Preparation method and application of a competitive ratiometric SERS aptamer sensor

Abstract

The present invention discloses a preparation method and application of a competitive ratio-type SERS aptasensor, belonging to the field of safety detection. The steps include: preparing a ratio-type SERS active substrate, preparing a SERS aptasensor, establishing a standard curve of the analyte, and detecting the analyte in a test sample. When the SERS aptasensor recognizes the target, the aptamer complementary strand with methylene blue separates from the sensor surface, resulting in a decrease in the intensity of the magnetically separated sensor at a Raman shift of 1612 cm-1, while the SERS intensity at a Raman shift of 1570 cm-1 remains unchanged. Among them, the magnetic separation strategy effectively simplifies the separation steps of the sensor; the ratio measurement strategy effectively improves the repeatability and stability of okadaic acid sensing. And by modifying the aptamer and the complementary strand, the constructed sensor can be used for the detection of different analytes, having good universality and practical prospects.

Description

Technical Field

[0001] The invention belongs to the field of safety detection, and in particular discloses a preparation method of a competitive ratiometric SERS aptamer sensor and an application thereof. Background Art

[0002] Surface enhanced Raman scattering (SERS) is an ultra-sensitive vibration fingerprint technology with remarkable characteristics such as fingerprint recognition, high resolution, and strong anti-interference ability, showing great application prospects in the field of detection. However, due to the low stability and repeatability of SERS signals, SERS sensors still face great challenges in detecting target substances in complex environments. This is mainly because the intensity of the SERS response depends not only on the concentration of the analyte, but also on the enhanced substrate. In fact, due to the complex matrix effect, it is almost impossible to control the uniformity of the electromagnetic field of the enhanced substrate, making it difficult to obtain stable and consistent SERS spectra. This means that a lot of efforts still need to be made to improve SERS-based strategies to obtain SERS sensors with high robustness and accuracy. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the present invention provides a method for preparing a competitive ratiometric SERS aptamer sensor for detecting okadaic acid and its application, wherein SERS reporter molecules are embedded in a SERS substrate as an internal standard to calibrate the Raman signal of the sensor, so as to solve the low reproducibility problem existing in the above-mentioned prior SERS technology, thereby realizing rapid and convenient detection of okadaic acid.

[0004] The technical solution of the present invention to solve the above technical problems is as follows:

[0005] A method for preparing a competitive ratiometric SERS aptamer sensor comprises the following steps:

[0006] Step 1, preparation of tetraaminothiophenol embedded core-shell nanomaterials:

[0007] (1) adding chloroauric acid aqueous solution to water, mixing, heating to boil, adding trisodium citrate dihydrate aqueous solution, stirring to obtain a mixed solution, keeping the solution boiling until the solution turns wine red, stopping heating, and obtaining a gold nanoparticle solution;

[0008] (2) After the gold nanoparticle solution is cooled to room temperature, a tetraaminothiophenol solution is added, and a first stirring reaction is carried out at room temperature. After the reaction, the solution is boiled, and after boiling, a trisodium citrate dihydrate solution is added, and stirring is continued for a period of time to obtain a tetraaminothiophenol-modified gold nanoparticle solution; finally, silver nitrate is added dropwise to the tetraaminothiophenol-modified gold nanoparticle solution, and a second stirring reaction is carried out to obtain a tetraaminothiophenol-embedded gold and silver core-shell nanoparticle solution;

[0009] Preferably, in step 1 (1), the volume ratio of the chloroauric acid aqueous solution, water and the trisodium citrate dihydrate aqueous solution is 1:100:1.5; the mass concentration of the chloroauric acid aqueous solution is 1%; and the mass concentration of the trisodium citrate dihydrate aqueous solution is 1%.

[0010] Preferably, the volume ratio of the gold nanoparticle solution, the tetraaminothiophenol solution, the trisodium citrate dihydrate solution and the silver nitrate in the tetraaminothiophenol-modified gold nanoparticle solution in step 1 (2) is 100:0.4-0.8:4:4; the concentration of the tetraaminothiophenol solution is 0.1 mM, the mass concentration of the trisodium citrate dihydrate solution is 1%, and the concentration of the silver nitrate is 4-8 mM;

[0011] The first stirring reaction time is 1 hour, and the stirring is continued for 5-10 minutes; the second stirring reaction time is 1 hour.

[0012] Step 2: Preparation of a magnetic composite SERS active substrate:

[0013] Ferric chloride hexahydrate, polyethylene glycol 6000, sodium acetate and ethylene glycol are mixed, and after stirring, the solution is cooled to room temperature and then magnetically separated to obtain magnetic nanospheres; the magnetic nanospheres are added to a polyethyleneimine ethanol solution to obtain ultrasonic dissolution of the solution, and amino-modified magnetic nanospheres are obtained by magnetic separation; subsequently, the amino-modified magnetic nanospheres are mixed with the gold-silver core-shell nano-solution embedded with tetraaminothiophenol synthesized in step 1, and the material obtained by magnetic separation after mechanical oscillation is washed with deionized water and ethanol for several times respectively, and then vacuum dried to obtain a composite SERS active substrate with magnetic properties;

[0014] Preferably, in step 2, the dosage ratio of ferric chloride hexahydrate, polyethylene glycol 6000, sodium acetate and ethylene glycol is 2.7g:1g:8g:80ml; the dosage ratio of the magnetic nanospheres to the polyethyleneimine ethanol solution is 0.04g:20ml, and the mass concentration of the polyethyleneimine ethanol solution is 3-8%; the dosage ratio of the magnetic microsphere solution to the gold and silver core-shell nano solution embedded with tetraaminothiophenol is 4g:10mL; the stirring time is 1h, the ultrasonic dissolution time is 2h, the mechanical oscillation time is 3h, and the vacuum drying temperature is 60°C.

