A method and apparatus for immunoassay of trace biomarkers based on gold magnetic nanoparticles and structurally graded plasmonic metasurfaces.

Abstract

This invention discloses a method and apparatus for immunodetection of trace biomarkers based on gold magnetic nanoparticles and a structurally graded plasmonic metasurface. The method enhances image sensing performance through coupling of gold magnetic nanoparticles and the structurally graded plasmonic metasurface. It significantly improves detection sensitivity through multiple techniques, including biomarker separation and purification, enhanced electric field coupling, image signal amplification, and signal-to-noise ratio enhancement, enabling rapid immunodetection of ultra-low concentrations and doses of tear biomarkers. Further coverage of more biomarkers suggests potential applications in screening and diagnosis of ophthalmological, cardiovascular, and neurodegenerative diseases. Based on this method, an apparatus for immunodetection of trace biomarkers is provided, which can be used for ultrasensitive and rapid immunodetection of trace tear biomarkers, completing the concentration detection of tear biomarkers within 3 minutes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of in vitro diagnosis, and particularly relates to a method and device for immunodetection of trace markers based on gold magnetic nanoparticles and a structure-graded plasmonic super surface. BACKGROUND

[0002] With the high-frequency use of electronic display products, the prevalence of eye diseases is showing an increasing trend year by year. For example, the global prevalence of dry eye syndrome ranges from 5% to 50% in different countries and regions, and grows at a rate of 10% per year. Functional vision impairment caused by eye diseases extends from hindering people's daily reading, driving, and visual experience to work efficiency decline, and even to harm the patient's mental state and even blindness. Therefore, routine examination of common eye diseases is of great significance for their prevention and timely treatment intervention.

[0003] Tear fluid, as an important component of maintaining the homeostasis of the ocular surface microenvironment, not only moisturizes and protects the ocular surface, but also contains a large amount of information about the health status of the eye. Among them, tear markers, including proteins, lipids and metabolites, have great potential for the diagnosis of eye diseases due to their significant diagnostic sensitivity and specificity. For example, matrix metalloproteinase 9 (MMP-9), an important tear marker for monitoring the prognosis of dry eye syndrome, is a proteinase secreted by corneal epithelium. When tear evaporation is rapid and tear film stability is weakened, the homeostasis of the ocular surface microenvironment is broken, leading to an increase in tear osmotic pressure and triggering the release of MMP-9, thereby causing a progressive inflammatory cycle. Therefore, detection of markers with indicative value or actual function can effectively assist in the judgment of eye disease-causing factors and disease screening. Currently, the main method for measuring tear markers is based on lateral flow immunochromatography. For example, Chinese patent CN113075414B authorizes a method and device for diagnosing ocular surface inflammation and dry eye disease, which qualitatively provides whether the marker exceeds the empirical safety value, but cannot detect the concentration to realize the severity grading of dry eye. The only commercially available InflammaDry device approved by the U.S. Food and Drug Administration for tear MMP-9 also has this problem. In addition, due to the small amount of tear fluid and the extremely low concentration of a considerable part of the markers (pg / mL), it also poses a great obstacle to the precise quantification of tear markers and disease diagnosis in the prior art. Therefore, there is an urgent need for a marker detection technology that can accurately quantify, is simple to operate, and responds to ultra-low concentrations, in order to make more accurate diagnoses of common eye diseases. SUMMARY

[0004] The purpose of the present application is to overcome the above-mentioned defects and shortcomings in the prior art, and to provide a method for immunodetection of trace markers based on gold magnetic nanoparticles and a structure-graded plasmonic super surface.

[0005] The second object of the present application is to provide a device for implementing the method.

