Surface enhanced Raman spectroscopy IL-6 immunoassay method and system and nano-label preparation method

Abstract

The invention relates to the technical field of surface-enhanced Raman scattering, in particular to a surface-enhanced Raman spectrum IL-6 immunoassay method and system and a nano-label preparation method, which are applied to data analysis of a low-antigen-concentration scene, firstly, a Raman scanning image is converted into a binary image according to a set threshold value, and quantification is performed by using a positive region proportion (RPP); an index relationship between a positive region proportion and an antigen concentration is utilized to verify that a single-molecule event obeys Poisson distribution. And finally, analyzing antigen concentration data by using a linear relationship between the product (RPP * ARI) of the proportion of the positive region and the average Raman intensity of the positive region and the concentration. When a clinical serum sample is tested, the immunoassay method is highly consistent with a standard clinical laboratory method. The nano-label prepared by the invention displays an obvious signal under the resolution of a single nano-particle, so that ultra-sensitive / single-molecule detection is realized.

Description

Technical Field

[0001] The invention relates to the technical field of surface enhanced Raman scattering, in particular to a surface enhanced Raman spectroscopy IL-6 immunoassay method and system and a nano-label preparation method. Background Art

[0002] Although SERS (surface enhancement of Raman scattering) has been widely used in various fields, the efficiency of SERS is often challenged by the uneven and sparse distribution of plasmon SERS hotspots. Although the hotspot area accounts for less than 0.007% of the surface area, it contributes up to 24% of the overall Raman signal, which poses a challenge to the positioning of single molecules in the hotspot area, especially at low target concentrations, which can lead to significant SERS intensity fluctuations, inevitably hindering the accuracy and repeatability of the results.

[0003] High-performance plasmon substrates with multiple hot spots, uniform distribution, and repeatability have always been sought after, which is also crucial for digital SERS detection. However, in existing technologies, solid substrates are usually heterogeneous, which poses challenges to batch-to-batch and intra-batch reproducibility even under strict manufacturing conditions. Therefore, reliable and accurate quantification at low concentrations may be limited. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a surface enhanced Raman spectroscopy IL-6 immunoassay method, system and nano-label preparation method to solve the problems in the background technology.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method of the present invention comprises the following steps:

[0007] The antigen-antibody sandwich structure was obtained by incubating a silicon chip functionalized with interleukin-6 antibody pre-modified by 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, interleukin-6 antigen and antibody-modified surface-enhanced Raman nanotags together;

[0008] Collect the surface enhanced Raman scattering spectrum of the silicon chip after the reaction;

[0009] Binarizing the surface enhanced Raman scattering spectra sequentially to obtain a binary image and using the binary image as a mask to obtain a surface enhanced Raman scattering digital image;

[0010] The Raman reporter molecule characteristic peaks of multiple partitions in the surface enhanced Raman scattering digital image are extracted, and the proportion of positive areas in the partitions is calculated according to the threshold method, and quantitative analysis is performed based on the proportion of the positive areas and the average Raman intensity of the positive areas to obtain antigen concentration data, wherein the positive area is the area where the characteristic Raman signal appears.

[0011] In one embodiment of the present application, the surface enhanced Raman scattering spectrum is sequentially binarized to obtain a binary image and used as a mask to obtain a digitized surface enhanced Raman scattering digital image, including:

[0012] Binarizing the surface enhanced Raman scattering spectrum based on a preset threshold to obtain a binary false color image with background information filtered out;

[0013] The binary false color image is used as a mask and convolved with the surface enhanced Raman scattering spectrum to obtain a surface enhanced Raman scattering digital image.

[0014] In one embodiment of the present application, quantitative analysis is performed based on the proportion of the positive area and the average Raman intensity of the positive area to obtain antigen concentration data, including:

[0015] Substitute the proportion of the positive area or the product of the proportion of the positive area and the average Raman intensity of the positive area into a pre-constructed quantitative formula to obtain antigen concentration data, wherein the quantitative formula includes an exponential quantitative formula and a linear quantitative formula, and the mathematical expressions of the exponential quantitative formula and the linear quantitative formula are:

[0016] y1=0.925-0.925*e- 0.03*x

[0017] y2=0.743*x+10.626

[0018] Wherein, y1 is the proportion of the positive area, and y2 is the product of the proportion of the positive area and the average Raman intensity of the positive area.

[0019] In one embodiment of the present application, the process of constructing the quantitative formula includes:

[0020] Assume that the number of antigens in each partition follows a Poisson distribution with parameter λ, and its probability expression is:

[0021]

[0022] p(0)=e -λ =1-E

[0023]

[0024]

[0025] In the formula, k represents the number of antigen molecules in each partition, and p(k) represents the probability that the number of antigen molecules in each partition is k;

[0026] When there is more than one antigen type in the partition, represents the average Raman intensity of the positive area, E is the percentage of the positive area, V d is the volume of each independent partition, C is the antigen concentration, and i0 is the average Raman intensity of a single nanometer;

[0027] The positive area ratio E and the volume of each independent partition V d , the average Raman intensity of a single nanometer i0, the average Raman intensity of the positive area Substitute the specific value of into the quantitative formula.

[0028] In one embodiment of the present application, the surface enhanced Raman scattering spectrum is collected by a Renishaw Raman microscope system with lasers of different wavelengths and powers.

[0029] In one embodiment of the present application, the spectrum of the surface enhanced Raman scattering spectrum is collected using a 100× objective lens, a power of 0.85 mW, an exposure time of 2 seconds, and a Raman scan data using a 633 nm excitation wavelength at 60×60 μm 2 Area collection with a step size of 3 μm.

