Surface-enhanced Raman spectroscopy IL-6 immunoassay method, system and nanotag preparation method

Abstract

This invention relates to the field of surface-enhanced Raman scattering (SERS), specifically to a surface-enhanced Raman spectroscopy (SERS) method, system, and nanotag preparation method for IL-6 immunoassay. Applied to data analysis in low antigen concentration scenarios, the Raman scan image is first converted into a binary image according to a set threshold, and quantification is performed using the percentage of positive regions (RPP). The exponential relationship between the RPP and antigen concentration verifies that single-molecule events follow a Poisson distribution. Finally, the linear relationship between the product of the RPP and the average Raman intensity of the positive regions (RPP*ARI) and the concentration is used to analyze antigen concentration data. When testing clinical serum samples, this immunoassay method shows strong consistency with standard clinical laboratory methods. The nanotags prepared in this application exhibit clear signals at single nanoparticle resolution, thereby achieving ultrasensitive / single-molecule detection.

Description

Technical Field

[0001] This invention relates to the field of surface-enhanced Raman scattering technology, specifically to a surface-enhanced Raman spectroscopy method, system, and nanotag preparation method for IL-6 immunoassay. Background Technology

[0002] Although SERS (surface enhancement of Raman scattering) has been widely used in various fields, its efficiency is often challenged by the uneven and sparse distribution of hotspots in plasma SERS. Although the hotspot region accounts for less than 0.007% of the surface area, it contributes up to 24% of the overall Raman signal. This poses a challenge to the localization of hotspot regions by individual molecules, especially at low target concentrations. This leads to significant fluctuations in SERS intensity, inevitably hindering the accuracy and reproducibility of the results.

[0003] High-performance plasmon substrates with multiple hot spots, uniform distribution, and reproducibility have long been sought after, as they are crucial for the detection of digital SERS. However, in existing technologies, solid substrates are typically heterogeneous, posing challenges to batch-to-batch and intra-batch reproducibility, even under stringent 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 this invention is to provide a surface-enhanced Raman spectroscopy method, system, and nanotag preparation method for IL-6 immunoassay, in order to solve the problems in the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0007] A silicon chip pre-modified with interleukin-6 antibody based on the 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, and surface-enhanced Raman nanotags modified with interleukin-6 antigen and antibody were co-incubated to obtain an antigen-antibody sandwich structure.

[0008] Surface-enhanced Raman scattering (SERS) spectra of the silicon chip after the reaction were collected.

[0009] The surface-enhanced Raman scattering (SERS) spectrum is sequentially binarized to obtain a binary image, which is then used as a mask to obtain a digital SERS image.

[0010] Raman reporter molecule characteristic peaks are extracted from multiple partitions in the surface-enhanced Raman scattering digital image, and the proportion of positive regions in the partitions is calculated according to the threshold method. Quantitative analysis is then performed based on the proportion of positive regions and the average Raman intensity of the positive regions to obtain antigen concentration data. The positive regions are those where characteristic Raman signals appear.

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

[0012] The surface-enhanced Raman scattering spectrum is binarized based on a preset threshold to obtain a binary false-color image with background information filtered out.

[0013] Using the binary false-color image as a mask, convolving it with the surface-enhanced Raman scattering spectrum yields a surface-enhanced Raman scattering digital image.

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

[0015] Substituting the proportion of positive regions or the product of the proportion of positive regions and the average Raman intensity of positive regions into a pre-constructed quantitative formula yields antigen concentration data. The quantitative formula includes an exponential quantitative formula and a linear quantitative formula, the mathematical expressions of which are:

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

[0017] y² = 0.743x + 10.626

[0018] In the formula, y1 is the proportion of the positive region, and y2 is the product of the proportion of the positive region and the average Raman intensity of the positive region.

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

[0020] The number of antigens in each partition is assumed to follow 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 type of antigen in the partition. The average Raman intensity represents the positive region, E is the percentage of the positive region, and V is the average Raman intensity. d For each independent partition, C is the antigen concentration, and i0 is the average Raman intensity per nanometer;

[0027] The percentage of positive areas (E) and the volume of each independent partition (V) are calculated. d Average Raman intensity i0 of a single nanometer, average Raman intensity of the positive region By substituting the specific values, we obtain a quantitative formula.

[0028] In one embodiment of this application, the surface-enhanced Raman scattering pattern is acquired using a Renishaw Raman microscope system with lasers of different wavelengths and powers.

[0029] In one embodiment of this application, the surface-enhanced Raman scattering spectrum was acquired using a 100× objective lens with a power of 0.85mW and an exposure time of 2 seconds. The Raman scan data were obtained using a 633nm excitation wavelength at 60×60μm. 2 Regional collection with a step size of 3 μm.

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

[0031] The silicon substrate with an oxide layer of the target thickness was cleaned using a piranha solution.

