Method and device for immunodetection of trace markers based on gold magnetic nanoparticles and structure gradient plasma metasurface

Abstract

The invention discloses a method and a device for immunodetection of trace markers based on gold magnetic nanoparticles and a structure gradient plasma metasurface. According to the method, the image sensing performance is enhanced based on coupling of the gold magnetic nanoparticles and the structure gradient plasma metasurface, and the detection sensitivity of the method is greatly improved through multiple means of marker separation and purification, electric field coupling enhancement, image signal amplification, signal-to-noise ratio improvement and the like. Rapid immunodetection of ultra-low-concentration and ultra-low-dose tear markers is realized, and further detection of more markers means that the kit is expected to be applied to screening and diagnosis of ophthalmology, cardiovascular and neurodegenerative diseases and the like. Based on the method, the invention provides a trace marker immunodetection device which can be used for ultrasensitive and rapid immunodetection of trace tear markers, and concentration detection of the tear markers can be completed within 3 minutes.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and specifically, relates to a method and device for immunodetection of trace markers based on gold magnetic nanoparticles and structure-gradient plasma supersurface. Background Art

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

[0003] Tears are an important component for maintaining the homeostasis of the ocular surface microenvironment. While moistening and protecting the ocular surface, they also contain a large amount of information reflecting the health status of the eyes. Among them, tear markers, including proteins, lipids and metabolites, provide great potential for the diagnosis of ocular diseases with their significant diagnostic sensitivity and specificity. For example, matrix metalloproteinase 9 (MMP-9), an important tear marker for monitoring the prognosis of dry eye, is a protease secreted by the corneal epithelium. When tears evaporate rapidly and the stability of the tear film weakens, the homeostasis of the ocular surface microenvironment is broken, resulting in an increase in tear osmotic pressure, which triggers the release of MMP-9, thereby inducing a progressive inflammatory cycle. Therefore, testing markers with indicative value or actual functions can effectively assist in the judgment of ophthalmic pathogenic factors and disease screening. At present, the determination method of tear markers is mainly based on lateral flow immunochromatography. For example, Chinese patent CN113075414B authorizes a method and device for diagnosing ocular surface inflammation and dry eye disease. Although it qualitatively provides whether the marker exceeds the empirical safety value, it cannot perform concentration detection to achieve the severity classification of dry eye. The only commercial instant detection device for tear MMP-9 approved by the U.S. Food and Drug Administration, InflammaDry, also has this problem. In addition, due to the small amount of tears and the extremely low concentration of a considerable number of markers (pg / mL), it also creates a major obstacle for the existing technology to accurately quantify tear markers and diagnose diseases. 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 ophthalmic diseases. Summary of the invention

[0004] The purpose of the present invention 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 structure-gradient plasma supersurfaces.

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

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention first provides a method for immunodetection of trace markers based on gold magnetic nanoparticles and structure-gradient plasma supersurfaces, comprising the following steps:

[0008] S1. Prepare a structured gradient plasma metasurface with surface-modified detection antibodies, use an optical imaging detection component to collect the initial transmission image of the structured gradient plasma metasurface, and record the position of the initial bright stripes;

[0009] S2. Adding gold magnetic nanoparticles modified with capture antibodies to the sample to be tested to obtain gold magnetic nanoparticles combined with markers, coupling the gold magnetic nanoparticles combined with markers with the structure-gradient plasma metasurface to obtain the structure-gradient plasma metasurface coupled with the gold magnetic nanoparticles combined with markers;

[0010] S3. Collect the transmission image of the structured gradient plasma metasurface coupled with gold magnetic particles with markers, record the position of the bright stripes, and calculate the marker concentration according to the moving distance value of the initial bright stripes;

[0011] Wherein, the structure gradient plasma supersurface is a periodic gradient plasma supersurface having a micro-nano structure with a geometric morphology that changes periodically at fixed intervals, or a morphology gradient plasma supersurface having a micro-nano structure with a geometric morphology that changes periodically at fixed intervals;

[0012] The particle size of the gold magnetic nanoparticles is smaller than the size of the micro-nanostructure in the structure-gradient plasma supersurface; the spectrum characteristic absorption peak range of the structure-gradient plasma supersurface includes the spectrum 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 nanoparticle and the size of the electric field attenuation layer of the structure-gradient plasma supersurface.

