Method for detecting an analyte

Abstract

The present invention provides a method for detecting an analyte in a sample, the method comprising the steps of: (i) providing a mixture comprising the sample and a reporter reagent to a device, the device comprising a substrate having optical components and binding components attached to the surface of the substrate; (ii) binding a proportion of the reporter reagent to the surface of the substrate by means of the binding components, the proportion being proportional to the concentration of the analyte; (iii) irradiating the device with electromagnetic radiation to be absorbed by the photosensitizer of the reporter reagent, such that the photosensitizer of the bound reporter reagent interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming a set of localized regions on the substrate of optical components having the second optical state; and (iv) detecting the set of localized regions on the substrate having the second optical state.

Description

Technical Field

[0001] The present invention relates to a method for detecting an analyte, in particular a method for detecting individual binding events due to the presence of an analyte in a sample.

Background Art

[0002] There are many available techniques for measuring biologically relevant parameters in human samples such as blood, plasma, serum, tissue, etc. A common method is to use a capture reagent that binds to a target of interest and a reporter reagent with some label. The capture reagent can be bound to a solid phase such as a microtiter plate, beads or a membrane. When the sample is incubated with the capture reagent, the analyte binds to the capture reagent. The reporter reagent also binds to the analyte. Next, the excess reporter can be removed (by washing), the amount of the reporter reagent is measurable, and thus a measurement of the amount of the analyte present in the sample is obtained. There are many different variants as to how these types of binding assays can be performed. For example, it is also possible to first bind the analyte to the capture reagent and then add the reporter in a separate step, or first bind the analyte to the reporter and then bind it to the capture agent.

[0003] A wide range of reagents can be used as capture and reporter in this type of binding assay, including nucleic acids, carbohydrates, antigens, peptides, proteins and antibodies. There are also a wide range of target analytes including peptides, proteins, antibodies, nucleic acids, cells, carbohydrates, small molecules, therapeutic drugs, drugs of abuse, steroids, hormones, lipids, etc.

[0004] Assays that use antibodies are generally called immunoassays. Immunoassays can take several forms. For example, when a capture antibody is used to capture an analyte and a reporter antibody is used to generate a measurable signal, this is generally called a sandwich immunoassay. Another form is known where the binder is bound to a solid phase and the target analyte in the liquid phase competes with a labeled reagent that also binds to the binder. In the absence of the analyte, a high level of the labeled reagent binds, resulting in a high signal. When the analyte is present, some of the binding sites are blocked, so less of the labeled reagent binds and the signal decreases. These assays are generally known as inhibition or competitive assays. Several types of competitive assays are known. For example, an antibody can be bound to a solid phase and a labeled analyte (or an analog of the analyte) can be made to compete around the binding site on the antibody. Alternatively, an analog of the analyte can be immobilized. A labeled antibody can be bound to this surface. When the analyte is present in the sample, it binds to the antibody in the liquid phase and prevents binding to the surface, and the signal decreases.

[0005] There are many formats for assays and many different types of labels that can be used. For example, assays can vary in that excess labels are removed, e.g., by use of a washing step, before the measurement is made. Removal of excess labels can also be achieved by flowing the sample and reporter over a capture region. This approach is used, for example, in immunochromatographic strips or lateral flow strips used in rapid tests for infectious diseases and pregnancy tests. Alternatively, heterogeneous assays are known in which the excess reporter is not removed. Heterogeneous assays tend to rely on the proximity of capture and reporter to produce some form of signal. An example of a heterogeneous assay is an agglutination assay, in which particles bind together in a liquid phase. The agglutinated particles produce light scattering, which can be measured by turbidimetry or nephelometry. A further example of a heterogeneous assay using particles is the LOCI method (luminescent oxygen channeling immunoassay), which is described in more detail below.

[0006] Another example of a heterogeneous assay is fluorescence resonance energy transfer (FRET), in which the capture and reporter reagents are a donor and acceptor fluorophore, respectively, and excitation of the donor results in energy transfer to the acceptor and subsequent light emission.

[0007] One heterogeneous assay format that functions with whole blood without removal of cellular material is the pyro-optical immunoassay. The capture antibody is coated on a pyroelectric polyvinylidene PVDF sensor, and carbon particles are used as the reporter. The signal is generated by irradiating the sample with light, causing local heating of the particles. What binds to the sensor transfers energy to the pyroelectric sensor, producing a thermal stress that is detected as an electrical signal. The greater the carbon binding, the greater the signal.

[0008] The label bound to the reporter binder can be a light-absorbing agent such as a dye, gold particles, or colored latex microspheres. In principle, the larger the particles, the more light they absorb and the larger the signal generated. However, as described in more detail below, there is a limit to the size at which particle labels become unusable for the assay. Luminescent labels such as fluorescent labels, chemiluminescent labels, bioluminescent labels, and electrochemiluminescent labels are also known. Luminescent labels are also encapsulated within particles in certain assays. Signal amplification may also be performed using an enzymatic reaction or a catalytic reaction. Enzymes can be used to convert a substrate from a leuco dye to a colored form, or to a fluorescent or luminescent form. It is common for excess enzyme to be removed using a washing step before adding the substrate, and thus the signal is generated only by the enzyme specifically bound to the analyte.

[0009] Immunoassays that do not use labels, such as assays that use surface plasmon resonance as a signal transduction method, are also known. However, label-free assays tend to reduce the sensitivity of assays that use labels to enhance the signal.

[0010] Further information in the field of immunoassays can be found in "The Immunoassay Handbook: 4th Edition: Theory and Applications of Ligand Binding, ELISA and Related Techniques", Ed. D. Wild, Elsevier Science, 2013.

[0011] All binding assays, including immunoassays, have limitations in terms of the minimum and maximum concentrations of analyte that can be reliably measured.

[0012] The signal maximum is generally limited by factors such as the total amount of capture antibody available for binding to the analyte and the total amount of reporter antibody to produce a signal. When the capture antibody is immobilized on a solid phase, the surface area of the solid phase can limit the upper detection limit. Further, some signal transduction techniques, such as colorimetric quantification, tend to reach saturation depending on the optical path length required for light to pass through the sample. Luminescence methods tend to have less of a tendency to reach saturation because the gain of the detector can be attenuated to handle higher levels of light emission. When all of the antibody binding sites in a heterogeneous assay are filled with analyte, the maximum signal is reached and the system saturates. Excess analyte is typically removed in a wash step before the reporter is added. Heterogeneous assays can also be subject to an effect known as the high-dose hook when the concentration is greater than the effective concentration of the capture antibody and / or reporter antibody. In this case, at extremely high concentrations, all of the binding sites on the capture and reporter may be blocked, and the assay signal may decrease, resulting in an incorrect result.

[0013] Lower-level detection is governed by several different factors. In general, all assays are affected by properties such as the quality (affinity and specificity) of the antibodies used and the cross-reactivity of the antibodies with the analyte of interest. The lower limit of detection also depends on factors that affect the signal / noise ratio of the assay setup and system design. For example, in a standard enzyme-linked immunosorbent assay (ELISA), capture antibodies are coated onto the surface of a 96-well microtiter plate, and then samples are incubated in those wells to obtain captured analytes. These wells are washed, and then an excess amount of a reporter that binds to the captured analyte is added. Next, the excess reporter is washed away, and a substrate that can react with the enzyme is added and converted to its active form. For example, a colorless leuco dye such as 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) can be changed to an oxidized green color by horseradish peroxidase in the presence of hydrogen peroxide. If the analyte is present in very small amounts (e.g., less than 1 picomole), very little of the enzyme that binds to the surface of the well will be present. ABTS reacts with the enzyme to produce a green form, which then disperses into the bulk of the fluid, resulting in a solution that is too dilute to be distinguishable from the background signal. Autoconversion of the substrate can also produce colors that interfere with the measurement. Similarly, other detection methods such as fluorescence also have problems with interfering factors and autofluorescence in the sample or reaction well.

