Methods for detecting an analyte

Abstract

The present invention provides a method for detecting an analyte in a sample, the method comprising: (i) providing a mixture comprising the sample and a reporter reagent to a device, the reporter reagent comprising a photosensitizer, the device comprising a substrate having an optical component and a binding component, the optical component and the binding component attached to a surface of the substrate; (ii) allowing a portion of the reporter reagent to bind to the surface of the substrate by the binding component in proportion to the concentration of the analyte; (iii) irradiating the device with electromagnetic radiation for absorption by the optical component and absorption by the photosensitizer of the reporter reagent, such that the optical component absorbs the electromagnetic radiation to produce an excited optical component, and the photosensitizer of the reporter reagent bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the excited optical component to change the excited optical component from a first optical state to a second optical state, thereby forming a series of localized regions on the substrate having the second optical state; and (iv) detecting the series of localized regions on the substrate having the second optical state.

Description

[Technical field]

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

[0002] There are many techniques available for the measurement of 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 the target of interest, and a reporter reagent that has some kind of 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. The excess reporter can then be removed (by washing) and the amount of reporter reagent can be measured, thus determining the amount of analyte present in the sample. There are many different variations in which these types of binding assays can be performed. For example, the analyte can be first bound to the capture reagent and then the reporter can be added in a separate step, or the analyte can be first bound to the reporter and then bound to the capture reagent.

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

[0004] Assays that use antibodies are commonly called immunoassays. Immunoassays can take several formats. For example, when a capture antibody is used to capture the analyte and a reporter antibody is used to generate a measurable signal, it is commonly called a sandwich immunoassay. Alternative formats are known in which a binder is attached to a solid phase and the target analyte competes in solution with a labeled reagent that also binds to the binder. In the absence of analyte, a high level of the labeled reagent binds, so a high signal is obtained. In the presence of analyte, some of the binding sites are blocked, so less of the labeled reagent binds and the signal is reduced. These assays are commonly 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 analogue of the analyte) can compete for binding sites on the antibody. Alternatively, an analogue of the analyte can be immobilized and a labeled antibody can be bound to this surface. In the presence of analyte in the sample, which binds to the antibody in solution and prevents it from binding to the surface, the signal is reduced.

[0005] There are many formats for assays and many different types of labels that can be utilized. For example, the assay can be heterogeneous, in which case excess label is removed before the measurement is performed, for example, by using a washing step. Removal of excess label can also be achieved by flowing the sample and reporter over a capture area. This approach is used in immunochromatography or lateral flow strips, for example in rapid tests for infectious disease testing and pregnancy testing. Alternatively, homogeneous assays are known in which excess reporter is not removed. Homogeneous assays tend to rely on the proximity of capture and reporter to create some form of signal. One example of a homogeneous assay is an agglutination assay, in which particles bind together in solution. Agglutinated particles cause light scattering, which can be measured turbidimetrically or nephelometrically. A further example of a homogeneous assay using particles is the luminescent oxygen channeling immunoassay (LOCI), which is described in more detail below.

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

[0007] One homogeneous assay format that works in whole blood without removing cellular material is the thermo-optical immunoassay. Capture antibodies are coated onto a pyroelectric polyvinylidene PVDF sensor and carbon particles are used as reporters. A signal is generated by illuminating the sample with light, causing localized heating of the particles. Particles that are bound to the sensor transfer energy to the pyroelectric sensor, causing thermal stress that is detected as an electrical signal. The more carbon bound, the stronger the signal.

[0008] The label attached to the reporter binding agent can be a light absorbing agent such as a dye, gold particles or dyed latex microspheres. Larger particles in particular absorb more light and generate more signal. However, as will be further detailed below, there is a size limit at which particulate labels are not practical for use in assays. Luminescent labels are also known, such as fluorescent, chemiluminescent, bioluminescent and electrochemiluminescent labels. Luminescent labels have also been specifically encapsulated in particles in certain assays. Amplification of the signal can also be performed using enzymatic or catalytic reactions. Enzymes can be used to convert the substrate from a leuco dye to a colored form, or to a fluorescent or luminescent form. Excess enzyme is typically removed using a washing step before adding the substrate, so that a signal is generated only by the enzyme that specifically binds to the analyte.

[0009] Label-free immunoassays are also known, such as assays that use surface plasmon resonance as a signaling method, but label-free assays tend to lack 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 constraints regarding 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 to bind the analyte and the total amount of reporter antibody generating the signal. If the capture antibody is immobilized on a solid phase, the surface area of ​​the solid phase may limit the upper limit of detection. In addition, some signaling techniques are prone to saturation such as colorimetric methods depending on the path length that the light must travel through the sample. Luminescence methods are less prone to saturation because the amplification of the detector may be attenuated to handle higher levels of emission. When all of the antibody binding sites in a heterogeneous assay are filled with analyte, a maximum signal is achieved and the system is saturated. Excess analyte is usually removed in a wash step before adding the reporter. Homogeneous assays may also suffer from an effect known as high-dose hook, where the concentration of analyte is higher than the effective concentration of the capture and / or reporter antibodies. In this case, at very high concentrations, all of the binding sites on the capture and reporter may be blocked and the assay signal may be reduced, resulting in erroneous results.

[0013] Lower level detection depends on several different factors. In general, all assays are influenced by attributes such as the quality (affinity and specificity) of the antibody used and the cross-reactivity of the antibody with the relevant analyte. The lower limit of detection also depends on factors that affect the signal-to-noise ratio of the assay setup and system design. For example, in a standard enzyme-linked immunosorbent assay (ELISA), a capture antibody is coated onto the surface of a 96-well microtiter plate, and then the sample is incubated in the well, resulting in the analyte being captured. The well is washed, and then a reporter is added in excess, which binds to the captured analyte. The excess reporter is then washed away, and a substrate is added that can react with the enzyme and convert it to its active form. For example, a non-colored leuco dye such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) can be converted with horseradish peroxidase to its oxidized substrate form under hydrogen peroxide. If the analyte is present in very low amounts (e.g., less than 1 picomole), only very small amounts of enzyme bound to the surface of the well are present. ABTS reacts with the enzyme to produce a substrate form that then diffuses into the bulk of the fluid, producing a solution that is so dilute that it cannot be distinguished from the background signal. Autoconversion of the substrate can also produce colors that interfere with the measurement. Similarly, other detection methods such as fluorescence can suffer from interferences and autofluorescence of components in the sample or reaction well.

[0014] Another confounding factor in immunoassays can be non-specific binding of the reporter reagent to the capture surface. For example, in the ELISA assay described above, the microtiter wells are coated with a layer of protein, some of which may be denatured during the coating process. It is not uncommon for the reporter to bind to a region of the capture surface during the assay. If this reporter is located on the substrate and contributes to the overall signal, it is not possible to distinguish the signal due to the specifically bound reporter from that which is non-specifically bound. Non-specific binding can also be promoted by many of the components present in the original sample that can bind to the capture surface during the initial incubation and modify the surface properties of the capture layer, creating a surface that can bind the reporter. Minimizing non-specific binding of the reporter involves careful optimization of all reagents and reaction conditions used during the assay, including antibodies, detergents, temperature and ionic strength.

[0015] In general, the detection limit of traditional immunoassays is about 0.1 picomolar to 1 nanomolar depending on the assay methodology. Developing an assay with a very low detection limit using traditional approaches often requires extensive optimization involving rigorous washing steps to mitigate non-specific binding and maximize signal-to-noise. In addition, the capture surface is often small compared to the sample volume to ensure that the signal is sufficiently high over the background.