[0014] The detection principle of the present invention is that when the spectral characteristic transmission peak of the nanostructure at a certain position of the structured gradient plasma supersurface matches the central wavelength of the narrow-band light, the transmission intensity at this position reaches a maximum value, and the optical imaging detection component collects the brightest area in the captured structured gradient plasma supersurface transmission intensity pattern image; the spectral characteristic transmission peak redshift caused by the coupling between the gold magnetic nanoparticles combined with the marker and the structured gradient plasma supersurface is then manifested as the movement of bright stripes in the transmission intensity pattern image, and finally the concentration of the tear marker is calculated according to the pixel movement of the bright stripes in the transmission intensity image; the step size of the redshift can be regulated by the particle size of the gold magnetic nanoparticles, the position of the spectral characteristic absorption peak and the coupling spacing.

[0015] Specifically, the present invention carries out structural design for plasma supersurfaces. After optical simulation and design, when the plasma supersurface with structure gradient interacts with light, the transmitted light intensity also changes gradually and continuously. After the micro-nano structures at different positions of the plasma supersurface with structure gradient interact with the light source of the optical imaging detection component, different characteristic spectra are exhibited, which are generally manifested as continuous gradual changes along the characteristic peaks of the spectrum corresponding to the structure gradient; when the optical imaging detection component shoots the plasma supersurface with structure gradient, since each pixel point on the image corresponds to the micro-nano structure of the plasma supersurface with structure gradient, the brightness on the image also changes with the law of the gradient structure. The more pixels, the higher the resolution, the slight changes caused by the entry of the marker to be tested in the sensitive area of ​​the plasma supersurface with structure gradient, and the movement of the bright stripes of the image can also be caused, so the sensitivity is also higher. The present invention calculates the concentration according to the moving distance of the bright stripes, and improves the imaging-based sensing sensitivity by optically amplifying the sensitive area of ​​the plasma supersurface with structure gradient. At the same time, when the gold magnetic nanoparticles combined with markers are captured by the structure gradient plasma supersurface, the refractive index near the structure gradient plasma supersurface changes significantly. At the same time, by modulating the gold magnetic nanoparticles to match the characteristic absorption wavelength of the structure gradient plasma supersurface array, and regulating the spacing between the gold magnetic nanoparticles and the structure gradient plasma supersurface array to achieve electric field coupling enhancement, the coupling effect of the gold magnetic particles makes the moving pixel points of the bright stripes more, further greatly improving the sensitivity of detection. That is, the present invention first realizes image sensing through the gradient structure of the plasma supersurface, and amplifies the signal through the image, so that it is much more sensitive than the ordered conventional array plasma supersurface; secondly, the gold magnetic nanoparticles further increase the moving pixel points of the bright stripes, further improving the detection sensitivity; the present invention provides a marker detection method that can be accurately quantified, simple to operate, and ultra-low concentration response, and its concentration response can be as low as pg / mL.

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

[0017] Preferably, the gold magnetic nanoparticles modified with the capture antibody bind to one binding site of the marker to obtain gold magnetic nanoparticles bound to the marker, and the gold magnetic nanoparticles bound to the marker are brought into contact with the structural gradient plasma supersurface modified with the detection antibody, wherein the detection antibody binds to another binding site on the marker, thereby achieving the coupling of gold magnetic nanoparticle-marker-structural gradient plasma supersurface.

