Portable nanochip for visual detection of cardiac troponin

Abstract

The application provides a portable nanochip for visual detection of cardiac troponin, which comprises a chip substrate and antibody-modified selenium nanoparticles coated on the chip substrate, the antibody-modified selenium nanoparticles can specifically capture target antigens, and the detection of the target antigens is realized by detecting the scattering light signal of the selenium nanoparticles. The application realizes label-free, portable and visual detection of protein biomarkers, and the detection mode is simple, the detection equipment is portable, and the detection is rapid and sensitive.

Description

Technical Field

[0001] This invention relates to the fields of optical nanoimaging and biomarker detection technology, and in particular to a portable, visual nanochip for detecting cardiac troponin. Background Technology

[0002] Cardiovascular disease is a major global health threat. Therefore, there is an urgent need to revolutionize cardiac biomarker detection technologies, enabling their application not only in the identification of rapid acute events (such as acute myocardial infarction) but also in routine cardiac health monitoring. Traditional enzyme-linked immunosorbent assays (ELISA) and more recent mass spectrometry (MS)-based analyses require specialized laboratory equipment and complex workflows, making them unsuitable for resource-constrained environments and non-invasive daily use. Nanoparticle-based optical biosensors, such as colloidal gold test strips, are easy to operate but fail to meet the clinical requirements for myocardial biomarker detection in terms of sensitivity and accuracy. Therefore, a critical trade-off exists between detection sensitivity and portability. Over the past decade, nanophotonic biosensors have emerged as one of the viable alternatives for point-of-care testing. For example, surface-enhanced Raman scattering and high-quality resonant modes of dielectric materials have achieved superior femtomolar sensitivity in rapid, label-free workflows. However, their reliance on spectrometers and complex optics reduces the portability and cost-effectiveness of device applications. To date, point-of-care biochemical cardiac assessment remains challenging. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a portable, visualized nanochip for detecting cardiac troponin, which has high sensitivity and high specificity.

[0004] In a first aspect, the present invention provides a nanochip comprising a chip substrate and antibody-modified selenium nanoparticles coated on the chip substrate, wherein the antibody-modified selenium nanoparticles are capable of specifically capturing target antigens, and the target antigens are detected by detecting the scattered light signal of the selenium nanoparticles.

[0005] In one embodiment of the present invention, the chip substrate can be a rigid substrate, such as a silicon substrate or a glass substrate, or a flexible substrate, such as a PET film.

[0006] In one embodiment of the present invention, the antibody-modified selenium nanoparticles are coated on the chip substrate by physical adsorption, chemical bonding or a biotin-streptavidin system.

[0007] In one embodiment of the present invention, the antibody is selected from one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nanobodies, or antibody fragments.

[0008] In one embodiment of the present invention, the antibody is an antibody against cardiac troponin.

[0009] In one embodiment of the present invention, the selenium nanoparticles are prepared by the following method: a reducing agent is added to a selenium solution to react and obtain selenium nanoparticles.

[0010] In one embodiment of the present invention, the reaction temperature is 90°C and the reaction time is 45-50 min.

[0011] In one embodiment of the present invention, the selenium solution includes a selenic acid solution, a selenate solution, a selenite solution, or a selenite solution. Preferably, the selenium solution is a sodium selenite solution. Preferably, the concentration of the selenium solution is 0.01-0.02 M.

[0012] In one embodiment of the present invention, the reducing agent is selected from one or more of ascorbic acid, sodium thiosulfate, sodium borohydride, or glucose. Preferably, the reducing agent is glucose. Preferably, the concentration of the reducing agent is 0.08-0.16M.

[0013] In one embodiment of the present invention, the molar ratio of selenic acid or selenate to reducing agent is 1:8.

[0014] In one embodiment of the present invention, the diameter of the selenium nanoparticles is 340nm ± 5nm.

[0015] In one embodiment of the present invention, the preparation process of selenium nanoparticles includes the following two stages:

[0016] 1) Selenium seed formation stage: Under the action of a reducing agent, selenium in the high valence state undergoes a reduction reaction to generate selenium seed crystals;

[0017] 2) Selenium nanosphere formation stage: Selenium seed crystals are directionally grown to a predetermined size, the reaction is terminated, and selenium nanospheres are obtained.

