A method for preparing a SERS probe for multiplex miRNA array detection

Abstract

The application discloses a preparation method of a SERS probe for multiplex miRNA array detection, and the silver nanoparticles (AgNPs) are obtained by being jointly modified by 4-mercapto-benzoic acid (MPBA) and 4-mercapto-benzonitrile (MBN). Gold film is used as a SERS substrate, and ssDNA with a-SH at the end is combined with the gold film substrate through Au-SH, so that a multifunctional capture SERS substrate is constructed. The modified SERS substrate can be combined with free miRNA through base complementary pairing. Meanwhile, the silver nanoparticles (AgNPs) are jointly modified by MPBA and MBN to obtain the SERS probe (mAgNPs), and the SERS probe can construct a SERS biosensor for multiplex miRNA array detection, so that the synchronous detection of multiple miRNA markers can be realized, and the detection efficiency and accuracy are improved.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, specifically to a method for preparing a SERS probe for multiplex miRNA array detection. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease and a leading cause of dementia in the elderly. AD patients not only face symptoms such as memory loss, cognitive decline, and behavioral abnormalities, severely impacting their quality of life, but also require long-term medical care and support, placing a tremendous strain on the economic and human resources of families and society. Simultaneously, the psychosomatic symptoms of AD patients, such as depression, anxiety, hallucinations, and delusions, also impose a heavy psychological burden on families and society. Therefore, AD has become one of the major challenges in global public health.

[0003] MicroRNAs (miRNAs), a class of non-coding RNAs approximately 22 nt in length, play a crucial role in the pathogenesis of Alzheimer's disease (AD) by regulating pathways such as synaptic plasticity, inflammatory responses, and oxidative stress, 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 activation and IL-6 induction, and is sufficient to disrupt normal hippocampal development; miR-146a can reduce amyloid-β protein deposition and tau protein hyperphosphorylation through the TLR / IRAK1 / TRAF6 pathway; in primary neurons, miR-125b overexpression leads to tau protein hyperphosphorylation and upregulates the p35, cdk5, and p44 / 42-MAPK signaling pathways, participating in the pathogenesis of AD; miR-34a expression is increased during aging and in AD, participating in the pathogenesis of AD through neuroinflammation and other pathways. Compared with traditional biomarkers, miRNAs have the following advantages as biomarkers for Alzheimer's disease (AD): 1) miRNAs are stable in body fluids, easy to obtain, and can be detected through samples such as peripheral blood and urine. This is a non-invasive test with good patient compliance; 2) Changes in miRNA expression occur earlier than the clinical symptoms and pathological changes of AD, which may enable early diagnosis of AD; 3) The combined detection of multiple miRNAs can improve the accuracy and specificity of diagnosis.

[0004] However, traditional detection methods (such as qRT-PCR and microarrays) are limited by low sensitivity (detection limit is usually at the pM level), poor multiplexing capability, and weak resistance to matrix interference, which cannot meet clinical needs. Summary of the Invention

[0005] The present invention aims to at least partially overcome the aforementioned technical problems and / or other potential problems existing in the prior art: The present invention provides a method for preparing a SERS probe for multiplex miRNA array detection. The detection process of the SERS probe prepared by the present invention is simple and rapid, usually taking only a few minutes, making it suitable for rapid clinical diagnosis. Furthermore, the SERS probe of the present invention can be used to construct a SERS biosensor for multiplex miRNA array detection, enabling simultaneous detection of multiple miRNA biomarkers, thereby improving detection efficiency and accuracy.

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

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

[0008] As an optimization, the 4-mercaptophenylboronic acid (MPBA), 4-mercaptobenzonitrile (MBN), and the silver nanoparticles (AgNPs) are covalently bonded by Ag-SH bonds.

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

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

[0011] As an optimization, 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.

[0012] The ssDNA and miRNA have complementary base pairing.

[0013] The MPBA forms a borate ester by covalently binding a hydroxyl group to the deoxyribose on ssDNA.

