Heterogeneous integrated single-molecule optical immunodetection chip and preparation method thereof

Abstract

The invention discloses a heterogeneous integrated single-molecule optical immunodetection chip and a preparation method thereof.The heterogeneous integrated single-molecule optical immunodetection chip comprises a micro-fluidic chip and a probe, the micro-fluidic chip is provided with a sample adding hole, a liquid flow channel communicated with the sample adding hole and a plurality of micron holes communicated with the liquid flow channel, and the probe is arranged in the micro-fluidic chip; each micrometer hole is communicated with a microchannel for liquid circulation, the probe comprises a rare earth light-emitting micrometer crystal modified with a primary antibody and a quenching agent modified with a secondary antibody, and photons emitted by at least one wave band of the rare earth light-emitting micrometer crystal can be quenched by the quenching agent. And each micron hole can accommodate at most one rare earth light-emitting micron crystal. According to the present invention, the detection can be improved by several times, the detection range of the to-be-detected molecule is improved, the chemical stability and the luminescence stability are high, and the method can be used for the one-time detection and the post review of the detection result.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a heterogeneous integrated single-molecule optical immunoassay chip and a preparation method thereof. Background Art

[0002] Optical sensing biochip is a microchip that integrates optical sensors and biochemical analyzers. It is mainly used to detect and analyze target molecules in biological samples and provide highly sensitive and selective detection results.

[0003] In the process of single-molecule immunoassay of biological molecules, Simoa technology is representative. Simoa technology basically includes: (1) incubating magnetic beads coated with primary antibodies with samples in containers such as EP tubes. The primary antibodies can capture the analyte in the sample, such as antigens, and thus capture the antigens onto the magnetic beads; (2) washing the magnetic beads, adding biotin-labeled secondary antibodies and avidin-coupled enzymes to obtain magnetic bead complexes, and coupling the enzymes to the magnetic beads. The coupled enzyme content is proportional to the antigen content; (3) washing the magnetic beads, and loading the magnetic beads onto the chip array. There are tens of thousands to hundreds of thousands of micrometer holes distributed on the array. The pore size and depth of each micrometer hole can only accommodate one magnetic bead. (4) After the magnetic beads are loaded into the micrometer holes, the enzyme-catalyzed luminescent substrate is dripped onto the array, so that the enzyme-coupled magnetic beads and the luminescent substrate are simultaneously present in the micrometer holes on the array. (5) After a period of catalysis, the CCD camera is used to capture the fluorescence signal in the microwell that exceeds the threshold and is marked as "yes" or 1, while the signal that does not exceed the threshold is marked as "no" or 0. Based on the Poisson distribution theory, the proportion of "yes" is calculated to achieve digital quantification of the molecules of the analyte.

[0004] In Simoa technology, the chip is only used as a carrier to hold luminescent substances, which can be used by the camera to capture and read the luminescence value (0 or 1) of each micron hole. Only two values, 0 or 1, are generated in each hole, and the data in the hole can be regarded as a "binary" variable. In addition, Simoa technology uses enzyme-catalyzed bioluminescence or dyes, which have the problem of unstable luminescence. Dyes have photobleaching phenomenon, and bioluminescence is irreversible over time, which will lead to inaccurate test results. Summary of the invention

[0005] Based on this, it is necessary to provide a heterogeneous integrated single molecule optical immunoassay chip. The heterogeneous integrated single molecule optical immunoassay chip of the present invention can improve detection several times, increase the detection range of the molecules to be tested, and has very high chemical and luminescent stability. It can be used not only for one-time detection but also for post-test review of the test results.

[0006] An embodiment of the present application provides a heterogeneously integrated single-molecule optical immunoassay chip.

[0007] A heterogeneous integrated single-molecule optical immunoassay chip comprises a microfluidic chip and a probe. The microfluidic chip is provided with a sample loading hole, a liquid flow channel communicating with the sample loading hole and a plurality of micrometer holes communicating with the liquid flow channel. Each of the micrometer holes is respectively connected to a microchannel for liquid circulation. The probe comprises a rare earth luminescent micrometer crystal modified with a primary antibody and a quencher modified with a secondary antibody. Photons emitted by at least one wavelength band of the rare earth luminescent micrometer crystal can be quenched by the quencher. Each of the micrometer holes can accommodate at most one rare earth luminescent micrometer crystal.

[0008] In some embodiments, the radial cross-sectional width of the micron hole is larger than the maximum particle size of one of the rare earth luminescent micron crystals and smaller than the maximum particle sizes of two of the rare earth luminescent micron crystals, so that each of the micron holes can accommodate one of the probes.

