A surface-enhanced Raman scattering probe and its preparation method and application

Abstract

The present invention discloses a surface-enhanced Raman scattering (SERS) probe, its preparation method, and application. The SERS probe comprises a nucleic acid probe and a SERS substrate, wherein the SERS substrate comprises gold nanorods and graphene quantum dots uniformly distributed around the gold nanorods. The SERS probe provided by the present invention uses graphene quantum dots as Raman signal molecules, gold nanorods to enhance characteristic SERS signals, and constructs a SERS substrate as a SERS substrate. Nucleic acid probes complementary to target miRNAs are immobilized on the graphene quantum dots, which serve as Raman reporter molecules. The gold nanorods are used to significantly enhance the Raman signal, effectively improving detection accuracy. The target molecule content in the sample is determined by analyzing changes in the intensity of characteristic peaks in the Raman spectrum.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a surface enhanced Raman scattering probe and a preparation method and application thereof. Background Art

[0002] Retinoblastoma (RB) is a common intraocular malignancy in infants and young children, making early diagnosis and effective monitoring of RB crucial. However, traditional detection methods typically rely on fundus examination, imaging analysis, and histopathological examination, which are complex, costly, and invasive. New detection methods are urgently needed to identify tumor-associated biomarkers for early diagnosis. MicroRNAs (miRNAs) are a class of noncoding single-stranded RNA molecules consisting of approximately 22 nucleotides. Numerous studies have demonstrated that these molecules are closely associated with the development, progression, and metastasis of RB. For example, miRNA-17 and miRNA-18a are significantly overexpressed in RB. Trace nucleic acid amplification techniques, including polymerase chain reaction, loop-mediated isothermal amplification, and recombinase polymerase amplification, have been commonly used to detect miRNAs. However, these techniques suffer from difficulties in extraction, complex procedures, stringent reaction conditions, and susceptibility to contamination. Summary of the Invention

[0003] The present invention aims to provide a surface-enhanced Raman scattering probe and its preparation method and application, which solves the problems faced in the prior art such as difficult extraction, complex operation, strict reaction conditions and susceptibility to contamination.

[0004] A first aspect of the present invention provides a surface-enhanced Raman scattering probe, comprising a nucleic acid probe and a surface-enhanced Raman scattering substrate, wherein the surface-enhanced Raman scattering substrate comprises gold nanorods and graphene quantum dots uniformly distributed around the gold nanorods.

[0005] In one embodiment of the present invention, the preparation of the surface enhanced Raman scattering substrate comprises the following steps:

[0006] S1, synthesizing gold seed solution using the seed growth method;

[0007] S2. HAuCl4 (tetrachloroauric acid) solution, AgNO3 solution, and HCl were sequentially added to a CTAB (hexadecyltrimethylammonium bromide) solution. After stirring at room temperature for 30 minutes, AA (ascorbic acid solution) was added dropwise and stirred vigorously to obtain a colorless solution. The gold seed solution prepared in step S1 was added to the mixture. After further stirring for 2 minutes, the mixture was allowed to stand at 30°C overnight. The obtained gold nanorods were washed and purified with ultrapure water and then dispersed in ultrapure water to obtain an AuNR solution.

[0008] S3, adding thiolated polyethylene glycol to the AuNR solution, stirring the mixture at room temperature for 24 hours, washing it twice with ultrapure water to obtain AuNR@PEG, and redispersing it in ultrapure water to obtain an AuNR@PEG suspension;

[0009] S4. Add GQDs to the AuNR@PEG suspension obtained in step S3, stir overnight, and wash twice with ultrapure water to obtain a surface-enhanced Raman scattering substrate.

[0010] In one embodiment of the present invention, the gold seed solution is prepared in step S1 as follows: HAuCl4 solution is added to CTAB solution, freshly prepared glacial NaBH4 is added while stirring, stirring is continued until a yellow solution is obtained, and the solution is allowed to stand at 30°C in the dark for 6 hours to obtain the gold seed solution.

[0011] In one embodiment of the present invention, the concentration of HAuCl4 solution is 0.01M, the concentration of CTAB solution is 0.1M, the concentration of glacial NaBH4 is 0.11M, and the volume ratio of HAuCl4 solution, CTAB solution, and glacial NaBH4 is 0.25:7.5:0.0.6.

[0012] In one embodiment of the present invention, in step S2, the concentration of the CTAB solution is 0.1 M, the concentration of the HAuCl4 solution is 0.01 M, the concentration of the AgNO3 solution is 0.01 M, the concentration of the AA solution is 0.1 M, and the volume ratio of the CTAB solution, HAuCl4 solution, AgNO3 solution, HCl, AA solution, and gold seed solution is 50:2.5:0.4:0.04:0.4:0.125.