[0030] In one embodiment of the present application, the configuration process of the silicon chip functionalized with interleukin 6 antibody includes:

[0031] Cleaning a silicon substrate having an oxide layer of target thickness using a piranha solution;

[0032] The cleaned silicon substrate is rinsed with ultrapure water and dried with a nitrogen flow;

[0033] The dried silicon substrate is subjected to plasma treatment to obtain a hydroxylated silicon substrate;

[0034] Treating the hydroxylated silicon substrate with 3-aminopropyltrimethoxysilane and baking the substrate to generate a dense organic silane layer to obtain a silanized silicon substrate;

[0035] The silanized silicon substrate is coupled with a glutaraldehyde solution, and after coupling, it is washed with ultrapure water and dried with a nitrogen flow to obtain an aldehyde-treated silicon substrate;

[0036] The aldehyde-modified silicon substrate is immersed in an interleukin-6 antibody capture solution for a certain target time, and then the surface of the aldehyde-modified silicon substrate is sealed using casein and a surfactant in a phosphate buffer solution to obtain an interleukin-6 antibody functionalized silicon chip.

[0037] The present application also provides a digital surface enhanced Raman spectroscopy interleukin-6 immunoassay system, including:

[0038] An experimental module for pre-modifying a silicon chip functionalized with an interleukin-6 antibody based on a 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, co-incubating an interleukin-6 antigen and an antibody-modified surface-enhanced Raman nanotag to obtain an antigen-antibody sandwich structure;

[0039] Raman spectrum acquisition module, used to collect surface enhanced Raman scattering spectrum of silicon chip after reaction;

[0040] An image processing module, used for sequentially binarizing the surface enhanced Raman scattering spectra to obtain a binary image and using it as a mask to obtain a surface enhanced Raman scattering digital image;

[0041] The quantitative analysis module is used to extract the characteristic peaks of Raman reporter molecules in multiple partitions in the surface enhanced Raman scattering digital image, calculate the proportion of positive areas in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive areas and the average Raman intensity of positive areas to obtain antigen concentration data, wherein the positive area is the area where the characteristic Raman signal appears.

[0042] The present application also provides a method for preparing a nano-tag, which is used to configure the antibody-modified surface-enhanced Raman nano-tag as described above, comprising:

[0043] Gold nanoparticles were obtained by reducing tetrachloroauric acid with citric acid;

[0044] The gold nanoparticles are used as seeds and silver ions are reduced by ascorbic acid to form an Ag shell;

[0045] Injection of Au into a solution containing an Ag shell I A solution and a reducing agent are used to prepare an Ag-Au alloy shell and a Raman reporter molecule is added to obtain a solution containing a surface enhanced Raman nano-label;

[0046] Purifying the solution containing the surface-enhanced Raman nano-tag to obtain the surface-enhanced Raman nano-tag;

[0047] The surface enhanced Raman nanotag is functionalized by using interleukin 6 antibody to obtain an antibody-modified surface enhanced Raman nanotag.

[0048] In one embodiment of the present application, the surface enhanced Raman nanotag is functionalized with an interleukin 6 antibody to obtain an antibody-modified surface enhanced Raman nanotag, comprising:

[0049] The surface enhanced Raman nano-tag is subjected to ultrasonic treatment in ultrapure water to obtain a suspension;

[0050] The suspension is coupled with a reaction buffer to obtain a resuspended suspension, wherein the reaction buffer comprises 0.2-0.6 mM CTAC, 1-5×10 -2 M Tris-HCl;

[0051] The nanotags in the resuspended suspension are incubated with the interleukin 6 antibody, stirred and mixed with a blocking solution to obtain a target solution, wherein the blocking solution comprises 1-3% BSA, 0.01-0.2% casein and 1-5×10 -2 M Tris-HCl;

[0052] The target solution is purified by centrifugation to obtain an antibody-modified surface-enhanced Raman nano-tag.

[0053] The beneficial effects of the present invention are as follows: the surface enhanced Raman spectroscopy IL-6 immunoassay method, system and nano-label preparation method of the present invention are applied to data analysis of low antigen concentration scenarios. First, the Raman scan image is converted into a binary image according to a set threshold, and the positive area ratio (RPP) is used for quantification. The exponential relationship between the positive area ratio and the antigen concentration verifies that the single molecule event obeys the Poisson distribution. Finally, the linear relationship between the product of the positive area ratio and the average Raman intensity of the positive area (RPP*ARI) and the concentration is used to analyze the antigen concentration data. Experimental results show that the present application is used for the quantitative detection of IL-6 in healthy human serum and saliva, with a recovery rate of 92.4% to 105.3%. Finally, when testing clinical serum samples, the immunoassay method has a strong consistency with the standard clinical laboratory method. In addition, the nano-label prepared by the present application shows a clear signal at a single nanoparticle resolution, thereby realizing ultrasensitive / single molecule detection. This provides a basis for antigen concentration analysis under low concentration conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0055] Figure 1 is a flow chart of a digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method shown in an embodiment of the present application;

[0056] Figure 2Schematic diagram of the construction process of digital SERS immunoassay in this application;

[0057] Figure 3 A schematic diagram of nanoparticle characterization in an embodiment of the present application;

[0058] Figure 4 A schematic diagram of the activity of a single nanoparticle in an embodiment of the present application;

[0059] Figure 5 These are the Raman scan binarization images at different IL-6 concentrations in this application;

[0060] Figure 6 This is the quantitative analysis result diagram of the binary image in this application;

[0061] Figure 7 These are the Raman scanned digital images at different IL-6 concentrations in this application;

[0062] Figure 8 It is a quantitative analysis result diagram under the digitized image in this application;

[0063] Fig. 9 This is a graph of the specificity evaluation results in this application;

[0064] Fig.10 This is a graph of the repeatability evaluation results in this application;

[0065] Fig.11 Schematic diagram of IL-6 measurement data in clinical samples in this application. DETAILED DESCRIPTION

[0066] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0067] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show the layers related to the present invention rather than being drawn according to the number, shape and size of the layers in actual implementation. In actual implementation, the type, quantity and proportion of each layer may be changed arbitrarily, and the layer layout may also be more complicated.

