Preparation method of SERS (Surface Enhanced Raman Scattering) probe for multiple miRNA array detection

Abstract

The invention discloses a preparation method of an SERS (Surface Enhanced Raman Scattering) probe for multiple miRNA (Micro Ribonucleic Acid) array detection. The SERS probe is obtained by jointly modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) and 4-mercaptobenzonitrile (MBN); according to the invention, a gold film is used as an SERS substrate and is combined with the gold film substrate through Au-SH by virtue of ssDNA of which the modification end is-SH, so that the multifunctional capture SERS substrate is constructed; according to the present invention, the modified SERS substrate can be combined with the free miRNA through the complementary base pairing, the SERS probe (mAgNPs) is obtained by jointly modifying the silver nanoparticles (AgNPs) with MPBA and MBN, and the SERS probe can be used for constructing the SERS biosensor for multiple miRNA array detection, and can achieve the synchronous detection of the multiple miRNA markers so as to improve the detection efficiency and the detection accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensors, in particular to a preparation method of a SERS probe for multiplex miRNA array detection. BACKGROUND

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease and the main cause of senile dementia. AD patients not only face symptoms such as memory decline, cognitive decline, and behavioral abnormalities, which seriously affect the quality of life, but also need long-term medical care and care, which causes great consumption of economic, human and other resources to families and society. At the same time, the psychiatric and behavioral symptoms of AD patients, such as depression, anxiety, hallucinations, and delusions, also bring heavy psychological burden to families and society. Therefore, AD has become one of the major challenges in the field of global public health.

[0003] MicroRNAs (miRNAs) as a class of non-coding RNAs with a length of about 22 nt play an important role in AD pathological process by regulating synaptic plasticity, inflammatory response and oxidative stress pathways, and are expected to become biomarkers for early diagnosis of AD. For example: miR-155 plays a key role in inflammation-induced neurogenesis defects through microglial cell activation and IL6 induction, and is sufficient to disrupt normal hippocampal development; miR-146a can reduce amyloid beta protein deposition and tau protein hyperphosphorylation through the TLR / IRAK1 / TRAF6 pathway; in primary neurons, overexpression of miR-125b leads to tau protein hyperphosphorylation and upregulation of p35, cdk5 and p44 / 42-MAPK signaling pathways, which are involved in the pathogenesis of AD; miR-34a is upregulated in the aging process and AD, and is involved in the pathogenesis of AD through pathways such as neuroinflammation. Compared with traditional biomarkers, miRNAs have the following advantages as biomarkers for AD: 1) miRNAs are stable in body fluids and easy to obtain, and can be detected through peripheral blood, urine and other samples, which belong to non-invasive examination and have good patient compliance; 2) the expression changes of miRNAs are earlier than the clinical symptoms and pathological changes of AD, which is expected to realize early diagnosis of AD; 3) the joint detection of multiple miRNAs can improve the accuracy and specificity of diagnosis.

[0004] However, traditional detection methods (such as qRT-PCR and microarray) are limited by low sensitivity (the detection limit is usually in the order of pM), poor multiplex detection capability, and weak anti-matrix interference capability, which cannot meet the clinical needs. SUMMARY

[0005] The present application aims to at least partially overcome the above technical problems and / or other potential problems existing in the prior art: the present application provides a preparation method of a SERS probe for multiplex miRNA array detection, the SERS probe prepared by the present application has a simple and rapid detection process, which can be completed in a few minutes, and is suitable for clinical rapid diagnosis. In addition, the SERS probe of the present application can construct a SERS biosensor for multiplex miRNA array detection, and can realize the synchronous detection of multiple miRNA markers, thereby improving the detection efficiency and accuracy.

[0006] The technical scheme of the present application is as follows: a preparation method of a SERS probe for multiplex miRNA array detection, which is obtained by modifying silver nanoparticles (AgNPs) with 4-mercaptobenzoic acid (MPBA) and 4-mercaptobenzonitrile (MBN).

[0007] As an optimization, the molar ratio of 4-mercaptobenzonitrile (MBN) to 4-mercaptobenzoic acid (MPBA) is 1:2.