[0014] Another confounding factor in immunoassays can be the non-specific binding of reporter reagents to the capture surface. For example, in the above-described ELISA assay, the microtiter wells are coated with a protein layer, but some of it may denature during the coating process. It is not uncommon for a reporter to bind to regions of the capture surface during the assay. If this reporter turns over the substrate and contributes to the overall signal, it is not possible to distinguish the signal from specifically bound reporters from that of non-specifically bound ones. Non-specific binding can also be enhanced by many components present in the original sample, which bind to the capture surface during the initial incubation, changing the surface properties of the capture layer and resulting in a surface that cannot bind the reporter. Minimizing non-specific binding of the reporter involves careful optimization of all reagents and reaction conditions used in the assay, including antibodies, surfactants, temperature, and ionic strength.

[0015] Generally, the detection limits of conventional immunoassays are around 0.1 picomoles to 1 nanomole depending on the assay method. Developing assays with extremely low detection limits using conventional approaches often requires stringent washing steps to reduce non-specific binding and maximize signal / noise, and considerable optimization. Additionally, the capture surface is often small relative to the sample volume to ensure that the signal is sufficiently high relative to the background.

[0016] One approach that has been used to overcome problems related to signal / noise and improve detection limits is to measure individual binding events and count these binding events as "on" or "off" events when their measured values exceed a localized threshold. In this way, much of the background noise can be eliminated. The digitization of acoustic or communication signals is similar. These digital assays have been shown to achieve detection limits that were previously impossible to achieve using conventional similar methods. For example, low femtomolar (10 -15 mol / L), and even attomolar (10-18 Detection limits in the range of mol / L have been reported. See, for example, “Evaluation of highly sensitive immunoassay technologies for quantitative measurements of sub-pg / mL levels of cytokines in human serum”, Yeung et al, Journal of Immunological Methods, 2016, 437, 53 and “Digital Detection of Biomarkers Assisted by Nanoparticles: Application to Diagnostics”, Cretich et al, Trends in Biotechnology, 2015, 33, 343.

[0017] The majority of labels / reporters used in assays (fluorophores, dyes, etc.) cannot be observed individually using wide-field microscopy, even at high magnification, because their sizes are below the diffraction limit of visible light. Thus, the presence of these labels can only be measured as a bulk phenomenon, rather than by counting each label. In contrast, particulate labels such as latex particles can, in theory, be visualized by wide-field optical microscopy if they exceed a certain size. Depending on the optical setup, visualization of the particles can be initiated when they have a diameter of several hundred microns and above, depending on the numerical aperture and type of microscope.

[0018] However, using particles of this size as labels for monitoring individual binding events (such as antibody-antigen interactions) on the capture surface is unrealistic for several reasons. For example, particles of this size diffuse extremely slowly compared to other types of labels, impairing the reaction rate on a flat surface. They also begin to exhibit macroscopic buoyancy effects, sedimenting or floating if the particle density is significantly different from that of the medium in which they are contained, which can also pose problems for the assay format. Particles of this size are particularly prone to non-specific binding to the surface, resulting in a high background and being difficult to remove. Finally, excess particles must be removed, requiring a washing step. However, large particles begin to experience shear effects in the presence of fluid flow, and if the shear force on the particle becomes greater than the rupture of the antibody-antigen interaction strength (approximately 60 - 250 pN), the particle will be washed away ("Rapid Femtomolar Bioassays in Complex Matrices Combining Microfluidics and Magnetoelectronics", Mulvaney et al, Biosensors and Bioelectronics, 2007, 23, 191).

[0019] Examples of digital assays include the Quanterix Single Molecular Array (SIMOA) system and the Singulex Single Molecular Counting (SMC) system.

[0020] The Quanterix SIMOA system uses antibody-coated paramagnetic beads to capture analytes from solution. Next, the magnetic beads are washed and a reporter antibody labeled with an enzyme is added. The amount of beads is sufficient to minimize the probability of having more than one analyte and reporter per bead. The beads are washed again and then added to an array of microwells that can hold only one bead per well. The volume of this microwell is on the femtoliter scale. If an enzyme is bound to the bead, the fluorescent substrate in the well turns over. The small size of the well prevents the fluorescent product from diffusing too far. Next, each well is counted as an "on" or "off" event if the fluorescence exceeds a threshold.

[0021] The SMC system is used with the Singulex Clarity instrument, as well as with the Merck Millipore Erenna system and the SMCxPRO system. In all three systems, the basic measurement technique is the same. To capture the target analyte in a sandwich assay, magnetic beads coated with a capture antibody are used. Also, a fluorescently labeled reporter antibody binds to the beads in the presence of the analyte. These beads are pulled towards a magnet and the excess fluorescently tagged reporter is washed away. Next, an elution buffer that causes dissociation of the sandwich complex is added, and then this is transferred to a measurement vessel. Next, the presence of the fluorescent tag is measured using a confocal fluorescence microscope that interrogates small volumes of the sample continuously to determine the presence or absence of the fluorescent tag. If the signal of an individual measurement exceeds a threshold, this is counted as an "on" event for that measurement.

[0022] Several independent academic reviews of ultrasensitive immunoassays have highlighted that a digital approach to immunoassays enables a previously unheard-of improvement in the limit of detection (see the above references Yeung and Cretich).

[0023] The detection limits of the Quanterix system and the Singulex system depend on the volume of the sample used in the assay. For a 10 microliter serum or plasma sample, the theoretical limit is the detection of a single binding event corresponding to 1 molecule. However, in terms of molar concentration, this corresponds to 100,000 molecules per liter of sample, or 0.16×10 -18 moles per liter (0.16 attomoles).

[0024] However, the above Quanterix system and Singulex system are complex and cumbersome, each requiring multiple washing and transfer steps. Furthermore, these assays can only be performed on samples that do not contain cellular material and require expensive instrumentation to achieve their required performance. Therefore, there is still a need for a simpler and more cost-effective high-sensitivity system. SUMMARY OF THE INVENTION

[0025] Thus, the present invention is a method for detecting an analyte in a sample, comprising (i) supplying a mixture comprising the sample and a reporter reagent to a device, the device comprising a substrate having an optical component and a binding component attached to the surface of the substrate; (ii) binding a proportion of the reporter reagent proportional to the concentration of the analyte to the surface of the substrate by means of the binding component; (iii) irradiating the device with electromagnetic radiation to cause the photosensitizer of the bound reporter reagent moiety to interact with the optical component and change the optical component from a first optical state to a second optical state, thereby forming on the substrate a set of local regions of the optical component having the second optical state; and (iv) detecting the set of local regions on the substrate having the second optical state and providing a method comprising.

[0026] Thus, the present invention provides a method for detecting an analyte in a sample, wherein only reporter reagents proximal to the surface of a substrate generate signals (local regions of optical components in a second optical state), and these signals (a set of local regions of optical components in a second optical state) are detected. Thus, the method of the present invention simplifies the digital detection of analytes. The method of the present invention facilitates heterogeneous assays for a range of samples, including those containing cellular material.

[0027] Hereinafter, the present invention will be described with reference to the drawings.

Brief Description of the Drawings

[0028]

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Mode for Carrying Out the Invention

[0029] The method of the present invention is used to detect an analyte in a sample (which may be through the detection of a complex or derivative of the analyte).

[0030] The components in Figure 1 are: analyte 1; photosensitizer 2; streptavidin-coated latex particles injected with photosensitizer 3; antibody-coated latex particles injected with photosensitizer 4; antibody 5; photosensitizer-labeled antibody 6; dye 7 in a fluorescent state; dye 8 in a parenteral state; polymerized streptavidin 9; polystreptavidin dye conjugate 10 in a fluorescent state; polystreptavidin dye conjugate 11 in a parenteral state; biotin-BSA conjugate 12; red blood cell 13; amino dextran-biotin-dye conjugate 14 in a fluorescent state; and amino dextran-biotin-dye conjugate 15 in a parenteral state.

[0031] Step (i) of the method of the present invention includes supplying a mixture comprising a sample and a reporter reagent to a device, which device comprises a substrate 17 having optical components and a binding component attached to the surface of the substrate 17. The sample and the reporter reagent may be premixed before adding the mixture to the device, or the sample and the reporter reagent may be sequentially added to the device to form the mixture. The mixture may also contain additional reagents, but preferably, the mixture consists of the sample and the reporter reagent.

