Blood typing method based on surface-enhanced raman spectroscopy

Abstract

The application discloses a blood typing method based on surface enhanced Raman spectroscopy. Three universal and stable SERS signal generation and detection systems are constructed, and various blood types can be rapidly and conveniently identified.

Description

Technical Field

[0001] This invention relates to the field of spectral detection and analysis, specifically to a blood typing method based on surface-enhanced Raman spectroscopy. Background Technology

[0002] Blood typing is a key technology in transfusion medicine, organ transplantation, and forensic medicine. Clinical transfusion medicine involves more than 40 known blood group systems, with the ABO and Rh systems being the most critical. Systems such as MNS, Kell, Duffy, Kidd, Diego, and Lewis also play important roles in transfusion compatibility testing. Accurate and rapid blood typing is crucial for ensuring transfusion safety. Traditional blood typing methods mainly include test tube methods, microplate methods, and microcolumn gel card methods. These methods are primarily based on the visible agglutination reaction between red blood cells and specific antibodies. However, they rely on manual interpretation and have limitations such as high subjectivity, difficulty in automation and quantitative analysis, insufficient sensitivity for weak antigens (e.g., weakly expressed RhD), inability to simultaneously detect multiple antigens, and typically only the ability to type a few systems. In recent years, molecular biology methods (such as PCR) have achieved high-sensitivity typing, but they suffer from problems such as complex operation, high cost, long testing cycles, and inability to directly detect antigen phenotypes.

[0003] Surface-enhanced Raman spectroscopy (SERS) has shown great potential in the field of biosensing due to its extremely high detection sensitivity (enabling single-molecule detection) and unique "fingerprint" characteristics. Studies have reported the application of SERS in pathogen detection and tumor marker detection. However, the direct and reliable application of SERS technology to clinical blood typing faces the following key unresolved technical challenges: Strong background interference in red blood cell samples: Red blood cells themselves have strong fluorescence and Raman background signals, which can severely interfere with the characteristic signals of Raman reporter molecules labeled on antibodies, resulting in low signal-to-noise ratio and high false negative / positive rates.

[0004] Spatial hindrance and signal uniformity issues in antigen-antibody binding: Erythrocyte antigens (such as ABO and Rh antigens) are located on the cell membrane surface and have a complex spatial structure. After traditional antibody-reporter molecule markers bind to antigens, the reporter molecules may not be able to effectively enter the "hotspot" regions of the SERS substrate (such as metal nanoparticles), resulting in unstable signal enhancement effects and poor reproducibility.

[0005] Lack of standardized SERS detection system applicable to blood typing: Existing studies are mostly principle validations, lacking systematic SERS detection scheme design for different types of blood group antigens (high density such as ABO, low density such as Rh), including substrate selection, antibody labeling strategy, incubation conditions, signal reading process, etc., and thus cannot form a stable and reliable standardized method.

[0006] Insufficient multiplex detection capabilities: Simultaneous identification of multiple blood group systems (such as ABO and Rh) in a single test is an urgent clinical need. Current SERS sensing research lacks mature protocols applicable to the complex biological sample of blood type, addressing issues such as the complexity of spectral coding and the avoidance of cross-interference from specific antibodies. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a blood typing method based on surface-enhanced Raman spectroscopy to overcome the deficiencies of the prior art. To achieve the above objective, this invention provides the following technical solution: This invention provides a method for preparing an enhancing reagent, the method comprising: 1) Add hydroxylamine hydrochloride solution to boiling AgNO3 water, and add NaOH dropwise while heating and stirring until the solution turns yellow. Then heat again, and keep the process dark to obtain Ag NPs stock solution. 2) Mix AgNO3, ascorbic acid, and CTAB to obtain a mixture; 3) Mix the Ag NPs stock solution with the mixed solution to obtain Ag NPs sol; 4) Heat the Ag NPs sol, add hydroxylamine hydrochloride and HAuCl4 in sequence, centrifuge, discard the supernatant, wash and resuspend to obtain Au@Ag NRs; 5) Disperse Au@Ag NRs in water, centrifuge, discard the supernatant, resuspend, and sonicate to obtain the enhancing reagent.

[0008] Furthermore, the method also includes: 6) Enhancer conjugation with antibody: The enhancer is mixed with the antibody corresponding to the blood group antigen; 7) Blocking and purification.