[0018] In one embodiment of the present invention, the selenium seed crystal formation stage takes 5-10 minutes, and the selenium nanosphere formation stage takes 45-50 minutes.

[0019] In one embodiment of the present invention, the predetermined size of the directional growth is 340nm ± 5nm.

[0020] In one embodiment of the present invention, the reaction is terminated by cooling, dilution, or adding a terminating agent. Preferably, the reaction is terminated by placing the reaction system in an ice bath.

[0021] In a second aspect, the present invention provides a method for preparing the above-mentioned chip, comprising coating antibody-modified selenium nanoparticles on a substrate.

[0022] In one embodiment of the present invention, the preparation method specifically includes the following steps:

[0023] (1) Coating: The selenium nanoparticle solution is coated onto the chip substrate and dried to form individual selenium nanospheres; then, antibody is added for incubation, and the antibody binds to the selenium nanospheres through electrostatic adsorption.

[0024] (2) Sealing treatment: A sealing liquid is added to the coated chip for sealing treatment.

[0025] In one embodiment of the present invention, the concentration of the selenium nanoparticle solution is 0.5 mg / ml, and the amount of selenium nanoparticle solution used for a 0.7 cm × 0.7 cm chip is 2.5 μL, resulting in a selenium nanoparticle distribution density of approximately 2.55 μg / cm² on the chip surface. 2 .

[0026] In one embodiment of the present invention, the drying temperature is 30°C and the drying time is 60 minutes.

[0027] In one embodiment of the present invention, the antibody concentration is approximately 50 μg / ml, and the amount of antibody used on a 0.7cm × 0.7cm chip is 50 μL, resulting in an antibody coating density of approximately 5.10 μg / cm² on the chip surface. 2 Preferably, the incubation time is 2 hours at 30°C.

[0028] In one embodiment of the present invention, the blocking solution comprises bovine serum albumin (BSA). Preferably, the concentration of the bovine serum albumin (BSA) solution is 1 mg / ml, and the blocking is performed at room temperature (30°C) for 30 minutes.

[0029] In one embodiment of the present invention, the effective antibody modification rate is >90%.

[0030] In one embodiment of the present invention, the chip substrate undergoes surface treatment before being coated with a selenium nanoparticle solution, including plasma cleaning and silanization treatment.

[0031] In one embodiment of the present invention, the method further includes a step of washing the sealed chip. Preferably, deionized water is used for washing, and the washing is performed 3-7 times.

[0032] In a third aspect, the present invention provides a method for antigen detection using the above-mentioned chip for non-diagnostic purposes, comprising contacting a sample containing a target antigen with the nanochip to generate a scattered light signal for detection.

[0033] In one embodiment of the present invention, the detection method specifically includes the following steps:

[0034] (1) The sample containing the target antigen is brought into contact with the nanochip so that the antibody-modified selenium nanoparticles specifically capture the target antigen;

[0035] (2) Rinse the nanochip to remove unbound material;

[0036] (3) The scattered light signal of the selenium nanoparticles on the nanochip was detected using a dark-field microscope;

[0037] (4) Analyze the scattered light signal to determine the presence or concentration of the target antigen.

[0038] Optionally, the analysis includes establishing a standard curve of scattered light signal intensity versus target antigen concentration using a target antigen standard solution, and comparing the scattered light signal intensity of the test sample with the standard curve to determine the concentration of the target antigen in the test sample.

[0039] In one embodiment of the present invention, a sample containing the target antigen is brought into contact with the nanochip and incubated at room temperature of about 25°C for 15 minutes.

[0040] In one embodiment of the present invention, the target antigen is selected from cardiac troponin (cTnT).

[0041] In one embodiment of the present invention, the sample includes serum, urine, and saliva.

[0042] In one embodiment of the present invention, the sample volume is 50 μL.

[0043] In one embodiment of the present invention, the rinsing step is performed using deionized water, and the rinsing is performed 3-7 times.