[0013] In one embodiment of the present invention, in step S3, the concentration of thiolated polyethylene glycol is 0.1 mM, the concentration of the AuNR solution is 1 nM, and the volume ratio of thiolated polyethylene glycol to the AuNR solution is 0.1:10.

[0014] In one embodiment of the present invention, in step S4, the concentration of GQDs is 1 mg / mL, and the volume ratio of AuNR@PEG suspension to GQDs is 10:0.0.

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned surface-enhanced Raman scattering probe, comprising the following steps: incubating the nucleic acid probe with a surface-enhanced Raman scattering substrate at room temperature overnight, and purifying unbound probes to obtain the surface-enhanced Raman scattering probe.

[0016] In one embodiment of the present invention, the surface-enhanced Raman scattering probe preparation method, the nucleic acid probe is a miRNA-17 detection probe or a miRNA-18a detection probe.

[0017] A third aspect of the present invention provides the use of the surface-enhanced Raman scattering probe in the preparation of a product for detecting the expression level of a retinoblastoma biomarker.

[0018] In one embodiment of the present invention, the product for detecting the expression level of retinoblastoma biomarkers includes a kit.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The surface-enhanced Raman scattering (SERS) probe provided by the present invention uses graphene quantum dots (GQDs) as Raman signal molecules and gold nanorods (AuNR) to enhance the characteristic SERS signal. A surface-enhanced Raman scattering substrate (AuNR@GQDs composite material) is constructed as a SERS substrate. Nucleic acid probes complementary to the target miRNAs are fixed on the Raman reporter molecules graphene quantum dots. The gold nanorods are used to achieve significant enhancement of the Raman signal, effectively improving the accuracy of detection. The target molecule content in the sample is determined by analyzing the changes in the characteristic peak intensity in its Raman spectrum.

[0021] 2. The surface-enhanced Raman scattering probe provided by the present invention is used to prepare products for detecting the expression level of retinoblastoma biomarkers. Since the Raman signal intensity generated by the probe has a good linear positive correlation with the concentration of the target molecule, it is suitable for the detection of trace nucleic acid molecules in liquid samples.

[0022] 3. The surface-enhanced Raman scattering probe provided by the present invention is used to detect the expression level of retinoblastoma biomarkers, which solves the problems faced by the existing technology such as difficult extraction, complex operation, strict reaction conditions and susceptibility to contamination.

[0023] Figures in the specification

[0024] Figure 1 is the relative expression level of miRNAs in Y79 cells in Example 1;

[0025] Figure 2 The graph is the characteristic test result of AuNR@GQDs in Example 2; Figure 2 A is the transmission electron microscopy images of AuNR, GQDs, and AuNR@GQDs; Figure 2 B is the UV-visible absorption spectra of AuNR, GQDs, and AuNR@GQDs; Figure 2 C is the Raman spectra of GQDs and AuNR@GQDs;

[0026] Figure 3 The results of the SERS detection probe in Example 3 for measuring the sensitivity and specificity of target miRNAs are shown in FIG. Figure 3 A is the SERS intensity corresponding to different concentrations of miR-17 and miR-18a; Figure 3 B and Figure 3 C is the linear regression analysis between SERS intensity and target miRNAs concentration; Figure 3 D is the SERS intensity map of target miRNAs and their corresponding single-base mutants;

[0027] Figure 4 This is a graph showing the results of the SERS detection probe detecting miRNAs in the supernatant of RB cells in Example 4; wherein, 1X: undiluted cell culture supernatant; 10X: 10-fold dilution; 100X: 100-fold dilution. DETAILED DESCRIPTION

[0028] In this document, the term "from a value to another value" is used as a summary to avoid listing all values ​​within the range. Therefore, a description of a specific numerical range encompasses any value within that range and any smaller numerical ranges defined by any value within that range, just as if the values ​​and smaller numerical ranges were explicitly stated in the specification.

[0029] In this application, unless otherwise stated, the use of “or” means “and / or.” In the case of multiple dependent claims, use of “or” is used only in the alternative to refer to more than one of the preceding independent or dependent claims.

[0030] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0031] The present invention is further described in detail below through specific examples.

[0032] Example 1

[0033] Screening of RB highly expressed miRNAs

[0034] Experimental Materials: Fetal bovine serum, RPMI-1640 medium, and DMEM medium were purchased from Gibco (USA); the retinoblastoma cell line Y79 and the normal control human retinal pigment epithelial cell line ARPE19 were purchased from the American Type Culture Collection (ATCC). The miRNA extraction kit (EZB-miRN1), miRNA reverse transcription kit (EZB-miRT4), and qPCR kit (EZB-Probe-R2) were purchased from EZbioscience (USA).

