Surface enhanced raman scattering detection kit for detecting acute myocardial infarction protein marker and preparation method and application thereof

Abstract

The application discloses a surface enhanced Raman scattering detection kit for detecting acute myocardial infarction protein markers and a preparation method and application thereof. The kit comprises a SERS detection chip, a first reagent, a second reagent and a third reagent. The SERS detection chip is a silver nanorod array substrate which is decorated with a hairpin type DNA single strand H1, the first reagent is Aptamer and Primer hybridization to form a recognition double-stranded Apt-P, the second reagent is a hairpin type DNA single strand H2, and the third reagent is a SERS probe. Through mutual cooperation of the SERS detection chip, the first reagent, the second reagent and the third reagent, Raman signals of the SERS probe on the SERS detection chip are detected, rapid and high-sensitivity detection of the acute myocardial infarction protein markers can be realized, and the existing antibody recognition-based immunodetection technology is overcome in terms of detection sensitivity, timeliness, portable operability and the like.

Description

Technical Field

[0001] This invention belongs to the fields of functional nanomaterials and biodetection, specifically relating to a surface-enhanced Raman scattering detection kit for detecting protein biomarkers of acute myocardial infarction, its preparation method, and its application. Background Technology

[0002] Acute myocardial infarction (AMI) is currently diagnosed primarily by detecting myocardial biomarkers such as cardiac troponin I (cTnI), creatine kinase isoenzyme (CK-MB), and myoglobin (Myo). Compared to other myocardial biomarkers, cTnI is more specific. Therefore, quantitative analysis of cTnI holds promise for effective early diagnosis of AMI. However, due to the delayed increase in its expression level in the blood, cTnI detection suffers from limitations in sensitivity and accuracy, necessitating the development of highly sensitive and selective cTnI detection methods. Many studies have employed techniques such as ELISA, chemiluminescence, fluorescence, and electrochemical methods for cTnI detection, but these methods suffer from low sensitivity and long detection times. In contrast, surface-enhanced Raman scattering (SERS) technology, due to its high sensitivity and rapid detection capabilities, has been widely used for the qualitative and quantitative analysis of trace molecules. Therefore, developing a highly sensitive and selective cTnI detection kit based on SERS technology offers unique technological advantages for effective early diagnosis of AMI.

[0003] In recent years, many SERS immunoassay strategies based on antibody recognition of cTnI have been reported. However, the protein properties of antibodies limit their production due to high costs, poor stability, lengthy production steps, and long incubation times. In contrast, aptamers, as single-stranded DNA, exhibit advantages such as better stability, stronger species recognition, easier storage, and more convenient synthesis. Therefore, using aptamer technology to recognize cTnI protein can provide an efficient, rapid, and reliable tool for the diagnosis of acute myocardial infarction (AMI). Catalytic hairpin assembly (CHA), as a nucleic acid-mediated signal amplification technique, features enzyme-free, isothermal reaction, and high signal amplification efficiency. Under target triggering, two hairpin DNAs hybridize and assemble to form a double-stranded structure, leading to target cycling and a hundredfold catalytic amplification. This provides a signal amplification strategy for constructing a highly sensitive surface-enhanced Raman scattering (SERS) detection kit for cardiac troponin (cTnI).

[0004] To address the shortcomings of antibody-based immunoassay techniques for recognizing cTnI, there is a need to develop a surface-enhanced Raman scattering (SERS) assay kit for detecting protein biomarkers in acute myocardial infarction. This kit should be simple to prepare and apply, and offer rapid detection with high sensitivity and specificity. Summary of the Invention

[0005] Purpose of the invention: In response to the significant need for effective early diagnosis of acute myocardial infarction (AMI) and the shortcomings of antibody-based immunoassay techniques in terms of detection sensitivity, timeliness, and portability, this invention discloses a surface-enhanced Raman scattering assay kit for detecting protein biomarkers of acute myocardial infarction, its preparation method, and its application.