[0044] In one embodiment of the present invention, the microscope can be a metallurgical microscope. The metallurgical objective lens has a magnification of 100x, and the imaging camera has a resolution of 16 megapixels. Alternatively, it can be a portable microscope and a mobile phone camera; the portable microscope has a magnification of 10x, and the smartphone camera has a resolution of 64 megapixels.

[0045] In one embodiment of the present invention, the binding of biomarkers to selenium nanospheres causes a color change in the nanospheres that is perceptible to the naked eye. Alternatively, RGB analysis of the acquired images can be performed using ImageJ software to quantify the concentration of the biomarkers by quantitatively characterizing the color intensity changes before and after biomarker binding.

[0046] In one embodiment of the present invention, the method for analyzing the scattered light signal includes:

[0047] (a) Acquire RGB color images of the biochip;

[0048] (b) Separate the RGB color image into three channels: red, green, and blue;

[0049] (c) Identify the core pixel region in each channel and extract its grayscale value;

[0050] (d) Calculate the average gray value of the core pixel region and compare the signal changes before and after capturing the antigen to determine the concentration of the target antigen.

[0051] In one embodiment of the present invention, the concentration of the target antigen is determined by identifying the intensity change of the scattered light signal in the red channel, thereby improving the accuracy of detection.

[0052] In one embodiment of the present invention, the detection method includes preparing target antigen standard solutions of different concentrations, contacting them with the nanochip, processing them according to steps (1) to (3), obtaining the corresponding scattered light signal intensity, and plotting a standard curve of scattered light signal intensity versus target antigen concentration.

[0053] In a fourth aspect, the present invention provides the application of the above-mentioned nanochip in the preparation of in vitro diagnostic reagents.

[0054] In one embodiment of the present invention, the reagent is used to detect cardiac troponin (cTnT).

[0055] In one embodiment of the present invention, the reagent can be used to detect acute myocardial infarction or predict myocardial injury.

[0056] In one embodiment of the present invention, the detection step includes:

[0057] Step a) Detect the expression level of cTnT in the sample to be tested;

[0058] Step b) Diagnose acute myocardial infarction or predict myocardial damage based on the test results of a).

[0059] In one specific embodiment of the present invention, the sample to be tested in step a) includes serum, urine and saliva.

[0060] In one specific embodiment of the present invention, step b) includes comparing the expression level of cTnT in the sample to be tested with a first threshold.

[0061] i. If the expression level of cTnT in the sample is significantly higher than the first threshold, the diagnosis is acute myocardial infarction;

[0062] ii. If the expression level of cTnT in the sample is significantly lower than the first threshold, acute myocardial infarction is excluded.

[0063] In one embodiment of the present invention, the first threshold is 108.65 pg / ml.

[0064] In one embodiment of the present invention, the more significant the increase in the expression level of cTnT compared to a first threshold, the greater the severity of acute myocardial infarction.

[0065] In one specific embodiment of the present invention, step b) includes predicting myocardial injury based on the expression level of cTnT in the sample to be tested:

[0066] i. If the expression level of cTnT in the sample to be tested is between the first threshold and the second threshold, the patient has a high level of cardiac damage;

[0067] ii. If the expression level of cTnT in the sample is significantly lower than the second threshold, the patient is excluded from the possibility of myocardial injury.

[0068] In one embodiment of the present invention, the second threshold is 41.6 pg / ml.

[0069] In a fifth aspect, the present invention provides a detection device comprising the aforementioned nanochip.

[0070] In one embodiment of the present invention, the detection device further includes:

[0071] a) A signal acquisition module for acquiring the scattered light signal of selenium nanoparticles, the signal acquisition module including a microscope and a smartphone with a camera function;

[0072] b) A signal analysis module, connected to the signal acquisition module, is used to analyze the acquired scattered light signal and calculate the detection result;

[0073] The smartphone is equipped with a data analysis application (Image J software), which is used to process scattered light signal data and generate detection results.