[0009] Furthermore, the antibody is selected from any one of anti-A, anti-A1, anti-B, anti-D, anti-E, and anti-C.

[0010] 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 2.

[0011] Further, step 6) specifically involves: slowly adding glutaraldehyde or mercapto polyethylene glycol to the enhancing reagent, stirring, centrifuging and washing, resuspending, slowly adding antibody, and stirring in the dark.

[0012] Furthermore, the blocking method involves adding a 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.

[0013] Furthermore, the purification method is as follows: centrifuge and wash the incubated enhancing reagent, resuspend, and add sodium azide.

[0014] Furthermore, the method also includes: 6) Modification of Au@Ag NRs using reporter molecules: Add reporter molecules to the colloidal solution of Au@Ag NRs, stir and protect from light; 7) With conjugated antibodies: 8) Blocking and purification.

[0015] Another aspect of the present invention provides an enhancing reagent prepared by the above method.

[0016] Another aspect of the present invention provides the application of the above-mentioned enhancing reagent in blood typing.

[0017] Another aspect of the present invention provides a method for identifying blood types, the method comprising: 5) Add protein L magnetic beads to the blood sample, mix well, and incubate; 6) Magnetic separation, discard the supernatant, add glycine-HCl to elute, pH 2.5, elution time less than 45 min, add Tris to neutralize; 7) Mix with the above-mentioned enhancing reagents; 8) Using a Raman spectrometer, the excitation wavelength was 785 nm, and characteristic peaks were obtained.

[0018] Compared with the prior art, the beneficial effects of the present invention are: The enhancing reagent provided by this invention can detect various blood types with stable characteristic peaks and high reproducibility. This method can not only distinguish conventional ABO blood types, but also sensitively identify the presence of anti-A1 antibodies in A2 subtype serum, providing higher resolution detection capabilities for clinical identification of difficult blood types and subtypes. Attached Figure Description

[0019] Figure 1 Raman spectra of four types of blood: A, B, O, and AB.

[0020] Figure 2 The spectra of the anti-A-SERS substrates prepared with 6 reagent ratios were used to detect the quality control samples of type A erythrocytes.

[0021] Figure 3 Raman spectra of type A Rh positive and type A Rh negative red blood cells.

[0022] Figure 4 The spectra of type A erythrocyte quality control samples were detected using a 4-ATP-anti-B-SERS probe prepared with 6 reagent groups.

[0023] Figure 5 The spectrum of the 2-ATP-anti-B-SERS probe detection system is shown. Detailed Implementation

[0024] Key improvements: Three universal and stable SERS signal generation and detection systems were constructed (critical). A. Improvements: A detection system based on unfunctionalized SERS substrates is proposed. Technical solution: Using an unfunctionalized SERS substrate without modifying any biomolecules, Au@Ag NRs are used as the SERS substrate for direct blood typing.

[0025] Detection procedure: A small amount of serum is incubated with protein L magnetic microspheres. Immunoglobulins are specifically adsorbed and then separated by a magnetic field to remove impurities. After mixing with silver nanosol, SERS spectra are collected. After preprocessing, the SERS spectral data are analyzed by partial least squares (PLS) and linear discriminant analysis (LDA) to complete the classification of blood type antibodies.

[0026] Corresponding advantages: Substrate preparation optimization: The AuAgNRs composite SERS substrate combines the excellent Raman enhancement properties 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 substrates, thus extending the substrate's service and storage time. The plasmon resonance coupling effect formed at the bimetallic interface further amplifies the Raman signal, significantly improving detection sensitivity. At the same time, the rich surface electronic structure optimizes the adsorption state of analyte molecules, resulting in better uniformity and reproducibility of the detection signal, better meeting the requirements of quantitative detection. This composite substrate has lower biotoxicity and is suitable for the detection of various complex biological samples such as serum and body fluids.

[0027] Significantly enhanced specificity: By replacing the broad-spectrum adsorption material with protein L, it only enriches antibodies and hardly adsorbs albumin, eliminating the main interference from serum at the source and greatly improving the specificity and reproducibility of SERS signals.

[0028] The elution system is mild and efficient: optimized pH 2.5 glycine elution + rapid Tris neutralization, and controlled elution time of 45 min, which ensures high recovery rate while avoiding antibody acid denaturation and improving detection stability.