[0006] This surface-enhanced Raman scattering (SERS) detection kit includes: a SERS detection chip, a first reagent, a second reagent, and a third reagent. The SERS detection chip is prepared by modifying a silver nanorod array substrate with a hairpin-type DNA single-stranded H1. The first reagent is a recognition double-stranded Apt-P formed by hybridization of Aptamer and Primer. The second reagent is a hairpin-type DNA single-stranded H2. The third reagent is a SERS probe, i.e., gold nanoparticles modified with a probe single strand and the Raman molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0007] The surface-enhanced Raman scattering (SERS) assay kit for detecting protein biomarkers of acute myocardial infarction disclosed in this invention is simple to prepare, rapid (60 minutes), highly sensitive (detection limit as low as 10 pg / mL), and highly specific (able to identify different protein biomarkers). By modifying the base sequence of the nucleic acid probe, it can detect biomarkers for various disease types, including acute myocardial infarction, providing a universal technology for the detection of disease-related biomarkers. It has significant advantages over existing immunoassay techniques and has outstanding application advantages in areas such as the early diagnosis of acute myocardial infarction.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A surface-enhanced Raman scattering (SERS) assay kit for detecting protein biomarkers in acute myocardial infarction includes: a SERS assay chip, a first reagent, a second reagent, and a third reagent.

[0010] The SERS detection chip was prepared by modifying the surface of a silver nanorod array substrate with hairpin-type DNA single-stranded H1;

[0011] The first reagent is the recognition double-stranded Apt-P formed by hybridization of Aptamer and Primer;

[0012] The second reagent is hairpin-type single-stranded DNA H2;

[0013] The third reagent is a SERS probe, namely gold nanoparticles with surface-modified Probe single chains and Raman molecules 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0014] The silver nanorod array was prepared using vacuum electron beam evaporation deposition technology, and a 3×10 array of micropores was formed on its surface using a polydimethylsiloxane (PDMS) film. Each micropore has a diameter of 3–5 mm and a depth of 0.8–1.2 mm. Preferably, the micropores have a diameter of 4 mm and a depth of 1 mm.

[0015] The gold nanoparticles (AuNP) have a particle size of 15–100 nm. Preferably, the gold nanoparticles have a particle size of 15 nm.

[0016] The hairpin DNA single-stranded H1 base sequence is shown in SEQ ID NO: 1, the Aptamer base sequence forming the double-stranded Apt-P is shown in SEQ ID NO: 2, the Primer base sequence is shown in SEQ ID NO: 3, the hairpin DNA single-stranded H2 base sequence is shown in SEQ ID NO: 4, and the Probe single-strand base sequence is shown in SEQ ID NO: 5.

[0017] Hairpin DNA single-stranded H1 (SEQ ID NO: 1):

[0018] 5'-AAT GCG CAA CCC TTT CTC CGC ATT CCA ATA GAG AAA GGG TTG TTT TTTTT-(CH2)6-SH-3'

[0019] Aptamer single chain (SEQ ID NO: 2):

[0020] 5'-CGT GCAGTACGC CAACCT TTC TCATGC GCT GCC CCT CTTA-3'

[0021] Primer single chain (SEQ ID NO: 3):

[0022] 5'-GAGAAA GGG TTG CGCATT-3'

[0023] Hairpin DNA single-stranded H2 (SEQ ID NO: 4):

[0024] 5'-TTT CTC TAT TGG AAT GCG GAG AAA GGG TGT CGC ATT CCA ATA ATG CCTCTAACC-3'

[0025] Probe single-stranded chain (SEQ ID NO: 5):

[0026] 5'-SH-(CH2)6-TTT TTT TTG GTTAGAGGCAT-3'.

[0027] The molar concentration ratio of Aptamer to Primer in forming the first reagent is 1:0.8 to 2, and preferably, the molar concentration ratio of Aptamer to Primer is 1:1.

[0028] The working concentration of the first reagent is 5–20 μM, and preferably, the concentration of the first reagent is 10 μM.

[0029] The working concentration of the second reagent is 5–20 μM, and preferably, the concentration of the second reagent is 10 μM.

[0030] The concentration of the third reagent is 1–10 nM, preferably 2.3 nM. Specifically, in the preparation of the third reagent, the amount of gold nanoparticles (AuNP) is 500 μL (2.3 nM), the amount of probe single chain is 10 μL (50 μM), and the amount of Raman molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is 10 μL (100 μM). After centrifugation and purification, the volume is adjusted to 50 μL to obtain the reagent.

[0031] The preparation method of the above-mentioned surface-enhanced Raman scattering detection kit for detecting protein biomarkers in acute myocardial infarction includes the following steps:

[0032] 1) Fabrication of SERS detection chip:

[0033] (1) Prepare a silver nanorod array substrate and rinse it multiple times with ultrapure water;

[0034] (2) Anneal the hairpin DNA single strand H1, wherein the annealing process is to heat at 95°C for 5 to 10 minutes and then cool it to 25 to 37°C in an ice water bath.

[0035] (3) Co-culture 10-20 μL of 500-2000 nM hairpin DNA single strand H1 with the sequence shown in SEQ ID NO: 1 with a silver nanorod array substrate. Culture conditions: 25-37℃, 60-80% humidity for 3-5 hours.

