Functionalized sers detection of abo and rh blood group system enhancing reagents and methods of making and use thereof

Abstract

The application discloses a functionalized SERS detection enhancement reagent for ABO and Rh blood type systems and a preparation method and application thereof. The application constructs Au@Ag NRs type gold-coated silver composite metal nanomaterials, connects blood type antigen antibodies, and combines SERS detection technology to detect blood types. The application has a remarkable enhancement effect, is good in repeatability and stability, and can realize integrated synchronous detection of ABO+Rh blood types.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to providing functionalized SERS detection enhancement reagents for ABO and Rh blood group systems, their preparation methods, and applications. Background Technology

[0002] ABO and Rh blood typing are fundamental core testing procedures in clinical transfusion, preoperative screening, prevention of neonatal hemolytic disease, organ transplantation, and forensic evidence identification. Accurate, rapid, and low-cost blood typing technologies play an irreplaceable role in clinical medical safety. Currently, mainstream clinical blood typing relies on traditional immunoagglutination techniques such as slide agglutination, microcolumn gel electrophoresis, and fully automated blood typing analyzers. The core principle of these techniques is based on the specific antigen-antibody binding between red blood cell antigens and corresponding blood type antibodies, producing an agglutination reaction that is visible to the naked eye or identifiable by instruments.

[0003] Surface-enhanced Raman scattering (SERS) spectroscopy, with its advantages of high fingerprint specificity, single-molecule level detection sensitivity, fast response speed, small sample volume, and ability to achieve micro-volume non-destructive detection, has been widely used in biorecognition, immunosensing, biochemical biomarker detection, and pathogen and biomolecule specific recognition. Gold and silver nanoparticles (AgNPs), as classic SERS activity enhancers, possess strong localized surface plasmon resonance effects, significant electromagnetic field enhancement effects, good biocompatibility, and ease of surface functionalization modification, making them the preferred enhancing reagent materials for constructing biological immunological SERS sensing platforms.

[0004] In theory, SERS can also be used for blood group antigen detection. For example, after labeling Ag metal nanoparticles with antibodies, they can be reacted with red blood cells, and the presence of antigens can be determined by detecting reporter molecule signals. However, the following key technical challenges remain unresolved in the direct and reliable application of SERS technology to clinical blood typing: 1. Insufficient SERS enhancement performance of enhancement reagents: The plasma resonance modulation range of single AgNPs is narrow, and the electromagnetic field enhancement effect is limited. The detection signal of trace red blood cell samples is weak and the signal-to-noise ratio is low. Low-concentration blood samples are prone to missed detection and false detection. Ordinary simple Ag composite particles have no ordered structure design, and the bimetallic coupling synergistic enhancement effect is not fully utilized, resulting in poor signal repeatability of enhancement reagents.

[0005] 2. Poor stability and specificity of antibody conjugation: Existing technologies mostly use physical adsorption to fix blood type antibodies, which are prone to detachment, changes in spatial conformation, and loss of antigen recognition activity; non-specific adsorption is serious, and they are prone to non-specific binding with impurities and non-target red blood cells in plasma, resulting in high background interference and decreased accuracy of blood type detection.

[0006] 3. Limited testing function and incompatibility with multiple blood typing systems: Most existing solutions can only detect ABO blood type or a single RhD antigen, and cannot achieve integrated simultaneous detection of ABO and Rh blood types. The testing process is cumbersome and the testing efficiency is low, making it difficult to meet the needs of clinical batch screening.

[0007] 4. Poor repeatability and precision, making quantification difficult: Traditional SERS enhancement reagents have inconsistent particle size and morphology between batches, uncontrollable antibody modification amount, and large RSD of SERS characteristic peak intensity between parallel samples. They can only achieve qualitative screening and cannot meet the standardized requirements of clinical testing precision and stability.

[0008] 5. Weak resistance to matrix interference: In actual testing, plasma proteins and impurity molecules in whole blood and diluted blood samples are easily adsorbed onto the surface of nano-enhanced reagents, masking antibody recognition sites, interfering with antigen-antibody specific binding, and generating stray Raman background peaks, which seriously affect the interpretation of results.