[0006] The above objects of the present application are achieved by the following technical solutions:

[0007] The present application first provides a method for detecting trace markers based on gold magnetic nanoparticles and structure-graded plasmonic super surface, comprising the following steps:

[0008] S1. Prepare a structure-graded plasmonic super surface with surface-modified detection antibodies, use an optical imaging detection assembly to collect an initial transmission image of the structure-graded plasmonic super surface, and record the position of the initial bright stripe;

[0009] S2. Add gold magnetic nanoparticles modified with capture antibodies to the sample to be tested, obtain gold magnetic nanoparticles combined with markers, couple the gold magnetic nanoparticles combined with markers with the structure-graded plasmonic super surface, and obtain a structure-graded plasmonic super surface coupled with gold magnetic nanoparticles combined with markers;

[0010] S3. Collect a transmission image of the structure-graded plasmonic super surface coupled with gold magnetic nanoparticles combined with markers, record the position of the bright stripe, and calculate the concentration of the marker according to the moving distance value of the initial bright stripe;

[0011] The structure-graded plasmonic super surface is a periodic-graded plasmonic super surface with fixed-interval-period-graded micro-nano structures on the surface or a topography-graded plasmonic super surface with fixed-interval-period micro-nano structures with graded topography;

[0012] The particle size of the gold magnetic nanoparticles is smaller than the size of the micro-nano structures in the structure-graded plasmonic super surface; and the spectral characteristic absorption peak range of the structure-graded plasmonic super surface contains the spectral characteristic absorption peak of the gold magnetic nanoparticles.

[0013] The size of the capture antibody, the marker and the detection antibody after combination is smaller than the sum of the particle size of the gold magnetic nanoparticles and the size of the electric field decay layer of the structure-graded plasmonic super surface.

[0014] The detection principle of the application is that when the spectral feature transmission peak of the nanostructure at a position of the structure-graded plasmonic metasurface matches the center wavelength of the narrow-band light, the transmission intensity at the position reaches a maximum value, the optical imaging detection component captures the brightest area in the captured structure-graded plasmonic metasurface transmission intensity mode image; the red shift of the spectral feature transmission peak caused by the coupling of the gold magnetic nanoparticles combined with the marker and the structure-graded plasmonic metasurface further appears as the movement of the bright stripe in the transmission intensity mode image, and finally the concentration of the tear marker is calculated according to the pixel movement of the bright stripe in the transmission intensity image; the step distance of the red shift can be regulated by the gold magnetic nanoparticle size, the spectral feature absorption peak position and the coupling distance.

[0015] Specifically, the application designs the structure of the plasmonic metasurface, and through optical simulation and design, when the structure-graded plasmonic metasurface interacts with light, the transmission light intensity also correspondingly changes continuously. The micro-nano structures at different positions of the structure-graded plasmonic metasurface interact with the light source of the optical imaging detection component, and show different characteristic spectra, which generally show a continuous change in the spectral feature peak along the structure-graded structure. When the optical imaging detection component photographs the structure-graded plasmonic metasurface, since each pixel point on the image corresponds to the micro-nano structure of the structure-graded plasmonic metasurface, the brightness on the image also changes regularly with the structure-graded structure. The more the pixel points, the higher the resolution, and the slight change caused by the entry of the to-be-measured marker into the sensitive area of the structure-graded plasmonic metasurface can also cause the movement of the bright stripe on the image, so the sensitivity is also higher. The application calculates the concentration according to the movement distance of the bright stripe, and improves the sensing sensitivity based on imaging by optically amplifying the sensitive area of the structure-graded plasmonic metasurface. At the same time, when the gold magnetic nanoparticles combined with the marker are captured by the structure-graded plasmonic metasurface, the refractive index near the structure-graded plasmonic metasurface changes significantly, and the coupling effect of the gold magnetic nanoparticles is further improved by matching the feature absorption wavelength of the gold magnetic nanoparticles and the structure-graded plasmonic metasurface array and regulating the distance between the gold magnetic nanoparticles and the structure-graded plasmonic metasurface array to realize the enhancement of the electric field coupling. The coupling effect of the gold magnetic nanoparticles makes the pixel points of the movement of the bright stripe more, and further greatly improves the detection sensitivity. That is, the application first realizes image sensing through the graded structure of the plasmonic metasurface, and greatly improves the sensitivity by amplifying the image signal compared with the ordered conventional array of the plasmonic metasurface; secondly, the gold magnetic nanoparticles further increase the moving pixel points of the bright stripe, and further improve the detection sensitivity; the application provides a marker detection method which can accurately quantify, is simple to operate and has ultra-low concentration response, and the concentration response can be as low as pg / mL.