[0009] In some embodiments, the plurality of micrometer holes are distributed in an array on the microfluidic chip, and each of the micrometer holes is respectively provided with a number.

[0010] In some of the embodiments, the maximum particle size of the rare earth luminescent micron crystals is 1 μm to 10 μm.

[0011] In some embodiments, the quencher includes one or more of nano-iron oxide microspheres, nano-gold microspheres, and dye microspheres.

[0012] In some embodiments, the microfluidic chip includes a base plate and a cover plate, and the cover plate can be fitted with the base plate. The base plate is provided with the micron hole, the liquid flow channel and the microchannel extending along the length or width direction of the base plate, and the cover plate is provided with the sample loading hole.

[0013] In some of the embodiments, the depth of the micropores and the depth of the microchannels are both greater than the maximum particle size of the rare earth luminescent micron crystals.

[0014] In some embodiments, the depth of the micropores is equal to the depth of the microchannels.

[0015] In some embodiments, the width of the microchannel is smaller than the minimum particle size of the rare earth luminescent micron crystals.

[0016] In some embodiments, adjacent micropores are connected to each other through the microchannels.

[0017] In some embodiments, the micron hole is a rectangular hole, and the side length of the micron hole is 1 μm to 15 μm.

[0018] In some embodiments, the micropore is a circular hole, and the diameter of the micropore is 1 μm to 15 μm.

[0019] In some embodiments, there are multiple liquid flow channels, and the multiple liquid flow channels are all connected to the sample loading hole, and the multiple liquid flow channels are respectively connected to different microchannels.

[0020] In some embodiments, the microchannels are interconnected to enable the micropores to be interconnected.

[0021] In some embodiments, the microfluidic chip includes a cover plate, an upper plate and a lower plate, the upper plate includes a plurality of the micron holes, the lower plate includes a plurality of the microchannels, the lower plate and the upper plate can be fitted together, the microchannels extend along the thickness direction of the lower plate, the micron holes can connect at least one of the microchannels, the cover plate is movably connected to the upper plate and can close each of the micron holes, a plurality of liquid adding holes are arranged on the cover plate, and when the cover plate is covered on the upper plate, each of the liquid adding holes can be connected to the micron holes respectively.

[0022] In some of the embodiments, the upper plate and the lower plate can be assembled by a snap-fit ​​structure.

[0023] In some embodiments, the upper plate and the lower plate are respectively provided with assembly holes, and the upper plate and the lower plate are assembled by fasteners passing through the assembly holes.

[0024] An embodiment of the present application also provides a method for preparing a heterogeneously integrated single-molecule optical immunoassay chip.

[0025] A method for preparing a heterogeneous integrated single-molecule optical immunoassay chip comprises the following steps:

[0026] A liquid flow channel, a plurality of micrometer holes communicating with the liquid flow channel, and a microchannel communicating with the micrometer holes are fabricated on a first glass wafer, wherein the microchannel extends along the length or width direction of the first glass wafer, or the microchannel extends along the thickness direction of the first glass wafer; wherein the liquid flow channel, the micrometer holes, and the microchannel are fabricated by photolithography etching and laser processing;

[0027] Furthermore, a sample addition hole is made on the second glass wafer, and the second glass wafer is bonded to the first glass wafer to form a microfluidic chip.

[0028] The above-mentioned heterogeneous integrated single-molecule optical immunoassay chip can improve the detection several times and enhance the detection range of the molecules to be tested. The chemical and luminescence stability are very high and it can be used not only for one-time detection but also for post-test review of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0030] In order to more completely understand the present application and its beneficial effects, the following description will be given in conjunction with the accompanying drawings. In the following description, the same reference numerals represent the same parts.

[0031] Figure 1 This is a schematic diagram of a heterogeneous integrated single-molecule optical immunoassay chip according to an embodiment of the present invention;

[0032] Figure 2 It is a side schematic diagram of a heterogeneous integrated single-molecule optical immunoassay chip according to another embodiment of the present invention;

[0033] Figure 3 It is a side schematic diagram of a heterogeneous integrated single-molecule optical immunoassay chip according to another embodiment of the present invention;

[0034] Figure 4 It is a side schematic diagram of a heterogeneous integrated single-molecule optical immunoassay chip according to another embodiment of the present invention.

[0035] Description of Reference Numerals

[0036] 100. microfluidic chip; 101. sample loading hole; 102. liquid flow channel; 103. micrometer hole; 104. microchannel; 105. upper plate; 106. lower plate; 107. cover plate; 200. rare earth luminescent micrometer crystal. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0038] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0039] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0040] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, a first feature being "above", "above" or "above" a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. A first feature being "below", "below" or "below" a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0041] In the description of the present invention, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0042] In this article, "optionally", "optional", "optional" means optional, that is, it means to be selected from any of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. In this application, the descriptions such as "optionally contain" and "optionally include" mean "contain or not contain".

