SERS (Surface Enhanced Raman Scattering) sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 as well as preparation method and application of SERS sensor

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 as well as a preparation method and application of the SERS sensor, and belongs to the technical field of biomedicine. The preparation method comprises the following steps: self-assembling AuNHs through an oil-water interface to obtain an AuNHs array, and modifying an aptamer chain cDNA on the surface of the array to obtain an SERS (Surface Enhanced Raman Scattering) substrate; the method comprises the following steps: modifying a Raman signal molecule 4-MBA and a nucleic acid aptamer complementary chain H1 to the surface of AuNTs to obtain an SERS probe 1; a Raman signal molecule DTNB and a nucleic acid aptamer complementary chain H2 are modified to the surface of AuNTs, and an SERS probe 2 is obtained; and combining the prepared SERS probe with a complementary basic group in the nucleic acid aptamer chain cDNA on the SERS substrate, and fixing on the AuNHs array to construct the SERS sensor.

Description

Technical Field

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

[0002] Diabetic retinopathy (DR) is a very common microvascular complication of diabetes, which poses a serious threat to visual health and is the leading cause of blindness in the working-age population. Studies have shown that the presence of DR poses a significant threat to patients' vision-related quality of life, and this quality of life further declines with the worsening of DR. 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 photo interpretation require the participation of doctors with expertise in diabetes-related eye diseases, fluorescein angiography is not suitable as a routine screening tool for DR in clinical practice. Therefore, it is extremely important to find relevant serum biomarker detection methods to help accurately diagnose early DR.

[0003] MicroRNAs (miRNAs) are a class of small, highly conserved noncoding RNAs that play important regulatory roles in a variety of biological processes. Some studies have shown that changes in miRNA levels in body fluids are closely related to the development of DR and are of great significance in the diagnosis of DR. 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 the expression level of PPARa. In addition, the study by AASaleh et al. also revealed that the expression of miR-152 in DR patients and healthy controls was significantly different, and it had good sensitivity and specificity for the diagnosis of DR. Serum miR-21 and miR-152 can be used as potential diagnostic markers for DR. To date, researchers have designed a variety of strategies to identify and analyze miRNAs closely related to the disease, including Northern blotting, quantitative real-time PCR (qRT-PCR), microarray analysis technology, etc. However, although these methods have shown potential in the field of biomedical research, they still face many problems, such as complex operation, high cost, time-consuming, low sensitivity, and are not suitable for rapid clinical testing. Notably, simultaneous monitoring and mapping of concentration changes of multiple miRNAs can reduce the occurrence of false positives and improve the precision of the analysis.

[0004] Surface enhanced Raman scattering spectroscopy (SERS) is a molecular spectroscopy technique that has developed rapidly in recent years. It has the advantages of high sensitivity, high precision, fingerprinting, and non-destructive testing. It plays an important role in many fields such as environmental monitoring, food safety, and biomedicine. The core mechanism of SERS is to use metal nanostructures to stimulate 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 with traditional Raman scattering, SERS has significant advantages, with an impact factor (EF) ≥ 10 8 . The microstructure of the SERS substrate greatly affects the distribution of "hot spots", which determines the sensitivity, repeatability and stability of the detection. The preparation of SERS substrates with high density and uniform distribution of "hot spots" has become a hot topic in current research. Gold trioctahedrons (AuNTs) have a unique geometric shape (a polyhedron with eight triangular pyramids), and their appearance of multiple sharp edges and nanogaps between particles provide high-density "hot spots" for SERS, thereby generating high-intensity and highly repeatable Raman signals. In recent years, the plasmonic characterization and application research of gold nanopolygons has developed rapidly and has aroused great interest. Among them, gold nanohexagonal plates (AuNHs) are characterized by their regular shape, many sharp corners and large specific surface area, which can provide more attachment sites for signal molecules, resulting in significant SERS enhancement. The AuNHs array is structurally stable and uniform, and the gaps between AuNHs and AuNHs edges contribute to the formation of "hot spots", which can achieve significant enhancement of local electromagnetic fields and can be used as excellent SERS substrates.