[0016] One approach used to circumvent the signal-to-noise problem and improve detection limits is to measure individual binding events and count these as "on" or "off" events depending on whether the measurement exceeds a local threshold. This allows much of the background noise to be eliminated. An analogy can be drawn with the digitization of speech or communication signals. These digital assays have been shown to reach detection limits previously unattainable using traditional analog methods. For example, low femtomolar (10 -15 mol / L) or even attomoles (10 -18Limits of detection in the range of 1000 to 1000 mol / L have been reported. See, e.g., "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] Most of the labels / reporters used in the assays (e.g. fluorophores, dyes, etc.) are not individually visible using wide-field microscopes even under high magnification, since their size is below the diffraction limit of visible light. Therefore, the presence of these labels is only measured as a bulk phenomenon, not by counting each label. In contrast, certain labels, such as latex particles, can theoretically be visualized by wide-field optical microscopes if they are larger than a certain size. Depending on the optical settings, it is possible to start to visualize particles when they are several hundred nanometers or larger in diameter, depending on the numerical aperture and type of microscope.

[0018] However, using particles of this size as labels to monitor individual binding events (e.g. antibody-antigen interactions) at capture surfaces is not practical for several reasons. For example, particles of this size diffuse very slowly compared to other types of labels, inhibiting the kinetics of reactions on flat surfaces. They also begin to exhibit macroscopic buoyancy effects, floating or sinking if the density of the particles differs significantly from the medium in which they are contained, which can also cause problems with the assay format. Particles of this size are particularly prone to non-specific binding to surfaces, resulting in high background that is difficult to remove. Eventually, excess particles must be removed, necessitating a washing step. However, large particles begin to experience shear effects in the presence of fluid flow, and the shear forces on the particles are greater than the disruptive strength of antibody-antigen interactions (approximately 60-250 pN), causing the particles to be washed away (see "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 Molecule Array (SIMOA) system and the Singulex Single Molecule Counting (SMC) system.

[0020] The Quanterix SIMOA system uses antibody-coated paramagnetic beads to capture the analyte from solution. The magnetic beads are then washed and an enzyme-labeled reporter antibody 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 loaded into an array of microwells that can hold only one bead per well. The volume of the microwells is on the femtoliter scale. When the beads are enzyme-attached, the fluorogenic substrate in the well is turned upside down. The small dimensions of the wells prevent the fluorescent product from diffusing too far. Each well is then counted as an "on" or "off" event if the fluorescence is higher than a threshold value.

[0021] The SMC system is used in Singulex transparency instruments, and in the Erenna and SMCxPRO systems manufactured by Merck Millipore. The basic measurement technology in all three systems is the same. Magnetic beads coated with a capture antibody are used to capture the target analyte in a sandwich assay. A fluorescently labeled reporter antibody also binds to the beads in the presence of analyte. The beads are pulled down with a magnet and excess fluorescently tagged reporter is washed away. An elution buffer is then added which causes dissociation of the sandwich complex, which is then transferred to the measurement vessel. The presence of the fluorescent tag is then measured using a confocal fluorescence microscope which sequentially probes small volumes of the sample to determine whether the fluorescent tag is present. If the signal for each individual measurement is greater than a threshold value, this is counted as an "on" event for that measurement.

[0022] Several independent scholarly reviews of high-sensitivity immunoassays have highlighted that digital approaches to immunoassays enable unexpected improvements to limits of detection (see references by Yeung and Cretich above).

[0023] The detection limit for the Quanterix and Singulex systems depends on the volume of sample used in the assay. For a 10 microliter serum or plasma sample, the theoretical limit is the detection of a single binding event, which corresponds to one molecule. However, in terms of molar concentrations, this corresponds to 100,000 molecules per liter of sample, or 0.16 × 10 per liter. -18 mole (0.16 attomole).

[0024] However, the Quanterix and Singulex systems mentioned above are complex and cumbersome, each requiring several washing and transfer steps. Furthermore, the assays can only be performed on samples that do not contain cellular material, and the systems require expensive equipment to achieve the performance offered. Thus, there is a need for a simpler, more cost-effective, highly sensitive system.

[0025] WO 2020 / 260865 describes a digital assay method that uses a photosensitizer reagent to convert an optical component from a first optical state to a second optical state. However, there remains a need for optimization / improvement of this conversion. Summary of the Invention

[0026] Accordingly, the present invention provides a method for detecting an analyte in a sample, comprising the steps of: (i) providing a mixture comprising a sample and a reporter reagent to a device, the reporter reagent comprising a photosensitizer, the device comprising a substrate having an optical component and a binding component, the optical component and the binding component attached to a surface of the substrate; (ii) allowing a portion of the reporter reagent to bind to the surface of the substrate by the binding moiety in proportion to the concentration of the analyte; (iii) illuminating the device with electromagnetic radiation for absorption by the optical moieties and by the photosensitizer of the reporter reagent, such that the optical moieties absorb the electromagnetic radiation to produce excited optical moieties, and the photosensitizer of the reporter reagent bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the excited optical moieties to change the excited optical moieties from a first optical state to a second optical state, thereby forming a series of localized regions on the substrate having the second optical state; (iv) detecting a set of localized regions on the substrate having a second optical state; The present invention provides a method comprising:

[0027] Thus, the present invention provides a method for detecting an analyte in a sample, where only reporter reagents proximate the surface of the substrate provide a signal, the signal being a localized region of the optical component in a second optical state. It is a set of localized regions of the optical component in the second optical state that are detected. Thus, the present invention simplifies the digital detection of analytes and aids in homogeneous assays for a range of samples, including those containing cellular material.

[0028] The inventors have surprisingly found that conversion of the optical moiety from a first optical state to a second optical state is more effective when the optical moiety is also excited with electromagnetic radiation. Specifically, conversion of the optical moiety occurs more rapidly when both the reporter reagent and the optical moiety are excited with electromagnetic radiation.

[0029] In addition, the intensity of electromagnetic radiation required for the conversion is reduced, making it possible to create distinct localized regions having a second optical state that are larger in size, reducing the complexity of the optical setup required to visualize these regions.

[0030] The present invention will now be described with reference to the drawings. [Brief description of the drawings]

[0031] [Figure 1]FIG. 1 illustrates various components that may be used in the methods of the present invention. [Diagram 2] FIG. 1 shows a device in which a reporter reagent is bound to the surface of a substrate prior to irradiation. [Diagram 3] FIG. 3 shows the device of FIG. 2 being illuminated. [Figure 4] FIG. 4 shows the device of FIG. 3 after irradiation. [Diagram 5] FIG. 1 shows a representative series of localized regions having a second optical state on a substrate, visible as bleached dark areas in the fluorescent layer. [Figure 6] FIG. 1 shows an optical setup for detection in which two electromagnetic radiation sources can be focused through an objective lens. [Figure 7] FIG. 1 shows a substrate and wells created for the method of the present invention. [Figure 8] FIG. 8 shows a sample chamber created using the substrate and well of FIG. 7. [Figure 9] 1 is a graph showing the rate of spot formation in a streptavidin AlexaFluor594 layer as a function of time and illumination source as described in Example 4. [Figure 10] 1 is a graph showing the rate of spot formation in a streptavidin Oregon Green 488 layer as a function of time and illumination source as described in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The methods of the invention are used to detect an analyte in a sample (which may be done through detection of a complex or derivative of the analyte).

[0033] The components in FIG. 1 are: photosensitizer 1; antibody-coated latex particles infused with photosensitizer 2 (hereinafter also referred to as photosensitizer-labeled antibody 2); antibody 3; fluorescent state optical component 4; non-fluorescent state optical component 5; streptavidin 6, streptavidin labeled with the fluorescent state optical component 7 (hereinafter also referred to as streptavidin-dye conjugate 7); streptavidin labeled with the non-fluorescent state optical component 8; and biotinylated polylysine 9 (hereinafter also referred to as polylysine-biotin conjugate 9).