[0068] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0069] The following is an explanation of the terms used in this manual:

[0070] SERS, surface-enhanced Raman scattering, surface enhanced Raman scattering;

[0071] Au@Ag-Au, gold core-silver shell nanoparticles;

[0072] IL-6, Interleukin-6, interleukin-6;

[0073] APTES–GA, 3-aminopropyltrimethoxysilane-glutaraldehyde;

[0074] This application proposes a digital surface enhanced Raman scattering (SERS) immunoassay method for detecting IL-6 using core-shell Au@Ag-Au nanotags with ultrasensitivity and high reliability. Low-cost biofunctionalized silica cores are used as capture substrates and novel SERS nanotags are used as amplification elements. These tags exhibit strong, robust and reproducible signals at single nanoparticle resolution. We propose two analytical methods to verify that single-molecule events follow a Poisson distribution and to quantify protein biomarkers over a wide linear dynamic range. The high consistency between theoretical and experimental results enhances the reliability of the method. The detection method in this application provides two readouts: colorimetric analysis by naked eye at high concentrations (>1ng / mL) and digital SERS analysis at low concentrations. After method optimization, we obtained a linear range (R 2 =0.994), with a limit of detection (LOD) of 12.4 fg / mL, which is suitable for clinical application. The method was used for the quantitative detection of IL-6 in healthy human serum and saliva, with a recovery rate of 92.4% to 105.3%. Finally, when testing clinical serum samples, the immunoassay method showed strong consistency with the standard clinical laboratory method. Therefore, our proposed digital SERS immunoassay is a promising tool for accurate clinical diagnosis of IL-6-related diseases or other conditions.

[0075] Figure 1 FIG. 1 is a flow chart of a digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method shown in an embodiment of the present application. Figure 1 As shown: The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method of this embodiment may include the following steps:

[0076] S110, a silicon chip functionalized with interleukin-6 antibody pre-modified by 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, interleukin-6 antigen and antibody-modified surface-enhanced Raman nanotags were co-incubated to obtain an antigen-antibody sandwich structure;

[0077] Among them, this application uses the classic APTES-GA coating method to form a sandwich structure of silicon chip-antibody-antigen.

[0078] By fixing the antibody on the surface of the silicon chip and introducing the antigen sample to be detected, the specific binding between the antigen and the antibody can form a sandwich structure. The nanoparticle structure can significantly enhance the signal strength, so that even trace amounts of antigen can be accurately detected. At the same time, the formation of the sandwich structure usually involves a rapid antigen-antibody binding reaction, which allows the entire detection process to be completed in a shorter time. For example, in some advanced biosensors, the formation of the sandwich structure and signal detection can be completed in a few minutes or even seconds. In addition, the design of the sandwich structure makes the detection process simpler and more intuitive. You only need to introduce the sample onto the chip and then observe the formation of the sandwich structure and the signal change. As a strong and stable substrate material, the silicon chip can provide good support and protection. This helps to maintain the stability and reliability of the sandwich structure, thereby improving the accuracy and repeatability of the detection.

[0079] S120, collecting a surface Raman scattering spectrum of the silicon chip after the reaction;

[0080] Surface enhanced Raman scattering (SERS) is a special type of Raman spectroscopy that significantly enhances the Raman signals of target molecules located near plasmonic metal nanostructures by utilizing the optical properties of nanostructures with plasmonic properties.

[0081] In this application, Raman spectra and SERS scan images were acquired using a Renishaw Raman microscope system (Renishaw, UK) equipped with lasers of different wavelengths and powers: 50 mW 532 nm diode laser, 17 mW 633 nm HeNe laser and 300 mW 785 nm diode laser. -1 The spectra were collected using a 100× objective (NA=0.85) with a power of 0.85 mW and an exposure time of 2 seconds. Raman scan data were collected using an excitation wavelength of 633 nm over a 60×60 μm2 area with a step size of 3 μm. According to the Abbe limit (i.e., d=1.22λ / NA), the theoretical laser spot size is 908.54 nm. Data analysis focused on the NBA Raman reporter molecule at 592 cm -1 The characteristic peak at .

[0082] s130, sequentially binarizing the surface enhanced Raman scattering spectra to obtain a binary image and using the binary image as a mask to obtain a surface enhanced Raman scattering digital image;

[0083] The specific process includes:

[0084] S131, binarizing the surface enhanced Raman scattering spectrum based on a preset threshold value to obtain a binary false color image with background information filtered out;

[0085] Binarization divides the image into positive area and background through thresholding. Since the positive area is the object or area of ​​interest, and the background is the area around these objects or areas, by setting the threshold reasonably, the background pixel value can be uniformly set to a level (such as black), thereby filtering out the background information.

[0086] The main purpose of binarization is to highlight the positive area information. Since the background information is uniformly set to one level during the binarization process, the positive area information becomes more prominent and clear in the image. This makes subsequent image processing and analysis easier and more accurate.

