Method for constructing SERS (Surface Enhanced Raman Scattering) aptamer sensor of carcino-embryonic antigen and application of SERS aptamer sensor

Abstract

The invention relates to a method for constructing an SERS (Surface Enhanced Raman Scattering) aptamer sensor of a CEA (Carcino-Embryonic Antigen) and application of the SERS aptamer sensor of the CEA, and the SERS aptamer sensor of the CEA is successfully constructed by taking a silver nanorod array chip of a modified CEA sulfydryl aptamer as an SERS capture substrate and CBBG as a Raman signal probe. In the range of 0.01-50 ng / mL, the CEA concentration and the Raman peak intensity ISERS have a good linear relationship, the linear equation is ISERS = 1245.45 LogC + 3428.05 (R2 = 0.9922), and the detection limit is 5.48 pg / mL (S / N = 3); a labeling test is carried out on CEA in serum, and the recovery rate is 98.11%-101.87%. The sensor has the advantages of high sensitivity, strong specificity, high detection speed and the like, has certain stability, and can provide a new detection method for clinical detection of CEA.

Description

Technical Field

[0001] The invention relates to the technical field of surface enhanced Raman detection, and in particular to a method for constructing a SERS aptamer sensor for carcinoembryonic antigen and its application. Background Art

[0002] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion and has been identified as a broad-spectrum tumor marker associated with lung cancer, breast cancer, rectal cancer, colon cancer, pancreatic cancer, etc. The normal level of CEA in the serum of healthy adults is less than 2.5 ng / mL, but it is generally less than 5.0 ng / mL in adult smokers. If the CEA level in human serum is higher than 5.0 ng / mL, it indicates the risk of cancer. Therefore, CEA levels are of great value in clinical diagnosis and efficacy evaluation, and it is necessary to develop new methods for sensitive, accurate and rapid CEA detection. At present, the main methods for detecting CEA include enzyme-linked immunosorbent assay, chemiluminescent immunoassay, electrochemical immunoassay, electrochemiluminescent immunoassay and fluorescent immunoassay.

[0003] Surface-enhanced Raman scattering (SERS) has the advantages of being sensitive, rapid, non-destructive, resistant to water interference, and capable of fingerprint recognition, and is widely used in analytical detection and biomedical fields. Aptamer is a single-stranded DNA or RNA fragment obtained by screening a random single-stranded oligonucleotide library in vitro using the SELEX technique. Compared with protein antibodies, aptamers have the advantages of simple preparation, good stability, strong specificity, high affinity, and easy modification. Therefore, aptamers are widely used as biorecognition elements and biosensors. At present, there have been reports on SERS immunobiosensors for CEA, but there are few reports on SERS aptamer sensors for CEA.

[0004] Song et al. modified CEA protein antibody on submicron gold in the shape of butterfly wings as SERS substrate and used R6G aptamer as signal probe to prepare CEA aptamer sensor. 4It shows a good linear relationship in the range of ng / ml, and the detection limit reaches 10 ng / ml. However, the sensor uses CEA protein antibody as the capture substrate, and the amide bond coupling of protein antibody is time-consuming. Based on core-shell structure nanoparticles (Au@4-MBA@Ag NPs), Lu et al. modified two different aptamer complementary chains, triggered the aggregation of Au@4-MBA@Ag NPs through the hybridization of aptamer and complementary chain, and the gap between nanoparticles provided abundant "hot spots". When the sample to be tested contains CEA, CEA interacts with the aptamer, resulting in the dissociation of the polymer and the reduction of SERS signal. This method shows a good linear relationship in the range of 0.01-1 ng / ml, and the detection limit reaches 3.24 pg / ml. However, the preparation of this sensor is relatively complicated, involving the steps of modifying Au@Ag NPs with aptamer complementary chain, hybridization of aptamer and complementary chain, and the formation of polymer. Liu et al. used a silicon plate coated with a layer of gold film modified with mercaptopropionic acid to modify CEA antibody through amide bonds as a SERS capture substrate, and modified Au NPs with an aptamer coupled to the Raman signal molecule 4-MBA as a SERS probe to construct a SERS sensor for CEA. This method showed a good linear relationship in the range of 1 ng / ml-10 μg / mL, and the detection limit reached 0.08 ng / ml. Wang connected a DNA tetrahedron modified with an internal standard Raman reporter molecule Rox and three other segments modified with different target protein aptamers to a bipyramidal nanogold through Au-S bonds as a capture substrate, and bound different SERS tags through complementary chains. When the target substance appears in the system, the target substance binds to the aptamer, and the corresponding SERS tag dissociates, and the quantification is performed based on the ratio of the Raman intensity of the SERS tag to the internal standard intensity. This method showed a good linear relationship in the range of 0.001-5 ng / mL, and the detection limit reached 0.08 ng / ml. This method introduces an internal standard and can detect multiple target proteins simultaneously, but the preparation process is relatively complicated. Summary of the invention