[0034] Step (i) of the method of the invention involves providing a mixture comprising a sample and a reporter reagent to a device, where the reporter reagent comprises a photosensitizer, and where the device comprises a substrate 11 having an optical component and a binding component, the optical component and the binding component being attached to a surface of the substrate 11. The sample and reporter reagent may be pre-mixed prior to adding the mixture to the device, or the sample and reporter reagent may be added to the device sequentially to form the mixture. The mixture may also contain further reagents, but preferably the mixture consists of the sample and the reporter reagent.

[0035] For the purpose of illustrating the principles underlying the present invention, FIG. 2 shows a device in which a reporter reagent is bound to the surface of a substrate prior to illumination. The device includes a substrate 11 and a sample chamber 10 for holding a sample containing a dissolved or suspended analyte. The substrate can be any substrate that allows for the detection of a series of localized regions having a second optical state on the substrate. Preferably, the substrate is planar. Preferably, the substrate is a transparent substrate, more preferably, the substrate is glass or plastic.

[0036] Substrate 11 has an antibody 3 bound to a streptavidin-dye conjugate 7 attached to the surface of substrate 11 via a polylysine-biotin conjugate 9. The dye acts as the optical moiety and the antibody acts as the binding moiety. The polylysine-biotin conjugate 9 is an inert macromolecule that aids in the attachment of the optical and binding moieties to the surface of substrate 11.

[0037] Although the optical and binding components are hereby illustrated, any technique for holding the optical and binding components in close proximity to the surface of the substrate 11 is applicable. For example, the optical and binding components can be a single reagent. The optical components can also be encapsulated within a polymer layer that is coated on the surface of the substrate 11, and the binding components can be 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 components.

[0038] Alternatively, a gel layer, for example a hydrogel layer, can be impregnated with the optical components, the gel / hydrogel layer can be coated onto the surface of substrate 11, and the binding components can be attached to the gel / hydrogel layer.

[0039] Step (ii) of the method of the invention involves allowing a proportion of the reporter reagent to bind to the surface of the substrate by the binding moiety in proportion to the concentration of analyte, which can be achieved by allowing the device to sit for a period of time, for example 10 minutes.

[0040] In Figure 2, photosensitizer-infused antibody-coated latex particles 2 are bound to the surface of a substrate 11 by antibody 3. For example, antibody 3 can be a mouse antibody and the antibody on the photosensitizer-infused antibody-coated latex particles 2 can be an anti-mouse antibody. The photosensitizer-infused antibody-coated latex particles 2 act as a reporter reagent.

[0041] The reporter reagent is hereby shown to be bound to the surface of the substrate 11; if the analyte is present, the reporter reagent will bind to the surface of the substrate in proportion to the concentration of the analyte. For example, if the binding moiety and reporter reagent are antibodies and the analyte is an antigen, the reporter reagent will bind to the binding moiety via the analyte to form a so-called "sandwich" complex. Other binding events such as antibody-hapten binding or nucleic acid binding are also possible.

[0042] All previous steps are performed in the absence of light. Step (iii) of the method of the present invention comprises irradiating the device with electromagnetic radiation for absorption by the optical moieties and by the photosensitizer of the reporter reagent, such that the optical moieties absorb the electromagnetic radiation to generate excited optical moieties, and the photosensitizer of the reporter reagent bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the excited optical moieties to change the excited optical moieties from a first optical state to a second optical state, thereby forming a series of localized regions having the second optical state on the substrate 11.

[0043] The electromagnetic radiation (typically referred to as "light") used to illuminate the device contains excitation wavelengths for both the optical moiety and the photosensitizer of the reporter reagent.

[0044] The use of electromagnetic radiation for absorption by the optical moiety and for absorption by the photosensitizer provides significant advantages over previous assays, where the device is only illuminated with electromagnetic radiation for absorption by the photosensitizer, in which case the change of the excited optical moiety from a first optical state to a second optical state occurs more quickly and more efficiently, and also produces a larger localized area in the second optical state. This allows assay results to be obtained in a shorter time and / or a lower intensity of light can be used to achieve the same results. Furthermore, a broadband light source can be used without the need for filters to remove undesirable portions of the electromagnetic spectrum, meaning that simpler equipment can be used.

[0045] Without wishing to be bound by theory, it is believed that the photosensitizer of the reporter reagent bound to the surface of the substrate absorbs electromagnetic radiation and interacts with the excited state of the optical moiety more rapidly due to the optical moiety being in a higher energy state.

[0046] In a preferred embodiment, the electromagnetic radiation for absorption by the optical moiety and the photosensitizer of the reporter reagent has the same wavelength. For example, the electromagnetic radiation used to illuminate the device can be of the excitation wavelength of both the optical moiety and the photosensitizer of the reporter reagent. The electromagnetic radiation used to illuminate the device can also be of a longer wavelength than the excitation wavelength of the photosensitizer of the reporter reagent and the optical moiety, for example, broad spectrum electromagnetic radiation.

[0047] When the electromagnetic radiation for absorption by the optical component and for absorption by the photosensitizer of the reporter reagent have the same wavelength, the device is irradiated with electromagnetic radiation for absorption by the optical component and for absorption by the photosensitizer of the reporter reagent simultaneously using one source of electromagnetic radiation.

[0048] The use of the same wavelength of electromagnetic radiation for absorption by the optical moiety and by the photosensitizer of the reporter reagent provides a simpler and more cost-effective method.

[0049] Alternatively, in a preferred embodiment, the electromagnetic radiation for absorption by the optical moiety and the photosensitizer of the reporter reagent have different wavelengths. For example, the electromagnetic radiation can consist essentially of the excitation wavelength of both the photosensitizer and the optical moiety of the reporter reagent. By "consist essentially of" we mean the narrowest possible window around the desired wavelength.

[0050] The use of different wavelengths of electromagnetic radiation for absorption by the optical moiety and for absorption by the photosensitizer of the reporter reagent provides a more controllable method.

[0051] In this regard, the electromagnetic radiation can originate from two or more sources.

[0052] When using two sources of electromagnetic radiation, one source for absorption by the optical component and the other source for absorption by the photosensitizer of the reporter reagent, the sources can be focused through the objective lens simultaneously. Thus, in a preferred embodiment, the electromagnetic radiation for absorption by the optical component and absorption by the photosensitizer of the reporter reagent arrives at the device from one direction, more preferably through one objective lens.

[0053] The electromagnetic radiation for absorption by the optical component and the photosensitizer of the reporter reagent can also reach the device from different directions. The electromagnetic radiation for absorption by the optical component can be focused through an objective lens, and the electromagnetic radiation for absorption by the photosensitizer does not have to be focused through an objective lens. Thus, in a preferred embodiment, the electromagnetic radiation for absorption by the optical component and the electromagnetic radiation for absorption by the photosensitizer of the reporter reagent reach the device from different directions, and more preferably, the electromagnetic radiation for absorption by the optical component reaches the device through an objective lens, and the electromagnetic radiation for the photosensitizer does not reach the device through an objective lens.

[0054] If the electromagnetic radiation for absorption by the optical moiety and for absorption by the photosensitizer of the reporter reagent have different wavelengths, the device may be irradiated with electromagnetic radiation for absorption by the photosensitizer of the reporter reagent and for absorption by the optical moiety simultaneously. Alternatively, the device may be irradiated with electromagnetic radiation for absorption by the optical moiety and for absorption by the photosensitizer of the reporter reagent sequentially. Thus, in a preferred embodiment, the device is irradiated with electromagnetic radiation for absorption by the optical moiety and for absorption by the photosensitizer of the reporter reagent simultaneously, or the device is irradiated with electromagnetic radiation for absorption by the optical moiety and for absorption by the photosensitizer of the reporter reagent sequentially.