[0005] At the sample detection stage, simple, accurate and quantitative strategies are needed to ensure clinical practicality. At present, the "sandwich" structure, HCR (hybridization chain reaction) and CHA (catalytic hairpin assembly) amplification strategies based on SERS technology are widely used, but they still face challenges such as complex design and strict reaction conditions. The competitive recognition strategy only requires one step of spiking reaction to achieve simultaneous and efficient detection of two miRNAs, which has the advantages of simplicity and practicality. As an integrated chip platform, microarray chips have the advantages of high throughput, low sample consumption, high safety, and compact and portable structure, which makes it possible for simple and rapid sample detection. However, there are no reports on the detection of serum miR-21 and miR-152 using SERS technology. Summary of the invention

[0006] The purpose of the present invention is to provide a SERS sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152, and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The present invention combines SERS, microarray chips and competitive recognition strategies to construct a new SERS sensor for the detection of miR-21 and miR-122 in DR rat serum. The present invention provides a preparation method and clinical application of a SERS sensor for simultaneous, rapid and highly sensitive detection of DR serum biomarkers miR-21 and miR-152.

[0007] To achieve the above object, the present invention provides the following solutions:

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

[0009] (1) AuNHs arrays were obtained by self-assembly of gold nanohexagonal plates at an oil-water interface, and nucleic acid aptamer chains cDNA were modified on the surface of the AuNHs arrays to obtain SERS substrates;

[0010] (2) The Raman signal molecule 4-MBA solution and the nucleic acid aptamer complementary chain H1 were modified to the gold trioctahedral AuNTs surface through Au-S bond coupling to obtain SERS probe 1AuNTs@4-MBA@H1; the Raman signal molecule DTNB and the nucleic acid aptamer complementary chain H2 were modified to the AuNTs surface through Au-S bond coupling to obtain SERS probe 2AuNTs@DTNB@H2;

[0011] (3) Combining 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 diabetic retinopathy serum biomarkers miR-21 and miR-152.

[0013] The third technical solution of the present invention is the use of the SERS sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 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 the 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] The present invention provides a method for preparing a SERS sensor for detecting DR-related serum biomarkers miR-21 and miR-152 and a sensor prepared based on the method. The invention firstly self-assembles AuNHs at an oil-water interface to obtain a uniform and densely arranged AuNHs array, modifies the nucleic acid aptamer chain cDNA on the array surface to obtain a SERS substrate; then, the Raman signal molecule 4-MBA and the nucleic acid aptamer complementary chain H1 are modified to the AuNTs surface through Au-S bond coupling to obtain a SERS probe 1 (AuNTs@4-MBA@H1); the Raman signal molecule DTNB and the nucleic acid aptamer complementary chain H2 are modified to the AuNTs surface through Au-S bond coupling to obtain a SERS probe 2 (AuNTs@DTNB@H2); the prepared SERS probes (AuNTs@4-MBA@H1 and AuNTs@DTNB@H2) are combined with complementary bases in the nucleic acid aptamer chain cDNA on the SERS substrate, fixed on the AuNHs array, and the preparation conditions are optimized to construct a SERS sensor. The sensor has the advantages of high sensitivity, strong specificity, fast detection speed, etc. The SERS sensor was embedded into the PDMS chip to obtain a microarray chip, and an advanced SERS analysis platform was constructed. When target miRNAs (miR-21 and miR-152) are present in the analyte, the target miRNAs show higher binding affinity to the aptamer, and the complementary chains of the aptamer (H1 and H2) show weaker binding affinity, so that the SERS probe initially bound to the capture substrate is replaced. This replacement process causes the 4-MBA and DTNB signal molecules to detach from the capture interface and the attenuation of the SERS signal. Therefore, by quantifying the attenuation degree of the SERS signal of 4-MBA and DTNB, the measurement of the levels of miR-21 and miR-152 in serum can be achieved. The results show that the SERS detection platform constructed by the present invention can be used for the simple, rapid and ultra-sensitive detection of 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 the present invention is simple, highly operable, and does not require special instruments and equipment. The experimental cost is low, and the required reagents are all commonly used chemical reagents. The SERS substrate prepared by the present invention has good repeatability 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 PPARa. In addition, existing studies have confirmed that miR-152 has significant differences in expression between DR patients and control groups, and has good sensitivity and specificity for the diagnosis of DR. MiR-21 and miR-152 may be biomarkers and key predictors for early detection of DR. At present, the "sandwich" structure, hybridization chain reaction (HCR), and catalytic hairpin self-assembly (CHA) signal amplification strategies based on SERS technology are widely used, but they still face challenges such as complex design and strict reaction conditions. The competitive recognition strategy only requires one-step sample addition reaction to achieve simultaneous and efficient detection of two miRNAs, which has the significant advantages of simplicity, rapidity, and strong practicality. The present invention combines the respective advantages of SERS technology and microarray chips to construct a high-throughput SERS microarray chip, which can achieve highly sensitive identification and rapid detection of miR-21 and miR-152. The present invention discloses a method for preparing a SERS sensor for detecting DR biomarkers miR-21 and miR-152. The method is to self-assemble AuNHs through the oil-water interface to obtain a uniform and densely arranged AuNHs array, and modify the nucleic acid aptamer chain cDNA on the array surface to obtain a SERS substrate; then, the Raman signal molecules (4-MBA and DTNB) and the nucleic acid aptamer complementary chains (H1 and H2) are modified to the AuNTs surface through Au-S bond coupling to obtain SERS probes (AuNTs@4-MBA@H1 and AuNTs@DTNB@H2); the prepared SERS probes are combined with the complementary bases in the nucleic acid aptamer chain cDNA on the SERS substrate and fixed on the AuNHs array to construct a SERS sensor, ultimately realizing the detection of DR biomarkers miR-21 and miR-152. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0020] Figure 2A is a SEM photograph of AuNTs synthesized in the present invention.