[0087] S132, using the binary false color image as a mask to perform convolution with the original surface enhanced Raman scattering spectrum to obtain a digitized surface enhanced Raman scattering image.

[0088] The purpose of the mask is to block a local area. In this application, by blocking the background part, the foreground ROI can be extracted, thereby obtaining the image data basis for subsequent analysis, namely, the surface enhanced Raman scattering digital image.

[0089] S140, extracting characteristic peaks of Raman reporter molecules from multiple partitions in the digitized surface enhanced Raman scattering image, calculating the proportion of positive areas in the partitions according to a threshold method, and performing quantitative analysis based on the proportion of positive areas and the average Raman intensity of positive areas to obtain antigen concentration data, wherein the positive area is an area where a characteristic Raman signal appears.

[0090] In this application, the core principle of digital SERS is to disperse the antigen into many independent reaction areas or pixels, which is equivalent to a beam of light when collecting signals in this application. The area where a specific Raman signal appears after the immune reaction is recorded as "1", and the area without such a signal is recorded as "0". By counting the proportion of positive areas and then substituting it into the quantitative formula in this application, the concentration data can be obtained. The quantitative formula in this application includes:

[0091] y1=0.925-0.925*e -0.03*x (1)

[0092] y2=0.743*x+10.626 (2)

[0093] Wherein, y1 is the proportion of the positive area, and y2 is the product of the proportion of the positive area and the average Raman intensity of the positive area.

[0094] Formula (1) is an exponential quantitative formula, and formula (2) is a linear quantitative formula. The derivation process of the above formula includes:

[0095] Since the number of antigens in each partition is discrete, it follows the Poisson distribution with parameter λ. The Poisson distribution is a discrete probability distribution, which is mainly used to describe the number of rare events in a fixed time or space. The main characteristics of the Poisson distribution include: discreteness, independence, rarity, stationarity, memorylessness, equal expectation and variance, asymmetry, and additivity of events. The probability of obeying the Poisson distribution can be expressed as:

[0096]

[0097] Where k represents the number of antigen molecules in each region, λ represents the average number of antigen molecules in each partition, and p(k) represents the probability that the number of antigen molecules in each partition is k;

[0098] p(0)=e -λ =1-E#(S2)

[0099] Where E represents the number of regions marked as "1".

[0100]

[0101] Where m represents the total number of antigen molecules, n represents the total number of partitions, C represents the concentration of antigen molecules, V d Represents the volume of each partition.

[0102] As concentration increases, each partition may contain more than one antigen. In this case:

[0103]

[0104] Where E represents the number of regions marked as "1", represents the average Raman intensity of the positive area, Represents the average Raman intensity of a single nanoparticle.

[0105] Combining the above formulas, two analysis methods can be obtained:

[0106]

[0107]

[0108] The ratio of positive area (E) and concentration have an exponential relationship, which is the ratio of positive area multiplied by the average Raman intensity of positive area. There is a linear function relationship between the concentration.

[0109] Finally, the positive area ratio E and the volume of each independent partition V d , the average Raman intensity of a single nanometer I0, the average Raman intensity of the positive area By substituting the specific value of into the quantitative formula, we can get the quantitative formula.

[0110] The immunoassay method proposed in this application utilizes beam partitioning, Poisson distribution, single-particle SERS activity and confocal SERS spectrum to achieve high-sensitivity detection of IL-6 antigen. Figure 2 Schematic diagram of the construction process of digital SERS immunoassay in this application, such as Figure 2 As shown in a, the aldehyde-based silicon chip was functionalized by combining the amino groups of the IL-6 capture antibody using the classic APTES-GA (3-aminopropyltrimethoxysilane-glutaraldehyde) coating method. A sandwich structure was formed by sequentially adding the IL-6 antigen and the antibody-modified SERS nanotag.

[0111] Subsequent SERS mapping was performed by setting a specific threshold to reduce the background signal, thereby generating a binary false color image. This binary image was used as a mask and combined with the original SERS image to generate a digital SERS image.

[0112] A key component of the above method is the customizable core-shell nanotags, which are composed of gold nanoparticles (AuNPs) as the core and Ag-Au alloy as the shell ( Figure 2 b). This structure combines the stability of gold (high oxidation resistance) and the electromagnetic field enhancement of silver (high molar extinction coefficient), resulting in uniform and multiple hot spots. These nanotags provide strong, stable and reproducible signals, Figure 3 Schematic diagram of nanoparticle characterization in one embodiment of the present application, such as Figure 3 As shown in a, its stability can last for at least 30 days. Figure 3 As shown in b, the Raman reporter is placed in the "gap" generated by the electrochemical replacement reaction and on the outer surface of the nanotag to form a monolayer through electrostatic adsorption, where Figure 3 b1 represents the transmission electron microscopy image of the nanoparticle structure. Figure 3 b2: S element energy spectrum, Figure 3 b3: Overlay of energy spectra of the three elements S-Au-Ag.

[0113] Then, using the 592cm -1The Raman reporter Nile Blue A (NBA) that emits a significant Raman signal at the site encodes the IL-6 antigen. Finally, colorimetry and digital SERS were used for qualitative and quantitative analysis, respectively. Figure 2 As shown in C, this digital SERS immunoassay enables direct observation and accurate quantification of low-abundance protein biomarkers over a wide dynamic range.