[0055] When the device is sequentially irradiated with electromagnetic radiation for absorption by the optical component and by the photosensitizer of the reporter reagent, the optical component and the photosensitizer of the reporter reagent can be irradiated in either order, as long as the photosensitizer of the reporter reagent that has absorbed electromagnetic radiation can interact with the excited optical component. This may depend on the lifetime of the excited optical component. However, preferably, the device is first irradiated with electromagnetic radiation for absorption by the photosensitizer of the reporter reagent, and then with electromagnetic radiation for absorption by the optical component. This ensures that the photosensitizer of the reporter reagent that has absorbed electromagnetic radiation can interact with the excited optical component.

[0056] Figure 3 shows the device of figure 2 illuminated with electromagnetic radiation, preferably visible light. The light source can be, for example, an LED 12. The light source illuminates the sample chamber 10 with light of a wavelength suitable for exciting the photosensitizer 2 and the dye component of the streptavidin-dye conjugate 7. The wavelength depends on the photosensitizer and the dye. The device is typically illuminated for at least 10 seconds. Preferably, the device is illuminated with electromagnetic radiation 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 change at the surface of the substrate, thus making it possible to distinguish between long-lived and transient binding events.

[0057] Figure 4 shows the device of Figure 3 after illumination. Photosensitizer 1 interacts with the illuminated dye optical moiety in streptavidin-dye conjugate 7, causing the dye to change from a fluorescent to a non-fluorescent state. The fluorescent state of streptavidin-dye conjugate 7 becomes the non-fluorescent state of streptavidin-dye conjugate 8. Only the dye in proximity to photosensitizer 1 changes from the first optical state to the second optical state.

[0058] The alternative first and second optical states may include changes in polarization, fluorescence lifetime, refractive index, light scattering (including Raman scattering), phosphorescence and other optical effects.

[0059] While Figures 2-4 illustrate one reporter reagent bound to the surface of the substrate 11, in reality multiple reporter reagents will be bound to the surface of the substrate 11 to create two or more localized regions on the substrate having a second optical state. Using the components of Figure 1, these localized regions on the substrate having a second optical state are visualized as distinct regions of the streptavidin-dye conjugate 8 in a non-fluorescent state. Such distinct regions can be seen in Figure 5, which shows a representative example of a series of localized regions on the substrate having a second optical state that are visible as bleached dark regions in the fluorescent layer.

[0060] Any photosensitizers in the vicinity of the excited optical component will interact with and change the excited optical component from a first optical state to a second optical state, whereby photosensitizers of the reporter reagent bound to the surface of the substrate will interact with the excited optical component and change the excited optical component from a first optical state to a second optical state, thereby forming a series of localized regions on the substrate 11 having a second optical state.

[0061] 2-4 also show the reporter reagent bound to the surface of substrate 11 via a binding moiety. However, when measuring analyte concentration, binding to the surface is mediated by the analyte, for example in a "sandwich" assay.

[0062] In addition, excess particles not bound to the surface may be suspended in the sample. If the sample is whole blood, the red blood cells are suspended in the medium. Preferably, the substrate 11 forms the upper portion of the sample chamber 10, allowing the red blood cells to settle and be removed from the substrate 11.

[0063] A percentage of the reporter reagent is bound to the surface of the substrate 11 by the binding moiety. Thus, the sample contains bound reporter reagent and unbound reporter reagent that is free in solution. The depth of the sample chamber 10 is designed to minimize the diffusion path length of the reporter reagent, allowing equilibrium to be reached quickly. Typically, the depth of the sample chamber is 50-200 μm.

[0064] In the method of the present invention, the sample chamber 10 is filled with a sample containing the analyte. A reporter reagent, such as an antibody 2 labeled with a photosensitizer, is also added to the sample chamber 10. If a whole blood sample is used, the sample may also contain further components, such as red blood cells.

[0065] Equilibrium is then reached. A reporter reagent, such as antibody 2 labeled with a photosensitizer, is bound to the surface of substrate 11 by a binding moiety, such as antibody 3, in proportion to the concentration of the analyte. Since an excess of reporter reagent is included, a significant proportion of the analyte forms a sandwich complex. Thus, a proportion of the reporter reagent is bound to the surface of substrate 11 by a binding moiety in proportion to the concentration of the analyte. The sample thus contains bound reporter reagent and unbound reporter reagent free in solution.

[0066] After irradiation, the photosensitizer interacts with the excited optical component, causing it to change from a first optical state to a second optical state. For example, streptavidin 7 labeled with a fluorescent optical component becomes streptavidin 8 labeled with a non-fluorescent optical component. Only the optical component in close proximity to the photosensitizer changes from the first optical state to the second optical state.

[0067] The reporter reagent must be permanently bound to the surface of substrate 11 for the entire period of illumination to achieve complete conversion from the first optical state to the second optical state. If the reporter reagent is only transiently bound to the surface for a portion of the illumination period, conversion to the second optical state will be incomplete and can be detected in the algorithm used to measure the size, shape and intensity of the distinct regions. Any unbound reporter reagent in solution will not significantly change the optical components from the first optical state to the second optical state.

[0068] This provides a significant advantage over other digital assay methods in that it removes the need for a washing step. This makes the method of the present invention a homogeneous assay. In conventional assays, unbound reporter reagent must be separated from bound reporter reagent before any measurement is made, as unbound reporter reagent will interfere with the signal generated by the bound reporter reagent. However, due to the localized surface changes provided by the present invention, it is possible to distinguish between bound and unbound reporter reagent. Indeed, the ability to distinguish between reporter reagent in close proximity to the surface of the substrate 11 (i.e., bound) and reporter reagent in bulk solution (i.e., unbound) is a particular advantage of the present invention. Preferably, steps (i)-(iii) are performed in the absence of a washing step, i.e., the method is carried out without removing the sample from the substrate in steps (i), (ii) and (iii).

[0069] The photosensitizer of the reporter reagent bound to the surface of the substrate may interact directly with the optical component to cause a change (e.g., the photosensitizer excites and transfers this energy directly to the excited optical component) or may interact indirectly with the optical component to cause a change via an additional reagent (e.g., the photosensitizer excites and transfers this energy to an additional component, which then transfers this energy to the excited optical component).

[0070] In a preferred embodiment, the photosensitizer of the reporter reagent absorbs electromagnetic radiation and interacts with a pre-activator reagent present in the mixture to generate an activator reagent that interacts with the excited optical component, changing the excited optical component from a first optical state to a second optical state. Thus, the reporter reagent is capable of generating an activator reagent from a pre-activator reagent present in the mixture upon absorption of electromagnetic radiation, and the excited optical component is capable of changing from a first optical state to a second optical state upon interaction with the activator reagent.

[0071] The preactivator reagent may be present in the sample or may be added as an additional reagent to the mixture of the sample and the reporter reagent. In a preferred embodiment, the preactivator reagent is triplet oxygen in the ground state. In another preferred embodiment, the activator reagent is a reactive oxygen species (ROS). Preferably, the ROS is selected from hydroxyl radicals, superoxides, peroxides, organic peroxides, peroxynitrite, singlet oxygen and mixtures thereof. More preferably, the ROS is singlet oxygen. Singlet oxygen is a preferred activator reagent because it has a short half-life and a finite diffusion path length (usually less than 1 micron under aqueous conditions).

[0072] It is believed that the photosensitizer absorbs light to generate singlet oxygen present in the sample, creating an excited state capable of intersystem crossing (ISC) with oxygen in close proximity to the reporter reagent. The singlet oxygen then proceeds to interact with the excited optical moiety as described below. In a particularly preferred embodiment, the preactivator reagent is ground state triplet oxygen and the activator reagent is singlet oxygen.

