A bifunctional nano-enzyme SERS detection platform for ultra-sensitive detection of cerebral infarction related miRNA and a preparation method thereof

Abstract

This invention provides a bifunctional nanozyme SERS detection platform and its preparation method for ultrasensitive detection of miRNAs related to cerebral infarction, belonging to the field of biomedical engineering technology. The preparation method includes the following steps: synthesizing gold nanorods (AuNRs) using a seed growth method, and growing Pt on the surface of the product to prepare gold-platinum nanorods (Au@Pt NRs); synthesizing gold nanohexagonal plates (AuNHs) using a seed growth method, and preparing an AuNHs array through self-assembly at an oil-water interface; modifying the Au@Pt NRs obtained in step 1) with the complementary strand (H1) of the nucleic acid aptamer via Au-S bonds. This is then combined with the functionalized nucleic acid aptamer (cDNA) corresponding to miR-106a-5p in step 2) of the assembled AuNHs array to construct the SERS detection platform. The target miR-106a-5p can specifically bind to cDNA, causing the Au@Pt NRs carrying H1 to detach from the surface of the AuNHs array, reducing TMB oxidation, and significantly decreasing the SERS signal. Based on the SERS signal intensity, the linear regression equation is used to achieve quantitative detection of miRNA.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a bifunctional nanozyme SERS detection platform and preparation method for ultrasensitive detection of cerebral infarction-related miRNAs. Background Technology

[0002] Cerebral infarction (CI), commonly known as ischemic stroke, is an irreversible damage to localized neurons caused by ischemia and hypoxia in brain tissue. Globally, stroke remains the second leading cause of disability and death, with CI accounting for 62.4% of all stroke events. The occurrence of CI is closely associated with a range of risk factors, including sex, age, obesity, hypertension, and diabetes. Early and accurate diagnosis is clinically crucial for improving patient prognosis, optimizing treatment plans, and increasing survival rates. Magnetic resonance imaging (MRI) and computed tomography (CT) are currently considered the gold standard for clinical diagnosis of CI. However, even when using CT and MRI in combination, limitations such as lower sensitivity and specificity, greater invasiveness, and operational complexity hinder large-scale screening in populations. Therefore, developing a convenient and ultrasensitive miRNA detection method is essential for efficient and accurate disease detection and meeting the need for early diagnosis of CI in large populations.

[0003] Surface-enhanced Raman scattering (SERS), due to its high sensitivity, strong specificity, rapid analysis capability, and non-invasive detection characteristics, has been widely used to detect the unique spectral "fingerprints" of various molecules. SERS has demonstrated its outstanding advantages and has become a key detection method in the fields of life sciences, chemistry, and materials science. Localized surface plasmon resonance (LSPR) generates multiple "hot spots" near the surface of noble metal nanomaterials, which is the main mechanism of SERS enhancement. Studies have shown that a superior SERS-active substrate is key to achieving the SERS enhancement effect. With the advancement of research, highly ordered gold nanohexagonal plates (AuNHs), with their regular shape, high aspect ratio, high stability, sharp corners, and neatly arranged nanoscale gaps between adjacent substrates, have become one of the most effective substrates for SERS-based biosensors, significantly improving the accuracy and reliability of detection. Gold nanorods (AuNRs), due to their superior LSPR properties, are widely used in optical sensing, cancer therapy, and imaging. Individual AuNRs exhibit relatively weak SERS signals, while bimetallic nanomaterials show a stronger SERS amplification effect. Furthermore, nanomaterials composed of noble metals such as platinum, gold, and palladium have attracted widespread attention due to their superior catalytic activity, stability, selectivity, and simple preparation process, far exceeding that of natural enzymes. Therefore, gold-platinum nanorods (Au@Pt NRs) with bimetallic structures have been extensively studied due to their highly efficient LSPR, the generation of dense "hot spots," and peroxidase-like (POD) activity. This structure not only enhances the binding ability with biomolecules but also significantly improves POD activity through the LSPR effect, overcoming the limitations of low sensitivity and interference in traditional colorimetric and electrochemical methods. Therefore, combining SERS technology with the enzyme-like properties of nanomaterials and introducing a recognition-competition strategy, a convenient and ultrasensitive bifunctional nanozyme SERS detection platform was designed. This not only solves the complexity and stringent conditions of traditional SERS-based methods such as double-antibody sandwich biosensors, catalytic hairpin assembly (CHA) amplification strategies, and qRT-PCR, but also provides an innovative and promising solution for biomarker detection. The integration of nanozyme-SERS technology has opened up new research directions and application potential for biomarker detection. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a dual-function nanozyme SERS detection platform and preparation method for ultrasensitive detection of infarction-related miRNAs, thus solving the problems mentioned in the background art.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a bifunctional nanozyme SERS detection platform for ultrasensitive detection of cerebral infarction-related miRNAs, wherein the cerebral infarction-related miRNA is miR-106a-5p, and the preparation method includes the following steps:

[0008] 1) AuNRs were synthesized using a seed-mediated method, and Pt was grown on the surface of the product to prepare gold-platinum nanorods Au@Pt NRs; the specific implementation method of step 1) is as follows:

[0009] 1.1) AuNRs were synthesized by seed growth method. 200 µL of chloroauric acid was added to 20 mL of cetyltrimethylammonium bromide solution, followed by 1.2 mL of sodium borohydride solution and stirring for 120 seconds. Then, the gold seed solution was allowed to stand at room temperature for 120 minutes.

[0010] 1.2) The growth solution was prepared by mixing 4 mL of chloroauric acid, 4.4 mL of silver nitrate, 200 mL of hexadecyltrimethylammonium bromide and 1.4 mL of ascorbic acid solution;

[0011] 1.3) The growth solution prepared in step 1.2) was mixed with 2 mL of gold seed solution and refluxed for 24 hours. Finally, AuNRs were obtained by centrifugation at 10000 rpm for 12 minutes.

[0012] 1.4) The AuNRs suspension was mixed with H2PtCl6 solution, and then 0.6 mL of ascorbic acid solution was added to the mixture. The mixture was refluxed at room temperature for 180 minutes. Finally, the mixture was centrifuged at 10,000 rpm for 12 minutes to obtain gold-platinum nanorods Au@Pt NRs. The volume ratio of AuNRs to H2PtCl6 in step 1.4) was 200:1.

[0013] 2) Gold nano-hexagonal plates (AuNHs) were synthesized using a seed growth method, and AuNHs arrays were prepared via an oil-water interface self-assembly method; the specific implementation method of step 2) is as follows:

[0014] 2.1) Dissolve 0.208 g of polyvinylpyrrolidone in 6 mL of ultrapure water at 35 °C and stir until the solution becomes transparent. Then, take 3 mL of the polyvinylpyrrolidone solution and quickly add it to 60 µL of chloroauric acid solution, and stir rapidly for 3 minutes to ensure that the two are fully mixed. Next, add 96 µL of freshly prepared ascorbic acid solution to the mixed solution and continue stirring for 30 minutes until the solution turns dark purple.

[0015] 2.2) To ensure complete reaction, the mixture was allowed to stand at room temperature for 10 hours, then centrifuged at 10,000 rpm for 18 minutes and washed three times each with anhydrous ethanol and deionized water to remove excess reactants from the surface. Finally, the product was dissolved in deionized water to obtain a gold nano-hexagonal plate AuNHs solution.

[0016] 2.3) Take the gold nano-hexagonal plate AuNHs solution obtained in step 2.1) and n-hexane into a beaker, then add ethanol solution and let stand for 180 seconds. At the oil-water interface, AuNHs self-assemble to form a tightly packed nanofilm with metallic luster; the volume ratio of AuNHs, n-hexane and ethanol in step 2.3) is 1:2:1.

[0017] 2.4) Use piranha solution to completely remove organic matter from the silicon wafer;

[0018] 2.5) Transfer the AuNHs monolayer film formed at the oil-water interface in step 2.3) onto the silicon wafer treated in step 2.4) and dry it under a thermostat;

[0019] 3) Modify the Au@Pt NRs obtained in step 1) with the complementary strand H1 of the nucleic acid aptamer via Au-S bonds, and bind it to the AuNHs array assembled in step 2) for functionalization of the nucleic acid aptamer cDNA corresponding to miR-106a-5p, thus constructing a bifunctional nanozyme SERS detection platform; the specific implementation method of step 3) is as follows:

[0020] 3.1) To ligate cDNA, 400 µL of cDNA was mixed with 40 µL of TCEP solution and incubated at room temperature for 30 minutes to activate the nucleic acid aptamers;

[0021] 3.2) The cDNA activated in step 3.1) was coupled to the surface of the AuNHs array and incubated at 37°C for 120 minutes to allow the cDNA to be completely bound to the AuNHs surface. The array was repeatedly rinsed with PBS buffer and deionized water. Finally, 15 mL of 1 wt% BSA was added to repair the non-specific binding sites on the particle surface to obtain the cDNA-functionalized AuNHs array AuNHs@cDNA.