[0114] Specifically, the interleukin 6 antibody functionalized silicon chip, interleukin 6 antigen and antibody modified surface enhanced Raman nano-label required for step S110 are configured as follows:

[0115] (1) Synthesis and purification of nanotags:

[0116] The Au@Ag-Au core-shell nanotags were synthesized in three steps. (1-1) First, HAuCl4 was reduced with citric acid to synthesize 15 nm AuNPs. (1-2) Then, AuNPs were used as seeds to reduce Ag by AA. + Finally, by injecting Au I Solution and reducing agent to prepare Ag-Au alloy shell.

[0117] During the purification process, ultrapure H2O (42.75 mL, 18.2 MΩ cm), HAuCl4 (1 mL, 2.5 × 10 -2 M), NaOH (1.25 mL, 0.1 M), and Na2SO3 (5 mL) were added to a glass beaker in sequence to prepare a gold growth solution until the solution changed from light yellow to colorless. The gold growth solution was stored at 4 °C for future use. To synthesize Au@Ag-Au nanotags, ultrapure H2O (80 mL, 18.2 MΩ·cm), CTAC (10 mL, 0.1 M), Au@AgNPs (10 mL, 0.2 nM), AA (1 mL, 0.1 M), and Raman reporter molecules (1 mL, 10 -3 M) was mixed under magnetic stirring at 40 rpm, and then the prepared gold growth solution (50 mL) was injected at a rate of 5 mL / min. After incubation for 4 hours, the sample was collected by centrifugation at 3000 rpm for 10 minutes and redispersed in CTAC (10 mL, 10 -2 M). In order to increase the yield of SERS nanotags, a purification process was performed to remove the nanorod byproducts using depletion-induced flocculation. The SERS nanotag solution was mixed with an equal volume of CTAC (0.1×10 -2The mixture was mixed with 10 mM CTAC (10 mM) to make the final concentration of CTAC reach 55 mM, and gravity sedimentation was carried out at 4°C for 12 h. The supernatant was collected and centrifuged at 2500 rpm for 10 min. The supernatant was replaced with CTAC (10 mM) to finally obtain a high-purity SERS nanotag, which was stored at 4°C for future use.

[0118] (2) Antibody-modified nanotags: To functionalize the SERS nanotags with antibodies, the nanotags (200 μL) were sonicated in ultrapure water for 2 min. The buffer was then replaced with reaction buffer (450 μL, 0.4 mM CTAC, 3 × 10 - 2 MTris-HCl, pH = 8.75). The resuspended nanotags were incubated with the detection antibody (2 μL, 1 mg / mL) for 1 h and then mixed with the blocking solution (2% BSA, 0.1% casein, 3×10-2MTris-HCl) for 3 h under gentle stirring. The functionalized nanotags were purified by centrifugation at 2500 rpm for 10 min to separate free antibody molecules, and the precipitate was resuspended in the optimized reaction buffer (400 μL, 2% BSA, 5×10 -2 MPB) for further use.

[0119] (3) Antibody functionalized silicon chip: A silicon substrate (5 mm × 5 mm) with a 300 nm oxide layer was cleaned with piranha solution (H2SO4 / H2O2, v / v, 3:1) at 80 °C for 2 h, thoroughly rinsed with ultrapure water and dried with a nitrogen stream, and then plasma treated for 2 min. The hydroxylated silicon substrate was then treated with a 2% APTES solution in ethanol for 2 h and baked at 120 °C for 20 min to produce a dense organosilane layer. Subsequently, the silanized silicon substrate was coupled with a 2.5% GA solution in PBS at room temperature for 45 min, rinsed with ultrapure water and dried with nitrogen. Next, the aldehyde-modified silicon substrate was immersed in a capture antibody solution (10 μg / mL) at 4 °C overnight. After the antibody-modified substrate was rinsed with ultrapure water, it was washed with PBS (10 -2 The antibody-modified substrate was blocked with casein (3%) and Tween-20 (0.05%) in PBS at 37° C. for 2 hours. Before use, the antibody-modified substrate was washed with ultrapure water.

[0120] Figure 4 This is a schematic diagram of the activity of a single nanoparticle in an embodiment of the present application. Figure 4 middle, Figure 4 (a) is the in-situ SEM image of nanoparticles. The red circle represents the location of the nanoparticles and the green circle represents the background location. Figure 4 (b) is the Raman spectrum of each position, with an excitation wavelength of 633nm, an exposure time of 2s, and an excitation power of 0.85mW. Figure 4As shown, the nanotags exhibit single-particle activity, making single-molecule detection possible.

[0121] The following are several specific embodiments of the present application:

[0122] (1) IL-6 standard test:

[0123] S11, IL-6 antigen was diluted with PBS to different concentrations of 100 fg / mL-1 μg / mL.

[0124] S12, the antibody-functionalized silicon chip was incubated with different concentrations of antigens at 37°C for 2 h, rinsed three times with ultrapure water after the reaction, and then the antibody-modified nanoparticles were added and incubated at 37°C for 0.5 h, rinsed three times with ultrapure water after the reaction, and then naturally air-dried for Raman scanning test.

[0125] S13, before testing, the device was calibrated using a silicon wafer. The spectrum was collected using an excitation wavelength of 633 nm, a 100× objective lens (NA=0.85), a power of 0.85 mW, and an exposure time of 2 seconds. The collection area was 60×60 μm2, the step size was 3 μm, and a total of 21×21 independent areas were collected.