[0035] The miRNA-17-92 cluster, miRNA-24, and miRNA-181c are highly expressed in RB. In this example, the expression of the above miRNAs was evaluated in RB cell lines and normal control cell lines using a real-time fluorescence quantitative nucleic acid amplification detection system (qPCR). Cells were treated with the lysis buffer in the miRNAs extraction kit (EZB-miRN1), and miRNAs were extracted after RNA binding, washing, and RNA elution. The miRNAs reverse transcription kit (EZB-miRT4) uses a method of simultaneous Poly (A) tailing reaction and reverse transcription reaction to synthesize the first chain of miRNA cDNA. The extracted miRNAs were mixed with 1 μL gDNA Remover, 5 μL miRNA RT Buffer, 2 μL miRNA RT Enzyme Mix, and Nuclease-free ddH2O in the kit to form a 20 μL system, and the corresponding first chain of cDNA was obtained after the reverse transcription reaction procedure. The cDNA obtained by reverse transcription, 0.4μL Specific forword qPCR Primer, 5μL EZ-Probe qPCR Master Mix for microRNA, 0.2μL Probe, 0.4μL Universal 3'qPCR Primer, and ddH2O in the qPCR kit were combined into a 10μL system. After the qPCR reaction procedure, the corresponding CT value was obtained and the relative expression of cellular miRNAs was calculated. The qPCR experimental data showed that miRNA-17 and miRNA-18a were significantly overexpressed in RB, while there was no significant difference in the expression levels of miRNA-24 and miRNA-181c ( Figure 1 Therefore, the present invention selects miRNA-17 and miRNA-18a as RB detection markers and develops SERS detection probes.

[0036] Example 2

[0037] This example provides a surface-enhanced Raman scattering substrate (AuNR@GQDs composite material) and its preparation, characterization, and property analysis.

[0038] Materials: Cetyltrimethylammonium bromide (CTAB), silver nitrate (AgNO₃), sodium borohydride (NaBH₄), ascorbic acid (AA), and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (Shanghai, China); tetrachloroauric acid trihydrate (HAuCl₄·3H₂O) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; graphene quantum dots (GQDs) (5 mg / mL) were purchased from Nanjing Jingtan Nanotechnology Co., Ltd.; and thiolated polyethylene glycol 200 (PEG200-SH) was purchased from Shanghai Tuoyang Biotechnology Co., Ltd. A 120 kV transmission electron microscope (Talos L120C G2, Thermo Fisher Scientific, USA) was used; a dispersive confocal Raman spectrometer (Senterra R200-L, Bruker, Germany) was used; and a UV-visible spectrophotometer (EV300, Thermo Fisher Scientific, USA) was used.

[0039] The preparation of the AuNR@GQDs composite material in this embodiment includes the following steps:

[0040] (1) Synthesis of gold nanorods by seed growth method: 0.25 mL of HAuCl4 solution (0.01 M) was added to CTAB solution (7.5 mL, 0.1 M). Freshly prepared glacial NaBH4 (0.06 mL, 0.1 M) was quickly added while stirring vigorously (600 rpm). Stirring was continued until the solution turned brownish yellow. The solution was kept in the dark at 30 °C for 6 h. The gold seed solution was set aside.

[0041] (2) 2.5 mL HAuCl4 (0.01 M), 0.4 mL AgNO3 (0.01 M), and 0.04 mL HCl (98 wt% concentrated hydrochloric acid) were added to 50 mL CTAB (0.1 M) solution in sequence. After stirring at room temperature for 30 minutes, 0.4 mL AA (0.1 M) was added dropwise and stirred vigorously (600 rpm) to obtain a colorless solution. 0.125 mL gold seed solution was added to the mixture, and stirring was continued for 2 minutes. The mixture was allowed to stand at 30 ° C overnight. The obtained AuNR was washed twice with ultrapure water, purified, and dispersed in 50 mL ultrapure water to obtain gold nanorods (AuNR solution) for subsequent experiments.

[0042] (3) Add 0.1 mL of 1 mM thiolated polyethylene glycol (PEG200-SH) to 10 mL of 1 nM AuNR solution, and stir the mixture at room temperature for 24 h. Wash the mixture twice with ultrapure water to obtain AuNR@PEG, which is then redispersed in 10 mL of water.

[0043] (4) 0.02 mL of 1 mg / mL GQDs was added to 10 mL of AuNR@PEG suspension, gently stirred (100 rpm) overnight, washed twice with ultrapure water, and the sample was resuspended in water to finally obtain AuNR@GQDs.

