Surface enhanced Raman scattering probe as well as preparation method and application thereof

Abstract

The invention discloses a surface-enhanced Raman scattering probe as well as a preparation method and application thereof, the surface-enhanced Raman scattering probe comprises a nucleic acid probe and a surface-enhanced Raman scattering substrate, and the surface-enhanced Raman scattering substrate comprises a gold nanorod and graphene quantum dots uniformly distributed at the periphery of the gold nanorod. According to the surface-enhanced Raman scattering probe provided by the invention, graphene quantum dots are used as Raman signal molecules, gold nanorods are used for enhancing characteristic SERS signals, a surface-enhanced Raman scattering substrate is constructed as an SERS substrate, and a nucleic acid probe complementarily paired with target miRNAs is fixed on the graphene quantum dots as Raman report molecules. Raman signals are remarkably enhanced by using the gold nanorods, the detection accuracy is effectively improved, and the content of target molecules in a sample is determined by analyzing the intensity change of a characteristic peak in a Raman spectrum of the gold nanorods.

Description

Technical Field

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

[0002] Retinoblastoma (RB) is the most common intraocular malignancy in infants and young children, with a global incidence of approximately one case per 15,000 to 20,000 infants and young children. In my country, there are approximately 1,100 new cases of the disease each year, and this disease seriously threatens children's vision and life safety. Therefore, early diagnosis and effective monitoring of RB are crucial. However, traditional detection methods usually rely on fundus examination, imaging analysis, and histopathological examination, which have problems such as complex procedures, high costs, and invasiveness. At present, new detection methods are urgently needed to identify tumor-related biomarkers for early diagnosis. MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules composed of approximately 22 nucleotides. A large number of studies have shown that these molecules are closely related to the occurrence, development, and metastasis of RB. For example, miRNA-17 and miRNA-18a are significantly overexpressed in RB. In the past, trace nucleic acid amplification techniques were usually used to detect miRNAs, including polymerase chain reaction, loop-mediated isothermal amplification, and recombinase polymerase amplification. However, these techniques face problems such as difficult extraction, complex operation, strict reaction conditions, and susceptibility to contamination. Summary of the invention

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

[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 seed growth method;

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

[0008] S3, adding thiolated polyethylene glycol to the AuNR solution, stirring the mixture at room temperature for 24 hours, washing 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 method for preparing the gold seed solution in step S1 is as follows: add HAuCl4 solution to CTAB solution, add freshly prepared glacial NaBH4 while stirring, continue stirring until a yellow solution is obtained, and stand at 30°C in the dark for 6 hours to obtain a 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 CTAB solution is 0.1M, the concentration of HAuCl4 solution is 0.01M, the concentration of AgNO3 solution is 0.01M, the concentration of AA solution is 0.1M, and the volume ratio of 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 AuNR solution is 1 nM, and the volume ratio of thiolated polyethylene glycol to 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 the surface enhanced Raman scattering substrate at room temperature overnight, purifying the unbound probe to obtain the surface enhanced Raman scattering probe.

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

[0017] The 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 probe provided by the present invention adopts graphene quantum dots (GQDs) as Raman signal molecules, gold nanorods (AuNR) to enhance characteristic SERS signals, and constructs a surface enhanced Raman scattering substrate (AuNR@GQDs composite material) as a SERS substrate. Nucleic acid probes complementary to target miRNAs are fixed on Raman reporter molecules graphene quantum dots, and gold nanorods are used to achieve significant enhancement of Raman signals, effectively improving the accuracy of detection, and the content of target molecules 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 a product 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 detecting 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 prior art, such as difficult extraction, complex operation, strict reaction conditions and susceptibility to contamination.

[0023] Instruction Manual

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

[0025] Figure 2 The characteristic test result diagram 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 result diagram of the sensitivity and specificity of the SERS detection probe for measuring target miRNAs in Example 3; wherein 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 graph of target miRNAs and their corresponding single-base mutants;

[0027] Figure 4 This is a result diagram of the SERS detection probe measuring miRNAs in the supernatant of RB cells in Example 4; wherein, 1X: undiluted cell culture supernatant; 10X: diluted 10 times; 100X: diluted 100 times. DETAILED DESCRIPTION

[0028] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

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

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

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

[0032] Example 1

[0033] Screening of RB highly expressed miRNA

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

[0035] 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 using a real-time fluorescence quantitative nucleic acid amplification detection system (qPCR) in RB cell lines and normal control cell lines. The cells were treated with the lysate 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 in which Poly (A) tailing reaction and reverse transcription reaction are performed simultaneously to synthesize the first chain of miRNAs cDNA. The extracted miRNAs and 1μL gDNA Remover, 5μL miRNA RT Buffer, 2μL miRNA RT Enzyme Mix, and Nuclease free ddH2O in the kit form a 20μL system, and the corresponding first chain of cDNA is 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 (EZB-Probe-R2) were combined into a 10μL system. After the qPCR reaction procedure, the corresponding CT value was obtained and the relative expression of miRNAs in the cells was calculated. The qPCR experimental data showed that miRNA-17 and miRNA-18a were significantly highly expressed 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 embodiment provides a surface enhanced Raman scattering substrate (AuNR@GQDs composite material) and its preparation, characterization and property analysis.

