A sers sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy and a preparation method and application thereof

Abstract

The application discloses a SERS sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy and a preparation method and application thereof, and belongs to the field of biomedical technology. AuNHs are self-assembled through an oil-water interface to obtain an AuNHs array, and a nucleic acid aptamer chain cDNA is modified on the surface of the array to obtain a SERS substrate; a Raman signal molecule 4-MBA and a nucleic acid aptamer complementary chain H1 are modified to the surface of AuNTs to obtain a SERS probe 1; a Raman signal molecule DTNB and a nucleic acid aptamer complementary chain H2 are modified to the surface of AuNTs to obtain a SERS probe 2; the prepared SERS probes are combined with complementary bases in the nucleic acid aptamer chain cDNA on the SERS substrate, are fixed on the AuNHs array, and a SERS sensor is constructed.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a SERS sensor for detecting serum biomarkers miR-21 and miR-152 in diabetic retinopathy, its preparation method, and its application. Background Technology

[0002] Diabetic retinopathy (DR) is a very common microvascular complication of diabetes, posing a serious threat to visual health and being a leading cause of blindness in working-age individuals. Studies have shown that the presence of DR significantly threatens patients' vision-related quality of life, and this quality of life declines further as DR worsens. Currently, fundus fluorescein angiography is the gold standard for early screening and diagnosis of diabetic retinopathy. However, due to its invasiveness, high cost, technical complexity, and allergy risk, as well as the fact that fundus examination and photograph interpretation require the involvement of physicians with expertise in diabetes-related eye diseases, fluorescein angiography is not suitable as a routine screening tool for DR in clinical practice. Therefore, finding relevant serum biomarker detection methods to help accurately diagnose early DR is extremely important.

[0003] Minimal RNAs (miRNAs) are a class of small, highly conserved non-coding RNAs that play important regulatory roles in various biological processes. Some studies have shown that changes in miRNA levels in body fluids are closely related to the development of diabetic retinopathy (DR) and are significant in DR diagnosis. MiR-21 is a miRNA that is significantly upregulated in the serum of DR patients and plays an important pathogenic role in DR by regulating PPARα expression levels. Furthermore, the study by AASaleh et al. revealed that miR-152 expression differs significantly between DR patients and healthy controls, demonstrating good sensitivity and specificity for DR diagnosis. Serum miR-21 and miR-152 can be used as potential diagnostic biomarkers for DR. To date, researchers have designed various strategies to identify and analyze miRNAs closely related to the disease, including Northern blotting, quantitative real-time PCR (qRT-PCR), and microarray analysis techniques. However, despite the potential of these methods in biomedical research, they still face many challenges, such as operational complexity, high cost, time consumption, low sensitivity, and unsuitability for rapid clinical testing. It is worth noting that simultaneously monitoring and mapping the concentration changes of multiple miRNAs can reduce false positives and improve the accuracy of the analysis.

[0004] Surface-enhanced Raman scattering (SERS) spectroscopy is a rapidly developing molecular spectroscopy technique in recent years, possessing advantages such as high sensitivity, high precision, fingerprinting capabilities, and non-destructive testing. It plays a crucial role in many fields, including environmental monitoring, food safety, and biomedicine. The core mechanism of SERS utilizes metal nanostructures to excite localized surface plasmon resonance (LSPR), a process that generates "hot spots" near the metal surface, which in turn greatly enhances the molecular Raman scattering signal. Compared to traditional Raman scattering, SERS exhibits significant advantages, with an impact factor (EF) ≥ 10. 8 The microstructure of the SERS substrate significantly influences the distribution of "hot spots," determining the sensitivity, repeatability, and stability of detection. Preparing SERS substrates with high density and uniform "hot spot" distribution has become a current research focus. Gold trioctahedrons (AuNTs) possess a unique geometry (polyhedrons with eight triangular pyramids). Their multiple sharp edges and nanoscale gaps between particles provide high-density "hot spots" for SERS, resulting in high-intensity, highly repeatable Raman signals. In recent years, the plasmonic characterization and application research of gold nanopolygons has developed rapidly, attracting considerable interest. Among them, gold nanohexagonal plates (AuNHs) are characterized by their regular shape, numerous sharp corners, and large specific surface area, providing more attachment sites for signal molecules, thus generating significant SERS enhancement. AuNH arrays are structurally stable and uniform, and the gaps between AuNHs edges facilitate the formation of "hot spots," which can achieve significant enhancement of the local electromagnetic field and can be used as excellent SERS substrates.

[0005] In the sample detection stage, simple, accurate, and quantitative strategies are needed to ensure clinical applicability. Currently, SERS-based "sandwich" structures, HCR (hybridization chain reaction), and CHA (catalytic hairpin assembly) amplification strategies are widely used, but they still face challenges such as complex design and stringent reaction conditions. Competitive recognition strategies, on the other hand, require only a single spiking reaction to achieve simultaneous and efficient detection of two miRNAs, offering advantages in simplicity and practicality. Microarray chips, as an integrated chip platform, offer the possibility of simple and rapid sample detection due to their high throughput, low sample consumption, high safety, and compact and portable structure. However, there are currently no reports on the use of SERS technology for the detection of serum miR-21 and miR-152. Summary of the Invention

[0006] The purpose of this invention is to provide a SERS sensor for detecting serum biomarkers miR-21 and miR-152 in diabetic retinopathy, along with its preparation method and applications, to address the problems existing in the prior art. This invention combines SERS, a microarray chip, and a competitive recognition strategy to construct a novel SERS sensor for detecting miR-21 and miR-152 in the serum of DR rats. This invention provides a method for preparing a SERS sensor for the simultaneous, rapid, and highly sensitive detection of the serum biomarkers miR-21 and miR-152 in DR rats, and its clinical applications.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] One of the technical solutions of this invention is a method for preparing a SERS sensor for detecting serum biomarkers miR-21 and miR-152 in diabetic retinopathy, comprising the following steps:

[0009] (1) AuNHs arrays were obtained by self-assembling gold nano-hexagonal plates through an oil-water interface, and SERS substrates were obtained by modifying the surface of the AuNHs arrays with nucleic acid aptamer strand cDNA.