[0044] The morphologies of AuNR, GQDs obtained in step (2) and AuNR@GQDs obtained in step (4) were characterized using transmission electron microscopy ( Figure 2 A), the results show that the average length of AuNR is about 50nm, the average particle size of GQD is 3nm, the morphology of AuNR@GQDs is uniform, the dispersion is good, and the GQDs are evenly dispersed around the AuNR. The UV-visible absorption spectrum of AuNR@GQDs has corresponding absorption peaks at 230nm, 520nm, and 800nm ​​( Figure 2 B), indicating that GQDs are deposited on the surface of AuNR. Compared with pure GQDs, the Raman peak of AuNR@GQDs is significantly enhanced, with 1300 cm -1 and 1600cm -1 Corresponding to the D peak and G peak of GQDs ( Figure 2 C), confirming that AuNR has excellent Raman signal enhancement properties.

[0045] Example 3

[0046] This embodiment provides a SERS detection probe and a preparation method, sensitivity and specificity evaluation of the probe.

[0047] Experimental Materials: MiRNA-17, miRNA-17 detection probe (miR-17d), miRNA-17 single-base mutant (miR-17m), miRNA-18a, miRNA-18a detection probe (miR-18ad), and miRNA-18a single-base mutant (miR-18am) nucleotide fragments were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Table 1).

[0048] Table 1 Probe miRNAs sequences

[0049] name sequence miRNA-17 SEQ ID No.1 5'-CAAAGUGCUUACAGUGCAGGUAG-3' Detection miR17 SEQ ID No. 2 5'-CUACCUGCACUGUAAGCACUUUG-3' Mutated miRNA-17 SEQ ID No. 3 5'-CAAAGUGCUUACAGUGCAGGUUG-3' miRNA-18a SEQ ID No.4 5'-UAAGGUGCAUCUAGUGCAGAUAG-3' Detection miR18a SEQ ID No.5 5'-CUAUCUGCACUAGAUGCACCUUA-3' Mutated miRNA-18a SEQ ID No.6 5'-UAAAGUGCAUCUAGUGCAGAUAG-3'

[0050] Experimental steps:

[0051] The detection probes (miRNA-17 detection probe (miR-17d), miRNA-18a detection probe (miR-18ad)) in the experimental materials were incubated with the AuNR@GQDs prepared in Example 2 at room temperature overnight, and the unbound probes were purified to prepare the SERS detection probe (AuNR@GQDs@miRNAd). The SERS signal was measured by a dispersive confocal Raman spectrometer (silicon wafer calibration instrument, spectral range 0-3000 cm -1 , the sample excitation wavelength is 532 nm, the actual laser power is 5 mW, and the accumulation time is 10 s).

[0052] Preparation 10 -6 The target miRNAs solution with a concentration of 10 M was obtained after gradient dilution. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 - 11 M, 10 -12 The concentration of miRNAs standard samples was quantified, and blank sample solutions were prepared as controls. The Raman spectroscopy results intuitively reflected that the characteristic peaks were concentration-dependent, and their intensity increased with the increase of the concentration of target miRNAs ( Figure 3 A). At extremely low concentrations (10-12M) of miRNAs, SERS signal changes can still be detected, and within a wide concentration range (10-12M to 10-6M), the Raman signal intensity maintains a good linear positive correlation with the miRNAs concentration ( Figure 3 B and Figure 3 C) confirmed that the SERS detection probe has high sensitivity and significantly reduces the detection limit without using any amplification method. When the miRNAs detected are single-base mismatched miR-17m and miR-18am, the detection limit is 1300 cm -1 , 1600cm -1 The characteristic SERS signal intensity at the Figure 3 D), it can be seen that this detection method has excellent potential in the specific determination of tumor-related miRNAs, ensuring the specific detection of target molecules.

[0053] Example 4

[0054] In this example, the application of the SERS detection probe prepared in Example 3 at the cellular level was verified.

[0055] Experimental materials: Fetal bovine serum and RPMI-1640 culture medium were purchased from Gibco (USA); retinoblastoma cell line Y79 was purchased from the American Type Culture Collection (ATCC).

[0056] Experimental steps: The supernatant of RB cell culture fluid was diluted 10 times and 100 times respectively to obtain RB cell culture fluid supernatant with three different miRNAs concentrations of 1X, 10X, and 100X. The SERS detection probe prepared in Example 3 was used to perform Raman spectroscopy detection on each sample. The results showed that the higher the concentration of miRNAs in the supernatant, the higher the signal intensity of the characteristic Raman peak ( Figure 4 ), which is consistent with the above test results. These data show that this detection probe is a promising application tool for measuring retinoblastoma biomarkers.