[0032] The cleaned silicon substrate was rinsed with ultrapure water and dried with nitrogen gas.

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

[0034] The hydroxylated silicon substrate was treated with 3-aminopropyltrimethoxysilane and baked to produce a dense organosilane layer, thereby obtaining a silanized silicon substrate.

[0035] The silanized silicon substrate was coupled with a glutaraldehyde solution, and then washed with ultrapure water and dried with a nitrogen stream to obtain an aldehyde-modified silicon substrate.

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

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

[0038] The experimental module is used to co-incubate a silicon chip pre-modified with interleukin-6 antibody based on the 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, and a surface-enhanced Raman nanotag modified with interleukin-6 antigen and antibody to obtain an antigen-antibody sandwich structure.

[0039] Raman spectroscopy acquisition module, used to acquire surface-enhanced Raman scattering spectra of silicon chips after reaction;

[0040] The image processing module is used to sequentially binarize the surface-enhanced Raman scattering (SMR) spectrum to obtain a binary image and to use it as a mask to obtain a digital SMR image.

[0041] The quantitative analysis module is used to extract Raman reporter molecule characteristic peaks from multiple partitions in the surface-enhanced Raman scattering digital image, calculate the proportion of positive regions in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive regions and the average Raman intensity of the positive regions to obtain antigen concentration data, wherein the positive regions are the regions where characteristic Raman signals appear.

[0042] This application also provides a method for preparing nanotags, for configuring antibody-modified surface-enhanced Raman nanotags as described above, including:

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

[0044] Using the gold nanoparticles as seeds, and reducing silver ions with ascorbic acid to form an Ag shell;

[0045] Au was injected into a solution containing an Ag shell. I An Ag-Au alloy shell was prepared by using a solution and a reducing agent, and a Raman reporter molecule was added to obtain a solution containing surface-enhanced Raman nanotags.

[0046] The solution containing the surface-enhanced Raman nanotag was purified to obtain the surface-enhanced Raman nanotag.

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

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

[0049] The surface-enhanced Raman nanotags were 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 and 1–5 × 10⁻⁶ CFU / mL. -2 M Tris-HCl;

[0051] The nanotags in the resuspended suspension were incubated together with the interleukin-6 antibody, stirred, and then mixed with a blocking solution to obtain the target solution. The blocking solution comprised 1–3% BSA, 0.01–0.2% casein, and 1–5 × 10⁻⁶ ppm of BSA. -2 M Tris-HCl;

[0052] The target solution was purified by centrifugation to obtain antibody-modified surface-enhanced Raman nanotags.

[0053] The beneficial effects of this invention are as follows: The surface-enhanced Raman spectroscopy (SERS) immunoassay method, system, and nanotag preparation method for IL-6 are applied to data analysis in low antigen concentration scenarios. First, the Raman scan image is converted into a binary image according to a set threshold, and quantification is performed using the percentage of positive regions (RPP). The exponential relationship between the RPP and antigen concentration verifies that single-molecule events follow a Poisson distribution. Finally, the linear relationship between the product of the RPP and the average Raman intensity of the positive regions (RPP*ARI) and the concentration is used to analyze antigen concentration data. Experiments have shown that this application can be 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, this immunoassay method shows strong consistency with standard clinical laboratory methods. Furthermore, the nanotags prepared in this application exhibit a clear signal at single nanoparticle resolution, thereby achieving ultrasensitive / single-molecule detection. This provides a basis for antigen concentration analysis in low-concentration scenarios. Attached Figure Description

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

[0055] Figure 1 This is a flowchart of a digital surface-enhanced Raman spectroscopy interleukin-6 immunoassay method shown in one embodiment of this application;

[0056] Figure 2This is a schematic diagram illustrating the construction process of the digital SERS immune analysis in this application;

[0057] Figure 3 This is a schematic diagram of nanoparticle characterization in one embodiment of this application;

[0058] Figure 4 This is a schematic diagram of the activity of a single nanoparticle in one embodiment of this application;

[0059] Figure 5 These are Raman scan binarized images of different IL-6 concentrations in this application.

[0060] Figure 6 This is a graph showing the quantitative analysis results under the binarized image of this application;

[0061] Figure 7 These are Raman scan digitized images of different IL-6 concentrations in this application;

[0062] Figure 8 This is a graph showing the quantitative analysis results under the digitized image in this application;

[0063] Figure 9 This is a diagram showing the specificity assessment results in this application;

[0064] Figure 10 This is a graph showing the repeatability evaluation results in this application;

[0065] Figure 11 This is a schematic diagram of IL-6 measurement data in the clinical samples used in this application. Detailed Implementation

[0066] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0067] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the layers related to the present invention and are not drawn according to the actual number, shape and size of the layers in the actual implementation. In the actual implementation, the form, number and proportion of each layer can be arbitrarily changed, and the layer layout may also be more complex.