[0018] Furthermore, the morphology-gradient plasma supersurface is a plasma supersurface whose micro-nanostructure morphology gradually changes from columnar, diamond, chessboard to hole shape from inside to outside.

[0019] Preferably, the structure-gradient plasma supersurface adopts nanoimprinting technology (including but not limited to thermal nanoimprinting and ultraviolet nanoimprinting) to replicate the micro-nano structure of the structure-gradient plasma supersurface in large areas and batches; the noble metal is deposited on the structure-gradient surface by electron beam evaporation or nano-electrodeposition and other processes to make it have a localized surface plasma resonance effect; the structure-gradient plasma supersurface can be transferred to a solid phase interface of any form by a nano-transfer process. Specifically, the plasma supersurface with morphology and periodic gradient can be realized by electron beam lithography exposure technology (EBL), and the morphology gradient can also be realized by coherent interference exposure. Among them, the morphology gradient law is to change along a certain specific angle based on a certain initial shape (such as a cube) (such as eventually becoming a cylinder); the periodic gradient can change periodically and gradually along the x direction or the x / y direction (that is, the structural distance of the two microscopic morphologies gradually changes).

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

[0021] Furthermore, the characteristic absorption peak of the structural gradient plasma metasurface is located in the visible light band.

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

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

[0024] Furthermore, the step S2 realizes coupling of the gold magnetic nanoparticles with markers and the structure-gradient plasma supersurface through reciprocating flow.

[0025] Preferably, the reciprocating flow is driven by air pressure to control the reciprocating flow of the liquid.

[0026] Preferably, the reciprocating flow is controlled by applying air pressure with periodic changes in size to control the reciprocating flow of the liquid.

[0027] Preferably, the air pressure variation cycle is 2 to 4 times per minute.

[0028] The present invention provides application of any of the above methods in detecting tear markers.

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

[0030] The present invention also provides a trace marker immunoassay device for implementing any of the above-mentioned 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 capture elements and a magnetic control component; the microfluidic chip detection module comprises a microfluidic chip with a structural gradient plasma supersurface having a surface-modified capture element fixed in a channel, a component for controlling the reciprocating flow of liquid, and an optical imaging detection component.

[0031] Furthermore, when the trace marker immunoassay device is used to detect tear markers, the sample collection module is a tear collector, and the tear collector is based on the capillary method. The tip of the collector is placed at the lower edge of the tear meniscus, and tears are collected from the conjunctival sac through capillary action; the sample pretreatment module is used to mix the tear sample with gold magnetic nanoparticles modified with capture elements, and the gold magnetic nanoparticles are combined with the markers to be tested in the sample, and the gold magnetic nanoparticles combined with the markers to be tested are obtained through magnetic separation.

[0032] Preferably, the tear collector collects tears from the conjunctival sac and then directly drops them into a tear diluent, which is then fully mixed; the tear diluent is a mixture of 0.1×PBS and gold magnetic nanoparticles modified with capture antibodies.

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

[0034] Furthermore, the microfluidic channel of the microfluidic chip has an inlet, a solution storage channel and a liquid outlet, and the inlet and the liquid outlet are separately located at two ends of the upper part of the solution storage channel.

[0035] Furthermore, the size of the microfluidic channel is determined after comprehensive analysis based on the size of the structure-gradient plasma metasurface, the volume of the sample to be tested, and the solution diffusion distance.

[0036] Preferably, the size of the microfluidic channel is larger than the structural gradient plasma supersurface sensing area.

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

[0038] Furthermore, the component for controlling the reciprocating flow of liquid realizes the reciprocating flow of liquid by controlling the air pressure.

[0039] Preferably, the air pressure variation cycle is 2 to 4 times per minute.

[0040] Preferably, the sample to be tested added to the microfluidic channel is controlled by the periodically changing air pressure at the injection port, and flows back and forth in the microfluidic channel, so that the markers bound to the gold magnetic particles quickly bind to the detection antibodies modified on the surface of the structure-gradient plasma supersurface array.