[0010] (2) The Raman signaling molecule 4-MBA solution and the complementary strand H1 of the nucleic acid aptamer were coupled to the surface of gold trioctahedral AuNTs via Au-S bond to obtain SERS probe 1 AuNTs@4-MBA@H1; the Raman signaling molecule DTNB and the complementary strand H2 of the nucleic acid aptamer were coupled to the surface of AuNTs via Au-S bond to obtain SERS probe 2 AuNTs@DTNB@H2;

[0011] (3) Combine the AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate to obtain the SERS sensor.

[0012] The second technical solution of the present invention is a SERS sensor prepared by the preparation method for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy.

[0013] The third technical solution of the present invention is the application of the SERS sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy in the preparation of products for detecting miR-21 and miR-152.

[0014] The fourth technical solution of the present invention is a SERS microarray chip, which is obtained by embedding the SERS sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy into a PDMS chip.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] This invention provides a method for preparing a SERS sensor for detecting DR-related serum biomarkers miR-21 and miR-152, and the sensor prepared based on this method. This invention first involves the self-assembly of AuNHs at an oil-water interface to obtain a uniformly and densely arranged AuNHs array. Nucleic acid aptamer strand cDNA is then modified onto the array surface to obtain a SERS substrate. Next, the Raman signaling molecule 4-MBA and the complementary nucleic acid aptamer strand H1 are coupled to the AuNTs surface via Au-S bonds to obtain SERS probe 1 (AuNTs@4-MBA@H1). Similarly, the Raman signaling molecule DTNB and the complementary nucleic acid aptamer strand H2 are coupled to the AuNTs surface via Au-S bonds to obtain SERS probe 2 (AuNTs@DTNB@H2). The prepared SERS probes (AuNTs@4-MBA@H1 and AuNTs@DTNB@H2) are then bound to complementary bases in the cDNA of the nucleic acid aptamer strand on the SERS substrate, immobilizing them on the AuNHs array. By optimizing the preparation conditions, a SERS sensor is constructed. This sensor exhibits advantages such as high sensitivity, strong specificity, and fast detection speed. An advanced SERS analysis platform was constructed by embedding a SERS sensor into a PDMS chip to obtain a microarray chip. When target miRNAs (miR-21 and miR-152) are present in the analyte, the target miRNAs exhibit higher binding affinity to the nucleic acid aptamers, while the complementary strands (H1 and H2) of the nucleic acid aptamers exhibit weaker binding affinity, causing the SERS probe initially bound to the capture substrate to be replaced. This replacement process leads to the detachment of 4-MBA and DTNB signaling molecules from the capture interface, resulting in the attenuation of the SERS signal. Therefore, by quantifying the degree of SERS signal attenuation of 4-MBA and DTNB, the levels of miR-21 and miR-152 in serum can be measured. The results show that the SERS detection platform constructed in this invention can be used for simple, rapid, and ultrasensitive detection of the DR biomarkers miR-21 and miR-152 in serum samples, providing technical support for expanding the wide application of SERS in the diagnosis and screening of DR.

[0017] The SERS substrate preparation method provided by this invention is simple, highly operable, and requires no special instruments or equipment. The experimental cost is low, and the required reagents are all commonly used chemical reagents. The SERS substrate prepared by this invention has excellent reproducibility and can be prepared on a large scale. MiR-21 is significantly upregulated in the serum of DR patients and plays a key pathogenic role in DR by regulating the expression level of PPARα. Furthermore, previous studies have confirmed that miR-152 expression differs significantly between DR patients and controls, showing good sensitivity and specificity for DR diagnosis. miR-21 and miR-152 may be biomarkers and key predictors for early detection of DR. Currently, the "sandwich" structure, hybridization chain reaction (HCR), and catalytic hairpin self-assembly (CHA) signal amplification strategies based on SERS technology are widely used, but still face challenges such as complex design and strict reaction conditions. The competitive recognition strategy requires only one sample addition reaction to achieve simultaneous and efficient detection of two miRNAs, offering significant advantages of simplicity, speed, and practicality. This invention combines the advantages of SERS technology with those of microarray chips to construct a high-throughput SERS microarray chip, enabling highly sensitive identification and rapid detection of miR-21 and miR-152. This invention also discloses a method for preparing a SERS sensor for detecting the DR biomarkers miR-21 and miR-152. The method involves self-assembling AuNHs at an oil-water interface to obtain a uniformly and densely arranged AuNHs array. Nucleic acid aptamer strand cDNA is then modified onto the array surface to obtain a SERS substrate. Next, Raman signal molecules (4-MBA and DTNB) and complementary strands of the nucleic acid aptamers (H1 and H2) are coupled to the AuNTs surface via Au-S bonds to obtain SERS probes (AuNTs@4-MBA@H1 and AuNTs@DTNB@H2). The prepared SERS probes are then bound to complementary bases in the nucleic acid aptamer strand cDNA on the SERS substrate and immobilized on the AuNHs array to construct a SERS sensor, ultimately enabling the detection of the DR biomarkers miR-21 and miR-152. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the fabrication process of the SERS microarray chip of the present invention and the competitive identification and detection of serum biomarkers miR-21 and miR-152 for diabetic retinopathy.