[0032] As an example of the principle underlying the present invention, FIG. 2 shows the device before irradiation, in which the reporter reagent is bound to the surface of the substrate. The device includes a substrate 17 and a sample chamber 16 for holding a sample containing the analyte in solution or suspension. The substrate can be any substrate that enables the detection of a set of local regions having a second optical state on the substrate. Preferably, the substrate is a transparent substrate, and more preferably, the substrate is glass.

[0033] The substrate 17 has an aminodextran-biotin-dye conjugate 14 attached to the surface of the substrate 17 via a biotin-BSA conjugate 12 and a polymerized streptavidin 9. The dye acts as an optical component and the biotin acts as a binding component. The biotin-BSA conjugate 12 and the polymerized streptavidin 9 are inert polymers that facilitate the attachment of the optical component and the binding component to the surface of the substrate 17. Since the optical component needs to be tethered to the surface of the substrate 17 for immobilization, this approach is used when the optical component is water-soluble.

[0034] Although the optical component and the binding component are shown in this way, any technique for holding the optical component and the binding component proximal to the surface of the substrate 17 is applicable. For example, the optical component and the binding component can be separate reagents, and the binding component may be attached to the optical component.

[0035] The optical component may also be encapsulated within a polymer layer coated on the surface of the substrate 17, and the binding component is attached to the polymer layer. The polymer can be silicone, polystyrene or polyisobutylene, or any other suitable polymeric plastic that can be used to encapsulate the optical component. This approach can be used when the optical component is water-insoluble.

[0036] Alternatively, the gel / hydrogel layer may be impregnated with the optical component in a gel / hydrogel layer coated on the surface of the substrate 17 and a binding component attached to the gel / hydrogel layer.

[0037] Step (ii) of the method of the present invention involves binding a reporter reagent in a proportion proportional to the concentration of the analyte to the surface of the substrate by means of a binding component. This can be achieved by leaving the device standing for a certain period of time, for example, 10 minutes.

[0038] In FIG. 2, streptavidin-coated latex particles impregnated with a photosensitizer 3 are bound to the surface of the substrate 17 by means of biotin on an aminodextran-biotin-dye conjugate 14. The streptavidin-coated latex particles impregnated with the photosensitizer 3 act as a reporter reagent.

[0039] The reporter reagent is thus shown bound to the surface of the substrate 17. However, when the analyte is present, the reporter reagent binds to the surface of the substrate in proportion to the concentration of the analyte. For example, when the binding component and the reporter reagent are antibodies and the analyte is an antigen, the reporter reagent binds to the binding component via the analyte to form a so-called "sandwich" complex as described later with reference to FIGS. 7 and 8.

[0040] All of the steps up to this point are performed in the absence of light. Step (iii) of the method of the present invention involves irradiating the device with electromagnetic radiation so that the photosensitizer of the bound reporter reagent portion interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming a set of local regions of the optical component having the second optical state on the substrate 17.

[0041] Figure 3 shows the device of Figure 2 irradiated with electromagnetic radiation (generally referred to as "light"), preferably visible light. The light source can be, for example, LED 18. This light source irradiates the sample chamber 16 with light of an appropriate wavelength to excite the photosensitizer 2. This wavelength varies depending on the photosensitizer, but a preferred wavelength is 680 nm. This device is generally irradiated for at least 30 seconds. Preferably, this device is exposed to the electromagnetic radiation source for more than 1 second, more preferably at least 2 seconds, more preferably at least 5 seconds, more preferably at least 20 seconds, and most preferably at least 30 seconds. This ensures that there is an irreversible optical conversion on the substrate surface, thus enabling the discrimination between long-lived binding events and transient binding events.

[0042] Figure 4 shows the device of Figure 3 after irradiation. The photosensitizer 2 interacts with the dye optical component of the aminodextran-biotin-dye conjugate 14 to change the dye from a fluorescent state to a non-fluorescent state. The aminodextran-biotin-dye conjugate 14 in the fluorescent state becomes the aminodextran-biotin-dye conjugate 15 in the parenteral state. Only the dye in close proximity to the photosensitizer 2 changes from the first optical state to the second optical state.

[0043] Although it is shown that the dye converts from a fluorescent state to a non-fluorescent state, other optical components undergo different changes. In one embodiment, the optical component in the first optical state absorbs light of one or more wavelengths, preferably visible light, and the optical component in the second optical state absorbs light of one or more other wavelengths, preferably visible light. 。

[0044] In this embodiment, the optical component is subject to color change.

[0045] Another first and second optical states can include changes in light polarization, fluorescence lifetime, refractive index, light scattering (including Raman scattering), and other optical effects.

[0046] Figure 5 shows the device before irradiation in the presence of a whole blood sample and a reporter reagent. The whole blood sample also contains additional components such as red blood cells 13. Preferably, the substrate 17 forms the upper part of the sample chamber 16 and allows the red blood cells 13 to sediment from the substrate 17.

[0047] A proportion of the reporter reagent binds to the surface of the substrate 17 by means of a binding component. Thus, the sample contains the bound reporter reagent and the unbound reporter reagent that is free in solution. The depth of the sample chamber 16 is designed to minimize the diffusion path length of the reporter reagent and enable easy achievement of equilibrium. Generally, the depth of the sample chamber is 50 - 200 μm.

[0048] Next, the device is irradiated as described above, and Figure 6 shows the device of Figure 5 after irradiation. All the photosensitizers proximal to the optical component interact to change the optical component from the first optical state to the second optical state. Thus, the photosensitizer of the bound reporter reagent portion interacts with the optical component to change the optical component from the first optical state to the second optical state, thereby forming a set of local regions of the optical component having the second optical state on the substrate 17.

[0049] Figure 7 shows the device used in the method of the present invention, where the reporter reagent is bound to the surface of the substrate in proportion to the concentration of the analyte by means of a binding component before irradiation.

[0050] In a typical sandwich immunoassay using the method of the present invention, the substrate 17 has a poly streptavidin - dye conjugate 11 attached to the surface of the substrate 17 via a biotin - BSA conjugate 12 and an antibody 5 attached to the poly streptavidin - dye conjugate 11. The dye acts as the optical component and the antibody acts as the binding component. Although the optical component and the binding component are shown as such, any technique for retaining the optical component and the binding component proximal to the surface of the substrate 17, such as those described above, is applicable.

[0051] In the method of the present invention, the sample chamber 16 is filled with a sample containing the analyte 1. A reporter reagent such as the photosensitizer-labeled antibody 6 is also added to the sample chamber 16. When a whole blood sample is used, the sample may also contain additional components such as red blood cells 13.

[0052] Next, equilibrium is achieved. The photosensitizer-labeled antibody 6 is bound to the surface of the substrate 17 by means of the antibody 5 in proportion to the concentration of the analyte 1. The photosensitizer-labeled antibody 6 acts as a reporter reagent. An excess amount of the photosensitizer-labeled antibody 6 is included so that all of the analyte 1 forms a sandwich complex. Thus, a proportion of the reporter reagent proportional to the concentration of the analyte 1 binds to the surface of the substrate 17 by means of the binding component. Accordingly, the sample contains the bound reporter reagent and the unbound reporter reagent free in the solution.

[0053] Figure 8 shows the device of Figure 7 after irradiation. The photosensitizer 2 interacts with the dye optical component of the polystreptavidin-dye conjugate 11 to convert the dye from a non-fluorescent state to a fluorescent state. The parenteral polystreptavidin-dye conjugate 11 becomes the fluorescent polystreptavidin-dye conjugate 10. Only the dye in proximity to the photosensitizer 2 changes from the first optical state to the second optical state.

[0054] In order to achieve a complete conversion from the first optical state to the second optical state, the reporter reagent must be permanently bound to the surface of the substrate 17 throughout the entire period of irradiation. The unbound reporter reagent in the solution does not change the optical component from the first optical state to the second optical state.