[0041] Furthermore, the optical imaging detection component includes a narrow-band light source, a camera and a CMOS image sensor.

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

[0043] S1. using an optical imaging detection component to collect an initial transmission image of a structured gradient plasma metasurface and record the position of bright stripes, wherein the structured gradient plasma metasurface is modified with a detection antibody;

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

[0045] S3. forming a seal at one end of the microchannel, applying a periodically changing air pressure through a component controlling the reciprocating flow of the liquid to control the solution sample to reciprocate in the microchannel for multiple times, so that the gold magnetic nanoparticles combined with the tear marker are coupled with the structure-gradient plasma supersurface of the surface-modified detection antibody;

[0046] S4. Applying air pressure to evacuate the solution in the microchannel by controlling the reciprocating flow of the liquid;

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

[0048] S6. The optical imaging detection component collects the transmission image of the structured gradient plasma metasurface coupled with the gold magnetic particles bound with markers, records the position of the bright stripes, and calculates the marker concentration based on the movement value of the bright stripe pixels.

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

[0050] 1. The present invention first provides a method for immunodetection of trace markers based on gold magnetic nanoparticles and structural gradient plasma metasurfaces. The method enhances the image sensing performance by coupling gold magnetic nanoparticles with structural gradient plasma metasurfaces. The method greatly improves the detection sensitivity of the method through multiple means such as marker separation and purification, electric field coupling enhancement, image signal amplification, and signal-to-noise ratio improvement, thereby achieving rapid immunodetection of tear markers at ultra-low concentrations and ultra-low doses. Further coverage of the detection of more markers means that the method is expected to be applied to the screening and diagnosis of ophthalmic, cardiovascular, and neurodegenerative diseases.

[0051] 2. The present invention further provides a trace marker immunoassay device based on the above method, which can be used for ultra-sensitive and rapid immunoassay of trace tear markers. After collecting tears, they are mixed with a gold magnetic nanoparticle solution and directly dripped onto a microfluidic chip, and the concentration of tear markers can be detected within 3 minutes. Accurate and rapid detection of tear marker concentrations is of great significance for routine examinations of common eye diseases and their prevention, intervention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a schematic diagram of the immunoassay device based on the structural gradient plasma supersurface of the present invention; wherein, 1-sample collection module, 2-sample pretreatment module, 3-microfluidic chip detection module, 21-gold magnetic nanoparticles modified with capture antibodies, 22-magnetic control component, 31-microfluidic chip, 32-component for controlling the reciprocating flow of liquid, 33-optical imaging detection component, 311-inlet, 312-solution storage channel, 313-liquid outlet, 314-structural gradient plasma supersurface array with surface modified detection antibodies.

[0053] Figure 2 This is a schematic diagram of the process of ultrasensitive and rapid immunodetection of trace tear markers based on the method of the present invention for enhancing image sensing performance based on the coupling of gold magnetic nanoparticles and structured gradient plasma supersurface.

[0054] Figure 3 The present invention regulates the gold magnetic nanoparticle size and coupling distance to enhance the electric field coupling (E / E0) 2 Finite-difference time-domain (FDTD) simulation results.

[0055] Figure 4 This is a schematic diagram of the morphology of the structural gradient plasma supersurface prepared by the present invention.

[0056] Figure 5 It is the pixel value of the movement of bright stripes in the transmission image caused by the combination of markers of different concentrations when the immunoassay device based on the structural gradient plasma supersurface of the present invention is coupled with or without gold magnetic nanoparticles.