[0015] Step 3: Synthesis of SERS aptamer sensor:

[0016] (1) mixing the composite SERS active substrate with magnetic properties obtained in step 2, streptavidin and phosphate buffer solution, shaking for a period of time and then performing magnetic separation to obtain a composite SERS substrate modified with streptavidin;

[0017] (2) mixing the streptavidin-modified composite SERS substrate, the biotin-modified okadaic acid aptamer single chain and a phosphate buffer solution, incubating, magnetically separating the product, and washing it several times with a Tween 20 aqueous solution to obtain a composite SERS substrate modified with an aptamer single chain;

[0018] (3) Finally, the composite SERS substrate modified with the aptamer single chain, the aptamer complementary chain modified with methylene blue and the phosphate buffer solution were mixed, cooled to room temperature after the first incubation, and incubated for the second time, and finally the SERS aptamer sensor was obtained after magnetic separation.

[0019] Preferably, in step three (1), the amount ratio of the magnetic composite SERS active substrate, streptavidin and phosphate buffer solution is 2 g: 0.1-0.3 g: 2 mL; the shaking temperature is 4° C., and the shaking time is 6 h.

[0020] Preferably, in step three (2), the dosage ratio of the streptavidin-modified composite SERS substrate, the biotin-modified okadaic acid aptamer single chain, the phosphate buffer solution and the Tween 20 aqueous solution is 1 g: 80-120 μL: 1 mL: 1 mL; the concentration of the biotin-modified okadaic acid aptamer single chain is 8-12 μM; the incubation time is 8 h, and the incubation temperature is 25° C.; the mass concentration of the Tween 20 aqueous solution is 0.02%.

[0021] Preferably, in step three (3), the dosage ratio of the aptamer single-chain modified composite SERS substrate, the methylene blue modified aptamer complementary chain and the phosphate buffer solution is 1g:80-120μL:1mL; the concentration of the methylene blue modified aptamer complementary chain is 8-12μM; the first incubation time is 3min, the incubation temperature is 95°C, and the cooling time is 1h; the second incubation time is 30min, and the incubation temperature is 25°C.

[0022] Preferably, the pH of the phosphate buffer solution used in step 3 is 7.4 and the concentration is 10 mmol / L.

[0023] The present invention also provides an application of a competitive ratiometric SERS aptamer sensor in detecting okadaic acid, comprising the following steps:

[0024] (1) Establishment of Okadaic Acid Standard Curve:

[0025] The SERS aptamer sensor prepared in step 3 is mixed with okadaic acid standard solutions of different concentrations, and the sensor and the okadaic acid standard solution are in a one-to-one correspondence. The obtained mixed solution is magnetically separated after incubation, and the SERS aptamer sensor after the reaction is collected and redissolved in aqueous solution, and SERS spectrum is collected; the characteristic value of the SERS intensity signal in the spectrum is recorded, and a standard curve is established by fitting with the okadaic acid concentration;

[0026] Preferably, the usage ratio of the SERS aptamer sensor, the okadaic acid standard solution and the aqueous solution in (1) is 1 g:1 mL;

[0027] The concentration range of the okadaic acid standard solution is 0-200 ng / mL; the incubation time is 50 min, and the incubation temperature is 25° C.; the dosage ratio of the SERS aptamer sensor collected after the magnetic separation reaction to the aqueous solution is 1 g:1 mL.

[0028] Preferably, the step of recording the characteristic values ​​of the SERS intensity signal in the spectrum in (1) is: respectively recording the characteristic values ​​of the SERS intensity signal at 1612 cm -1 With 1570cm -1 The SERS intensity value at the former and the latter is I 1612 / 1570 is the characteristic value of the SERS intensity signal in the spectrum.

[0029] (2) Detection of okadaic acid in food and environmental samples:

[0030] The food or environmental sample is pretreated to obtain an extract containing okadaic acid; the SERS aptamer sensor prepared in step 3 is mixed with the extract, incubated, magnetically separated, and the reaction SERS aptamer sensor is collected and redissolved in an aqueous solution, and a SERS spectrum is collected; the characteristic value of the SERS intensity signal in the spectrum is recorded, and the obtained result is brought into the standard curve obtained in step (1) to detect the content of okadaic acid in the sample.

[0031] Preferably, the usage ratio of the SERS aptamer sensor to the okadaic acid extract in (2) is 1 g:1 ml;

[0032] The incubation time is 50 minutes, and the incubation temperature is 25° C.; the amount ratio of the SERS aptamer sensor collected after the magnetic separation reaction to the aqueous solution is 1 g:1 mL.

[0033] Preferably, the environmental sample in step (2) includes water and soil, and the pretreatment method of the food or environmental sample is: if it is a liquid, the liquid sample is directly tested after coarse filtration; if it is a solid, the solid sample is first homogenized, methanol is added and mixed and shaken, and the supernatant is taken after centrifugation and recorded as solution A, the remaining residue is mixed with methanol again and shaken, and the supernatant is taken after centrifugation and recorded as solution B, solution A and solution B are mixed and fixed to volume with methanol, and the mixed solution is taken and dried with nitrogen and then formic acid and aqueous solution are added for re-dissolution;

[0034] In the pretreatment method, the dosage ratio of the solid sample, methanol in solution A, and methanol in solution B is 2g:9mL:9mL; the shaking time is 1 minute; the fixed volume of the mixed solution A and solution B is 20mL; the volume ratio of the mixed solution, formic acid and water is 5:0.1:0.99mL; the centrifugal speed of solution A and solution B is 12000rpm, and the centrifugation time is 3min.