[0068] Numerous details are explored in the following description to provide a more thorough explanation of embodiments of the invention; however, it will be apparent to those skilled in the art that embodiments of the invention may be practiced without these specific details.

[0069] The following is a definition of the terms used in this manual:

[0070] SERS, 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 presents a digital surface-enhanced Raman scattering (SERS) immunoassay method for detecting IL-6 using core-shell Au@Ag-Au nanotags, exhibiting high sensitivity and reliability. A low-cost, biofunctionalized silicon core is used as the capture substrate, and novel SERS nanotags are employed as amplification elements. These tags demonstrate strong, robust, and reproducible signals at single-nanoparticle resolution. We propose two analytical methods to validate that single-molecule events follow a Poisson distribution and to quantify protein biomarkers over a wide linear dynamic range. High agreement between theoretical and experimental results enhances the reliability of the method. The detection method in this application provides two readings: colorimetric analysis by visual inspection at high concentrations (>1 ng / mL) and digital SERS analysis at low concentrations. After method optimization, we obtained a linear range (R0) from 100 fg / mL to 1 ng / mL. 2 With a detection limit (LOD) of 12.4 fg / mL and a relative optimum (0.994), this method is suitable for clinical applications. It was used for the quantitative detection of IL-6 in serum and saliva of healthy individuals, with recoveries ranging from 92.4% to 105.3%. Finally, when testing clinical serum samples, this immunoassay showed strong concordance with standard clinical laboratory methods. Therefore, our proposed digital SERS immunoassay is a promising tool for the accurate clinical diagnosis of IL-6-related diseases or other conditions.

[0075] Figure 1 This is a flowchart illustrating a digital surface-enhanced Raman spectroscopy interleukin-6 immunoassay method in one embodiment of this application, as shown below. Figure 1 As shown: The digital surface-enhanced Raman spectroscopy interleukin-6 immunoassay method of this embodiment may include the following steps:

[0076] S110, based on the 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, pre-modified interleukin-6 antibody functionalized silicon chip, interleukin-6 antigen and antibody modified surface-enhanced Raman nanotag were co-incubated to obtain antigen-antibody sandwich structure.

[0077] In this application, the classic APTES–GA coating method is used to form a silicon chip-antibody-antigen sandwich structure.

[0078] By immobilizing antibodies on the surface of a silicon chip and introducing the antigen sample to be detected, the specific binding between the antigen and antibody can form a sandwich structure. The nanoparticle structure can significantly enhance signal intensity, allowing even trace amounts of antigen to be accurately detected. Simultaneously, the formation of the sandwich structure typically involves a rapid antigen-antibody binding reaction, enabling the entire detection process to be completed in a short time. For example, in some advanced biosensors, sandwich structure formation and signal detection can be completed within minutes or even seconds. Furthermore, the sandwich structure design makes the detection process simpler and more intuitive. Simply introduce the sample onto the chip and observe the formation of the sandwich structure and signal changes. The silicon chip, as a robust and stable substrate material, provides excellent support and protection. This helps maintain the stability and reliability of the sandwich structure, thereby improving the accuracy and repeatability of the detection.

[0079] S120, Collect the 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 signal of target molecules located near plasmonic metallic nanostructures by utilizing the optical properties of nanostructures with plasmonic properties.

[0081] In this application, the Raman spectra and SERS scan images were obtained using a Renishaw Raman microscopy system (Renishaw, UK) equipped with lasers of different wavelengths and powers: a 50mW 532nm diode laser, a 17mW 633nm helium-neon laser, and a 300mW 785nm diode laser. A 520.5cm... -1 The silicon wafer was calibrated. Spectroscopy was acquired using a 100× objective lens (NA = 0.85), with a power of 0.85 mW and an exposure time of 2 seconds. Raman scan data were collected in a 60×60 μm² region with an excitation wavelength of 633 nm and a step size of 3 μm. Based on 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. -1 The characteristic peak at that location.

[0082] s130, the surface-enhanced Raman scattering pattern is sequentially binarized to obtain a binary image and used as a mask to obtain a surface-enhanced Raman scattering digital image;

[0083] The specific process includes:

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

[0085] Binarization divides an image into positive regions and background using a threshold. Since the positive regions are the objects or areas of interest, while the background is the area surrounding these objects or areas, by setting a reasonable threshold, the background pixel values ​​can be uniformly set to a single level (such as black), thereby filtering out background information.

[0086] The main purpose of binarization is to highlight positive areas. Because background information is uniformly set to a single level during binarization, positive areas become more prominent and clearer in the image. This makes subsequent image processing and analysis easier and more accurate.

[0087] S132, the binary false-color image is used as a mask and convolved with the original surface-enhanced Raman scattering spectrum to obtain a digital surface-enhanced Raman scattering image.