[0036] (4) After cleaning the substrate with reaction buffer, add 10-20 μL of 0.1-1 mM 6-mercaptohexanol (MCH) to the surface of the substrate and react in a constant temperature mixer at 25-37°C for 10 min.

[0037] (5) The substrate was washed repeatedly with reaction buffer and ultrapure water to obtain the SERS detection chip;

[0038] 2) Preparation of the first reagent: The Aptamer single chain with the sequence shown in SEQ ID NO: 2 and the Primer single chain with the sequence shown in SEQ ID NO: 3 are mixed at a concentration ratio of 1:0.8 to 2 and annealed at 90 to 95°C for 5 to 10 minutes to obtain the reagent.

[0039] 3) Preparation of the second reagent: Based on the hairpin DNA single-stranded H1, a hairpin DNA single-stranded H2 with the sequence shown in SEQ ID NO.4 was designed and synthesized;

[0040] 4) Preparation of the third reagent:

[0041] (1) Mix 1-10 μL of 10-100 μM Probe single strand with 100-500 μL of 1-10 nM AuNP solution in 0.5×TBE solution and incubate overnight at 200-400 rpm at 25-37℃;

[0042] (2) Add 5, 10, 15 and 20 μL of 1-3M NaCl solution slowly in 4 portions every 30 minutes to form a mixture. The final concentration of NaCl is 100-300 mM. Incubate overnight at 200-400 rpm at 25-37°C.

[0043] (3) Add 10-100 μL of 10-100 μM Raman molecule DTNB and react for 2-4 hours;

[0044] (4) Finally, remove the supernatant by centrifugation, disperse the centrifuged sediment with 0.5×TBE solution and make up to 10-100 μL to obtain the final product.

[0045] In steps 1)-3), the working concentration of the first reagent is 5-20 μM, the working concentration of the second reagent is 5-20 μM, and the working concentration of the third reagent is 1-10 nM.

[0046] The application of the surface-enhanced Raman scattering assay kit for detecting protein markers in acute myocardial infarction is as follows (taking the detection of cardiac troponin I (cTnI), a protein marker for acute myocardial infarction, for non-disease diagnostic purposes as an example):

[0047] 1) Mix the first, second, and third reagents with sample solutions containing different concentrations of target cardiac troponin (cTnI) and add them to the surface of the SERS detection chip for co-culture; the concentration range of cardiac troponin (cTnI) is 10 pg / mL to 500 ng / mL;

[0048] 2) After cleaning the SERS detection chip with ultrapure water multiple times, SERS tests were performed to obtain the SERS spectra and characteristic signal intensity values ​​corresponding to different concentrations of target cTnI. The working curve of the surface-enhanced Raman scattering detection kit was obtained by plotting the logarithm of the target cTnI concentration on the x-axis and the intensity value of the SERS characteristic peak on the y-axis. The detection limit of the surface-enhanced Raman scattering detection kit for cTnI was calculated based on the working curve.

[0049] 3) The sample to be tested is mixed with the first reagent, the second reagent and the third reagent and then dropped onto the surface of the SERS detection chip for co-cultivation. After washing the chip with ultrapure water multiple times, the SERS test is performed to obtain the SERS spectrum and its characteristic signal intensity value. The concentration of the target cTnI in the sample to be tested is calculated according to the working curve.

[0050] The culture conditions in steps 1) and 3) are as follows: culture at 25-37°C and 200-400 rpm for 60-80 min in a constant temperature mixer.

[0051] The detection principle of this invention (taking the detection of cardiac troponin I, a protein marker for acute myocardial infarction, as an example for non-disease diagnostic purposes):

[0052] Hairpin-type DNA single-stranded H1 is fixed on the surface of a silver nanorod array substrate by forming covalent bonds with silver through thiol groups. Then, 6-mercaptohexanol (MCH) is dropped onto the substrate surface to seal it, reducing non-specific adsorption on the substrate, thus obtaining a SERS detection chip.

[0053] When the target cTnI is present, due to the specific binding of Aptamer and the cTnI protein, the Primer recognizing the double-stranded Apt-P is released from Apt-P, triggering a catalytic hairpin assembly (CHA) reaction between the hairpin-type DNA single strands H1 and H2. Several H1-H2 double strands are formed on the surface of the SERS detection chip, accompanied by the release of the Primer, for recycling.

[0054] After adding the first, second, and third reagents simultaneously to the SERS detection chip, the single strand of the Probe on the third reagent hybridizes with the sticky ends of the H1-H2 double strand, capturing the SERS probe onto the SERS detection chip.