[0008] As an optimization, the 4-mercaptobenzoic acid (MPBA), 4-mercaptobenzonitrile (MBN) and the silver nanoparticles (AgNPs) are combined through Ag-SH covalent bond.

[0009] As an optimization, the particle size of the silver nanoparticles (AgNPs) is 45-55 nm.

[0010] The present application also provides a SERS biosensor for multiplex miRNA array detection, which comprises a SERS probe, a SERS substrate and ssDNA.

[0011] As an optimization, the SERS substrate is a gold film substrate; the ssDNA has a thiol-SH modified end, one end of the ssDNA is combined with the gold film substrate through Au-SH bond, and the other end is combined with the SERS probe.

[0012] The ssDNA is complementary to the miRNA base.

[0013] The MPBA is covalently combined with the deoxyribose on the ssDNA through the hydroxyl group to generate borate ester.

[0014] The preparation method of the SERS biosensor for multiplex miRNA array detection specifically comprises the following steps: 1) Synthesis of AgNPs: AgNPs are synthesized using Lee-Meisel scheme; 2) Preparation of SERS probe mAgNPs: Add MPBA and MBN with a molar ratio of 2:1 to the AgNPs solution, stir uniformly at room temperature, then centrifuge to fix MPBA and MBN on the silver nanoparticles, and prepare mAgNPs; 3) Preparation of SERS substrate: immerse clean glass pieces in a solution containing HAuCl4, KHCO3 and glucose, soak at 40-50°C for 3-4 h, until a dense gold film is formed on the surface of each glass piece to obtain a SERS substrate; 4) Preparation of SERS biosensor for array miRNA detection: drop different thiolated ssDNA on the gold film of the SERS substrate, which are complementary to multiple miRNAs, then incubate at room temperature for 12-24 hours, and finally wash to obtain a SERS biosensor for array miRNA detection.

[0015] The beneficial effects of the present application are: the present application uses a gold film as a SERS substrate, and modifies ssDNA with a-SH to bind to the gold film substrate through Au-SH, thereby constructing a multifunctional capture SERS substrate; the modified SERS substrate can bind to free miRNA through base complementary pairing to form a double-stranded structure. Meanwhile, the present application uses 4-mercapto phenylboronic acid (MPBA) and 4-mercapto benzonitrile (MBN) to modify silver nanoparticles (AgNPs) to obtain a SERS probe (mAgNPs), wherein MPBA can bind to the other end of the deoxyribonucleic acid of ssDNA, and MBN acts as a Raman reporter molecule to output SERS signals. Through the construction of a sandwich structure SERS biosensor, high-sensitivity array detection of miRNA in the plasma of AD patients can be achieved. The SERS biosensor of the present application plays a role in amplifying SERS signals, and the detection level can reach aM level. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a schematic diagram for the preparation of the SERS probe in the embodiment.

[0017] Figure 2 It is an electron microscope image of the SERS substrate in Example 1, wherein Figure 2 A in the above is a SEM scanning image of the morphology of the SERS substrate without capturing target molecules; Figure 2 B in the above is a SEM image of the sandwich structure formed by mAgNPs, miRNA-ssDNA and the gold film substrate; Figure 2 C in the above is Figure 2 B in the above is an enlarged view of B.

[0018] Figure 3 It is a SERS and Uv-vis spectrum characterization diagram of the SERS probe in Example 2, whereinFigure 3 A is the SERS spectra of MBN-AgNPs before and after the addition of Raman reporter MBN; Figure 3 B is the SERS spectra of MPBA-AgNPs before and after the addition of Raman reporter MPBA; Figure 3 C is the SERS spectra of MBN and MPBA co-modified AgNPs before and after the addition of Raman reporter MBN and MPBA; Figure 3 D is the Uv-vis absorption spectra of AgNPs, MBN-AgNPs, MPBA-AgNPs and Raman nanotags.

[0019] Figure 4 is the SERS spectra in Example 3, where Figure 4 A is the SERS spectra of SERS substrate capturing target miRNAs without the addition of SERS probes; Figure 4 B is the SERS spectra of SERS substrate capturing target miRNAs with the addition of SERS probes.