[0035] Beneficial effects:

[0036] 1. The present invention integrates the ratio probe and the magnetic separation feature in the same sensor, so that the constructed sensor has good self-correction ability and can quickly separate the sensor in a complex matrix, simplifying the reaction steps.

[0037] 2. The present invention applies the core-shell structure to the components of the SESR sensor, thereby amplifying the SERS enhancement effect on the sensor surface.

[0038] 3. The present invention uses tetraaminothiophenol as an internal standard molecule and embeds it into the core-shell structure of the substrate, which not only effectively avoids competitive adsorption caused by indirect contact with the target analyte, but also compensates for signal fluctuations caused by different degrees of substrate aggregation and measurement conditions.

[0039] 4. The present invention uses tetraaminothiophenol and methylene blue as the internal standard molecule and the response molecule, respectively. Both have obvious SERS responses, and some of the SERS peaks of the two do not overlap, which is conducive to the construction of a ratio sensor.

[0040] 5. The aptamer in the present invention is bound to the substrate surface through the streptavidin-biotin interaction, and the binding is tight and stable.

[0041] 6. The present invention proposes a competitive ratiometric SERS aptamer sensor for okadaic acid detection. The sensor is simple to operate, has a short detection time, and provides accurate results, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the construction and detection principle diagram of the competitive ratiometric SERS aptamer sensor in Example 1.

[0043] Figure 2 Transmission electron microscopy and element distribution map of the gold and silver core-shell embedded with tetraaminothiophenol constructed in Example 1;

[0044] Figure A is a transmission electron microscopy of gold and silver core-shell nanoparticles embedded with tetraaminobenzenethiophenol, and Figures BC are the element distribution maps of Au, Ag and S respectively.

[0045] Figure 3 Transmission electron microscopy and element distribution diagram of the magnetic composite SERS active substrate constructed in Example 1;

[0046] Figure A is the transmission electron microscopy image of the magnetic composite SERS active substrate, and Figure BE are the corresponding distribution maps of Fe, Au, S, and Ag elements, respectively.

[0047] Figure 4 This is a characterization diagram of the self-calibration capability of the SERS sensor prepared in Example 1;

[0048] Among them, A is the detection of any 15 sets of sensors at 1612cm -1 SERS intensity at the Raman shift; B is the SERS intensity at 1612 and 1570 cm for any 15 groups of sensors -1 Ratiometric SERS intensity at the Raman shift.

[0049] Figure 5 The standard curve established after testing the standard solutions of different concentrations of the analyte in Example 1;

[0050] Among them, A is the Raman signal diagram of the SERS aptamer sensor for detecting different concentrations of okadaic acid in seawater samples in Example 1; B is the standard curve established by the SERS aptamer sensor for detecting different concentrations of okadaic acid in seawater samples in Example 1. DETAILED DESCRIPTION

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0054] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0055] The biotin-modified okadaic acid aptamer single chain is a conventional well-known reagent material purchased from Sangon Biotech (Shanghai) Co., Ltd., and the sequence is:

[0056] 5'Biotin-GGTCACCAACAACAGGGAGCGCTACGCGAAGGGTCAATGTGACGTCATGCGGATGTGTGG-3';

[0057] The methylene blue modified aptamer complementary chain is a conventional well-known reagent material purchased from Sangon Biotech (Shanghai) Co., Ltd., and the sequence is: 5'Methylene Blue-CCCTTCGCGTAGCGCTCCCTGTTGTTGGTG-3'.

[0058] Example 1: Detection of Okadaic Acid in Seawater

[0059] Figure 1 The construction and detection principle diagram of the competitive ratiometric SERS aptamer sensor in Example 1;

[0060] Step 1, preparation of tetraaminothiophenol embedded core-shell nanomaterials:

[0061] Add 1% chloroauric acid aqueous solution into 100mL water and boil it, add 1.5mL trisodium citrate dihydrate aqueous solution with a mass concentration of 1% after heating and boiling, keep the solution boiling until the solution turns into wine red, stop heating, and obtain a gold nano solution; after the gold nano solution is cooled to room temperature, add 0.4mL tetraaminothiophenol solution with a concentration of 0.1mM, stir at room temperature for 1h, boil the solution after reaction, add 4mL trisodium citrate dihydrate solution with a mass concentration of 1% after boiling, continue stirring for 5min, and obtain a tetraaminothiophenol-modified gold nano solution; finally, add 4mL silver nitrate with a concentration of 4mM dropwise into the tetraaminothiophenol-modified gold nano solution, stir for 1h, and obtain a gold-silver core-shell nano material embedded with tetraaminothiophenol;

[0062] Figure 2 The transmission electron microscopy and element distribution map of the gold-silver core-shell nanoparticles embedded with tetraaminothiophenol constructed in Example 1, wherein Figure A is the transmission electron microscopy of the gold-silver core-shell nanoparticles embedded with tetraaminothiophenol, and Figures BC are the element distribution maps of Au, Ag and S respectively; Figure 2 It can be seen that the gold-silver core-shell structure is spherical with a diameter of 27.50±3.00nm; taking the S element as the representative of the internal standard molecule (tetraaminothiophenol), the results show that the tetraaminothiophenol molecules are evenly distributed between the gold core and the silver shell.