[0043] In the present invention, unless otherwise stated, the sum of the parts of each component in the composition can be 100 parts by weight. Unless otherwise specified, the basis of the percentage (including weight percentage) of the present invention is the total weight of the composition, and "wt%" herein means mass percentage.

[0044] In this article, unless otherwise stated, each reaction step may be carried out in the order described herein or may not be carried out in the order described herein. For example, other steps may be included between each reaction step, and the order of the reaction steps may also be appropriately swapped. This can be determined by the technician based on common knowledge and experience. Preferably, the reaction method herein is carried out sequentially.

[0045] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical range disclosed in the present application should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows for a broad range of quantitative intervals such as percentage intervals, ratio intervals, and ratio intervals.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0047] The embodiment of the present application provides a heterogeneous integrated single-molecule optical immunoassay chip to solve the problem that the simoa technology uses enzyme-catalyzed bioluminescence or dyes to emit light, which has unstable luminescence and inaccurate detection results. The heterogeneous integrated single-molecule optical immunoassay chip will be described below in conjunction with the accompanying drawings.

[0048] The heterogeneous integrated single-molecule optical immunoassay chip provided in the present application is exemplary, see Figure 1 As shown, Figure 1This is a schematic diagram of the structure of the heterogeneous integrated single molecule optical immunoassay chip provided in the embodiment of the present application. The heterogeneous integrated single molecule optical immunoassay chip of the present application can be used for single molecule biological detection.

[0049] In order to more clearly illustrate the structure of the heterogeneous integrated single-molecule optical immunoassay chip, the heterogeneous integrated single-molecule optical immunoassay chip will be introduced below with reference to the accompanying drawings.

[0050] For example, see Figure 1 As shown, a heterogeneous integrated single-molecule optical immunoassay chip includes a microfluidic chip 100 and a probe. The microfluidic chip 100 is provided with a sample addition hole 101, a liquid flow channel 102 communicating with the sample addition hole 101, and a plurality of micrometer holes 103 communicating with the liquid flow channel 102. Each micrometer hole 103 is respectively connected to a microchannel 104 for liquid circulation. The probe includes a rare earth luminescent micron crystal 200 modified with a primary antibody and a quencher modified with a secondary antibody. Photons emitted by at least one band of the rare earth luminescent micron crystal 200 can be quenched by the quencher. Each micrometer hole 103 can accommodate at most one rare earth luminescent micron crystal 200.

[0051] The above-mentioned heterogeneous integrated single-molecule optical immunoassay chip can improve detection several times and increase the detection range of the molecules to be tested. The chemical and luminescent stability are very high, which can be used not only for one-time detection but also for post-test review of the test results. In this application, by changing "two-classification" to "multi-classification", such as "ten-classification", the detection range can be increased, and the probe is physically fixed through the micron hole 103. After the molecule of the object to be tested is identified, the luminescence in each micron hole 103 is maintained at a fixed value, which can realize the post-test review of the test results.

[0052] In some embodiments, the radial cross-sectional width of the micrometer hole 103 is greater than the maximum particle size of one rare earth luminescent micrometer crystal 200 and less than the maximum particle size of two rare earth luminescent micrometer crystals 200, so that each micrometer hole 103 can accommodate a probe. It should be noted that, since the rare earth luminescent micrometer crystal 200 is not necessarily a regular shape, the above-mentioned maximum particle size refers to the maximum length of the rare earth luminescent micrometer crystal 200 in any direction. Among all the lengths, the maximum value is the above-mentioned maximum particle size.

[0053] In some embodiments, a plurality of micrometer holes 103 are distributed in an array on the microfluidic chip 100 , and each micrometer hole 103 is respectively provided with a number.

[0054] In some embodiments, the maximum particle size of the rare earth luminescent micron crystals 200 is 1 μm-10 μm.

[0055] In some embodiments, the quencher includes one or more of nano iron oxide microspheres, nano gold microspheres, and dye microspheres. For example, under the excitation of a 980nm light source, NaYF4:Yb,Er micron crystals emit green light with a maximum wavelength of 540nm, red light with a maximum wavelength of 654nm, and near infrared light with a maximum wavelength of 1532nm. The nano gold quencher has a strong absorption capacity at 525nm, which can quench the green light of the rare earth luminescent micron crystal 200, and thus can be used as a quencher for the rare earth luminescent micron crystal 200.