[0009] 6. High operational threshold and poor portability: Existing solutions mostly rely on large Raman spectrometers and complex sample pretreatment, making it difficult to achieve bedside instant detection and rapid on-site screening, which limits their application in primary medical institutions and emergency rescue scenarios. Summary of the Invention

[0010] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide functionalized SERS detection enhancement reagents for ABO and Rh blood group systems, their preparation methods and uses, to fill the gap in the prior art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: One aspect of the present invention provides an enhancing reagent for blood type identification based on SERS technology. The enhancing reagent is a metal nanoparticle linked to blood type antigens and antibodies, and the enhancing reagent is an Au@Ag NRs type gold-coated silver composite metal nanomaterial.

[0012] Furthermore, the blood type antigen antibody is selected from any one of anti-A, anti-A1, anti-B, anti-D, anti-E, and anti-C.

[0013] Furthermore, the amino acid sequences of the heavy chain variable regions and light chain variable regions of the anti-A, anti-A1, anti-B, anti-D, anti-E, and anti-C are shown in Table 1.

[0014] Furthermore, the preparation method of the enhancing reagent includes the following steps: 1) Preparation of Au@Ag NRs: a. Add hydroxylamine hydrochloride solution to boiling AgNO3, and add NaOH dropwise while heating and stirring until the solution turns yellow. Then heat again, keeping the process dark throughout, to obtain Ag NPs stock solution. b. Mix AgNO3, ascorbic acid and CTAB to obtain a mixture; c. Mix the Ag NPs stock solution with the mixed solution to obtain Ag NPs sol; d. Heat the Ag NPs sol, add hydroxylamine hydrochloride and HAuCl4 in sequence, centrifuge, discard the supernatant, wash and resuspend to obtain Au@Ag NRs; e. Disperse Au@Ag NRs in water, centrifuge, discard the supernatant, resuspend, and sonicate to obtain the enhancing reagent; 2) Coupling of antibodies with Au@Ag NRs: The enhancing reagent is mixed with the blood group antigen antibody; 3) Blocking and purification.

[0015] Further, step 2) specifically involves slowly adding mercapto polyethylene glycol to the enhancing reagent, stirring, centrifuging and washing, resuspending, slowly adding antibody, and stirring in the dark.

[0016] Furthermore, the blocking method is as follows: adding blocking solution to the enhancing reagent of the conjugated antibody and incubating; the blocking solution is selected from any one of BSA, ethanolamine, glycine, casein, and skim milk powder.

[0017] Furthermore, the purification method is as follows: centrifuge and wash the incubated enhancing reagent, resuspend, and add sodium azide.

[0018] Another aspect of the present invention provides the use of the above-described enhancing reagent for blood type identification.

[0019] Another aspect of the present invention provides a product for identifying blood types, the product containing the above-mentioned enhancing reagent.

[0020] Furthermore, the product may also contain auxiliary reagents, such as 0.02% sodium azide, which is an antibacterial and preservative agent.

[0021] Another aspect of the present invention provides a method for identifying ABO and Rh blood types, wherein the method involves mixing a blood sample with the aforementioned enhancing reagent, detecting the sample using SERS technology, obtaining characteristic peaks, and determining the blood type based on the characteristic peaks.

[0022] Furthermore, the SERS technology is used to set the Raman spectroscopy excitation wavelength to 785 nm and the power to 0-500 mW. Compared with the prior art, the beneficial effects of this invention are: This invention provides a functionalized SERS detection enhancement reagent for ABO and Rh blood group systems, which has strong enhancement ability, good repeatability, and good stability, and can realize integrated simultaneous detection of ABO and Rh blood groups. Attached Figure Description

[0023] Figure 1The spectra of A-SERS enhanced reagents were prepared for the detection of quality control samples of type A erythrocytes using 6 reagent groups.

[0024] Figure 2 Raman spectra of A1, B, A2B, and O-type red blood cell quality controls and A-type whole blood samples used as anti-A-SERS enhancement reagents.

[0025] Figure 3 Raman spectra of A1, B, and O type erythrocyte quality controls and AB type whole blood samples used as anti-B-SERS enhancement reagents.

[0026] Figure 4 Raman spectra of R1R1, R2R2, and rr red blood cell quality control and Rh-whole blood samples as anti-D-SERS enhancement reagents.