[0088] The purpose of masking is to block local areas. In this application, by blocking the background, the mid-foreground region of interest (ROI) can be extracted, thereby obtaining the image data basis for subsequent analysis, namely, the surface-enhanced Raman scattering digital image.

[0089] S140, extract the Raman reporter molecule characteristic peaks of multiple partitions in the digital surface-enhanced Raman scattering image, calculate the proportion of positive regions in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive regions and the average Raman intensity of the positive regions to obtain antigen concentration data, wherein the positive regions are the regions where characteristic Raman signals appear.

[0090] In this application, the core principle of digital SERS is to disperse the antigen into many independent reaction regions or pixels, which in this invention is equivalent to a beam of light during signal acquisition. Regions exhibiting a specific Raman signal after the immune response are denoted as "1," while regions without such a signal are denoted as "0." By statistically analyzing the proportion of positive regions and then substituting it into the quantitative formula in this application, 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] In the formula, y1 is the proportion of the positive region, and y2 is the product of the proportion of the positive region and the average Raman intensity of the positive region.

[0094] Equation (1) is the exponential quantitative formula, and equation (2) is the linear quantitative formula. The derivation process of the above formulas includes:

[0095] Since the number of antigens in each partition is discrete, it follows a Poisson distribution with parameter λ. The Poisson distribution is a discrete probability distribution primarily used to describe the frequency of rare events occurring within a fixed time or space. The main characteristics of the Poisson distribution include: discreteness, independence, rarity, stationarity, memorylessness, equality of expectation and variance, asymmetry, and additivity of events. The probability following a 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 regions, C represents the concentration of antigen molecules, and V d This represents the volume of each partition.

[0102] As the concentration increases, each partition may contain more than one antigen. At this point:

[0103]

[0104] Where E represents the number of regions marked as "1". The average Raman intensity representing the positive region, This represents the average Raman intensity of a single nanoparticle.

[0105] Based on the above formulas, two analytical methods can be derived:

[0106]

[0107]

[0108] The proportion (E) of positive regions has an exponential relationship with the concentration, which is calculated by multiplying the proportion of positive regions by the average Raman intensity of the positive regions. The relationship between concentration and concentration is a linear function.

[0109] Finally, the percentage of positive areas (E) and the volume of each independent partition (V) are calculated. d Average Raman intensity I0 of a single nanometer, average Raman intensity of the positive region By substituting the specific values, a quantitative formula can be obtained.

[0110] The immunoassay method proposed in this application achieves high-sensitivity detection of IL-6 antigen by utilizing beam partitioning, Poisson distribution, single-particle SERS activity, and confocal SERS mapping. Figure 2 This is a schematic diagram of the construction process of the digital SERS immunoassay in this application, as shown below. Figure 2 As shown in Figure a, the aldehyde-based silicon chip was functionalized by binding 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 IL-6 antigen and antibody-modified SERS nanotags.

[0111] The subsequent SERS image is used to reduce the background signal by setting a specific threshold, thereby generating a binary false-color image. This binary image is 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 a customizable core-shell nanotag, with gold nanoparticles (AuNPs) as the core and an Ag-Au alloy as the shell. Figure 2 (As shown in b). This structure combines the stability of gold (high oxidation resistance) with the electromagnetic field enhancement of silver (high molar extinction coefficient), enabling the generation of uniform and multiple hot spots. These nanotags can provide strong, stable, and reproducible signals. Figure 3 This is a schematic diagram of nanoparticle characterization in one embodiment of this application, as shown below. Figure 3 As shown in a, its stability lasts for at least 30 days. (Example: Single-particle element) Figure 3 As shown in b, the Raman reporter is placed within the "voids" generated by the electrochemical displacement reaction and on the outer surface of the nanotag, forming a monolayer through electrostatic adsorption. Figure 3 b1 represents a transmission electron microscope (TEM) image of the nanoparticle structure. Figure 3 b2: S element energy spectrum Figure 3 b3: Superimposed energy spectra of S, Au, and Ag.

[0113] Subsequently, using 592cm -1The Raman reporter Nile Blue A (NBA), which emits a distinct Raman signal, 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 configuration process of the interleukin-6 antibody-functionalized silicon chip, the interleukin-6 antigen, and the antibody-modified surface-enhanced Raman nanotag required for step S110 is as follows:

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

[0116] Au@Ag-Au core-shell nanotags were synthesized in three steps. (1-1) First, 15 nm AuNPs were synthesized by reducing HAuCl4 with citric acid. (1-2) Second, using AuNPs as seeds, Ag was reduced with AA. + An Ag shell is formed. (1-3) Finally, Au is injected. I Preparation of Ag-Au alloy shells using solutions and reducing agents.