[0055] This provides a significant advantage over other digital assay methods in eliminating the need for a washing step. Thus, the method of the present invention is a heterogeneous assay. In conventional assays, unbound reporter reagent interferes with the signal generated by the bound reporter reagent, so the unbound reporter reagent must be separated from the bound reporter reagent before measurement. However, due to the local surface changes provided by the present invention, the bound and unbound reporter reagents are distinguishable. In fact, the ability to distinguish between reporter reagents proximal to the surface of substrate 17 (i.e., bound) and reporter reagents in the bulk solution (i.e., unbound) is a particular advantage of the present invention. Preferably, steps (i)-(iii) are performed without a washing step, i.e., the method is performed without removing the sample from the substrate in steps (i), (ii), and (iii).

[0056] The photosensitizer of the bound reporter reagent portion interacts directly with the optical component to cause a change (e.g., the photosensitizer is excited and this energy is directly transferred to the optical component) or indirectly with the optical component to cause a change via an additional reagent (e.g., the photosensitizer is excited and this energy is transferred to an additional component and then this energy is transferred to the optical component).

[0057] In a preferred embodiment, the absorption by the photosensitizer is for the purpose of generating an activator reagent by interaction with a pre-activator reagent present in the mixture, and the activator reagent is for the purpose of causing a change from a first optical state to a second optical state by interaction with the optical component.

[0058] The pre-activator reagent may be present in the sample, or the pre-activator reagent may be added as an additional reagent to the mixture of the sample and the reporter reagent. In a preferred embodiment, the pre-activator reagent is triplet oxygen. In another preferred embodiment, the activator reagent is a reactive oxygen species (ROS). Preferably, the ROS is selected from hydroxyl radicals, superoxide, peroxide, organic peroxide, peroxynitrite, singlet oxygen, and mixtures thereof. More preferably, the activator reagent is singlet oxygen. Singlet oxygen is a preferred activator reagent because it has a short half-life and a limited diffusion path length (up to 200 nm under aqueous conditions).

[0059] While not wishing to be bound by any particular theory, it is believed that the photosensitizer absorbs light to generate an excited state, which can undergo intersystem crossing (ISC) with oxygen present in the sample and proximal to the reporter reagent to generate singlet oxygen. The singlet oxygen will then interact with the optical components as described below. In a particularly preferred embodiment, the pre-activator reagent is triplet oxygen and the activator reagent is singlet oxygen.

[0060] Singlet oxygen has been used in immunoassays in luminescent oxygen channeling immunoassay (LOCI) heretofore. The LOCI immunoassay is a homogeneous non-digital assay using donor and acceptor beads. Donor beads generate singlet oxygen upon irradiation at 680 nm, and acceptor beads generate a chemiluminescent signal when activated by singlet oxygen. The binding of the donor to the acceptor beads is facilitated by antibody-antigen binding. The reaction mixture is generally irradiated for 0.5 - 1.0 seconds, and then the luminescent signal is measured for 0.5 - 1.0 seconds. Critically, the measurement is performed in the presence of all unbound donor and acceptor beads. Spatial separation of the beads minimizes the background signal, but the LOCI assay cannot distinguish between long-lived and transient binding events due to the short measurement time. The assay can achieve a detection limit of around 1 - 5 pg / mL for its most sensitive assays, such as interleukin 6 (IL-6) or thyroid stimulating hormone (TSH). The method of the present invention further minimizes the background signal because, for a signal to be detected, the "donor" particles, the reporter reagent, need to be in proximity to the surface of the substrate 17 rather than in solution and also need to be present there during the duration of the irradiation period. Thus, the method of the present invention is more sensitive than the LOCI assay and can detect low concentrations of analytes.

[0061] In a preferred embodiment, the optical component is a dye. Preferably, the optical component is selected from one of the following dyes and mixtures thereof.

Chemical formula

[0062] The dyes (1)-(5) are known and are used as cell probes for monitoring ROS formation in cells under oxidative stress. However, these dyes are not known for use in either standard immunoassays or digital immunoassays. Dye (6) is a common fluorescent dye. There are also other extensive fluorescent dyes commercially available and suitable for use in the present invention, including Alexa Fluor dyes, BODIPY dyes, rhodamine dyes, Texas Red, Oregon Green, Cascade Yellow, Pacific Blue, etc. For further examples, refer to the Molecular Probes Handbook, Thermo Fisher Scientific.

[0063] The dye singlet oxygen sensor green (SOSG) (1) reacts with singlet oxygen and converts it from a weak fluorescence form to a high fluorescence form. Singlet oxygen reacts with the anthracenyl group to form an endoperoxide. In a preferred embodiment, the optical component is SOSG.

[0064] The dye boron-dipyrromethene 581 / 591 (BODIPY 581 / 591 )(2) reacts with reactive oxygen species such as singlet oxygen and hydroxyl radicals, resulting in a hypsochromic shift in the fluorescence excitation / emission maximum.

[0065] The dyes (3)-(5) share the same core structure and react with common oxidants containing singlet oxygen to convert them into a fluorescent form. Thus, these dyes can be converted from a leuco state to a fluorescent state.

[0066] The pigment (6) has been found to react with singlet oxygen and convert it from a fluorescent form to a non-fluorescent form. When using fluorescein, it has surprisingly been found that the fluorescence proximal to the photosensitizer can be completely eliminated to create a dark non-fluorescent region on the fluorescent substrate. Thus, in a preferred embodiment, the optical component is fluorescein. More preferably, the optical component is fluorescein, the first optical state is fluorescent, and the second optical state is non-fluorescent. Most preferably, the optical component is fluorescein, the first optical state is fluorescent, the second optical state is non-fluorescent, and the set of local regions is the dark non-fluorescent regions on the substrate.

[0067] Preferably, the optical component is fluorescent when in one of the first and second optical states, and the optical component is non-fluorescent when in the other of the first and second optical states. In one embodiment, the optical component is non-fluorescent when in the first optical state and the optical component is fluorescent when in the second optical state. However, more preferably, the optical component is fluorescent when in the first optical state and the optical component is non-fluorescent when in the second optical state.

[0068] In another preferred embodiment, the optical component is fluorescent at one or more wavelengths when in the first optical state, and the optical component is fluorescent at one or more other wavelengths when in the second optical state. In this embodiment, the optical component shifts its fluorescence excitation / emission maximum.

[0069] Preferably, the change from the first optical state to the second optical state is irreversible. This enables subsequent scanning of the substrate to identify regions where the change in optical state has occurred.

[0070] Step (iv) of the method of the present invention includes detecting a set of local regions having the second optical state on the substrate.

[0071] The optical component having the second optical state forms a set of local regions on the substrate. Advantageously, the local regions having the second optical state can be counted as individual binding events. Thus, the method of the present invention is suitable for performing digital assays. However, if there are a large number of binding events and as a result, most of the optical components are in the second optical state, a bulk change can be detected.

[0072] In a preferred embodiment, the set of local regions having the second optical state on the substrate can be detected by counting the local regions in the set of local regions having the second optical state on the substrate or by measuring the set of local regions having the second optical state on the substrate as a bulk property. More preferably, the set of local regions having the second optical state on the substrate is detected by counting the local regions in the set of local regions having the second optical state on the substrate.

[0073] The local regions having the second optical state may need to exceed a threshold corresponding to the background signal depending on the first and second optical states. For example, the sample may have some background autofluorescence, but due to the digital nature of the assay, if this does not exceed the threshold, it should not affect the signal. Background chemiluminescence does not tend to occur.

[0074] Furthermore, the surface of the substrate may be scanned before irradiation with the photosensitizer to obtain a two-dimensional image of the surface, and then scanned after irradiation to reduce interference from artifacts, contaminants, and any autofluorescence on the surface or from the sample itself by background subtracting the pre-irradiation image from the post-irradiation image. Thus, in a preferred embodiment, the method of the present invention further comprises eliminating any components having a second optical state on the substrate detected prior to irradiating the device with electromagnetic radiation from the set of local regions having a second optical state on the substrate detected in step (iv). In this way, the background-subtracted image shows only changes in the optical properties of the substrate (e.g., changes in fluorescence intensity).