[0063] Step 2: Preparation of a magnetic composite SERS active substrate:

[0064] 2.7 g of ferric chloride hexahydrate, 1 g of polyethylene glycol 6000 and 8 g of sodium acetate were mixed with 80 mL of ethylene glycol, and after stirring for 1 hour, the solution was cooled to room temperature and then magnetically separated to obtain magnetic nanospheres; 0.04 g of magnetic nanospheres were added to 20 mL of 3% polyethyleneimine ethanol solution, the solution was ultrasonically dissolved for 2 hours, and amino-modified magnetic nanospheres were obtained by magnetic separation; subsequently, 4 g of amino-modified magnetic nanospheres were mixed with 10 mL of tetraaminothiophenol-embedded gold-silver core-shell nanosolution synthesized in step 1, mechanically vibrated for 3 hours, and the material obtained by magnetic separation was washed with deionized water and ethanol for 3 times respectively, and then dried in a vacuum oven at 60°C to obtain a composite SERS active substrate with magnetic properties;

[0065] Figure 3 The transmission electron micrograph and element distribution diagram of the composite SERS active substrate with magnetic properties constructed in Example 1, wherein Figure A is the transmission electron micrograph of the composite SERS active substrate with magnetic properties, and Figure BE are the corresponding element distribution diagrams of Fe, Au, S, and Ag, respectively; Figure 3 From the element distribution, it can be seen that the gold and silver core-shell have been evenly fixed on the surface of the magnetic nanospheres.

[0066] Step 3: Synthesis of SERS aptamer sensor:

[0067] (1) 2 g of a magnetic composite SERS active substrate, 0.1 g of streptavidin and 2 mL of phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, and the mixture was shaken at 4 °C for 6 h and then magnetically separated to obtain a streptavidin-modified composite SERS substrate;

[0068] (2) 1 g of streptavidin-modified composite SERS substrate, 80 μL of 8 μM biotin-modified okadaic acid aptamer single chain and 1 mL of phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, incubated at 25° C. for 8 h, magnetically separated the product, and washed twice with 1 mL of 0.02% Tween 20 aqueous solution to obtain a composite SERS substrate modified with a single chain aptamer;

[0069] (3) Finally, 1 g of the composite SERS substrate modified with a single-chain aptamer, 80 μL of an aptamer complementary chain modified with 8 μM methylene blue, and 1 mL of a phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, incubated at 95 °C for 3 min, cooled for 1 h, and then incubated at 25 °C for 30 min. After magnetic separation, the SERS aptamer sensor was finally obtained.

[0070] Figure 4 Figure 1 is a characterization diagram of the self-calibration capability of the SERS aptamer sensor prepared in Example 1; Figure A is a characterization diagram of the self-calibration capability of the SERS aptamer sensor prepared in Example 1 at 1612 cm -1 SERS intensity histogram at Raman shift. B is the SERS intensity histogram of 15 batches of SERS aptamer sensors self-calibrated at 1612 cm -1 SERS intensity histogram at the Raman shift; from Figure 4 It can be seen that the RSD of the peak intensity of any 15 spectra of the sensor before and after self-calibration is reduced from 9.54% to 4.6%, which greatly improves the reproducibility of the constructed SERS aptamer sensor.

[0071] Detection of Okadaic Acid in Seawater Samples:

[0072] (1) Establishment of Okadaic Acid Standard Curve:

[0073] Prepare 0, 0.5, 5, 10, 20, 40, 60, 80, 100, 200 ng / mL okadaic acid standard solutions, mix 1 g of the SERS aptamer sensor prepared in step 3 with 1 mL of okadaic acid standard solutions of different concentrations, incubate the mixture at 25 °C for 50 min, collect the reacted SERS aptamer sensor after magnetic separation, redissolve it in 1 mL of aqueous solution, and collect SERS spectra ( Figure 5 A); record the characteristic value of the SERS intensity signal in the spectrum and fit it with the concentration of okadaic acid to establish a standard curve ( Figure 5 B);

[0074] The steps of the characteristic value of the SERS intensity signal in the spectrum are as follows: respectively record the intensity of the sensor at 1612 cm under the excitation of 785 nm excitation light. -1 With 1570cm -1 The SERS intensity value at the former and the latter is I 1612 / 1570is the characteristic value of the SERS intensity signal in the spectrum.

[0075] Figure 5 A is a Raman signal diagram of the SERS aptamer sensor for detecting different concentrations of okadaic acid in seawater samples in Example 1; Figure 5 B is the standard curve established by the SERS aptamer sensor for detecting different concentrations of okadaic acid in seawater samples in Example 1; Figure 5 It can be seen that in the range of 0.5-100 ng / mL, I 1612 / 1570 The relationship between the ratio and the concentration of okadaic acid showed a negative correlation (y = 0.7005-0.0042x), R 2 =0.9866; in addition, the limit of detection (LOD) was estimated to be 0.21 ng / mL (S / N=3);

[0076] (2) Detection of okadaic acid content in seawater samples:

[0077] Take 0.9 mL of seawater sample and mix it with 0.1 mL of 500 ng / mL okadaic acid standard solution, and filter the mixed solution to obtain a pretreated seawater sample; mix 1 g of the SERS aptamer sensor prepared in step 3 with 1 mL of the pretreated seawater sample, incubate at 25° C. for 50 min, collect the reacted SERS aptamer sensor after magnetic separation, redissolve it in 1 mL of aqueous solution, and collect SERS spectra; record the characteristic value of the SERS intensity signal in the spectrum, and substitute the obtained result into the standard curve obtained in step (1) to obtain the amount of the analyte 50 ng / mL.