[0055] Finally, by testing the Raman signal of the SERS probe on the SERS detection chip, rapid, highly sensitive and specific detection of cardiac troponin I (cTnI), a protein marker of acute myocardial infarction, was achieved.

[0056] Beneficial effects: Compared with existing antibody-based immunoassay technologies, this invention has the following advantages:

[0057] This invention discloses a surface-enhanced Raman scattering (SERS) detection kit for detecting protein biomarkers of acute myocardial infarction, its preparation method, and its application. The SERS detection kit comprises a SERS detection chip, a first reagent, a second reagent, and a third reagent. The SERS detection chip is prepared by modifying a silver nanorod array substrate with a hairpin-type single-stranded DNA H1. The first reagent is a recognition double-stranded Apt-P formed by hybridization of Aptamer and Primer. The second reagent is a hairpin-type single-stranded DNA H2. The third reagent is a SERS probe, i.e., gold nanoparticles surface-modified with a probe single strand and the Raman molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0058] The surface-enhanced Raman scattering (SERS) assay kit for detecting protein biomarkers in acute myocardial infarction disclosed in this invention is simple to prepare, rapid (60 minutes), highly sensitive (detection limit as low as 10 pg / mL), and highly specific (able to identify different protein biomarkers). By modifying the base sequence of the nucleic acid probe, it can detect biomarkers for various disease types, including acute myocardial infarction, providing a universal technology for the detection of disease-related biomarkers. It has significant advantages over existing immunoassay techniques and has outstanding application advantages in areas such as the early diagnosis of acute myocardial infarction. Attached Figure Description

[0059] Figure 1 This is a schematic diagram illustrating the preparation of a surface-enhanced Raman scattering assay kit for detecting cardiac troponin I (cTnI), a protein marker used in acute myocardial infarction.

[0060] Figure 2 This is the optimization experiment of the optimal culture (detection) time for detecting cTnI using the surface-enhanced Raman scattering detection kit in Example 1;

[0061] Figure 3 This is the working curve of the surface-enhanced Raman scattering detection kit used in Example 2 for detecting different concentrations of cTnI;

[0062] Figure 4 This is a specific characterization of the cTnI detection using the surface-enhanced Raman scattering detection kit in Example 3;

[0063] Figure 5 Example 4 describes the uniformity characterization of cTnI detected by the surface-enhanced Raman scattering detection kit. Detailed Implementation

[0064] The present invention will be further described below with reference to specific embodiments, but the content of the present invention is not limited to the embodiments described.

[0065] This invention provides a surface-enhanced Raman scattering assay kit for detecting protein biomarkers of acute myocardial infarction, comprising:

[0066] 1. SERS detection chip

[0067] The SERS detection chip uses a silver nanorod array as a substrate, and hairpin-type DNA single strand H1 is modified on the surface of the substrate. The hairpin-type DNA single strand H1 is fixed on the substrate surface by forming covalent bonds with silver through thiol groups. Then, 6-mercaptohexanol (MCH) is dropped onto the substrate surface for sealing, reducing non-specific adsorption of the substrate, thereby obtaining the SERS detection chip. In this invention, the silver nanorod array is prepared by vacuum electron beam evaporation coating technology (based on the literature CYSong, JLAbell, YPHe, SHMurph, YPCui, YPZhao. Gold-modified silver nanorodarrays: growth dynamics and improved SERS properties. Journal of Materials Chemistry, 2012, 22(3): 1150-1159). and its surface is encapsulated with a polydimethylsiloxane (PDMS) film. The PDMS film includes a 3×10 array of small holes, each with a diameter of 3-5 mm and a depth of 0.8-1.2 mm.

[0068] 2. The first reagent (i.e., the recognition double-stranded Apt-P formed by hybridization of Aptamer and Primer) is obtained by mixing Aptamer single strands and Primer single strands at a molar concentration ratio of 1:0.8-2 and annealing at 90-95°C. Preferably, the molar concentration ratio of Aptamer to Primer is 1:1. The working concentration of the first reagent is 5-20 μM, and preferably, the concentration of the first reagent is 10 μM.

[0069] 3. The second reagent (i.e., hairpin DNA single-stranded H2) has a working concentration of 5-20 μM, preferably 10 μM.