[0075] The set of local regions having a second optical state on the substrate are generally discrete regions on the substrate. However, there are also local regions that are excluded from detection due to their morphology. Local regions corresponding to individual binding events tend to be uniform and circular, but there are also local regions with irregular shapes corresponding to artifacts. Furthermore, there are larger local regions where particles have agglomerated together. Thus, in a preferred embodiment, only uniform and circular local regions having a second optical state on the substrate are detected.

[0076] A set of local regions on a substrate can be detected using optical means. In a preferred embodiment, a set of local regions having a second optical state on the substrate is detected using an optical microscope. A suitable optical setting for the detection is shown in FIG. 9. More preferably, a set of local regions having a second optical state on the substrate is detected using a wide-field microscope. A wide-field microscope is the simplest form of microscope, whereby the entire sample is irradiated and imaged simultaneously, as opposed to more complex techniques such as confocal microscopes where only one single focus is irradiated and recorded at a time. The advantages of confocal microscopes are that the contrast is increased by removing out-of-focus blur and that an image stack can be obtained up to the depth of the sample. Also known are super-resolution microscopy techniques such as photoactivated localization microscopy (PALM or FPALM) and stochastic optical reconstruction microscopy (STORM). These methods are more complex and more costly than the simple wide-field method.

[0077] When using dyes (1) and (3) to (5), fluorescence hot spots (or an enhancement of fluorescence if multiple binding events are present) are seen on the surface of the substrate where the optical component has been converted to the second optical state. To detect this fluorescence, the substrate can be irradiated at the excitation wavelength of the dye and the surface of the substrate can be scanned with respect to light emission.

[0078] When using dye (2), either a decrease in fluorescence emission at 610 nm or an increase in fluorescence emission at 515 nm can be measured, or both wavelengths can be monitored.

[0079] When using the pigment (6), dark spots (or a dark image if there are multiple binding events) are seen on the surface of the substrate where the optical component has been converted to the second optical state. To detect these dark spots or images, a wide-field fluorescence microscope equipped with a light source (e.g., an LED) and a light detector (e.g., a camera such as a photomultiplier tube or a CCD) can be used with excitation and emission filters suitable for the detection of fluorescein (e.g., excitation at 490 nm and emission at 520 nm).

[0080] When a binding event occurs, the photosensitizer is proximal to the substrate. That is, the photosensitizer is sufficiently close to the surface of the substrate to interact with the optical component and convert it from the first optical state to the second optical state upon irradiation of the device. However, the actual distance between the photosensitizer and the surface of the substrate varies depending on the size and nature of the photosensitizer, the binding component, the size and nature of the reporter reagent and the analyte, as well as the nature of the sample medium.

[0081] The binding component has binding sites that can bind to the reporter reagent in proportion to the concentration of the analyte in the sample. This proportionality is important for the function of the assay because for a meaningful measurement of the concentration of the analyte to be determined, the binding must depend on the concentration of the analyte. This binding may be directly proportional or inversely proportional to the concentration of the analyte depending on the type of assay being performed. In the case of a non-competitive assay, such as an immunoassay, the binding is directly proportional to the concentration of the analyte, while in the case of a competitive assay, the binding is inversely proportional to the concentration of the analyte.

[0082] One particular type of competitive assay is proposed where an antibody to the analyte is immobilized on the substrate and a labeled analog of the analyte is introduced into the sample. The analyte and the labeled analog of the analyte then "compete" around the antibody on the surface. And in the absence of the analyte, the labeled analog binds at the maximum possible rate. However, in the presence of the analyte, the antibody on the substrate becomes occupied with the analyte and the binding rate of the analog decreases.

[0083] The binding component can be adapted to bind to the analyte, or a complex or derivative of the analyte, in which case the reporter reagent binds to the binding component in the presence of the analyte, or a complex or derivative of the analyte. In this case, the binding component has a binding site that can bind to the reporter reagent in the presence of the analyte or a complex or derivative of the analyte. However, the binding still is proportional to the concentration of the analyte.

[0084] Alternatively, the binding component itself may be an analog of the analyte, and the reporter reagent binds directly to the binding component (since it is bound to the surface of the substrate via a covalent or non-covalent interaction, it is an analog). In this case, the binding component competes with unbound analyte, or an unbound complex or derivative of the analyte, for binding to the reporter reagent. Thus, the binding component can readily bind to the reporter reagent.

[0085] Determining the extent of binding of the reporter reagent to the binding component (either directly or through the intervention of the analyte / analyte complex or derivative) gives a measurement of the concentration of the analyte in the sample.

[0086] The assay also requires the presence of the reporter reagent. The reporter reagent comprises a photosensitizer. The photosensitizer can absorb electromagnetic radiation and interact with an optical component. It is this interaction that changes the optical component from a first optical state to a second optical state.

[0087] Thus, the photosensitizer can be composed of any material that can interact with electromagnetic radiation in this way. Suitable photosensitizers are known as PDT reagents from photodynamic therapy (PDT). PDT reagents are used to destroy cells upon irradiation in cancer therapy and dermatology (Shafirstein et al, Cancers, 2017, 9, 12; Wan and Lin, Clinical, Cosmetic and Investigational Dermatology, 2014, 7, 145).

[0088] When irradiated with electromagnetic radiation, the PDT reagent is promoted to the excited triplet state. This excited triplet state can interact directly with cell components, which is called a type I process, or can interact with oxygen, which is called a type II process. Both the type I process and the type II process can lead to the formation of ROS. In the type II process, the main product is singlet oxygen through an intersystem crossing mechanism.

[0089] Singlet oxygen is an excited state of highly reactive oxygen. It can undergo many reactions including Diels-Alder reactions and ene reactions until it decays. It also undergoes general oxidation reactions by sulfur- and nitrogen-containing compounds. This non-discriminatory reactivity of singlet oxygen is one of the reasons why it is used in photodynamic therapy.

[0090] A wide range of photosensitizer compounds are known, including porphyrins, chlorins (e.g., pyropheophorbide-a), phthalocyanines, and other polycyclic aromatic species (see, for example, Antibody-Directed Phototherapy, Pye et al, Antibodies, 2013, 2, 270).

[0091] In one embodiment, the photosensitizer is selected from porphyrins, chlorins, phthalocyanines, and other polycyclic aromatic species. In a preferred embodiment, the photosensitizer is pyropheophorbide-a (PPa). PPa has the following structure.

Chemical formula

[0092] Other photosensitizers that catalyze the reaction without generating ROS can also be used. These include Ru(II)-tris(2,2'-bipyridyl) dichloride that catalyzes the reduction of dichlorodiphenyltrichloroethane in the presence of a reducing agent; benzophenone / eosin that catalyzes the cis-trans isomerization of stilbene; and chlorophyll that catalyzes the reaction of carbon dioxide and water to produce carbohydrates. Photosensitizers are also known that can catalyze polymerization reactions that can bring about changes in the optical properties of the substrate. See, for example, Dyes as Photoinitiators or Photosensitisers of Polymerisation Reactions, Fouassier et al, Materials, 2010, 3, 5130.

[0093] The nature of the binding component and the reporter reagent varies depending on the nature of the analyte, but they are preferably antibodies. The method of the present invention has particular applicability to immunoassays. In a particularly preferred embodiment, the binding component is an antibody produced against the analyte or a complex or derivative of the analyte, and the reporter reagent comprises an antibody produced against the analyte or a complex or derivative of the analyte. In principle, a single molecule can be used for each reagent, but in practice, the binding component and the reporter reagent are populations of molecules. The term "antibody" preferably includes within its scope Fab fragments, single-chain variable fragments (scFv), and recombinant binding fragments.

[0094] As an alternative to the antibody-antigen reaction, the binding component, the reporter reagent, and the analyte may be a first and a second nucleic acid whose first and second nucleic acids are complementary, or a reagent containing avidin or a derivative thereof and an analyte containing biotin or a derivative thereof, or vice versa. The binding component and the reporter reagent may also be aptamers. This system is not limited to biological assays and may be applied, for example, to the detection of heavy metals in water. This system also does not need to be limited to liquids, and any fluid system can be used, such as the detection of enzymes, cells, and viruses in air.