[0027] Figure 5 Raman spectra of R1R1, R2R2, and rr red blood cell quality control and Rh-whole blood samples as anti-C-SERS enhancement reagents.

[0028] Figure 6 Raman spectra of R1R1, R2R2, rr red blood cell quality control and Rh-whole blood samples as anti-E-SERS enhancement reagents. Detailed Implementation

[0029] By conjugating and functionalizing Ag metal nanoparticles with ABO and Rh blood type-specific antibodies, a specific SERS detection enhancement reagent can be constructed. This method can overcome the limitations of traditional agglutination methods, which rely on visual interpretation, are susceptible to human subjective error, have insignificant agglutination in low-concentration samples, and are difficult to achieve accurate quantification and rapid on-site screening of trace samples. It provides a new technical approach for real-time blood typing and non-invasive identification of trace blood samples, and has significant clinical application and industrialization value.

[0030] 1. Improvement 1: Construct Au@Ag NRs composite SERS enhancement reagent to replace traditional AgNRs single-structure enhancement reagent. Advantages: The Au@Ag NRs composite SERS enhancer combines the excellent Raman enhancement performance of silver with the good chemical stability and biocompatibility of gold, effectively improving the problems of easy oxidation and weak corrosion resistance of traditional single AgNRs enhancers, and extending the use and storage time of the enhancer. The plasmon resonance coupling effect formed by the bimetallic interface further amplifies the Raman signal, significantly improving detection sensitivity. At the same time, the rich surface electronic structure can optimize the adsorption state of the analyte molecules, making the uniformity and reproducibility of the detection signal better, and better meeting the requirements of quantitative detection. This composite enhancer has lower biotoxicity, is suitable for the detection of various complex biological samples such as serum and body fluids, and has a wider adjustable range of plasmon resonance peaks, making it compatible with a variety of probe molecules and detection systems, and thus has greater overall versatility.

[0031] 2. Improvement point two: Use cross-linking agents to directionally couple and modify blood group antibodies to achieve controllable functionalization of the enhancing reagent. Advantages: It abandons the traditional physical adsorption method and uses mercapto-polyethylene glycol crosslinking reagent to activate and modify the metal surface, directionally immobilizing ABO and Rh specific blood group antibodies. The antibodies are firmly immobilized and do not easily fall off, and can maintain their natural spatial conformation, retaining complete antigen recognition activity. At the same time, it enhances the blocking of non-specific adsorption sites on the reagent surface, effectively inhibits the non-specific binding of plasma proteins and impurities, significantly reduces background interference, and improves the specificity and accuracy of blood typing.

[0032] 3. Improvement point three: Optimize and enhance the anti-contamination modification of reagent surfaces to improve the anti-interference ability of complex blood sample matrices. Corresponding advantages: By using blocking agents such as ethanolamine to passivate the surface of functionalized enhancing reagents, a bio-antifouling interface is formed, which effectively resists the adsorption interference of impurities such as proteins and lipids in whole blood and hemolyzed samples. It is suitable for direct detection of raw blood samples without complex pretreatment, simplifies sample pretreatment steps, and lowers the threshold for detection operation.

[0033] 4. Improvement point four: Enhance reagent compatibility with portable Raman spectroscopy devices to suit on-site, real-time detection scenarios. Corresponding advantages: The functionalized SERS enhancement reagent of this invention requires no complicated pretreatment and requires very little sample volume. It can be used with a portable small Raman spectrometer, eliminating the dependence on large instruments. It is suitable for scenarios such as primary hospitals, pre-hospital emergency care, emergency blood collection in the field, and rapid bedside blood typing, with a wider range of applications and greater practicality.

[0034] 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.

[0035] Table 1. Amino acid sequence of blood type antibodies in each example

[0036] Example 1: Preparation of Functionalized SERS Enhancer The first step is to prepare Au@Ag NRs 1. Add 2 mL of 1% hydroxylamine hydrochloride solution to 100 mL of 0.005% AgNO3 boiling water, and add 1% NaOH dropwise while heating and stirring until the solution turns yellow. Then heat at 85°C for 5-10 min, avoiding light throughout the process. Cool the resulting Ag NPs stock solution for later use.