[0029] The SERS signal quality has been significantly improved: after enrichment and purification, the antibody concentration is higher, the background is lower, and the characteristic peaks are more obvious, solving the problems of weak SERS peaks, heavy interference, and unreliable modeling in direct detection of serum.

[0030] The analysis process is simpler and faster: no labeling, large instruments, or complex chromatographic separation are required throughout the process. Only a small amount of serum can be detected, and the entire process can be completed within 30 minutes, making it suitable for rapid typing.

[0031] It enables more precise blood type and subtype determination: This method can not only distinguish between conventional ABO blood types, but also sensitively identify the presence of anti-A1 antibodies in A2 subtype serum, providing higher resolution detection capabilities for the identification of difficult blood types and subtypes in clinical practice.

[0032] B. Improvements: Propose a detection system based on a functionalized SERS substrate. Technical solution: Based on the above solution, we will firmly couple blood type antigen-specific recognition elements (such as antibodies and aptamers) to the surface of SERS active nanoparticles (such as gold nanoparticles and silver nanoparticles) through chemical bonds, and use them directly for blood type detection.

[0033] More specific: Relying on the specific binding of recognition elements such as antibodies and aptamers, it can accurately target the target blood type antigen, greatly reduce the interference of impurities and non-target substances in the sample, and effectively avoid false positive and false negative results.

[0034] Higher sensitivity: The recognition element can enrich the target antigen, increase the local concentration of the analyte on the substrate surface, amplify the SERS signal, and is more suitable for the detection of low-concentration samples. Its detection capability is superior to that of non-functionalized substrates.

[0035] Enhanced resistance to matrix interference: Biological samples have complex compositions. Functionalized substrates can reduce non-specific adsorption, resulting in better detection stability in real samples such as serum and whole blood.

[0036] Better repeatability and stability: The chemical bonding method fixes the recognition element, making the structure less prone to detachment, resulting in smaller deviations in test results between batches and parallel samples, and higher method reliability.

[0037] Wider range of applications: Corresponding recognition elements can be customized for different blood type antigens, which facilitates the simultaneous detection and differentiation of multiple samples and multiple blood types, thus expanding the application scope of the method.

[0038] C. Improvements: A direct detection system using "pre-assembled functionalized SERS probes" is proposed. Technical Solution: Based on the above solution, we have made further improvements. We pre-couple blood group antigen-specific recognition elements (such as antibodies and aptamers) to the surface of SERS active nanoparticles (such as gold nanoparticles and silver nanoparticles) through chemical bonds, and at the same time, we also immobilize Raman reporter molecules on the nanoparticles to form a stable "three-in-one" composite probe.

[0039] Detection procedure: Mix this pre-assembled probe with a red blood cell sample. Subsequent processing can employ two main methods: Separation and Enrichment Mode: The probe complex specifically bound to the target erythrocytes is separated and enriched by centrifugation, magnetic separation (if using magnetic nanoparticles), or by transferring the reaction system to a solid substrate and washing, followed by SERS detection. Direct Liquid Chromatography Detection Mode: In an optimized buffer system, no complex separation is required after the reaction; SERS detection is performed directly in the liquid phase, utilizing spectral differences caused by changes in probe aggregation state or microenvironment before and after binding.

[0040] Corresponding advantages: Revolutionary improvement in signal stability: The reporter molecule is pre- and firmly anchored near the "hot spot" of the nanoparticle, where the enhancement environment is constant and optimized. Regardless of whether an antigen-antibody binding event occurs, the reporter molecule itself can generate a strong and stable SERS signal ("signal on" mode) or will change the signal intensity through binding events ("signal enhancement / decrease" mode). This completely solves the problem of random signal fluctuations.

[0041] Extremely low background interference: The pre-assembled probe itself is pure and homogeneous. Free probes not bound to red blood cells can be easily removed from the solution through efficient separation and enrichment steps (such as magnetic separation), thereby minimizing nonspecific adsorption background.

[0042] Suitable for multiple detection modes: This probe can be used for both homogeneous liquid phase detection and solid phase chip detection (the probe is pre-spotted on the chip, and after incubation with the sample, it is cleaned), providing flexibility for different application scenarios.

[0043] Precise control of reaction conditions: The three-dimensional parameter space of probe to red blood cell ratio (1:50000 to 50000:1), incubation time (0-30 minutes), and temperature (25-37℃) was optimized.