[0126] S14, binarize each region according to whether the Raman spectrum of each region has a characteristic peak (592 cm-1) of the Raman reporter molecule (Nile Blue, NB), with the characteristic peak being recorded as "1" and the absence of the characteristic peak being recorded as "0". Figure 5 The Raman scan binarization images at different IL-6 concentrations in this application are as follows: Figure 5 As shown, a binary image is obtained through the above process;

[0127] S15, according to formula S5, the ratio of "1" is exponentially related to the antigen concentration, Figure 6 This is the quantitative analysis result diagram of the binary image in this application, such as Figure 6 As shown, the analysis results are consistent with the actual quantitative results;

[0128] S16, then the binary image is used as a mask, and the Raman intensity is added to generate a digital image. Figure 7 The Raman scanned digitized images at different IL-6 concentrations in this application are as follows: Figure 7 shown.

[0129] S17, according to formula S6, the product of the positive area ratio and the average Raman intensity of the positive area is linearly related to the antigen concentration. Figure 8 This is a quantitative analysis result diagram under the digitized image in this application, such as Figure 8 As shown, the analysis results are consistent with the actual quantitative results.

[0130] S18, the experiment was repeated three times for each concentration.

[0131] (2) IL-6 clinical sample testing:

[0132] S21, serum samples of unknown concentration were diluted 10-fold with PBS to reduce matrix effects.

[0133] S22, and then incubated with the antibody-functionalized silicon chip and the antibody-modified nanoparticles, respectively (same as above).

[0134] S23, the silicon wafer after immune reaction is subjected to Raman test, and the test process is consistent with the previous standard. The test result (the product of the positive area ratio and the average Raman intensity of the positive area) is substituted into the standard curve obtained by the standard, such as Figure 8 As shown, the concentration of the diluted sample is obtained, and then the result is multiplied by 10 to obtain the true concentration of the unknown serum sample.

[0135] S24, each sample was subjected to three replicate experiments.

[0136] (3) Specificity test:

[0137] S31, 1ng / mL of different antigen molecules IL-6, alpha-fetoprotein (AFP), novel coronavirus N protein (SARS-CoV-2N), C-reactive protein (CRP), PBS buffer and diluted serum and saliva samples of healthy people were used for specific experiments, of which IL-6 was used as the experimental group and the rest were set as the control group. The detection process was consistent with the above standard test. Fig. 9 This is a specificity evaluation result diagram in this application, such as Fig. 9 As shown, the final experimental results reflect that this method has good specificity.

[0138] S32, each sample was subjected to three replicate experiments.

[0139] (3) Repeatability test:

[0140] IL-6 antigen (10 pg / mL) was tested 10 times using 10 different antibody-functionalized chips, and the relative standard deviation (RSD) was calculated to be 6.45%. Fig.10 This is a graph of the repeatability evaluation results in this application, such as Fig.10 It is shown that this method has good repeatability.

[0141] The beneficial effects of this application are mainly:

[0142] (1) It does not rely on micro-nano processing technology and uses silicon oxide wafers as the capture substrate, thus reducing the technical cost;

[0143] (2) The nanotags have high reproducibility and maintain signal stability for at least 30 days;

[0144] (3) Nanotags exhibit single-particle activity, making single-molecule detection possible;

[0145] (4) Compared with the existing nano-tag based digital detection method which takes more than 5 hours, the entire process of this method only takes 2 hours, thus improving time efficiency.

[0146] (5) This method was validated using clinical samples and was highly consistent with the clinical standard method.

[0147] The present invention first proves that the prepared nano-tags have single-particle activity on silicon wafers. Figure 4 As shown in the figure, by combining scanning electron microscopy and Raman microscopy, Raman signals were collected from a single nanoparticle and the silicon wafer background. It can be seen that a single particle has a clearly distinguishable characteristic peak, proving that the nanotag has single-particle activity. Next, we used this method to detect IL-6 standard samples, with detection concentrations ranging from 100fg / mL to 1ng / mL, as shown in the figure. Figure 5 As shown in , the Raman scan image is first converted into a binary image according to a set threshold, and the positive area percentage (RPP) is used for quantification, as shown in Figure 6 As shown, after calibration, it can be seen that there is an obvious exponential function relationship, which is consistent with the theoretical results. The binary image is used as a mask and the corresponding Raman intensity is added in the positive area, such as Figure 7 As shown, a digital Raman image was generated and quantified using the product of the positive area percentage and the average Raman intensity of the positive area (RPP×ARI), as shown in Figure 8 As shown in the figure, there is an obvious linear relationship between the two, which is consistent with the theoretical results, and the detection limit is as low as 12.4fg / mL (0.5fM). Then, we explored the specificity and repeatability of this method. Fig. 9 As shown in Figure 2, the method showed good specificity for IL-6 relative to interferences such as the SARS-CoV-2 N protein and alpha-fetoprotein. The reproducibility was evaluated by measuring 10 pg / mL of IL-6 on 10 chips using the same protocol. The results are shown in Figure 2. Fig.10 As shown, the relative standard deviation (RSD) of RPP×ARI was 6.45%.

[0148] The present invention has passed the evaluation of both pseudo-clinical samples and real clinical samples. In order to verify the reliability and feasibility of the digital SERS method, standard IL-6 was added to commercially available healthy human serum and saliva from healthy donors at three concentrations (1, 10, 100 pg / mL). As shown in Table 1, the recoveries of spiked serum and saliva samples were 92.4% to 105.3%, with RSDs less than 10%. These results indicate that this method has reliable accuracy in complex human biological fluids. In order to verify the practicality and feasibility of the present invention in quantifying IL-6 in clinical biological samples, we detected normal concentrations (n=1) and abnormal concentrations (n=5) of IL-6 in serum. These results were compared with the ELISA method commonly used in clinical practice. Fig.11 This is a schematic diagram of the IL-6 measurement data in the clinical samples in this application, such as Fig.11 As shown, the slope and R2 of the regression equation in the inset are close to 1, indicating that the two methods have strong consistency. Although there are large fluctuations at low concentrations, which may be due to the high protein content in serum affecting the quantitative accuracy, the recovery rate and RSD show good reliability and stability at abnormal concentrations. Therefore, the digital method proposed in this paper has good applicability for the detection of IL-6-related diseases in human serum.