[0005] The technical problem to be solved by the present invention is: to provide a method for constructing a SERS aptamer sensor for carcinoembryonic antigen and its application. The construction method uses a thiol aptamer (Aptamer-SH) of CEA to modify a silver nanoarray chip (Chip) as a SERS capture substrate, selects Coomassie Brilliant Blue (CBBG) as a Raman signal molecule, and establishes a new type of CEA SERS aptamer sensor. The sensor has the advantages of sensitive and rapid detection of CEA and can be used for CEA detection.

[0006] The technical solution to solve the above technical problem is: a method for constructing a SERS aptamer sensor for carcinoembryonic antigen, comprising the following steps:

[0007] A. Preparation of SERS capture substrate: 15-25 μL of 0.5-1.5 μM Aptamer-SH and 2-6 μL of 0.5-1.5 mM TCEP solution were mixed and treated at room temperature for 0.8-1.2 h to reduce the formation of disulfide bonds, and then all of the above solutions were added dropwise to the Chip, reacted in a sealed environment at room temperature for 5.5-6.5 h, and then the Chip was rinsed with PBS to remove the unconnected Aptamer-SH to obtain Chip@Apt; finally, 5-15 μL of 0.5-1.5 mM MCH was added dropwise to the Chip@Apt, reacted at room temperature for 4-6 min, blocked the unbound sites on the Chip, and the Chip@Apt was rinsed with PBS to remove the unreacted MCH to obtain the SERS capture substrate, which was stored at 3-5°C for future use;

[0008] B. Preparation of the sensor: Take 5-15 μL of CEA standard solution or human serum and add it to the prepared SERS capture substrate. After reacting for 0.8-1.2 hours, rinse with PBS, then add 15-25 μL of 0.1-0.3 mg / mL CBBG solution. After reacting for 10-20 minutes at room temperature, rinse with PBS to wash away the unbound CBBG solution. After it is naturally dried, the SERS aptamer sensor of carcinoembryonic antigen is obtained.

[0009] Furthermore, the nucleotide sequence of Aptamer-SH is: 5'-SH-(CH2)6-ATA CCA GCT TAT TCAATT-3'.

[0010] Another technical solution of the present invention is: the application of the SERS aptamer sensor of carcinoembryonic antigen constructed by the above method comprises the following steps:

[0011] (1) Drawing a standard curve: Take a number of CEA standard solutions with different concentrations to construct a SERS aptamer sensor for carcinoembryonic antigen and perform SERS detection to draw a CEA standard curve;

[0012] (2) Sample detection: The SERS aptamer sensor for carcinoembryonic antigen constructed from the sample to be tested is used for SERS detection, and the concentration of carcinoembryonic antigen is calculated using the CEA standard curve.

[0013] Furthermore, the CEA standard curve in step (1) is: SERS =1245.45LogC+3428.05, R 2 =0.9922, where I SERS represents the Raman intensity of CEA on the substrate material, and C represents the mass concentration of carcinoembryonic antigen.

[0014] Coomassie Brilliant Blue G-250 (CBBG) is a methyl-substituted triphenylmethane blue dye, which can be divided into G-250 (CBBG) and R-250 (CBBR) in terms of structure. In 1976, Bradford used CBBG to establish a protein detection method, namely the Coomassie Brilliant Blue method. The principle of CBBG binding to protein is that the anions in CBBG easily form water-insoluble complexes with aromatic and amino acid residues on the surface of proteins through non-covalent binding (hydrophobic interaction and van der Waals force). Among them, although CBBG is not as sensitive as CBBR in protein staining, it binds to proteins more stably and for a shorter time. The color development by the interaction between protein and Coomassie Brilliant Blue and the detection of protein concentration by absorbance or fluorescence intensity has the advantages of fast detection time and simple operation, but this method requires a large amount of sample to be tested and can only detect proteins with higher concentrations. CBBG has Raman activity and can achieve high-sensitivity detection of proteins in combination with SERS technology. The aptamer is used to recognize and capture the target protein, the target protein is quantitatively bound to CBBG, and the Raman signal of CBBG can be used to detect the target protein.