[0020] Figure 5 is the SERS spectra in Example 4, where Figure 5 A is the SERS spectra of SERS substrate before (blue line) and after (red line) capturing target miRNAs with MBN-AgNPs as SERS probes; Figure 5 B is the SERS spectra of SERS substrate before (blue line) and after (red line) capturing target miRNAs with MPBA-AgNPs as SERS probes.

[0021] Figure 6 is the SERS spectra and linear fitting in Example 5, where Figure 6 A is the SERS spectra of SERS substrate capturing target miRNA-125b (1 nM) with the addition of Raman nanotags; Figure 6 B is the SERS spectra of miRNA-125b at different dilution concentrations; Figure 6 C is the linear fitting of the intensity of the characteristic peak at 2227 cm -1 of miRNA-125b at different concentrations; Figure 6 D is the SERS spectra of miRNA-125b and other non-complementary ssDNA and PBS.

[0022] Figure 7 is the SERS spectra of simultaneous detection of four miRNAs, miRNA-155, miRNA-146a, miRNA-125b and miRNA-34a in Example 6. DETAILED DESCRIPTION

[0023] Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are intended to be illustrative only and are not intended to limit the scope of the present application. Where specific conditions are not mentioned in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagent or instrument is not mentioned, it is a conventional product that can be obtained by commercial purchase.

[0024] Example 1 The purpose of this example is to construct a SERS biosensor to realize the array detection of AD-related miRNA (miRNA-155, miRNA-146a, miRNA-125b and miRNA-34) markers.

[0025] The SERS biosensor is prepared according to the following steps: 1) Synthesis of AgNPs Before starting the experiment, the glassware used is thoroughly washed with aqua regia, then washed with a large amount of ultrapure water, and then oven dried at 100-110°C for 1-2 h. Lee and Meisel's protocol is used to synthesize AgNPs with a particle size of 50±5 nm. Briefly, AgNO3(36 mg) is dissolved in 200 mL water and boiled under continuous stirring. Then, 4 milliliters of 1% (w / v) trisodium citrate are added. The mixture is stirred and boiled for about 1 hour, and then naturally cooled to room temperature. Before use, the solution is stored at 4°C. The resulting solution is characterized by ultraviolet-visible spectroscopy, and its maximum absorption is recorded at about 420 nm.

[0026] 2) Preparation of SERS probe mAgNPs 20 μL of 10 mM MPBA and 10 μL of 10 mM MBN solution (2:1, V / V) are added to 1 mL of AgNPs solution, and stirred at room temperature for 1 h, then centrifuged at 15000 rpm for 10 min to fix MPBA and MBN on silver nanoparticles, to prepare mAgNPs. The precipitate is resuspended in 1 mL of water. The resulting solution is stored in a 4°C refrigerator.

[0027] 3) Preparation of SERS substrate The glass sheet is first soaked in ethanol, then ultrasonically cleaned in an ultrasonic instrument for 15 minutes, then washed with ultrapure water three times, and then naturally air-dried at room temperature. The clean glass sheet is immersed in a solution containing 12 mM of HAuCl4, 0.5 M of KHCO3 and 25 mM of glucose, and soaked at 45°C for 3-4 h until a dense gold layer is formed on the surface of each glass sheet. Then wash with pure water three times, then wash with anhydrous ethanol three times, and naturally air-dry at room temperature for standby.

[0028] 4) Preparation of SERS biosensor for array miRNA detection The prepared gold film substrate was divided into different rows and columns with a marker pen, and 100 ul of four different thiolated ssDNA was added dropwise in the 1st, 2nd, 3rd and 4th columns, respectively. The four thiolated ssDNA were the complementary DNA of miRNA-155, miRNA-146a, miRNA-125b and miRNA-34, respectively. Then, the substrate was incubated in a closed humid box at room temperature for 12 hours, and then washed slowly with distilled water twice to obtain the SERS biosensor for array miRNA detection.