[0014] The method for preparing the SERS biosensor for multiplex miRNA array detection specifically includes the following steps:

[0015] 1) Synthesis of AgNPs: AgNPs were synthesized using the Lee-Meisel method;

[0016] 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.

[0017] 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.

[0018] 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.

[0019] The beneficial effects of this invention are as follows: This invention uses a gold membrane as the SERS substrate and constructs a multifunctional SERS-capturing substrate by binding ssDNA with a modified -SH end to the gold membrane substrate via Au-SH. The modified SERS substrate can bind to free miRNA through base complementary pairing to form a double-stranded structure. Simultaneously, this invention uses 4-mercaptophenylboronic acid (MPBA) and 4-mercaptobenzonitrile (MBN) to co-modify silver nanoparticles (AgNPs) to obtain SERS probes (mAgNPs). MPBA can bind to the deoxyribonucleic acid at the other end of the ssDNA, while MBN acts as a Raman reporter molecule to output the SERS signal. Through the construction of a sandwich-structured SERS biosensor, highly sensitive array detection of miRNA in the plasma of AD patients can be achieved. The SERS biosensor of this invention amplifies the SERS signal, achieving a detection level at the aM level. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the fabrication principle of the SERS probe in this embodiment.

[0021] Figure 2 Here is an electron microscope image of the SERS substrate in Example 1, where Figure 2 In the figure, A represents the SERS substrate morphology of the target molecules not captured by SEM scanning; Figure 2 B in the image is a SEM image of the sandwich structure formed by mAgNPs, miRNA-ssDNA, and the gold membrane substrate. Figure 2 C in the text is Figure 2 A magnified view of B in the image.

[0022] Figure 3The images show the SERS and UV-Vis spectra of the SERS probe in Example 2. Figure 3 In the figure, A represents the SERS spectra of AgNP before and after modification with the Raman reporter molecule MBN; Figure 3 B in the figure represents the SERS spectra of AgNPs before and after MPBA modification; Figure 3 C in the figure represents the SERS spectra of AgNPs before and after co-modification with MBN and MPBA; Figure 3 D in the figure represents the UV-Vis absorption spectra of AgNPs, MBN-AgNPs, MPBA-AgNPs, and Raman nanotags.

[0023] Figure 4 The SERS spectrum in Example 3 is shown below. Figure 4 In the figure, A represents the SERS spectrum collected after the target miRNA was captured by the SERS substrate without the addition of the SERS probe; Figure 4 B in the figure represents the SERS spectrum of the target miRNA captured by the SERS substrate after the addition of the SERS probe.

[0024] Figure 5 The SERS spectrum is shown in Example 4, where Figure 5 In the diagram, A represents AgNPs modified with only MBN labeling, and the SERS spectra of the SERS substrate before (blue line) and after (red line) capture of the target miRNA are tested. Figure 5 B in the diagram represents AgNPs modified with MPBA label only, and the SERS spectra of the SERS substrate before (blue line) and after (red line) capture of the target miRNA.

[0025] Figure 6 The image shows the SERS spectrum and linear fit plot from Example 5. Figure 6 In the figure, A represents the SERS spectrum of the target miRNA-125b (1 nM) captured by the SERS substrate after the addition of Raman nanotags; Figure 6 B in the figure represents the concentration-dependent SERS spectra of miRNA-125b at different dilution concentrations; Figure 6 In the figure, C represents different concentrations of miRNA-125b at 2227 cm⁻¹. -1 Linear fitting of the characteristic peak intensity at the location; Figure 6 D in the image represents the SERS spectra of miRNA-125b with other non-complementary ssDNA and PBS.

[0026] Figure 7 The SERS spectra of four miRNAs, miRNA-155, miRNA-146a, miRNA-125b and miRNA-34a, were detected simultaneously in Example 6. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] Example 1

[0029] The purpose of this embodiment is to construct a SERS biosensor to achieve array detection of AD-related miRNA (miRNA-155, miRNA-146a, miRNA-125b and miRNA-34) biomarkers.

