Method and apparatus for measuring target substance in solution, and electronic device

Abstract

A method and apparatus for measuring a target substance in a solution, and an electronic device. The target substance measurement method comprises: obtaining at least one optical image, collected in chronological order, of a preset spatial region (S11), wherein the preset spatial region comprises a spatial region in a solution in which a preset localized light field is located, the solution comprises at least one target substance, the target substance is dispersed and moves in the solution as opposed to being fixed, bound, or retained on any solid phase surface, and the target substance located within the preset spatial region generates luminescence; identifying a luminescence process of a single target substance on the basis of the optical image (S12); and measuring the target substance on the basis of the luminescence process of the single target substance (S13). The measurement method is simpler, and the measurement efficiency and accuracy are also improved.

Description

Method and device for detecting target in solution, and electronic device

[0001] This application claims priority to Chinese patent application CN202411260444.9, filed on September 9, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the technical field of detection, and particularly relates to a method and device for detecting target in solution, an electronic device, a storage medium, a computer program product, and a system for detecting target in solution. BACKGROUND

[0003] Various means for quantitatively or qualitatively measuring the types and concentrations of protein molecules in biomedical samples have played a crucial role in the development of life science research and medical diagnosis. Traditional detection methods are based on measuring all signals emitted by the entire sample, such as various colorimetric methods, fluorescence methods, and chemiluminescence methods. In such methods, the signal to be detected is mixed with background signals and noise signals, so when the concentration of the target protein molecules to be detected is very low, the signal to be detected will be overwhelmed by other irrelevant signals and cannot be identified. In order to achieve accurate detection of extremely low concentration (<1 pM) biomedical samples, various digital detection methods have appeared in the prior art, the basic principle of which is based on the recognition and counting of individual target protein molecules, greatly improving the signal-to-noise ratio of detection.

[0004] In the above background, single molecule fluorescence detection techniques have been developed and applied. One of the methods uses a sensing element with a solid surface such as a microsphere to detect molecules with fluorescent groups bound to the solid surface. Specifically, first, recognition molecules are immobilized on a large number of microspheres; then the recognition molecules specifically capture target molecules to be detected, and since the concentration of the target molecules is very low, the number of target molecules that can be captured by each microsphere is much less than 1; then the microspheres bound to the target molecules are made to emit fluorescence by catalysis or other means; finally, the light-emitting microspheres are counted to obtain the quantitative measurement result of the target molecules. This kind of technology has played a role in the diagnosis of diseases such as Alzheimer's disease. This solid surface-based sensing technology uses a complex process of specific capture of target molecules and amplification of fluorescence signals, involving multiple steps such as binding, elution and catalysis, and the sample processing process is relatively cumbersome. Another method uses a confocal microscope to detect molecules with fluorescent groups in solution, thereby avoiding various problems and cumbersome processing techniques caused by the solid surface. Specifically, the confocal microscope focuses a laser on the solution to form a very small light spot, and when a molecule with a fluorescent group passes through the focused light spot, the fluorescence signal excited by the laser is detected. Since the number of molecules with fluorescent groups in the light spot is much less than 1, the concentration of molecules with fluorescent groups can be obtained by counting the fluorescence events. However, for very low concentration samples, the detection time of this method is very long, and it may take more than a few hours.

[0005] Therefore, how to realize a single molecule fluorescence detection technique that can directly detect protein molecules in solution while being simple and fast has important research and application value for the development of life science and medical diagnosis technology, and is also an important technical problem to be solved by those skilled in the art. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the above-mentioned defects in the prior art, and to provide a solution target detection method and device, electronic equipment, storage medium, computer program product and solution target detection system.

[0007] The technical problem to be solved by the present application is to overcome the above-mentioned defects in the prior art, and to provide a solution target detection method and device, electronic equipment, storage medium, computer program product and solution target detection system.

[0008] The first aspect of the present application provides a solution target detection method, comprising the following steps:

[0009] acquiring at least one optical image of a preset spatial region collected in time sequence; wherein the preset spatial region is included in a spatial region where a preset local optical field is located in a solution, the solution includes at least one target object, the target object is dispersed and moves in the solution without being fixed, combined or staying on any solid surface, and the target object in the preset spatial region generates luminescence;

[0010] identifying a luminescence process of a single target object according to the optical image; wherein the luminescence process of the single target object is a process from entering the preset spatial region to leaving the preset spatial region of the single target object;

[0011] detecting the target object according to the luminescence process of the single target object.

[0012] Optionally, the step of detecting the target object according to the luminescence process of the single target object specifically includes:

[0013] detecting the concentration or the moving speed of the target object according to the number or frequency of the luminescence process of the single target object;

[0014] And / or, detecting the residence time of the target object in the preset spatial region according to the brightness of the luminescence process of the single target object;

[0015] And / or, distinguishing target objects with different diffusion coefficients or moving speeds according to the brightness of the luminescence process of the single target object;

[0016] And / or, detecting the diffusion coefficient or the moving speed change of the target object according to the brightness and / or the number or frequency of the luminescence process of the single target object;

[0017] Wherein, the brightness of the luminescence process of the single target object is determined according to the brightness distribution of the corresponding interval of the optical image corresponding to the luminescence process of the single target object.

[0018] Optionally, the target object is the product of the combination of the to-be-tested object and the marker, and the step of distinguishing target objects with different diffusion coefficients or moving speeds according to the brightness of the luminescence process of the single target object specifically includes:

[0019] Using markers with different diffusion coefficients or moving speeds to combine different types of to-be-tested objects, so that different types of to-be-tested objects correspond to target objects with different diffusion coefficients or moving speeds;

[0020] Distinguishing target objects with different diffusion coefficients or moving speeds according to the brightness of the luminescence process of the single target object, so as to distinguish different types of to-be-tested objects.

[0021] Optionally, the optical image is divided into a plurality of units in time and space, each unit corresponding to a pixel, and the optical image comprises at least two spatially adjacent pixels; and the step of identifying the light-emitting process of a single target object according to the optical image comprises:

[0022] determining, as first target pixels, the pixels in the optical image having luminance within a first preset luminance range;

[0023] identifying each first target pixel or each first pixel array as a light-emitting process of a single target object; wherein the first pixel array is an array of first target pixels that are identical in time and spatially continuously adjacent, or an array of first target pixels that are continuously adjacent in time and space;

[0024] wherein,

[0025] two pixels are adjacent in time if the optical images in which the two pixels are located are adjacent frames in acquisition time, or the acquisition time interval of the optical images in which the two pixels are located is less than a first preset value;

[0026] two pixels are adjacent in space if the two pixels differ by 1 in row number and are identical in column number, or the two pixels are identical in row number and differ by 1 in column number, or the two pixels differ by 1 or less in both row number and column number, or the spatial distance between the two pixels is less than a second preset value;

[0027] two pixels are adjacent in time and space if the two pixels are identical or adjacent in both time and space;

[0028] a plurality of pixels are connected to each other through spatially adjacent pixels to form a connected pixel array, and the pixels and the pixel array are spatially continuously adjacent;

[0029] a plurality of pixels are connected to each other through spatially adjacent pixels to form a connected pixel array, and the pixels and the pixel array are spatially continuously adjacent.

[0030] Optionally, after the step of determining, as first target pixels, the pixels in the optical image having luminance within a first preset luminance range, the method further comprises:

[0031] in response to a first pixel set comprising any of the first target pixels not satisfying a first preset condition, updating the first target pixel to not belong to the first target pixels.

[0032] Optionally, the first pixel set comprises a second pixel array;

[0033] The second pixel array is composed of the first target pixel and a second target pixel, and the second target pixel includes and only includes all pixels that satisfy a preset brightness condition, are identical in time to the first target pixel, and are continuously adjacent in space to the second pixel array.

[0034] The second pixel array is composed of the first target pixel and a second target pixel, and the second target pixel includes and only includes all pixels that satisfy a preset brightness condition and are continuously adjacent in time and space to the second pixel array.

[0035] The preset brightness condition is that the brightness of the second target pixel in the optical image is within a second preset brightness interval.

[0036] When the second target pixel does not exist, the second pixel array is equivalent to the first target pixel. Optionally, the first preset condition equivalently includes at least one of the following conditions or a logical combination of several conditions, or a logical combination of one or more of the following conditions and other conditions:

[0037] The number of elements in the second pixel array that are adjacent in time and space to the first target pixel is within a first preset range.

[0038] The number of elements in the second pixel array that are identical in time and adjacent in space to the first target pixel is within a second preset range.

[0039] The number of elements in the second pixel array that are adjacent in time and identical in space to the first target pixel is within a third preset range.

[0040] The number of elements in the second pixel array is within a fourth preset range.

[0041] The overall or partial spatial length of the second pixel array is within a fifth preset range.

[0042] The overall or partial time length of the second pixel array is within a sixth preset range.

[0043] The second pixel array is a subset or proper subset of a first preset array structure.

[0044] The second pixel array is not a subset or proper subset of a second preset array structure.

[0045] The second pixel array contains or properly contains a third preset array structure.

[0046] The second pixel array does not contain or properly contain a fourth preset array structure.

[0047] the sum of the brightness of each pixel in the second pixel array, the weighted sum of the brightness of each pixel, or the function value with the brightness of each pixel as the variable is in a seventh preset range;

[0048] In a specified frame or a plurality of consecutive frames, the second pixel array satisfies the at least one condition.

[0049] Optionally, the first preset condition equivalently includes at least one of the following conditions or a logical combination of a plurality of conditions, or a logical combination of the following one or more conditions and other conditions:

[0050] The number of elements of the second pixel array in the same frame as the first target pixel is greater than or equal to a, where a is a natural number greater than or equal to 2;

[0051] The number of elements of the second pixel array in the same frame as the first target pixel is less than or equal to b, where b is a natural number;

[0052] The second pixel array in the same frame as the first target pixel contains or is contained in a preset square or rectangular arrangement of spatially continuous adjacent pixel arrays;

[0053] The maximum spatial length of the entire second pixel array in the same frame as the first target pixel is less than or equal to f, where f is a natural number;

[0054] The number of elements of the second pixel array that are spatially identical to the first target pixel and temporally adjacent to the first target pixel is greater than or equal to 1 or 2;

[0055] The number of elements of the second pixel array that are spatially identical to the first target pixel and temporally adjacent to the first target pixel is less than or equal to 0 or 1;

[0056] The overall time length of the second pixel array is greater than or equal to g, where g is a natural number greater than or equal to 2;

[0057] The overall time length of the second pixel array is less than or equal to h, where h is a natural number.