[0029] The SERS biosensor constructed in this example has a sandwich structure, and the preparation principle of the SERS probe mAgNPs is shown in Figure 1 The synthesis of the mAgNPs mainly includes two steps: first, AgNPs are synthesized, and then deoxyribose binding ligand MPBA and Raman reporter MBN are modified on the surface of the AgNPs through the covalent bond of Ag-SH. In the actual detection and analysis process, MPBA is covalently combined with the deoxyribose on the ssDNA through the hydroxyl group to form a borate ester, forming the upper layer of the sandwich structure, and the ssDNA is covalently combined with the gold film substrate through Au-SH to form the lower layer of the sandwich structure. The sandwich structure of the fixed ssDNA forms a double-stranded structure by base complementary pairing of the target miRNA, and the Raman reporter MBN shows enhanced SERS signal under the action of laser at 2227 cm -1 .

[0030] First, the gold film SERS substrate was characterized by field emission scanning electron microscopy, as shown in Figure 2 A of FIG. 1, uniform and large-sized block-shaped AuNPs were formed on the clean glass surface, and the roughness of the gold surface was sufficient to meet the SERS detection requirements of ssDNA functionalization. Figure 2 B of FIG. 1 and Figure 2 C of FIG. 1 are SEM images after the functionalized SERS substrate captures miRNA and combines with the SERS probe to form a sandwich structure. From Figure 2 B of FIG. 1 and Figure 2 C of FIG. 1, it can be seen that the surface of the gold film is covered with a layer of silver-white particles, which are the SERS probes mAgNPs covalently linked to the surface of the gold film. The mAgNPs, miRNA-ssDNA and the functionalized gold film substrate form a sandwich structure, which amplifies the SERS signal.

[0031] Example 2 To further verify whether MPBA and MBN modified AgNPs, SERS spectral characterization was performed in this embodiment. First, SERS characterization was performed on AgNPs modified with MBN alone. Figure 3 As shown in A, compared with the Bare AgNPs group, MBN-AgNPs were at 2227 cm⁻¹ -1 The appearance of a unique peak for cyanide derivatives confirms the successful modification of AgNPs with MBN. Similarly, this example also performed SERS characterization on MPBA after modification of AgNPs alone, as shown... Figure 3 As shown in B, compared with the Bare AgNPs group, MPBA-AgNPs were found to have a lower concentration at 998 cm⁻¹. -1 The presence of distinct SERS characteristic peaks confirms the successful modification of AgNPs with MPBA. Furthermore, in this embodiment, AgNPs were modified with MBN : MPBA at a ratio of 1 : 2, followed by SERS testing. Figure 3 As shown in C, MBN is at 2227 cm. -1 A distinctive Raman peak for cyanide derivatives appeared in the interference-free region, with MPBA at 998 cm⁻¹. -1 The presence of distinct SERS characteristic peaks indicates that MPBA and MBN have been successfully modified onto AgNPs.

[0032] Furthermore, in this embodiment, UV-Vis spectroscopy was used to characterize AgNPs, MBN-modified AgNPs, MPBA-modified AgNPs, and AgNPs co-modified with MBN and MPBA, respectively. The results are as follows: Figure 3 As shown in Figure D, the UV-vis spectra of AgNPs, MBN-modified AgNPs, and MPBA-modified AgNPs show almost no change. However, when MPBA and MBN are co-modified onto AgNPs, the LSPR peak of the nanoparticles red-shifts towards higher wavenumbers, indicating that MPBA and MBN have been successfully modified onto AgNPs.