[0015] The key to the application of SERS technology in detection is to solve the problem of substrate activity and signal uniformity. The structure and morphology of the substrate affect the intensity and uniformity of the SERS signal. Liu et al. found that the Ag nanorod array substrate has a significant and uniform enhancement effect on the Raman signal, and also proved that the SERS signal of the Ag nanorod array is much stronger than that of the Au nanopillar array.

[0016] The present invention selects CEA Aptamer as the recognition element of CEA, utilizes the CEA thiol aptamer modified silver nanoarray chip as the SERS capture substrate, uses CBBG as the Raman signal molecule, and the captured CEA is quickly combined with CBBG to realize the SERS detection of CEA. Compared with the currently reported CEA SERS sensor, the SERS aptamer sensor constructed by this method has the advantages of simplicity, sensitivity, and rapidity.

[0017] The results of the investigation of the performance of the aptamer sensor of the present invention show that in the range of 0.01-50 ng / mL, the SERS signal intensity (I SERS ) has a good linear relationship with the logarithmic concentration of CEA, I SERS =1245.45LogC+ 3428.05(R 2=0.9922), detection limit 5.48 pg / mL (S / N=3); spiked test of CEA in serum, recovery rate 98.11%-101.87%, RSD ≤ 6.89% (n=3). The sensor has the advantages of sensitive and rapid detection of CEA, and has application value in the clinical detection of CEA. This strategy can also be used to construct SERS biosensors for other proteins.

[0018] The technical features of a method for constructing a SERS aptamer sensor for carcinoembryonic antigen according to the present invention are further described below in conjunction with the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the detection principle of the aptamer sensor of the present invention.

[0020] Figure 2 The graphs are the influence of the concentration of the Aptamer solution and the reaction time. (a) is the influence of the concentration of the Aptamer, and (b) is the influence of the reaction time of the Aptamer and the chip.

[0021] Figure 3 The graphs are the influence curves of CBBG concentration, dosage, and binding time with CEA. (a) is the influence curve of CBBG concentration, (b) is the influence curve of CBBG dosage, and (c) is the influence curve of CBBG binding time with CEA.

[0022] Figure 4 The SERS spectra and CEA standard curve of the sensor detecting different concentrations of CEA standard solutions. (a) is the SERS spectra of the sensor detecting different concentrations of CEA standard solutions; (b) is the standard curve of CEA in the concentration range of 0.01 - 50 ng / mL.

[0023] Figure 5 Different proteins and mixtures at 1 ng / mL at 464 cm -1 SERS peak intensity diagram at .

[0024] Figure 6 Figures 2 and 3 show the uniformity test results and the reproducibility test results of the SERS aptamer sensor, where (a) is the uniformity test result of the SERS aptamer sensor at 10 pg / mL; (b) is the reproducibility test result of the SERS aptamer sensor at 10 ng / mL.

[0025] Figure 7 This is the sensor stability test result diagram. DETAILED DESCRIPTION

[0026] Tests and materials used in the present invention:

[0027] CEA aptamer (Aptamer-SH: 5'-SH-(CH2)6-ATA CCA GCT TAT TCA ATT-3') (Shanghai Biotech Co., Ltd.), CEA protein (CEA, Shanghai Lingchao Biotechnology Co., Ltd.), nanosilver nanorod array chip (Chip) (Guangxi Sanhuan High-Tech Raman Chip Technology Co., Ltd.), Coomassie Brilliant Blue G-250 (G-250, Shanghai MacLean Biochemical Technology Co., Ltd.), 6-mercaptohexanol (MCH, Shanghai MacLean Biochemical Technology Co., Ltd.), phosphate buffer (PBS, Huankai Microbiological Technology Co., Ltd.), tris(2-carboxyethyl)phosphine hydrochloride (0.5 M TCEP solution, Shanghai Biyuntian Biotechnology Co., Ltd.), rhodamine 6G (R6G, Sinopharm Group Chemical Reagent Co., Ltd.). All reagents were of analytical grade, and all water used was ultrapure water.