[0058] Optionally, at least one of the pixels includes at least two physical pixels in at least one frame of the optical image;

[0059] Alternatively, at least one of the pixels includes at least two physical pixels in at least two frames of the optical image;

[0060] Alternatively, at least one of the pixels includes at least two physical pixels in the optical image that are not spatially adjacent;

[0061] Alternatively, at least one of the pixels includes at least two physical pixels in at least two frames of the optical image that are not temporally adjacent.

[0062] Alternatively, at least two of the pixels comprise at least one spatially and temporally identical physical pixel.

[0063] The values of the brightness, the acquisition time and / or the spatial position of the pixels are determined according to the spatiotemporal interval covered by the pixels and the brightness distribution of the physical pixels in the spatiotemporal interval.

[0064] Optionally, the pixels comprise a spatially continuous array of adjacent physical pixels arranged in a square or rectangular array in the optical image, wherein the brightness of the pixels is determined according to the brightness of the physical pixels in the array.

[0065] Optionally, the target object is an object capable of producing luminescence or luminescence of a specific wavelength band in the predetermined spatial region, which is formed by specific binding or reaction of the analyte and the label in the solution.

[0066] Optionally, the analyte comprises at least one of the following: a chemical molecule, a protein molecule, a nucleic acid molecule and other biological molecules.

[0067] Optionally, the label comprises at least one of the following: a fluorescently labeled molecule, a nucleic acid aptamer-based molecular beacon, a molecule that produces fluorescence resonance energy transfer after binding with the analyte, a substance capable of cutting other substances to produce fluorescence.

[0068] The second aspect of the present application provides a method for detecting a target object in a solution, comprising the following steps:

[0069] Obtaining at least one optical image of a predetermined spatial region acquired in time sequence; wherein the predetermined spatial region is included in a spatial region at which a predetermined local optical field in the solution is located, the solution comprises at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the predetermined spatial region produces luminescence.

[0070] Detecting the concentration of the target object or the diffusion coefficient or the change in the movement speed of the target object according to the features of the pixel brightness distribution function of the optical image; wherein the pixel brightness distribution function is a function with the brightness of the pixels in the optical image as the independent variable and the number of pixels in a unit brightness range as the dependent variable.

[0071] Optionally, the features of the pixel brightness distribution function comprise at least one of the following: the sum of the brightness of all the pixels, the centroid of the pixel brightness distribution function, the independent variable and / or the dependent variable at the peak value of the pixel brightness distribution function, the area of the pixel brightness distribution function with the independent variable in a first range, and the sum of the brightness of all the pixels with the independent variable in a second range.

[0072] Optionally, in the detection method according to the first aspect or the second aspect, the object plane or the object surface of the imaging device corresponding to the optical image comprises a continuous first sub-region, the first sub-region is included in the preset spatial region, and the light emitted by a single target object occurring at any position of the first sub-region is imaged in the optical image.

[0073] The area of the first sub-region is greater than or equal to a first preset area.

[0074] Optionally, the first preset area is 10 times or 100 times the square of the light wavelength of the target object, or the first preset area is 5 μm 2 or 50 μm 2 .

[0075] Optionally, in the detection method according to the first aspect or the second aspect, the object plane or the object surface of the imaging device corresponding to the optical image comprises a continuous second sub-region, and the second sub-region is a regular polygon or a regular polygon with pixel discretization after imaging by the imaging device, and the area of the second sub-region is greater than or equal to a second preset area.

[0076] The part of the second sub-region overlapping with the preset spatial region comprises a third sub-region, and the ratio of the area of the third sub-region to the area of the second sub-region is greater than or equal to a preset area ratio, and the light emitted by a single target object occurring at any position of the third sub-region is imaged in the optical image.

[0077] Optionally, the preset area ratio is 1 / 5 or 1 / 10, and / or,

[0078] The second preset area is 10 times or 50 times the square of the light wavelength of the target object, or the second preset area is 5 μm 2 or 25 μm 2 .

[0079] Optionally, in the detection method according to the first aspect or the second aspect, the preset local optical field comprises at least one of the following: a surface evanescent optical field located at the interface between an optical material and the solution, a surface plasmon optical field located at the interface between a metal material and the solution.

[0080] The interface between the optical material or the metal material and the solution is provided with a material resistant to adsorption of the target object.

[0081] Optionally, in the detection method according to the first aspect or the second aspect, the target object is a product of the combination of the to-be-detected object and the label, and the solution contains a non-target object capable of generating light emission in the preset spatial region.

[0082] The non-target objects include at least one of the following: a label that does not bind with the target object but produces light emission by mistake, and a light-emitting substance that falls off from the label;

[0083] The diffusion coefficient or the movement speed of the target object is much smaller than that of the non-target object.

[0084] Optionally, in the detection method according to the first aspect or the second aspect, the target object is a target molecule or a product of the target molecule binding with a label, and the diffusion coefficient or the movement speed of the target object changes correspond to the binding or dissociation of the target molecule with other molecules.

[0085] The third aspect of the present application provides a detection device for a target object in a solution, comprising:

[0086] The first acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; the preset spatial region is included in a spatial region at a preset local light field in a solution, the solution includes at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or staying on any solid surface, and the target object in the preset spatial region produces light emission;

[0087] The light emission recognition module is configured to recognize a light emission process of a single target object according to the optical images; the light emission process of the single target object is a process from entering the preset spatial region to leaving the preset spatial region of the single target object;

[0088] The first detection module is configured to detect the target object according to the light emission process of the single target object.

[0089] The fourth aspect of the present application provides a detection device for a target object in a solution, comprising:

[0090] The second acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; the preset spatial region is included in a spatial region at a preset local light field in a solution, the solution includes at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or staying on any solid surface, and the target object in the preset spatial region produces light emission;

[0091] The second detection module is configured to detect the concentration of the target object or the diffusion coefficient or the movement speed change of the target object according to the features of the pixel brightness distribution function of the optical images; the pixel brightness distribution function is a function with the brightness of the pixels in the optical images as the independent variable and the number of pixels in a unit brightness range as the dependent variable.

[0092] The fifth aspect of the present application provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the detection method according to the first aspect or the second aspect.

[0093] The sixth aspect of the present application provides a computer readable storage medium, which stores a computer program, wherein the computer program is executable on a processor to implement the detection method according to the first aspect or the second aspect.

[0094] The seventh aspect of the present application provides a computer program product, comprising a computer program, wherein the computer program is executable on a processor to implement the steps of the detection method according to the first aspect or the second aspect.

[0095] The eighth aspect of the present application provides a detection system for a target in a solution, comprising:

[0096] an excitation device configured to excite the target in a preset spatial region in the solution to generate luminescence, wherein the preset spatial region is included in a spatial region at a preset local optical field in the solution, the solution comprises at least one target, the target is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target in the preset spatial region generates luminescence;

[0097] an imaging device configured to sequentially acquire at least one optical image of the preset spatial region in time;

[0098] a detection device configured to execute the steps of the detection method according to the first aspect or the second aspect.

[0099] Optionally, the excitation device comprises an excitation light source, and the preset local optical field is a surface plasmon polariton field or a surface plasmon field formed at an interface between the solution and a solid object by light emitted by the excitation light source and directed to the interface.

[0100] In the interface between the solid object and the solution, a material resistant to adsorption of the target is arranged.

[0101] On the basis of common knowledge in the art, the above optional conditions can be combined arbitrarily, i.e., the preferred embodiments of the present application are obtained.

[0102] The positive progress effect of the present application is that:

[0103] Compared with the traditional technology of detecting all signals emitted by the whole sample, the technology of detecting the luminescence process of a single target in the present application, especially the single molecule fluorescence detection technology, can not only effectively remove the influence of background signal, stray light signal and detection noise, thereby obtaining higher detection signal-to-noise ratio, but also can avoid the quantitative measurement error caused by the change of molecular fluorescence intensity with experimental conditions through the way of discrete counting.

[0104] Compared with the existing single molecule fluorescence detection technology using solid surface to fix the recognition molecule and capture the molecules to be detected, in the present application, the molecules to be detected (for example, the molecules to be detected) and the labels (for example, the recognition molecules or the labeled molecules) specifically bind in a free and relaxed state in a liquid environment. This binding mode avoids the non-specific binding effect caused by the solid surface, so there is no need to design a complex binding and elution process to ensure the specificity of the detection; at the same time, the binding reaction between the molecules dispersed in the solution can be much faster than the molecular binding reaction on the solid surface, and these two characteristics make the detection process of the present application have the characteristics of high simplicity and rapidity. On the other hand, in the present application, the binding reaction of molecules in the solution is not affected by the space steric hindrance and molecular deformation effect of the solid surface, has better specific recognition ability and higher binding efficiency, and also avoids the difficulty of solid surface molecular design; for example, after the aptamer is directly fixed on the solid surface, it often loses the ability to bind the molecules to be detected, while the binding in the solution is not restricted by this difficulty.

[0105] Compared with the existing detection technology of measuring single molecule fluorescence in solution by confocal microscope, the preset local light field of the present application, such as surface evanescent light field, has much larger area compared with the focused spot of confocal microscope; the surface evanescent light field of the present application can be imagined as the parallel operation of many confocal detection regions, so the detection efficiency of the former is improved by orders of magnitude compared with the latter. The present application can detect protein molecules with fM concentration in a few minutes, which significantly embodies the advantages of high sensitivity and high efficiency, while the single molecule fluorescence measurement method based on confocal microscope needs to take several hours.