[0149] The present application also provides a digital surface enhanced Raman spectroscopy interleukin-6 immunoassay system, including:

[0150] An experimental module for pre-modifying a silicon chip functionalized with an interleukin-6 antibody based on a 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, co-incubating an interleukin-6 antigen and an antibody-modified surface-enhanced Raman nanotag to obtain an antigen-antibody sandwich structure;

[0151] Raman spectrum acquisition module, used to collect surface enhanced Raman scattering spectrum of silicon chip after reaction;

[0152] An image processing module, used for sequentially binarizing the surface enhanced Raman scattering spectra to obtain a binary image and using it as a mask to obtain a surface enhanced Raman scattering digital image;

[0153] The quantitative analysis module is used to extract the characteristic peaks of Raman reporter molecules in multiple partitions in the surface enhanced Raman scattering digital image, calculate the proportion of positive areas in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive areas and the average Raman intensity of positive areas to obtain antigen concentration data, wherein the positive area is the area where the characteristic Raman signal appears.

[0154] The surface enhanced Raman spectroscopy IL-6 immunoassay method, system and nano-label preparation method of the present invention are applied to data analysis of low antigen concentration scenarios. First, the Raman scan image is converted into a binary image according to a set threshold, and the positive area ratio (RPP) is used for quantification. The exponential relationship between the positive area ratio and the antigen concentration verifies that the single molecule event obeys the Poisson distribution. Finally, the linear relationship between the product of the positive area ratio and the average Raman intensity of the positive area (RPP*ARI) and the concentration is used to analyze the antigen concentration data. Experimental results show that the present application is used for the quantitative detection of IL-6 in healthy human serum and saliva, with a recovery rate of 92.4% to 105.3%. Finally, when testing clinical serum samples, the immunoassay method has a strong consistency with the standard clinical laboratory method. In addition, the nano-label prepared by the present application shows a clear signal at a single nanoparticle resolution, thereby realizing ultrasensitive / single molecule detection. And then provide a basis for antigen concentration analysis under low concentration conditions.

[0155] This embodiment also provides an electronic terminal, including: a processor and a memory;

[0156] The memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory, so that the terminal executes any one of the methods in this embodiment.

[0157] The computer-readable storage medium in this embodiment can be understood by ordinary technicians in this field: all or part of the steps of implementing the above-mentioned method embodiments can be completed by hardware related to the computer program. The aforementioned computer program can be stored in a computer-readable storage medium. When the program is executed, the steps of the above-mentioned method embodiments are executed; and the aforementioned storage medium includes: ROM, RAM, magnetic disk or optical disk and other media that can store program codes.

[0158] The electronic terminal provided in this embodiment includes a processor, a memory, a transceiver and a communication interface. The memory and the communication interface are connected to the processor and the transceiver and complete communication with each other. The memory is used to store computer programs, the communication interface is used to communicate, and the processor and the transceiver are used to run computer programs so that the electronic terminal executes each step of the above method.

[0159] In this embodiment, the memory may include a random access memory (RAM), and may also include a non-volatile memory (non-volatile memory), such as at least one disk memory.

[0160] The above-mentioned processor can be a general-purpose processor, including a central processing unit (CPU), a graphics processing unit (GPU), a network processor (NP), etc.; it can also be a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components.

[0161] In the above-mentioned embodiments, although the present invention has been described in conjunction with the specific embodiments of the present invention, many replacements, modifications and variations of these embodiments will be apparent to those of ordinary skill in the art based on the foregoing description. The embodiments of the present invention are intended to cover all such replacements, modifications and variations falling within the broad scope of the appended claims.

[0162] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method, characterized in that: Includes steps: The antigen-antibody sandwich structure was obtained by incubating a silicon chip functionalized with interleukin-6 antibody pre-modified by 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, interleukin-6 antigen and antibody-modified surface-enhanced Raman nanotags together; Collect the surface enhanced Raman scattering spectrum of the silicon chip after the reaction; Binarizing the surface enhanced Raman scattering spectra sequentially to obtain a binary image and using the binary image as a mask to obtain a surface enhanced Raman scattering digital image; The Raman reporter molecule characteristic peaks of multiple partitions in the surface enhanced Raman scattering digital image are extracted, and the proportion of positive areas in the partitions is calculated according to the threshold method, and quantitative analysis is performed based on the proportion of the positive areas and the average Raman intensity of the positive areas to obtain antigen concentration data, wherein the positive area is the area where the characteristic Raman signal appears.

2. The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method according to claim 1, characterized in that: The surface enhanced Raman scattering spectrum is sequentially binarized to obtain a binary image and used as a mask to obtain a digitized surface enhanced Raman scattering digital image, including: Binarizing the surface enhanced Raman scattering spectrum based on a preset threshold to obtain a binary false color image with background information filtered out; The binary false color image is used as a mask and convolved with the surface enhanced Raman scattering spectrum to obtain a surface enhanced Raman scattering digital image.