[0095] The maximum observable signal is the maximum signal achievable when monitoring a photosensitizer that binds to the surface. The binding of particles to the substrate is governed by the diffusion rates of the analyte and reporter reagent and is then strongly governed by the hydrodynamic radii of these components and the viscosity / temperature of the sample.

[0096] The device used in the method of the present invention may further comprise a control that compensates for natural variations in the components of the measurement, variations in the sample being measured, and variations in the environmental conditions during the measurement. This can be achieved by exposing the sample to the reagents on the surface of the substrate. Generally, different reagents are disposed in different regions of the surface of the substrate, and these regions are coated with different reagents. These controls are defined as "negative" and "positive" controls in the sense that the negative control approximates the signal expected in the absence of the analyte and the positive control approximates the signal expected when the analyte saturates the system.

[0097] To achieve detection with these controls, the device of the present invention preferably comprises a binding component, a negative control reagent, and a positive control reagent, each of which is attached to the surface of the substrate as described above.

[0098] The binding component is as described above.

[0099] The negative control reagent has a lower affinity for the reporter reagent than the binding component under the conditions of the assay. Thus, the negative control reagent provides a negative control. It is important that the affinity be considered under the conditions of that assay. This is because in the case of a non-competitive assay, the presence of the analyte, or a complex or derivative of the analyte, intervenes in the affinity of the binding component for the reporter reagent. Thus, in the absence of the analyte, or a complex or derivative of the analyte, neither the binding component nor the negative control reagent has an affinity for the reporter reagent. However, in the presence of the analyte, or a complex or derivative of the analyte, the negative control reagent has a lower affinity for the reporter reagent than the binding component.

[0100] Furthermore, in embodiments where the binding component binds to the analyte, or a complex or derivative of the analyte, the negative control reagent preferably has a higher affinity for the analyte or, if used, the complex or derivative of the analyte than the binding component. The negative control reagent is preferably a protein, more preferably an antibody. The negative control reagent generally has chemical and physical properties similar to those of the binding component but shows little or no affinity for the reporter reagent under the conditions of the assay. In particularly preferred embodiments, the negative control reagent has substantially no affinity for the reporter reagent under the conditions of the assay. Preferably, the negative control reagent provides substantially no affinity for the analyte or a complex or derivative of the analyte. That is, the binding of the reporter reagent, or, where applicable, the analyte or a complex or derivative of the analyte, to the negative control reagent is non-specific. Thus, the negative control reagent can offset non-specific binding of the reporter reagent to the binding component.

[0101] The positive control reagent binds to the reporter reagent and has an affinity for the reporter reagent such that the influence of the concentration of the analyte, or if used, the complex or derivative of the analyte, in the sample is less than that of the binding component, thus providing a positive control. Preferably, the positive control reagent has an affinity for the reporter reagent that is substantially independent of the concentration of the analyte or the complex or derivative of the analyte. More preferably, the positive control reagent has a higher affinity for the reporter reagent than the binding component under the assay conditions. Thus, the positive control reagent measures the maximum expected signal in the system.

[0102] To increase the dynamic range and improve the accuracy of the assay implemented in the present invention, it is preferred to have the binding component at a plurality of locations on the substrate. These locations can be adjusted to different sensitivities by varying the concentration of the binding component at each location. Each location may also have its own negative and positive control reagents for functioning as a control for different dynamic ranges. This is particularly applicable to competitive assays that are particularly sensitive to the respective concentrations of the individual components constituting the system.

[0103] The above description allows the assay to reach equilibrium before activating the photosensitizer, but the kinetics of the binding event can also be monitored by irradiating the photosensitizer during discrete periods of a time course and monitoring the binding events that occur during the reaction process before equilibrium is achieved.

[0104] The analyte can be a macromolecule or a small molecule. Macromolecules are generally proteins, such as protein-based hormones, and can also be part of large particles such as viruses, bacteria, cells (e.g., red blood cells) or prions. Small molecules can be drugs.

[0105] As used herein, the term "small molecule" is a technical term used to distinguish its molecules from macromolecules such as proteins and nucleic acids. Small molecules are often referred to as "haptens" in the field of immunoassays, can elicit an immune response when attached to a large carrier molecule such as a protein, and include small molecules such as hormones and synthetic drugs. This type of small molecule generally has a molecular weight of 2,000 or less, often 1,000 or less, and even more preferably 500 or less. The binding component can be adapted to bind to the analyte itself, but the analyte can undergo a chemical reaction or an initial complex formation event before binding to the binding component. For example, the analyte can be protonated / deprotonated at the pH of the assay conditions. Thus, the analyte bound to the binding component can be the analyte itself or a derivative of the analyte, both of which are included within the scope of the present invention.

[0106] In a preferred embodiment, the present invention may be used to simultaneously detect multiple analytes in the same sample. Different binding components can be used at different locations on the substrate for the measurement of each analyte. Sandwich assays and competitive assays can be performed in parallel, and these assays may use the same negative and positive controls as described above, or there may be a separate control for each analyte being measured.

[0107] A sample suspected of containing the analyte of interest is generally a fluid sample, e.g., a liquid sample, usually a biological sample such as a body fluid, e.g., blood, plasma, saliva, serum, intraocular fluid, cerebrospinal fluid or urine. The sample may contain suspended particles and may be whole blood. In a preferred embodiment, the sample is untreated, and more preferably, an untreated fluid. Untreated means that the sample / fluid is not pretreated by filtration, dilution or any other pretreatment step before being mixed with the reporter reagent and other assay components. The advantage of the method of the present invention is that the assay can be performed on a sample containing suspended particles without unduly affecting the assay results.

[0108] In a preferred embodiment, the sample is whole blood. It is surprising that the components of whole blood do not interfere with the detection method of the present invention. In order to measure fluorescence in the plasma or serum components of blood, it is usually necessary to remove red blood cells from the blood due to unpredictable scattering of light by different cell components for each sample. However, in the method of the present invention, since fluorescence is measured on the substrate and individual binding events are measurable, measurements can be performed using whole blood.

[0109] The sample is generally on the order of microliters (e.g., 1 - 100 μL, preferably 1 - 10 μL). To hold the fluid sample, the substrate is preferably placed in a sample chamber having one or more side walls, a top surface, and a bottom surface. Thus, the device used in the method of the present invention preferably further comprises a chamber for contacting and holding a sample containing an analyte with the substrate.

[0110] A potential additional background interference source is the sedimentation of suspended particles onto the substrate surface, including the reporter reagent and cell components of the sample. This interference source can be reduced by placing the substrate above the bulk solution, e.g., on the top surface of the reaction chamber. Thus, even if sedimentation occurs, it does not interfere with the substrate. Preferably, as shown in the figure, the substrate forms the top surface. Clearly, the optical components and the binding components are on the inner surface of the chamber to enable contact with the sample. This modification and other modifications are included within the scope of the present invention.

[0111] The sample can be easily held, for example, within a capillary channel by surface tension.

[0112] The reporter reagent and optionally one or more additional reagents are preferably stored in a chamber incorporated into the device used in the method of the present invention.

[0113] The method of the present invention is particularly useful in point-of-care (POC) testing. POC testing is defined as a diagnostic test at or near the clinical site, i.e., bedside testing. POC testing enables convenient and rapid testing, and improves decision-making and triage while better allocating hospital resources such as in accidents and emergency medicine as well as hospital beds. This is in contrast to conventional testing where samples are taken at the clinical site and then sent to the laboratory for testing. In such tests, it often takes hours or even hours to obtain results, during which time medical treatment has to continue without the desired information. POC testing often uses test kits in combination with portable instruments.

[0114] The method of the present invention is particularly useful for monitoring the concentration or presence / absence of analytes that are usually present in extremely low amounts. Potential applications include the measurement of biomarkers in heart disease (e.g., high-sensitivity troponin), infectious diseases (e.g., hepatitis C core antigen), aging / cognitive impairment (e.g., Alzheimer's disease markers amyloid β and tau), cytokines, and oncology (e.g., circulating tumor markers).