[0106] The positive progress effect of the present application is also that for the target with smaller molecular weight, faster diffusion motion or flow, and lower concentration, because its fluorescence brightness is weak, it cannot be distinguished in the background signal and noise in the traditional fluorescence detection technology or total internal reflection fluorescence microscopy, but the present application can clearly identify its single molecule fluorescence process.

[0107] Further, because different molecular weights of the target objects have different diffusion coefficients or movement speeds, they have different residence times in the preset local light field, so the brightness of the light emission process of the individual target objects is also different; the application can distinguish different molecular weights of fluorescent objects based on this rule, so as to avoid free fluorophores or other fluorescent impurities in the reagent from being counted into the detection signal. At the same time, because the probability of the same target object entering the preset local light field repeatedly and staying for a long time is low, the application can also effectively reduce the quantitative measurement error caused by fluorescence bleaching.

[0108] The above advantages make it possible to accurately identify and count individual target objects entering the preset local light field, and thus to realize accurate and rapid detection of extremely low concentration samples.

[0109] In addition, when the molecular weight of the target object changes, its diffusion coefficient or movement speed will also change, resulting in a change in the brightness of the light emission process of the individual target object; the application can measure the change in the molecular weight of the target object based on this rule, for example, measure the binding or dissociation kinetics of the molecules. Compared with the traditional method of measuring the binding and dissociation process of molecules on a solid surface, the application avoids many problems such as non-specific adsorption, steric hindrance, mass transfer barrier, and change of molecular conformation caused by the solid surface.

[0110] Finally, regarding the detection method of the application for detecting the concentration or diffusion coefficient or movement speed change of the target object according to the pixel brightness distribution function of the optical image of the preset spatial region, although it does not identify the light emission process of the individual target object, it is actually equivalent to a kind of statistics of the light emission process of the individual target object, so it has many same or similar positive effects as the above-mentioned light emission process detection technology of the individual target object, including: simple, fast and accurate detection method, avoiding the negative effects of the solid surface, simpler molecular design than solid surface experiment, effectively removing the effects of fluorescent impurities, stray light, background signal and equipment noise, effectively reducing the measurement error caused by fluorescence bleaching, and being able to measure the binding or dissociation kinetics of molecules in solution, etc. BRIEF DESCRIPTION OF DRAWINGS

[0111] FIG. 1 is a flowchart of a detection method of a target object in a solution provided by an embodiment of the application.

[0112] FIG. 2 is a schematic diagram of the binding process of a molecular beacon based on a nucleic acid aptamer and a target molecule provided by an embodiment of the application.

[0113] FIG. 3 is a structural schematic diagram of a total internal reflection fluorescence microscopy imaging device provided by an embodiment of the application.

[0114] Fig. 4 is a schematic diagram of exciting fluorescence of a target object by surface evanescent light field in a total internal reflection fluorescence microscopy imaging device according to an embodiment of the present application.

[0115] Fig. 5 is a flow chart of step S12 according to an embodiment of the present application.

[0116] Fig. 6 is a flow chart of step S13 according to an embodiment of the present application.

[0117] Fig. 7 is a schematic diagram of an image of a single target object in a fluorescence process according to an embodiment of the present application.

[0118] Fig. 8 is a schematic diagram of a measurement result of a relationship between a single molecule fluorescence light emitting process count and cTnl concentration and a linear fitting result according to an embodiment of the present application.

[0119] Fig. 9 is a schematic diagram of a comparison result of background fluorescence signals of surfaces of closed containers made of different materials according to an embodiment of the present application.

[0120] Fig. 10 is a structural block diagram of a detection device for a target object in a solution according to an embodiment of the present application.

[0121] Fig. 11 is a flow chart of a detection method for a target object in a solution according to an embodiment of the present application.

[0122] Fig. 12 is a schematic diagram of a pixel brightness distribution function corresponding to a cTnl sample concentration of 10 pM in a histogram according to an embodiment of the present application.

[0123] Fig. 13 is a schematic diagram of a pixel brightness distribution function corresponding to a cTnl sample concentration of 100 pM in a histogram according to an embodiment of the present application.

[0124] Fig. 14 is a structural block diagram of a detection device for a target object in a solution according to an embodiment of the present application.

[0125] Fig. 15 is a structural block diagram of a detection system for a target object in a solution according to an embodiment of the present application.

[0126] Fig. 16 is a structural diagram of an electronic device according to an embodiment of the present application. DETAILED DESCRIPTION

[0127] The present application will be further described below by way of examples, but the present application is not limited to the examples.

[0128] Embodiment 1

[0129] Fig. 1 is a flowchart of a method for detecting a target in a solution according to an embodiment of the present application. The method can be performed by a device for detecting a target in a solution, which can be implemented in software and / or hardware. The device can be part or all of an electronic device.

[0130] The method for detecting a target in a solution according to an embodiment of the present application will be described below with an electronic device as an execution subject. As shown in Fig. 1, the method for detecting a target in a solution according to an embodiment of the present application can include the following steps S11-S13.

[0131] In step S11, at least one optical image of a preset spatial region collected in time sequence is acquired. The preset spatial region is included in a spatial region where a preset local optical field in a solution is located. The solution includes at least one target. The target is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target in the preset spatial region generates light emission.

[0132] The target enters or exits the preset spatial region in a diffusion or flow manner. When the target enters the preset spatial region, it generates light emission or is excited more strongly to generate a stronger light emission signal.

[0133] In a specific implementation, the preset local optical field can be a surface evanescent optical field located at an interface between an optical material and the solution. The interface between the optical material and the solution is provided with a material resistant to adsorption of the target. The optical material is part or all of a container storing the solution. In a specific example, the surface evanescent optical field is formed by a light wave such as a laser or an LED from the optical material to the interface between the optical material and the solution and satisfying total internal reflection conditions. In another specific example, the optical material constitutes an optical waveguide, and the surface evanescent optical field is located at an interface between the optical waveguide and the solution and is formed by transmission of a light wave in the optical waveguide. In another specific example, the surface evanescent optical field is not formed by excitation of an external light source, but appears in the form of vacuum fluctuation of an optical field. When the target enters a spatial region of the surface evanescent optical field, light emission of the target is enhanced, for example, fluorescence radiation lifetime is shortened.

[0134] The preset local optical field can also be a surface plasmon optical field located at an interface between a metal material and the solution. The interface between the metal material and the solution is provided with a material resistant to adsorption of the target.

[0135] The material capable of resisting adsorption of the target object can include a blocking protein, milk powder, polyethylene glycol, a zwitterionic material, a detergent, serum, plasma, or other material capable of increasing the hydrophilicity of a surface, which can effectively reduce the physical adsorption of biomolecules, fluorescent molecules, and other substances on the surface of a solid phase, thereby improving the accuracy of detection of the target object.

[0136] In an optional embodiment, the target object is an object capable of emitting light or light of a specific wavelength band in the pre-set spatial region, which is formed by specific binding or reaction of the target object and a label in the solution. For example, the target object emits fluorescence at a specific wavelength band under excitation of light of a specific wavelength. The target object can include at least one of a chemical molecule, a protein molecule, a nucleic acid molecule, and other biological molecules. The label can include at least one of a fluorescently labeled molecule, a nucleic acid aptamer-based molecular beacon, a molecule capable of producing fluorescence resonance energy transfer after binding with the target object, and a substance capable of cutting other substances to produce fluorescence. The nucleic acid aptamer-based molecular beacon is a nucleic acid aptamer with a fluorescent group. When the nucleic acid aptamer is not combined with the target object, the fluorescent group is quenched. When the nucleic acid aptamer is specifically combined with the target object, the fluorescent group can effectively emit fluorescence of a specific wavelength band. The fluorescence excitation or emission wavelength of the molecule of resonance energy transfer changes before and after the molecule is combined with the target object.

[0137] In a specific example, as shown in FIG. 2, the nucleic acid aptamer-based molecular beacon is composed of five components, including a fluorescent group F, a quenching group Q, a nucleic acid aptamer, a short-chain DNA (deoxyribonucleic acid), and a polyethylene glycol linker (PEG linker). In the molecular beacon that is not combined with the target molecule, a hairpin structure is formed by complementary pairing of the short-chain DNA and the nucleic acid aptamer, so that the fluorescent group F and the quenching group Q are close to each other and the latter effectively quenches the fluorescence emission of the former. When the molecular beacon is specifically combined with the target molecule through the nucleic acid aptamer, the fluorescent group F and the quenching group Q are separated, and the unquenched fluorescent group F is obtained. The combination of the molecular beacon containing the unquenched fluorescent group F and the target molecule is the target object, which can emit fluorescence of a specific wavelength band.

[0138] In an optional embodiment, more than one label can be used, and different types of labels are combined with different types of target objects to form different types of target objects. Each label has a unique light-emitting group or a combination of light-emitting groups, so that the corresponding target object has a unique light excitation spectrum, a unique light emission spectrum, and / or a unique combination of light excitation and light emission spectrum. Based on the above characteristics, different types of target objects can be distinguished by excitation and / or emission spectrum, so as to measure more than one target object, and to better distinguish the target object from background signals and fluorescent impurities.