[0073] In the embodiment in which the preactivator reagent is converted to an activator reagent, if the activator reagent has a finite lifespan, there will be a short time after the irradiation of the photosensitizer that the activator reagent is still in the vicinity of the binding event, depending on the half-life of the activator reagent.Therefore, the irradiation of the photosensitizer and the optical component may be slightly time-shifted.In the extreme case, the photosensitizer can be first illuminated to generate the activator reagent, and then the optical component can be illuminated subsequently for the lifespan of the activator reagent.

[0074] Singlet oxygen has been used previously in immunoassays such as luminescent oxygen channeling immunoassay (LOCI). LOCI immunoassays are homogeneous non-digital assays that use donor and acceptor beads to measure bulk events. The donor beads generate singlet oxygen upon illumination at 680 nm, and the acceptor beads generate a chemiluminescent signal when activated with singlet oxygen. Binding of the donor beads to the acceptor beads is driven by antibody-antigen binding. The reaction mixture is typically illuminated for 0.5-1.0 s, and then the luminescent signal is measured for 0.5-1.0 s. Importantly, measurements are performed in the presence of all unbound donor and acceptor beads. Although the spatial separation of the beads minimizes background signal, the short measurement period of the LOCI assay does not allow for distinguishing between long-lived and transient binding events. The assay can achieve detection limits of about 1-5 pg / mL for its most sensitive assays, e.g., interleukin 6 (IL-6) or thyroid stimulating hormone (TSH). The method of the present invention further minimizes background signal because the reporter reagent, the "donor" particle, must be in close proximity to the surface of the substrate 11, rather than a particle in solution, for a signal to be detected, and must be present there for the duration of illumination to generate a signal above (or below) the threshold criteria used in the image analysis software. Thus, the method of the present invention is more sensitive and can detect analytes at lower concentrations than LOCI assays.

[0075] In a preferred embodiment, the optical moiety is a dye. Preferably, the optical moiety is selected from one of the following fluorescent dyes and mixtures thereof:

[0076] [ka]

[0077] Dyes (1), (2) and (3) are common organic fluorophores that can be attached to proteins and other macromolecules by attachment to an N-hydroxysuccinimide group, which reacts with amine groups to form covalent amide bonds. These dyes, along with a wide range of other fluorophores, can be irreversibly converted to a non-fluorescent state by the methods of the present invention.

[0078] Preferably, 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. In one embodiment, the optical component in the first optical state is fluorescent and the optical component in the second optical state is non-fluorescent, or the optical component in the first state is non-fluorescent and the optical component in the second optical state is fluorescent. However, more preferably, the optical component in the first optical state is fluorescent and the optical component in the second optical state is non-fluorescent.

[0079] Preferably, the change from the first optical state to the second optical state is irreversible. This allows for subsequent scanning of the substrate to identify areas where a change in optical state has taken place.

[0080] Step (iv) of the method of the present invention comprises detecting a series of localized regions on the substrate having a second optical state. Step (iv) is preferably performed by image analysis software capable of distinguishing real binding events from transient binding events and surface artifacts by analyzing surface brightness and morphology images. Step (iv) can be a separate step performed after the optical component transforms from the first optical state to the second optical state. Alternatively, step (iv) can be performed during the transformation to the second optical state.

[0081] Any method that can successfully identify individual binding events can be used, for example, an initial image of the surface can be obtained using first an excitation wavelength of the optical moiety, followed by illumination with electromagnetic radiation that excites both the photosensitizer and the optical moiety, followed by a final step of illumination with only the excitation wavelength of the optical moiety.

[0082] Alternatively, it may be possible to capture a series of images (video files) of the surface while irradiating it with electromagnetic radiation that excites both the optical component and the photosensitizer simultaneously. Any combination of optical filters, dichroic mirrors and illumination sequences that allows one to identify binding events is acceptable.

[0083] The second optical state forms a series of localized regions on the substrate. Advantageously, the localized regions with the second optical state can be counted as individual binding events. Therefore, the method of the present invention is suitable for carrying out digital assays. However, when there are a large number of binding events in which the majority of optical components are in the second optical state, a large change is detected.

[0084] In a preferred embodiment, the set of local regions on the substrate having the second optical state is detected by counting local regions in the set of local regions on the substrate having the second optical state or by measuring the set of local regions on the substrate having the second optical state as a bulk property. More preferably, the set of local regions on the substrate having the second optical state is detected by counting local regions in the set of local regions on the substrate having the second optical state.

[0085] The localized region having the second optical state may need to be greater than a threshold to represent background signal depending on the first and second optical states, for example, a sample may have some background autofluorescence, but if this is not greater than a threshold it should not affect the signal due to the digital nature of the assay.

[0086] A series of local regions having a second optical state on a substrate are typically separate regions on the substrate. However, some local regions can be excluded from detection due to their morphological image. The local regions corresponding to individual binding events tend to be uniformly circular, while some local regions may be irregular in shape, which corresponds to artifacts. Furthermore, some local regions may be larger than others when particles are aggregated together, or smaller when only transient binding events occur. Therefore, in a preferred embodiment, a uniformly circular local region having a second optical state on a substrate is detected.

[0087] A series of localized regions on a substrate can be detected using simple optical means. In a preferred embodiment, a series of localized regions on a substrate having a second optical state are detected using an optical microscope. A suitable optical setup for detection is shown in FIG. 6. More preferably, a series of localized regions on a substrate having a second optical state are detected using a wide-field microscope. Wide-field microscopes are the simplest microscopes, as they illuminate and image the entire sample simultaneously, compared to more complex techniques such as confocal microscopes, which only illuminate a single focus and record in time. The advantages of confocal microscopes are the high contrast due to the elimination of out-of-focus haze, and the ability to obtain stacks of images through the depth of the sample. Super-resolution microscopy techniques are also known, such as photoactivated localization microscopy (PALM or FPALM) and stochastic optical reconstruction microscopy (STORM). These methods add complexity and are more expensive than simple wide-field methods.

[0088] To detect a series of localized regions on a substrate, a wide-field fluorescence microscope equipped with a light source (e.g., an LED) and a photodetector (e.g., a photomultiplier tube or a camera, e.g., a CCD) can be used with excitation and emission filters suitable for detection of specific optical components.

[0089] The photosensitizer is in close proximity to the substrate when a binding event occurs. That is, the photosensitizer is close enough to the surface of the substrate to interact with the excited optical component and convert it from a first optical state to a second optical state upon irradiation of the device. However, the actual distance between the photosensitizer and the surface of the substrate depends on several variables, such as the size and nature of the photosensitizer, the size and nature of the binding component, the reporter reagent and the analyte, and the nature of the sample medium.

[0090] The binding component has binding sites capable of binding to the reporter reagent in proportion to the concentration of the analyte in the sample. Proportionality is important to the function of the assay since binding must depend on the concentration of the analyte for any meaningful measurement of the concentration of the analyte to be determined. Binding may be directly proportional or indirectly proportional to the concentration of the analyte depending on the type of assay being performed. In non-competitive assays, such as immunometric assays, binding is directly proportional to the concentration of the analyte, whereas in competitive assays, binding is indirectly proportional to the concentration of the analyte.

[0091] We present one particular type of competitive assay in which an antibody against an analyte is immobilized on a substrate and a labeled analog of the analyte is introduced into the sample. The analyte and the labeled analog of the analyte then "compete" for the antibody on the surface. In the absence of analyte, the labeled analog then binds at the maximum possible rate. However, in the presence of analyte, the antibody on the substrate is filled with analyte and the rate of binding of the analog decreases.

[0092] The binding moiety can be adjusted to bind to the analyte or a complex or derivative of the analyte, where the reporter reagent binds to the binding moiety in the presence of the analyte or a complex or derivative of the analyte, where the binding moiety has a binding site that allows it to bind to the reporter reagent in the presence of the analyte or a complex or derivative of the analyte, but where binding remains proportional to the concentration of the analyte.