[0078] Example 2: Detection of okadaic acid in shellfish meat

[0079] Step 1, preparation of tetraaminothiophenol embedded core-shell nanomaterials:

[0080] Add 1% chloroauric acid aqueous solution into 100mL water and boil it, add 1.5mL trisodium citrate dihydrate aqueous solution with a mass concentration of 1% after heating and boiling, keep the solution boiling until the solution turns into wine red, stop heating, and obtain a gold nano solution; after the gold nano solution is cooled to room temperature, add 0.6mL tetraaminothiophenol solution with a concentration of 0.1mM, stir at room temperature for 1h, boil the solution after reaction, add 4mL trisodium citrate dihydrate solution with a mass concentration of 1% after boiling, continue stirring for 7min, and obtain a tetraaminothiophenol-modified gold nano solution; finally, add 4mL silver nitrate with a concentration of 4mM dropwise into the tetraaminothiophenol-modified gold nano solution, stir for 1h, and obtain a gold-silver core-shell nano material embedded with tetraaminothiophenol;

[0081] Step 2: Preparation of a magnetic composite SERS active substrate:

[0082] 2.7 g of ferric chloride hexahydrate, 1 g of polyethylene glycol 6000 and 8 g of sodium acetate were mixed with 80 mL of ethylene glycol, and after stirring for 1 hour, the solution was cooled to room temperature and magnetic nanospheres were obtained by magnetic separation; 0.04 g of magnetic nanospheres were added to 20 mL of 5% polyethyleneimine ethanol solution, and the solution was ultrasonically dissolved for 2 hours, and amino-modified magnetic nanospheres were obtained by magnetic separation; then, 4 g of amino-modified magnetic nanospheres were mixed with 10 mL of tetraaminothiophenol-embedded gold-silver core-shell nanosolution synthesized in step 1, mechanically vibrated for 3 hours, and the material obtained by magnetic separation was washed with deionized water and ethanol for 3 times respectively, and then dried in a vacuum oven at 60°C to obtain a composite SERS active substrate with magnetic properties; Step 3, synthesis of SERS aptamer sensor: 2 g of composite SERS active substrate was dispersed in 2 mL of phosphate buffer solution ( The composite SERS substrate was prepared by adding 100 μL of 10 μM aptamer PBS buffer solution (pH=7.4) to a PBS buffer solution (pH=7.4) containing 200 μL of 0.5 mg / mL streptavidin, and the mixed solution was continuously shaken at 4°C for 6 hours and then magnetically separated; finally, 1 mL of 10 μM aptamer PBS buffer solution (pH=7.4) was added to 1 mL of 1 mg / mL streptavidin-modified composite SERS substrate and incubated for 8 hours; after magnetic separation, the composite SERS substrate was washed twice with 0.02% Tween 20 aqueous solution to remove the uncross-linked aptamer, and the obtained particles were uniformly dispersed in a PBS buffer solution (pH=7.4) at a concentration of 1 mg / ml; finally, 100 μL of PBS buffer solution (pH=7.4) containing a methylene blue-modified aptamer complementary chain was added to the above solution, incubated at 95°C for 3 minutes, and cooled to room temperature within 1 hour; thereafter, the mixture was incubated at room temperature for 30 minutes to finally obtain a SERS aptamer sensor;

[0083] Step 3: Synthesis of SERS aptamer sensor:

[0084] (1) 2 g of a magnetic composite SERS active substrate, 0.2 g of streptavidin and 2 mL of a phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, and the mixture was shaken at 4°C for 6 h and then magnetically separated to obtain a streptavidin-modified composite SERS substrate;

[0085] (2) 1 g of streptavidin-modified composite SERS substrate, 100 μL of 10 μM biotin-modified okadaic acid aptamer single chain and 1 mL of phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, incubated at 25° C. for 8 h, magnetically separated the product, and washed twice with 1 mL of 0.02% Tween 20 aqueous solution to obtain a composite SERS substrate modified with a single chain aptamer;

[0086] (3) Finally, 1 g of the composite SERS substrate modified with a single chain of aptamer, 100 μL of aptamer complementary chain modified with 10 μM methylene blue, and 1 mL of phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, incubated at 95 °C for 3 min, cooled for 1 h, and then incubated at 25 °C for 30 min. After magnetic separation, the SERS aptamer sensor was finally obtained.

[0087] Detection of okadaic acid content in shellfish meat samples:

[0088] (1) Establishment of Okadaic Acid Standard Curve:

[0089] Prepare 0, 0.5, 5, 10, 20, 40, 60, 80, 100, 200 ng / mL okadaic acid standard solutions, mix 1 g of the SERS aptamer sensor prepared in step 3 with 3 mL of okadaic acid standard solutions of different concentrations, incubate the mixture at 25°C for 50 min, collect the reacted SERS aptamer sensor after magnetic separation, redissolve it in 1 mL of aqueous solution, and collect SERS spectra; record the characteristic value of the SERS intensity signal in the spectrum, and establish a standard curve by fitting with the okadaic acid concentration; in the range of 0.5-100 ng / mL, I 1612 / 1570 The relationship between the ratio and the concentration of okadaic acid showed a negative correlation (y = 0.7000-0.0050x), R 2 was 0.9896; in addition, the limit of detection (LOD) was estimated to be 0.18 ng / mL (S / N = 3);