[0139] The optical image is an image of the whole of the preset spatial region. In the implementation of step S11, the optical image can be acquired by using an imaging device, and the acquired optical image can be sent to an electronic device. In one specific example, the imaging device shown in FIG. 3 is a total internal reflection fluorescence microscopic imaging device, which includes an excitation light source 21, a narrow bandpass filter 22, a beam splitter 23, an oil immersion lens 24, and a camera 25. As shown in FIGS. 3 and 4, the excitation light source 21 is configured to emit laser light of a specific wavelength, for example, 635 nm, which passes through the narrow bandpass filter 22 and the oil immersion lens 24 in sequence and is irradiated on a container containing a solution at a specific total internal reflection angle, and total internal reflection occurs at the interface between the container and the solution. Under the irradiation of the laser light, a surface evanescent light field extending into the solution is formed on the surface of the container, and the depth of the surface evanescent light field can be adjusted in a range of 100-250 nm. When the target object containing the unquenched fluorescent group is located in the preset spatial region where the surface evanescent light field is formed, the unquenched fluorescent group is excited by the laser light and emits a strong fluorescent signal. These fluorescent signals are collected by the same oil immersion lens, and after passing through the beam splitter 23 and the narrow bandpass filter 22, the laser light and other stray optical signals can be effectively filtered out, and finally imaged on the camera 25. In FIGS. 3 and 4, the thick arrows represent the direction of the light beam. By taking a picture by using the camera, a sequence of at least one frame of optical images of the preset spatial region acquired in time sequence is obtained for subsequent data analysis. In actual applications, a high-sensitivity and low-noise electronic intensified CCD camera or CMOS camera can be used.

[0140] In the implementation, in order to distinguish the fluorescent signal from the background signal, that is, in order to obtain a maximum fluorescent signal and a minimum background signal, the integration time of the camera for taking each frame of optical image should be close to or appropriately longer than the average residence time of the target object in the preset spatial region. The integration time refers to the time for the camera to collect the light signal at each pixel. In general, the integration time of the camera for taking each frame of optical image ranges from 1 ms to 100 ms.

[0141] In an optional embodiment, before the optical image is acquired, the solution is diluted by using a preset solvent to control the diffusion coefficient and / or other movement speed of the target object, the marker and the target object in the detection process. The dilution multiple can be greater than or equal to 5 times. For example, human serum is diluted by 10-100 times by using standard serum, so as to avoid the difference in viscosity of serum of different people from affecting the diffusion coefficient or movement speed of the target object.

[0142] In step S12, the light-emitting process of the single target object is identified according to the optical image. The light-emitting process of the single target object is the process from entering the preset space region to leaving the preset space region, i.e., the whole process of wandering in the preset space region, and the light-emitting image can be dispersed in one or more frames of the optical image of the preset space region.

[0143] The medical sample is complex. For example, when detecting the fluorescence signal of a target object in serum, the serum itself contains substances that can generate fluorescence, which are often not the detection target. For another example, the aptamer solution contains free fluorescent groups that can also generate fluorescence, which are also not the detection target. In order to retain the light-emitting image of the single target object and exclude the interference of other substances, the light-emitting brightness, the residence time in the preset space region, and the time and space criteria of the optical image can be used for screening.

[0144] In the embodiment, the optical image is divided into a plurality of units in time and space, each unit corresponds to a pixel, and the optical image includes at least two adjacent pixels in space, as shown in FIG. 5, step S12 specifically includes steps S121-S122.

[0145] In step S121, the pixels in the optical image with brightness in the first preset brightness interval are determined as first target pixels.

[0146] The pixel can be a physical camera pixel or a generalized pixel. In a specific implementation, one generalized pixel can include at least two physical pixels in at least one frame of the optical image, or at least two physical pixels in at least two frames of the optical image, or at least two physical pixels in the optical image that are not adjacent in space, or at least two physical pixels in at least two frames of the optical image that are not adjacent in time; two pixels can include at least one physical pixel that is the same in space and time. The values of the brightness, acquisition time, and / or spatial position of the generalized pixel are determined according to the time and space interval covered by the generalized pixel and the brightness distribution of the physical pixels in the time and space interval according to a certain function, wherein different generalized pixels can use different functions.

[0147] In a specific example, the physical pixels of the camera are arranged in a square or rectangular array, and the image of the light-emitting process of a single target object includes more than one physical pixel. In this case, the pixels are defined as an m x n physical pixel array to better identify the light-emitting process of a single target object. In a specific implementation, the pixel can include a square or rectangular array of spatially continuous adjacent physical pixels composed of at least two physical pixels in the optical image, and the brightness of the pixel is determined according to the brightness of each physical pixel in the physical pixel array. For example, the sum of the brightness of each physical pixel can be taken as the brightness of the pixel, or the brightness of each physical pixel can be weighted and summed, and the weighted sum result is taken as the brightness of the pixel.

[0148] In step S122, each first target pixel or each first pixel array is identified as a light-emitting process of a single target object. The first pixel array is an array of first target pixels that are spatially continuous adjacent in time or an array of first target pixels that are spatially continuous adjacent in time and space.

[0149] It should be noted that if a first target pixel has no spatially adjacent first target pixel, the corresponding first pixel array that is spatially continuous adjacent in time includes only the first target pixel. If a first target pixel has no spatially and temporally adjacent first target pixel, the corresponding first pixel array that is spatially and temporally continuous adjacent includes only the first target pixel.

[0150] In the above description, two pixels are adjacent in time if the optical images in which the two pixels are located are adjacent frames in the collection time, or the interval between the collection times of the optical images in which the two pixels are located is less than a first preset value. Two pixels are adjacent in space if the row numbers of the two pixels differ by 1 and the column numbers are the same, or the row numbers of the two pixels are the same and the column numbers differ by 1, or the row and column numbers of the two pixels differ by 1 or less, or the spatial distance between the two pixels is less than a second preset value. Two pixels are adjacent in time and space if the two pixels are the same or adjacent in time and space. If a plurality of pixels are connected to each other through spatially adjacent pixels to form a connected pixel array, the pixels and the pixel array are spatially continuous adjacent. If a plurality of pixels are connected to each other through temporally and spatially adjacent pixels to form a connected pixel array, the pixels and the pixel array are temporally and spatially continuous adjacent.

[0151] In an optional embodiment, the above step S121 is followed by the following steps:

[0152] Step S1212, in response to the first pixel set including any of the first target pixels not satisfying the first preset condition, updating the first target pixel as not belonging to the first target pixel. In this embodiment, by screening out the first target pixel not satisfying the first preset condition, the accuracy of detecting the target object can be improved.

[0153] In an optional embodiment, the first pixel set includes a second pixel array. Specifically, the second pixel array can be composed of the first target pixel and a second target pixel, the second target pixel can include and only include all pixels satisfying a preset brightness condition, being the same in time and being spatially continuous adjacent to the first target pixel so as to make the second pixel array continuous adjacent, and the second target pixel can also include and only include all pixels satisfying the preset brightness condition and being continuous adjacent in time and space. Wherein, the preset brightness condition is that the brightness of the second target pixel in the optical image is within a second preset brightness interval. Wherein, when there is no second target pixel, the second pixel array is equivalent to the first target pixel.

[0154] In a specific implementation, the second preset brightness interval can be set as the same for each pixel, or can be set as different according to the spatial and temporal position relationship of each pixel with the first target pixel.

[0155] It should be noted that the lower limit of the second preset brightness interval can be set to be less than or equal to the lower limit of the first preset brightness interval. In one specific example, the second preset brightness interval is uniformly set as the same as the first preset brightness interval. In another specific example, the second preset brightness interval is greater than or equal to a preset lower limit threshold, wherein the preset lower limit threshold decreases with the increase of the distance from the first target pixel.

[0156] In an optional embodiment, the first preset condition equivalently comprises at least one of the following conditions or a logical combination of several conditions, or a logical combination of the following one or more conditions and other conditions: the number of elements in the second pixel array that are spatially and temporally adjacent to the first target pixel is within a first preset range, the number of elements in the second pixel array that are temporally identical and spatially adjacent to the first target pixel is within a second preset range, the number of elements in the second pixel array that are temporally adjacent and spatially identical to the first target pixel is within a third preset range, the number of elements of the second pixel array is within a fourth preset range, the overall or partial spatial length of the second pixel array is within a fifth preset range, the overall or partial temporal length of the second pixel array is within a sixth preset range, the second pixel array is a subset or proper subset of a first preset array structure, the second pixel array is not a subset or proper subset of a second preset array structure, the second pixel array contains or properly contains a third preset array structure, the second pixel array does not contain or properly contain a fourth preset array structure, the sum of the brightness of each pixel, the weighted sum of the brightness of each pixel, or the function value with the brightness of each pixel as the variable in the second pixel array is within a seventh preset range, and the second pixel array satisfies at least one of the above conditions within a specified frame or a plurality of consecutive frames.

[0157] In another optional embodiment, the first preset condition equivalently comprises at least one of the following conditions or a logical combination of several conditions, or a logical combination of the following one or more conditions and other conditions: the number of elements in the second pixel array that are in the same frame as the first target pixel is greater than or equal to a, where a is a natural number greater than or equal to 2; the number of elements in the second pixel array that are in the same frame as the first target pixel is less than or equal to b, where b is a natural number; the second pixel array that is in the same frame as the first target pixel contains or properly contains a spatially continuous and adjacent pixel array arranged in a preset square or rectangular manner; the maximum spatial length of the second pixel array as a whole that is in the same frame as the first target pixel is less than or equal to f, where f is a natural number; the number of elements in the second pixel array that are spatially identical and temporally adjacent to the first target pixel is greater than or equal to 1 or 2; the number of elements in the second pixel array that are spatially identical and temporally adjacent to the first target pixel is less than or equal to 0 or 1; the overall temporal length of the second pixel array is greater than or equal to g, where g is a natural number greater than or equal to 2; and the overall temporal length of the second pixel array is less than or equal to h, where h is a natural number.

[0158] It should be noted that the steps of determining the first target pixel and the light-emitting process of the single target object, including steps S121, S122 and / or step S1212, can be repeatedly executed multiple times to further screen the determined first target pixel. And each time the execution is repeated, the specific way and / or parameters of the various operations of the division, selection, definition and preset of the pixel, its brightness / collection time / space position, the preset brightness interval, the preset condition, the preset range, the preset array structure, etc. can be changed.

[0159] In an optional embodiment, the target object is the product of the combination of the to-be-tested substance and the marker, and the solution contains non-target objects capable of emitting light in the preset spatial region. The diffusion coefficient or movement speed of the target object is much smaller than that of the non-target object. The non-target object can include at least one of the following: a marker that does not combine with the to-be-tested substance but mistakenly emits light, and a light-emitting substance that falls off from the marker.