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

[0022] Figure 2C This is a local enlargement of the high-resolution TEM image of the AuNTs synthesized by the present invention.

[0023] Figure 2D It is the UV-visible absorption spectrum of AuNTs synthesized by the present invention.

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

[0025] Figure 3A is a SEM photograph of AuNHs synthesized in the present invention.

[0026] Figure 3B This is a local enlargement of the high-resolution TEM image of the AuNHs synthesized in the present invention.

[0027] Figure 3C is the UV-visible absorption spectrum of AuNHs synthesized in the present invention.

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

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

[0030] Figure 3F 1 and 2 are top views and side views of the microarray chip prepared by the present invention.

[0031] Figure 3G These are the SERS spectra of 8 points randomly selected on the surface of the 4-MBA labeled AuNHs array.

[0032] Figure 3H 88 points randomly selected from the surface of the 4-MBA labeled AuNHs array at 1082 cm -1 The corresponding SERS intensity histogram at .

[0033] Fig. 3I The 4-MBA labeled AuNHs array was stored at room temperature for different days (0 day, 7 day, 14 day) at 1078 cm -1 The corresponding SERS spectra.

[0034] Figure 4AThe cDNA concentration of the SER sensor prepared by the present invention is optimized.

[0035] Figure 4B The detection time of the SER sensor prepared by the present invention is optimized.

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

[0037] Figure 4D The SERS microarray chip prepared by the present invention detects miR-21 and miR-152. Only miR-21 and miR-152 exist, and no two target miRNAs are present at 1082 cm -1 and 1339cm -1 SERS intensity histogram.

[0038] Figure 5A The SERS spectra of serum samples with different concentrations of miR-21 and miR-152 detected by the SERS sensor constructed by the present invention are shown.

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

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

[0041] Fig. 6A is the average body weight curve of rats.

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

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

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

[0045] Figure 7B The average SERS spectrum of serum during diabetic retinopathy in rats at 1082 cm -1 and 1339cm -1 . SERS intensity histogram at . DETAILED DESCRIPTION

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

[0047] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0051] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

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

[0053] (1) AuNHs arrays were obtained by self-assembly of gold nanohexagonal plates at an oil-water interface, and nucleic acid aptamer chains cDNA were modified on the surface of the AuNHs arrays to obtain SERS substrates;

[0054] (2) The Raman signal molecule 4-MBA solution and the nucleic acid aptamer complementary chain H1 were modified to the gold trioctahedral AuNTs surface through Au-S bond coupling to obtain SERS probe 1AuNTs@4-MBA@H1; the Raman signal molecule DTNB and the nucleic acid aptamer complementary chain H2 were modified to the AuNTs surface through Au-S bond coupling to obtain SERS probe 2AuNTs@DTNB@H2;

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

[0056] In some specific embodiments, the preparation method of the gold nanohexagonal plate AuNHs is: mixing a polyvinyl pyrrolidone solution with a HAuCl4 solution, adding an AA solution to the mixture, stirring for 20 minutes, standing at room temperature for 12 hours, and obtaining AuNHs after centrifugal purification;

[0057] The density of the polyvinyl pyrrolidone 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 polyvinyl pyrrolidone solution, HAuCl4 solution and AA solution is (1-2):20:32.