[0057] Figure 6 It is the pixel value of the movement of bright stripes in the transmission image caused by the combination of markers of different concentrations under the conditions of with and without controlling the reciprocating flow of tear samples in the microchannel in the method of the present invention based on the coupling of gold magnetic nanoparticles and structured gradient plasma supersurface to enhance image sensing performance. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0060] Example 1

[0061] This embodiment provides an ultra-sensitive rapid immunoassay device for detecting trace tear markers ( Figure 1 ), the device includes a sample collection module 1, a sample pre-treatment module 2 and a microfluidic chip detection module 3; the sample pre-treatment 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, a liquid outlet 313, and a structured gradient plasma supersurface array 314 with surface-modified detection antibodies; the structured gradient plasma supersurface 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 based on coupling gold magnetic nanoparticles with structured gradient plasma metasurfaces to enhance image sensing performance is used for ultrasensitive and rapid immunoassay of trace tear markers. Figure 2 As shown, gold magnetic nanoparticles modified with capture antibodies are used to separate and purify markers in tear samples. Through reciprocating flow, the tear markers bind to the detection antibodies on the structural gradient plasmonic metasurface, thereby coupling the gold magnetic particles bound to the tear markers to the structural gradient plasmonic metasurface.

[0063] The electric field coupling enhancement can be achieved by regulating the distance between the gold magnetic nanoparticles and the structured gradient plasma metasurface array. Generally speaking, the particle size of the gold magnetic nanoparticles is best within 100nm, and the coupling distance between the gold magnetic nanoparticles and the structured gradient plasma metasurface array can be determined according to the size of the capture antibody, marker, and detection antibody. Therefore, before the specific implementation, the electric field coupling enhancement (E / E0) of gold magnetic nanoparticles with different particle sizes and coupling distances was simulated with FDTD. 2The appropriate particle size and antibody were then selected; the results were Figure 3 As shown, the coupling enhancement effect is most obvious when the gold magnetic nanoparticles have larger particle sizes and shorter coupling distances.

[0064] In this embodiment, a commercial tear collector is first used to collect tears based on the capillary method, and then the tear sample is added dropwise to a solution containing capture antibody-modified gold magnetic nanoparticles to mix, and finally the mixed solution is loaded into the injection port of the microfluidic chip.

[0065] Further in this embodiment, the tear collection and pre-treatment process follows the following steps:

[0066] Place the tip of the collector at the lower edge of the tear meniscus and collect tears from the conjunctival sac through capillary action. It should be noted that the operation should be gentle to avoid the production of reflex tears. Then add the collected tears to 50 μL of a colloidal gold magnetic nanoparticle solution with a mass fraction of 8 parts per thousand (the gold magnetic nanoparticles are coated with capture antibodies, with a particle size of 100 nm, of which the gold particle size is 20 nm), mix well and keep it away from light for 30 minutes, then place a strong magnet at the bottom of the mixture to separate the gold magnetic nanoparticles bound to the marker to be tested, pour out the supernatant, add a certain volume of PBS solution and mix with the gold magnetic nanoparticles bound to the marker to be tested, and then directly add it to the injection port of the microfluidic chip.

[0067] In this embodiment, the characteristic absorption wavelength of the structure-gradient plasma supersurface coupled with the gold magnetic particles combined with biomarkers is in the visible light band. The structure-gradient plasma supersurface is manufactured in large areas and batches using nanoimprint technology, nanoelectrodeposition and nanotransfer processes; the silicon master plate used for nanoimprinting is prepared using laser interference lithography and reactive ion etching. The prepared plasma supersurface array is located at the center of the bottom of the microfluidic channel and is tightly adhered to its inner wall.

[0068] Specifically, the plasma metasurface with gradual morphology and periodicity can be realized by electron beam lithography (EBL), and the gradual morphology can also be realized by coherent interference exposure. Among them, the morphology gradual change rule is to change along a certain angle based on a certain initial shape (such as a cube) (such as eventually becoming a cylinder); the periodic gradual change can be periodically changed along the x direction or the x / y direction (that is, the structural distance of the two microscopic morphologies gradually changes).