[0028] Bovine serum albumin (BSA) and immunoglobulin G (IgG) (Aladdin Reagent Co., Ltd.), human serum albumin (HSA) and lipoprotein lipase (LPL) (Nanjing Dulai Biotechnology Co., Ltd.), human serum zinc and calcium protein I (cTnI) (Hangzhou Qitai Biotechnology Co., Ltd.), human C-reactive protein (CRP, Shanghai Biotech Co., Ltd.), human serum samples (Shanghai Jingkang Bioengineering Co., Ltd.).

[0029] Embodiment 1:

[0030] A method for constructing a SERS aptamer sensor for carcinoembryonic antigen comprises the following steps:

[0031] A. Preparation of SERS capture substrate: First, 20 μL of Aptamer-SH (1 μM) and 4 μL of TCEP solution (1 mM) were mixed and treated at room temperature for 1 h to reduce the formation of disulfide bonds. Then, all of the above solutions were added dropwise to the Chip, and after reacting for 6 h in a sealed environment at room temperature, the Chip was rinsed 3 times with PBS to remove the unconnected Aptamer-SH and obtain Chip@Apt. Finally, 10 μL of 1 mM MCH was added dropwise to Chip@Apt and reacted at room temperature for 5 min to block the unbound sites on the surface of the Ag nanorod (Chip). Chip@Apt was rinsed 3 times with PBS to remove the unreacted MCH and stored at 4°C for future use.

[0032] B. Preparation of the sensor: Take 10 μL of CEA standard solution or human serum and drop it onto the prepared SERS capture substrate. After reacting for 1 hour, rinse it with PBS three times to obtain Chip@Apt@CEA. Then add 20 μL of CBBG solution (0.2 mg / mL) and react at room temperature for 20 min. Rinse it with PBS three times to wash away the unbound CBBG solution. After it is dried naturally, the SERS aptamer sensor of carcinoembryonic antigen (Chip@Apt@CEA@CBBG) is obtained.

[0033] Embodiment 2:

[0034] The application of the SERS aptamer sensor for carcinoembryonic antigen prepared in Example 1, using the SERS aptamer sensor for carcinoembryonic antigen to detect carcinoembryonic antigen, comprises the following steps:

[0035] (1) Drawing a standard curve: Take a number of different concentrations (10pg / mL, 100pg / mL, 1ng / mL, 10ng / mL, 50ng / mL) of CEA standard solutions to construct a SERS aptamer sensor for carcinoembryonic antigen for SERS detection and draw a CEA standard curve; the CEA standard curve is: I SERS =1245.45LogC+3428.05, R 2 =0.9922, where I SERS represents the Raman intensity of CEA on the substrate material, and C represents the mass concentration of carcinoembryonic antigen.

[0036] (2) Sample detection: The SERS aptamer sensor of carcinoembryonic antigen constructed by the sample to be tested was used for SERS detection. The detection conditions were: excitation wavelength of 638 nm, 1200 g / mm grating, 10% power, and 10 s integration time. The concentration of carcinoembryonic antigen was calculated using the CEA standard curve.

[0037] The principle of the sensor of the present invention to detect CEA is as follows Figure 1 As shown. Aptamer-SH was first modified onto the Chip by dripping, and used as a SERS capture substrate. The unbound sites on the Chip were blocked by MCH. When the sample solution contained CEA, the aptamer on the SERS capture substrate recognized and captured CEA (PBS was used to wash and remove unbound CEA). CBBG solution was dripped, and CBBG and CEA were quickly bound (PBS was used to wash and remove unbound CBBG). After it was naturally dried, the SERS of CBBG was detected. The signal intensity of SERS was linearly related to the logarithm of the CEA concentration in the sample solution.