[0117] During the purification process, ultrapure H2O (42.75 mL, 18.2 MΩ·cm) and HAuCl4 (1 mL, 2.5 × 10⁻⁶ MΩ·cm) were added under magnetic stirring (40 rpm). -2 To prepare the gold growth solution, add NaOH (1.25 mL, 0.1 M) and Na₂SO₃ (5 mL) sequentially to a glass beaker until the solution changes from pale yellow to colorless. Store the gold growth solution at 4 °C for later use. To synthesize Au@Ag-Au nanotags, add 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 a Raman reporter molecule (1 mL, 10 nM) to a glass beaker. -3 M) was mixed with magnetic stirring at 40 rpm, and then injected into the prepared gold growth solution (50 mL) 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 mL). -2 In order to improve the yield of SERS nanotags, a purification process was carried out, and exhaustion-induced flocculation was used to remove nanorod byproducts. The SERS nanotag solution was mixed with an equal volume of CTAC (0.1 × 10⁻⁶ m³). -2Mix M) to achieve a final concentration of 55 mM of CTAC, allow to settle by gravity at 4 °C for 12 h, collect the supernatant, centrifuge at 2500 rpm for 10 min, replace the supernatant with CTAC (10 mM) to obtain high-purity SERS nanotags, and store at 4 °C for later use.

[0118] (2) Antibody-modified nanotags: To functionalize SERS nanotags with antibodies, the nanotags (200 μL) were sonicated in ultrapure water for 2 minutes. The buffer solution was then replaced with reaction buffer (450 μL, 0.4 mM MCTAC, 3 × 10⁻⁶ ppm). - 2 MTris-HCl, pH = 8.75). The resuspended nanotags were incubated with the detection antibody (2 μL, 1 mg / mL) for 1 hour, and then mixed with blocking solution (2% BSA, 0.1% casein, 3 × 10⁻² MTris-HCl) for 3 hours with gentle stirring. The functionalized nanotags were purified by centrifugation at 2500 rpm for 10 minutes to separate free antibody molecules, and the precipitate was resuspended in optimized reaction buffer (400 μL, 2% BSA, 5 × 10⁻² MTris-HCl, pH = 8.75). -2 For further use in MPB).

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

[0120] Figure 4 This is a schematic diagram of the activity of a single nanoparticle in one embodiment of this application. Figure 4 middle, Figure 4 (a) is an in-situ SEM image of nanoparticles, with red circles representing the location of nanoparticles and green circles representing the background. Figure 4 (b) shows the Raman spectra at various locations, with an excitation wavelength of 633 nm, an exposure time of 2 s, and an excitation power of 0.85 mW. Figure 4As shown, the nanotags exhibit single-particle activity, enabling single-molecule detection.

[0121] The following are some specific embodiments of this application:

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

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

[0124] S12, antibody-functionalized silicon chips were co-incubated with antigens of different concentrations at 37°C for 2 hours. After the reaction, the chips were rinsed three times with ultrapure water. Then, antibody-modified nanoparticles were added and co-incubated at 37°C for 0.5 hours. After the reaction, the chips were rinsed three times with ultrapure water and air-dried before Raman scanning was performed.

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

[0126] S14. Based on whether the Raman spectrum of each region contains the characteristic peak (592 cm⁻¹) of the Raman reporter molecule (Nell Blue, NB), each region is binarized. The presence of the characteristic peak is marked as "1", and the absence of the characteristic peak is marked as "0". Figure 5 The Raman scan binarized images of different IL-6 concentrations in this application are as follows: Figure 5 As shown, a binary image is obtained through the above process;

[0127] According to formula S15, the proportion of "1" is exponentially related to the antigen concentration. Figure 6 This is a graph showing the quantitative analysis results under the binarized image of this application, such as... Figure 6 As shown, the analytical results are consistent with the actual quantitative results;

[0128] S16, then using the binary image as a mask, Raman intensity is added to generate a digital image. Figure 7 The digital images generated are Raman scans at different IL-6 concentrations in this application, as shown below. Figure 7 As shown.

[0129] S17, According to formula S6, the product of the percentage of positive areas and the average Raman intensity of positive areas is linearly related to the antigen concentration. Figure 8 This is a graph showing the quantitative analysis results under the digitized image in this application, such as... Figure 8 As shown, the analytical results are consistent with the actual quantitative results.

[0130] For S18, each concentration was tested in triplicate.

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

[0132] S21. Dilute serum samples of unknown concentration 10 times with PBS to reduce matrix effect.

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

[0134] S23. After the immunoassay, the silicon wafer undergoes Raman testing, following the same procedure as the previous standard test. The test results (the product of the percentage of positive areas and the average Raman intensity of the positive areas) are then substituted into the standard curve obtained from the standard test. 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 repeated three times.

[0136] (3) Specificity test:

[0137] S31. Specificity experiments were conducted using 1 ng / mL of different antigen molecules IL-6, alpha-fetoprotein (AFP), SARS-CoV-2 N protein, C-reactive protein (CRP), PBS buffer, and diluted serum and saliva samples from healthy individuals. IL-6 was used as the experimental group, and the others were set as control groups. The detection process was consistent with the standard assays described above. Figure 9 This is a diagram showing the specificity assessment results in this application, such as... Figure 9 As shown, the final experimental results demonstrate that this method has good specificity.