[0020] Figure 2A This is a SEM image of AuNTs synthesized in this invention.

[0021] Figure 2B This is a TEM image of the AuNTs synthesized in this invention.

[0022] Figure 2C This is a partial magnification of the high-resolution TEM image of AuNTs synthesized in this invention.

[0023] Figure 2D This is the UV-Vis absorption spectrum of the AuNTs synthesized in this invention.

[0024] Figure 2E These are Raman spectra of 4-MBA and 4-MBA labeled AuNTs.

[0025] Figure 3A This is a SEM image of the AuNHs synthesized in this invention.

[0026] Figure 3B This is a partial magnification of a high-resolution TEM image of AuNHs synthesized in this invention.

[0027] Figure 3C This is the UV-Vis absorption spectrum of the AuNHs synthesized in this invention.

[0028] Figure 3D This is a SEM image of the AuNHs array prepared according to the present invention.

[0029] Figure 3E These are SERS spectra of 4-MBA and 4-MBA labeled AuNHs arrays.

[0030] Figure 3F These are top and side views of the microarray chip prepared according to the present invention.

[0031] Figure 3G These are SERS spectra of eight randomly selected points on the surface of a 4-MBA labeled AuNHs array.

[0032] Figure 3H These are 88 randomly selected points (1082 cm⁻¹) on the surface of a 4-MBA labeled AuNHs array. -1 The corresponding SERS intensity histogram.

[0033] Figure 3I The 1078 cm⁻¹ of 4-MBA labeled AuNHs arrays stored at room temperature for different numbers of days (0 days, 7 days, 14 days) -1 The corresponding SERS spectrum.

[0034] Figure 4AThis invention optimizes the cDNA concentration of the SER sensor prepared in this invention.

[0035] Figure 4B This invention optimizes the detection time of the SER sensor prepared in this invention.

[0036] Figure 4C The SERS microarray chip prepared in this invention detects miR-21 and miR-152, and only miR-21 and miR-152 are present. There are no SERS spectra of the two target miRNAs.

[0037] Figure 4D The SERS microarray chip prepared in this invention detects miR-21 and miR-152, showing only miR-21 and only miR-152 present, with neither of the two target miRNAs present at 1082 cm⁻¹. -1 and 1339cm -1 SERS intensity histogram.

[0038] Figure 5A This invention describes the SERS spectra of serum samples with different concentrations of miR-21 and miR-152 detected by the SERS sensor constructed in this invention.

[0039] Figure 5B It is the logarithm of miR-21 concentration and 1082 cm⁻¹ -1 Linear fitting plot of SERS intensity at the location.

[0040] Figure 5C It is the logarithm of miR-152 concentration and 1339 cm⁻¹ -1 Linear fitting plot of SERS intensity at the location.

[0041] Figure 6A This is the average weight curve of rats.

[0042] Figure 6B This is the average blood glucose curve of rats.

[0043] Figure 6C This is an image of HE staining of a rat retina.

[0044] Figure 7A This is the SERS spectrum of miR-21 and miR-152 in the serum of rats during diabetic retinopathy.

[0045] Figure 7B The mean SERS spectrum of serum during diabetic retinopathy in rats at 1082 cm⁻¹ -1 and 1339cm -1 The SERS intensity histogram at [location]. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0050] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0051] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0052] This invention provides a method for preparing a SERS sensor for detecting serum biomarkers miR-21 and miR-152 in diabetic retinopathy, comprising the following steps:

[0053] (1) AuNHs arrays were obtained by self-assembling gold nano-hexagonal plates through an oil-water interface, and SERS substrates were obtained by modifying the surface of the AuNHs arrays with nucleic acid aptamer strand cDNA.

[0054] (2) The Raman signaling molecule 4-MBA solution and the complementary strand H1 of the nucleic acid aptamer were coupled to the surface of gold trioctahedral AuNTs via Au-S bond to obtain SERS probe 1 AuNTs@4-MBA@H1; the Raman signaling molecule DTNB and the complementary strand H2 of the nucleic acid aptamer were coupled to the surface of AuNTs via Au-S bond to obtain SERS probe 2 AuNTs@DTNB@H2;

[0055] (3) Combine the AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate to obtain the SERS sensor.

[0056] In some specific implementations, the preparation method of the gold nano-hexagonal plate AuNHs is as follows: a polyvinylpyrrolidone solution is mixed with a HAuCl4 solution, an AA solution is added to the mixture, the mixture is stirred for 20 min, allowed to stand at room temperature for 12 h, and AuNHs is obtained after centrifugation and purification.

[0057] The density of the polyvinylpyrrolidone solution is 50-55 mg / ml; the concentration of the HAuCl4 solution is 50 mM; the concentration of the AA solution is 15 mM; and the volume ratio of the polyvinylpyrrolidone solution, HAuCl4 solution and AA solution is (1-2):20:32.