[0056] In some embodiments, the microfluidic chip 100 includes a base plate and a cover plate, which can be fitted with the base plate. The base plate is provided with micrometer holes 103, liquid flow channels 102 and microchannels 104 extending along the length or width direction of the base plate, and the cover plate is provided with sample addition holes 101. Figure 1 As shown, the liquid flow channel 102 extends along the length direction of the bottom plate, and the plurality of microchannels 104 can extend along the length direction and width direction of the bottom plate to form a crisscross distribution. After the liquid enters the micropores 103, it flows along the length and width direction of the bottom plate.

[0057] In some of the embodiments, the depth of the micro-hole 103 and the depth of the micro-channel 104 are both greater than the maximum particle size of the rare earth luminescent micro-crystal 200 .

[0058] In some embodiments, the depth of the micro-pores 103 and the depth of the micro-channels 104 are equal.

[0059] In some embodiments, the width of the microchannel 104 is smaller than the minimum particle size of the rare earth luminescent micron crystal 200. It should be noted that, since the rare earth luminescent micron crystal 200 is not necessarily a regular shape, the above-mentioned minimum particle size refers to the minimum length of the rare earth luminescent micron crystal 200 in any direction. Among all the lengths, the minimum value is the above-mentioned minimum particle size.

[0060] In some embodiments, adjacent micropores 103 are connected to each other through microchannels 104 .

[0061] In some embodiments, the micro-hole 103 is a rectangular hole, and the side length of the micro-hole 103 is 1 μm-15 μm.

[0062] In some embodiments, the micropore 103 is a circular hole, and the diameter of the micropore 103 is 1 μm-15 μm.

[0063] In some of the embodiments, it is not difficult to understand that in other examples, the shape of the micro-hole 103 can also be other regular shapes, such as a regular hexagon, an ellipse, etc.

[0064] In some embodiments, there are multiple liquid flow channels 102 , and the multiple liquid flow channels 102 are all connected to the sample loading hole 101 , and the multiple liquid flow channels 102 are respectively connected to different microchannels 104 .

[0065] In some embodiments, the microchannels 104 are connected to each other so that the micropores 103 are connected to each other.

[0066] When the plurality of microchannels 104 extend along the length direction and the width direction of the bottom plate, the method for preparing the microfluidic chip 100 includes the following steps:

[0067] The base plate can be made by micro-nano processing methods such as spin coating, photolithography and etching, wherein during photolithography preparation: (1) Base plate treatment: ultrasonically treat the glass wafer in organic solvents acetone and isopropanol in an ultrasonic machine for 5 minutes to 10 minutes, then rinse with deionized water and blow dry with nitrogen; use a plasma degumming machine to apply glue in an oxygen atmosphere for 10 minutes to 15 minutes; (2) Spinning glue: place the intermediate of step (1) in an HMDS device and run it for at least 10 minutes to coat the surface with a layer of HMDS (hexamethyldisilazane, Hexamethyldisilazane, HMDS) solvent, and then apply S1805 photoresist at a speed of 4000 for at least 30 seconds; (3) Pre-baking: after the intermediate is evenly coated, place it on a hot plate and bake it for at least 1 minute; (4) Photolithography: use an MA6 contact photolithography machine to align and expose for 2 seconds; (5) Post-baking: after the photolithography is completed, dry it on the hot plate for at least 1 minute; (6) Development: add 2.38wt% of the developer Put TMAH (tetramethylammonium hydroxide) into a container, put the intermediate in a basket and put it into the container, let it stand for at least 25 seconds, then shake the basket evenly for 5 seconds, take it out, rinse it with water, blow it dry, and examine it under a microscope. (7) Hard film: After microscopic examination, place it on a hot plate and bake it for 3 minutes; (8) ICP etching: Apply silicone oil evenly on the back of the intermediate, put the intermediate into the chamber of the etching machine and adjust the program for etching, and take it out after etching is completed. (9) Degumming: Use organic solvents acetone and isopropanol in an ultrasonic machine for 5 minutes each, then rinse with deionized water, blow it dry with nitrogen, and finally examine it under a microscope. (10) Cutting: Cut the intermediate by high-speed rotation of the grinding wheel to obtain a microfluidic chip 100. The above method is only one etching, and multiple etching methods can also be used to obtain a larger micropore 103 depth and micropores 103 with different shapes. In addition to the above methods for processing micropores 103, femtosecond laser processing and other methods can also be used.

[0068] It should be noted that HMDS equipment can be a dedicated evaporation system or a part integrated into other process steps. The operating principle of HMDS equipment is usually to heat HMDS liquid to evaporate it, and then guide the vapor to the surface of the substrate to be treated, where HMDS will condense and form a monomolecular layer. This process is usually carried out in a vacuum environment to ensure good control and uniform coating.