[0033] Example 3 To verify the SERS response of the sandwich-structured SERS biosensor to the target miRNA, this embodiment first constructed a miRNA capture substrate using a gold membrane modified with ssDNA complementary to miRNA-125b. In the presence of the target miRNA-125b, miRNA-125b was captured by hybridization with ssDNA through base complementarity pairing. Subsequent SERS testing yielded the following results: Figure 4 As shown in A, since it did not bind to the SERS probe, it was at 2227 cm⁻¹ -1The characteristic peaks of the Raman reporter molecule did not appear. When the SERS probe (mAgNPs) was added, the MPBA on the mAgNPs formed a sandwich structure with the deoxyribose on the ssDNA through covalent bonds, and the Raman reporter molecule MBN appeared at 2227 cm -1 The characteristic peaks of the Raman reporter molecule did not appear. When the SERS probe (mAgNPs) was added, the MPBA on the mAgNPs formed a sandwich structure with the deoxyribose on the ssDNA through covalent bonds, and the Raman reporter molecule MBN appeared at 2227 cm Figure 4 The characteristic peaks of the Raman reporter molecule did not appear. When the SERS probe (mAgNPs) was added, the MPBA on the mAgNPs formed a sandwich structure with the deoxyribose on the ssDNA through covalent bonds, and the Raman reporter molecule MBN appeared at 2227 cm The characteristic peaks of the Raman reporter molecule did not appear. When the SERS probe (mAgNPs) was added, the MPBA on the mAgNPs formed a sandwich structure with the deoxyribose on the ssDNA through covalent bonds, and the Raman reporter molecule MBN appeared at 2227 cm

[0034] The characteristic peaks of the Raman reporter molecule did not appear. When the SERS probe (mAgNPs) was added, the MPBA on the mAgNPs formed a sandwich structure with the deoxyribose on the ssDNA through covalent bonds, and the Raman reporter molecule MBN appeared at 2227 cm Example 4 Figure 5 In order to evaluate the non-specific binding SERS signal background of the SERS probe (mAgNPs), this example only used the Raman reporter molecule MBN to modify the AgNPs. In the presence of the ssDNA functionalized substrate, the Raman test results showed that whether the target miRNA was present or not, the AgNP-MBN had no SERS response, as shown in

[0035] Similarly, this example only used MPBA to modify the AgNPs. Since the hydroxyl group on the MPBA can bind to the deoxyribose on the ssDNA, if the target miRNA is present, the SERS characteristic peaks of the MPBA will appear at 998 cm -1 , as shown in Figure 5 This is because the MPBA binds to the deoxyribose on the ssDNA, which shortens the distance between the AgNP and the SERS substrate, resulting in a significant SERS effect. When the target miRNA is not present, this example found that the SERS peaks shifted significantly, because although the AgNP-MPBA and the ssDNA functionalized capture substrate can also form a sandwich structure, the absence of the target molecule causes the SERS peaks to shift significantly.

[0036] Example 5 In order to evaluate the sensitivity of the SERS biosensor in miRNA detection, this example selected miRNA-125b associated with AD as the target analyte, and based on the cDNA modified plasmonic array library detection scheme, this example conducted SERS spectral sensitivity tests, and the results are shown in Figure 6 A, in the presence of the target analyte miRNA-125b, the SERS spectrum appeared at 998 cm -1 and 2227 cm -1 , and the characteristic peaks were obvious.

[0037] To verify the relationship between miRNA concentration and SERS signal intensity, this example performed SERS detection on miRNA-125b under different concentration conditions, such as... Figure 6 As shown in B, at 2227 cm -1 The Raman characteristic peak intensity at 2227 cm⁻¹ gradually increased with increasing target miRNA concentration. The Raman reporter molecule at 2227 cm⁻¹... -1 The characteristic peak intensity at a given location was used as a function of the logarithm of the target miRNA concentration. Data was fitted using a dose-response function, and a corresponding standard curve was plotted. The results are as follows: Figure 6 As shown in C. According to the fitted curve, the ssDNA-functionalized substrate has excellent targeting ability for miRNAs, and the SERS substrate has a targeting ability of 0–10. 7 aM(y = 648.36x + 308.75, R) 2 The SERS spectrum exhibited good linearity within the range of 0.98, providing a solid foundation for the quantitative analysis of miRNAs. Furthermore, to verify the specificity of the SERS capture substrate, this example also compared the SERS spectra of non-coding cDNA, antisense miRNA, and PBS after interaction with the SERS capture substrate. The results showed that only miRNA-125b exhibited linearity within the range of 2227 cm⁻¹. -1 Raman characteristic peaks appeared at such locations, such as Figure 6 As shown in D in the diagram. Therefore, the SERS biosensor and the corresponding miRNA in this embodiment have excellent targeting effects and extremely high sensitivity.