[0058] In some specific implementations, the method for obtaining the AuNHs array through self-assembly at the oil-water interface is as follows: AuNHs, n-hexane and anhydrous ethanol are mixed and allowed to stand, and the AuNHs are closely arranged at the oil-water interface and self-assemble to form an AuNHs array.

[0059] The volume ratio of AuNHs, n-hexane, and anhydrous ethanol is 1:2:1.

[0060] In some specific implementations, the method of modifying the surface of the AuNHs array with nucleic acid aptamer chain cDNA is as follows: the activated nucleic acid aptamer chain cDNA solution is dropped onto the surface of the AuNHs array, and then incubated to obtain a nucleic acid aptamer chain-coupled AuNHs array, which is the SERS substrate;

[0061] The concentration of the cDNA solution is 10 mM; the nucleic acid aptamer strand cDNA includes cDNA1 as shown in SEQ ID NO.3 and cDNA2 as shown in SEQ ID NO.4; the molar ratio of cDNA1 to cDNA2 is 1:1;

[0062] The activation method is as follows: mix 1M TCEP solution with 0.1mM cDNA solution and reduce at room temperature for 0.5h to activate the nucleic acid aptamer;

[0063] The volume ratio of the nucleic acid aptamer chain cDNA to the AuNHs array is 1:2;

[0064] The incubation conditions are: incubation at 37°C for 2 hours.

[0065] In some specific implementations, the method for modifying the surface of gold trioctahedral AuNTs by coupling the Raman signaling molecule 4-MBA solution and the nucleic acid aptamer complementary strand H1 via Au-S bond is as follows: the AuNTs solution is mixed with the Raman signaling molecule 4-MBA solution, and then the resulting AuNTs@4-MBA is added to the activated nucleic acid aptamer complementary strand H1 solution for incubation; after incubation, BSA solution is added to the reaction solution to carry out the reaction, thereby obtaining the SERS nanoprobe 1 AuNTs@4-MBA@H1;

[0066] The concentration of the 4-MBA solution is 10 mM; the concentration of the AuNTs solution is 0.3 mM; and the volume ratio of the 4-MBA solution to the AuNTs solution is 1:50.

[0067] The activation method for the complementary strand H1 of the nucleic acid aptamer is as follows: activation is performed using TCEP buffer.

[0068] The incubation conditions were: 37°C and 80% humidity for 2 hours.

[0069] In some specific implementations, the method for modifying the surface of AuNTs by coupling the Raman signaling molecule DTNB and the complementary nucleic acid aptamer strand H2 via Au-S bonds is as follows: the AuNTs solution is mixed with the Raman signaling molecule DTNB solution, and then the resulting AuNTs@DTNB is added to the activated complementary nucleic acid aptamer strand H2 solution for incubation; after incubation, BSA solution is added to the reaction solution to react and obtain the SERS nanoprobe 2AuNTs@DTNB@H2;

[0070] The concentration of the DTNB solution is 10 mM; the concentration of the AuNTs solution is 0.3 mM; and the volume ratio of the DTNB solution to the AuNTs solution is 1:50.

[0071] The activation method for the complementary strand H2 of the nucleic acid aptamer is as follows: activation is performed using TCEP buffer.

[0072] The incubation conditions were: 37°C and 80% humidity for 2 hours.

[0073] In some specific implementations, the method for combining AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate is as follows: AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 are dropped onto the surface of the SERS substrate, followed by incubation to obtain the SERS sensor;

[0074] The molar ratio of AuNTs@4-MBA@H1, AuNTs@DTNB@H2 to the SERS substrate is 1:1:1;

[0075] The incubation conditions are: 37℃ for 1.5 hours.

[0076] This invention also provides a SERS sensor prepared by the above preparation method for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy.

[0077] This invention also provides the application of the SERS sensor for detecting the serum biomarkers miR-21 and miR-152 of diabetic retinopathy in the preparation of products for detecting miR-21 and miR-152.

[0078] This invention also provides a SERS microarray chip, which is obtained by embedding the SERS sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy into a PDMS chip.

[0079] The scanning electron microscope (TEM) images were obtained using an S-4800II field emission scanning electron microscope manufactured by Hitachi, Japan.

[0080] The transmission electron microscope (SEM) images were obtained using a Tecnai 12 transmission electron microscope manufactured by Philips in the Netherlands.

[0081] High-resolution TEM (HRTEM) and selected-area electron diffraction (SAED) images were obtained using a JEM-2100Plus emission transmission electron microscope manufactured by JEOL Ltd., Japan.

[0082] Raman spectroscopy and SERS imaging were performed using a DXRxi micro Raman spectrometer manufactured by Thermo Fisher Scientific, USA. The test conditions were: laser wavelength 785 nm, exposure time 10 s, laser intensity 50 mW, and 50× objective lens.

[0083] The UV-Vis absorption spectrum was measured using a Cary UV-5000 ultraviolet absorption spectrometer manufactured by Agilent Technologies, Inc.

[0084] Example 1

[0085] Preparation of SERS probes and characterization of AuNTs

[0086] 1) Under slow stirring, HAuCl4 solution (60 mL, 0.6 mM) was added to hexadecyltrimethylammonium chloride (CTAC, prepared by dissolving 0.192 g of CTAC in 60 mL of deionized water). Freshly prepared ascorbic acid AA (2.4 mL, 0.1 M) was then rapidly added to the mixture. After gentle stirring, the mixture was placed at 30 °C for 20 min, during which time the color of the mixture gradually changed from colorless to light pink. Finally, the precipitate obtained by centrifugation at 6000 r / min for 15 min was washed three times with ultrapure water to remove CTAC. Ultrapure water was then added and the volume was adjusted to 12 mL to obtain AuNTs.