[0057] The above disclosure is merely a preferred embodiment of the present invention. The preferred embodiment does not exhaustively describe all details, nor does it limit the invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

[0058] Under the guidance of the present invention and the above-mentioned embodiments, it is easy for those skilled in the art to foresee that the raw materials or their equivalent substitutes, the processing methods or their equivalent substitutes listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of the raw materials and processing methods can realize the present invention. The embodiments are not listed one by one here.

Claims

1. A surface-enhanced Raman scattering probe, characterized in that: The method comprises a nucleic acid probe and a surface-enhanced Raman scattering substrate, wherein the surface-enhanced Raman scattering substrate comprises gold nanorods and graphene quantum dots uniformly distributed around the gold nanorods; The preparation of the surface enhanced Raman scattering substrate comprises the following steps: S1, synthesizing gold seed solution using the seed growth method; S2. HAuCl4 solution, AgNO3 solution, and HCl were sequentially added to the hexadecyltrimethylammonium bromide solution, and the mixture was stirred at room temperature for 30 minutes. Then, ascorbic acid solution was added dropwise and stirred vigorously to obtain a colorless solution. The gold seed solution prepared in step S1 was added to the mixture, and the mixture was stirred for 2 minutes. The mixture was allowed to stand at 30°C overnight. The obtained gold nanorods were washed and purified with ultrapure water, and then dispersed in ultrapure water to obtain a gold nanorod solution. S3, adding thiolated polyethylene glycol to the gold nanorod solution, stirring the mixture at room temperature for 24 hours, washing it twice with ultrapure water to obtain AuNR@PEG, and redispersing it in ultrapure water to obtain an AuNR@PEG suspension; S4, adding graphene quantum dots to the AuNR@PEG suspension obtained in step S3, stirring overnight, and washing twice with ultrapure water to obtain a surface-enhanced Raman scattering substrate; The preparation of the surface-enhanced Raman scattering probe comprises the following steps: incubating the nucleic acid probe with the surface-enhanced Raman scattering substrate at room temperature overnight, and purifying the unbound probe to obtain the surface-enhanced Raman scattering probe.

2. The surface-enhanced Raman scattering probe according to claim 1, characterized in that: The gold seed solution in step S1 is prepared as follows: HAuCl4 solution is added to hexadecyltrimethylammonium bromide solution, freshly prepared glacial NaBH4 is added while stirring, and stirring is continued until a yellow solution is obtained. The solution is allowed to stand at 30°C in the dark for 6 hours to obtain the gold seed solution.

3. The surface-enhanced Raman scattering probe according to claim 2, characterized in that: The concentration of HAuCl4 solution is 0.01M, the concentration of hexadecyltrimethylammonium bromide solution is 0.1M, the concentration of glacial NaBH4 is 0.11M, and the volume ratio of HAuCl4 solution, hexadecyltrimethylammonium bromide solution, and glacial NaBH4 is 0.25:7.5:0.0.

6.

4. The surface-enhanced Raman scattering probe according to claim 1, characterized in that: In step S2, the concentration of the cetyltrimethylammonium bromide solution is 0.1 M, the concentration of the HAuCl4 solution is 0.01 M, the concentration of the AgNO3 solution is 0.01 M, and the concentration of the ascorbic acid solution is 0.1 M. The volume ratio of the cetyltrimethylammonium bromide solution, HAuCl4 solution, AgNO3 solution, concentrated HCl, ascorbic acid solution, and gold seed solution is 50:2.5:0.4:0.04:0.4:0.

125.

5. The surface-enhanced Raman scattering probe according to claim 1, characterized in that: In step S3, the concentration of thiolated polyethylene glycol is 0.1 mM, the concentration of the gold nanorod solution is 1 nM, and the volume ratio of thiolated polyethylene glycol to the gold nanorod solution is 0.1:

10.

6. The surface-enhanced Raman scattering probe according to claim 1, wherein: In step S4, the concentration of graphene quantum dots was 1 mg / mL, and the volume ratio of AuNR@PEG suspension to graphene quantum dots was 10:0.

02.

7. The method for preparing a surface-enhanced Raman scattering probe according to claim 1, wherein: The nucleic acid probes are miRNA-17 detection probes and miRNA-18a detection probes.

8. Use of the surface-enhanced Raman scattering probe according to claim 1 in the preparation of a product for detecting the expression level of a retinoblastoma biomarker.

9. The use according to claim 8, characterized in that The product for detecting the expression level of retinoblastoma biomarkers includes a kit.

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