[0016] Further, the marker is a tear marker.

[0017] Preferably, the gold magnetic nanoparticles of the modified capture antibody are combined with one binding site of the marker to obtain gold magnetic nanoparticles combined with the marker, and the gold magnetic nanoparticles combined with the marker are contacted with a structure-gradual plasmonic metasurface modified with a detection antibody, wherein the detection antibody is combined with another binding site on the marker to achieve coupling of the gold magnetic nanoparticles-marker-structure-gradual plasmonic metasurface.

[0018] Further, the morphology-gradual plasmonic metasurface is a plasmonic metasurface whose micro-nano structure morphology gradually changes from a column shape, a rhombus shape, a chessboard shape to a hole shape from inside to outside.

[0019] Preferably, the structure-gradual plasmonic metasurface is replicated in large area and batch by nanoimprint technology (including but not limited to thermal nanoimprint and ultraviolet nanoimprint); the noble metal is deposited on the structure-gradual surface by electron beam evaporation or nano-electrodeposition process to make it have a localized surface plasmon resonance effect; and the structure-gradual plasmonic metasurface can be transferred to any form of solid interface by nano-transfer process. Specifically, the morphology- and period-gradual plasmonic metasurface can be realized by electron beam lithography exposure technology (EBL), and the morphology-gradual plasmonic metasurface can also be realized by coherent interference exposure. Among them, the morphology-gradual law is to change along a certain specific angle (such as becoming a cylindrical shape finally) on the basis of a certain initial shape (such as a cube); the period-gradual can gradually change in the x direction or the x / y direction (i.e., the structure distance of two micro-morphologies gradually changes).

[0020] Further, the spectral characteristic absorption peak of the gold magnetic nanoparticles is controlled by the particle size and shape.

[0021] Further, the characteristic absorption peak of the structure-gradual plasmonic metasurface is located in the visible light band.

[0022] Further, the optical detection assembly in step S1 includes a narrow-band light source and a camera device.

[0023] Further, the camera device is a camera and a CMOS image sensor; preferably, the camera device can be replaced by a smart phone.

[0024] Further, the step S2 realizes the coupling of the gold magnetic nanoparticles with the marker and the structure-gradual plasmonic metasurface by reciprocating flow.

[0025] Preferably, the reciprocating flow is the reciprocating flow of the liquid controlled by gas pressure driving.

[0026] Preferably, the reciprocating flow is the reciprocating flow of the liquid controlled by applying a gas pressure with a size and period change.

[0027] Preferably, the air pressure magnitude change cycle is 2-4 times per minute.

[0028] The application provides application of any of the above methods in detection of tear markers.

[0029] Further, the tear marker is matrix metalloproteinase 9 (MMP-9).

[0030] The application also provides a micro-marker immunodetection device for implementing any of the above methods, comprising a sample collection module, a sample pretreatment module and a microfluidic chip detection module; the sample pretreatment module comprises gold magnetic nanoparticles modified with a capture element and a magnetic control component; the microfluidic chip detection module comprises a microfluidic chip with a structure-gradient plasmonic super surface fixed with a surface-modified capture element in a channel, an assembly for controlling liquid reciprocating flow, and an optical imaging detection assembly.

[0031] Further, when the micro-marker immunodetection device is used for detecting tear markers, the sample collection module is a tear collector, the tear collector is based on capillary method, the tip of the collector is placed at the lower edge of the tear river, and tear fluid is collected from the conjunctival sac through capillary action; the sample pretreatment module is used for mixing the tear sample with gold magnetic nanoparticles modified with a capture element, the gold magnetic nanoparticles are combined with the marker to be detected in the sample, and gold magnetic nanoparticles combined with the marker to be detected are obtained through magnetic separation.