[0087] 2) Mix 200 mL of Raman signal molecule 4-mercaptobenzoic acid (4-MBA) solution (10 mM) and 10 mL of AuNTs obtained in step 1) (1.18 μg / mL), stir vigorously at 700 r / min for 2 hours at room temperature, centrifuge, and then add 100 μL of 150 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 100 μL of 30 mM N-hydroxysuccinimide (NHS) dropwise to obtain AuNTs@4-MBA; the same experimental procedure was used to obtain AuNTs@DTNB.

[0088] 3) Activate the complementary strand of the nucleic acid aptamer (H1) with 5 mL of freshly prepared TCEP buffer (1M). Then, add the AuNTs@4-MBA from step 2) to the activated H1 solution, with an excess of H1. Incubate at 37°C and 80% humidity for 2 hours. Add 15 mL of 1% BSA solution to the incubated reaction solution and react for 3 hours. After centrifugation and purification, the SERS probe AuNTs@4-MBA@H1 is obtained. The same experimental procedure can be used to obtain the SERS probe AuNTs@DTNB@H2.

[0089] H1 (SEQ ID NO.1): SH-TAG CTT ATC A;

[0090] H2 (SEQ ID NO. 2): SH-AGG TTC TGT G.

[0091] 4) Morphology of AuNTs and characterization of the SERS effect

[0092] The morphology and structure of AuNTs were detected by SEM, TEM and HRTEM.

[0093] UV-Vis absorption spectra were measured using an Agilent Cary UV-5000 UV-Vis spectrometer; SERS was performed using a Thermo Fisher Scientific DXRxi micro Raman spectrometer with the following settings: 50× objective lens, 785 nm laser wavelength, 5 mW power, and 10 s data acquisition time. To ensure data validity, the spectra of each sample were averaged from five random points during the reaction process and preprocessed using Origin 2021 software to correct the baseline and smooth the spectra for subsequent analysis.

[0094] See results Figure 2A and 2B The images are SEM and TEM images of the synthesized AuNTs, showing that the prepared AuNTs have regular morphology and clear outlines, and are dispersed in the field of view at different angles and directions. Figure 2C This is a magnified HRTEM image showing that the lattice fringe spacing of AuNTs is 0.230 nm. Figure 2D The results show that AuNTs exhibit high surface plasmon resonance absorption in the 500-800 nm range, with a peak value of 621 nm in the UV-Vis absorption spectrum. The inset image shows that the prepared AuNTs are pink in color. Figure 2E Comparison of 4-MBA (10 -2 M) and marked 4-MBA(10 -8 Raman spectra of AuNTs (M) show that AuNTs have excellent SERS enhancement effect.

[0095] Example 2

[0096] Preparation of SERS substrate and characterization of AuNHs array

[0097] 1) Add 0.154 g of polyvinylpyrrolidone (PVP) to 3 mL of ultrapure water and stir at 30 °C to dissolve and obtain a uniformly dispersed PVP solution. Mix the PVP solution with HAuCl4 solution (60 mL 50 mM) under rapid stirring at 700 r / min, and add freshly prepared AA solution (96 mL 15 mM) to the mixture. After stirring for 20 min, let stand at room temperature for 12 h, and then centrifuge to purify and obtain AuNHs.

[0098] 2) Add AuNHs (1 mL) and n-hexane (2 mL) to the same dry beaker, add anhydrous ethanol (1 mL) and let stand for 3 minutes. At this time, AuNHs are tightly arranged at the oil-water interface and self-assemble to form an AuNHs array.

[0099] 3) Use piranha solution (V 98％H2SO4: V 35％H2O2A clean silicon wafer, treated with a 7:3 ratio of hydrophilicity (80℃ for 2 h), was used to extract the AuNHs array prepared in step 2). After drying, the silicon wafer was immersed in 20 mL of a solution containing freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 150 mM) and N-hydroxysuccinimide (NHS, 30 mM). 20 mL of TCEP solution (1 M) was mixed with 200 μL of cDNA1 and cDNA2 (total concentration of cDNA1 and cDNA2 was 0.1 mM, molar ratio of cDNA1 to cDNA2 was 1:1), and the mixture was reduced at room temperature for 0.5 h to activate the nucleic acid aptamers. Subsequently, the activated nucleic acid aptamers were dropped onto the surface of the AuNHs array (excess activated nucleic acid aptamer cDNA), and incubated at 37℃ for 2 h to obtain the capture substrate.

[0100] cDNA1 (SEQ ID NO.3): SH-TCA ACA TCA GTC TGA TAA GCT A;

[0101] cDNA2 (SEQ ID NO. 4): SH-AGT CGG AGT GTA TCA CAG AAC CT.

[0102] 4) Morphology of AuNHs, morphology of AuNHs arrays, and characterization of SERS effect.

[0103] The morphology and structure of AuNTs were detected by SEM, TEM and HRTEM.

[0104] UV-Vis absorption spectra were measured using an Agilent Cary UV-5000 UV-Vis spectrometer; SERS was performed using a Thermo Fisher Scientific DXRxi micro Raman spectrometer with the following settings: 50× objective lens, 785 nm laser wavelength, 5 mW power, and 10 s data acquisition time. To ensure data validity, the spectra of each sample were averaged from five random points during the reaction process and preprocessed using Origin 2021 software to correct the baseline and smooth the spectra for subsequent analysis.