[0037] 2. Mix 0.25 mL of 10 mM AgNO3, 0.5 mL of 100 mM ascorbic acid, and 10 mL of 80 mM CTAB.

[0038] 3. Add Ag NPs stock solution to the above mixture, shake gently, and let stand to react to obtain Ag NPs sol.

[0039] 4. Dilute the Ag NRs sol with pure water at a ratio of 1:5, and gently stir and heat in a 60°C water bath.

[0040] 5. Add 30 μL of 2 mM hydroxylamine hydrochloride and 90 μL of 0.25 mM HAuCl4 dropwise in sequence, at 8000 rpm for 15 min. Discard the supernatant, wash twice with ultrapure water, and resuspend to obtain Au@Ag NRs.

[0041] 6. Disperse Au@Ag NRs in 10 mL of ultrapure water, centrifuge at 8000 rpm for 15 min, and discard the supernatant.

[0042] 7. Resuspend in 0.1 mM PVP solution, sonicate for 5 min, and store at 4°C for later use.

[0043] The second step involves the conjugation of the antibody with Au@Ag NRs. 1. Take 10 mL of the prepared Au@Ag NRs, centrifuge at 12,000 g for 20 min at 4 °C, wash twice, and resuspend in 0.05 M PBS to 10 mL.

[0044] 2. Slowly add 100 μL of 1% mercapto polyethylene glycol and stir at room temperature in the dark for 2 hours.

[0045] 3. Centrifuge at 10,000g for 15 min at 4°C, wash twice, and resuspend in 0.05M PBS (pH 7.2) to 10 ml.

[0046] 4. Slowly add 0.1-1 mg / mL of anti-A monoclonal antibody (refer to Table 2). After the addition is complete, continue stirring at room temperature in the dark for 4 hours.

[0047] The third step is sealing and purification (sealing agents: BSA / ethanolamine / glycine / casein / skim milk powder). 1. Add 100 μL of 0.1 M ethanolamine (pH 7.4) and incubate at room temperature for 30 min to block residual aldehyde groups.

[0048] 2.12,000g×20min, centrifuge at 4℃, wash twice, and resuspend in 0.05M PBS (pH7.2) to 10mL.

[0049] 3. Add 0.02% sodium azide (antibacterial preservative) to obtain the final anti-D-SERS enhancing reagent.

[0050] 4. Aliquot into sterile brown centrifuge tubes (1.5 mL / tube) and store at 4°C protected from light.

[0051] result: All the anti-A-SERS enhancing reagents prepared according to the reagent ratios could specifically recognize the A antigen on the surface of type A erythrocyte quality control samples. Among them, the anti-A-SERS enhancing reagent prepared according to the fifth reagent ratio showed the best effect. Figure 1 Subsequent specific enhancement reagents were prepared using the reagent ratios from group 5, and the same methods were used to prepare anti-A1 / anti-B / anti-D / anti-E / anti-C-SERS enhancement reagents.

[0052] Table 2. Partial optimization of the main reagent ratios for anti-A-SERS enhancement reagents.

[0053] Collect red blood cell quality control samples of type A1, type B, type A2B, type O, type A R1R1 (CCDee), type O R2R2 (ccDEE), and type B rr (ccdee), as well as whole blood samples identified as type A, type B, type O, type AB, and type A Rh-, for later use.

[0054] Example 2: Rapid Detection of Type A Blood Using Anti-A-SERS Enhancer and Anti-A1-SERS Enhancer. 1. Take out the centrifuge tubes containing the prepared anti-A-SERS enhancement reagent and anti-A1-SERS enhancement reagent and vortex them. Take 150uL each of A1 type, B type, A2B type, O type red blood cell quality control and A type whole blood sample and mix them with 150uL of enhancement reagent. Incubate at room temperature for 20min.

[0055] Centrifuge at 3000g for 3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0056] 3. Transfer 300 μL of the mixture to a 96-well plate, set the parameters of the 785 nm Raman spectrometer to 0-500 mW, and start the detection.