[0069] In some embodiments, the microfluidic chip includes a cover plate 107, an upper plate 105 and a lower plate 106. The upper plate 105 includes a plurality of micrometer holes 103, and the lower plate 106 includes a plurality of microchannels 104. The upper plate 105 and the lower plate 106 can be fitted and assembled, and the microchannels 104 extend along the thickness direction of the lower plate 106. The micrometer holes 103 can connect at least one microchannel 104. The cover plate 107 is movably connected to the upper plate 105 and can close each micrometer hole 103. The cover plate 107 is provided with a plurality of liquid adding holes. When the cover plate 107 is covered on the upper plate 105, each liquid adding hole can be connected to the micrometer hole 103 respectively. The liquid adding holes are used to add cleaning liquid for cleaning in preparation for detection. See Figure 2 As shown, Figure 2 It is a side view of the microfluidic chip. The liquid flow channel 102 extends along the length direction of the upper plate 105 , and the multiple microchannels 104 can be along the thickness direction of the lower plate 106 . After the liquid enters the micropores 103 , it flows along the thickness direction of the upper plate 105 .

[0070] In some embodiments, when the micropore 103 is able to connect to at least one microchannel 104, one micropore 103 may correspond to one microchannel 104, one micropore 103 may correspond to multiple microchannels 104, or multiple micropores 103 may correspond to one microchannel 104, which will be explained in detail below.

[0071] In some of the embodiments, the upper plate 105 and the lower plate can be assembled by a snap-fit ​​structure.

[0072] In some of the embodiments, the upper plate 105 and the lower plate 106 are respectively provided with assembly holes, and the upper plate 105 and the lower plate 106 are assembled by fasteners passing through the assembly holes.

[0073] When the plurality of microchannels 104 extend along the thickness direction of the upper plate 105, the method for preparing the microfluidic chip 100 includes the following steps:

[0074] See also Figure 2As shown, the microfluidic chip 100 is divided into two layers, the upper plate 105 is the core, the upper plate 105 has micron holes 103, the lower part represents the microchannel 104, the solid black represents the rare earth luminescent micron crystal 200, the microchannel 104 allows the liquid to flow from the micron hole 103 along the vertical direction from top to bottom, but the size of the rare earth luminescent micron crystal 200 is larger than the microchannel 104, so the rare earth luminescent micron crystal 200 will be physically confined in the micron hole 103, the upper plate 105 can be a glass plate, the size of the upper plate 105 is thin, the thickness is between 5μm and 30μm, the upper plate 105 can be processed by the above-mentioned photolithography method, and then processed by the chip thinning process.

[0075] The lower plate 106 is a large through-hole glass array, which is made of glass wafers commonly used in the market. The thickness of the lower plate 106 is between 100μm and 1000μm. The upper plate 105 is thin and easy to break during operation. Therefore, the lower plate 106 serves as a support for the upper plate 105. At the same time, the large through-hole diameter of the glass array of the lower plate 106 is set to be greater than 40μm, which can provide a faster flow rate for the liquid. The processing method of the lower plate 106 can be selected from laser etching, femtosecond laser etching, photolithography / etching, etc., with many options. When in use, the upper plate 105 and the lower plate 106 are overlapped and connected together. When in use, the upper plate 105 and the lower plate 106 are overlapped, rare earth luminescent micron crystals 200 are added to the upper plate 105, and then a glass slide with a flat surface is used to slide closely against the upper plate 105 to bring the rare earth luminescent micron crystals 200 into the micron holes 103, and at the same time, the rare earth luminescent micron crystals 200 that have not been transferred into the micron holes 103 are removed; then the upper plate 105 and the lower plate 106 are buckled together, and the cover plate 107 is covered on the upper plate 105. After cleaning with cleaning liquid added through the liquid filling hole of the cover plate 107, the detection can be carried out.