[0032] Preferably, after the tear collector collects tear fluid from the conjunctival sac, the tear fluid is directly added to tear diluent and fully mixed; the tear diluent is a mixture of 0.1x PBS and gold magnetic nanoparticles modified with a capture antibody.

[0033] Preferably, the magnetic separation is to place a strong magnet at the bottom of the diluent, to obtain gold magnetic nanoparticles combined with the marker to be detected through magnetic separation, to pour out the supernatant, to add a certain volume of diluent to the gold magnetic nanoparticles combined with the marker to be detected, to mix, and to directly add to the microfluidic chip detection module; the diluent is a PBS solution.

[0034] Further, the microfluidic channel of the microfluidic chip has a sample inlet, a solution storage channel and a liquid outlet, and the sample inlet and the liquid outlet are separate at both ends of the upper part of the solution storage channel.

[0035] Further, the size of the microfluidic channel is determined based on the size of the structure-gradient plasmonic super surface, the volume of the sample to be detected and the solution diffusion distance.

[0036] Preferably, the size of the microfluidic channel is greater than that of the sensing area of the structure-gradient plasmonic super surface.

[0037] Preferably, the height of the microfluidic channel is greater than the diffusion distance of the gold magnetic nanoparticles in solution.

[0038] Further, the component for controlling the reciprocating flow of the liquid controls the reciprocating flow of the liquid by controlling the size of the air pressure.

[0039] Preferably, the size of the air pressure changes periodically 2-4 times per minute.

[0040] Preferably, the sample to be tested added to the microfluidic channel is controlled by the periodically changing size of the air pressure at the sample inlet, and the sample to be tested flows reciprocally in the microfluidic channel, so that the markers combined with the gold magnetic particles rapidly combine with the detection antibodies modified on the surface of the gradient-structured plasmonic metasurface array.

[0041] Further, the optical imaging detection component includes a narrowband light source, a camera, and a CMOS image sensor.

[0042] The present application also provides a method for detecting markers using the above-mentioned micro-marker immunoassay device, comprising the following steps:

[0043] S1. Collect the initial transmission image of the gradient-structured plasmonic metasurface using the optical imaging detection component, and record the position of the bright stripes, wherein the gradient-structured plasmonic metasurface is modified with detection antibodies;

[0044] S2. Collect the sample to be tested using the sample collection module, separate and purify the marker to be tested of the sample to be tested using the gold magnetic particles modified with capture antibodies in the sample pretreatment module, and obtain a solution sample of gold magnetic nanoparticles combined with the marker to be tested, and add the solution sample to the sample inlet of the microfluidic channel of the microfluidic chip;

[0045] S3. Form a seal at one end of the microchannel, and control the reciprocating flow of the solution sample in the microchannel by applying periodically changing air pressure to the component for controlling the reciprocating flow of the liquid, so that the gold magnetic nanoparticles combined with the tear marker couple with the gradient-structured plasmonic metasurface modified with detection antibodies;

[0046] S4. Empty the solution in the microchannel by applying air pressure to the component for controlling the reciprocating flow of the liquid.

[0047] S5. Add ultrapure water to the sample inlet of the microchannel of the microfluidic system, and repeatedly clean the residual solution in the microchannel.

[0048] S6. Collect the transmission image of the gradient-structured plasmonic metasurface coupled with the gold magnetic particles combined with the marker using the optical imaging detection component, record the position of the bright stripes, and calculate the concentration of the marker based on the movement value of the bright stripe pixels.