[0090] (2) Detection of okadaic acid content in shellfish meat samples:

[0091] 0.1 mL of 200 ng / mL okadaic acid standard solution was added to 2 g of shellfish sample, and the obtained sample was homogenized. The homogenized shellfish sample was then mixed with 9 mL of methanol, and the mixture was rotated for 1 minute and centrifuged at 12000 g for 3 min at 4°C to obtain supernatant A. The remaining residue was re-extracted into 9 mL of methanol, and the mixture was rotated for 1 minute and centrifuged at 12000 g for 3 min at 4°C to obtain supernatant B. Supernatants A and B were mixed, and the mixed solution was fixed to 20 mL with methanol. 5 mL of the mixed solution was taken. The mixture was evaporated to dryness under nitrogen, and then 0.1 mL of formic acid and 0.9 mL of aqueous solution were added to dissolve the mixture to obtain a pretreated shellfish meat sample; 1 g of the SERS aptamer sensor prepared in step 3 was mixed with 1 mL of the pretreated shellfish meat sample, and the mixture was incubated at 25° C. for 50 min. After magnetic separation, the SERS aptamer sensor after the reaction was collected and redissolved in 1 mL of aqueous solution, and a SERS spectrum was collected; the characteristic value of the SERS intensity signal in the spectrum was recorded, and the result was substituted into the standard curve obtained in step (1), and the okadaic acid content in the shellfish meat was obtained to be 51 ng / mL.

[0092] Example 3: Detection of Okadaic Acid in Fish

[0093] Step 1, preparation of tetraaminothiophenol embedded core-shell nanomaterials:

[0094] Add 100 mL of water with a mass concentration of 1% chloroauric acid aqueous solution and boil it. After heating and boiling, add 1 mL of 1% trisodium citrate dihydrate aqueous solution, keep the solution boiling until the solution turns wine red, stop heating, and obtain a gold nano solution; after the gold nano solution is cooled to room temperature, add 0.8 mL of 0.1 mM tetraaminothiophenol solution, stir at room temperature for 1 hour, boil the solution after the reaction, add 4 mL of 1% trisodium citrate dihydrate solution after boiling, and continue stirring for 10 minutes to obtain a tetraaminothiophenol-modified gold nano solution; finally, add 4 mL of 4 mM silver nitrate dropwise to the tetraaminothiophenol-modified gold nano solution, stir for 1 hour, and obtain a tetraaminothiophenol-embedded gold and silver core-shell nanomaterial;

[0095] Step 2: Preparation of a magnetic composite SERS active substrate:

[0096] 2.7 g of ferric chloride hexahydrate, 1 g of polyethylene glycol 6000 and 8 g of sodium acetate were mixed with 80 mL of ethylene glycol, and after stirring for 1 hour, the solution was cooled to room temperature and then magnetically separated to obtain magnetic nanospheres; 0.04 g of magnetic nanospheres were added to 20 mL of 8% polyethyleneimine ethanol solution, the solution was ultrasonically dissolved for 2 hours, and amino-modified magnetic nanospheres were obtained by magnetic separation; subsequently, 4 g of amino-modified magnetic nanospheres were mixed with 10 mL of tetraaminothiophenol-embedded gold-silver core-shell nanosolution synthesized in step 1, mechanically vibrated for 3 hours, and the material obtained by magnetic separation was washed with deionized water and ethanol for 3 times respectively, and then dried in a vacuum oven at 60°C to obtain a composite SERS active substrate with magnetic properties;

[0097] Step 3: Synthesis of SERS aptamer sensor:

[0098] (1) 2 g of a magnetic composite SERS active substrate, 0.3 g of streptavidin, and 2 mL of a phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, and the mixture was shaken at 4 °C for 6 h and then magnetically separated to obtain a streptavidin-modified composite SERS substrate;

[0099] (2) 1 g of streptavidin-modified composite SERS substrate, 120 μL of 12 μM biotin-modified okadaic acid aptamer single chain and 1 mL of phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, incubated at 25° C. for 8 h, and the product was magnetically separated and washed twice with 1 mL of 0.02% Tween 20 aqueous solution to obtain a composite SERS substrate modified with a single chain aptamer;

[0100] (3) Finally, 1 g of the composite SERS substrate modified with a single-chain aptamer, 120 μL of the aptamer complementary chain modified with 12 μM methylene blue, and 1 mL of phosphate buffer solution (pH = 7.4, 10 mmol / L) were mixed, incubated at 95 °C for 3 min, cooled for 1 h, and then incubated at 25 °C for 30 min. After magnetic separation, the SERS aptamer sensor was finally obtained.