[0022] 3.3) By adding 400 μL of TCEP-activated H1 to the Au@Pt NRs prepared in step 1), incubating at 37°C for 2 hours, and finally adding 100 μL of 1wt% BSA solution to the solution to block non-specific binding sites on the particle surface, the solution was centrifuged at 10000 rpm for 10 minutes to remove excess reagents and nucleic acid chains, thus obtaining Au@Pt NRs@H1 functionalized with H1.

[0023] 3.4) The Au@Pt NRs@H1 prepared in step 3.3) was added to the surface of the cDNA-functionalized AuNHs array prepared in step 3.2), and incubated in an incubator at 37°C for 2 hours to prepare a bifunctional nanozyme SERS detection platform for detecting miR-106a-5p. The nucleotide sequence of H1 is SH-AAAAGTGCTT; the nucleotide sequence of cDNA is SH-CTACCTGCACTGTAAGCACTTTT.

[0024] A bifunctional nanozyme SERS detection platform for detecting cerebral infarction-related miRNAs is characterized in that it is prepared based on the preparation method of the bifunctional nanozyme SERS detection platform for detecting cerebral infarction-related miRNAs as described above.

[0025] (III) Beneficial Effects

[0026] This invention provides a bifunctional nanozyme SERS detection platform and preparation method for ultrasensitive detection of infarction-related miRNAs, which has the following beneficial effects:

[0027] 1. This invention incorporates the highly efficient catalytic properties of nanozymes into a competitive identification strategy, demonstrating higher sensitivity and specificity in detecting CI-related miRNAs, and providing a new research approach for the early diagnosis of CI.

[0028] 2. This invention enables single-step, ultrasensitive detection of target miRNAs in patient serum, with a limit of detection (LOD) of 0.96 fM, consistent with the results of quantitative real-time polymerase chain reaction (qRT-PCR). It provides a novel method for the effective detection of miRNAs and has significant clinical value for the early detection of clinical complications (CI).

[0029] 3. This invention develops a convenient and ultrasensitive miRNA detection method, which can more efficiently and accurately detect diseases and meet the needs of early diagnosis of CI in large populations.

[0030] In summary, this study successfully developed a bifunctional nanozyme SERS detection platform for the ultrasensitive detection of CI-related miRNAs. By designing an Au@Pt NRs@H1-AuNHs@cDNA composite structure and utilizing a competitive recognition strategy, a nanozyme-functional SERS detection platform was developed, achieving rapid and accurate detection of CI-related miR-106a-5p. Furthermore, the highly efficient catalytic properties of Au@Pt NRs were incorporated into the competitive recognition strategy, effectively improving detection sensitivity. In addition, this method enables rapid detection of target miRNAs in patient serum with a LOD of 0.96 fM, consistent with qRT-PCR results. In conclusion, this bifunctional nanozyme SERS detection platform provides a novel approach for the early detection of CI. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the detection process and principle of a bifunctional nanozyme SERS detection platform; (A) is the synthesis of Au@Pt NRs, (B) is the fabrication of the AuNHs array and the construction of the detection platform, and (C) is the clinical application process of the detection platform.

[0032] Figure 2 These are transmission electron microscope (TEM) images of Au@Pt nanorods;

[0033] Figure 3 This is a magnified high-resolution transmission electron microscope (HR TEM) image of Au@Pt nanorods.

[0034] Figure 4 These are the energy dispersive X-ray spectroscopy (EDX) spectra and elemental mapping images of Au@Pt nanorods;

[0035] Figure 5 These are ultraviolet-visible (UV-vis) absorption spectra and solution color images under three different conditions;

[0036] Figure 6 It is 4-MBA (1×10 -8 Au@Pt nanorods labeled with M) and pure 4-MBA (1×10⁻⁶) -2 Surface-enhanced Raman scattering (SERS) spectral images of M);

[0037] Figure 7 These are transmission electron microscope (TEM) images of AuNHs;

[0038] Figure 8 These are magnified high-resolution transmission electron microscope (HRTEM) images of AuNHs.