[0069] Further in this embodiment, the processing technology of the plasma metasurface array with a gradual morphology adopts the following steps:

[0070] The pre-prepared ormostamp material master plate (array of nanopore structure with gradient morphology) was batch-copied by hot nanoimprinting process, thereby obtaining a complementary nanopillar structure array on the hot melt adhesive 652 (pressure 0.3 tons, duration 5 minutes); then the array was subjected to nanoelectrodeposition process (deposition current 10 mA, duration 2 minutes) to obtain a complementary gold nanopore structure. Figure 4 That is, the morphology-gradient plasma supersurface prepared by the chip, and the morphology gradually changes from column-diamond-chessboard-hole from the inside to the outside.

[0071] In this embodiment, the microfluidic channel integrated with the metasurface array and capable of controlling the reciprocating flow of the sample to be tested has a length, width, and height of 3 cm, 5 mm, and 150 μm, respectively. The channel is formed by inverting a pre-prepared mother plate with polydimethylsiloxane (PDMS) and then curing it; the metasurface array is then placed at the center of the bottom of the microfluidic channel and tightly adhered to its inner wall; the component for controlling the reciprocating flow of the sample in the microfluidic chip channel is driven by air pressure to apply large and small periodic changes to achieve the reciprocating flow of the liquid; the optical detection component includes a narrow-band light source, a camera, and a CMOS image sensor.

[0072] The method for ultrasensitive and rapid immunoassay of tear markers using the above-mentioned device comprises the following steps:

[0073] S1: using CMOS to collect an initial transmission image of a structured gradient plasma metasurface and record the position of bright stripes, wherein the structured gradient plasma metasurface is modified with a detection antibody;

[0074] S2: Use gold magnetic particles to separate and purify the sample to be tested: first, mix the gold magnetic nanoparticles coated with the capture antibody with the tear sample, pour out the supernatant after magnetic separation, mix the gold magnetic nanoparticles combined with the tear marker with PBS buffer to obtain a mixed solution, and add the mixed solution to the injection port of the microchannel in the microfluidic chip;

[0075] S3: A seal is formed at one end of the microchannel, and the mixed liquid is controlled to flow back and forth in the microchannel multiple times by applying a periodically changing air pressure through air pressure drive, so that the gold magnetic nanoparticles combined with tear markers are coupled with the structure-gradient plasma metasurface of the surface-modified detection antibody;

[0076] S4: applying air pressure to empty the liquid in the microchannel by air pressure driving;

[0077] S5: adding ultrapure water to the inlet of the microchannel in the microfluidic chip to repeatedly clean the residual liquid in the microchannel;

[0078] S6: Collect the transmission image of the structured gradient plasma metasurface coupled with the gold magnetic particles bound with markers, record the position of the bright stripes, and calculate the biomarker concentration based on the movement value of the bright stripe pixels.

[0079] The rapid immunoassay device based on the coupling of gold magnetic nanoparticles and structure-gradient plasma metasurface manufactured in this embodiment can be used for the detection of various markers in tears and the screening of markers for common ophthalmic diseases.

[0080] Taking the detection of MMP-9, a tear marker for dry eye syndrome, as an example, the feasibility of the application of the developed ultra-sensitive and rapid immunoassay chip for tear markers based on gold magnetic nanoparticle-mediated structure-gradient supersurface plasmon resonance enhancement was tested. First, the optical imaging system was used to record the position of the bright stripes in the initial image of the structure-gradient plasmon supersurface, and the tears were collected and mixed with a PBS solution containing gold magnetic nanoparticles in one step, and then the sample was added to a microchannel of transparent material. Subsequently, a periodically varying air pressure was applied to the microchannel to control the reciprocating flow of the tear sample in the microfluidic channel for 10 times, and then the sample in the channel was emptied. Finally, ultrapure water was added to the microchannel to rinse it back and forth, and then the chip spectrum was tested. The biomarker concentration ( Figure 5 ).