[0049] Compared with the prior art, the present application has the following beneficial effects:

[0050] 1. The present application first provides a method for immunodetection of trace markers based on gold magnetic nanoparticles and structure-graded plasmonic metasurfaces. The method is based on the coupling of gold magnetic nanoparticles and structure-graded plasmonic metasurfaces to enhance image sensing performance. Through multiple means such as marker separation and purification, electric field coupling enhancement, image signal amplification, and signal-to-noise ratio improvement, the detection sensitivity of the method is greatly improved, realizing rapid immunodetection of tear markers at ultra-low concentration and ultra-low dose. Further covering more marker detection means that it is expected to be applied to screening and diagnosis of ophthalmology, cardiovascular and neurodegenerative diseases, etc.

[0051] 2. The present application further provides a trace marker immunodetection device based on the above method, which can be used for ultra-sensitive and rapid immunodetection of trace tear markers. After collecting the tear, the gold magnetic nanoparticle solution is mixed and directly added to the microfluidic chip. The concentration of tear markers can be detected within 3 minutes. Precise and rapid detection of tear marker concentration is of great significance for the routine examination, prevention, intervention and treatment of common eye diseases. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 It is a schematic diagram of the immunodetection device based on the structure-graded plasmonic metasurface of the present application; wherein 1 is a sample collection module, 2 is a sample pretreatment module, 3 is a microfluidic chip detection module, 21 is a gold magnetic nanoparticle modified with a capture antibody, 22 is a magnetic control component, 31 is a microfluidic chip, 32 is a component for controlling the reciprocating flow of liquid, 33 is an optical imaging detection component, 311 is a sample inlet, 312 is a solution storage channel, 313 is a liquid outlet, and 314 is a structure-graded plasmonic metasurface array modified with a detection antibody.

[0053] Figure 2 It is a schematic diagram of the method for coupling and enhancing image sensing performance of gold magnetic nanoparticles and structure-graded plasmonic metasurfaces, which is used for ultra-sensitive and rapid immunodetection of trace tear markers.

[0054] Figure 3 It is the time-domain finite difference (FDTD) simulation result of the present application for regulating the particle size and coupling distance of gold magnetic nanoparticles to electric field coupling enhancement (E / E0) 2 .

[0055] Figure 4 It is a schematic diagram of the structure-graded plasmonic metasurface prepared by the present application.

[0056] Figure 5 It is the pixel value of the bright stripe movement in the transmission image caused by the binding of different concentrations of markers when the immunodetection device based on the structure-graded plasmonic metasurface of the present application is coupled with or without gold magnetic nanoparticles.

[0057] Figure 6 This invention relates to a method for enhancing image sensing performance based on the coupling of gold magnetic nanoparticles and structurally graded plasma metasurfaces. Under conditions of controlled reciprocating flow of tear samples within microchannels, the pixel values ​​of bright stripe movement in transmission images caused by the combination of different concentrations of markers are measured. Detailed Implementation

[0058] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0059] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0060] Example 1

[0061] This embodiment provides a highly sensitive and rapid immunoassay device for detecting trace tear biomarkers. Figure 1 The device includes a sample acquisition module 1, a sample pretreatment module 2, and a microfluidic chip detection module 3. The sample pretreatment module 2 includes gold magnetic nanoparticles 21 modified with capture antibodies and a magnetic control component 22. The microfluidic chip detection module 3 includes a microfluidic chip 31, a component 32 for controlling the reciprocating flow of liquid, and an optical imaging detection component 33. The microfluidic chip includes an inlet 311, a solution storage channel 312, an outlet 313, and a structure-gradient plasma metasurface array 314 with surface-modified detection antibodies. The structure-gradient plasma metasurface array 314 is integrated in the center of the bottom of the solution storage channel 312 and is tightly adhered to its inner wall.

[0062] A method for enhancing image sensing performance based on coupling gold magnetic nanoparticles with a structurally graded plasmonic metasurface is illustrated in the schematic diagram of a highly sensitive and rapid immunoassay process for micro-tear biomarkers. Figure 2 As shown, gold magnetic nanoparticles modified with capture antibodies are used to separate and purify biomarkers in tear samples. Through reciprocating flow, tear biomarkers bind to detection antibodies on a structure-gradient plasmonic metasurface, thereby coupling the gold magnetic nanoparticles bound with tear biomarkers onto the structure-gradient plasmonic metasurface.