[0138] S32, each sample was repeated three times.

[0139] (3) Repeatability testing:

[0140] The IL-6 antigen (10 pg / mL) was tested 10 times using 10 different antibody-functionalized chips. The relative standard deviation (RSD) was calculated to be 6.45%. Figure 10 The graph shows the repeatability evaluation results in this application, such as... Figure 10 As shown, this demonstrates that the method has good repeatability.

[0141] The main benefits of this application are:

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

[0143] (2) The nanotags have high repeatability 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 digital detection method based on nanotags, which takes more than 5 hours, this method only takes 2 hours for the entire process, thus improving time efficiency.

[0146] (5) This method was validated using clinical samples and is in good agreement with standard clinical methods.

[0147] This invention first demonstrates that the prepared nanotags all exhibit single-particle activity on silicon wafers. For example... Figure 4 As shown, using combined scanning electron microscopy and Raman microscopy, Raman signals were acquired from the position of a single nanoparticle against the silicon wafer background. This revealed distinct characteristic peaks in each nanoparticle, demonstrating the single-particle activity of the nanotag. Next, we used this method to detect IL-6 standard samples at concentrations ranging from 100 fg / mL to 1 ng / mL. Figure 5 As shown, the Raman scan image is first converted into a binary image according to a set threshold, and then quantified using the positive region percentage (RPP). Figure 6 As shown, after calibration, a clear exponential function relationship can be observed, which is consistent with the theoretical results. Using the binary image as a mask, corresponding Raman intensities are added to the positive regions, such as... Figure 7 As shown, a digital Raman image is generated, and quantification is performed using the product of the percentage of positive regions and the average Raman intensity of the positive regions (RPP×ARI). Figure 8 As shown, a clear linear relationship exists between the two, consistent with the theoretical results, with a detection limit as low as 12.4 fg / mL (0.5 fM). We then investigated the specificity and reproducibility of this method. Figure 9 As shown, this method exhibits good specificity for IL-6 compared to interfering factors such as the SARS-CoV-2 N protein and alpha-fetoprotein. Reproducibility was assessed by measuring 10 pg / mL of IL-6 on 10 chips using the same protocol, and the results are as follows. Figure 10 As shown, the relative standard deviation (RSD) of RPP×ARI is 6.45%.

[0148] This invention was evaluated using both spurious and real clinical samples. To verify the reliability and feasibility of the digital SERS method, standard IL-6 was added at three concentrations (1, 10, and 100 pg / mL) to commercially available healthy human serum and healthy donor saliva. As shown in Table 1, the recoveries of the spiked serum and saliva samples ranged from 92.4% to 105.3%, with RSDs below 10%. These results demonstrate the reliable accuracy of this method in complex human biological fluids. To verify the practicality and feasibility of quantifying IL-6 in clinical biological samples, we detected normal (n=1) and abnormal (n=5) concentrations of IL-6 in serum. These results were compared with commonly used clinical ELISA methods. Figure 11 This is a schematic diagram of IL-6 measurement data in clinical samples used in this application, as shown below. Figure 11 As shown in the illustration, the slope and R² of the regression equation are close to 1, indicating strong consistency between the two methods. Although there are large fluctuations at low concentrations, possibly due to the high protein content in serum affecting quantitative accuracy, the recovery rate and RSD show good reliability and stability at abnormal concentrations. Therefore, the digital method proposed in this paper is well applicable to the detection of IL-6-related diseases in human serum.

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

[0150] The experimental module is used to co-incubate a silicon chip pre-modified with interleukin-6 antibody based on the 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, and a surface-enhanced Raman nanotag modified with interleukin-6 antigen and antibody to obtain an antigen-antibody sandwich structure.

[0151] Raman spectroscopy acquisition module, used to acquire surface-enhanced Raman scattering spectra of silicon chips after reaction;

[0152] The image processing module is used to sequentially binarize the surface-enhanced Raman scattering (SMR) spectrum to obtain a binary image and to use it as a mask to obtain a digital SMR image.

[0153] The quantitative analysis module is used to extract Raman reporter molecule characteristic peaks from multiple partitions in the surface-enhanced Raman scattering digital image, calculate the proportion of positive regions in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive regions and the average Raman intensity of the positive regions to obtain antigen concentration data, wherein the positive regions are the regions where characteristic Raman signals appear.