[0058] In some specific embodiments, the method for obtaining the AuNHs array by oil-water interface self-assembly is: AuNHs, n-hexane and anhydrous ethanol are mixed and allowed to stand, and the AuNHs are closely arranged and self-assembled at the oil-water interface to form the AuNHs array;

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

[0060] In some specific embodiments, the method for modifying the aptamer chain cDNA on the surface of the AuNHs array is: dropping the activated aptamer chain cDNA solution onto the surface of the AuNHs array, followed by incubation to obtain the aptamer chain-coupled AuNHs array, which is the SERS substrate;

[0061] The concentration of the cDNA solution is 10 mM; the nucleic acid aptamer chain 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: mixing 1M TCEP solution with 0.1mM cDNA solution, and reducing at room temperature for 0.5h to activate the nucleic acid aptamer;

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

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

[0065] In some specific embodiments, the method of coupling and modifying the Raman signal molecule 4-MBA solution and the nucleic acid aptamer complementary chain H1 to the gold trioctahedral AuNTs surface through Au-S bond is as follows: the AuNTs solution is mixed with the Raman signal molecule 4-MBA solution, and then the obtained AuNTs@4-MBA is added to the activated nucleic acid aptamer complementary chain H1 solution for incubation; after the incubation is completed, the BSA solution is added to the reaction solution obtained by the incubation to react, and the SERS nanoprobe 1AuNTs@4-MBA@H1 is obtained;

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

[0067] The activation method of the nucleic acid aptamer complementary chain H1 is: activating with TCEP buffer;

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

[0069] In some specific embodiments, the method of coupling and modifying the Raman signal molecule DTNB and the nucleic acid aptamer complementary chain H2 to the AuNTs surface through the Au-S bond is as follows: the AuNTs solution is mixed with the Raman signal molecule DTNB solution, and then the obtained AuNTs@DTNB is added to the activated nucleic acid aptamer complementary chain H2 solution for incubation; after the incubation is completed, the BSA solution is added to the reaction solution obtained by the incubation to react, and the SERS nanoprobe 2AuNTs@DTNB@H2 is obtained;

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

[0071] The activation method of the nucleic acid aptamer complementary chain H2 is: activating with TCEP buffer;

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

[0073] In some specific embodiments, the method of combining the AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate is: dropping AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 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 and the SERS substrate is 1:1:1;

[0075] The incubation conditions are: incubation at 37° C. for 1.5 h.

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

[0077] The embodiment of the present invention also provides the use of the SERS sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 in preparing a product for detecting miR-21 and miR-152.

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

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

[0080] Transmission electron microscope (SEM) images were obtained using a Tecnai 12 transmission electron microscope produced by Philips of the Netherlands.

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

[0082] Raman spectra and SERS imaging were measured by a DXRxi Raman microscope produced by Thermo Fisher Scientific Inc. The test conditions were a laser wavelength of 785 nm, an exposure time of 10 s, a laser intensity of 50 mW, and a 50× objective lens.

[0083] The UV-Vis absorption spectrum was measured by a Cary UV-5000 ultraviolet absorption spectrometer produced by Agilent Corporation of the United States.

[0084] Example 1

[0085] Preparation of SERS probes and characterization of AuNTs

[0086] 1) 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) under slow stirring, and newly prepared ascorbic acid AA (2.4 mL, 0.1 M) was quickly added to the above mixed solution. After slight stirring, the mixture was placed at 30 ° C for 20 min, and the color of the mixture was observed to gradually change from colorless to light pink. Finally, the above solution was centrifuged at 6000 r / min for 15 min to obtain a precipitate, and washed three times with ultrapure water to remove CTAC, and ultrapure water was 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 (1.18 μg / mL) obtained in step 1), stir vigorously at 700 r / min at room temperature for 2 hours, centrifuge, and then drop 100 μL of 150 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 100 μL of 30 mM N-hydroxysuccinimide (NHS) into the mixture in sequence to obtain AuNTs@4-MBA; the same experimental process is used to obtain AuNTs@DTNB.