[0115] The present invention also provides a device for detecting an analyte in a sample, the device comprising a substrate having an optical component and a binding component bound to the surface of the substrate, the optical component changing from a first optical state to a second optical state in response to the interaction of a reporter reagent bound to the surface of the substrate with an irradiated photosensitizer in proportion to the concentration of the analyte in the sample, thereby forming a set of local regions of the optical component having the second optical state on the substrate.

[0116] The features of the device are as described above with respect to the device used in the method of the present invention.

[0117] In a preferred embodiment, the device further comprises a chamber for holding a mixture of the sample and the reporter reagent.

[0118] The device of the present invention may include a radiation source adapted to generate electromagnetic radiation and a detector adapted to detect an optical component in a second optical state, thereby enabling an accurate determination of the position of the photosensitizer relative to the substrate.

[0119] The device of the present invention may take the form of a cartridge for use with another reader. The radiation source and the detector may be incorporated into the reader. The reader is preferably a portable reader. Preferably, the device comprises a cartridge, the substrate is within the cartridge, and the device further comprises a detector for detecting a set of local regions having a second optical state on the substrate. The present invention also provides a cartridge comprising a substrate, an optical component and a binding component as defined above. This cartridge is preferably a disposable cartridge.

[0120] The present invention also provides a system for detecting an analyte in a sample, the system comprising: the device of the present invention; and a reporter reagent for forming a mixture comprising the sample, the reporter reagent comprising a photosensitizer, the photosensitizer being capable of absorbing electromagnetic radiation and interacting with the optical component to change the optical component from a first optical state to a second optical state.

[0121] Preferably, the photosensitizer is capable of absorbing electromagnetic radiation and interacting with a pre-activator reagent present in the mixture to generate an activator reagent, the activator reagent being capable of interacting with the optical component to change the optical component from a first optical state to a second optical state.

[0122] In a preferred embodiment, the system of the present invention consists essentially of the above features. "Consists essentially of" means that no other features are required to perform the assay.

[0123] The present invention will now be described with reference to examples, which are not intended to be limiting.

Example

[0124] Biotinylated BSA was prepared according to techniques known in the art. Alphascreen donor beads encapsulated with singlet oxygen photosensitizer were supplied by Perkin Elmer.

[0125] Example 1 70 kD amino dextran-biotin-fluorescein conjugate 10 mg of amino dextran (70 kD) was weighed into a glass vial and made up to 2 mg / mL with 100 mM potassium phosphate buffer. 250 μL of this solution was dispensed into a 2 mL cryotube. A 4 mg / mL biotin-N-hydroxysuccinimide (NHS) ester solution was prepared in DMSO, and 16.2 μL of this was added to the cryotube. The sample was mixed on a roller at 20 °C for 30 minutes, during which time a 20 mg / mL fluorescein-NHS ester solution was prepared in DMSO.

[0126] After 30 minutes of reaction with biotin-NHS ester, 8.5 μL of the 20 mg / mL fluorescein-NHS ester solution was added and it was mixed on a roller at 20 °C for a further 60 minutes. The reaction was then quenched by adding 11.3 μL of a 10 mg / mL glycine solution and mixed on a roller at 20 °C for 20 minutes. The sample was desalted in 50 mM phosphate buffer using a Sephadex G-25 PD10 column. Absorbance was measured at 280 nm and 495 nm using a Nanodrop2000 spectrophotometer to determine the concentration and fluorescein incorporation. The sample was then filtered to 0.2 μm using a Minisart (Sartorius) filter. The calculated incorporations of biotin and fluorescein were 1.2 and 4.3 respectively.

[0127] Example 2 Polymerized streptavidin (polystreptavidin) Conjugated streptavidin was prepared according to techniques known in the art. Briefly, an aliquot of streptavidin (1 mg / mL) was activated with 9.7 molar equivalents of SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) for 1 hour, after which the reaction was quenched with glycine and the product was desalted on a PD10 size exclusion column. In parallel, another aliquot of streptavidin was reacted with 9.7 molar equivalents of SATA (N-succinimidyl-s-acetyl-thioacetate) for 1 hour, then desalted with hydroxylamine and purified on a PD10 size exclusion column.

[0128] SATA-streptavidin and SMCC-streptavidin were combined at a molar ratio of 3:2 for 30 minutes, after which NEM (N-ethylmaleimide) was added to quench the reaction. The product was then purified on a G25 Sephadex column to obtain conjugated streptavidin. The concentration of the product was measured by UV-vis spectroscopy and adjusted to 1.0 mg / mL. Further characterization was not performed.

[0129] Example 3 Fabrication of Substrate Surface 1 A 200-μm-thick, 1 cm × 1 cm piece of pressure-sensitive adhesive 19 with a 6-mm-diameter hole cut out was adhered to a 22 mm × 22 mm cover glass 20 (Brand, catalog number 4700 55) to create a shallow well 21.

[0130] Next, 30 μL of biotinylated BSA (10 μg / mL in 40 mM phosphate buffer) was added to this well, incubated for 2 hours, and then washed with wash buffer (40 mM phosphate, 2% sucrose, 0.9% NaCl, 0.03% BSA). Then, 30 μL of polystreptavidin (10 μg / mL in 40 mM phosphate buffer) was added to this well, incubated for 60 minutes, and then washed 3 times with wash buffer. Then, 30 μL of 70 kD aminodextran-biotin-fluorescein conjugate (10 μg / mL in 40 mM phosphate buffer) was incubated for 30 minutes, then washed 3 times with wash buffer, and air-dried at room temperature.

[0131] Example 4 Binding of streptavidin beads to the surface Streptavidin-coated Alphascreen donor beads (Perkin Elmer catalog number 6760002S, 5 mg / mL) were diluted 1 / 1000 in 40 mM phosphate buffer, and then 20 μL of this solution was added to the substrate from Example 3, incubated for 2 hours, then washed 3 times with wash buffer, and dried. This was performed under low illumination conditions with a green filter on the illuminator. The beads coated with streptavidin bind to the free biotin groups on the aminodextran-biotin-fluorescein conjugate as shown in Figure 2.

[0132] Example 5 Generation of non-fluorescent spots in the fluorescent layer 20 μL of H2O was added to the substrate, and the entire surface was irradiated at 680 nm using a red LED, and the total emission output was such that the light flux on the surface was approximately 0.5 mW / mm as shown in Figure 3. 2It was set to 15 mW corresponding to [the relevant value]. After 5 minutes of irradiation, the liquid was removed and the surface was dried. Next, the substrate was inverted and attached onto a slide glass such as that shown in FIG. 11, and then an image was obtained using a 100x oil immersion lens and a fluorescein set with a Leica DMR wide-field fluorescence microscope equipped with a CoolLED pE-300 irradiation source and a Leica DFC9000GT digital camera (2048×2048 pixels). FIG. 9 shows the optical setup.

[0133] FIG. 4 shows how the dye molecules on the aminodextran in proximity to the conjugated beads were converted from their fluorescent form 14 to their non-fluorescent form 15 due to the singlet oxygen flux generated by bead 3 when irradiated at 680 nm as described above.

[0134] FIG. 12 is an image of a part of the substrate surface taken by the microscope camera. Regions with discrete dark spots can be observed, where beads are attached. There are also somewhat long spots, where beads are in clusters. However, the smaller, uniform spots are clearly distinguishable as individual binding events due to the beads held in proximity to the surface during the 5-minute irradiation period.

[0135] Example 6 As described in Examples 3 and 4 above, the substrate surface was prepared and beads were attached to the surface. Next, 20 μL of human plasma was added to the surface, and excitation and imaging of the beads were performed as described in Example 5 above. A microscope camera image of a part of the substrate surface is shown in FIG. 13. Discrete dark regions corresponding to the individually attached beads are seen on the substrate surface, indicating that the components of human plasma do not interfere with or quench the reaction of singlet oxygen with the active dye during the irradiation stage.