[0039] Figure 9This is the ultraviolet-visible-near-infrared (UV-Vis-NIR) spectral image of AuNHs;

[0040] Figure 10 These are scanning electron microscope (SEM) images of the AuNHs array;

[0041] Figure 11 It is 4-MBA (1×10 -8 AuNHs arrays labeled with M) and 4-MBA (1×10⁻⁶) -2 Raman spectra of M);

[0042] Figure 12 It is 4-MBA (1×10 -2 SERS spectral images of six different locations randomly selected on the surface of the AuNHs array labeled with M);

[0043] Figure 13 yes Figure 12 1073cm -1 Histogram of signal strength at the location;

[0044] Figure 14 It is 4-MBA (1×10 -2 The AuNHs array labeled M stores SERS spectra at different times (0 days, 7 days, 14 days);

[0045] Figure 15 yes Figure 14 1073cm -1 A line graph showing the signal strength at a given location;

[0046] Figure 16 Au@Pt NRs catalyzes the oxidation of TMB to generate oxTMB signaling molecules at 1601 cm⁻¹. -1 A graph showing the relationship between signal strength and temperature.

[0047] Figure 17 Au@Pt NRs catalyzes the oxidation of TMB to generate oxTMB signaling molecules at 1601 cm⁻¹. -1 A graph showing the relationship between signal intensity and pH.

[0048] Figure 18 Au@Pt NRs catalyzes the oxidation of TMB to generate oxTMB signaling molecules at 1601 cm⁻¹. -1 A graph showing the relationship between signal strength and incubation time;

[0049] Figure 19 Au@Pt NRs catalyzes the oxidation of TMB to generate oxTMB signaling molecules at 1601 cm⁻¹. -1 A graph showing the relationship between signal intensity and TMB concentration at a given location;

[0050] Figure 20 Au@Pt NRs catalyzes the oxidation of TMB to generate oxTMB signaling molecules at 1601 cm⁻¹. -1 A graph showing the relationship between signal intensity and H2O2 concentration;

[0051] Figure 21 The Michaelis equation and double-inverted curve plot for Au@Pt NRs;

[0052] Figure 22 This is a diagram of the SERS detection platform for six different batches of nanozymes.

[0053] Figure 23 yes Figure 22 1601cm -1 A line graph showing the signal strength at a given location;

[0054] Figure 24 The nanozyme SERS detection platform is used to detect the SERS spectra of different miRNA sequences;

[0055] Figure 25 yes Figure 24 1601cm -1 Histogram of signal strength at the location;

[0056] Figure 26 These are SERS spectra of miR-106a-5p in serum at different concentrations;

[0057] Figure 27 yes Figure 26 1601cm -1 Calibration curves of characteristic peak intensity and logarithmic miR-106a-5p concentration;

[0058] Figures 28 to 30 These are MRI images of healthy individuals;

[0059] Figures 31 to 33 These are MRI images of a patient with CI (collapse).

[0060] Figure 34 This is the SERS spectrum of miR-106a-5p in the serum of healthy subjects and CI patients;

[0061] Figure 35 Healthy subjects and CI patients in Figure 34 1601cm -1 Histogram of signal strength at the location. Detailed Implementation

[0062] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] The instruments, equipment, and testing conditions used in this invention are as follows:

[0064] The scanning electron microscope (SEM) images were obtained using a Hitachi S-4800II field emission scanning electron microscope from Japan. The test conditions were 10.0 kV.

[0065] The transmission electron microscope (TEM) images were obtained using a Philips Tecnai 12 transmission electron microscope. The test conditions were 120 kV.

[0066] High-resolution transmission electron microscopy (HRTEM) images and selected area electron diffraction (SAED) patterns were obtained using a Tecnai G2F30 S-TWIN, 152 FEI field emission transmission electron microscope. The testing conditions were an accelerating voltage of 200 kV.

[0067] The UV-Vis spectral data were measured using a Cary 60 UV-Vis spectrophotometer manufactured by Agilent Technologies.

[0068] The Raman spectra were measured using a DXRxi miniature Raman spectrometer manufactured by Thermo Fisher Scientific.

[0069] like Figure 1 As shown, a method for preparing a bifunctional nanozyme SERS detection platform for ultrasensitive detection of infarction-related miRNAs is disclosed. This method includes the following steps:

[0070] 1) AuNRs were synthesized using a seed-mediated method, and Pt was grown on the surface of the product to prepare gold-platinum nanorods Au@Pt NRs;

[0071] 2) Gold nano-hexagonal plates (AuNHs) were synthesized using a seed growth method, and AuNHs arrays were prepared via an oil-water interface self-assembly method;

[0072] 3) Modify the complementary strand H1 of the nucleic acid aptamer to the Au@Pt NRs obtained in step 1) via Au-S bond, and bind it to the AuNHs array assembled in step 2) of the nucleic acid aptamer cDNA functionalization of miR-106a-5p to construct a bifunctional nanozyme SERS detection platform.