[0070] 4. The third reagent (i.e., the SERS probe)

[0071] The third reagent is a SERS probe used in conjunction with the SERS detection chip. Gold nanoparticles, within a certain size and density range, can achieve the maximum SERS enhancement effect, with a particle size of 15–100 nm, preferably 15 nm. The concentration of the third reagent is 1–10 nM, preferably 2.3 nM. Specifically, in the preparation of the third reagent, the amount of gold nanoparticles (AuNP) is 500 μL (2.3 nM), the amount of probe single chain is 10 μL (50 μM), and the amount of Raman molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is 10 μL (100 μM). After centrifugation purification, the volume is adjusted to 50 μL to obtain the final reagent.

[0072] All DNA base sequence fragments used above were artificially synthesized by Sangon Biotech (Shanghai) Co., Ltd. The base sequences of hairpin DNA single-stranded H1, Aptamer, and Primer, and hairpin DNA single-stranded H2 and Probe are as follows:

[0073] Hairpin DNA single-stranded H1 (SEQ ID NO: 1):

[0074] 5'-AAT GCG CAA CCC TTT CTC CGC ATT CCA ATA GAG AAA GGG TTG TTT TTTTT-(CH2)6-SH-3'

[0075] The hairpin-type DNA single strand H1 has a -(CH2)6-SH group attached to its 3' end;

[0076] Aptamer single chain (SEQ ID NO: 2):

[0077] 5'-CGT GCAGTACGC CAACCT TTC TCATGC GCT GCC CCT CTTA-3'

[0078] Primer single chain (SEQ ID NO: 3):

[0079] 5'-GAGAAA GGG TTG CGCATT-3'

[0080] Hairpin DNA single-stranded H2 (SEQ ID NO: 4):

[0081] 5'-TTT CTC TAT TGG AAT GCG GAG AAA GGG TGT CGC ATT CCA ATA ATG CCTCTAACC-3'

[0082] Probe single-stranded chain (SEQ ID NO: 5):

[0083] 5'-SH-(CH2)6-TTT TTT TTG GTTAGAGGCAT-3'

[0084] The 5' end of the Probe single chain is connected to a -(CH2)6-SH group.

[0085] Please see Figure 1 This diagram illustrates the working principle of the surface-enhanced Raman scattering (SERS) detection kit for detecting protein biomarkers in acute myocardial infarction according to the present invention. The first, second, and third reagents are mixed with the sample to be tested and then dropped onto the surface of the SERS detection chip. If the sample contains cardiac troponin I (cTnI), the Aptamer of the first reagent specifically binds to cTnI, releasing the Primer from it. The Primer hybridizes with H1. The second reagent can compete with the Primer already hybridized to H1 for hybridization, releasing the Primer and the H1-H2 double strand. The released Primer can be recycled. The Probe single strand on the third reagent hybridizes with the sticky ends of the H1-H2 double strand, capturing the SERS probe onto the SERS detection chip. Therefore, by detecting the Raman signal of the SERS probe on the SERS detection chip, rapid, highly sensitive, and specific detection of cardiac troponin I (cTnI) is achieved.

[0086] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0087] Preparation of a surface-enhanced Raman scattering detection kit for detecting protein biomarkers in acute myocardial infarction

[0088] The surface-enhanced Raman scattering (SERS) detection kit contains: a SERS detection chip, a first reagent, a second reagent, and a third reagent.

[0089] 1. SERS detection chip

[0090] The silver nanorod array is used as a substrate, and the surface of the substrate is modified with hairpin-type single-stranded DNA H1 at a concentration of 500 nM. The silver nanorod array substrate includes 3×10 array-type micropores, each micropore having a diameter of 4 mm and a depth of 1 mm.

[0091] 2. The first reagent (i.e., the recognition double-stranded Apt-P formed by the hybridization of Aptamer and Primer)

[0092] It is obtained by mixing Aptamer single-chain and Primer single-chain in a concentration ratio of 1:1 and annealing at 95°C. The working concentration of the first reagent is 10 μM.

[0093] 3. Second reagent (i.e., hairpin-type DNA single-stranded H2)

[0094] Hairpin-type DNA single-stranded H1 was designed and synthesized into hairpin-type DNA single-stranded H2, with the working concentration of the second reagent being 10 μM.

[0095] 4. The third reagent (i.e., the SERS probe)

[0096] 500 μL of 2.3 nM gold nanoparticles were modified with 10 μL of 50 μM Probe single chain and 10 μL of 100 μM Raman molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), with the gold nanoparticles having a particle size of 15 nm.

[0097] The preparation method of the surface-enhanced Raman scattering detection kit for detecting protein biomarkers in acute myocardial infarction in this embodiment includes the following steps:

[0098] 1. Fabrication of SERS detection chip

[0099] (1) Prepare silver nanorod arrays and rinse them multiple times with ultrapure water;

[0100] (2) Anneal the hairpin DNA single strand H1, wherein the annealing process is to heat at 95°C for 5 min and then cool to 25°C in an ice water bath.