[0160] Based on the diffusion coefficient or movement speed of the target object being much smaller than that of the non-target object, the single residence time of the target object in the preset spatial region is much longer than that of the non-target object, so that the total brightness of the single light-emitting process of the target object is much larger than that of the non-target object, thereby achieving the purpose of identifying the single light-emitting process of the target object.

[0161] In a specific example, a molecular beacon based on an aptamer is used to identify the to-be-tested substance cardiac troponin I (cTnI) molecule; the molecular weight of the aptamer is much smaller than that of the cTnI molecule, and the molecular weight of the fluorescent group carried by the former is also smaller. Because the diffusion coefficient or movement speed of an object with a larger molecular weight is smaller, the average residence time of cTnI in the preset spatial region is significantly longer than that of the aptamer and its free fluorescent group. Therefore, by screening the pixels and / or pixel arrays with light-emitting brightness higher than a certain threshold, the strong fluorescent signal of the aptamer combined with cTnI can be retained, and the weak fluorescent signal emitted by substances not combined with cTnI can be excluded.

[0162] In a specific implementation, when the detected solution sample is human serum, the sample is diluted by 10-100 times with standard serum before the optical image is acquired, so as to avoid the difference in the diffusion coefficient or movement speed of the target object formed by the combination of cTnI and the aptamer due to the difference in the viscosity of the serum of different people.

[0163] In another optional embodiment, in order to eliminate or reduce the background brightness difference and / or signal brightness difference of the acquired optical image in different spatial and / or temporal regions, the acquired optical image can be pre-processed by Rolling Ball method, normalization method or other methods before step S12. In the normalization method, the brightness of all pixels is divided by a specific non-uniform brightness distribution. The sources of the above-mentioned background brightness include but are not limited to noise of the measuring device, stray light and light from outside the preset spatial region.

[0164] Step S13, detecting the target according to the light-emitting process of the single target.

[0165] In the embodiment, the behavior of the target moving in the solution includes diffusion movement. The larger the volume of the target formed by the combination of the analyte and the label, the smaller the diffusion coefficient of the target in the solution, and the longer the average length of stay of the target in the preset spatial region, and the higher the brightness of the light-emitting process of the single target, and the less or smaller the frequency of the light-emitting process of the single target. In the specific implementation, the targets with different characteristics can be distinguished according to the length of stay, and the different characteristics include the size, mass, shape and viscous resistance of the target.

[0166] In an optional embodiment of step S13, the concentration or the movement speed of the target is detected according to the frequency of the light-emitting process of the single target.

[0167] In another optional embodiment of step S13, the length of stay of the target in the preset spatial region is detected according to the brightness of the light-emitting process of the single target.

[0168] In another optional embodiment of step S13, the targets with different diffusion coefficients or movement speeds are distinguished according to the brightness of the light-emitting process of the single target. As shown in FIG. 6, the specific steps S131-S132 can include the following steps:

[0169] Step S131, using the labels with different diffusion coefficients or movement speeds to combine with different types of analytes respectively, so that the different types of analytes correspond to the targets with different diffusion coefficients or movement speeds;

[0170] Step S132, distinguishing the targets with different diffusion coefficients or movement speeds according to the brightness of the light-emitting process of the single target, so as to distinguish the different types of analytes.

[0171] In another optional embodiment of step S13, the change of the diffusion coefficient or the movement speed of the target is detected according to the brightness and / or the frequency of the light-emitting process of the single target.

[0172] In an optional embodiment, the target object is a molecule to be detected or a product of the molecule to be detected combined with a label, and the change in diffusion coefficient or movement speed of the target object corresponds to a chemical reaction of the molecule to be detected or a combination or dissociation of the molecule to be detected with other molecules. In this embodiment, when the molecule to be detected undergoes a chemical reaction or a combination, dissociation or other kinetic process with other molecules, the change in molecular weight of the molecule to be detected will cause a change in diffusion coefficient or movement speed, and the change in the number of occurrences, frequency and brightness of the luminescence process caused thereby can be used to detect the kinetic process.

[0173] It should be noted that the brightness of the luminescence process of the single target object is determined according to the brightness distribution of the corresponding interval of the optical image corresponding to the luminescence process of the single target object; for example, if the luminescence process of the single target object corresponds to one pixel of the optical image, the brightness of the luminescence process is determined as the brightness of the pixel, and if the luminescence process of the single target object corresponds to multiple pixels of the optical image, the brightness of the luminescence process is determined as the sum, weighted sum or other function of the brightness of the corresponding pixels.

[0174] In an optional embodiment, the object plane or object surface of the imaging device corresponding to the optical image includes a continuous first sub-region, the first sub-region is included in the preset spatial region, and the luminescence of the single target object occurring at any position of the first sub-region is imaged in the optical image; the area of the first sub-region is greater than or equal to a first preset area.

[0175] In a specific implementation, the first preset area can be 10 times or 100 times the square of the luminescence wavelength of the target object, or can be 5 μm 2 or 50 μm 2 .

[0176] In another optional embodiment, the object plane or object surface of the imaging device corresponding to the optical image includes a continuous second sub-region, and the second sub-region is a regular polygon or a regular polygon with edges discretized by pixels after being imaged by the imaging device, and the area of the second sub-region is greater than or equal to a second preset area; the part of the second sub-region overlapping with the preset spatial region includes a third sub-region, and the ratio of the area of the third sub-region to the area of the second sub-region is greater than or equal to a preset area ratio, and the luminescence of the single target object occurring at any position of the third sub-region is imaged in the optical image.

[0177] In a specific implementation, the preset area ratio can be 1 / 5 or 1 / 10. The second preset area can be 10 times or 50 times the square of the luminescence wavelength of the target object, or can be 5 μm2 Or 25μm 2 .

[0178] In specific implementations, the height range of the aforementioned preset spatial region along the direction perpendicular to the object plane or object curved surface of the aforementioned imaging device can be within the range of [1nm, 10000nm], [10nm, 3000nm], or [20nm, 300nm].

[0179] The following is a specific example illustrating the target detection method provided in this embodiment.

[0180] In this nucleic acid aptamer-based molecular beacon, the fluorescent group used is Alexa Flour 647, the quencher group is Black Hole Quencher 3 (BHQ3), and the nucleic acid aptamer is a DNA sequence that specifically binds to the analyte. The analyte is cTnI, an important myocardial biomarker closely related to the development of myocardial diseases. After binding to cTnI, the molecular beacon forms a target analyte capable of emitting fluorescence in a specific wavelength band upon excitation.

[0181] Fetal bovine serum solution was used as the solution, with a molecular beacon concentration of 1 nM. Gold particles with a diameter of 100 nm were pre-adsorbed onto the surface of the storage container. Imaging these gold particles allowed adjustment of the working distance of the oil-immersion lens in the imaging device. Furthermore, bovine serum albumin (BSA) molecules were used to seal the container surface to prevent the adsorption of the molecular beacon, analyte, target analytes formed by the combination of the molecular beacon and analyte, and fluorescent impurities onto the container surface.

[0182] Specifically, a camera with a 512×512 square array of physical pixels acquires 5000 frames of optical images sequentially over time, with each physical pixel divided into one pixel. A 635nm wavelength laser shines through an oil-immersed lens onto the container surface, forming a surface evanescent light field at the interface between the container and the solution. The area in the surface evanescent light field that is imaged onto the optical image is a preset spatial region. When a target object is within the preset spatial region, it emits fluorescence. Based on a rough estimate, the residence time of a single target object in the preset spatial region is approximately less than 1ms, and the integration time for each frame of fluorescence imaging is set to 20ms. When the concentration of the target object is sufficiently low, the probability of overlapping or coinciding optical images of different targets within the same frame is negligible. In other words, we can achieve the identification and counting of individual target objects entering the preset spatial region.

[0183] Fig. 7 is a schematic diagram showing an image of a single target object actually observed in a fluorescence emission process, which is a part of a frame of optical images. In this experiment, the theoretical fluorescence imaging area of a single target object is slightly larger than a single pixel, accordingly, the optical image of a single target object as shown in Fig. 7 is composed of 3 spatially continuous adjacent brighter pixels. In order to identify the optical image of a single target object and filter out the background and noise signals in the optical image, first, a higher lower limit of brightness is set for the first preset brightness interval, so that only the brightness of the brightest pixel in Fig. 7 is within the first preset brightness interval and is determined as the first target pixel. Second, a lower lower limit of brightness is set for the second preset brightness interval, so that the brightness of the two less bright pixels in Fig. 7 is within the second preset brightness interval and is determined as the second target pixel, thereby forming a second pixel array including 3 spatially continuous adjacent pixels together with the first target pixel. Further, the first preset condition is set as the number of elements of the second pixel array is greater than or equal to 3; if the number of elements of the second pixel array is 1 or 2, it is considered that the first target pixel comes from the background or noise signal, and it is updated as not belonging to the first target pixel. After the above steps, the first target pixel and the second pixel array in Fig. 7 are identified as the fluorescence signal from the target object.

[0184] In a specific implementation, in order to help exclude the case that the target object is adsorbed on the surface of the container and exclude other interference signals, the optical image of a single target object obtained after the above identification steps can be further processed. For example, the first target pixel satisfying the following conditions can be updated as not belonging to the first target pixel: (1) in a frame of optical images, spatially continuous adjacent pixels with brightness greater than a certain brightness threshold are connected together to form a spatial array structure including the first target pixel, which is greater than or equal to 3x3; (2) the first target pixel appears in the same or adjacent spatial position in 3 or more consecutive frames of optical images; (3) the first target pixel has brightness greater than or equal to twice the lower limit of the first preset brightness interval.