[0076] In some of these embodiments, see Figure 3As shown, the microfluidic chip includes a cover plate 107, an upper plate 105 and a lower plate 106. In this embodiment, the size of the microchannel 104 on the lower plate 106 is relatively large, and one microchannel 104 can collect the liquid flow in multiple micropores 103, thereby improving the liquid flow efficiency. At this time, the cover plate 107 is penetrated with nanometer-level holes, which can be used to add cleaning liquid to clean the micropores 103 and the rare earth luminescent micron crystals 200. The material used for the cover plate 107 is a nanoporous anodized aluminum film. After the rare earth luminescent micron crystals 200 are scraped into the micropores 103 of the upper plate 105 with a glass slide, the cover plate 107 is covered. The nanometer-level holes on the cover plate 107 are smaller than the micropores 103 of the upper plate 105. On the one hand, the rare earth luminescent micron crystals 200 can be confined in the micropores 103; on the other hand, liquid can be dripped through the nanometer-level holes on the cover plate 107 to perform operations such as washing or reaction. The upper plate 105 can be processed by more than two photolithography-etching processes, and the upper plate 105 can also be divided into two layers, and the micron hole 103 is roughly T-shaped ( Figure 2-Figure 4 The upper micropore 103 is larger in size to accommodate a rare earth luminescent micron crystal 200, and the lower micropore 103 is smaller in size to prevent the rare earth luminescent micron crystal 200 from falling. In this embodiment, the purpose of adding the microchannel 104 extending along the thickness direction is to speed up the liquid flow rate and facilitate the full washing and reaction of the probe.

[0077] In some of these embodiments, see Figure 4 As shown, the microfluidic chip includes a cover plate 107, an upper plate 105 and a lower plate 106. In this embodiment, the size of the microchannel 104 on the lower plate 106 is relatively small, and one micron hole 103 can correspond to multiple microchannels 104. Multiple microchannels 104 can collect the liquid flow in one micron hole 103 at the same time, thereby improving the liquid flow efficiency. At this time, the cover plate 107 is penetrated with nanometer-level holes, which can be used to add cleaning liquid to clean the micron hole 103 and the rare earth luminescent micron crystal 200. The material used for the cover plate 107 is a nanoporous anodized aluminum film. After the rare earth luminescent micron crystal 200 is scraped into the micron hole 103 of the upper plate 105 with a glass slide, the cover plate 107 is covered. The lower plate 106 is similar to the cover plate 107, and also has a plurality of nanopores penetrated therethrough. The material of the lower plate 106 is a nanoporous anodized aluminum film. The thickness of the lower plate 106 is greater than that of the cover plate 107. The thicker lower plate 106 can play a supporting role.

[0078] An embodiment of the present application also provides a method for preparing a heterogeneously integrated single-molecule optical immunoassay chip.

[0079] A method for preparing a heterogeneous integrated single-molecule optical immunoassay chip comprises the following steps:

[0080] A liquid flow channel 102, a plurality of micrometer holes 103 communicating with the liquid flow channel 102, and a microchannel 104 communicating with the micrometer holes 103 are manufactured on a first glass wafer. The microchannel 104 extends along the length or width direction of the first glass wafer, or the microchannel 104 extends along the thickness direction of the first glass wafer. The liquid flow channel 102, the micrometer holes 103, and the microchannel 104 are manufactured by photolithography, etching, and laser processing.

[0081] Furthermore, a sample addition hole 101 is made on the second glass wafer, and the second glass wafer is bonded to the first glass wafer to form the microfluidic chip 100 .

[0082] When the heterogeneous integrated single-molecule optical immunoassay chip of the present application is used for detection, the detection method comprises the following steps:

[0083] The sample to be tested is added to the sample well 101 on the microfluidic chip 100, and flows into the rare earth luminescent micron crystal 200 modified with the primary antibody through the liquid flow channel 102, so that the molecules to be tested in the sample are combined with the antibodies on the rare earth luminescent micron crystal 200, and then a buffer solution is added to the sample well 101 to wash the rare earth luminescent micron crystal 200. Then, the quencher modified with the secondary antibody is added to the sample well 101, and flows into the rare earth luminescent micron crystal 200 combined with the molecules to be tested through the liquid flow channel 102. Through the specific binding of the secondary antibody to the molecules to be tested, part of the quencher is connected to the rare earth luminescent micron crystal 200, and the amount of the quencher on the rare earth luminescent micron crystal 200 is proportional to the amount of the molecules to be tested.

[0084] After the rare earth luminescent micron crystal 200 is fixed in the array micron hole 103 of the microfluidic chip 100, the luminescent brightness of each micron hole 103 on the microfluidic chip 100 is scanned and photographed by a laser confocal microscope or other equipment, and the luminescent brightness value of each micron hole 103 is extracted, and the luminescent brightness value of each micron hole 103 is automatically recorded one by one according to the number of each micron hole 103. It is assumed that the brightness in the micron hole 103 numbered A12-B31 is recorded as I0; then after the operations of adding recognition, binding, washing, recognition of the quencher modified with the secondary antibody, washing, etc. of the molecule to be tested, the luminescent brightness of each micron hole 103 is photographed again in turn according to the coordinates of each micron hole 103, and the luminescent brightness value of each micron hole 103 is extracted, and the luminescent brightness of the micron hole 103 numbered A12-B31 is recorded as I1, then the difference of I1-I0 is the brightness change value in the micron hole 103 numbered A12-B31, and the number or concentration of the molecule to be tested is obtained according to the detection standard curve. Each micrometer hole 103 on the microfluidic chip 100 is uniquely numbered by two-dimensional coordinates, so the chip can be easily positioned when taking two photos, ensuring that the values ​​in each micrometer hole 103 correspond one to one. When designing and processing the micrometer hole 103, the stepping parameters of the confocal microscope are considered to ensure that each micrometer hole 103 can be accurately scanned and detected.