[0105] Figure 3A The image shows a TEM image of AuNHs, which indicates that the prepared AuNHs have a uniform shape, with an average parallel side distance and thickness of 150 nm and 40 nm, respectively. The edges of a single AuNH surface appear to be sharp. Figure 3B The image is an HRTEM image of AuNHs, showing lattice fringes with a spacing of 0.236 nm. The SAED depicted in the inset is the result of a high-energy electron beam guided perpendicular to AuNHs, confirming the presence of a single crystal in the incident region. Figure 3CThe UV-Vis absorption spectrum of AuNHs shows that AuNHs has a strong absorption peak at 799 nm. The inset image shows that the prepared AuNHs is green. Figure 3D These are SEM images of the AuNHs array. To evaluate the SERS activity of the AuNHs array, Figure 3E Using 4-MBA (1×10) -2 M) and 4-MBA (1×10 -9 The Raman signal enhancement effect of the AuNHs array labeled with M was further investigated. The Raman enhancement effect of the AuNHs array can be expressed by the equation EF = (I SERS / C SERS ) / (I Raman / C Raman The value is calculated to be 1.42 × 10⁻⁶. 9 The AuNHs array is embedded in a microarray chip. The top and side views of the three-layer microarray chip are shown below. Figure 3F As shown, the uniformity and stability of the AuNHs array significantly affect the detection performance of the microarray chip. Eight points were randomly selected on the surface of the 4-MBA modified AuNHs array, and SERS spectra were generated as follows. Figure 3G As shown, the results exhibit good reproducibility. The difference is at 1082 cm. -1 The corresponding histogram of the average signal strength at the location Figure 3H As shown, the relative standard deviation (RSD) is 4.58%, indicating that the AuNHs array has excellent uniformity. Meanwhile, Figure 3I The stability of the AuNHs arrays was evaluated, and the SERS spectra of 4-MBA-labeled AuNHs arrays stored at room temperature for different numbers of days (0 days, 7 days, and 14 days) are shown. Compared with newly tested samples, the intensity of the SERS spectra obtained from the array surface after 14 days of storage showed only a slight decrease of about 5.51%, demonstrating its excellent stability.

[0106] Example 3

[0107] Fabrication of SERS sensors and microarray chips

[0108] 1) The SERS probes prepared in Example 1 (AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 in a volume ratio of 1:1) were added in excess to the surface of the AuNHs array on the SERS substrate prepared in Example 2. After incubation in a constant temperature incubator at 37°C for 1.5 h, the substrate was washed with PBS buffer. The SERS probes and nucleic acid aptamer chains partially paired complementary bases, which anchored the SERS nanoprobes on the AuNHs array, and the SERS sensor was successfully prepared.

[0109] 2) First, the SERS sensor prepared in step 1) is embedded into a glass substrate using laser etching, with its size and distribution matching the micropores on the chip. Next, using oxygen plasma technology, the glass substrate containing the SERS sensor only in the SERS testing area and the PDMS sandwich structure containing 9 micropores are placed together in the working chamber of a plasma cleaner for 1 minute of surface treatment. After removal, the two are precisely aligned and immediately bonded. Finally, a PDMS cover plate is placed on top to successfully construct the SERS micropore array chip. (The vacuum-treated mixture (PDMS:crosslinking agent = 10:1, where the crosslinking agent is a platinum catalyst) is poured into the microarray mold, vacuum-treated again, and then baked and cured in an 80°C oven for 3 hours to obtain the PDMS chip. After cooling to room temperature, the PDMS chip is perforated, cleaned, dried, and surface-modified according to the design. The SERS sensor prepared in Example 3 is then embedded into the lower glass substrate of the prepared PDMS chip to obtain the designed SERS microarray chip.)

[0110] Example 4

[0111] Experimental optimization and specificity testing

[0112] 1) A systematic screening of the total concentration of complementary strands of nucleic acid aptamers (cDNA) was conducted. The experimental design covered multiple cDNA concentration gradients (1 nM, 10 nM, 100 nM, 1 mM, 10 mM, 100 mM), which were used to modify the AuNHs array surface to prepare SERS substrates. SERS probes were then paired and bound to these substrates to prepare SERS sensors for detecting the SERS spectra of specific biomarkers miR-21 (SEQ ID NO.5: TAG CTT ATC AGA CTG ATG TTG A) and miR-152 (SEQ ID NO.6: AGGTTC TGT GAT ACA CTC CGA CT).

[0113] 1082cm -1 and 1339cm -1 The average SERS signal strength at the location. For example... Figure 4A As shown, with increasing cDNA concentration, 1082 cm⁻¹ -1 and 1339cm -1 The average SERS signal intensity at each location gradually increased. When the cDNA concentration reached 10 mM, the increasing trend of SERS intensity tended to stabilize. Therefore, the optimal concentration of cDNA at 10 mM was selected for the preparation of the subsequent SERS sensor.

[0114] 2) The serum sample to be tested was mixed with the SERS sensor and subjected to a competitive recognition reaction in an incubator for different reaction times (0 min, 2 min, 4 min, 6 min, 8 min) to obtain a 1082 cm⁻¹ sample. -1 and 1339cm -1 The average SERS signal strength at the location. For example... Figure 4B As shown, corresponding to 1082cm -1 and 1339cm -1 The SERS intensity of 4-MBA and DTNB gradually decreased with increasing test time. When the reaction time reached 8 minutes, the SERS intensity of 4-MBA and DTNB no longer decreased further with increasing time, indicating that the optimal reaction time had been reached. Therefore, the optimal reaction time for competitive identification in this invention is 8 minutes.