[0088] 3) Take 5 mL of freshly prepared TCEP buffer (1 M) to activate the complementary chain of the nucleic acid aptamer (H1), then add the AuNTs@4-MBA from step 2) to the activated H1 solution, with an excess of H1; incubate in a constant temperature incubator 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 centrifugal purification, the SERS probe AuNTs@4-MBA@H1 is obtained. The same experimental process 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) Characterization of AuNTs morphology and SERS effect

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

[0093] The UV-Vis absorption spectra were measured using the Cary UV-5000 ultraviolet absorption spectrometer produced by Agilent; the SERS detection was performed using the Thermo Fisher DXRxi micro-Raman spectrometer, and the parameters were set as follows: objective lens 50×, laser wavelength 785nm, power 5mW, and data acquisition time 10s. To ensure the validity of the data, the spectrum of each sample was averaged at 5 random points during the reaction process, and was preprocessed using Origin 2021 software to correct the baseline and smooth the spectrum for subsequent analysis.

[0094] Results Figure 2A and 2B , are SEM and TEM photos of the synthesized AuNTs, respectively, showing that the prepared AuNTs have regular morphology and clear outlines, and are dispersed in the field of view at different angles. Figure 2C This is a locally enlarged HRTEM image, showing that the lattice fringe spacing of AuNTs is 0.230nm. Figure 2D It shows that AuNTs exhibit high surface plasmon resonance absorption in the range of 500-800nm, with a peak at 621nm in the UV-Vis absorption spectrum, and the inset image shows that the color of the prepared AuNTs is pink. Figure 2E Compared with 4-MBA (10 -2 M) and labeled 4-MBA (10 -8 M) Raman spectrum of AuNTs, showing 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 polyvinyl pyrrolidone (PVP) to 3 mL ultrapure water and stir to dissolve at 30°C to 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. Stir for 20 min and let stand at room temperature for 12 h. Purify by centrifugation to obtain AuNHs.

[0098] 2) AuNHs (1 mL) and n-hexane (2 mL) were added to the same dry beaker, and anhydrous ethanol (1 mL) was added and allowed to stand for 3 minutes. At this time, the AuNHs were closely arranged at the oil-water interface and self-assembled to form an AuNHs array.

[0099] 3) Use piranha solution (V 98％H2SO4: V 35％H2O2=7:3) hydrophilic treatment (80℃ treatment for 2h) of clean silicon wafer, take out the AuNHs array prepared in step 2). After drying, soak the silicon wafer in 20mL of a solution containing freshly prepared 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC, 150mM) and N-hydroxysuccinimide (NHS, 30mM). Mix 20mL of TCEP solution (1M) with 200μL of cDNA1 and cDNA2 (the total concentration of cDNA1 and cDNA2 is 0.1mM, and the molar ratio of cDNA1 and cDNA2 is 1:1), and reduce at room temperature for 0.5h to activate the nucleic acid aptamer. Subsequently, the activated nucleic acid ligand is added dropwise to the surface of the AuNHs array (the activated nucleic acid ligand cDNA is in excess), and incubated at 37℃ for 2 hours to obtain a 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) Characterization of the morphology of AuNHs, AuNHs arrays and SERS effect

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

[0104] The UV-Vis absorption spectra were measured using the Cary UV-5000 ultraviolet absorption spectrometer produced by Agilent; the SERS detection was performed using the Thermo Fisher DXRxi micro-Raman spectrometer, and the parameters were set as follows: objective lens 50×, laser wavelength 785nm, power 5mW, and data acquisition time 10s. To ensure the validity of the data, the spectrum of each sample was averaged at 5 random points during the reaction process, and was preprocessed using Origin 2021 software to correct the baseline and smooth the spectrum for subsequent analysis.