[0185] Finally, the first target pixels in 5000 frames of optical images taken by the camera that meet the above requirements are counted, i.e., the fluorescence emission process of a single target object is counted, to obtain the number of single molecule fluorescence emission processes of the cTnI to be detected. Through detection of fetal bovine serum solutions with different cTnI concentrations, we obtain the corresponding relationship between the counting value of the single molecule fluorescence emission process and the cTnI concentration, as shown in Fig. 8, wherein the circular data points and error bars in Fig. 8 are the results of multiple measurements, and the straight line is the linear fitting result. It can be seen that the relationship between the counting value of the single molecule fluorescence emission process and the cTnI concentration is a linear relationship, wherein the R 2= 0.9919. Therefore, the detection method provided in this embodiment can realize reliable and high-sensitivity detection of the concentration of the protein molecules. As shown in FIG. 8, quantitative measurement of the cTnI molecules in the fetal bovine serum solution at a low fM concentration level is realized within a total integration time of only 100 s.

[0186] When detecting a sample with complex components, such as human serum or plasma, it is very important to use a container surface material with good anti-physical adsorption properties, which is to reduce the concentration of the analyte, the label and / or the target in the solution due to adsorption to the container surface, and to reduce the background fluorescence signal from impurities. In another example, a zwitterionic material, i.e., poly (methacrylic acid sulfobetaine) (pSBMA), with stronger anti-physical adsorption ability is used to replace BSA to block the surface of the solution container. For comparison, the surfaces of the solution containers are blocked with pSBMA and BSA respectively, and the single-molecule fluorescence emission processes of multiple undiluted human serum samples are counted; the experiment does not add molecular beacons, and the detected single-molecule fluorescence emission processes belong to background fluorescence signals. The specific detection comparison results are shown in FIG. 9. It can be seen that, compared with the surface of the solution container blocked with BSA, the surface of the solution container blocked with pSBMA can significantly reduce the background fluorescence signal.

[0187] The embodiment also provides a detection device for a target in a solution, as shown in FIG. 10, which comprises a first acquisition module 61, a luminescence recognition module 62 and a first detection module 63.

[0188] The first acquisition module 61 is used to acquire at least one optical image of a preset spatial region acquired in time sequence; wherein the preset spatial region is included in a spatial region at a preset local optical field in a solution, the solution comprises at least one target, the target is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target in the preset spatial region produces luminescence.

[0189] The luminescence recognition module 62 is used to recognize the luminescence process of a single target according to the optical image; wherein the luminescence process of the single target is the process of the single target from entering the preset spatial region to leaving the preset spatial region.

[0190] The first detection module 63 is used to detect the target according to the luminescence process of the single target.

[0191] In an optional implementation, the first detection module is specifically used to detect the concentration or the moving speed of the target according to the number or frequency of the luminescence process of the single target;

[0192] In an optional implementation, the first detection module is specifically configured to detect the residence time of the target object in the preset spatial region according to the brightness of the light emission process of the single target object.

[0193] In an optional implementation, the first detection module is specifically configured to distinguish target objects with different diffusion coefficients or motion speeds according to the brightness of the light emission process of the single target object. In a specific example, the target object is a product of the binding of the analyte and the label, and the first detection module is specifically configured to use labels with different diffusion coefficients or motion speeds to bind different types of analytes, so that different types of analytes correspond to target objects with different diffusion coefficients or motion speeds; and distinguish target objects with different diffusion coefficients or motion speeds according to the brightness of the light emission process of the single target object, thereby distinguishing different types of analytes.

[0194] In an optional implementation, the first detection module is specifically configured to detect the diffusion coefficient or motion speed change of the target object according to the brightness and / or the number of occurrences or frequency of the light emission process of the single target object.

[0195] It should be noted that the brightness of the light emission process of the single target object is determined according to the brightness distribution of the corresponding interval of the optical image corresponding to the light emission process of the single target object.

[0196] In an optional implementation, the optical image is divided into a plurality of units in time and space, each unit corresponds to a pixel, and the optical image includes at least two spatially adjacent pixels. The light emission identification module is specifically configured to determine the pixels in the optical image with brightness in a first preset brightness interval as first target pixels; and identify each first target pixel or each first pixel array as a light emission process of a single target object; wherein the first pixel array is an array of first target pixels that are the same in time and continuously adjacent in space, or an array of first target pixels that are continuously adjacent in time and space.

[0197] In an optional implementation, after the light emission identification module determines the pixels in the optical image with brightness in a first preset brightness interval as first target pixels, in response to a first pixel set including any first target pixel not meeting a first preset condition, the first target pixel is updated to not belong to the first target pixel.

[0198] It should be noted that the detection device of the target object in the solution in this embodiment can be a separate chip, a chip module or an electronic device, or a chip or a chip module integrated in an electronic device.

[0199] The modules / units included in the solution target detection device described in this embodiment can be software modules / units, hardware modules / units, or partly software modules / units and partly hardware modules / units.

[0200] Embodiment 2

[0201] FIG. 11 is a flowchart of a solution target detection method provided in this embodiment, which can be executed by a solution target detection device, which can be implemented in software and / or hardware, and can be part or all of an electronic device.

[0202] The solution target detection method provided in this embodiment is described below with an electronic device as the execution subject. As shown in FIG. 11, the solution target detection method provided in this embodiment can include the following steps S21-S22:

[0203] In step S21, at least one optical image of a preset spatial region collected in time sequence is acquired. The preset spatial region is included in a spatial region where a preset local optical field in a solution is located. The solution includes at least one target object. The target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence.

[0204] The specific implementation of step S21 can refer to step S11 in Embodiment 1.

[0205] In step S22, the concentration of the target object or the diffusion coefficient or the change in the moving speed of the target object is detected according to a feature of a pixel brightness distribution function of the optical image. The pixel brightness distribution function is a function with the brightness of a pixel in the optical image as the independent variable and the number of pixels in a unit brightness range as the dependent variable.

[0206] The feature of the pixel brightness distribution function can include at least one of the following: the sum of the brightness of all pixels, the centroid of the pixel brightness distribution function, the independent variable and / or the dependent variable at the peak value of the pixel brightness distribution function, the area of the pixel brightness distribution function with the independent variable in a first range, and the sum of the brightness of all pixels with the independent variable in a second range. In a specific implementation, the first range can be higher than a first threshold, and the second range can be higher than a second threshold.

[0207] FIGS. 12 and 13 show pixel intensity distribution functions of different cTnl sample concentrations in histogram form, respectively. As can be seen from FIGS. 12 and 13, as the cTnl sample concentration increases from 10 pM to 100 pM, the centroid of the pixel intensity distribution function moves towards high pixel intensity, the position of the peak of the pixel intensity distribution function moves to the lower right, and the area of the pixel intensity distribution function and the sum of pixel intensities in the range of greater than 1000 are also changed.

[0208] In an optional embodiment, in order to eliminate or reduce the background intensity, the difference in background intensity, and / or the difference in average luminescence intensity of the acquired optical image in different spatial and / or temporal regions, the acquired optical image can be pre-processed using the Rolling Ball method, normalization method, background removal method, or other methods before step S22. Among them, the normalization method divides the brightness of all pixels by a specific non-uniform brightness distribution. The sources of the above-mentioned background intensity include but are not limited to non-target fluorescent light, measurement device noise, stray light, and luminescence from non-pre-set spatial regions, etc.

[0209] In an optional embodiment, the target object is the product of the combination of the to-be-measured object and the label, and there is a non-target object in the solution that can produce luminescence in the pre-set spatial region. The non-target object can include at least one of the following: a label that does not combine with the to-be-measured object but erroneously produces luminescence, and a luminescent substance that falls off from the label; wherein the diffusion coefficient or movement speed of the target object is much smaller than that of the non-target object. In this embodiment, by setting the diffusion coefficient or movement speed of the target object to be much smaller than that of the non-target object, the target object can have a long enough residence time relative to the non-target object in the pre-set spatial region, and then under the condition that the integration time of the camera collecting the luminescence image is appropriate, the luminescence signal of the target object can be strong enough relative to the non-target object, thereby facilitating the differentiation of the luminescence signal of the target object by brightness.

[0210] In an optional embodiment, the target object is a to-be-measured molecule or the product of the combination of the to-be-measured molecule and the label, and the change in the diffusion coefficient or movement speed of the target object in step S22 corresponds to the to-be-measured molecule undergoing a chemical reaction or combining or dissociating with other molecules. In this embodiment, when the to-be-measured molecule undergoes a chemical reaction or combines or dissociates with other molecules, the change in the molecular weight of the target object will cause a change in its diffusion coefficient or movement speed, and the pixel intensity change caused thereby can be used to detect these kinetic processes.

[0211] In an optional embodiment, the object plane or object surface of the imaging device corresponding to the optical image comprises a continuous first sub-region, the first sub-region is included in the preset spatial region, and the light emitted by a single target object occurring at any position of the first sub-region is imaged in the optical image; wherein the area of the first sub-region is greater than or equal to a first preset area.

[0212] In a specific implementation, the first preset area can be 10 times or 100 times the square of the light wavelength of the light emitted by the target object, and can also be 5 μm 2 or 50 μm 2 .

[0213] In another optional embodiment, the object plane or object surface of the imaging device corresponding to the optical image comprises a continuous second sub-region, and the second sub-region is a regular polygon or a regular polygon with edges discretized by pixels after imaging by the imaging device, and the area of the second sub-region is greater than or equal to a second preset area; the part of the second sub-region overlapping with the preset spatial region comprises a third sub-region, and the ratio of the area of the third sub-region to the area of the second sub-region is greater than or equal to a preset area ratio, and the light emitted by a single target object occurring at any position of the third sub-region is imaged in the optical image.

[0214] In a specific implementation, the preset area ratio can be 1 / 5 or 1 / 10. The second preset area can be 10 times or 50 times the square of the light wavelength of the light emitted by the target object, and can also be 5 μm 2 or 25 μm 2 .

[0215] The embodiment also provides a detection device for a target object in a solution, as shown in FIG. 14, comprising a second acquisition module 71 and a second detection module 72.