[0093] Alternatively, the binding moiety may itself be an analog of the analyte, and the reporter reagent binds directly to the binding moiety (which is an analog because it is bound to the surface of the substrate through either covalent or non-covalent interactions). In this case, the binding moiety competes with unbound analyte or an unbound complex or derivative of the analyte for binding of the reporter reagent. Thus, the binding moiety is simply allowed to bind to the reporter reagent.

[0094] Determining the extent of binding of the reporter reagent to the binding moiety (either direct or mediated by an analyte / analyte complex or derivative) measures the concentration of the analyte in the sample. Typically, the system is pre-calibrated at the time of manufacture to generate a calibration curve that is used to convert the instrument signal to the analyte concentration measured in the sample.

[0095] The assay also requires the presence of a reporter reagent. The reporter reagent comprises a photosensitizer. The photosensitizer absorbs electromagnetic radiation allowing it to interact with the excited optical component, an interaction that changes the excited optical component from a first optical state to a second optical state.

[0096] Thus, the photosensitizer may consist of any substance by which it is possible to interact with electromagnetic radiation. Suitable photosensitizers are known as PDT agents from photodynamic therapy (PDT). PDT agents are used in cancer therapy and dermatology, destroying cells upon illumination (Shafirstein et al., Cancers, 2017, 9, 12; Wan and Lin, Clinical, Cosmetic and Investigational Dermatology, 2014, 7, 145).

[0097] Upon irradiation with electromagnetic radiation, the PDT agent is promoted to an excited triplet state. This excited triplet state can either interact directly with cellular components (called the type I process) or with oxygen (called the type II process). Both type I and type II processes can result in the formation of ROS. In the type II process, the predominant product is singlet oxygen through the mechanism of intersystem crossing.

[0098] Singlet oxygen is an excited state of oxygen that is highly reactive. It can undergo several reactions before decaying, including the Diels-Alder and ene reactions. It also undergoes common oxidation reactions with sulfur- and nitrogen-containing compounds. The promiscuous reactivity of singlet oxygen is one of the reasons it is used in photodynamic therapy.

[0099] A wide range of photosensitizer compounds are known, including porphyrins, chlorins (e.g., pyropheophorbide-a), phthalocyanines and other polyaromatic species (see, e.g., Antibody-Directed Phototherapy, Pye et al., Antibodies, 2013, 2, 270).

[0100] In one embodiment, the photosensitizer is selected from porphyrins, chlorins, phthalocyanines and other polyaromatic species. In a preferred embodiment, the photosensitizer is a silicon phthalocyanine derivative, such as the examples shown below.

[0101] [ka]

[0102] The nature of the binding moiety and the reporter reagent depends on the nature of the analyte, but is preferably an antibody. The method of the present invention has particular applicability to immunoassays. In a particularly preferred embodiment, the binding moiety is an antibody raised against the analyte or a complex or derivative of the analyte, and the reporter reagent comprises an antibody raised against the analyte or a complex or derivative of the analyte. Essentially, a single molecule may be used for each reagent, but in practice the binding moiety and the reporter reagent are a collection of molecules. The term "antibody" preferably includes within its scope Fab fragments, single chain variable fragments (scFv), and recombinant binding fragments.

[0103] As an alternative to antibody-antigen reactions, the binding moiety, reporter reagent and analyte can be a first and a second nucleic acid, which can be complementary or a reagent containing avidin or a derivative thereof and an analyte containing biotin or a derivative thereof, or vice versa. The binding moiety and reporter reagent can also be aptamers. The system is also not limited to biological assays, and can be applied to, for example, the detection of heavy metals in water. The system also does not need to be limited to liquids, and can be used in any fluid system, such as the detection of enzymes, cells and viruses in air.

[0104] The maximum observable signal is the maximum signal that can be achieved when monitoring photosensitizer binding to a surface. Binding of particles to a substrate depends on the diffusion rates of the analyte and reporter reagents, which in turn depend strongly on the hydrodynamic radii of these components and the viscosity / temperature of the sample.

[0105] The device used in the method of the present invention may further include controls that compensate for natural variability in the components of the measuring system, variability in the sample being measured, and variability under environmental conditions during the measurement. This can be achieved by exposing the sample to a reagent on the surface of the substrate. Different reagents are typically located in different areas of the surface of the substrate, and these areas are coated with different reagents. These controls are defined as "negative" and "positive" controls, meaning that the negative control should approximate the expected signal in the absence of analyte, and the positive control should approximate the expected signal when analyte fills the system.

[0106] To achieve detection of these controls, the devices of the invention preferably include a binding moiety, a negative control reagent and a positive control reagent, each of which is attached to the surface of the substrate as described above.

[0107] The binding components are as described above.

[0108] The negative control reagent has a lower affinity for the reporter reagent under the conditions of the assay than the binding component. Thus, the negative control reagent provides a negative control. It is important that affinity is considered under the conditions of the assay, because in a non-competitive assay, the affinity of the binding component for the reporter reagent is mediated by the presence of the analyte or analyte complex or derivative. Thus, in the absence of the analyte or analyte complex or derivative, neither the binding component nor the negative control reagent has affinity for the reporter reagent. However, in the presence of the analyte or analyte complex or derivative, the negative control reagent has a lower affinity for the reporter reagent than the binding component.

[0109] Additionally, in embodiments in which the binding moiety binds to the analyte or analyte complex or derivative, the negative control reagent preferably has a lower affinity for the analyte or, if used, analyte complex or derivative than the binding moiety. The negative control reagent is preferably a protein, more preferably an antibody. The negative control reagent typically has similar chemical and physical properties as the binding moiety, but provides little or no affinity for the reporter reagent under the conditions of the assay. In a particularly preferred embodiment, the negative control reagent has essentially no affinity for the reporter reagent under the conditions of the assay. Preferably, the negative control reagent has essentially no affinity for the analyte or analyte complex or derivative. That is, the binding of the reporter reagent, or, if applicable, the analyte or analyte complex or derivative, to the negative control reagent is non-specific. This allows the negative control reagent to compensate for non-specific binding of the reporter reagent to the binding moiety. In certain embodiments, the software algorithm uses data from the negative control group as part of the calculation to obtain the analyte concentration.

[0110] The positive control reagent binds to the reporter reagent and has an affinity for the reporter reagent that is less affected by the concentration in the sample of the analyte or, if used, the analyte complex or derivative, than the binding moiety, thus providing a positive control. Preferably, the positive control reagent has an affinity for the reporter reagent that is essentially independent of the concentration of the analyte or the analyte complex or derivative. More preferably, the positive control reagent has a higher affinity for the reporter reagent than the binding moiety under the conditions of the assay. The positive control reagent thereby measures the maximum expected signal in the system.

[0111] For both the positive and negative control measurements, the software algorithm is capable of detecting abnormal binding patterns which can result in an error message and interrupt the measurement process.

[0112] To increase the dynamic range of the assay performed according to the present invention while also improving the accuracy, it is preferable to have the binding component at multiple 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 can also have its own negative and positive control reagents that act as controls for different dynamic ranges. This is particularly applicable to competitive assays that are particularly sensitive to the concentration of each of the individual components that make up the system.

[0113] While the above description allows for the assay to reach equilibrium before activating the photosensitizer, it also allows for the kinetics of the binding events to be monitored by irradiating the photosensitizer for discrete periods of time over a time course and monitoring the binding events that take place over the course of the reaction before reaching equilibrium.

[0114] The analyte can be a macromolecule or a small molecule. Macromolecules are typically proteins, such as protein-based hormones, but can also be part of larger particles, such as viruses, bacteria, cells (e.g. red blood cells) or prions. Small molecules can be drugs.