[0105] Figure 3A is the TEM image of AuNHs, which shows that the prepared AuNHs have uniform shape, with an average parallel edge distance and thickness of 150 nm and 40 nm, respectively, and the edge of a single AuNH surface appears to be sharp. Figure 3B is a HRTEM image of AuNHs, showing lattice fringes with a lattice spacing of 0.236 nm. The SAED depicted in the inset is the result of directing a high-energy electron beam perpendicular to the AuNH, confirming the presence of single crystals within the incident region. Figure 3Cis the UV-Vis absorption spectrum of AuNHs. It can be seen that AuNHs has a strong absorption peak at 799 nm, and the inset image shows that the color of the prepared AuNHs is green. Figure 3D is the SEM image of the AuNHs array. In order to evaluate the SERS activity of the AuNHs array, Figure 3E Use 4-MBA (1×10 -2 M) and 4-MBA (1×10 -9 M) labeled AuNHs arrays to further study the Raman signal enhancement effect. The Raman enhancement effect of the AuNHs array can be expressed by the equation EF = (I SERS / C SERS ) / (I Raman / C Raman ) is calculated as 1.42×10 9 The AuNHs array is embedded in the microarray chip. The top view and side view of the microarray chip with a three-layer structure are shown in Figure 3F The uniformity and stability of the AuNHs array will have a significant impact on the detection effect of the microarray chip. Eight points were randomly selected on the surface of the 4-MBA-modified AuNHs array to generate SERS spectra as shown in Figure 2. Figure 3G As shown, the results show good reproducibility. -1 The corresponding bar graph of the average signal intensity at Figure 3H As shown, the relative standard deviation (RSD) value is 4.58%, indicating that the AuNHs array has excellent uniformity. Fig. 3I The stability of the AuNHs array was evaluated, and the SERS spectra of the 4-MBA labeled AuNHs array stored at room temperature for different days (0 days, 7 days, and 14 days) were shown. Compared with the newly tested sample, the intensity of the SERS spectrum 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] Preparation of SERS sensors and microarray chips

[0108] 1) The SERS probe prepared in Example 1 (the volume ratio of AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 was 1:1) was excessively added to the AuNHs array surface of the SERS substrate prepared in Example 2, incubated in a constant temperature incubator at 37°C for 1.5 h, and then rinsed with PBS buffer. The SERS probe was partially complementary to the bases of the nucleic acid aptamer chain, so that the SERS nanoprobe was anchored on the AuNHs array, and the SERS sensor was successfully prepared.

[0109] 2) First, the SERS sensor prepared in step 1) is embedded in a glass substrate by laser etching, and its size and distribution match the micropores on the chip. Then, by oxygen plasma technology, the glass substrate containing the SERS sensor only in the SERS test area and the PDMS sandwich structure containing 9 micropores are placed in the working chamber of the plasma cleaning machine, surface treated for 1 minute, and then taken out to align the two accurately and fit them immediately. Finally, the PDMS cover plate is covered to successfully construct the SERS micropore array chip. (The vacuum-treated mixture (PDMS: cross-linking agent = 10:1, the cross-linking agent is a platinum catalyst) is poured into the microarray mold, vacuum-treated again, and placed in an 80°C oven for baking and curing for 3 hours to obtain a PDMS chip. After cooling to room temperature, the PDMS chip is punched, cleaned, dried, and surface modified according to the design drawing, and the SERS sensor prepared in Example 3 is embedded in 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) The total concentration of aptamer complementary chain (cDNA) was systematically screened, and the experimental design covered multiple cDNA concentration gradients (1nM, 10nM, 100nM, 1mM, 10mM, 100mM), which were used to modify the surface of AuNHs array to prepare SERS substrate. SERS probes were combined with their paired counterparts to prepare SERS sensors, which were used to detect 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] Gain 1082cm -1 and 1339cm -1 The average SERS signal intensity at the position. Figure 4A As shown, with the increase of cDNA concentration, the 1082cm -1 and 1339cm -1 The average SERS signal intensity at the position gradually increased. When the cDNA concentration reached 10mM, the growth trend of the SERS intensity tended to be stable, so the cDNA with the optimal concentration of 10mM was selected to prepare the subsequent SERS sensor.