[0074] In a sixth aspect, the present invention provides a reagent kit comprising: the above-described chip and deionized water.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] This invention enables label-free, portable, and visualized detection of protein biomarkers. The detection method is simple, the equipment is portable, and the detection is rapid and sensitive. This invention eliminates the need for complex instruments or specialized data analysis software; patients can complete home testing using only a common optical microscope. This is of great significance for patients in remote areas or those lacking professional knowledge. Taking cardiac troponin T (cTnT) as an example, this invention enables real-time detection of acute myocardial infarction and early warning of cardiac damage based on various body fluids (serum, urine, or saliva).

[0077] This invention is based on the fact that after high-refractive-index selenium nanospheres specifically adsorb target protein biomarkers on their surface, the scattering signal changes significantly, and the detection results can be observed with the naked eye after imaging with a camera, without the need for complex detection instruments and analysis software.

[0078] Compared to other methods for detecting protein biomarkers, this invention requires only 50 microliters of sample to provide results. It uses a small sample volume and requires no complex instruments or specialized data processing software. The results are visualized using an optical microscope and can be viewed with the naked eye, making it simple and convenient. Furthermore, this invention eliminates the need for fluorescent markers, reducing complex sample processing steps and interference from background fluorescence. This invention provides a convenient method for portable and rapid detection of biomarkers, which is of great significance in early warning of cardiovascular diseases, rapid diagnosis of acute myocardial infarction, and postoperative monitoring, making home testing for diseases including, but not limited to, cardiovascular diseases possible.

[0079] Compared to selenium nanoparticle-based flumetometry assays reported in the literature, this invention has a lower detection limit (from 1 ng / ml to 0.4 pg / ml) and is suitable for non-invasive bodily fluids (such as urine and saliva), avoiding blood collection and blood sample processing, making it more suitable for daily home use. Attached Figure Description

[0080] Figure 1 This is a schematic diagram of the present invention. Figure 1 Selenium nanospheres were synthesized via the glucose reduction of sodium selenite. Figure 1 b is the preparation of the biodetection chip. A selenium nanosphere solution of appropriate concentration is dropped onto a hydrophilic substrate, evaporated to dryness to form a single-sphere selenium nanosphere dispersion array, and then an antibody is incubated on the surface of the selenium sphere. Due to electrostatic interaction, the antibody will be adsorbed on the selenium sphere to form an antibody layer. Figure 1 c is sample detection, in which body fluid containing the antigen of the protein to be detected is dropped onto the detection chip, incubated, and then rinsed to remove non-specific adsorption; Figure 1 d represents direct optical detection and result readout. Through direct observation using a regular optical microscope, the selenium nanospheres that specifically capture antigens produce a visible change in scattering color. The entire detection process is simple, fast, and portable, and the results are intuitive.

[0081] Figure 2 These are optical images of a single selenium nanosphere captured by a camera under dark-field light. The selenium nanospheres exhibit significant color changes when capturing antibodies and recognizing antigens of different concentrations. ImageJ software was used to analyze and quantify the color intensity changes in different channels.

[0082] Figure 3 It is a standard curve for the quantitative detection of cTnT protein using nanochips based on the intensity of the red channel.

[0083] Figure 4 These are the test results from the nanochip on clinical samples. The nanochip can enable rapid diagnosis of acute myocardial infarction based on various bodily fluids (serum, urine, saliva), and can also provide early warning of heart damage in the general population.

[0084] Figure 5 The results are ROC curve analysis of the detection results of the nanochip on clinical samples.

[0085] Figure 6 It is an integrated mobile phone detection device that uses a portable microscope to image the scattering signals of selenium nanospheres on a mobile phone, and processes and analyzes the images through a built-in mobile phone APP to read out the results. Detailed Implementation

[0086] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0087] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0088] Example 1

[0089] 1. Preparation method of selenium nanospheres as follows: Figure 1 As shown in a, the specific steps include the following:

[0090] (1) Preparation of selenium seed crystals by reduction reaction:

[0091] A certain amount of sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a target concentration of 0.01M. Under stirring conditions, a glucose solution with a concentration of 0.08M was added to the above solution as a reducing agent. The molar ratio of sodium selenite solution to glucose solution was 1:8. The system temperature was controlled at 90℃ and the reaction time was controlled at 5min to achieve the reduction reaction of sodium selenite and form red selenium seed crystals.