[0073] Example 1

[0074] Preparation and characterization of Au@Pt NRs@H1

[0075] 1) Synthesis of AuNRs via seed growth method. 200 µL (25 mM) chloroauric acid (HAuCl4) was added to 20 mL (0.1 M) hexadecyltrimethylammonium bromide (CTAB) solution, followed by the addition of 1.2 mL (0.01 M) sodium borohydride (NaBH4) solution and stirring for 120 seconds. The gold seed solution was then allowed to stand at room temperature for 120 minutes.

[0076] 2) The growth solution was prepared by mixing 4 mL (25 mM) HAuCl4, 4.4 mL (4 mM) silver nitrate (AgNO3), 200 mL (0.1 M) CTAB and 1.4 mL (0.788 mM) ascorbic acid (L-AA) solution.

[0077] 3) Mix the growth medium prepared in step 1.2) with 2 mL of gold seed solution and reflux for 24 hours. Finally, obtain AuNRs by centrifugation at 10000 rpm for 12 minutes.

[0078] 4) Mix 1 mL of AuNRs suspension with 5 µL (0.01 M) H₂PtCl₆ solution. Then add 0.6 mL (0.1 M) L-AA solution to the mixture and reflux at room temperature for 180 minutes. Finally, centrifuge at 10,000 rpm for 12 minutes to obtain Au@PtNRs.

[0079] 5) such as Figure 2 The TEM image of Au@Pt NRs shows that the morphology of Au@Pt NRs is a regular rod shape. Figure 3 This is a magnified image of a portion of Au@Pt NRs. Figure 4 Energy scattering spectroscopy (EDX) and elemental mapping confirmed that gold and platinum are the main components of Au@Pt NRs, while copper doping plays a crucial role in controlling their growth and shape. Figure 5 In the study, the Au@Pt NRs solution showed a distinct absorption peak at 725 nm. When H₂O₂ and TMB were present simultaneously, the solution became colorless. Upon simultaneous addition of Au@Pt NRs, the solution changed from colorless to blue, exhibiting a distinct absorption peak at 651 nm. Figure 6 In China, 4-MBA (1×10 -8The M-labeled Au@Pt NRs significantly enhanced the Raman peak of 4-MBA, but the Raman signal intensity of 4-MBA was extremely low, demonstrating that Au@Pt NRs have excellent SERS enhancement performance. The EF value of Au@Pt NRs was calculated to be 1.2 × 10⁻⁶ using the formula: EF = IS / IR∙CR / CS. 7 .

[0080] Example 2

[0081] Au@Pt NRs@cDNA preparation and characterization

[0082] 2.1) Dissolve 0.208 g of polyvinylpyrrolidone (PVP) in 6 mL of ultrapure water at 35 °C and stir until the solution becomes clear. Then, quickly add 3 mL of PVP solution to 60 µL (50 mM) HAuCl4 solution and stir rapidly for 3 minutes to ensure thorough mixing. Next, add 96 µL (15 mM) freshly prepared AA solution to the mixture and continue stirring for 30 minutes until the solution turns deep purple.

[0083] 2.2) To ensure complete reaction, the mixture was allowed to stand at room temperature for 10 hours. Afterward, excess reactants were removed from the surface by centrifugation at 10,000 rpm for 18 minutes and washing three times each with anhydrous ethanol and deionized water. Finally, the product was dissolved in deionized water to obtain a gold nano-hexagonal plate (AuNHs) solution.

[0084] 2.3) Take 2 mL of the AuNHs solution obtained in step 2.1) and 4 mL of n-hexane and add them to a beaker, followed by 2 mL of ethanol solution and let stand for 180 seconds. At the oil-water interface, AuNHs self-assemble to form a tightly packed nanofilm with a metallic luster.

[0085] 2.4) Use piranha solution (formed by mixing concentrated sulfuric acid and 30% hydrogen peroxide in a 7:3 ratio) to completely remove all organic matter from the silicon wafer.

[0086] 2.5) Transfer the AuNHs monolayer film formed at the oil-water interface in step 2.3) onto the silicon wafer treated in step 2.4) and dry it under a thermostat.