[0030] The SERS biosensor was prepared according to the following steps:

[0031] 1) Synthesis of AgNPs

[0032] Before starting the experiment, all glassware was thoroughly washed with aqua regia, rinsed with plenty of ultrapure water, and then dried in an oven at 100-110°C for 1-2 hours. AgNPs with a particle size of 50±5 nm were synthesized using the Lee and Meisel method. In short, AgNO3 (36 mg) was dissolved in 200 mL of water and boiled with continuous stirring. Then, 4 mL of 1% (w / v) trisodium citrate was added. The mixture was stirred and boiled for about 1 hour, then allowed to cool naturally to room temperature. The solution was stored at 4°C before use. The resulting solution was characterized by UV-Vis spectroscopy, and its maximum absorbance was recorded at approximately 420 nm.

[0033] 2) Preparation of SERS probe mAgNPs

[0034] 20 µL of 10 mM MPBA and 10 µL of 10 mM MBN solution (2:1, v / v) were added to 1 mL of AgNPs solution and stirred at room temperature for 1 h. The mixture was then centrifuged at 15000 rpm for 10 min to immobilize MPBA and MBN on the silver nanoparticles, yielding mAgNPs. The precipitate was resuspended in 1 mL of water. The resulting solution was stored at 4 °C.

[0035] 3) Preparation of SERS substrate

[0036] The glass slides were first soaked in ethanol, then ultrasonically cleaned for 15 minutes, rinsed three times with ultrapure water, and then air-dried at room temperature. The clean glass slides were then immersed in a solution containing 12 mM HAuCl4, 0.5 M KHCO3, and 25 mM glucose at 45°C for 3-4 hours until a dense gold layer formed on the surface of each slide. They were then rinsed three times with pure water, then washed three times with anhydrous ethanol, and air-dried at room temperature for later use.

[0037] 4) Fabrication of a SERS biosensor for array miRNA detection

[0038] The prepared gold film substrate was divided into different rows and columns using a marker. 100 μL of four different thiolated ssDNAs were added to columns 1, 2, 3, and 4, respectively. These four thiolated ssDNAs were complementary DNAs of miRNA-155, miRNA-146a, miRNA-125b, and miRNA-34. The substrate was then incubated at room temperature in a sealed humidified chamber for 12 hours, followed by two slow washes with distilled water to obtain the SERS biosensor for array miRNA detection.

[0039] The SERS biosensor constructed in this embodiment has a sandwich structure, and the preparation principle of the SERS probe mAgNPs is as follows: Figure 1 As shown, the synthesis of mAgNPs mainly includes two steps: first, AgNPs are synthesized, and then the deoxyribose-binding ligand MPBA and the Raman reporter molecule MBN are modified onto the surface of AgNPs via covalent bonding of Ag-SH. In the actual detection and analysis process, MPBA covalently binds to the deoxyribose on ssDNA via its hydroxyl groups to form a borate ester, forming the upper layer of a sandwich structure. ssDNA covalently binds to the gold membrane substrate via Au-SH, forming the lower layer of the sandwich structure. The immobilized ssDNA forms the sandwich structure's interlayer, which targets the corresponding miRNA through base complementarity pairing to form a double-stranded structure. The Raman reporter molecule MBN, under the influence of a laser, is activated at 2227 cm⁻¹. -1 It exhibits an enhanced SERS signal.

[0040] First, the gold film SERS substrate was characterized using field emission scanning electron microscopy, such as... Figure 2 As shown in Figure A, large, uniformly dispersed AuNPs were formed on a clean glass surface, and the roughness of the gold surface was sufficient to meet the SERS detection requirements for ssDNA functionalization. Figure 2 B and Figure 2 C in the image represents a SEM image of a functionalized SERS substrate capturing miRNA and binding to an SERS probe to form a sandwich structure. Figure 2 B and Figure 2As shown in Figure C, a layer of silvery-white particles covers the surface of the gold membrane. These silvery-white particles are SERS probes mAgNPs covalently linked to the surface of the gold membrane. mAgNPs, miRNA-ssDNA, and the functionalized gold membrane substrate form a sandwich structure, which amplifies the SERS signal.

[0041] Example 2

[0042] 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 be 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.