[0115] 3) Under optimal experimental conditions, the ability of the detection platform to reliably and effectively distinguish between miR-21 and miR-152 was evaluated. SERS spectra of various targets (both target chains present, miR-21 only, miR-152 only, and neither target chain present) were obtained using a SERS microarray chip. The relevant spectra are shown... Figure 4C In the middle, the signal strength is displayed Figure 4D In the middle. Compared with the case where both target chains are missing, when both target chains are present, at 1082 cm⁻¹. -1 and 1339cm -1 A weak Raman signal was generated at the location. Furthermore, when miR-21 or miR-152 was present alone, the signal intensity of the corresponding peaks was significantly reduced, and there was no undesirable overlap. The results validate the feasibility and target recognition specificity of this microarray chip for the simultaneous detection of miR-21 and miR-152, and suggest its high practical applicability.

[0116] Example 5

[0117] Quantitative analysis of miR-21 and miR-152

[0118] 1) Prepare SERS microarray chip as in Example 3

[0119] 2) such as Figure 5A As shown, different concentrations (10 -14 -10 -8miR-21 and miR-152 (miR-21 and miR-152 = 1:1) were dispersed in serum, and SERS spectra of different concentrations of miR-21 and miR-152 in serum were obtained. This demonstrated that as the concentration of the target nucleic acid in serum increased, the SERS signal intensity at the corresponding position decreased. Furthermore, a significant linear correlation was found between the SERS intensity of the unique peaks at appropriate positions and the logarithm of the concentrations of the two target miRNAs. Figure 5B SERS was displayed at 1082cm -1 The calibration curve between the intensity at a given location and the logarithm of the miR-21 concentration in serum exhibits a linear regression equation: y = -2405.64x - 17993.52 (R²). 2 =0.9865). Similarly, such as Figure 5C As shown, the logarithm of miR-152 concentration and 1339 cm⁻¹ -1 The trend between SERS intensities follows a linear regression equation: y = -3020.40x - 22766.18 (R²) 2 =0.9890). Based on this, the LOD of miR-21 and miR-152 in serum are 2.76 × 10⁻⁶. -14 M and 1.94×10 -14 M. Compared with other experimental methods (Table 1), this method has the advantages of low detection limit and rapid analysis, and can simultaneously quantify two DR-related miRNAs, making it suitable for rapid diagnosis and real-time monitoring of clinical samples.

[0120] Table 1. Comparison of this method with other miRNA biomarker detection methods.

[0121]

[0122] Example 6

[0123] Establishment and characterization of a rat model of diabetic retinopathy

[0124] 1) Before surgery, rats were fasted for 12 hours. Rats in the model group were intraperitoneally injected with streptozotocin (STZ, purchased from Aladdin Reagent) at a dose of 55 mg / kg, while the control group received the same dose of sodium citrate buffer (purchased from Shanghai Sinopharm Group). Three days after STZ injection, fasting blood glucose was measured in the model group rats, and a blood glucose level greater than 13.5 mmol / L was selected as the criterion for successful modeling, and the rats were included in the formal experiment. After successful modeling, eight rats were divided into a control group, a 2-week STZ induction group, a 4-week STZ induction group, and a 6-week STZ induction group. At each time point, the rat eyes were paraffin-embedded and cut into retinal sections for hematoxylin-eosin (HE) staining. In addition, serum was collected from the eight rats before STZ induction and at 2, 4, and 6 weeks after induction. Changes in body weight and blood glucose levels in each rat were also recorded.

[0125] 2) Figure 6A and Figure 6B The curves showing the changes in blood glucose levels and body weight over time in the same DR-induced rat are presented. These curves all indicate that the model is effective and that serum samples can be obtained for actual sample testing. Figure 6C HE staining results are shown to assess the structural integrity of rat retinal tissue induced by DR. Before STZ induction, the layers in the rat retinal tissue were structurally ordered with well-defined boundaries and tightly packed, orderly cell arrangement. Retinal tissues from DR rats at 2, 4, and 6 weeks post-induction showed distinct pathological features, with looser interlayer structures, indistinct boundaries, tissue deformation, edema, and more pronounced proliferation of retinal capillary endothelial cells and fibrous tissue.

[0126] Example 7

[0127] 1) Prepare SERS microarray chip as in Example 3

[0128] 2) The collected serum samples were spotted onto the SERS microarray chip and reacted in a 37°C incubator. After 8 minutes, the samples were removed and subjected to SERS testing to detect the SERS signal. The SERS microarray chip created in this invention was used to measure the levels of miR-21 and miR-152 in rat blood samples at four different time periods (0W, 2W, 4W, and 6W) to further confirm the reliability of this SERS analysis platform. Figure 7A and 7B The results show that as DR progresses, 4-MBA reaches 1082 cm⁻¹. -1 And DTNB at 1339cm -1 The peak intensity decreased significantly. Then, a linear regression equation between the logarithm of the target miRNA concentration and the SERS intensity at the characteristic peak was used to calculate the actual concentrations of miR-21 and miR-152 in rat blood. Furthermore, the accuracy of the data was confirmed by comparison with qRT-PCR results (Table 2). The results confirm the reliability of this SERS analysis platform as a first-class analytical method and demonstrate its superior accuracy in biomarker identification.