[0044] 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.

[0045] Example 1: ABO blood group identification based on "unfunctionalized SERS matrix" (System A) 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 6 min, avoiding light throughout the process. Cool the resulting Ag NPs stock solution for later use.

[0046] 2. Mix 0.25 mL of 10 mM AgNO3, 0.5 mL of 100 mM ascorbic acid, and 10 mL of 80 mM CTAB.

[0047] 3. Add Ag NPs stock solution to the above mixture, shake gently, and let stand to react to obtain Ag NPs sol.

[0048] 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.

[0049] 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.

[0050] 6. Disperse Au@Ag NRs in 10 mL of ultrapure water, centrifuge at 8000 rpm for 15 min, and discard the supernatant.

[0051] 7. Resuspend in 0.1 mM PVP solution, sonicate for 5 min, and store at 4°C for later use.

[0052] 8. 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. 9. Take 50 μL of serum from blood types A, B, O and AB respectively, dilute with 200 μL of PBS, add 50 μg of Pierce Protein L magnetic beads (Thermo Scientific 88849), vortex to mix, and incubate at room temperature for 20 min at 25°C.

[0053] 10. Perform magnetic separation for 1 min, discard the supernatant, and wash twice with PBS.

[0054] 11. Add 50 μL of glycine-HCl (pH 2.5) to elute for 5 min (500 rpm), and immediately add 5 μL of 1M Tris to neutralize.

[0055] 12. Mix with AuAgNRs.

[0056] 13. Transfer the mixture to a 96-well plate, set the 785nm Raman spectrometer parameters to 0-500mW, and start the detection.

[0057] 14. Data preprocessing: Remove fluorescence baseline and normalize the full spectrum to eliminate systematic errors.

[0058] 15. Statistical Modeling: Partial Least Squares (PLS) dimensionality reduction is used to extract latent features. The optimal components are imported into Linear Discriminant Analysis (LDA) to construct a PLS-LDA classification model. Leave-one-out cross-validation is used to evaluate the model performance.

[0059] result: 260 Raman spectra of serum identified as type A blood, 256 for type B blood, 561 for type O blood, and 120 for type AB blood were obtained for reference. Figure 1 .

[0060] The PLS-LDA model achieved cluster separation for four blood types, with a cross-validation classification accuracy of ≥95%.

[0061] Example 2: Rapid Detection of Rh Blood Group Based on "Functionalized SERS Base" (System B) The first step is to prepare Au@Ag NRs colloidal solution. 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 obtained Ag NPs stock solution for later use.

[0062] 2. Mix 0.25 mL of 10 mM AgNO3, 0.5 mL of 100 mM ascorbic acid, and 10 mL of 80 mM CTAB.

[0063] 3. Add Ag NPs stock solution to the above mixture, shake gently, and let stand to react to obtain Ag NPs sol.

[0064] 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.

[0065] 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.

[0066] 6. Disperse Au@Ag NRs in 10 mL of ultrapure water, centrifuge at 8000 rpm for 15 min, and discard the supernatant.

[0067] 7. Resuspend in 0.1 mM PVP solution, sonicate for 5 min, and store at 4°C for later use.

[0068] The second step is the conjugation of antibodies with AgNPs. 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.

[0069] 2. Slowly add 100 μL of 1% glutaraldehyde and stir at room temperature in the dark for 2 hours.

[0070] 3.10,000g×15min, centrifuge at 4℃, wash twice, and resuspend in 0.05M PBS (pH7.2) to 10ml.

[0071] Slowly add 0.1-1 mg / mL of anti-A / anti-A1 / anti-B / anti-D / anti-E / anti-C monoclonal antibody. After the addition is complete, continue stirring at room temperature in the dark for 4 hours.

[0072] The concentrations of various antibodies are illustrated using the anti-A antibody concentrations in Table 1 as an example. The concentrations of other antibodies are also shown in Table 1. The amino acid sequences of the variable regions of the heavy chain and light chain of each antibody are shown in Table 2.

[0073] Table 1

[0074] Table 2

[0075] Step 3: Blocking + Purification (Blocking agent: ethanolamine) 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.

[0076] 2.12,000g×20min, centrifuge at 4℃, wash twice, and resuspend in 0.05M PBS (pH7.2) to 10mL.