[0057] result: like Figure 2Raman spectra of A1, B, A2B, and O type erythrocyte controls and type A whole blood samples were obtained using the anti-A-SERS enhancing reagent. The A1, A2B, and type A erythrocyte controls and type A whole blood samples showed obvious Raman characteristic peaks in the anti-A-SERS enhancing reagent detection system; however, the B and O type erythrocyte controls did not show obvious characteristic peaks. This indicates that the A antigen on the surface of A1, A2B, and type A whole blood erythrocytes successfully and specifically binds to the anti-A-SERS probe, producing a clear Raman signal. It also indicates that the type A whole blood sample is the A1 subtype, and that the anti-A-SERS and anti-A1-SERS enhancing reagents can distinguish between the A1 and A2 blood group subtypes.

[0058] Example 3: Rapid detection of type B blood using anti-B-SERS enhancement reagent. 1. Take out the centrifuge tube containing the prepared anti-B-SERS enhancement reagent and vortex it. Take 150uL each of A1 type, B type, O type red blood cell quality control and AB type whole blood sample and mix them with 150uL of enhancement reagent. Incubate at room temperature for 20min.

[0059] Centrifuge at 3000g for 2-3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0060] Transfer 300 μL of the mixture to a 96-well plate, set the 785 nm Raman spectrometer parameters to 0-500 mW, and begin detection.

[0061] result: like Figure 3 Raman spectra of A1, B, and O type erythrocyte quality controls and AB type whole blood samples were obtained using the anti-B-SERS enhancer. The B type erythrocyte quality controls and AB type whole blood samples showed obvious Raman characteristic peaks in the anti-B-SERS enhancer detection system, while the A1 and O type erythrocyte quality controls did not. This indicates that the B antigen on the surface of B and AB type erythrocytes successfully and specifically binds to the 4-ATP-anti-B-SERS enhancer, and the anti-D-SERS enhancer is successfully captured, producing a clear Raman signal.

[0062] Example 4: Rapid detection of Rh blood using anti-D-SERS enhancement reagent Remove the centrifuge tube containing the prepared anti-D-SERS enhancement reagent and vortex it. Take 150 μL each of R1R1 (CCDee), R2R2 (ccDEE), rr (ccdee) red blood cell quality control and Rh-whole blood sample and mix them with 150 μL of enhancement reagent. Incubate at room temperature for 20 min.

[0063] Centrifuge at 3000g for 2-3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0064] Transfer 300 μL of the mixture to a 96-well plate, set the 785 nm Raman spectrometer parameters to 0-500 mW, and begin detection.

[0065] result: like Figure 4 Raman spectra of R1R1 (CCDee), R2R2 (ccDEE), and rr (ccdee) erythrocyte quality controls and Rh-whole blood samples were obtained using the anti-D-SERS enhancer. The R1R1 (CCDee) and R2R2 (ccDEE) erythrocyte quality controls showed obvious Raman characteristic peaks in the anti-D-SERS enhancer detection system; however, the rr (ccdee) erythrocyte quality control and Rh-whole blood samples did not show obvious characteristic peaks. This indicates that the D antigen on the surface of R1R1 (CCDee) and R2R2 (ccDEE) erythrocytes successfully and specifically bound to the anti-D-SERS enhancer, producing a clear Raman signal.

[0066] Example 5: Rapid Detection of Rh Blood Using Anti-C-SERS Enhancer Reagent 1. Take out the centrifuge tube containing the prepared anti-C-SERS enhancement reagent and vortex it. Take 150uL each of R1R1 (CCDee), R2R2 (ccDEE), rr (ccdee) red blood cell quality control and Rh-whole blood sample and mix them with 150uL of enhancement reagent. Incubate at room temperature for 20min.

[0067] Centrifuge at 3000g for 2-3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0068] 3. Transfer 300 μL of the mixture to a 96-well plate, set the 785 nm Raman spectrometer parameters to 0-500 mW, and start the detection.

[0069] result: like Figure 5 Raman spectra of R1R1 (CCDee), R2R2 (ccDEE), and rr (ccdee) erythrocyte quality controls and Rh-whole blood samples were obtained using the anti-C-SERS enhancer. The R1R1 (CCDee) erythrocyte quality control showed a distinct Raman characteristic peak in the anti-C-SERS enhancer detection system; however, the R2R2 (ccDEE), rr (ccdee) erythrocyte quality controls and Rh-whole blood samples did not show obvious characteristic peaks. This indicates that the C antigen on the surface of R1R1 (CCDee) erythrocytes successfully and specifically binds to the anti-C-SERS enhancer, producing a clear Raman signal.