[0085] The method for preparing the detection standard curve comprises the following steps:

[0086] (1) Prepare 10 to 20 concentration gradients of different concentrations of the molecule to be tested. The operation of each gradient molecule to be tested is the same, so only the steps at one concentration are described below.

[0087] (2) At a certain concentration of the molecule to be tested, the microfluidic chip 100 is first cleaned, and rare earth luminescent micron crystals 200 are added and fixed in the micropores 103. Each micropore 103 contains a rare earth luminescent micron crystal 200. The luminescence brightness of the rare earth luminescent micron crystal 200 in each micropore 103 is photographed in sequence according to the coordinate number of the micropore 103. Then, the standard operation of detecting a certain concentration of the molecule to be tested is performed until the nano-gold quencher is connected to the rare earth luminescent micron crystal 200. After washing, the luminescence brightness of the rare earth luminescent micron crystal 200 in each micropore 103 is photographed again according to the coordinate number. At the same time, the scanning electron microscope (SEM) element content (EDS) and micro-Raman spectrum of the rare earth luminescent micron crystal 200 in each micropore 103 are photographed according to the coordinate number. The characteristic peaks of the nano-gold are used to determine how many nano-golds are connected to the surface of each micro-crystal.

[0088] (3) Through the luminescence intensity value, EDS and Raman value, a relationship between the luminescence brightness of a rare earth luminescent micron crystal 200 connected with different numbers of gold nanoparticles and the number of connected gold nanoparticles is established. In this way, the number of connected quenchers can be inferred based on the change in luminescence brightness of the rare earth luminescent micron crystal 200 before and after the actual test. The number of connected quenchers is proportional to the number of molecules to be tested, thereby indirectly calculating the number of molecules to be tested.

[0089] (4) Draw a detection standard curve based on the data obtained from 10 to 20 concentration gradients of different concentrations of the test molecule solution.

[0090] In summary, compared with the traditional technology, the heterogeneous integrated single-molecule optical immunoassay chip of the present application has the following beneficial effects:

[0091] (1) In each micron hole 103 on the heterogeneous integrated single-molecule optical immunoassay chip, the judgment is not a "binary classification" of "luminescence" or "no" luminescence, but a "multi-classification" luminescence intensity change of >2, which can improve the detection range of the molecules to be tested.

[0092] (2) The array of microfluidic chips 100 is not only a reading carrier of luminescent signals, but also a place for the reaction and identification of probes and molecules of the analyte. Since the volume in each micron hole 103 can be set to 10 -12 ~10 -15 L level, so if there are molecules of the test object in the micron hole 103, their concentration will be very high, and high concentration means a fast reaction speed, so the speed of the recognition reaction of the molecules of the test object can be accelerated. Compared with the traditional sioma and other technologies, the recognition and binding reaction of the molecules of the test object is carried out in a relatively large container such as an EP tube, and the reaction speed of the present application can be greatly improved.

[0093] (3) This application uses the physical structure of the micron hole 103 to restrict the probe on the chip to perform operations such as identification, binding, and washing of the molecules to be tested, rather than through chemical modification and other methods, which makes the operation simpler and more reliable. Since the precision limit of microfluidic array processing in traditional technology is about 1 micron, and the size of nanomaterials is a few nanometers to hundreds of nanometers, it is difficult to restrict heterogeneous probes on the chip through physical architecture. This application uses micron-level luminescent materials to achieve operations such as identification, binding, and washing of the molecules to be tested in the micron hole 103. All these operations are performed on the chip surface, which makes the experiment simpler and more efficient.

[0094] (4) Rare earth micron crystals are used as luminescent probes. Their chemical and luminescent stability are very high, much higher than that of dyes and chemiluminescence. They can be used not only for one-time detection, but also for post-detection review of test results.