[0092] (2) Seed-induced growth to form selenium nanospheres:

[0093] The reaction system temperature was maintained at 90℃, and the reaction continued to achieve the directional growth of selenium seeds and gradually form spherical selenium nanoparticles. Stirring was maintained throughout the reaction, and the reaction time was controlled at 50 min to regulate the particle size to 340 nm.

[0094] (3) Reaction termination treatment:

[0095] After the reaction is complete, the reaction system is immediately subjected to rapid cooling (e.g., placed in an ice bath) to terminate the further growth of selenium nanoparticles and obtain a selenium nanoparticle suspension with stable particle size and good dispersibility.

[0096] (4) Centrifugation and washing:

[0097] The reaction solution was transferred to a centrifuge tube and the selenium nanoparticles were collected by centrifugation (10,000 rpm, 10 min). After discarding the supernatant, the precipitate was washed multiple times (e.g., 2-3 times) with deionized water to remove any residual soluble impurities and byproducts from the reaction.

[0098] (5) Low-temperature storage:

[0099] The washed selenium nanoparticles were resuspended in deionized water or a suitable buffer solution to obtain a colloidal solution with a concentration of 0.1 mg / ml. The solution was then sealed and stored at 4°C for later use.

[0100] 2. The fabrication method of the bio-detection chip is as follows: Figure 1 As shown in b, the specific steps include:

[0101] (1) Loading and drying of nanoparticle solution

[0102] 2.5 μl of a colloidal solution containing selenium nanoparticles (SeNPs) (concentration 0.5 mg / ml) was dropped onto the surface of the chip substrate and allowed to stand at 30 °C for 1 hour to evaporate to dryness. The SeNPs on the chip were uniformly distributed and formed a single-particle dispersion.

[0103] (2) Antibody modification

[0104] A capture antibody solution (Anti-cTnT antibody, purchased from Xiamen Tongrenxin Biotechnology Co., Ltd.) with a concentration of 50 μg / ml was prepared. 50 μl of the antibody solution was added to the prepared selenium nanoparticle chip and incubated at 30℃ for 2 h. Due to electrostatic interaction, the antibody was adsorbed onto the surface of the selenium nanospheres. The effective modification rate of the antibody was >90% as determined by fluorescence method.

[0105] (3) Sealing treatment

[0106] After antibody modification, a blocking solution containing bovine serum albumin (BSA) is added, and the mixture is incubated at 30°C for 0.5 hours to block the active and non-specific binding sites of the unbound antibody and reduce background interference.

[0107] 3. Marker detection and signal analysis

[0108] like Figure 1As shown in Figure c, a solution containing the myocardial infarction biomarker cTnT (purchased from Bio-Tech Biotechnology Co., Ltd.) was added to the chip for specific recognition. The recognition time was 15 min at a temperature of 25°C. After capture, the detection chip was rinsed three times with deionized water, dried, and observed under an optical microscope.

[0109] like Figure 1 As shown in d, under a dark-field incident light source, the camera collects the scattered light signal from the selenium nanospheres. After the selenium nanospheres capture the antigen, ImageJ software automatically identifies all single nanospheres within the entire field of view and extracts their average pixel values. Non-single-particle scattered points can be excluded based on roundness and area. High-throughput identification effectively increases the number of parallel experiments, ensuring high reliability and repeatability of the experimental results. The specific method is as follows: the RGB color image is separated into three channels: red, green, and blue; the core pixel region is identified in each channel, and its grayscale value is extracted; the average pixel value of each nanosphere is calculated; and the signal changes before and after capture are compared to reflect the change in scattering intensity as antigen binding occurs. Figure 2 As shown in the figure, the histogram displays the quantitative grayscale values ​​of the R, G, and B channels analyzed by ImageJ software. The intensity of the red channel shows a significant change, with a marked decrease in intensity.