[0087] 2.6) Figure 7 Transmission electron microscopy images of AuNHs show that individual AuNHs have regular morphology, sharp edges, and good dispersion, which enables them to better capture miRNAs. Figure 8 This is an HRTEM image of AuNHs. Figure 9 The ultraviolet-visible-near-infrared absorption spectrum of AuNHs is shown, with a distinct absorption peak appearing near 797 nm. Figure 10The scanning electron microscope images showed uniformly arranged, regularly shaped gold nanohexagonal plates. Furthermore, Figure 11 It uses 4-MBA (1×10 -2 AuNHs arrays labeled with M) and 4-MBA (1×10⁻⁶) -8 A comparison of the Raman spectra of M) showed that its EF = 1.8 × 10⁻⁶. 8 This indicates that the AuNHs array has a significant SERS enhancement effect. Figure 12 The SERS spectra of six randomly selected locations on the surface of a 4-MBA labeled AuNHs array are shown, exhibiting high reproducibility. Figure 13 The histogram in the middle and 1073cm -1 The signal strength corresponds at different locations. The AuNHs array exhibits significant uniformity and stability, with a relative standard deviation (RSD) of 5.98%. Figure 14 and Figure 15 The SERS spectra of a 4-MBA labeled AuNHs array stored at room temperature for 0, 7, and 14 days are shown. Compared to a newly prepared array, the signal intensity on the array surface decreases, but this change gradually stabilizes over time, indicating that the array has good stability.

[0088] Example 3

[0089] Optimized preparation of a bifunctional nanozyme SERS detection platform

[0090] 1) Synthesize Au@Pt NRs as in Example 1.

[0091] 2) Synthesize AuNHs arrays as in Example 2.

[0092] 3) To ligate cDNA, 400 µL of cDNA (0.1 mM) was mixed with 40 µL of TCEP (1 M) solution and reacted at room temperature for 30 minutes to activate the nucleic acid aptamers. The activated cDNA was coupled to the surface of the AuNHs array and incubated at 37 °C for 120 minutes to ensure complete cDNA binding to the AuNHs surface. The array was repeatedly rinsed with PBS buffer and deionized water, and finally 15 mL of 1 wt% BSA was added to repair non-specific binding sites on the particle surface to obtain a cDNA-functionalized AuNHs array (AuNHs@cDNA).

[0093] 4) 400 μL of TCEP-activated H1 (1 M, 40 μL) was added to the prepared Au@Pt NRs and incubated at 37 °C for 2 hours. Finally, 100 μL of 1 wt% BSA solution was added to the solution to block non-specific binding sites on the particle surface. The mixture was then centrifuged at 10,000 rpm for 10 minutes to remove excess reagents and nucleic acid chains, yielding H1-functionalized Au@PtNRs (Au@PtNRs@H1).

[0094] 5) The prepared Au@Pt NRs@H1 was added to the surface of the prepared cDNA-functionalized AuNHs array and incubated at 37°C for 2 hours to prepare a SERS detection platform for detecting CI-related miRNA biomarkers. The nucleotide sequence of H1 is SH-AAAAGTGCTT; the nucleotide sequence of cDNA is SH-CTACCTGCACTGTAAGCACTTTT; the target miRNA to be detected is the CI biomarker miR-106a-5p.

[0095] 6) This study analyzed the effects of temperature, pH, incubation time, TMB concentration, and H2O2 concentration to obtain the optimal performance of the nanozyme SERS detection platform. For example... Figure 16 , 17 As shown, when the pH and temperature of the experiment were set to 4.5 and 30℃ respectively, the Au@Pt NRs-catalyzed oxidation of TMB to generate oxTMB signaling molecules at 1601 cm⁻¹ -1 The signal strength is highest at that location. For example... Figure 19 and 20 The results showed that when the TMB concentration was 0.9 mM and the H2O2 concentration was 0.5 M, the Au@Pt NRs-catalyzed oxidation of TMB to generate the oxTMB signal molecule occurred at 1601 cm⁻¹. -1 The signal strength is highest at this location. Furthermore, incubation time is also an important parameter affecting target detection, such as... Figure 18 As shown, the solution turned completely blue after 20 minutes, and the signal intensity gradually stabilized. Figure 21 To determine the peroxidase-like reaction kinetics of Au@PtNRs nanozymes, the Michaelis-Menten equation and double reciprocal curves were obtained by fitting the nanozymes, with Vmax = 0.5147 μMs. -1 With a Km of 0.275 mM, it exhibits excellent peroxidase activity. See Table 1.

[0096]

[0097] Example 4

[0098] Platform specificity and reproducibility tests of the bifunctional nanozyme SERS platform

[0099] 1) A bifunctional nanozyme SERS detection platform was prepared in the same manner as in Example 3.