[0114] 2) The serum sample to be tested was mixed with the SERS sensor and a competitive recognition reaction was carried out in a constant temperature box for different reaction times (0 min, 2 min, 4 min, 6 min, 8 min) to obtain 1082 cm -1 and 1339cm -1 The average SERS signal intensity at the position. Figure 4B As shown, corresponding to 1082cm -1 and 1339cm -1 The SERS intensity of 4-MBA and DTNB gradually decreases with the extension of the test time. When the reaction time reaches 8 minutes, the SERS intensity of 4-MBA and DTNB no longer decreases with the extension of time, which indicates that the optimal reaction time has been reached. It can be seen that the optimal reaction time for competitive recognition of the present 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 were obtained for various targets (both target chains present, miR-21 only, miR-152 only, and neither target chain present) studied using the SERS microarray chip. The relevant spectra are shown in Figure 4C In the Figure 4D Compared with the absence of both target chains, when both target chains are present, the -1 and 1339cm -1 In addition, when miR-21 or miR-152 existed alone, the signal intensity of the corresponding different peaks was significantly reduced, and there was no unfavorable overlap. The results verified the feasibility and target recognition specificity of the microarray chip for simultaneous detection of miR-21 and miR-152, and it is believed that it has high practical use.

[0116] Example 5

[0117] Quantification of miR-21 and miR-152

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

[0119] 2) If Figure 5A As shown, different concentrations (10 -14 -10 -8The SERS spectra of miR-21 and miR-152 at different concentrations in serum were obtained, which proved that when the concentration of target nucleic acid in serum increased, the SERS signal intensity at the corresponding position decreased. In addition, it was found that the SERS intensity of the unique peak at the appropriate position showed a significant linear correlation with the logarithm of the concentration of the two target miRNAs. Figure 5B The SERS at 1082 cm -1 The calibration curve between the intensity at 100 μg / cm2 and the logarithm of the miR-21 concentration in serum has a linear regression equation y=-2405.64x-17993.52 (R 2 =0.9865). Similarly, Figure 5C As shown, the logarithm of miR-152 concentration and 1339 cm -1 The trend of SERS intensity at different locations follows the linear regression equation y = -3020.40x -22766.18 (R 2 =0.9890). Based on this, the LODs of miR-21 and miR-152 in serum were 2.76×10 -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 quantitatively detect two DR-related miRNAs simultaneously, which is 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 diabetic retinopathy rat model

[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, and 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 blood glucose greater than 13.5 mmol / L was selected as the standard for successful modeling and included in the formal experiment. After successful modeling, 8 rats were divided into control group, STZ induced 2-week group, 4-week group and 6-week group. At each of the above time points, the rats' eyes were paraffin-embedded and cut into retinal sections for hematoxylin-eosin (HE) staining. In addition, serum was collected from 8 rats before STZ induction and 2, 4 and 6 weeks after induction. Changes in body weight and blood glucose levels of each rat were also recorded.

[0125] 2) Fig. 6A and Figure 6B Curves tracking the blood glucose level and body weight of the same DR-induced rat over time are shown. These curves all indicate that the model is valid and that serum for actual sample testing can be obtained. Figure 6C HE staining results are shown to evaluate the structural integrity of DR-induced rat retinal tissue. Before STZ induction, the layers in rat retinal tissue were structurally orderly, with well-defined boundaries, and the cells were tightly and neatly arranged. The retinal tissue of DR rats at 2, 4, and 6 weeks after induction showed obvious pathological characteristics, with loose and unclear boundaries between the layers, tissue deformation and edema, and more obvious 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, reacted in a 37°C incubator, and taken out for SERS testing after 8 minutes to detect the SERS signal of the serum samples. The SERS microarray chip created by the present 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. Fig. 7A and 7B It was shown that as DR progressed, 4-MBA was at 1082cm -1 and DTNB at 1339cm -1 The peak intensity was significantly reduced. The actual concentrations of miR-21 and miR-152 in rat blood were then calculated using a linear regression equation between the logarithm of the target miRNA concentration and the SERS intensity at the characteristic peak. At the same time, the correctness of the data was confirmed by further comparison with the qRT-PCR results (Table 2). The results confirm the reliability of this SERS analysis platform as a first-class analysis method and demonstrate its excellent 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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a SERS sensor for detecting serum biomarkers of diabetic retinopathy miR-21 and miR-152, characterized in that: The following steps are involved: (1) AuNHs arrays were obtained by self-assembly of gold nanohexagonal plates at an oil-water interface, and nucleic acid aptamer chains cDNA were modified on the surface of the AuNHs arrays to obtain SERS substrates; (2) The Raman signal molecule 4-MBA solution and the nucleic acid aptamer complementary chain H1 were modified to the gold trioctahedral AuNTs surface through Au-S bond coupling to obtain SERS probe 1AuNTs@4-MBA@H1; the Raman signal molecule DTNB and the nucleic acid aptamer complementary chain H2 were modified to the AuNTs surface through Au-S bond coupling to obtain SERS probe 2AuNTs@DTNB@H2; (3) Combining 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 preparation method of the gold nanohexagonal plate AuNHs is as follows: a polyvinyl pyrrolidone solution is mixed with a HAuCl4 solution, an AA solution is added to the mixture, the mixture is stirred for 20 minutes, and then allowed to stand at room temperature for 12 hours, and AuNHs is obtained after centrifugal purification; The density of the polyvinyl pyrrolidone 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 polyvinyl pyrrolidone solution, HAuCl4 solution and AA solution is (1-2):20:

32.

3. The preparation method according to claim 1, characterized in that: The method for obtaining the AuNHs array by 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-assembled to form the AuNHs array; The volume ratio of AuNHs, n-hexane and anhydrous ethanol is 1:2:

1.

4. The preparation method according to claim 1, characterized in that: The method for modifying the nucleic acid aptamer chain cDNA on the surface of the AuNHs array is: dropping the activated nucleic acid aptamer chain cDNA solution onto the surface of the AuNHs array, and then incubating to obtain the AuNHs array coupled with the nucleic acid aptamer chain, which is the SERS substrate; The concentration of the cDNA solution is 10 mM; the nucleic acid aptamer chain 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: mixing 1M TCEP solution with 0.1mM cDNA solution, and reducing at room temperature for 0.5h to activate the nucleic acid aptamer; The volume ratio of the aptamer chain cDNA to the AuNHs array is 1:2; The incubation conditions are: incubation at 37° C. for 2 h.

5. The preparation method according to claim 1, characterized in that: The method for coupling and modifying the Raman signal molecule 4-MBA solution and the nucleic acid aptamer complementary chain H1 to the gold trioctahedral AuNTs surface through Au-S bond is as follows: the AuNTs solution is mixed with the Raman signal molecule 4-MBA solution, and then the obtained AuNTs@4-MBA is added to the activated nucleic acid aptamer complementary chain H1 solution for incubation; after the incubation is completed, the BSA solution is added to the reaction solution obtained by the incubation for reaction to obtain the SERS nanoprobe 1AuNTs@4-MBA@H1; The concentration of the 4-MBA solution is 10 mM; the concentration of the AuNTs solution is 0.3 mM; the volume ratio of the 4-MBA solution to the AuNTs solution is 1:50; The activation method of the nucleic acid aptamer complementary chain H1 is: activating with TCEP buffer; The incubation conditions are: 37° C. and 80% humidity for 2 hours.

6. The preparation method according to claim 1, characterized in that: The method for coupling and modifying the Raman signal molecule DTNB and the nucleic acid aptamer complementary chain H2 to the AuNTs surface through the Au-S bond is as follows: the AuNTs solution is mixed with the Raman signal molecule DTNB solution, and then the obtained AuNTs@DTNB is added to the activated nucleic acid aptamer complementary chain H2 solution for incubation; after the incubation is completed, the BSA solution is added to the reaction solution obtained by the incubation to react, and the SERS nanoprobe 2AuNTs@DTNB@H2 is obtained; The concentration of the DTNB solution is 10 mM; the concentration of the AuNTs solution is 0.3 mM; the volume ratio of the DTNB solution to the AuNTs solution is 1:50; The activation method of the nucleic acid aptamer complementary chain H2 is: activating with TCEP buffer; The incubation conditions are: 37° C. and 80% humidity for 2 hours.

7. The preparation method according to claim 1, characterized in that: The method of combining the AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 with the SERS substrate is as follows: dropping AuNTs@4-MBA@H1 and AuNTs@DTNB@H2 onto the surface of the SERS substrate, followed by incubation to obtain the SERS sensor; The molar ratio of AuNTs@4-MBA@H1, AuNTs@DTNB@H2 and the SERS substrate is 1:1:1; The incubation conditions are: incubation at 37° C. for 1.5 h.

8. A SERS sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the SERS sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 as claimed in claim 8 in preparing a product for detecting miR-21 and miR-152.

10. A SERS microarray chip, characterized in that: The SERS sensor for detecting diabetic retinopathy serum biomarkers miR-21 and miR-152 as claimed in claim 8 is embedded in a PDMS chip.

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