[0101] (3) Co-culture 20 μL of 500 nM hairpin DNA single strand H1 with silver nanorod array substrate. Culture conditions: 37℃, 80% humidity for 3 hours.

[0102] (4) After cleaning the substrate with reaction buffer, add 20 μL of 1 mM 6-mercaptohexanol (MCH) to the surface of the substrate and place it in a 25°C constant temperature mixer for 10 min.

[0103] (5) The substrate was washed repeatedly with reaction buffer and ultrapure water to obtain the SERS detection chip;

[0104] 2. Preparation of the first reagent

[0105] Aptamer single chain and Primer single chain were mixed at a concentration ratio of 1:1 and annealed at 95°C for 5 min to obtain the working concentration of the first reagent, which was 10 μM.

[0106] 2. Preparation of the second reagent

[0107] The hairpin-shaped DNA single-stranded H2, designed and synthesized based on hairpin-shaped DNA single-stranded H1, is the second reagent. The working concentration of the second reagent is 10 μM.

[0108] 3. Preparation of the third reagent

[0109] (1) Mix 10 μL of 50 μM Probe single strand with 500 μL of 2.3 nM AuNP solution in 0.5 × TBE solution and incubate overnight at 37 °C and 300 rpm.

[0110] (2) Add 5, 10, 15 and 20 μL of 2M NaCl solution slowly in 4 portions every 30 minutes to form a mixture. The final concentration of NaCl is 200 mM. Incubate overnight at 37°C and 300 rpm.

[0111] (3) Add 10 μL of 100 μM Raman molecule DTNB and react for 3 hours;

[0112] (4) Finally, the supernatant was removed by centrifugation, the centrifuged sediment was dispersed with 0.5×TBE solution and the volume was adjusted to 50μL to obtain the final product.

[0113] Example 1: Optimization Experiment of Optimal Culture (Detection) Time for Detection of cTnI Using Surface Enhanced Raman Scattering Detection Kit

[0114] 8 μL of 10 μM reagent I, 2 μL of 10 μM reagent II, 5 μL of reagent III, and 4 μL of 10 ng / mL cTnI were mixed in 20 μL of reaction buffer and added dropwise to the surface of the SERS detection chip. The mixture was then incubated at 37℃ and 300 rpm for 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 min, respectively. Afterward, the wells were washed with reaction buffer and ultrapure water, and SERS detection was performed. The SERS spectrum was obtained (see [reference needed]). Figure 2 As can be seen, by detecting the SERS signal of the chip after different time periods of cultivation from 10 to 100 min, it was observed that the SERS intensity gradually increased within 10 to 60 min, and the SERS signal reached the maximum saturation value at 60 min of co-cultivation, indicating that 60 min is the optimal cTnI detection time.

[0115] Example 2: Working curves of surface-enhanced Raman scattering detection kit for detecting different concentrations of cTnI

[0116] 8 μL of 10 μM reagent 1, 2 μL of 10 μM reagent 2, 5 μL of reagent 3, and 4 μL of cTnI at different concentrations (10 pg / mL to 500 ng / mL) were mixed in 20 μL of reaction buffer and added to the surface of the SERS detection chip. After incubation at 37℃ and 300 rpm for 60 min, the wells were washed repeatedly with reaction buffer and ultrapure water. After air drying, SERS testing was performed on the SERS detection chip (Raman testing conditions: scan time 1 s, laser power 1%, objective magnification 20×, cumulative number of times 1, excitation wavelength 785 nm). The SERS spectrum and its characteristic signal intensity values ​​were obtained. A working curve was plotted with the logarithm of the target cTnI concentration on the x-axis and the characteristic peak intensity value of the SERS probe on the y-axis. The detection limit of the surface-enhanced Raman scattering detection kit for cTnI was calculated based on the working curve. (Refer to...) Figure 3 As shown, (a) is the SERS spectrum obtained by detecting different concentrations of cTnI, and (b) is the spectrum of each spectral line at 1331 cm⁻¹. -1 The SERS peak intensity corresponding to the location. For detecting cTnI, the working curve is I. 1331 =1689×lg C T +6664(R 2 =0.998), and the detection limit was calculated to be 3.5 pg / mL.