[0063] Enhanced electric field coupling can be achieved by adjusting the spacing between gold magnetic nanoparticles and a graded-scale plasmonic metasurface array. Generally, a particle size of less than 100 nm is optimal for the gold magnetic nanoparticles. The coupling spacing between the gold magnetic nanoparticles and the graded-scale plasmonic metasurface array can be determined based on the sizes of the captured antibody, biomarker, and detection antibody. Therefore, before implementation, FDTD simulations were used to study the effect of different particle sizes and coupling distances on the enhancement of electric field coupling (E / E0). 2The influence of this was considered, and then appropriate particle size and antibody were selected; the results were as follows: Figure 3 As shown, the coupling enhancement effect is most obvious when the gold magnetic nanoparticles have a larger particle size and a shorter coupling distance.

[0064] In this embodiment, tears are first collected using a commercially available tear collector based on the capillary method. Then, the tear sample is added dropwise to a solution containing gold magnetic nanoparticles modified with capture antibodies and mixed. Finally, the mixture is loaded into the inlet of the microfluidic chip.

[0065] Furthermore, in this embodiment, the tear collection and pretreatment process follows these steps:

[0066] Place the tip of the collector at the lower edge of the tear duct and collect tears from the conjunctival sac through capillary action. Care should be taken to handle the tears gently to avoid reflexive tearing. The collected tears are then added dropwise to 50 μL of a 0.08% (w / w) colloidal gold magnetic nanoparticle solution (the gold magnetic nanoparticles are coated with a capture antibody, with a particle size of 100 nm, of which the gold particles are 20 nm in diameter). After mixing and allowing to stand in the dark for 30 minutes, a strong magnet is placed at the bottom of the mixture to separate the gold magnetic nanoparticles bound to the target biomarker. The supernatant is poured off, and a certain volume of PBS solution is added to mix with the gold magnetic nanoparticles bound to the target biomarker. This mixture is then directly added to the injection port of the microfluidic chip.

[0067] In this embodiment, the characteristic absorption wavelength of the structured graded plasmon metasurface coupled with the biomarker-coated gold magnetic particles is located in the visible light band. The structured graded plasmon metasurface is manufactured in large areas and batches using nanoimprint lithography, nanoelectrodeposition, and nanotransfer processes; the silicon substrate used for nanoimprint lithography is prepared using laser interference lithography and reactive ion etching. The prepared plasmon metasurface array is located at the center of the bottom of the microfluidic channel and is tightly adhered to its inner wall.

[0068] Specifically, plasma metasurfaces with morphological and periodic gradients can be achieved using electron beam lithography (EBL), while morphological gradients can be achieved using coherent interference lithography. The morphological gradient follows a pattern of change along a specific angle from an initial shape (e.g., eventually becoming a cylinder); the periodic gradient can gradually change along the x-direction or both x and y directions (i.e., the structural distance between pairs of micromorphic features gradually changes).

[0069] Furthermore, in this embodiment, the fabrication process of the plasma metasurface array with gradually changing morphology adopts the following steps:

[0070] The pre-prepared ormostamp material mother plate (array of nano-pore structure with gradually changing morphology) is batch-replicated by using thermal nano-imprinting process to obtain a complementary nano-pillar structure array on the hot melt adhesive 652 (pressure of 0.3 tons, time of 5 minutes); then the array is subjected to nano-electrodeposition process (deposition current of 10 mA, time of 2 minutes) to obtain a complementary gold nano-pore structure, Figure 4 That is, the gradually changing morphology plasmonic metasurface prepared for the chip, the gradually changing morphology being gradually changed from a column to a rhombus to a chessboard to a hole from inside to outside.