[0115] The term "small molecule" as used herein is a term of the art and is used to distinguish molecules from macromolecules such as proteins and nucleic acids. Small molecules are often referred to in the field of immunoassays as "haptens" and are small molecules that can trigger an immune response when attached to a large carrier molecule such as a protein, including molecules such as hormones and synthetic drugs. The molecular weight of this type of small molecule is typically 2,000 or less, often 1,000 or less, or even 500 or less. The binding moiety may be adjusted to bind the analyte itself, but the analyte may undergo a chemical reaction or an initial complexation event before binding to the binding moiety. For example, the analyte may be protonated / deprotonated at the pH of the assay conditions. Thus, the analyte that binds to the binding moiety may be the analyte itself or a derivative of the analyte, both of which are included within the scope of the present invention.

[0116] In a preferred embodiment, the present invention can be used to detect the presence of multiple analytes in the same sample simultaneously. Different binding moieties can be used at different locations on the substrate for the measurement of each analyte. Sandwich and competitive assays can be performed in parallel, and the assays can use the same negative and positive controls as described above, or can be different controls for each analyte being measured.

[0117] The sample that may contain the analyte of interest is generally a fluid sample, e.g. a liquid sample, usually a biological sample, e.g. a body fluid such as 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 unprocessed, more preferably an unprocessed fluid. Unprocessed means that the sample / fluid has not been pretreated by filtration, dilution or any other pretreatment step before mixing the reporter reagent with the other components of the assay. An 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 outcome of the assay.

[0118] In a preferred embodiment, the sample is whole blood. It is surprising that the components of whole blood do not interfere with the method of detection of the present invention. It is usual to remove red blood cells from blood and measure fluorescence in the plasma or serum components of blood, since light is scattered unpredictably by cellular components, which varies from sample to sample. However, in the method of the present invention, the measurement can be performed in whole blood, since the fluorescence is measured at the substrate using an imaging system with a shallow depth of field, allowing individual binding events to be measured.

[0119] The sample is typically in the order of microliters (e.g., 1-100 μL, preferably 1-10 μL). To hold the fluid sample, the substrate is preferably located in a sample chamber having one or more side walls, an upper surface, and a lower surface. Thus, the device used in the method of the present invention preferably further comprises a chamber for holding a sample containing an analyte in contact with the substrate.

[0120] An additional possible source of background interference is the precipitation of suspended particles on the surface of the substrate, including the reporter reagents and cellular components of the sample. This source of interference can be reduced by placing the substrate higher than the bulk solution, for example on the top surface of the reaction chamber. Thus, if any precipitation occurs, it does not interfere with the substrate. Preferably, the substrate forms the top surface as shown in the figure. Preferably, the substrate is substantially planar. More preferably, the substrate is planar. By "substantially planar" we mean that the substrate deviates from planarity only to a point where it remains functional in the present invention, for example, so that the entire substrate remains within a single focal range when imaging. Obviously, the optical and binding components are on the interior surface of the chamber that allows contact with the sample. This and other modifications are within the scope of the present invention.

[0121] The sample may, for example, simply be held by surface tension within a capillary channel.

[0122] The reporter reagent and optionally one or more additional reagents are preferably stored in a chamber incorporated into a device used in the methods of the invention.

[0123] The method of the present invention is particularly useful for point-of-care (POC) testing. POC testing is defined as diagnostic testing at or near the point of care, i.e. bedside testing. POC testing allows for convenient and rapid testing, improving decision-making and triage while allowing for better allocation of hospital resources such as accident and emergency care and hospital beds. This is in contrast to traditional testing, where samples are taken at the point of care and then sent to a laboratory for testing. Such testing often requires hours or days to obtain results, during which treatment must continue without the desired information. POC testing often uses a test kit in combination with a portable device.

[0124] The methods of the invention are particularly useful for monitoring the concentration or presence of analytes that are usually in very small amounts. Potential applications include measuring biomarkers in cardiovascular disease (e.g., high-sensitivity troponin), infectious diseases (e.g., Hepatitis C core antigen), aging / dementia (e.g., Alzheimer's disease markers amyloid beta and tau), cytokines, and oncology (e.g., circulating tumor markers).

[0125] The 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, the optical component and the binding component attached to a surface of the substrate, the optical component capable of being excited in response to irradiation with electromagnetic radiation, the excited optical component capable of changing from a first optical state to a second optical state in response to interaction with an illuminated 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 series of localized regions on the substrate having the second optical state.

[0126] The characteristics of the device are as described above for the device used in the method of the invention.

[0127] In a preferred embodiment, the device further comprises a chamber for holding a mixture of the sample and the reporter reagent.

[0128] The device may include one or more radiation sources that are adjusted to generate electromagnetic radiation, and a detector that is adjusted to detect a second optical state, thereby allowing precise determination of the position of the photosensitizer relative to the substrate.

[0129] The device can take the form of a cartridge for use in another reader. The reader can incorporate a radiation source and a detector. The reader is preferably a handheld reader. Preferably, the device comprises a cartridge, the substrate is in the cartridge, and the device further comprises a detector for detecting a series of localized regions on the substrate having a second optical state. The present invention can also provide a cartridge comprising the substrate as defined herein and optical and binding components. The cartridge is preferably a disposable cartridge.

[0130] The invention may also provide a system for detecting an analyte in a sample, comprising: a device as described above; and a reporter reagent for forming a mixture with the sample, the reporter reagent comprising a photosensitizer, the photosensitizer absorbing electromagnetic radiation and interacting with an excited optical component enabling the excited optical component to change from a first optical state to a second optical state.

[0131] Preferably, the photosensitizer is capable of absorbing electromagnetic radiation and interacting with a preactivator reagent present in the mixture to generate an activator reagent, which is capable of interacting with an excited optical component to change the excited optical component from a first optical state to a second optical state.

[0132] In a preferred embodiment, the system of the invention consists essentially of the features described above, by "essentially" it is meant that no other features are required to perform the assay.

[0133] The invention will now be described with reference to the following examples, which are not intended to be limiting. These examples are simplified to have direct binding of the reporter reagent to the binding moiety. However, it should be understood that in various assay formats, it is envisioned that binding of the reporter reagent to the binding moiety is either promoted or inhibited by the presence of analyte in solution. The examples are provided to illustrate the mode of conversion of an excited optical moiety from a first optical state to a second optical state. EXAMPLES

[0134] material Polylysine hydrochloride (MW 30 kD), Innolink biotin 354S, biotin-NHS ester, silicon 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide, goat anti-mouse antibody and other general laboratory reagents were supplied by Merck Millipore.

[0135] AlexaFluor594 streptavidin conjugate and AlexaFluor Oregon Green488 conjugate were supplied by Thermo Fisher.

[0136] Carboxypolystyrene particles (100 nm diameter) were supplied by Bangs Laboratories.

[0137] The 5409 anti-TSH monoclonal antibody was supplied by Medix Biochemica.

[0138] Biotinylated polylysine hydrochloride (5 biotins per polylysine) was prepared by reaction of Innolink Biotin 354S with polylysine by methods known in the art. Biotin incorporation was measured by absorbance at 354 nm. Biotinylation of the 5409 antibody was carried out by methods known in the art. Biotin incorporation (approximately 2) was measured by HABA assay.

[0139] Carboxypolystyrene particles were impregnated with a silicon phthalocyanine reagent using the method described by Ullman et al. (Ullman et al, Clinical Chemistry, 1996, 42, 1518-1526) Goat anti-mouse antibodies were covalently coupled to these particles using EDC / NHS coupling reagents by methods known in the art.

[0140] [Example 1] Preparation of AlexaFluor594-based chips A 22 mm glass cover slip (Brand GMBH) was cleaned using a Henniker Cirrus atmospheric pressure plasma etcher to remove surface impurities from the glass. 50 microliters of biotin-polylysine conjugate (at 50 micrograms / mL in deionized water) was then deposited on the surface of the cover slip for 10 minutes, then washed with deionized water and dried. A 1 cm x 1 cm, 200 μm thick pressure sensitive adhesive 13 with a 6 mm diameter hole cut out was then glued to the cover slip to create a shallow well 15 as shown in FIG. 7.