[0216] The second acquisition module 71 is configured to acquire at least one frame of optical image of a preset spatial region acquired in time sequence; wherein the preset spatial region is included in a spatial region at which a preset local optical field in a solution is located, the solution comprises at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or staying on any solid surface, and the target object in the preset spatial region emits light.

[0217] The second detection module 72 is configured to detect the concentration of the target object or the diffusion coefficient or the change of the movement speed of the target object according to the characteristics of the pixel brightness distribution function of the optical image; wherein the pixel brightness distribution function is a function with the brightness of the pixel in the optical image as the independent variable and the number of pixels in a unit brightness range as the dependent variable.

[0218] It should be noted that the solution target detection device in this embodiment can be a single chip, a chip module or an electronic device, or a chip or a chip module integrated in an electronic device.

[0219] The various modules / units included in the solution target detection device described in this embodiment can be software modules / units, hardware modules / units, or part software modules / units and part hardware modules / units.

[0220] Embodiment 3

[0221] This embodiment provides a solution target detection system, as shown in FIG. 15, which includes an excitation device 81, an imaging device 82 and a detection device 83. The excitation device 81 is used to excite the target in a preset spatial region in the solution to generate luminescence; the preset spatial region is included in a spatial region at a preset local optical field in the solution, the solution includes at least one target, the target is dispersed and moves in the solution without being fixed, combined or staying on any solid surface, and the target in the preset spatial region generates luminescence. The imaging device 82 is used to acquire at least one frame of optical image of the preset spatial region in time sequence. The detection device 83 is used to execute the steps of the detection method provided in Embodiment 1 or 2.

[0222] In an optional implementation, the excitation device includes an excitation light source, and the preset local optical field is a surface plasmon polariton field or a surface plasmon field formed at the interface between the solution and a solid object when light emitted by the excitation light source is shot to the interface; wherein the interface between the solid object and the solution is provided with a material resistant to adsorption of the target.

[0223] Embodiment 4

[0224] FIG. 16 is a structural schematic diagram of an electronic device provided in this embodiment. The electronic device includes at least one processor and a memory in communication connection with the at least one processor. Wherein the memory stores a computer program which can be run by the at least one processor, and the computer program is executed by the at least one processor to enable the at least one processor to execute the detection method provided in Embodiment 1 or 2. The electronic device 3 shown in FIG. 16 is only an example, and should not bring any limitation to the functions and use range of the embodiments of the present application.

[0225] The components of the electronic device 3 can include but are not limited to the above-mentioned at least one processor 4, the above-mentioned at least one memory 5, a bus 6 connecting different system components including the memory 5 and the processor 4.

[0226] The bus 6 includes a data bus, an address bus and a control bus.

[0227] The memory 5 can include volatile memory, such as random access memory (RAM) 51 and / or cache memory 52, and / or non-volatile memory, such as read only memory (ROM) 53.

[0228] The memory 5 can also include a program / utility 55 having a set (at least one) of program modules 54, including but not limited to an operating system, one or more application programs, other program modules, and program data, each of which

[0229] The processor 4, through the running of the computer program stored in the memory 5, executes various function applications and data processing, such as the detection method provided in Embodiment 1 or 2.

[0230] The electronic device 3 can also communicate with one or more external devices 7, such as a keyboard, a pointing device, etc. through an input / output (I / O) interface 8. Further, the electronic device 3 can communicate with one or more networks, such as a local area network (LAN), a wide area network (WAN), and / or a public network, such as the Internet, through a network adapter 9. As shown in FIG. 16, the network adapter 9 communicates with the other modules of the electronic device 3 through the bus 6. It should be appreciated that although not shown in FIG. 16, other hardware and / or software modules can be used in conjunction with the electronic device 3, including but not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID (Redundant Array of Independent Disks) systems, tape drives, and data backup storage systems, etc.

[0231] It should be noted that although several units / modules or sub-units / modules of the electronic device are mentioned in the foregoing detailed description, such division is merely exemplary and not mandatory. In fact, according to the embodiments of the present application, the features and functions of two or more units / modules described above can be embodied in one unit / module. Conversely, the features and functions of one unit / module described above can be further divided into multiple units / modules.

[0232] Embodiment 5

[0233] The present embodiment provides a computer readable storage medium storing a computer program, which, when executed by a processor, implements the detection method provided in Embodiment 1 or 2.

[0234] Wherein, the readable storage medium can include but is not limited to: portable disc, hard disk, random access memory, read only memory, erasable programmable read only memory, optical storage device, magnetic storage device or any suitable combination of the above.

[0235] In possible implementation manners, the present application can also be implemented in the form of a computer program product, which comprises a computer program for implementing the steps of the detection method provided in Embodiments 1 or 2 when executed by a processor.

[0236] The computer program for executing the present application can be written in any combination of one or more programming languages, and can be executed completely on an electronic device, partially on an electronic device, as an independent software package, partially on an electronic device and partially on a remote device, or completely on a remote device.

[0237] Although the specific implementation manners of the present application are described above, those skilled in the art should understand that this is only an example, and the protection scope of the present application is defined by the appended claims. Those skilled in the art can make various changes, modifications or combinations to these implementation manners without departing from the principles and essence of the present application, and these changes, modifications and combinations all fall within the protection scope of the present application.

Claims

1. A method for detecting a target analyte in a solution, characterized in that, The method comprises the following steps: acquiring at least one optical image of a preset spatial region collected in time sequence; wherein the preset spatial region is included in a spatial region where a preset local optical field is located in a solution, the solution includes at least one target object, the target object is dispersed and moves in the solution without being fixed, combined or staying on any solid surface, and the target object in the preset spatial region generates luminescence; identifying a luminescence process of a single target object according to the optical image; wherein the luminescence process of the single target object is a process of the single target object from entering the preset spatial region to leaving the preset spatial region; detecting the target object according to the luminescence process of the single target object.

2. The detection method of claim 1, wherein, The step of detecting the target object according to the luminescence process of the single target object specifically comprises: detecting the concentration or the moving speed of the target object according to the number or frequency of the luminescence process of the single target object; and / or, detecting the residence time of the target object in the preset spatial region according to the brightness of the luminescence process of the single target object; and / or, distinguishing the target objects with different diffusion coefficients or moving speeds according to the brightness of the luminescence process of the single target object; and / or, detecting the change of the diffusion coefficient or the moving speed of the target object according to the brightness and / or the number or frequency of the luminescence process of the single target object; wherein the brightness of the luminescence process of the single target object is determined according to the brightness distribution of the corresponding interval of the optical image corresponding to the luminescence process of the single target object.

3. The detection method of claim 2, wherein, The target object is a product of the combination of a to-be-tested object and a label, and the step of distinguishing the target objects with different diffusion coefficients or moving speeds according to the brightness of the luminescence process of the single target object specifically comprises: adopting labels with different diffusion coefficients or moving speeds to combine different types of to-be-tested objects, so that the different types of to-be-tested objects correspond to the target objects with different diffusion coefficients or moving speeds; distinguishing the different types of to-be-tested objects according to the brightness of the luminescence process of the target objects with different diffusion coefficients or moving speeds.

4. The detection method according to any one of claims 1 to 3, wherein The optical image is divided into a plurality of units in time and space, each unit corresponds to a pixel, and the optical image includes at least two spatially adjacent pixels; and the step of identifying the luminescence process of the single target object according to the optical image specifically comprises: determining the pixels with brightness in a first preset brightness interval in the optical image as first target pixels; identifying each first target pixel or each first pixel array as a luminescence process of a single target object; wherein the first pixel array is an array formed by the first target pixels which are the same in time and continuously adjacent in space, or an array formed by the first target pixels which are continuously adjacent in time and space; wherein, two pixels are adjacent in time, which means that the optical images where the two pixels are located are adjacent frames in collection time, or the interval of the collection time of the optical images where the two pixels are located is less than a first preset value; Two pixels are spatially adjacent if the row number of one pixel is one more than that of the other pixel and the column number of the two pixels is the same, or the row number of one pixel is the same as that of the other pixel and the column number of the two pixels is one more than the other, or the row number and the column number of the two pixels are both within one, or the spatial distance between the two pixels is less than a second preset value; Two pixels are spatio-temporally adjacent if the two pixels are the same or adjacent in time and space; If a plurality of pixels are connected to each other through spatially adjacent pixels to form a connected pixel array, the pixels and the pixel array are spatially continuously adjacent; If a plurality of pixels are connected to each other through spatio-temporally adjacent pixels to form a connected pixel array, the pixels and the pixel array are spatio-temporally continuously adjacent.

5. The detection method of claim 4, wherein, After the step of determining the pixels in the optical image whose brightness is within a first preset brightness interval as first target pixels, the method further comprises: updating the first target pixels to not belong to the first target pixels in response to a first pixel set comprising any of the first target pixels not satisfying a first preset condition.

6. The detection method of claim 5, wherein, The first pixel set comprises a second pixel array; wherein the second pixel array is composed of the first target pixels and second target pixels, and the second target pixels comprise and only comprise all pixels satisfying a preset brightness condition, being the same in time and spatially making the second pixel array continuously adjacent to the first target pixels; or, the second pixel array is composed of the first target pixels and second target pixels, and the second target pixels comprise and only comprise all pixels satisfying a preset brightness condition and being spatio-temporally making the second pixel array continuously adjacent; the preset brightness condition is that the brightness of the second target pixels in the optical image is within a second preset brightness interval; wherein, when there is no second target pixel, the second pixel array is equivalent to the first target pixel.