[0154] This invention discloses a surface-enhanced Raman spectroscopy (SERS) method, system, and nanotag preparation method for IL-6 immunoassay. Applied to data analysis in low antigen concentration scenarios, the Raman scan image is first converted into a binary image according to a set threshold, and quantification is performed using the percentage of positive regions (RPP). The exponential relationship between the RPP and antigen concentration verifies that single-molecule events follow a Poisson distribution. Finally, the linear relationship between the product of the RPP and the average Raman intensity of the positive regions (RPP*ARI) and the concentration is used to analyze antigen concentration data. Experiments have shown that this application can be 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, this immunoassay method shows strong consistency with standard clinical laboratory methods. Furthermore, the nanotags prepared in this application exhibit a clear signal at single nanoparticle resolution, thereby achieving ultrasensitive / single-molecule detection. This provides a basis for antigen concentration analysis in low-concentration scenarios.

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

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

[0157] As will be understood by those skilled in the art, the computer-readable storage medium described in this embodiment allows for the implementation of all or part of the steps in the above method embodiments by computer program-related hardware. The aforementioned computer program can be stored in a computer-readable storage medium. When executed, the program performs the steps of the above method embodiments; and the aforementioned storage medium includes various media capable of storing program code, such as ROM, RAM, magnetic disks, or optical disks.

[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 between them. The memory is used to store computer programs, the communication interface is used to perform communication, and the processor and the transceiver are used to run the computer programs, so that the electronic terminal performs the various steps of the above method.

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

[0160] The processors mentioned above can be general-purpose processors, including central processing units (CPUs), graphics processing units (GPUs), network processors (NPs), etc.; they can also be digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components.

[0161] In the above embodiments, although the invention has been described in conjunction with specific embodiments thereof, many substitutions, modifications, and variations of these embodiments will be apparent to those skilled in the art from the foregoing description. The embodiments of the invention are intended to cover all such substitutions, 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 invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A digital surface-enhanced Raman spectroscopy method for interleukin-6 immunoassay, characterized in that, Including the following steps: A silicon chip pre-modified with interleukin-6 antibody based on the 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, and surface-enhanced Raman nanotags modified with interleukin-6 antigen and antibody were co-incubated to obtain an antigen-antibody sandwich structure. Surface-enhanced Raman scattering (SERS) spectra of the silicon chip after the reaction were collected. The surface-enhanced Raman scattering (SERS) spectrum is sequentially binarized to obtain a binary image, which is then used as a mask to obtain a digital SERS image. Raman reporter molecule characteristic peaks are extracted from multiple partitions in the surface-enhanced Raman scattering digital image, and the proportion of positive regions in the partitions is calculated according to the threshold method. Quantitative analysis is then performed based on the proportion of positive regions and the average Raman intensity of the positive regions to obtain antigen concentration data. The positive regions are those where characteristic Raman signals appear. The step of sequentially binarizing the surface-enhanced Raman scattering (SERS) spectrum to obtain a binary image and using it as a mask to obtain a digital SERS image includes: The surface-enhanced Raman scattering spectrum is binarized based on a preset threshold to obtain a binary false-color image with background information filtered out. Using the binary false-color image as a mask, convolving it with the surface-enhanced Raman scattering pattern yields a surface-enhanced Raman scattering digital image. The antigen concentration data is obtained through quantitative analysis based on the proportion of positive regions and the average Raman intensity of the positive regions. This includes substituting the product of the proportion of positive regions and the average Raman intensity of the positive regions into a pre-constructed quantitative formula to obtain the antigen concentration data. The quantitative formula includes a linear quantitative formula, the mathematical expression of which is: y² = 0.

743. x+10.626 In the formula, x represents the antigen concentration data, and y2 is the product of the percentage of positive regions and the average Raman intensity of the positive regions. The methods used to prepare surface-enhanced Raman nanotags include: Gold nanoparticles were obtained by reducing tetrachloroauric acid with citric acid. Using the gold nanoparticles as seeds, and reducing silver ions with ascorbic acid to form an Ag shell; Au was injected into a solution containing an Ag shell. I An Ag-Au alloy shell was prepared by using a solution and a reducing agent, and a Raman reporter molecule was added to obtain a solution containing surface-enhanced Raman nanotags. The solution containing the surface-enhanced Raman nanotag was purified to obtain the surface-enhanced Raman nanotag. The surface-enhanced Raman nanotag was functionalized using an interleukin-6 antibody to obtain an antibody-modified surface-enhanced Raman nanotag.

2. The digital surface-enhanced Raman spectroscopy method for interleukin-6 immunoassay according to claim 1, characterized in that, The surface-enhanced Raman scattering spectra were acquired using a Renishaw Raman microscopy system with lasers of different wavelengths and powers.

3. The digital surface-enhanced Raman spectroscopy method for interleukin-6 immunoassay according to claim 1, characterized in that, The surface-enhanced Raman scattering (SERS) spectra were acquired using a 100× objective lens with a power of 0.85 mW and an exposure time of 2 seconds. The Raman scan data were obtained using a 633 nm excitation wavelength at 60 × 60 μm. 2 Regional collection with a step size of 3 μm.