[0117] Example 3: Specific characterization of cTnI detection using a surface-enhanced Raman scattering detection kit

[0118] The target cTnI was diluted to 2.5 ng / mL, and the bovine serum albumin (BSA), brain natriuretic peptide (BNP), cardiac troponin C (cTnC), and cardiac troponin T (cTnT) samples were diluted to 20 ng / mL. A reaction buffer without any added biomolecules was used as a blank control. 8 μL of 10 μM reagent I, 2 μL of 10 μM reagent II, and 5 μL of reagent III were mixed with 4 μL of 2.5 ng / mL cTnI, 4 μL of 20 ng / mL BSA, BNP, cTnC, and cTnT protein samples, and a blank sample (reaction buffer), respectively. The mixture was then added dropwise to the surface of the SERS detection chip. After incubation at 37°C and 300 rpm for 60 min, the wells were washed sequentially with reaction buffer and ultrapure water. After air drying, SERS measurements were performed on the SERS detection chip to obtain the SERS spectrum and its characteristic peak intensities. (Reference) Figure 4 As shown, (a) is the SERS spectrum of different protein samples, and (b) is the spectrum of their spectral lines at 1331 cm⁻¹. -1The SERS peak intensity corresponding to the location. The prepared surface-enhanced Raman scattering (SERS) detection kit can distinguish the target cTnI from other protein samples well, indicating that the SERS detection kit has good specificity.

[0119] Example 4: Uniformity characterization of cTnI detection using a surface-enhanced Raman scattering detection kit

[0120] 8 μL of 10 μM reagent I, 2 μL of 10 μM reagent II, 5 μL of reagent III, and 4 μL of 10 ng / mL cTnI were mixed in 20 μL of reaction buffer and added to the surface of the SERS detection chip. The mixture was then incubated at 37°C and 300 rpm for 60 min, followed by rinsing with reaction buffer and ultrapure water. The SERS signals at 15 random points on the SERS detection chip were recorded to study the uniformity of the silver nanorod array substrate modified with hairpin-type DNA single-stranded H1. (Reference) Figure 5 As shown, (a) is the SERS spectrum of 10 ng / mL cTnI detected at 15 random points, and (b) is the spectrum of its spectral line at 1331 cm⁻¹. -1 The SERS peak intensity corresponding to the location. The relative standard deviation (RSD) of the SERS peak intensity corresponding to 15 random points for cTnI detection is very small (RSD = 6.14%), which indicates that the proposed surface-enhanced Raman scattering detection kit has good uniformity in detecting cTnI.

Claims

1. A surface enhanced Raman scattering detection kit for the detection of the acute myocardial infarction protein marker cardiac troponin cTn characterized in that: The surface-enhanced Raman scattering (SERS) detection kit comprises a SERS detection chip, a first reagent, a second reagent, and a third reagent; The SERS detection chip is a silver nanorod array substrate with a hairpin-type DNA single-stranded H1 sequence as shown in SEQ ID NO: 1 on its surface; The first reagent is a recognition double-stranded Apt-P formed by hybridization of Aptamer single strand with sequence as shown in SEQ ID NO: 2 and Primer single strand with sequence as shown in SEQ ID NO: 3; The second reagent is a hairpin-type single-stranded DNA H2 with the sequence shown in SEQ ID NO: 4; The third reagent is a SERS probe used in conjunction with a silver nanorod array substrate; the SERS probe is a gold nanoparticle with a Probe single chain as shown in SEQ ID NO: 5 and a Raman molecule 5,5'-dithiobis(2-nitrobenzoic acid) on its surface.

2. The surface-enhanced Raman scattering detection kit for detecting cardiac troponin cTn, a protein marker in acute myocardial infarction, according to claim 1, is characterized in that: The first reagent is obtained by mixing Aptamer single strand with sequence as shown in SEQ ID NO: 2 and Primer single strand with sequence as shown in SEQ ID NO: 3 and annealing at 90~95 °C; the concentration ratio of Aptamer to Primer is 1:0.8~2; In the third reagent, the concentration ratio of the Probe single chain to DTNB is 1:1~3.

3. The surface-enhanced Raman scattering detection kit for detecting cardiac troponin cTn, a protein biomarker for acute myocardial infarction, according to claim 1, is characterized in that: The hairpin-type DNA single strand H1 has a -(CH2)6-SH group attached to its 3' end; the Probe single strand has a -(CH2)6-SH group attached to its 5' end.