[0110] Example 2

[0111] Prepare cTnT protein solutions of different concentrations (10 -14 g / mL, 10 -13 g / mL, 10 -12 g / mL, 10 -11 g / mL, 10 -10 g / mL, 10 -9 g / mL, 10 -8 g / mL, 10 -7 A concentration of g / mL was added dropwise onto a parallel-prepared selenium nanosphere biodetector chip. 50 μl of sample was added to the chip, incubated at room temperature for 15 min, rinsed, and the chip was observed under a 100x objective lens of an optical microscope. Dark-field optical images were taken under dark-field illumination. The optical images showed that as the concentration of the recognized antigen gradually increased, the grayscale value of the red channel in the optical image of the selenium nanospheres decreased significantly, such as... Figure 3 This method detects substances containing 10. -14 g / ml to 10 -7 Post-sample color analysis results of g / ml cTnT protein are as follows: Figure 3As shown, a standard curve was obtained using a four-parameter fitting method. The detection limit was calculated to be 0.4 pg / ml based on the 3σ principle, and the linear detection range was 0.4–1134 pg / ml. This method offers short detection time, high sensitivity, and high reliability. Furthermore, it requires no complex labeling or equipment, making it easy to operate. It comprehensively improves the portability, timeliness, and sensitivity of myocardial infarction biomarkers.

[0112] Example 3

[0113] A selenium nanosphere biodetector chip was prepared based on the method described in Example 1, and a cTnT capture antibody was modified for it. Clinically obtained serum, urine, and saliva samples were used for detection. For detection, 50 μl of centrifuged body fluid sample was added to the detection chip, incubated at 25°C for 15 min, rinsed with deionized water, and dried. The identified chip was placed under a dark-field optical microscope to acquire dark-field optical images of the selenium nanospheres. The average value of the red channel pixels of all selenium nanospheres within the analytical field of view was automatically extracted using ImageJ software for quantitative analysis. Analysis revealed significant differences in cTnT levels between patients with acute myocardial infarction and healthy individuals in serum, urine, and saliva. Figure 4 The areas under the ROC curves were 0.997, 0.987, and 0.962, respectively, demonstrating high sensitivity and specificity. Figure 5 This demonstrates that it is feasible to use our method for rapid diagnosis of acute myocardial infarction using two non-invasive bodily fluids: urine and saliva.

[0114] Next, we measured the cTnT concentration in saliva samples from different populations using the same method as above. Some individuals in the general population had elevated cTnT levels, exceeding the 75% threshold (41.6 pg / ml), which may indicate a higher risk of cardiac damage. Figure 4 Saliva-based cTnT testing avoids the need for medical equipment and sample processing associated with venous blood collection. Combined with the advantages of our method—speed, sensitivity, and portability—it makes routine home-based cardiac biochemical assessments possible.

[0115] Example 4

[0116] This method also developed a smartphone-based microscope observation device. The chip fabrication and sample testing procedures are the same as above; however, instead of using a metallurgical microscope camera, the integrated observation device of a smartphone is used to acquire optical images during signal acquisition. For example... Figure 6As shown, the device comprises the hardware of a mobile phone and a portable microscope, as well as a data analysis app built into the phone. First, locate the smartphone's main camera and install a clamp aligned with it. The clamp's circular hole and the interface on the top of the microscope interlock to connect the phone to the microscope. Second, place the saliva-based detection chip for acute myocardial infarction and healthy individuals to be observed in the center of the sample holder, which is compatible with the bottom interface of the microscope. Third, turn on the LED light source of the portable microscope and capture a dark-field optical image of the selenium nanospheres on the chip. Finally, upload the acquired optical image to the app for automatic reading of the red intensity value and evaluation results. Verification shows that this mobile phone-integrated device can effectively differentiate and interpret acute myocardial infarction and healthy individuals, with a diagnostic sensitivity of 100%, specificity of 85%, and accuracy of 92.5%.

[0117] This method is not limited to the detection of cardiac troponin cTnT, but can also enable the portable and ultrasensitive detection of other related biomarkers, such as cancer protein biomarkers, viruses, and bacteria.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nanochip, characterized in that: The device includes a chip substrate and antibody-modified selenium nanoparticles coated on the chip substrate. The antibody-modified selenium nanoparticles can specifically capture target antigens, and the target antigens can be detected by detecting the scattered light signal of the selenium nanoparticles.