[0038] Effect of Aptamer solution concentration and reaction time:

[0039] The effect of Aptamer solution concentration (0.1, 0.5, 1, 5, 10 μM) on SERS signal was investigated. Figure 2 (a). Figure 2 (a) It can be seen that in the range of 0.1 to 10 μM, as the Aptamer concentration increases, the amount of Aptamer-SH modified to the Chip surface through the S-Ag bond gradually increases, and the amount of captured CEA and bound CBBG also increases accordingly, so the SERS signal is enhanced. When the Aptamer concentration reaches 1 μM, the SERS signal is the highest. As the Aptamer concentration continues to increase, the SERS signal shows a downward trend. This is because the amount of Aptamer modification is too large, resulting in a steric hindrance effect, which is not conducive to the capture of CEA molecules. Therefore, the 1 μM Aptamer concentration is determined to be the optimal amount.

[0040] The effect of reaction time (4, 5, 6, 7, 8 h) of Aptamer-modified chip on SERS signal was investigated. Figure 2 (b) is shown. Figure 2 (b) It can be seen that with the extension of reaction time, the SERS signal gradually increases. The Raman signal intensity is the highest at 6 h, and then decreases. Therefore, 6 h is the best time for aptamer modification of the chip.

[0041] Optimization of the concentration and dosage of Raman signal molecule CBBG:

[0042] To investigate the effect of CBBG concentration on SERS signals, CBBG with different concentrations (0.05, 0.1, 0.15, 0.2, and 0.25 mg / mL) was added to Chip@Apt@CEA and reacted for 10 min. Figure 3 (a). Figure 3 (a) It can be seen that when the CBBG concentration reaches 0.2 mg / mL, the SERS signal is the largest, and the SERS signal tends to be stable when the concentration is further increased. Therefore, the optimal concentration of CBBG is determined to be 0.2 mg / mL.

[0043] The effect of the volume of CBBG at a concentration of 0.2 mg / mL on the SERS signal was investigated. Different volumes (10, 15, 20, 25, 30 μL) of 0.2 mg / mL CBBG solution were added dropwise to Chip@Apt@CEA and reacted for 10 minutes. The results are shown in the figure. Figure 3 (b) is shown. Figure 3(b) As can be seen, as the volume increases, the amount of CBBG bound to CEA gradually increases, and the SERS signal increases. When the volume is 20 μL, the Raman peak intensity reaches the maximum. If the volume continues to increase, the SERS signal begins to decrease. Therefore, 20 μL CBBG is determined to be the optimal amount.

[0044] At room temperature, 0.2 mg / mL CBBG solution (20 μL) was added to Chip@Apt@CEA. The effect of the interaction time (5, 10, 20, 30 min) of CBBG and CEA on the SERS signal was shown in the figure. Figure 3 (c) is shown. Figure 3 (c) It can be seen that with the extension of the action time, the Raman peak intensity continues to increase, and the SERS signal tends to be stable at 20 min. Therefore, the optimal time for the action of CBBG and CEA is determined to be 20 min.

[0045] Investigation of the performance of the SERS aptamer sensor prepared by the present invention:

[0046] Working curve and detection limit of SERS aptamer sensor. Figure 4 (a) SERS spectra of the sensor detecting CEA standard solutions with different concentrations. As the CEA concentration increases, the -1 The SERS signal at the position gradually increases. The standard curve is shown in Figure 4 As shown in (b), in the range of 0.01–50 ng / mL, the SERS intensity and the CEA logarithm value show a good linear relationship, and the linear fitting equation is I SERS =1245.45LogC +3428.05, correlation coefficient R 2 =0.9922. Detection limit LOD=5.48 pg / mL (S / N=3). As shown in Table 1, compared with the CEA detection method reported in the literature, this method has a wider detection range and a lower detection limit.

[0047]

[0048] Investigation of the selectivity and specificity of SERS aptamer sensors. This method was used to detect 1 ng / mL CEA, LPL, IgG, HSA, CRP, cTn I standard solutions and blank samples (PBS). At the same time, a mixed solution containing CEA, LPL, IgG, HSA, CRP, and cTnI (the concentration of each substance was 1 ng / mL) was prepared, and the Raman signal intensity was detected using this method. Figure 5It can be seen that the SERS signal of the sensor detecting CEA is the strongest, much larger than the signals of other interfering proteins, and the SERS signals of the blank sample and other protein samples are not much different. The Raman peak intensity of the mixed sample solution is similar to that of the CEA standard solution, indicating that the sensor has good selectivity and specificity for the detection of CEA.