[0101] Detection of Okadaic Acid in Fish Samples:

[0102] (1) Establishment of Okadaic Acid Standard Curve:

[0103] Prepare 0, 0.5, 5, 10, 20, 40, 60, 80, 100, 200 ng / mL okadaic acid standard solutions, mix 1 g of the SERS aptamer sensor prepared in step 3 with 3 mL of okadaic acid standard solutions of different concentrations, incubate the mixture at 25°C for 50 min, collect the reacted SERS aptamer sensor after magnetic separation, redissolve it in 1 mL of aqueous solution, and collect SERS spectra; record the characteristic value of the SERS intensity signal in the spectrum, and establish a standard curve by fitting with the okadaic acid concentration; in the range of 0.5-100 ng / mL, I 1612 / 1570 The relationship between the ratio and the concentration of okadaic acid showed a negative correlation (y = 0.7005-0.0042x), R 2 was 0.9866; in addition, the limit of detection (LOD) was estimated to be 0.21 ng / mL (S / N = 3);

[0104] (2) Detection of okadaic acid content in fish samples:

[0105] 0.1 mL of 200 ng / mL okadaic acid standard solution was added to 2 g of fish sample, and the obtained sample was homogenized. The homogenized shellfish sample was then mixed with 9 mL of methanol, and the mixture was rotated for 1 minute and centrifuged at 12000 g for 3 min at 4 °C to obtain supernatant A. The remaining residue was re-extracted into 9 mL of methanol, and the mixture was rotated for 1 minute and centrifuged at 12000 g for 3 min at 4 °C to obtain supernatant B. Supernatants A and B were mixed, and the mixture was fixed to 20 mL with methanol. 5 mL of the mixture was taken. The mixture was evaporated to dryness under nitrogen, and then 0.1 mL of formic acid and 0.9 mL of aqueous solution were added to dissolve the mixture to obtain a pretreated fish meat sample; 1 g of the SERS aptamer sensor prepared in step 3 was mixed with 1 mL of the pretreated fish meat sample, and the mixture was incubated at 25° C. for 50 min. After magnetic separation, the SERS aptamer sensor after the reaction was collected and redissolved in 1 mL of aqueous solution, and a SERS spectrum was collected; the characteristic value of the SERS intensity signal in the spectrum was recorded, and the result was substituted into the standard curve obtained in step (1), and the okadaic acid content in the shellfish meat was obtained to be 49 ng / mL.

[0106] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a competitive ratiometric SERS aptamer sensor, characterized in that: Here are the steps: Step 1, preparation of tetraaminothiophenol embedded core-shell nanomaterials: (1) adding chloroauric acid aqueous solution to water, mixing, heating to boil, adding trisodium citrate dihydrate aqueous solution, stirring to obtain a mixed solution, keeping the solution boiling until the solution turns wine red, stopping heating, and obtaining a gold nanoparticle solution; (2) After the gold nanoparticle solution is cooled to room temperature, a tetraaminothiophenol solution is added, and a first stirring reaction is carried out at room temperature. After the reaction, the solution is boiled, and after boiling, a trisodium citrate dihydrate solution is added, and stirring is continued for a period of time to obtain a tetraaminothiophenol-modified gold nanoparticle solution; finally, silver nitrate is added dropwise to the tetraaminothiophenol-modified gold nanoparticle solution, and a second stirring reaction is carried out to obtain a tetraaminothiophenol-embedded gold and silver core-shell nanoparticle solution; Step 2: Preparation of a magnetic composite SERS active substrate: Ferric chloride hexahydrate, polyethylene glycol 6000, sodium acetate and ethylene glycol are mixed, and after stirring, the solution is cooled to room temperature and then magnetically separated to obtain magnetic nanospheres; the magnetic nanospheres are added to a polyethyleneimine ethanol solution to obtain ultrasonic dissolution of the solution, and amino-modified magnetic nanospheres are obtained by magnetic separation; subsequently, the amino-modified magnetic nanospheres are mixed with the gold-silver core-shell nano-solution embedded with tetraaminothiophenol synthesized in step 1, and the material obtained by magnetic separation after mechanical oscillation is washed with deionized water and ethanol for several times respectively, and then vacuum dried to obtain a composite SERS active substrate with magnetic properties; Step 3: Synthesis of SERS aptamer sensor: (1) mixing the composite SERS active substrate with magnetic properties obtained in step 2, streptavidin and phosphate buffer solution, shaking for a period of time and then performing magnetic separation to obtain a composite SERS substrate modified with streptavidin; (2) mixing the streptavidin-modified composite SERS substrate, the biotin-modified okadaic acid aptamer single chain and a phosphate buffer solution, incubating, magnetically separating the product, and washing it several times with a Tween 20 aqueous solution to obtain a composite SERS substrate modified with an aptamer single chain; (3) Finally, the composite SERS substrate modified with the aptamer single chain, the aptamer complementary chain modified with methylene blue and the phosphate buffer solution were mixed, cooled to room temperature after the first incubation, and incubated for the second time, and finally the SERS aptamer sensor was obtained after magnetic separation.

2. The method for preparing the competitive ratiometric SERS aptamer sensor according to claim 1, characterized in that: The volume ratio of the chloroauric acid aqueous solution, water and the trisodium citrate dihydrate aqueous solution in step 1 (1) is 1:100:1.5; the mass concentration of the chloroauric acid aqueous solution is 1%; and the mass concentration of the trisodium citrate dihydrate aqueous solution is 1%.

3. The method for preparing the competitive ratiometric SERS aptamer sensor according to claim 1, characterized in that: The volume ratio of the gold nanoparticle solution, the tetraaminothiophenol solution, the trisodium citrate dihydrate solution and the silver nitrate in the tetraaminothiophenol-modified gold nanoparticle solution in step 1 (2) is 100:0.4-0.8:4:4; the concentration of the tetraaminothiophenol solution is 0.1 mM, the mass concentration of the trisodium citrate dihydrate solution is 1%, and the concentration of the silver nitrate is 4-8 mM; The first stirring reaction time is 1 hour, and the stirring is continued for 5-10 minutes; the second stirring reaction time is 1 hour.