[0071] In the embodiment, the microfluidic channel integrated with the metasurface array and capable of controlling reciprocating flow of the sample has a length, width and height of 3 cm, 5 mm and 150 μm respectively, the channel is formed by pouring a pre-prepared mother plate with polydimethylsiloxane (PDMS) and then curing; then the metasurface array is placed at the center of the bottom of the microfluidic channel and tightly adhered to the inner wall thereof; the component for controlling reciprocating flow of the sample in the microfluidic chip is a gas pressure driving device for applying a periodically changing gas pressure to realize reciprocating flow of the liquid; the optical detection assembly includes a narrowband light source, a camera and a CMOS image sensor.

[0072] The method for performing super-sensitive and rapid immune detection of tear markers by using the above-described device includes the following steps:

[0073] S1: CMOS is used to collect an initial transmission image of the gradually changing structure plasmonic metasurface, and the position of the bright stripe is recorded, the gradually changing structure plasmonic metasurface being modified with a detection antibody;

[0074] S2: Gold magnetic particles are used to separate and purify the sample to be tested: gold magnetic nanoparticles wrapped with a capture antibody are mixed with the tear sample, the mixture is magnetically separated, the supernatant is poured out, the gold magnetic nanoparticles combined with the tear markers are mixed with PBS buffer to obtain a mixed solution, and the mixed solution is added to the sample inlet of the microchannel of the microfluidic chip;

[0075] S3: A seal is formed at one end of the microchannel, a periodically changing gas pressure is applied by a gas pressure driving device to control reciprocating flow of the mixed solution in the microchannel multiple times, and the gold magnetic nanoparticles combined with the tear markers are coupled with the gradually changing structure plasmonic metasurface modified with the detection antibody;

[0076] S4: The liquid in the microchannel is emptied by applying a gas pressure by a gas pressure driving device;

[0077] S5: Ultra-pure water is added to the sample inlet of the microchannel of the microfluidic chip to repeatedly clean the residual liquid in the microchannel;

[0078] S6: Collecting the transmission image of the structure-graded plasmonic superstructure coupled with the gold magnetic nanoparticles bound with the biomarker, recording the position of the bright fringe, and calculating the concentration of the biomarker by combining the moving value of the bright fringe pixel.

[0079] The rapid immune detection device based on the coupling of gold magnetic nanoparticles and structure-graded plasmonic superstructure manufactured in this embodiment can be used for the detection of various markers in tear fluid and the screening of markers for common eye diseases.

[0080] Taking the detection of the tear marker MMP-9 of dry eye as an example, the feasibility of the developed gold magnetic nanoparticle-mediated structure-graded superstructure plasmonic resonance-enhanced tear marker ultra-sensitive and rapid immune detection chip application is verified. First, the position of the bright fringe in the initial image of the structure-graded plasmonic superstructure is recorded by an optical imaging system, and the tear fluid is mixed with the PBS solution containing gold magnetic nanoparticles in one step, and then added to the microchannel of transparent material. Subsequently, the size and period of the air pressure applied to the microchannel are controlled to control the reciprocating flow of the tear sample in the microfluidic channel for 10 times, and then the sample in the channel is emptied. Finally, after adding ultrapure water to the microchannel and reciprocally flushing it clean, the chip spectrum is tested. According to the moving value of the bright fringe pixel caused by the MMP-9 bound with the gold magnetic nanoparticles, the concentration of the biomarker is calculated. Figure 5

[0081] Comparative Example 1

[0082] This comparative example is basically the same as Example 1, the only difference being that the tear sample is not mixed with gold magnetic nanoparticles before testing. The results of this method and the method of mixing the tear fluid with gold magnetic nanoparticles before testing are compared as follows Figure 5 , which shows that the coupling of gold magnetic nanoparticles and structure-graded plasmonic superstructure array greatly improves the sensitivity of detection.

[0083] Comparative Example 2

[0084] This comparative example is basically the same as Example 1, the only difference being that after adding the tear sample to be tested into the microchannel, no air pressure of varying size and period is applied to the microchannel, but it is left to stand for 30 minutes before testing. The results of this method and the method of controlling the reciprocating flow of the tear sample for 10 times (2 minutes) are compared as follows Figure 6 , which shows that controlling the reciprocating flow of the tear sample can accelerate the capture of gold magnetic nanoparticles bound with the structure-graded plasmonic superstructure array, thereby shortening the detection time.

