chip according to any one of claims 10-12, wherein the chamber contains a sample and the interface between the transparent window and the chamber containing a sample is defined as the chip-sample interface.

14. An optical chip as claimed in any one of claims 10 to 13 , wherein the optical element is capable of directing an incoming light beam towards the interface between the transparent window and the chamber to hit this interface with an angle above 50 degrees.

15. An optical chip according to claim 14, wherein the incoming light beam hits the interface with an angle of at least 55 degrees.

16. An optical chip according to claim 15, wherein the incoming light beam hits the interface with an angle of about 60 degrees.

17. An optical chip as claimed in any one of the claims 10-16, in combination with a sample, wherein the optical element is capable of directing an incoming light beam towards the chip-sample interface with an angle above the critical angle when the chamber comprises the sample.

18. An optical chip as claimed in any one of the claims 10-17, wherein the optical element is capable of directing an incoming light beam towards the interface between the transparent window and the chamber to hit this interface with an angle above the critical angle when the chamber comprises an aqueous sample.

19. An optical chip according to any one of the claims 10-18, wherein the chip is a microfluidic device.

20. An optical chip according to any one of the claims 10-19, wherein the chip comprises a upper and a lower surface, the chamber being arranged between the upper and the lower surface, the transparent window preferably being arranged in one or both of the upper and lower surfaces of the optical chip.

21. An optical chip according to any one of the claims 10-20, wherein the chip additionally comprises an integrated lens forming a transparent window into the chamber.

22. An optical chip according to claim 21, wherein the lens is arranged on the same side of the optical chip as the optical element capable of directing an incoming light.

23. An optical chip according to claim 21, wherein the lens is arranged on the side of the optical chip opposite the optical element capable of directing an incoming light.

24. An optical system according to any one of claims 1-9, comprising an optical chip according to any one of claims 10-23.

25. A method of using an optical chip for an analytical operation of a sample, comprising the steps of

• providing an optical chip with a chamber comprising a transparent window into the chamber, wherein the material of the window has a refractive index which is higher than the refractive index of the sample;

• directing a light beam towards the chip-sample interface between the transparent window and a sample with a light incident higher than the critical angle to generate an evanescent field; • arranging a lens and a detector to collect light from objects in the sample induced by the evanescent field, the lens being arranged between the optical chip and the detector.

26. The method of claim 25, wherein the light from objects in the sample induced by the evanescent field is fluorescence or scattered light.

27. The method of claim 25 or claim 26, wherein the optical chip is a micro fluidic device.

28. The method of claim 27, wherein the optical chip comprises an optical element.

29. The method of claim 25 or claim 26, wherein the optical chip is the optical chip according to any one of claims 10-23.