4. The method for preparing the competitive ratiometric SERS aptamer sensor according to claim 1, characterized in that: The dosage relationship of ferric chloride hexahydrate, polyethylene glycol 6000, sodium acetate and ethylene glycol in step 2 is 2.7g:1g:8g:80ml; the dosage ratio of the magnetic nanospheres to the polyethyleneimine ethanol solution is 0.04g:20ml, and the mass concentration of the polyethyleneimine ethanol solution is 3-8%; the dosage ratio of the magnetic microsphere solution to the gold and silver core-shell nano solution embedded with tetraaminothiophenol is 4g:10mL; the stirring time is 1h, the ultrasonic dissolution time is 2h, the mechanical oscillation time is 3h, and the vacuum drying temperature is 60°C.

5. The method for preparing the competitive ratiometric SERS aptamer sensor according to claim 1, characterized in that: The amount ratio of the magnetic composite SERS active substrate, streptavidin and phosphate buffer solution in step 3 (1) is 2 g: 0.1-0.3 g: 2 mL; the shaking temperature is 4° C., and the shaking time is 6 h; In step 3 (2), the amount ratio of the streptavidin-modified composite SERS substrate, the biotin-modified okadaic acid aptamer single chain, the phosphate buffer solution and the Tween 20 aqueous solution is 1 g: 80-120 μL: 1 mL: 1 mL; the concentration of the biotin-modified okadaic acid aptamer single chain is 8-12 μM; the incubation time is 8 h, the incubation temperature is 25° C.; the mass concentration of the Tween 20 aqueous solution is 0.02%; In step three (3), the dosage ratio of the aptamer single-chain modified composite SERS substrate, the methylene blue modified aptamer complementary chain and the phosphate buffer solution is 1g:80-120μL:1mL; the concentration of the methylene blue modified aptamer complementary chain is 8-12μM; the first incubation time is 3min, the incubation temperature is 95°C, and the cooling time is 1h; the second incubation time is 30min, and the incubation temperature is 25°C.

6. The method for preparing the competitive ratiometric SERS aptamer sensor according to claim 1, characterized in that: The pH of the phosphate buffer solution used in step 3 is 7.4, and the concentration is 10 mmol / L.

7. Use of the competitive ratiometric SERS aptamer sensor prepared according to any one of claims 1 to 6 in detecting okadaic acid, characterized in that: Here are the steps: (1) Establishment of Okadaic Acid Standard Curve: The SERS aptamer sensor prepared in step 3 is mixed with okadaic acid standard solutions of different concentrations, and the sensor and the okadaic acid standard solution are in a one-to-one correspondence. The obtained mixed solution is magnetically separated after incubation, and the SERS aptamer sensor after the reaction is collected and redissolved in aqueous solution, and SERS spectrum is collected; the characteristic value of the SERS intensity signal in the spectrum is recorded, and a standard curve is established by fitting with the okadaic acid concentration; (2) Detection of okadaic acid in food and environmental samples: The food or environmental sample is pretreated to obtain an extract containing okadaic acid; the SERS aptamer sensor prepared in step 3 is mixed with the extract, incubated, magnetically separated, and the reaction SERS aptamer sensor is collected and redissolved in an aqueous solution, and a SERS spectrum is collected; the characteristic value of the SERS intensity signal in the spectrum is recorded, and the obtained result is brought into the standard curve obtained in step (1) to detect the content of okadaic acid in the sample.

8. The use according to claim 7, characterized in that In step (1), the dosage ratio of the SERS aptamer sensor, the okadaic acid standard solution and the aqueous solution is 1 g:1 mL; The concentration range of the okadaic acid standard solution is 0-200 ng / mL; the incubation time is 50 min, and the incubation temperature is 25° C.; the amount ratio of the SERS aptamer sensor collected after the magnetic separation reaction to the aqueous solution is 1 g:1 mL; The step of recording the characteristic value of the SERS intensity signal in the spectrum is: respectively recording the characteristic value of the sensor at 1612cm under the excitation of 785nm excitation light -1 With 1570cm -1 The SERS intensity value at the former and the latter is I 1612 / 1570 is the characteristic value of the SERS intensity signal in the spectrum.

9. The use according to claim 7, characterized in that: The usage ratio of the SERS aptamer sensor to the okadaic acid extract in (2) is 1 g:1 ml; The incubation time is 50 minutes, and the incubation temperature is 25° C.; the amount ratio of the SERS aptamer sensor collected after the magnetic separation reaction to the aqueous solution is 1 g:1 mL.

10. The use according to claim 7, characterized in that The environmental sample in step (2) includes water and soil, and the pretreatment method of the food or environmental sample is as follows: if it is a liquid, the liquid sample is directly tested after coarse filtration; if it is a solid, the solid sample is first homogenized, methanol is added and mixed and shaken, and the supernatant is taken after centrifugation and recorded as solution A, the remaining residue is mixed with methanol again and shaken, and the supernatant is taken after centrifugation and recorded as solution B, solution A and solution B are mixed and fixed to volume with methanol, and the mixed solution is taken and dried with nitrogen and then formic acid and aqueous solution are added for re-dissolution; In the pretreatment method, the dosage ratio of the solid sample, methanol in solution A, and methanol in solution B is 2g:9mL:9mL; the shaking time is 1 minute; the fixed volume of the mixed solution A and solution B is 20mL; the volume ratio of the mixed solution, formic acid and water is 5:0.1:0.99mL; the centrifugal speed of solution A and solution B is 12000rpm, and the centrifugation time is 3min.

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