[0085] ​Obviously, the above embodiments of the present application are merely exemplary but not intended to limit the embodiments of the present application. Based on the above description, any other variations or changes can be made by those skilled in the art without departing from the spirit and principles of the present application. It is not necessary to list all the embodiments here. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall fall within the scope of the claims of the present application.

Claims

1. A method for immunoassay of micro-marks based on gold magnetic nanoparticles and a structure-graded plasmonic metasurface, characterized in that, The method comprises the following steps: S1. Preparing a structure-gradient plasmonic super surface with surface-modified detection antibodies, collecting an initial transmission image of the structure-gradient plasmonic super surface using an optical imaging detection assembly, and recording the initial bright fringe position; S2. Adding gold-magnetic nanoparticles modified with capture antibodies to the sample to be tested, obtaining gold-magnetic nanoparticles combined with markers, coupling the gold-magnetic nanoparticles combined with markers with the structure-gradient plasmonic super surface to obtain a structure-gradient plasmonic super surface coupled with gold-magnetic nanoparticles combined with markers; S3. Collecting a transmission image of the structure-gradient plasmonic super surface coupled with gold-magnetic nanoparticles combined with markers, recording the bright fringe position, and calculating the marker concentration according to the moving distance value of the initial bright fringe. The structure-gradient plasmonic super surface is a periodic-gradient plasmonic super surface with fixed-interval-period-gradient micro-nano structures on the surface or a morphology-gradient plasmonic super surface with fixed-interval-period micro-nano structures with gradient geometry. The particle size of the gold-magnetic nanoparticles is smaller than the size of the micro-nano structures in the structure-gradient plasmonic super surface, and the spectral characteristic absorption peak range of the structure-gradient plasmonic super surface contains the spectral characteristic absorption peak of the gold-magnetic nanoparticles. The size of the combination of the capture antibodies, the markers and the detection antibodies is smaller than the sum of the particle size of the gold-magnetic nanoparticles and the size of the electric field decay layer of the structure-gradient plasmonic super surface.

2. The method of claim 1, wherein, The morphology-gradient plasmonic super surface is a plasmonic super surface with micro-nano structure morphology gradually changing from columnar, rhombic, chessboard-shaped to hole-shaped from inside to outside.

3. The method of claim 1, wherein, The characteristic absorption peak of the structure-gradient plasmonic super surface is located in the visible light band.

4. The method of claim 1, wherein, The optical detection assembly in step S1 comprises a narrow-band light source and a camera device.

5. The method of claim 4, wherein, The camera device is a camera and a CMOS image sensor.

6. The method of claim 1, wherein, The step S2 realizes the coupling of the gold-magnetic nanoparticles combined with markers with the structure-gradient plasmonic super surface through reciprocating flow.

7. The method of claim 6, wherein, The reciprocating flow is controlled by gas pressure to control the reciprocating flow of the liquid.

8. The method according to any one of claims 1-7 for detecting tear markers.

9. A micro- marker immunoassay device for implementing the method of any one of claims 1 to 7, characterized in that, The device comprises a sample collection module (1), a sample pretreatment module (2) and a microfluidic chip detection module (3); the sample pretreatment module (2) comprises gold-magnetic nanoparticles (21) modified with capture antibodies and a magnetic control component (22); the microfluidic chip detection module (3) comprises a microfluidic chip (31) with a structure-gradient plasmonic super surface array (314) fixed in the channel and modified with surface detection antibodies, an assembly (32) for controlling the reciprocating flow of the liquid, and an optical imaging detection assembly (33).

10. The micro-marker immunoassay device of claim 9, wherein, The assembly (32) for controlling the reciprocating flow of the liquid realizes the reciprocating flow of the liquid by controlling the gas pressure.

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