[0129] Table 2 Comparison of SERS and qRT-PCR detection results

[0130]

[0131] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a SERS sensor for detecting serum biomarkers miR-21 and miR-152 in diabetic retinopathy, characterized in that, Includes the following steps: (1) AuNHs arrays were obtained by self-assembling gold nano-hexagonal plates through an oil-water interface, and SERS substrates were obtained by modifying the surface of the AuNHs arrays with complementary nucleic acid strand cDNA. The preparation method of the gold nano-hexagonal plate AuNHs is as follows: Polyvinylpyrrolidone solution and HAuCl4 solution are mixed, AA solution is added to the mixture, stirred for 20 min, and then allowed to stand at room temperature for 12 h. After centrifugation and purification, AuNHs are obtained. The density of the polyvinylpyrrolidone solution is 50-55 mg / ml; the concentration of the HAuCl4 solution is 50 mM; the concentration of the AA solution is 15 mM; the volume ratio of the polyvinylpyrrolidone solution, HAuCl4 solution, and AA solution is (1-2):20:

32. The method of obtaining the AuNHs array through self-assembly at the oil-water interface is as follows: AuNHs, n-hexane, and anhydrous ethanol are mixed and allowed to stand. The AuNHs are tightly arranged at the oil-water interface and self-assemble to form an AuNHs array; the volume ratio of the AuNHs, n-hexane, and anhydrous ethanol is 1:2:

1. The AuNHs has an average parallel side distance of 150 nm, a thickness of 40 nm, and a lattice spacing of 0.236 nm. The method for modifying the surface of the AuNHs array with complementary nucleic acid cDNA is as follows: the activated complementary nucleic acid cDNA solution is dropped onto the surface of the AuNHs array, and then incubated to obtain the AuNHs array coupled with complementary nucleic acid, which is the SERS substrate. The concentration of the cDNA solution is 10 mM; the complementary nucleic acid strand cDNA includes cDNA1 as shown in SEQ ID NO.3 and cDNA2 as shown in SEQ ID NO.4; the molar ratio of cDNA1 to cDNA2 is 1:1; The activation method is as follows: mix 1M TCEP solution with 0.1mM cDNA solution and reduce at room temperature for 0.5h to activate the complementary nucleic acid strands; The volume ratio of the complementary nucleic acid strand cDNA to the AuNHs array is 1:2; The incubation conditions are: incubation at 37°C for 2 hours; (2) The Raman signaling molecule 4-MBA solution and the nucleic acid complementary strand H1 were coupled to the surface of gold trioctahedral AuNTs via Au-S bond to obtain SERS probe 1 AuNTs@4-MBA@H1; the Raman signaling molecule DTNB and the nucleic acid complementary strand H2 were coupled to the surface of AuNTs via Au-S bond to obtain SERS probe 2 AuNTs@DTNB@H2; (3) Combine the AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate to obtain the SERS sensor.

2. The preparation method according to claim 1, characterized in that, The method for modifying the surface of gold trioctahedral AuNTs by coupling Raman signal molecule 4-MBA solution and nucleic acid complementary strand H1 via Au-S bond is as follows: the AuNTs solution is mixed with the Raman signal molecule 4-MBA solution, and then the resulting AuNTs@4-MBA is added to the activated nucleic acid complementary strand H1 solution for incubation; after incubation, BSA solution is added to the reaction solution to react and obtain the SERS nanoprobe 1 AuNTs@4-MBA@H1; The concentration of the 4-MBA solution is 10 mM; the concentration of the AuNTs solution is 0.3 mM; and the volume ratio of the 4-MBA solution to the AuNTs solution is 1:

50. The activation method for the nucleic acid complementary strand H1 is as follows: activation is performed using TCEP buffer. The incubation conditions were: 37°C and 80% humidity for 2 hours.

3. The preparation method according to claim 1, characterized in that, The method for modifying the surface of AuNTs by coupling Raman signaling molecule DTNB and nucleic acid complementary strand H2 via Au-S bond is as follows: the AuNTs solution is mixed with the Raman signaling molecule DTNB solution, and then the resulting AuNTs@DTNB is added to the activated nucleic acid complementary strand H2 solution for incubation; after incubation, BSA solution is added to the reaction solution to react and obtain the SERS nanoprobe 2AuNTs@DTNB@H2; The concentration of the DTNB solution is 10 mM; the concentration of the AuNTs solution is 0.3 mM; and the volume ratio of the DTNB solution to the AuNTs solution is 1:

50. The activation method for the complementary nucleic acid strand H2 is as follows: activation is performed using TCEP buffer. The incubation conditions were: 37°C and 80% humidity for 2 hours.

4. The preparation method according to claim 1, characterized in that, The method for combining AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate is as follows: AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 are dropped onto the surface of the SERS substrate, and then incubated to obtain the SERS sensor. The molar ratio of AuNTs@4-MBA@H1, AuNTs@DTNB@H2 to the SERS substrate is 1:1:1; The incubation conditions are: 37℃ for 1.5 hours.

5. A SERS sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy, prepared by the preparation method according to any one of claims 1-4.

6. The use of the SERS sensor as described in claim 5 for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy in the preparation of products for detecting miR-21 and miR-152.

7. A SERS microarray chip, characterized in that, The SERS sensor for detecting serum biomarkers miR-21 and miR-152 of diabetic retinopathy as described in claim 5 is obtained by embedding it into a PDMS chip.

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