[0100] 2) Under optimized experimental conditions, this study evaluated the reproducibility and specificity of the detection platform. For example... Figure 22 As shown, six nanozyme SERS detection platforms were prepared, and the SERS spectra of different batches of platforms were recorded. Figure 23 yes Figure 22 At 1601cm -1 A scatter plot of the average signal strength at the location, RSD=3.53%. Figure 24 and Figure 25 To detect the target miR-106a-5p using a nanozyme SERS detection platform, the corresponding SERS spectra were measured with MT1, MT3, random RNA sequences, and a blank as interferences. The SERS signal intensity was significantly reduced in the presence of the target analyte. In contrast, all three strands (interference, random, and blank) exhibited significant SERS signals in their presence. This indicates that the SERS detection platform constructed in this study has good reproducibility and specificity.

[0101] Example 5

[0102] Quantitative detection of miR-106a-5p using a SERS detection platform

[0103] 1) A bifunctional nanozyme SERS detection platform was prepared in the same manner as in Example 3.

[0104] 2) Under the optimized experimental conditions described above, the SERS platform was used to detect different concentrations (10) in serum. -15 M-10 -9 miR-106a-5p (M). Figure 26 As shown, the platform detected different concentrations of serum (concentration range from 10) -15 M to 10 -9 miR-106a-5p (M). Figure 26 As shown, with increasing target miRNA concentration, Au@Pt NRs continuously detach from the platform, nanozyme concentration continuously decreases, oxTMB oxidation gradually decreases, and SERS signal gradually weakens. Figure 27 As shown, miR-106a-5p at 1601 cm⁻¹ -1 The SERS intensity at a given location follows a linear regression equation: y = -2949.16x - 23837.15 (R²). 2 =0.9825), and the LOD calculated according to this equation is as low as 0.96fM, as shown in Table 2.

[0105]

[0106] Example 6 Characterization of clinical samples

[0107] Characterization of samples from healthy individuals and patients with cerebral infarction using MRI. Figures 28 to 30 MRI images of healthy individuals, Figures 31 to 33 The patient's MRI image. (Example) Figure 29 and Figure 32 As shown, by comparing the DWI images of normal individuals, the DWI images of this patient showed patchy high signal in the left parietal lobe, indicating acute ischemia, cytotoxic edema, and restricted water molecule diffusion in the brain tissue. Figure 28 and Figure 31 The apparent diffusion coefficient (ADC) is mainly used to describe the speed and range of molecular diffusion in different directions in DWI. It can quantitatively calculate the motion information of water molecules. In the acute cerebral infarction area, it usually shows a weakened signal and is lower than normal. Figure 30 and Figure 33 For magnetic resonance imaging fluid-enhanced inversion recovery sequence (FLAIR), in which Figure 33 The findings showed that, in addition to the acute cerebral infarction lesion, the patient also had scattered multiple ischemic infarction lesions in the bilateral lateral parietal ventricles, the subcortical anterior parietal lobe, and the coronal region of the radiating corona.

[0108] Example 7

[0109] The levels of miR-106a-5p in the serum of healthy individuals and patients with cerebral infarction were detected using a bifunctional nanozyme SERS detection platform.

[0110] 1) A bifunctional nanozyme SERS detection platform was prepared in the same manner as in Example 3.

[0111] 2) The SERS spectra of serum samples from 15 healthy subjects and 15 patients with cerebral infarction were tested using a bifunctional nanozyme SERS detection platform. The miRNA content was calculated by substituting the results into a linear regression equation and compared with the results of qRT-PCR to analyze the reliability of the platform in detecting miR-106a-5p in real clinical samples. Figure 34 This is the SERS spectrum of serum from CI patients. Figure 35 yes Figure 34 At 1601 cm -1 Histograms of signal intensity were plotted. The results of miR-106a-5p detection by the SERS platform were compared with those of qRT-PCR (Table 3). The relative error between the two was very small, demonstrating that the proposed bifunctional nanozyme SERS detection platform has high accuracy in detecting miR-106a-5p. Therefore, this bifunctional nanozyme SERS detection platform can serve as a reliable tool for biomarker detection and has good potential for clinical application.

[0112]

[0113] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a bifunctional nanozyme SERS detection platform for detecting cerebral infarction-related miRNAs, characterized in that: The infarction-related miRNA is miR-106a-5p, and the preparation method includes the following steps: 1) AuNRs were synthesized using a seed-mediated method, and Pt was grown on the surface of the product to prepare gold-platinum nanorods Au@Pt NRs; the specific implementation method of step 1) is as follows: 1.1) AuNRs were synthesized by seed growth method. 200 µL of chloroauric acid was added to 20 mL of cetyltrimethylammonium bromide solution, followed by 1.2 mL of sodium borohydride solution and stirring for 120 seconds. Then, the gold seed solution was allowed to stand at room temperature for 120 minutes. 1.2) The growth solution was prepared by mixing 4 mL of chloroauric acid, 4.4 mL of silver nitrate, 200 mL of hexadecyltrimethylammonium bromide and 1.4 mL of ascorbic acid solution; 1.3) The growth solution prepared in step 1.2) was mixed with 2 mL of gold seed solution and refluxed for 24 hours. Finally, AuNRs were obtained by centrifugation at 10000 rpm for 12 minutes. 1.4) The AuNRs suspension was mixed with H2PtCl6 solution, and then 0.6 mL of ascorbic acid solution was added to the mixture. The mixture was refluxed at room temperature for 180 minutes. Finally, the mixture was centrifuged at 10,000 rpm for 12 minutes to obtain gold-platinum nanorods Au@Pt NRs. The volume ratio of AuNRs to H2PtCl6 in step 1.4) was 200:

1. 2) Gold nano-hexagonal plates (AuNHs) were synthesized using a seed growth method, and AuNHs arrays were prepared via an oil-water interface self-assembly method; the specific implementation method of step 2) is as follows: 2.1) Dissolve 0.208 g of polyvinylpyrrolidone in 6 mL of ultrapure water at 35 °C and stir until the solution becomes transparent. Then, take 3 mL of the polyvinylpyrrolidone solution and quickly add it to 60 µL of chloroauric acid solution, and stir rapidly for 3 minutes to ensure that the two are fully mixed. Next, add 96 µL of freshly prepared ascorbic acid solution to the mixed solution and continue stirring for 30 minutes until the solution turns dark purple. 2.2) To ensure complete reaction, the mixture was allowed to stand at room temperature for 10 hours, then centrifuged at 10,000 rpm for 18 minutes and washed three times each with anhydrous ethanol and deionized water to remove excess reactants from the surface. Finally, the product was dissolved in deionized water to obtain a gold nano-hexagonal plate AuNHs solution. 2.3) Take the gold nano-hexagonal plate AuNHs solution obtained in step 2.1) and n-hexane into a beaker, then add ethanol solution and let stand for 180 seconds. At the oil-water interface, AuNHs self-assemble to form a tightly packed nanofilm with metallic luster; the volume ratio of AuNHs, n-hexane and ethanol in step 2.3) is 1:2:

1. 2.4) Use piranha solution to completely remove organic matter from the silicon wafer; 2.5) Transfer the AuNHs monolayer film formed at the oil-water interface in step 2.3) onto the silicon wafer treated in step 2.4) and dry it under a thermostat; 3) Modify the Au@Pt NRs obtained in step 1) with the complementary strand H1 of the nucleic acid aptamer via Au-S bonds, and bind it to the AuNHs array assembled in step 2) for functionalization of the nucleic acid aptamer cDNA corresponding to miR-106a-5p, thus constructing a bifunctional nanozyme SERS detection platform; the specific implementation method of step 3) is as follows: 3.1) To ligate cDNA, 400 µL of cDNA was mixed with 40 µL of TCEP solution and incubated at room temperature for 30 minutes to activate the nucleic acid aptamers; 3.2) The cDNA activated in step 3.1) was coupled to the surface of the AuNHs array and incubated at 37°C for 120 minutes to allow the cDNA to be completely bound to the AuNHs surface. The array was repeatedly rinsed with PBS buffer and deionized water. Finally, 15 mL of 1 wt% BSA was added to repair the non-specific binding sites on the particle surface to obtain the cDNA-functionalized AuNHs array AuNHs@cDNA. 3.3) By adding 400 μL of TCEP-activated H1 to the Au@Pt NRs prepared in step 1), incubating at 37°C for 2 hours, and finally adding 100 μL of 1wt% BSA solution to the solution to block non-specific binding sites on the particle surface, the solution was centrifuged at 10000 rpm for 10 minutes to remove excess reagents and nucleic acid chains, and Au@PtNRs@H1 functionalized Au@Pt NRs were obtained. 3.4) The Au@Pt NRs@H1 prepared in step 3.3) was added to the surface of the cDNA-functionalized AuNHs array prepared in step 3.2) and incubated in an incubator at 37°C for 2 hours to prepare a bifunctional nanozyme SERS detection platform for detecting miR-106a-5p. The nucleotide sequence of H1 is SH-AAAAGTGCTT; the nucleotide sequence of cDNA is SH-CTACCTGCACTGTAAGCACTTTT.

2. A bifunctional nanozyme SERS detection platform for detecting infarction-related miRNAs, characterized in that: This invention is prepared based on the method for preparing a bifunctional nanozyme SERS detection platform for detecting cerebral infarction-related miRNAs as described in claim 1.

Images