7. The detection method of claim 6, wherein, The first preset condition equivalently comprises at least one of the following conditions or a logical combination of a plurality of conditions, or a logical combination of one or more of the following conditions and other conditions: the number of elements in the second pixel array which are spatio-temporally adjacent to the first target pixels is within a first preset range; the number of elements in the second pixel array which are the same in time and spatially adjacent to the first target pixels is within a second preset range; the number of elements in the second pixel array which are adjacent in time and the same in space to the first target pixels is within a third preset range; the number of elements in the second pixel array is within a fourth preset range; the overall or partial spatial length of the second pixel array is within a fifth preset range; the overall or partial time length of the second pixel array is within a sixth preset range; the second pixel array is a subset or proper subset of a first preset array structure; the second pixel array is not a subset or proper subset of a second preset array structure; the second pixel array contains or properly contains a third preset array structure; the second pixel array does not contain or properly contain a fourth preset array structure; the sum of the brightness of each pixel in the second pixel array, the weighted sum of the brightness of each pixel, or the function value with the brightness of each pixel as the variable is within a seventh preset range; In a specified frame or a plurality of consecutive frames, the second pixel array satisfies at least one of the above conditions.

8. The detection method of claim 6, wherein, The first preset condition equivalently includes at least one of the following conditions or a logical combination of a plurality of conditions, or a logical combination of one or more of the following conditions and other conditions: The number of elements of the second pixel array in the same frame as the first target pixel is greater than or equal to a, where a is a natural number greater than or equal to 2; The number of elements of the second pixel array in the same frame as the first target pixel is less than or equal to b, where b is a natural number; The second pixel array in the same frame as the first target pixel contains or is contained in a preset square or rectangular arrangement of spatially continuous adjacent pixel arrays; The maximum spatial length of the entire second pixel array in the same frame as the first target pixel is less than or equal to f, where f is a natural number; The number of elements of the second pixel array that are spatially identical to the first target pixel and temporally adjacent to the first target pixel is greater than or equal to 1 or 2; The number of elements of the second pixel array that are spatially identical to the first target pixel and temporally adjacent to the first target pixel is less than or equal to 0 or 1; The overall time length of the second pixel array is greater than or equal to g, where g is a natural number greater than or equal to 2; The overall time length of the second pixel array is less than or equal to h, where h is a natural number.

9. The detection method of any one of claims 4-8, wherein: At least one of the pixels includes at least two physical pixels in at least one of the optical images; Or, at least one of the pixels includes at least two physical pixels in at least two optical images; Or, at least one of the pixels includes at least two physical pixels that are not spatially adjacent in the optical image; Or, at least one of the pixels includes at least two physical pixels in at least two optical images that are not temporally adjacent; Or, at least two of the pixels include at least one physical pixel that is spatially and temporally identical; The values of the brightness, acquisition time, and / or spatial position of the pixel are determined according to the spatiotemporal interval covered by the pixel and the brightness distribution of the physical pixels in the spatiotemporal interval.

10. The assay of any one of claims 4-8, wherein, The pixel includes a square or rectangular arrangement of spatially continuous adjacent physical pixel arrays composed of at least two physical pixels in the optical image, and the brightness of the pixel is determined according to the brightness of each physical pixel in the physical pixel array.

11. The assay of any one of claims 1-8, wherein, The target object is an object capable of producing luminescence or luminescence of a specific light wave band in the preset spatial region formed by specific binding or reaction of the test object and the label in the solution.

12. The detection method of claim 11, wherein, The test object includes at least one of the following: chemical molecules, protein molecules, nucleic acid molecules, and other biological molecules.

13. The method of claim 11, wherein the detecting is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. The label includes at least one of the following: a molecule with a fluorescent label, a molecular beacon based on a nucleic acid aptamer, a molecule that produces fluorescence resonance energy transfer after binding with the test object, and a substance capable of cutting other substances to produce fluorescence.

14. A method for detecting a target analyte in a solution, characterized in that, The method includes the following steps: acquire at least one optical image of a preset spatial region collected in time sequence; wherein the preset spatial region is included in a spatial region where a preset local optical field is located in a solution, the solution includes at least one target object, the target object is dispersed and moves in the solution without being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; detect the concentration of the target object or the diffusion coefficient or the change of the moving speed of the target object according to the characteristics of the pixel brightness distribution function of the optical image; wherein the pixel brightness distribution function is a function with the brightness of the pixel in the optical image as the independent variable and the number of pixels in the unit brightness range as the dependent variable.

15. The detection method as described in claim 14, characterized in that, The characteristics of the pixel brightness distribution function include at least one of the following: the sum of the brightness of all pixels, the centroid of the pixel brightness distribution function, the independent variable and / or dependent variable at the peak value of the pixel brightness distribution function, the area of the pixel brightness distribution function with the independent variable in the first range, and the sum of the brightness of all pixels with the independent variable in the second range.

16. The assay of claim 1 or 14, wherein, The object plane or object surface of the imaging device corresponding to the optical image includes a continuous first sub-region, the first sub-region is included in the preset spatial region, and the luminescence of a single target object occurring at any position in the first sub-region is imaged in the optical image. The area of the first sub-region is greater than or equal to a first preset area.

17. The assay method of claim 16, wherein, The first preset area is 10 times or 100 times of the square of the light-emitting wavelength of the target object, or the first preset area is 5 μm 2 or 50 μm 2 .

18. The assay of claim 1 or 14, wherein, The object plane or object surface of the imaging device corresponding to the optical image includes a continuous second sub-region, and the second sub-region is a regular polygon or a regular polygon with pixel discretization after imaging by the imaging device, and the area of the second sub-region is greater than or equal to a second preset area. The part of the second sub-region overlapping with the preset spatial region includes a third sub-region, and the ratio of the area of the third sub-region to the area of the second sub-region is greater than or equal to a preset area ratio, and the luminescence of a single target object occurring at any position in the third sub-region is imaged in the optical image.

19. The assay method of claim 18, wherein, The preset area ratio is 1 / 5 or 1 / 10, and / or The second preset area is 10 times or 50 times of the square of the light-emitting wavelength of the target object, or the second preset area is 5 μm 2 or 25 μm 2 .

20. The assay of claim 1 or 14, wherein The preset local optical field includes at least one of the following: surface evanescent optical field at the interface between optical material and the solution, surface plasmon optical field at the interface between metal material and the solution; The interface between the optical material or metal material and the solution is provided with a material resistant to adsorption of the target object.

21. The assay of claim 1 or 14 wherein, The target object is the product of the combination of the to-be-tested object and the label, and the solution contains a non-target object capable of generating luminescence in the preset spatial region; The non-target object includes at least one of the following: a label that does not combine with the to-be-tested object but mistakenly generates luminescence, a luminescent substance that falls off from the label; The diffusion coefficient or the moving speed of the target object is much smaller than that of the non-target object.

22. The assay method of claim 2 or 14, wherein The target object is a to-be-tested molecule or the product of the combination of the to-be-tested molecule and the label, and the change of the diffusion coefficient or the moving speed of the target object corresponds to the combination or dissociation of the to-be-tested molecule and other molecules.

23. A device for detecting a target in a solution, comprising: It includes: The first acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; wherein the preset spatial region is a spatial region at a preset local optical field in a solution, the solution contains at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; The luminescence identification module is configured to identify a luminescence process of a single target object according to the optical images; wherein the luminescence process of the single target object is a process from entering the preset spatial region to leaving the preset spatial region of the single target object; The first detection module is configured to detect the target object according to the luminescence process of the single target object.

24. A device for detecting a target in a solution, comprising: The second acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; wherein the preset spatial region is a spatial region at a preset local optical field in a solution, the solution contains at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; The second detection module is configured to detect a concentration of the target object or a diffusion coefficient or a change in a moving speed of the target object according to a feature of a pixel brightness distribution function of the optical images; wherein the pixel brightness distribution function is a function with brightness of a pixel in the optical images as an independent variable and with a number of pixels in a unit brightness range as a dependent variable. The processor executes the computer program to implement the detection method of any one of claims 1-22.

25. An electronic device comprising a memory, a processor, and a computer program stored on the memory and executable on the processor, wherein, The computer program is executed by the processor to implement the detection method of any one of claims 1-22.

26. A computer readable storage medium having stored thereon a computer program, characterized in that, The computer program is executed by the processor to implement the steps of the detection method of any one of claims 1-22.

27. A computer program product comprising a computer program, characterized in that, The first acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; wherein the preset spatial region is a spatial region at a preset local optical field in a solution, the solution contains at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; 28. A system for detecting a target in a solution, the system comprising: The luminescence identification module is configured to identify a luminescence process of a single target object according to the optical images; wherein the luminescence process of the single target object is a process from entering the preset spatial region to leaving the preset spatial region of the single target object; The first detection module is configured to detect the target object according to the luminescence process of the single target object. The second acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; wherein the preset spatial region is a spatial region at a preset local optical field in a solution, the solution contains at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; The second detection module is configured to detect a concentration of the target object or a diffusion coefficient or a change in a moving speed of the target object according to a feature of a pixel brightness distribution function of the optical images; wherein the pixel brightness distribution function is a function with brightness of a pixel in the optical images as an independent variable and with a number of pixels in a unit brightness range as a dependent variable. The processor executes the computer program to implement the detection method of any one of claims 1-22.

29. The detection system of claim 28, wherein, The computer program is executed by the processor to implement the detection method of any one of claims 1-22. The computer program is executed by the processor to implement the steps of the detection method of any one of claims 1-22. The first acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; wherein the preset spatial region is a spatial region at a preset local optical field in a solution, the solution contains at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; The luminescence identification module is configured to identify a luminescence process of a single target object according to the optical images; wherein the luminescence process of the single target object is a process from entering the preset spatial region to leaving the preset spatial region of the single target object; The first detection module is configured to detect the target object according to the luminescence process of the single target object. The second acquisition module is configured to acquire at least one frame of optical images of a preset spatial region collected in time sequence; wherein the preset spatial region is a spatial region at a preset local optical field in a solution, the solution contains at least one target object, the target object is dispersed and moves in the solution rather than being fixed, combined or stayed on any solid surface, and the target object in the preset spatial region generates luminescence; The second detection module is configured to detect a concentration of the target object or a diffusion coefficient or a change in a moving speed of the target object according to a feature of a pixel brightness distribution function of the optical images; wherein the pixel brightness distribution function is a function with brightness of a pixel in the optical images as an independent variable and with a number of pixels in a unit brightness range as a dependent variable.

Images