2. The nanochip as described in claim 1, characterized in that: The chip substrate can be a rigid substrate, such as a silicon substrate or a glass substrate, or it can be a flexible substrate, such as a PET film. Optionally, the antibody-modified selenium nanoparticles are coated on the chip substrate by physical adsorption, chemical bonding, or a biotin-streptavidin system; Optionally, the antibody is selected from one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nanobodies, or antibody fragments; Optionally, the antibody is an antibody against cardiac troponin.

3. The nanochip as described in claim 1, characterized in that: The selenium nanoparticles are prepared by the following method: adding a reducing agent to a selenium solution and reacting to obtain selenium nanoparticles; Optionally, the reaction temperature is 90℃ and the reaction time is 45-50 min; Optionally, the selenium solution includes a selenic acid solution, a selenate solution, a selenite solution, or a selenite solution. Preferably, the selenium solution is a sodium selenite solution, and the concentration of the selenium solution is preferably 0.01-0.02M. Optionally, the reducing agent is selected from one or more of ascorbic acid, sodium thiosulfate, sodium borohydride, or glucose; preferably, the reducing agent is glucose; preferably, the concentration of the reducing agent is 0.08-0.16M. Optionally, the molar ratio of the selenic acid or selenate to the reducing agent is 1:8; Optionally, the preparation process of selenium nanoparticles includes the following two stages: 1) Selenium seed formation stage: Under the action of a reducing agent, selenium in the high valence state undergoes a reduction reaction to generate selenium seed crystals; 2) Selenium nanosphere formation stage: Selenium seed crystals are directionally grown to the predetermined size, the reaction is terminated, and selenium nanospheres are obtained; Optionally, the selenium seed crystal formation stage takes 5-10 minutes, and the selenium nanosphere formation stage takes 45-50 minutes. Optionally, the predetermined size for the directional growth is 340 nm ± 5 nm.

4. A method for preparing the chip according to any one of claims 1-3, comprising coating antibody-modified selenium nanoparticles onto a substrate; Optionally, the preparation method specifically includes the following steps: (1) Coating: The selenium nanoparticle solution is coated onto the chip substrate and dried to form individual selenium nanospheres; then, antibody is added for incubation, and the antibody binds to the selenium nanospheres through electrostatic adsorption. (2) Sealing treatment: A sealing liquid is added to the coated chip for sealing treatment; Optionally, the concentration of the selenium nanoparticle solution is 0.5 mg / ml; Optionally, the drying temperature is 30°C and the time is 60 minutes; Optionally, the antibody concentration is approximately 50 μg / ml; the antibody coating density on the chip surface is approximately 5.10 μg / cm³. 2 Preferred incubation time is 2 hours at 30°C. Optionally, the blocking solution includes bovine serum albumin (BSA), preferably with a concentration of 1 mg / ml, and the blocking is performed at room temperature of 30°C for 30 min. Optionally, the chip substrate undergoes surface treatment before being coated with the selenium nanoparticle solution, including plasma cleaning and silanization treatment. Optionally, the process may also include a step of washing the sealed chip; preferably, deionized water is used for washing, and the number of washing cycles is 3-7.

5. A method for antigen detection using the chip described in any one of claims 1-3 for non-diagnostic purposes, characterized in that: This includes contacting a sample containing the target antigen with the nanochip to generate a scattered light signal for detection; Optionally, the detection method specifically includes the following steps: (1) The sample containing the target antigen is brought into contact with the nanochip so that the antibody-modified selenium nanoparticles specifically capture the target antigen; (2) Rinse the nanochip to remove unbound material; (3) The scattered light signal of the selenium nanoparticles on the nanochip was detected using a dark-field microscope; (4) Analyze the scattered light signal to determine the presence or concentration of the target antigen. Optionally, the analysis includes establishing a standard curve of scattered light signal intensity versus target antigen concentration using a target antigen standard solution, and comparing the scattered light signal intensity of the test sample with the standard curve to determine the concentration of the target antigen in the test sample.