[0038] Experimental materials: Hexadecyltrimethylammonium 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) was purchased from Nanjing Jingtan Nanotechnology Co., Ltd.; thiol-polyethylene glycol 200 (PEG200-SH) was purchased from Shanghai Tuoyang Biotechnology Co., Ltd. 120 kV transmission electron microscope (Talos L120C G2, Thermo Fisher Scientific, USA); dispersive confocal Raman spectrometer (Senterra R200-L, Bruker, Germany); UV-visible spectrophotometer (EV300, Thermo Fisher Scientific, USA).

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

[0040] (1) Synthesize gold nanorods using the seed growth method: add 0.25 mL of HAuCl4 solution (0.01 M) to CTAB solution (7.5 mL, 0.1 M), and quickly add freshly prepared glacial NaBH4 (0.06 mL, 0.1 M) while stirring vigorously (600 rpm). Continue stirring until the solution turns brownish yellow, and stand at 30 °C in the dark for 6 hours. The gold seed solution is ready for use.

[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. After stirring 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) 0.1 mL of 1 mM thiolated polyethylene glycol (PEG200-SH) was added to 10 mL of 1 nM AuNR solution, and the mixture was stirred at room temperature for 24 h. The AuNR@PEG was washed twice with ultrapure water and redispersed in 10 mL of water;

[0043] (4) Add 0.02 mL of 1 mg / mL GQDs to 10 mL of AuNR@PEG suspension, gently stir (100 rpm) overnight, wash twice with ultrapure water, and resuspend the sample in water to finally obtain AuNR@GQDs.

[0044] The AuNR, GQDs obtained in step (2) and the AuNR@GQDs obtained in step (4) were characterized by 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 peaks of AuNR@GQDs are significantly enhanced, among which 1300cm -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: The nucleotide fragments of 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) 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 0-3000cm -1 , sample excitation wavelength is 532nm, actual laser power is 5mW, and accumulation time is 10s).

[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 detected miRNAs are single-base mismatched miR-17m and miR-18am, the detection limit is 1300 cm -1 、1600cm -1 The characteristic SERS signal intensity at cannot be enhanced ( 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: Take the supernatant of RB cell culture fluid, dilute it 10 times and 100 times respectively, and obtain RB cell culture fluid supernatant with three different miRNAs concentrations of 1X, 10X, and 100X. Use the SERS detection probe prepared in Example 3 to perform Raman spectrum detection on each sample. The results show 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 the detection probe is a promising application tool for retinoblastoma biomarker measurement.

[0057] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific implementation method described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of 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 each raw material or its equivalent substitute, each processing method or its equivalent substitute 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 each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A surface enhanced Raman scattering probe, characterized in that: The invention 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.

2. The surface enhanced Raman scattering probe according to claim 1, characterized in that: The preparation of the surface enhanced Raman scattering substrate comprises the following steps: S1, synthesizing gold seed solution using seed growth method; S2, HAuCl4 solution, AgNO3 solution, and HCl were added to the CTAB solution in sequence, and after stirring at room temperature for 30 minutes, AA solution was added dropwise, and stirred vigorously to obtain a colorless solution, and the gold seed solution prepared in step S1 was added to the mixture, and after continuing to stir for 2 minutes, the mixture was allowed to stand at 30°C overnight, and the obtained gold nanorods were washed and purified with ultrapure water and then dispersed in ultrapure water to obtain AuNR solution; S3, adding thiolated polyethylene glycol to the AuNR solution, stirring the mixture at room temperature for 24 hours, washing twice with ultrapure water to obtain AuNR@PEG, and redispersing it in ultrapure water to obtain an AuNR@PEG suspension; 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.

3. The surface enhanced Raman scattering probe according to claim 2, characterized in that: The preparation method of the gold seed solution in step S1 is as follows: add HAuCl4 solution to CTAB solution, add freshly prepared glacial NaBH4 while stirring, continue stirring until a yellow solution is obtained, and stand at 30°C in the dark for 6 hours to obtain the gold seed solution.

4. The surface enhanced Raman scattering probe according to claim 3, characterized in that: 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.

5. The surface enhanced Raman scattering probe according to claim 2, characterized in that: In step S2, the concentration of CTAB solution is 0.1M, the concentration of HAuCl4 solution is 0.01M, the concentration of AgNO3 solution is 0.01M, the concentration of AA solution is 0.1M, and the volume ratio of CTAB solution, HAuCl4 solution, AgNO3 solution, concentrated HCl, AA solution, and gold seed solution is 50:2.5:0.4:0.04:0.4:0.

125.

6. The surface enhanced Raman scattering probe according to claim 2, characterized in that: In step S3, the concentration of thiolated polyethylene glycol is 0.1 mM, the concentration of AuNR solution is 1 nM, and the volume ratio of thiolated polyethylene glycol to AuNR solution is 0.1:

10.

7. The surface enhanced Raman scattering probe according to claim 2, characterized in that: In step S4, the concentration of GQDs was 1 mg / mL, and the volume ratio of AuNR@PEG suspension to GQDs was 10:0.

0.

8. The method for preparing a surface enhanced Raman scattering probe according to any one of claims 1 to 7, characterized in that: The following steps are involved: The nucleic acid probe is incubated with the surface-enhanced Raman scattering substrate at room temperature overnight, and unbound probes are purified to obtain the surface-enhanced Raman scattering probe.

9. The method for preparing a surface enhanced Raman scattering probe according to claim 8, characterized in that: The nucleic acid probes are miRNA-17 detection probes and miRNA-18a detection probes.

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

11. The use according to claim 10, characterized in that: The product for detecting the expression level of retinoblastoma biomarkers includes a kit.

Images