4. The digital surface-enhanced Raman spectroscopy method for interleukin-6 immunoassay according to claim 1, characterized in that, The configuration process of the silicon chip functionalized with interleukin-6 antibody includes: The silicon substrate with an oxide layer of the target thickness was cleaned using a piranha solution. The cleaned silicon substrate was rinsed with ultrapure water and dried with nitrogen gas. The dried silicon substrate was subjected to plasma treatment to obtain a hydroxylated silicon substrate; The hydroxylated silicon substrate was treated with 3-aminopropyltrimethoxysilane and baked to produce a dense organosilane layer, thereby obtaining a silanized silicon substrate. The silanized silicon substrate was coupled with a glutaraldehyde solution, and then washed with ultrapure water and dried with nitrogen gas to obtain an aldehyde-modified silicon substrate. The aldehyde-modified silicon substrate is immersed in a solution of interleukin-6 antibody for a certain target time, and then the surface of the aldehyde-modified silicon substrate is sealed with casein and surfactant in phosphate buffer solution to obtain a silicon chip functionalized with interleukin-6 antibody.

5. The digital surface-enhanced Raman spectroscopy method for interleukin-6 immunoassay according to claim 1, characterized in that, The surface-enhanced Raman nanotag was functionalized using an interleukin-6 antibody to obtain an antibody-modified surface-enhanced Raman nanotag, comprising: The surface-enhanced Raman nanotags were subjected to ultrasonic treatment in ultrapure water to obtain a suspension; The buffer solution of the suspension was replaced with a reaction buffer solution to obtain a resuspended suspension, wherein the reaction buffer solution comprised 0.2–0.6 mM CTAC and 1–5 × 10⁻⁶ mg / L. -2 M Tris-HCl; The nanotags in the resuspended suspension were incubated together with the interleukin-6 antibody, stirred, and then mixed with a blocking solution to obtain the target solution. The blocking solution comprised 1–3% BSA, 0.01–0.2% casein, and 1–5 × 10⁻⁶ ppm. -2 M Tris-HCl; The target solution was purified by centrifugation to obtain antibody-modified surface-enhanced Raman nanotags.

6. A digital surface-enhanced Raman spectroscopy interleukin-6 immunoassay system, characterized in that, include: The experimental module is used to co-incubate a silicon chip pre-modified with interleukin-6 antibody based on the 3-aminopropyltrimethoxysilane-glutaraldehyde coating method, and a surface-enhanced Raman nanotag modified with interleukin-6 antigen and antibody to obtain an antigen-antibody sandwich structure. Raman spectroscopy acquisition module, used to acquire surface-enhanced Raman scattering spectra of silicon chips after reaction; The image processing module is used to sequentially binarize the surface-enhanced Raman scattering (SMR) spectrum to obtain a binary image and to use it as a mask to obtain a digital SMR image. The quantitative analysis module is used to extract the Raman reporter molecule characteristic peaks of multiple partitions in the surface-enhanced Raman scattering digital image, calculate the proportion of positive regions in the partitions according to the threshold method, and perform quantitative analysis based on the proportion of positive regions and the average Raman intensity of the positive regions to obtain antigen concentration data, wherein the positive regions are the regions where characteristic Raman signals appear; The step of sequentially binarizing the surface-enhanced Raman scattering (SERS) spectrum to obtain a binary image and using it as a mask to obtain a digital SERS image includes: The surface-enhanced Raman scattering spectrum is binarized based on a preset threshold to obtain a binary false-color image with background information filtered out. Using the binary false-color image as a mask, convolving it with the surface-enhanced Raman scattering pattern yields a surface-enhanced Raman scattering digital image. The antigen concentration data is obtained through quantitative analysis based on the proportion of positive regions and the average Raman intensity of the positive regions. This includes substituting the product of the proportion of positive regions and the average Raman intensity of the positive regions into a pre-constructed quantitative formula to obtain the antigen concentration data. The quantitative formula includes a linear quantitative formula, the mathematical expression of which is: y2=0.743 x+10.626 In the formula, x represents the antigen concentration data, and y2 is the product of the percentage of positive regions and the average Raman intensity of the positive regions. The surface-enhanced Raman nanotag preparation methods used include: Gold nanoparticles were obtained by reducing tetrachloroauric acid with citric acid. Using the gold nanoparticles as seeds, and reducing silver ions with ascorbic acid to form an Ag shell; Au was injected into a solution containing an Ag shell. I An Ag-Au alloy shell was prepared by using a solution and a reducing agent, and a Raman reporter molecule was added to obtain a solution containing surface-enhanced Raman nanotags. The solution containing the surface-enhanced Raman nanotag was purified to obtain the surface-enhanced Raman nanotag. The surface-enhanced Raman nanotag was functionalized using an interleukin-6 antibody to obtain an antibody-modified surface-enhanced Raman nanotag.

Images