[0070] Example 6: Rapid Detection of Rh Blood Using Anti-E-SERS Enhancement Reagent 1. Take out the centrifuge tube containing the prepared anti-E-SERS enhancement reagent and vortex it. Take 150uL each of R1R1 (CCDee), R2R2 (ccDEE), rr (ccdee) red blood cell quality control and Rh-whole blood sample and mix them with 150uL of enhancement reagent. Incubate at room temperature for 20min.

[0071] Centrifuge at 3000g for 2-3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0072] 3. Transfer 300 μL of the mixture to a 96-well plate, set the 785 nm Raman spectrometer parameters to 0-500 mW, and start the detection.

[0073] result: like Figure 6 Raman spectra of R1R1 (CCDee), R2R2 (ccDEE), and rr (ccdee) erythrocyte quality controls and Rh-whole blood samples were obtained using the anti-E-SERS enhancer. The R2R2 (ccDEE) erythrocyte quality control showed a distinct Raman characteristic peak in the anti-E-SERS enhancer detection system; however, the R1R1 (CCDee), rr (ccdee) erythrocyte quality controls and Rh-whole blood samples did not show obvious characteristic peaks. This indicates that the E antigen on the surface of R2R2 (ccDEE) erythrocytes successfully and specifically binds to the anti-E-SERS enhancer, producing a clear Raman signal.

[0074] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An enhancing reagent for blood typing based on SERS technology, characterized in that, The enhancing agent is a metal nanoparticle-linked blood group antigen-antibody enhancing agent, which is an Au@Ag NRs type gold-coated silver composite metal nanomaterial.

2. The enhancing agent according to claim 1, characterized in that: The blood type antigens and antibodies are selected from any one of anti-A, anti-A1, anti-B, anti-D, anti-E, and anti-C.

3. The enhancing agent according to claim 2, characterized in that: The amino acid sequences of the heavy chain variable regions and light chain variable regions of the anti-A, anti-A1, anti-B, anti-D, anti-E, and anti-C antibodies are shown in Table 1.

4. The enhancing agent according to claim 1, characterized in that: The preparation method of the enhancing reagent includes the following steps: 1) Preparation of Au@Ag NRs: a. Add hydroxylamine hydrochloride solution to boiling AgNO3, and add NaOH dropwise while heating and stirring until the solution turns yellow. Then heat again, keeping the process dark throughout, to obtain Ag NPs stock solution. b. Mix AgNO3, ascorbic acid and CTAB to obtain a mixture; c. Mix the Ag NPs stock solution with the mixed solution to obtain Ag NPs sol; d. Heat the Ag NPs sol, add hydroxylamine hydrochloride and HAuCl4 in sequence, centrifuge, discard the supernatant, wash and resuspend to obtain Au@Ag NRs; e. Disperse Au@Ag NRs in water, centrifuge, discard the supernatant, resuspend, and sonicate to obtain the enhancing reagent; 2) Coupling of antibodies with Au@Ag NRs: The enhancing reagent is mixed with the blood group antigen antibody; 3) Blocking and purification.

5. The enhancing agent according to claim 4, characterized in that: Step 2) Specifically, slowly add mercapto polyethylene glycol to the enhancing reagent, stir, centrifuge and wash, resuspend, slowly add antibody, and stir in the dark.

6. The enhancing agent according to claim 4, characterized in that: The blocking method is as follows: adding blocking solution to the enhancing reagent of the conjugated antibody and incubating; the blocking solution is selected from any one of BSA, ethanolamine, glycine, casein, and skim milk powder.

7. The enhancing agent according to claim 4, characterized in that: The purification method is as follows: centrifuge and wash the incubated enhancing reagent, resuspend it, and add sodium azide.

8. Use of the enhancing reagent as described in any one of claims 1-7 for blood type identification.

9. A product for identifying ABO and Rh blood types, characterized in that: The product contains the enhancing agent as described in any one of claims 1-7.

10. A method for identifying ABO and Rh blood types, characterized in that: The method involves mixing the enhancing reagent as described in any one of claims 1-7 with a blood sample, detecting it using SERS technology, obtaining characteristic peaks, and determining the blood type based on the characteristic peaks.

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