[0095] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0096] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A heterogeneous integrated single-molecule optical immunoassay chip, characterized in that: The invention comprises a microfluidic chip and a probe, wherein the microfluidic chip is provided with a sample loading hole, a liquid flow channel connected to the sample loading hole and a plurality of micrometer holes connected to the liquid flow channel, each of the micrometer holes is respectively connected to a microchannel for liquid circulation, the probe comprises a rare earth luminescent micrometer crystal modified with a primary antibody and a quencher modified with a secondary antibody, photons emitted by at least one wavelength band of the rare earth luminescent micrometer crystal can be quenched by the quencher, and each of the micrometer holes can accommodate at most one rare earth luminescent micrometer crystal.

2. The heterogeneous integrated single-molecule optical immunoassay chip according to claim 1, characterized in that: The radial cross-sectional width of the micron hole is larger than the maximum particle size of one of the rare earth luminescent micron crystals and smaller than the maximum particle sizes of two of the rare earth luminescent micron crystals, so that each of the micron holes can accommodate one of the probes.

3. The heterogeneous integrated single-molecule optical immunoassay chip according to claim 1, characterized in that: The plurality of micrometer holes are distributed in an array on the microfluidic chip, and each of the micrometer holes is respectively provided with a number.

4. The heterogeneous integrated single-molecule optical immunoassay chip according to claim 1, characterized in that: The heterogeneous integrated single-molecule optical immunoassay chip also satisfies at least one of the following conditions: (1) The maximum particle size of the rare earth luminescent micron crystal is 1 μm to 10 μm; (2) The quencher includes one or more of nano iron oxide microspheres, nano gold microspheres and dye microspheres.

5. The heterogeneous integrated single-molecule optical immunoassay chip according to any one of claims 1 to 4, characterized in that: The microfluidic chip includes a base plate and a cover plate, the cover plate and the base plate can be fitted together, the base plate is provided with the micron hole, the liquid flow channel and the microchannel extending along the length direction or width direction of the base plate, and the cover plate is provided with the sample addition hole.

6. The heterogeneous integrated single-molecule optical immunoassay chip according to claim 5, characterized in that: The heterogeneous integrated single-molecule optical immunoassay chip also satisfies at least one of the following conditions: (1) The depth of the micron hole and the depth of the microchannel are both greater than the maximum particle size of the rare earth luminescent micron crystal; (2) The depth of the micropore is equal to the depth of the microchannel; (3) The width of the microchannel is smaller than the minimum particle size of the rare earth luminescent micron crystal; (4) Adjacent micropores are connected to each other through the microchannels.

7. The heterogeneous integrated single-molecule optical immunoassay chip according to claim 5, characterized in that: The heterogeneous integrated single-molecule optical immunoassay chip also satisfies at least one of the following conditions: (1) The micropore is a rectangular hole, and the side length of the micropore is 1 μm to 15 μm; (2) The micropore is a circular hole, and the diameter of the micropore is 1 μm to 15 μm; (3) There are multiple liquid flow channels, all of which are connected to the sample loading hole, and each of which is connected to different microchannels; (4) The microchannels are interconnected to realize interconnection of the micropores.

8. The heterogeneous integrated single-molecule optical immunoassay chip according to any one of claims 1 to 4, 6 to 7, characterized in that: The microfluidic chip includes a cover plate, an upper plate and a lower plate, the upper plate includes a plurality of the micron holes, the lower plate includes a plurality of the microchannels, the lower plate and the upper plate can be fitted together, the microchannels extend along the thickness direction of the lower plate, the micron holes can connect at least one of the microchannels, the cover plate is movably connected to the upper plate and can close each of the micron holes, a plurality of liquid adding holes are arranged on the cover plate, and when the cover plate is covered on the upper plate, each of the liquid adding holes can be connected to the micron holes respectively.

9. The heterogeneous integrated single-molecule optical immunoassay chip according to claim 8, characterized in that: The upper plate and the lower plate can be assembled by means of a snap-fit ​​structure; Alternatively, the upper plate and the lower plate are respectively provided with assembly holes, and the upper plate and the lower plate are assembled by fasteners passing through the assembly holes.

10. A method for preparing the heterogeneous integrated single-molecule optical immunoassay chip according to any one of claims 1 to 9, characterized in that: The steps include: A liquid flow channel, a plurality of micrometer holes communicating with the liquid flow channel, and a microchannel communicating with the micrometer holes are fabricated on a first glass wafer, wherein the microchannel extends along the length or width direction of the first glass wafer, or the microchannel extends along the thickness direction of the first glass wafer; wherein the liquid flow channel, the micrometer holes, and the microchannel are fabricated by photolithography etching and laser processing; Furthermore, a sample addition hole is made on the second glass wafer, and the second glass wafer is bonded to the first glass wafer to form a microfluidic chip.

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