[0043] 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.

[0044] Example 3

[0045] 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 -1 No Raman reporter molecule characteristic peaks were observed at this location. However, upon addition of the SERS probe (mAgNPs), the MPBA on the mAgNPs covalently binds to the deoxyribose on the ssDNA, forming a sandwich structure. Simultaneously, the Raman reporter molecule MBN was observed at 2227 cm⁻¹. -1 Distinctive characteristic peaks appeared, such as Figure 4 As shown in B in the diagram.

[0046] Example 4

[0047] To assess the SERS signaling background caused by non-specific binding of SERS probes (mAgNPs), this embodiment only modified AgNPs with the Raman reporter molecule MBN. In the presence of an ssDNA-functionalized substrate, Raman spectroscopy results showed that AgNP-MBN did not exhibit a SERS response regardless of the presence or absence of the target miRNA. Figure 5 As shown in A, the absence of the ligand molecule prevents AgNP-MBN from forming a special sandwich structure with the functionalized SERS substrate, thus hindering the reduction of the distance between AgNP and the substrate. Therefore, no SERS response occurs even in the presence of the reporter molecule.

[0048] Similarly, in this embodiment, only MPBA is used to modify AgNPs. Since the hydroxyl groups on MPBA can bind to the deoxyribose on ssDNA, if the target miRNA is present, it will be able to bind at 998 cm⁻¹. -1 The characteristic SERS peak of MPBA appeared at [location], such as [example]. Figure 5 As shown in B in the figure. This is because MPBA binds to the deoxyribose on ssDNA, bringing AgNP closer to the SERS substrate and producing a significant SERS effect. When the target miRNA is absent, this embodiment found a significant shift in the SERS peak. This is because although the AgNP-MPBA and ssDNA functionalized capture substrate can also form a sandwich structure, the absence of the target molecule leads to a significant shift in the SERS peak.

[0049] Example 5

[0050] To evaluate the sensitivity of the SERS biosensor in miRNA detection, this embodiment selected AD-related miRNA-125b as the target analyte. Based on a cDNA-modified plasmonic array library detection scheme, SERS spectral sensitivity tests were performed. The results are as follows: Figure 6 As shown in Figure A, in the presence of the target analyte miRNA-125b, the SERS spectrum at 998 cm⁻¹ -1 and 2227 cm -1 All showed obvious characteristic peaks.

[0051] 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.

[0052] Example 6

[0053] 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. -1 Different intensities of SERS enhancement were observed at the locations. The SERS biosensor constructed in this embodiment exhibits excellent specificity and sensitivity for four miRNAs: miRNA-155, miRNA-146a, miRNA-125b, and miRNA-34a. By constructing an array-type multiplex capture SERS substrate, simultaneous detection of multiple miRNA biomarkers can be achieved, thereby improving detection efficiency and accuracy.

[0054] The above are merely exemplary embodiments of the present invention and do not constitute any limitation on the scope of protection of the present invention. All technical solutions formed by equivalent exchange or substitution fall within the scope of protection of the present invention.

Claims

1. A SERS biosensor for multiplex miRNA array detection, characterized in that, The device includes a SERS probe, a SERS substrate, and ssDNA; 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 via an Au-SH bond, and the other end binds to the SERS probe; the SERS probe is obtained by co-modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) and 4-mercaptobenzonitrile (MBN); the 4-mercaptophenylboronic acid (MPBA), 4-mercaptobenzonitrile (MBN) are covalently bonded to the silver nanoparticles (AgNPs) via Ag-SH bonds.

2. The SERS biosensor 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 SERS biosensor for multiplex miRNA array detection according to claim 2, characterized in that, The silver nanoparticles (AgNPs) have a particle size of 45-55 nm.

4. The SERS biosensor for multiplex miRNA array detection according to claim 3, characterized in that, The ssDNA and miRNA have complementary base pairing.

5. The SERS biosensor for multiplex miRNA array detection according to claim 4, characterized in that, The MPBA forms a borate ester by covalently binding a hydroxyl group to the deoxyribose on ssDNA.

6. 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; 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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