[0038] Example 6 Multiple capture SERS substrates were constructed by modifying the gold membrane surface with various corresponding ssDNAs (each ssDNA being complementary to the corresponding target miRNA bases). These substrates can specifically target miRNA-155, miRNA-146a, miRNA-125b, and miRNA-34a, respectively. Subsequently, the four target miRNAs were mixed with the multiple capture SERS substrates. After the target miRNAs were captured by the corresponding ssDNAs on the gold membrane substrate surface, SERS probes (mAgNPs) were added to form a sandwich complex, enabling multiplex detection and analysis of the four miRNA arrays. The SERS test results are shown below. Figure 7 As shown, the four miRNAs miRNA-155, miRNA-146a, miRNA-125b, and miRNA-34a were at 2227 cm⁻¹. -1The SERS biosensor constructed in the embodiment shows different intensities of SERS enhancement. The SERS biosensor constructed in the embodiment has good specificity and sensitivity to four miRNAs, i.e., miRNA-155, miRNA-146a, miRNA-125b and miRNA-34a. By constructing an array type multiplexed capture SERS substrate, simultaneous detection of multiple miRNA markers can be realized, so that the detection efficiency and accuracy are improved.

[0039] The above merely describes the characteristic embodiments of the present application, and does not constitute any limitation on the protection scope of the present application. Any technical solution formed by equivalent exchange or equivalent replacement falls within the protection scope of the present application.

Claims

1. A method for preparing a SERS probe for multiplex miRNA array detection, characterized in that, The silver nanoparticles (AgNPs) were obtained by co-modifying silver nanoparticles with 4-mercaptophenylboronic acid (MPBA) and 4-mercaptobenzonitrile (MBN).

2. The method for preparing SERS probes for multiplex miRNA array detection according to claim 1, characterized in that, The molar ratio of 4-mercaptobenzonitrile (MBN) to 4-mercaptophenylboronic acid (MPBA) is 1:

2.

3. The method for preparing SERS probes for multiplex miRNA array detection according to claim 2, characterized in that, The 4-mercaptophenylboronic acid (MPBA), 4-mercaptobenzonitrile (MBN), and the silver nanoparticles (AgNPs) are bonded together by Ag-SH covalent bonds.

4. The method for preparing SERS probes for multiplex miRNA array detection according to claim 3, characterized in that, The silver nanoparticles (AgNPs) have a particle size of 45-55 nm.

5. A SERS biosensor for multiplex miRNA array detection, characterized in that, This includes SERS probes, SERS substrates, and ssDNA.

6. The SERS biosensor for multiplex miRNA array detection according to claim 5, characterized in that, The SERS substrate is a gold membrane substrate; the ssDNA has a thiol-SH modified end, one end of the ssDNA binds to the gold membrane substrate through an Au-SH bond, and the other end binds to the SERS probe.

7. The SERS biosensor for multiplex miRNA array detection according to claim 6, characterized in that, The ssDNA and miRNA have complementary base pairing.

8. The SERS biosensor for multiplex miRNA array detection according to claim 7, characterized in that, The MPBA forms a borate ester by covalently binding a hydroxyl group to the deoxyribose on ssDNA.

9. A method for preparing a SERS biosensor for multiplex miRNA array detection, characterized in that, Specifically, the following steps are included: 1) Synthesis of AgNPs: AgNPs were synthesized using the Lee-Meisel method; 2) Preparation of SERS probe mAgNPs: MPBA and MBN in a molar ratio of 2:1 were added to the AgNPs solution and stirred evenly at room temperature. Then, the mixture was centrifuged to fix MPBA and MBN on the silver nanoparticles to obtain mAgNPs. 3) Preparation of SERS substrate: Clean glass slides are immersed in a solution containing HAuCl4, KHCO3 and glucose at 40-50℃ for 3-4 h until a dense gold film is formed on the surface of each glass slide to obtain the SERS substrate. 4) Preparation of SERS biosensor for array miRNA detection: Different thiolated ssDNAs are dropped onto a gold membrane on a SERS substrate. These thiolated ssDNAs are complementary to various miRNAs. The membrane is then incubated at room temperature for 12-24 hours and finally washed to obtain the SERS biosensor for array miRNA detection.

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