4. The surface-enhanced Raman scattering detection kit for detecting cardiac troponin cTn, a protein marker for acute myocardial infarction, according to claim 1, is characterized in that: The silver nanorod array substrate was prepared using vacuum electron beam evaporation coating technology. It includes a 3×10 array of small holes, each with a diameter of 3~5 mm and a depth of 0.8~1.2 mm. The gold nanoparticles have a particle size of 15~100 nm; The working concentration of the first reagent is 5~20 μM, the working concentration of the second reagent is 5~20 μM, and the working concentration of the third reagent is 1~10 nM.

5. The preparation method of the surface-enhanced Raman scattering detection kit for detecting cardiac troponin cTn, a protein marker of acute myocardial infarction, as described in claim 1 or 2, characterized in that: Includes the following steps: (I) Fabrication of SERS detection chip: (1) Prepare silver nanorod array substrate and rinse repeatedly with ultrapure water; (2) Anneal the hairpin DNA single strand H1, wherein the annealing process is to heat at 90~95 ℃ for 5~10 min and then cool it down to 25~37 ℃ in an ice water bath; (3) Co-culture 10-20 μL of hairpin DNA single strand H1 with a sequence of 500-2000 nM as shown in SEQ ID NO: 1 with a silver nanorod array substrate. Culture conditions: 25-37 ℃, 60-80% humidity for 3-5 hours. (4) After cleaning the substrate with reaction buffer, add 10~20 μL of 0.1~1 mM 6-mercaptohexanol (MCH) to the surface of the substrate and place it in a constant temperature mixer at 25~37 ℃ for reaction; (5) The substrate was washed repeatedly with reaction buffer and ultrapure water to obtain the SERS detection chip; (II) Preparation of the first reagent: The Aptamer single strand with the sequence shown in SEQ ID NO: 2 and the Primer single strand with the sequence shown in SEQ ID NO: 3 are mixed at a concentration ratio of 1:0.8~2 and annealed at 90~95 °C for 5~10 min to obtain the product. (III) Preparation of the second reagent: Based on the hairpin DNA single-stranded H1, a hairpin DNA single-stranded H2 with the sequence shown in SEQ ID NO. 4 was designed and synthesized. (iv) Preparation of the third reagent: (1) Mix 1~10 μL of 10~100 μM Probe single strand with 100~500 μL of 1~10 nM AuNP solution in 0.5×TBE solution and incubate overnight at 200~400 rpm at 25~37 ℃; (2) Add 5, 10, 15 and 20 μL of 1~3 M NaCl solution slowly in 4 portions every 30 minutes to form a mixture. The final concentration of NaCl is 100~300 mM. Incubate overnight at 200~400 rpm at 25~37 ℃. (3) Add 10~100 μL of 10~100 μM Raman molecule DTNB and react for 2~4 hours; (4) Finally, remove the supernatant by centrifugation, disperse the centrifuged sediment with 0.5×TBE solution and make up to 10~100 mL to obtain the final product.

6. The method for preparing the surface-enhanced Raman scattering detection kit for detecting cardiac troponin cTn, a protein marker in acute myocardial infarction, according to claim 5, is characterized in that: The reaction buffer consisted of 10 mM tris-HCl and 1 mM MgCl2, pH 8.

0.

7. The application of the surface-enhanced Raman scattering assay kit for detecting cardiac troponin cTn, a protein marker for acute myocardial infarction, as described in any one of claims 1 to 4, for non-disease diagnostic purposes.

8. The application according to claim 7, characterized in that: The steps are as follows: 1) Mix the first, second, and third reagents with sample solutions containing different concentrations of target acute myocardial infarction protein markers, and add them to the surface of the SERS detection chip for co-incubation; 2) After washing the SERS detection chip with ultrapure water multiple times, SERS testing was performed to obtain the SERS spectra and characteristic signal intensity values ​​corresponding to different concentrations of the target acute myocardial infarction protein biomarkers. The working curve of the surface-enhanced Raman scattering detection kit was obtained by plotting the logarithm of the concentration of the target acute myocardial infarction protein biomarker on the x-axis and the intensity value of the SERS characteristic peak on the y-axis. The detection limit of the surface-enhanced Raman scattering detection kit for detecting acute myocardial infarction protein biomarkers was calculated based on the working curve. 3) After mixing the sample to be tested with the first, second and third reagents, the mixture is dropped onto the surface of the SERS detection chip for co-culture. The chip is then washed with ultrapure water multiple times before SERS testing is performed to obtain the SERS spectrum and its characteristic signal intensity values. The concentration of the target acute myocardial infarction protein marker in the sample to be tested is calculated based on the working curve.

9. The application according to claim 8, characterized in that: The culture conditions for steps 1) and 3) are as follows: culture in a constant temperature mixer at 25-37℃ and 200-400 rpm for 60-80 min.

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