[0049] The signal uniformity and reproducibility of the SERS aptamer sensor were investigated. The SERS signals of 10 different points were collected by detecting the 10 pg / mL CEA standard solution and summarizing the SERS spectra. Figure 6 (a) shows that the relative standard deviation (RSD) is 5.36%, indicating that the SERS signal has good uniformity. Five sensors were prepared to detect 10 ng / mL CEA to test the reproducibility of the sensor ( Figure 6 (b)), the relative standard deviation (RSD) is 4.12%, indicating that the SERS aptamer sensor has good reproducibility.

[0050] The stability of SERS aptamer sensor was investigated. The SERS peak value change of 1 ng / mL CEA protein was detected within 20 days. Figure 7 As shown. Figure 7 It can be seen that when SERS detection was performed on it at 0, 1, 3, 5, 7, 10, and 15 days of storage (n=3), no obvious changes occurred, but the SERS peak intensity decreased significantly on the 20th day, indicating that the sensor has good stability within 15 days.

[0051] Serum sample detection and spike test. First, the serum sample was tested and the CEA concentration in the serum was measured to be 12.09 pg·mL -1 Subsequently, different amounts of CEA were added to the serum samples to a final concentration of 18.14 pg mL -1 , 24.18 pg·mL -1 、30.23 pg·mL -1 , and the spike test was carried out (the results are shown in Table 2). The spike recovery rate was between 98.11% and 101.87%, and the relative standard deviation RSD was ≤ 6.89% (n = 3).

[0052]

[0053] It can be seen from Table 2 that the sensor has good reliability and can be applied to the detection of CEA in actual samples.

Claims

1. A method for constructing a SERS aptamer sensor for carcinoembryonic antigen, characterized in that: The following steps are involved: A. Preparation of SERS capture substrate: 15-25 μL of 0.5-1.5 μM Aptamer-SH and 2-6 μL of 0.5-1.5 mM TCEP solution were mixed at room temperature for 0.8-1.2 h to reduce the formation of disulfide bonds, and then all of the above solutions were added dropwise to the Chip. After reacting for 5.5-6.5 h in a sealed environment at room temperature, the Chip was rinsed with PBS to remove the unconnected Aptamer-SH to obtain Chip@Apt; finally, 5-15 μL of 0.5-1.5 mM MCH was added dropwise to the Chip@Apt, reacted at room temperature for 4-6 min to block the unbound sites on the Chip, and the Chip@Apt was rinsed with PBS to remove the unreacted MCH to obtain the SERS capture substrate, which was stored at 3-5°C for future use; B. Preparation of the sensor: Take 5-15 μL of CEA standard solution or human serum and add it to the prepared SERS capture substrate. After reacting for 0.8-1.2 hours, rinse with PBS, then add 15-25 μL of 0.1-0.3 mg / mL CBBG solution. After reacting for 10-20 minutes at room temperature, rinse with PBS to wash away the unbound CBBG solution. After it is naturally dried, the SERS aptamer sensor of carcinoembryonic antigen is obtained.

2. The method for constructing a SERS aptamer sensor for carcinoembryonic antigen according to claim 1, characterized in that: The nucleotide sequence of Aptamer-SH is: 5'-SH-(CH2)6-ATA CCA GCT TAT TCA ATT-3'.

3. Application of the SERS aptamer sensor for carcinoembryonic antigen constructed according to claim 1 or 2, characterized in that: The following steps are involved: (1) Drawing a standard curve: Take a number of CEA standard solutions with different concentrations to construct a SERS aptamer sensor for carcinoembryonic antigen and perform SERS detection to draw a CEA standard curve; (2) Sample detection: The SERS aptamer sensor for carcinoembryonic antigen constructed from the sample to be tested is used for SERS detection, and the concentration of carcinoembryonic antigen is calculated using the CEA standard curve.

4. The use of the SERS aptamer sensor for carcinoembryonic antigen according to claim 3, characterized in that: The CEA standard curve in step (1) is: SERS =1245.45LogC+3428.05, R 2 =0.9922, where I SERS represents the Raman intensity of CEA on the substrate material, and C represents the mass concentration of carcinoembryonic antigen.

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