[0077] 3. Add 0.02% sodium azide to obtain the final anti-D-SERS substrate.

[0078] 4. Aliquot into sterile brown centrifuge tubes (1.5 mL / tube) and store at 4°C protected from light.

[0079] Step 4: Base-specific verification (taking anti-D-SERS base as an example) 1. Take out the prepared anti-D-SERS substrate centrifuge tube and vortex it. Take 150uL of type A Rh positive red blood cells and type A Rh negative red blood cells respectively and mix them with 150uL of substrate. Incubate at room temperature for 20min.

[0080] Centrifuge at 3000g for 3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0081] 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.

[0082] 4. Data analysis.

[0083] result: All the anti-A-SERS substrates prepared with different reagent ratios could specifically recognize the A antigen on the surface of A1 type erythrocyte quality control samples. Among them, the anti-A-SERS substrate prepared with reagent ratio 5 showed the best effect. Figure 2 Subsequent preparations of specific probes all used the reagent ratios from group 5.

[0084] like Figure 3 Raman spectra of A-Rh positive and A-Rh negative erythrocytes on an anti-D-SERS substrate were obtained. A-Rh positive erythrocytes showed a distinct characteristic peak, while A-Rh negative erythrocytes did not. This indicates that the D antigen on the surface of A-Rh positive erythrocytes successfully and specifically binds to the anti-D-SERS substrate, producing a clear Raman signal.

[0085] Example 3: Rapid ABO Blood Group Detection Based on "Pre-assembled Functionalized SERS Probes" (System C) 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 8 min, avoiding light throughout the process. Cool the resulting Ag NPs stock solution for later use.

[0086] 2. Mix 0.25 mL of 10 mM AgNO3, 0.5 mL of 100 mM ascorbic acid, and 10 mL of 80 mM CTAB.

[0087] 3. Add Ag NPs stock solution to the above mixture, shake gently, and let stand to react to obtain Ag NPs sol.

[0088] 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.

[0089] 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.

[0090] 6. Disperse Au@Ag NRs in 10 mL of ultrapure water, centrifuge at 8000 rpm for 15 min, and discard the supernatant.

[0091] 7. Resuspend in 0.1 mM PVP solution, sonicate for min, and store at 4℃ for later use.

[0092] The second step reports the modification of Au@Ag NRs molecules. 1. Take 100 mL of the prepared Au@Ag NRs colloidal solution into a brown beaker, stir magnetically at room temperature (300 r / min), add 100 μL of 0.2-10 mmol / L reporter molecule stock solution, and continue stirring for 4 h at room temperature in the dark.

[0093] 2. Transfer the modified AgNPs colloidal solution to a centrifuge tube, centrifuge at 4°C (8000 r / min) for 20 min, and discard the supernatant.

[0094] 3. Resuspend the precipitate in 10 mL of 0.01 mol / L PBS (pH=7.4), centrifuge and wash twice, resuspend to 10 mL, and set aside.

[0095] Table 3

[0096] Step 3: Antibody-directed conjugation 1. Add 10 mL of 2 mmol / L EDC solution to each of the reporter molecule-modified Au@Ag NRs colloidal solutions, gently invert and mix, and stir at room temperature in the dark for 4 h.

[0097] 2. After activation, centrifuge at 6000 rpm and 4℃ for 8 min, and discard the supernatant.

[0098] 3. Resuspend the precipitate in 10 mL PBS buffer (pH=7.4) and sonicate for 3 min.

[0099] 4. In a sterile laminar flow hood, slowly add 1 mL of 0.1-1 mg / mL anti-A / anti-A1 / anti-B / anti-D / anti-E / anti-C antibody solution to the activated modified AgNPs colloidal solution, and gently invert to mix, avoiding vigorous stirring to prevent damage to antibody activity.

[0100] Incubate at 5.4℃ for 12 hours, then turn on the magnetic stirrer at 150 rpm.

[0101] Step 4: Blocking + Purification (Blocking agent: BSA) 1. Add 2 mL of 5% BSA to the coupled system, stir at 4°C in the dark for 4 h to block non-specific sites.

[0102] 2. Aliquot the system into centrifuge tubes and centrifuge at 4°C using a gradient centrifugation method. First, centrifuge at 8000 rpm for 10 min and discard the precipitate to remove a small amount of aggregated substrate. Then centrifuge at 12000 rpm for 20 min and discard the supernatant to remove free antibody, BSA, and EDC impurities.