[0081] Comparative Example 1

[0082] This comparative example is basically the same as Example 1, except that the tear sample was not mixed with the gold magnetic nanoparticles before the test. Figure 5 It can be seen that the coupling of gold magnetic nanoparticles and the structured gradient plasma metasurface array greatly improves the detection sensitivity.

[0083] Comparative Example 2

[0084] This comparative example is basically the same as Example 1, except that after the tear sample to be tested is added to the microchannel, the air pressure with periodic changes in size is not applied to the microchannel, but the test is performed after standing for 30 minutes. The test results after using this method and controlling the tear sample to flow back and forth 10 times (2 minutes) are compared. Figure 6 , which can control the reciprocating flow of tear samples and accelerate the capture of gold magnetic nanoparticles by the structural gradient plasma metasurface array, thereby shortening the detection time.

[0085] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for immunodetection of trace markers based on gold magnetic nanoparticles and structure-gradient plasma supersurfaces, characterized in that: The following steps are involved: S1. Prepare a structured gradient plasma metasurface with surface-modified detection antibodies, use an optical imaging detection component to collect the initial transmission image of the structured gradient plasma metasurface, and record the position of the initial bright stripes; S2. Adding gold magnetic nanoparticles modified with capture antibodies to the sample to be tested to obtain gold magnetic nanoparticles combined with markers, coupling the gold magnetic nanoparticles combined with markers with the structure-gradient plasma metasurface to obtain the structure-gradient plasma metasurface coupled with the gold magnetic nanoparticles combined with markers; S3. Collect the transmission image of the structured gradient plasma metasurface coupled with gold magnetic particles with markers, record the position of the bright stripes, and calculate the marker concentration according to the moving distance value of the initial bright stripes; Wherein, the structure gradient plasma supersurface is a periodic gradient plasma supersurface having a micro-nano structure with a geometric morphology that changes periodically at fixed intervals, or a morphology gradient plasma supersurface having a micro-nano structure with a geometric morphology that changes periodically at fixed intervals; The particle size of the gold magnetic nanoparticles is smaller than the size of the micro-nanostructure in the structure-gradient plasma supersurface; the spectrum characteristic absorption peak range of the structure-gradient plasma supersurface includes the spectrum characteristic absorption peak of the gold magnetic nanoparticles; 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 nanoparticle and the size of the electric field attenuation layer of the structure-gradient plasma supersurface.

2. The method according to claim 1, characterized in that: The morphology-gradient plasma supersurface is a plasma supersurface whose micro-nanostructure morphology gradually changes from columnar, diamond, chessboard to hole shape from inside to outside.

3. The method according to claim 1, characterized in that: The characteristic absorption peak of the structural gradient plasma supersurface is located in the visible light band.

4. The method according to claim 1, characterized in that: The optical detection component in step S1 includes a narrow-band light source and a camera device.

5. The method according to claim 4, characterized in that: The imaging device is a camera and a CMOS image sensor.

6. The method according to claim 1, characterized in that: The step S2 realizes coupling of the gold magnetic nanoparticles with markers and the structure-gradient plasma supersurface through reciprocating flow.

7. The method according to claim 6, characterized in that: The reciprocating flow is driven by air pressure to control the reciprocating flow of the liquid.

8. Use of the method according to any one of claims 1 to 7 in detecting tear markers.

9. A trace marker immunoassay device for implementing the method according to any one of claims 1 to 7, characterized in that: The invention comprises a sample collection module (1), a sample pre-processing module (2) and a microfluidic chip detection module (3); the sample pre-processing 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) having a structured gradient plasma supersurface array (314) with surface-modified detection antibodies fixed in a channel, a component (32) for controlling the reciprocating flow of liquid, and an optical imaging detection component (33).

10. The trace marker immunoassay device according to claim 9, characterized in that: The component (32) for controlling the reciprocating flow of liquid realizes the reciprocating flow of liquid by controlling the air pressure.

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