3. The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method according to claim 1, characterized in that: Quantitative analysis is performed based on the proportion of the positive area and the average Raman intensity of the positive area to obtain antigen concentration data, including: Substitute the proportion of the positive area or the product of the proportion of the positive area and the average Raman intensity of the positive area into a pre-constructed quantitative formula to obtain antigen concentration data, wherein the quantitative formula includes an exponential quantitative formula and a linear quantitative formula, and the mathematical expressions of the exponential quantitative formula and the linear quantitative formula are: y1=0.925-0.925*e -0.03*x y2=0.743*x+10.626 Wherein, y1 is the proportion of the positive area, x is the antigen concentration data, and y2 is the product of the proportion of the positive area and the average Raman intensity of the positive area.

4. The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method according to claim 3, characterized in that: The process of constructing the quantitative formula includes: The number of antigens in each partition is assumed to follow a Poisson distribution with parameter λ, and its probability expression is: p(0)=and -λ =1-E In the formula, k represents the number of antigen molecules in each partition, and p(k) represents the probability that the number of antigen molecules in each partition is k; When there is more than one antigen type in the partition, represents the average Raman intensity of the positive area, E is the percentage of the positive area, V d is the volume of each independent partition, C is the antigen concentration, and I0 is the average Raman intensity of a single nanometer; The positive area ratio E and the volume of each independent partition V d , the average Raman intensity of a single nanometer I0, the average Raman intensity of the positive area Substitute the specific value of into the quantitative formula.

5. The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method according to claim 1, characterized in that: The surface enhanced Raman scattering patterns are collected by a Renishaw Raman microscope system using lasers of different wavelengths and powers.

6. The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method according to claim 1, characterized in that: The spectrum of the surface enhanced Raman scattering spectrum was collected using a 100× objective lens, a power of 0.85 mW, an exposure time of 2 seconds, and Raman scanning data using an excitation wavelength of 633 nm at 60×60 μm 2 Area collection with a step size of 3 μm.

7. The digital surface enhanced Raman spectroscopy interleukin-6 immunoassay method according to claim 1, characterized in that: The configuration process of the silicon chip functionalized with IL-6 antibody includes: Cleaning a silicon substrate having an oxide layer of target thickness using a piranha solution; The cleaned silicon substrate is rinsed with ultrapure water and dried with a nitrogen flow; The dried silicon substrate is subjected to plasma treatment to obtain a hydroxylated silicon substrate; Treating the hydroxylated silicon substrate with 3-aminopropyltrimethoxysilane and baking the substrate to generate a dense organic silane layer to obtain a silanized silicon substrate; The silanized silicon substrate is coupled with a glutaraldehyde solution, and after coupling, it is washed with ultrapure water and dried with a nitrogen flow to obtain an aldehyde-treated silicon substrate; The aldehyde-modified silicon substrate is immersed in an interleukin-6 antibody capture solution for a certain target time, and then the surface of the aldehyde-modified silicon substrate is sealed using casein and a surfactant in a phosphate buffer solution to obtain an interleukin-6 antibody functionalized silicon chip.

8. Digital surface enhanced Raman spectroscopy interleukin-6 immunoassay system, characterized in that: include: An experimental module for pre-modifying a silicon chip functionalized with an interleukin-6 antibody based on a 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, co-incubating an interleukin-6 antigen and an antibody-modified surface-enhanced Raman nanotag to obtain an antigen-antibody sandwich structure; Raman spectrum acquisition module, used to collect surface enhanced Raman scattering spectrum of silicon chip after reaction; An image processing module, used for sequentially binarizing the surface enhanced Raman scattering spectra to obtain a binary image and using it as a mask to obtain a surface enhanced Raman scattering digital image; The quantitative analysis module is used to extract the characteristic peaks of Raman reporter molecules in multiple partitions in the surface enhanced Raman scattering digital image, calculate the proportion of positive areas in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive areas and the average Raman intensity of positive areas to obtain antigen concentration data, wherein the positive area is the area where the characteristic Raman signal appears.

9. A method for preparing a nanotag, for preparing the antibody-modified surface-enhanced Raman nanotag according to claim 1, characterized in that: include: Gold nanoparticles were obtained by reducing tetrachloroauric acid with citric acid; The gold nanoparticles are used as seeds and silver ions are reduced by ascorbic acid to form an Ag shell; Injection of Au into a solution containing an Ag shell I A solution and a reducing agent are used to prepare an Ag-Au alloy shell and a Raman reporter molecule is added to obtain a solution containing a surface enhanced Raman nano-label; Purifying the solution containing the surface-enhanced Raman nano-tag to obtain the surface-enhanced Raman nano-tag; The surface enhanced Raman nanotag is functionalized by using interleukin 6 antibody to obtain an antibody-modified surface enhanced Raman nanotag.

10. The method for preparing a nano tag according to claim 9, characterized in that: The surface enhanced Raman nanotag is functionalized with interleukin 6 antibody to obtain an antibody-modified surface enhanced Raman nanotag, comprising: The surface enhanced Raman nano-tag is subjected to ultrasonic treatment in ultrapure water to obtain a suspension; The suspension is coupled with a reaction buffer to obtain a resuspended suspension, wherein the reaction buffer comprises 0.2-0.6 mM CTAC, 1-5×10 -2 M Tris-HCl; The nanotags in the resuspended suspension are incubated with the interleukin 6 antibody, stirred and mixed with a blocking solution to obtain a target solution, wherein the blocking solution comprises 1-3% BSA, 0.01-0.2% casein and 1-5×10 -2 M Tris-HCl; The target solution is purified by centrifugation to obtain an antibody-modified surface-enhanced Raman nano-tag.

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