6. The method as described in claim 5, characterized in that: The sample containing the target antigen was brought into contact with the nanochip and incubated at room temperature of approximately 25°C for 15 minutes. Optionally, the target antigen is selected from cardiac troponin (cTnT); Optionally, the samples include serum, urine, and saliva; Optionally, the sample volume is 50 μL; Optionally, the rinsing step is performed using deionized water, and the number of rinsing cycles is 3-7. Optionally, the microscope can be a metallurgical microscope, wherein the metallurgical objective lens has a magnification of 100x and the imaging camera has a resolution of 16 megapixels; or it can be a portable microscope and a mobile phone camera, wherein the portable microscope has a magnification of 10x and the smartphone camera has a resolution of 64 megapixels.

7. The method as described in claim 5, characterized in that: The binding of biomarkers to selenium nanospheres can cause a color change in the nanospheres that is visible to the naked eye; or the concentration of biomarkers can be quantified by performing RGB analysis on the acquired images using ImageJ software and quantitatively characterizing the color intensity changes before and after the binding of biomarkers.

8. The method as described in claim 5, characterized in that: The method for analyzing the scattered light signal includes: (a) Acquire RGB color images of the biochip; (b) Separate the RGB color image into three channels: red, green, and blue; (c) Identify the core pixel region in each channel and extract its grayscale value; (d) Calculate the average gray value of the core pixel region and compare the signal changes before and after capturing the antigen to determine the concentration of the target antigen; Optionally, the concentration of the target antigen can be determined by identifying changes in the intensity of the scattered light signal from the red channel; Optionally, the detection method includes preparing target antigen standard solutions of different concentrations, contacting them with the nanochip, processing them according to steps (1) to (3), obtaining the corresponding scattered light signal intensity, and plotting a standard curve of scattered light signal intensity versus target antigen concentration; Optionally, the detection method has the following performance parameters: a detection limit of 0.4 pg / mL and a linear detection range of 0.4-1134 pg / mL, thereby achieving high sensitivity and a wide linear detection range.

9. The use of the nanochip according to any one of claims 1-3 in the preparation of in vitro diagnostic reagents; Optionally, the reagent is used to detect cardiac troponin (cTnT); Optionally, the reagent can be used to detect acute myocardial infarction or predict myocardial damage; Optionally, the detection steps include: Step a) Detect the expression level of cTnT in the sample to be tested; Step b) Diagnose acute myocardial infarction or predict myocardial damage based on the test results of a); Optionally, the sample to be tested in step a) includes serum, urine, and saliva; Optionally, step b) includes comparing the expression level of cTnT in the sample to be tested with a first threshold. i. If the expression level of cTnT in the sample is higher than the first threshold, it is diagnosed as acute myocardial infarction; ii. If the expression level of cTnT in the sample is lower than the first threshold, acute myocardial infarction is excluded. Optionally, the first threshold is 108.65 pg / ml; Optionally, the more significant the increase in cTnT expression level compared to the first threshold, the greater the severity of acute myocardial infarction; Optionally, step b) includes predicting myocardial injury based on the expression level of cTnT in the test sample: i. If the expression level of cTnT in the sample to be tested is between the first threshold and the second threshold, the patient has a high level of cardiac damage; ii. If the expression level of cTnT in the sample is below the second threshold, the patient is excluded from the possibility of myocardial injury; Optionally, the second threshold is 41.6 pg / ml.

10. A detection device or kit, said detection device comprising the nanochip according to any one of claims 1-3; Optional, also includes: a) A signal acquisition module for acquiring the scattered light signal of selenium nanoparticles, the signal acquisition module including a microscope and a smartphone with a camera function; b) A signal analysis module, connected to the signal acquisition module, is used to analyze the acquired scattered light signal and calculate the detection result; The smartphone is equipped with a data analysis application (Image J software), which is used to process scattered light signal data and generate detection results. And / or the kit comprising: the nanochip according to any one of claims 1-3, and deionized water.

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