[0136] Example 7 As described in Examples 3 and 4 above, the substrate surface was prepared and beads were coupled to the surface. Next, 20 μL of fresh human blood was added to the surface, and excitation and imaging of the beads were performed as described in Example 5 above. A microscopic camera image of a part of the substrate surface is shown in FIG. 14. Discrete dark regions corresponding to the individually coupled beads are seen on the substrate surface, indicating that in the irradiation stage, the cellular components of human blood do not interfere with or quench the reaction of singlet oxygen with the active dye.

[0137] Example 8 Polystreptavidin-singlet oxygen sensor green (SOSG) conjugate Six vials of singlet oxygen sensor green (Thermo Fisher, catalog number S36002, 100 μg / vial) are taken out of the freezer and thawed at room temperature for 30 minutes. Next, these are reconstituted with methanol and pooled to a total volume of 200 μL. 19.1 μL of N-hydroxysuccinimide (6 mg / mL in 25 mM MES buffer), then 31.9 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (6 mg / mL in 25 mM MES buffer) are added, and the solution is mixed on a roller at 20 °C for 15 minutes to activate the SOSG. 1 mL of 1 mg / mL polystreptavidin solution (in 100 mM phosphate buffer) is dispensed into a cryotube.

[0138] After 15 minutes of incubation, 90.7 μL of the activated SOSG is added, and the sample is mixed on a roller at 20 °C for about 65 hours. Next, the sample is desalted with a Sephadex G-25 PD10 column using 50 mM phosphate buffer. The absorbance is measured at 280 nm and 520 nm using a Nanodrop2000 spectrophotometer to determine the concentration and the incorporation of SOSG. 25 μL of Proclin 950 is added to the sample, and then it is filtered to 0.2 μm using a Minisart filter. The incorporation of SOSG is calculated to be 1.8 moles per mole of streptavidin monomer.

[0139] Example 11 Fabrication of the substrate surface 2 A 200-μm-thick, 1-cm × 1-cm piece of pressure-sensitive adhesive 19 with a 6-mm-diameter hole cut out is adhered to a 22-mm × 22-mm cover glass 20 (Brand, catalog number 4700 55) to create a shallow well 21 as shown in Fig. 10.

[0140] Next, 30 μL of biotinylated BSA (10 μg / mL in 40 mM phosphate buffer) is placed in this well, incubated for 2 hours, and then washed away with wash buffer (40 mM phosphate, 2% sucrose, 0.9% NaCl, 0.03% BSA). Next, 30 μL of polystreptavidin-SOSG conjugate (10 μg / mL in 40 mM phosphate buffer) is added to the well, incubated for 60 minutes, and then washed 3 times with wash buffer. Next, 30 μL of biotinylated anti-TSH antibody 5409 (recognizing the junction between the α and β subunits of TSH, dissociation constant 50 pM) (2 μg / mL in 40 mM phosphate buffer) is incubated for 30 minutes, then washed 3 times with wash buffer, and air-dried at room temperature.

[0141] Example 12 Preparation of the pyropheophorbide-a conjugate of anti-TSH antibody 5407 Antibody 5407 (recognizing the β subunit of TSH, dissociation constant 180 pM) is supplied by Medix Biochemica, and pyropheophorbide-a (PPa) is supplied by Frontier Scientific (Logan, Utah). PPa is converted to the succinimidyl ester and conjugated to the antibody using the technique described in "Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments, Bhatti et al, Int. J. Cancer, 2008, 122, 1155". The molar incorporation of PPa is calculated to be 3.5 per mole of antibody.

[0142] Example 13 Generation of discrete fluorescence spots After reversing the substrate surface 17 and removing the adhesive release liner, attach the substrate to an acrylic sheet 22 that has two small holes 23 on one side of the substrate well as shown in the profile of FIG. 11.

[0143] Prepare a reaction mixture containing 5407-PPa antibody conjugate (10 ng / mL) and TSH-free whole blood supplemented with a defined concentration range (1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM) of TSH by serial dilution. After pipetting the mixture (approximately 6 μL) onto the substrate from one of the holes 23, seal the hole 23 with grease.

[0144] After incubating the chamber in the dark for 10 minutes, irradiate at 680 nm using a red LED with a total emission output of 15 mW. After 2 minutes of irradiation, obtain an image of the substrate surface using the optical setup described above and shown in FIG. 9. Discrete fluorescent regions where PPa has bound to the surface of the substrate are seen over the entire 2 minutes. TSH can be quantified when it exceeds the background signal at an estimated concentration of 50 femtomoles (3 times the standard deviation of the spot count in the analyte-free sample).

Claims

1. A method for detecting an analyte in a sample, comprising: (i) supplying to a device a mixture comprising the sample and a reporter reagent, said device comprising a substrate having an optical component and a binding component attached to the surface of said substrate; (ii) binding a proportion of said reporter reagent proportional to the concentration of said analyte to the surface of said substrate by means of said binding component; (iii) irradiating the device with electromagnetic radiation to cause the photosensitizer of the bound reporter reagent moiety to interact with said optical component, changing said optical component from a first optical state to a second optical state, thereby forming on said substrate a set of local regions of the optical component having the second optical state; and (iv) detecting the set of local regions on said substrate having the second optical state wherein the change from the first optical state to the second optical state is irreversible, and the local regions having the second optical state are counted as individual binding events. A method.

2. The method according to claim 1, wherein the absorption by the photosensitizer is for interaction with a preactivator reagent present in the mixture, thereby generating an activator reagent which interacts with said optical component to change it from the first optical state to the second optical state.

3. The method according to claim 2, wherein said activator reagent is a reactive oxygen species.

4. The method according to claim 3, wherein said reactive oxygen species is singlet oxygen.

5. The method according to any one of claims 1 to 4, wherein the set of local regions on said substrate having the second optical state is detected using an optical microscope.

6. The method according to any one of claims 1 to 5, wherein the optical component in the first optical state absorbs light at one or more wavelengths and the optical component in the second optical state absorbs light at one or more other wavelengths.

7. The method according to any one of claims 1 to 5, wherein the optical component in one of the first and second optical states is fluorescent and the optical component in the other of the first and second optical states is non-fluorescent.

8. The method according to any one of claims 1 to 7, wherein steps (i) to (iii) are performed without a washing step.

9. The method according to any one of claims 1 to 8, wherein the sample is untreated.

10. The method according to any one of claims 1 to 9, wherein the device is irradiated with electromagnetic radiation for more than 1 second.

11. The method according to any one of claims 1 to 10, wherein the binding component has a binding site capable of binding to the reporter reagent in proportion to the concentration of the analyte in the sample.

12. The method according to any one of claims 1 to 11, wherein the optical component in the first optical state is fluorescent and the optical component in the second optical state is non-fluorescent.

13. A device for detecting an analyte in a sample, comprising a substrate having an optical component and a binding component attached to the surface of the substrate, wherein the optical component changes from a first optical state to a second optical state in response to an interaction with an irradiated photosensitizer of a reporter reagent bound to the surface of the substrate in proportion to the concentration of the analyte in the sample, thereby forming a set of local regions of the optical component having the second optical state on the substrate, the change from the first optical state to the second optical state is irreversible, and the local regions having the second optical state are counted as individual binding events, device.

14. The device according to claim 13, comprising a cartridge, wherein the substrate is within the cartridge, and the device further comprises a detector for detecting a set of local regions having the second optical state on the substrate.

15. The device according to claim 13 or 14, wherein the binding component has a binding site capable of binding to the reporter reagent in proportion to the concentration of the analyte in the sample.

16. The device according to any one of claims 13 to 15, wherein the optical component in the first optical state is fluorescent and the optical component in the second optical state is non-fluorescent.

17. A system for detecting an analyte in a sample, comprising the device according to any one of claims 13 to 16; and a reporter reagent for forming a mixture comprising the sample, wherein the reporter reagent comprises the photosensitizer, wherein the photosensitizer can absorb electromagnetic radiation and interact with the optical component to change the optical component from a first optical state to a second optical state. A system comprising

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