[0141] Thirty microliters of streptavidin AlexaFluor594 conjugate (10 micrograms / mL) was then incubated in the shallow wells for 30 minutes, then washed off and replaced with 30 microliters of biotinylated 5409 antibody for an additional 30 minutes. The surface was washed several times with phosphate buffer containing 0.05% BSA and 2% sucrose, then air-dried at room temperature.

[0142] The release liner was then removed from the PSA and the substrate was inverted and adhered to a piece of acrylic sheet 16 with two small holes 17 drilled through it to create the reaction chambers, as shown in FIG.

[0143] [Example 2] Preparation of Oregon Green 488 substrate chips The surface was prepared as described in Example 1, substituting the AlexaFluor streptavidin conjugate for the Oregon Green 488 streptavidin conjugate at the same concentration.

[0144] [Example 3] Incubation of beads with AlexaFluor594 substrate or Oregon Green488 substrate Eight microliters of a 1:2000 dilution of photosensitizer beads (beads at 0.5% solids) coated with goat anti-mouse antibody in phosphate buffer, 0.05% BSA, 0.05% Tween-20 was added to the reaction chambers from Examples 1 and 2 under low light conditions. The two holes in the plastic cartridge were sealed with clear adhesive tape and incubated for 30 minutes.

[0145] [Example 4] AlexaFluor594-based illumination and imaging Several chips were activated and imaged using a home-built prototype instrument as shown in Figure 5. The instrument has two high-power LED light sources that can each be focused onto the substrate surface through a 63x objective. The 680nm red LED is centered on the excitation wavelength of the photosensitizer beads, and the 560nm LED matches the excitation wavelength of the AlexaFluor594 fluorophore. Each LED can be controlled separately and the drive current (i.e. light intensity) can be adjusted separately. The maximum drive current for continuous use of the 680nm LED is 0.6A, and for the 560nm LED the maximum drive current is 1.0A. Images are collected with a camera chip connected to a PC that controls the focus, exposure and gain of the camera.

[0146] Four data sets were collected. For each data set, the surface was illuminated with either the 680 nm LED, the 560 nm LED or both LEDs. Images were taken of the surface at several separate time points. Images were taken of the surface using illumination with the 560 nm LED only. Dark spots began to form over time under different illumination conditions. For each captured image, the diameter of several representative spots was measured using ImageJ software to obtain the average size of the spots at a particular time point. The size of the spots was given in pixels, with 20 pixels being approximately 1 micron.

[0147] As can be seen in Figure 9, the speed of spot formation varies depending on the illumination conditions. Only when illuminated with 560 nm (1.0 A) does the spot reach a size of approximately 6 pixels after 10 minutes and a maximum size of approximately 8 pixels after approximately 1 hour. When illuminated with exactly 680 nm light (0.6 A), the spot formation is more rapid, reaching 10 pixels in 6 minutes and a maximum of 18 pixels after approximately 1 hour. When illuminated with both wavelengths simultaneously, the spot formation is more rapid, reaching 10 pixels in 1 minute. In addition, when the driving current on the LED is reduced to 0.5 A and 0.3 A for the 560 nm and 680 nm LEDs, respectively, the speed of spot formation remains roughly the same. It was surprising that the spots formed exactly at 560 nm, since the photosensitizer has low absorption at this wavelength. However, since little effort was made to eliminate ambient and / or stray light that was filtered, it is presumed that there was enough light present to partially activate the beads. It can be clearly seen that the speed of spot formation is improved when using both light sources versus using the light sources independently. Additionally, the intensity of each light source can be reduced without affecting spot formation when both LEDs are used.

[0148] [Example 5] Illuminating and Imaging Oregon Green 488 Substrate The experimental setup was the same as in Example 4, except that a 470 nm LED was used instead of a 560 nm LED. The 470 nm LED had a maximum drive current of 1.0 A for continuous use. The results are shown in FIG. 10. Oregon Green 488 is less photostable and has a faster rate of spot formation compared to Alexa Fluor 594. Also, spots were only formed using the 470 nm LED, but these were very small. When illuminated by the 680 nm LED by itself, spot formation was relatively rapid, reaching 10 pixels in about 90 seconds. When both LEDs were used simultaneously, spot formation was extremely rapid, occurring within seconds, with spots with diameters greater than 15 pixels formed in less than 20 seconds. [Explanation of symbols]

[0149] 1. Photosensitizers 2. Photosensitizer-injected antibody-coated latex particles 3 Antibodies 4 Optical components of the fluorescent state 5. Optical components in the non-fluorescent state 6 Streptavidin 7. Streptavidin labeled with a fluorescent optical component 8. Streptavidin labeled with a non-fluorescent optical moiety 9. Biotinylated polylysine 10 Sample Chamber 11 Base material 12 LED 13 Pressure Sensitive Adhesives 14 15 Shallow Well 16 Acrylic Sheet 17 2 small holes

Claims

1. A method for detecting analytes in a sample, (i) Providing a mixture comprising a sample and a reporter reagent to a device, wherein the reporter reagent comprises a photosensitizer, and the device comprises a substrate having an optical component and a binding component, wherein the optical component and the binding component are attached to the surface of the substrate; (ii) a step that enables a portion of the reporter reagent to bind to the surface of the substrate in proportion to the concentration of the analyte by the binding component; (iii) Irradiating the device with electromagnetic radiation for absorption by the optical component and absorption by the photosensitizer of the reporter reagent, so that the optical component absorbs the electromagnetic radiation and generates an excited optical component, the photosensitizer of the reporter reagent bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the excited optical component to change the excited optical component from a first optical state to a second optical state, thereby forming a series of local regions having the second optical state on the substrate; (iv) the step of detecting a series of local regions having the second optical state on the substrate; Methods that include...

2. The method according to claim 1, wherein the electromagnetic radiation for absorption by the optical component and for absorption of the reporter reagent by the photosensitizer has the same wavelength.

3. The method according to claim 1, wherein the electromagnetic radiation for absorption by the optical component and absorption by the photosensitizer has different wavelengths.

4. The method according to claim 3, wherein the electromagnetic radiation for absorption by the optical component and absorption of the reporter reagent by the photosensitizer reaches the device from one direction.

5. The method according to claim 3, wherein the electromagnetic radiation for absorption by the optical component and absorption by the photosensitizer arrives at the device from different directions.

6. The method according to claim 3, wherein the device is simultaneously irradiated with electromagnetic radiation for absorption by the optical component and absorption of the reporter reagent by the photosensitizer, or the device is irradiated first with electromagnetic radiation for absorption of the reporter reagent by the photosensitizer and then with electromagnetic radiation for absorption by the optical component.

7. The method according to claim 1, wherein the photosensitizer of the reporter reagent absorbs electromagnetic radiation and interacts with a preactivator reagent present in the mixture to generate an activator reagent that interacts with the excited optical component, thereby changing the excited optical component from the first optical state to the second optical state.

8. The method according to claim 7, wherein the activator reagent is a reactive oxygen species.

9. The method according to claim 8, wherein the reactive oxygen species is singlet oxygen.

10. The method according to claim 1, wherein the first optical state is fluorescent and the second optical state is non-fluorescent.

11. The method according to claim 1, wherein the change from the first optical state to the second optical state is irreversible.

12. The method according to claim 1, wherein steps (i) to (iii) are performed in the absence of a washing step.

13. The method according to claim 1, wherein the sample is untreated.

14. The method according to claim 1, wherein the device is irradiated with electromagnetic radiation for a longer period than one second.

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