[0103] 3. Resuspend the purified precipitate in 0.01 mol / L PBS (pH=7.4, containing 0.02% sodium azide), sonicate for 5 min (100W), and restore to 10 mL to obtain the final SERS probes containing anti-A / anti-A1 / anti-B / anti-D / anti-E / anti-C. Aliquot into sterile brown centrifuge tubes (1.5 mL / tube) and store at 4°C protected from light for later use.

[0104] Step 5: Probe specificity verification (taking a 4-ATP modified anti-B-SERS probe as an example) 1. Take out the prepared 4-ATP-anti-B-SERS probe centrifuge tube and vortex it. Take 150uL of A1 type, B type, O type red blood cell quality control and O type whole blood sample respectively and mix them with 150uL of probe. Incubate at room temperature for 20min.

[0105] Centrifuge at 3000g for 3 minutes, discard the supernatant, wash twice with PBS, and resuspend.

[0106] 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.

[0107] result: The 4-ATP-anti-B-SERS probes prepared using each reagent ratio could specifically recognize the A antigen on the surface of type A erythrocyte quality control samples. Among them, the 4-ATP-anti-B-SERS probe prepared using the fifth reagent ratio showed the best effect. Figure 4 Subsequent preparations of specific probes all used the reagent ratios from group 5.

[0108] Raman spectra of A1, B, and O type erythrocyte quality controls and O type whole blood samples containing the 4-ATP-anti-B-SERS probe were obtained. The B type erythrocyte quality control showed a distinct characteristic peak, while the A1, O type erythrocyte quality controls and O type whole blood samples did not. This indicates that the B antigen on the surface of B type erythrocytes successfully and specifically binds to the 4-ATP-anti-B-SERS probe, the probe is successfully captured, and the reporter molecule produces a distinct Raman signal, such as... Figure 5 .

[0109] 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. A method for preparing an enhancing agent, the method comprising: 1) Add hydroxylamine hydrochloride solution to boiling AgNO3 water, and add NaOH dropwise while heating and stirring until the solution turns yellow. Then heat again, and keep the process dark to obtain Ag NPs stock solution. 2) Mix AgNO3, ascorbic acid, and CTAB to obtain a mixture; 3) Mix the Ag NPs stock solution with the mixed solution to obtain Ag NPs sol; 4) Heat the Ag NPs sol, add hydroxylamine hydrochloride and HAuCl4 in sequence, centrifuge, discard the supernatant, wash and resuspend to obtain Au@Ag NRs; 5) Disperse Au@Ag NRs in water, centrifuge, discard the supernatant, resuspend, and sonicate to obtain the enhancing reagent.

2. The method according to claim 1, characterized in that, Also includes: 6) Enhancer conjugation with antibody: The enhancer is mixed with the antibody corresponding to the blood group antigen; 7) Blocking and purification.

3. The method according to claim 2, characterized in that: The antibody is selected from any one of anti-A, anti-A1, anti-B, anti-D, anti-E, and anti-C.

4. The method according to claim 2, characterized in that: Step 6) specifically involves: slowly adding glutaraldehyde or mercapto polyethylene glycol to the enhancing reagent, stirring, centrifuging and washing, resuspending, slowly adding antibody, and stirring in the dark.

5. The method according to claim 2, 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.

6. The method according to claim 2, characterized in that: The purification method is as follows: centrifuge and wash the incubated enhancing reagent, resuspend it, and add sodium azide.

7. The method according to claim 1, characterized in that: The method also includes: 6) Modification of Au@Ag NRs using reporter molecules: Add reporter molecules to the colloidal solution of Au@Ag NRs, stir and protect from light; 7) With conjugated antibodies; 8) Blocking and purification.

8. The enhancing reagent prepared by the method according to any one of claims 1-7.

9. The application of the enhancing reagent as described in claim 8 in blood typing.

10. A method for identifying blood type, the method comprising: 1) Add protein L magnetic beads to the blood sample, mix well, and incubate; 2) Magnetic separation, discard the supernatant, add glycine-HCl to elute, pH 2.5, elution time less than 45 min, add Tris to neutralize; 3) Mix with the above-mentioned enhancing reagents; 4) Using a Raman spectrometer, the excitation wavelength was 785 nm, and characteristic peaks were obtained.

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