Ratio biosensor for detection of marine biotoxins and preparation and use thereof

Abstract

The application discloses a ratio biosensor for marine biological toxin detection and preparation and application thereof, and the ratio biosensor comprises a specific detection biomagnetic element FA, a catalytic reaction solution and a SERS enhanced nanosubstrate; wherein, magnetic particles (Fe3O4-Apt) anchored by aptamers are combined with gold-labeled short complementary DNA (Au-cDNA) to generate the biomagnetic element (FA). In the detection, if the target exists, the Au-cDNA is dissociated, the separated Au cDNA is used as a catalyst to initiate a reduction reaction of 4-nitrothiophenol (4-NTP) in the presence of a hydrogen source, and a ratio change of SERS spectrum of 4-NTP is caused. The change is finally collected on the SERS enhanced nanosubstrate. The design of the ratio biosensor can effectively overcome unstable detection response caused by spectral fluctuation, and has self-calibration ability and high robustness.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food safety detection, and particularly discloses a ratio biosensor for marine biotoxin detection and preparation and application thereof. BACKGROUND

[0002] Marine biotoxins are a class of active compounds with toxicity, which can accumulate in fish or shellfish and cause various human diseases. Marine biotoxin outbreaks are often closely related to the proliferation of some toxic phytoplankton species or the outbreak of harmful algal blooms. In particular, in the past few decades, the frequency and range of toxic algal blooms have increased significantly, making marine biotoxin safety an increasingly important focus of attention at home and abroad. Considering the long-term persistence, bioaccumulation and high toxicity of marine biotoxins, it is urgent to provide a detection method that is sensitive and reliable enough to implement appropriate regulatory restrictions on the toxin content in marine products for human consumption.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a detection technique that uses metal nano-substrates to significantly enhance the Raman signal of target objects. Due to its rapidity, traceability, high sensitivity and other advantages, SERS has become an important tool for detecting trace amounts of marine biotoxins. However, due to the highly concentrated electromagnetic hot spots, the non-uniformity of the SERS substrate and the measurement conditions, the reproducibility and reliability are low. Therefore, the use of SERS for quantitative analysis of analyte concentration based on a single characteristic peak has been controversial. Especially for the detection of trace amounts of marine biotoxins, this signal instability increases the uncertainty even more. SUMMARY

[0004] In order to solve the problems existing in the prior art, the present application aims to provide a ratio biosensor for marine biotoxin detection and preparation and application thereof. The ratio biosensor comprises a biomagnetic element, a catalytic reaction solution and a SERS-enhanced nano-substrate. When marine biotoxins are present, the biomagnetic element releases gold nanoparticles, which catalyze the catalytic reaction solution, resulting in a ratio change in the SERS spectral signal of the catalytic reaction solution, which is collected on the surface of the SERS-enhanced nano-substrate.

[0005] The object of the present application can be achieved by the following technical solutions:

[0006] A ratio biosensor for marine biotoxin detection preparation method, the ratio biosensor comprises a biomagnetic element FA with specific detection, a catalytic reaction solution and a SERS-enhanced nano-substrate;

[0007] The preparation method of the biomagnetic element FA is: synthesizing carboxylated magnetic nanoparticles Fe3O4-COOH, sequentially combining the same with streptavidin SA and 5'-end biotin-labeled aptamer Apt to form a Fe3O4-Apt complex; grafting a fragment complementary chain cDNA of the aptamer to a surface of gold nanoparticles AuNPs to form an Au-cDNA complex; hybridizing the Au-cDNA with the Fe3O4-Apt through a base pair complementary bond to form the biomagnetic element FA;

[0008] The preparation method of the catalytic reaction solution is: mixing 4-nitrothiophenol 4-NTP and sodium borohydride to obtain the catalytic reaction solution.

[0009] The preparation method of the SERS enhanced nanosubstrate is: depositing gold nanoparticles on a surface of a solid phase carrier to form the SERS enhanced nanosubstrate.

[0010] Further, the synthesis step of the biomagnetic element FA comprises:

[0011] 1) synthesizing carboxylated magnetic nanoparticles through a thermal decomposition method, mixing ferric chloride hexahydrate, trisodium citrate and ethylene glycol and fully stirring; then, adding ammonium acetate and continuously stirring for 25-35 minutes; subsequently, transferring the uniform precursors into a polytetrafluoroethylene-lined autoclave and heating at 200℃ for 8 hours; after completion, slowly cooling the Fe3O4 product to room temperature and rinsing with ethanol and water; the mass ratio of the ferric chloride hexahydrate, the trisodium citrate, the ethylene glycol and the ammonium acetate is 1.0802:0.2:20:1.2 g;

[0012] 2) the synthesis Fe3O4-Apt step is: adding the prepared Fe3O4, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysulfosuccinimide into a 0.01M MES buffer and mechanically stirring for 3 hours; then, separating the carboxyl-activated Fe3O4 from the mixture through magnetic separation and washing with water and ethanol for three times; then, dispersing streptavidin SA into the carboxyl-activated Fe3O4 and incubating at 37℃ for 12 hours; thoroughly purifying the obtained Fe3O4-SA by washing with a PBS buffer for three times; subsequently, adding 0.8-1.2 μM of an Apt solution, incubating at 37℃ for 6-10 hours, separating the reacted Fe3O4-Apt complex through magnetic separation and washing with a PBS buffer to remove excess aptamer; the ratio of the Fe3O4, the EDC, the NHS, the MES, the SA and the Apt is 0.01 g:0.04 g:0.02 g:20 g:0.1 g:100 μL;

[0013] 3) The Au-cDNA synthesis step is: synthesis of AuNPs with different sizes by trisodium citrate reduction method; 1% gold chloride tetrahydrate is dissolved in water and stirred vigorously for about 1 minute; then, 1% trisodium citrate solution is quickly added to the boiling solution and continuously refluxed for 30 minutes; the prepared AuNPs are mixed with 50 nM cDNA and shaken gently at 37°C overnight to obtain Au-cDNA complex; the amount ratio of gold chloride tetrahydrate, water, trisodium citrate solution, and cDNA is 50 μL: 10 mL: 0.5-1 mL: 5 mL;

[0014] 4) The FA synthesis step includes: injecting 50 μL, 0.8-1.2 mg / mL Fe3O4-Apt into 2 mL Au-cDNA and mixing at 37°C for 8 hours to form FA.

[0015] Further, the volume ratio of 4-NTP and sodium borohydride in S2 is 200: 20 μL, and the concentration ratio is 10: 1 mM.

[0016] Further, 4-NTP in S2 is reduced to 4-aminothiophenol (4-ATP) under the catalysis of gold nanoparticles and in the environment of sodium borohydride as a hydrogen source; during the reduction process, the SERS spectrum of 4-NTP is significantly reduced at 1336 cm – 1 , and the intensity at 1077 cm – 1 is basically unchanged.

[0017] Further, the solid phase carrier of the SERS enhanced nanosubstrate in S3 includes but is not limited to silicon wafer, glass, polydimethylsiloxane PDMS, polymethyl methacrylate PMMA, polyethylene terephthalate PET, polyethylene PE, or transparent tape.

[0018] Further, the step of depositing gold nanoparticles on the surface of the solid phase carrier in S3 includes: immersing the solid phase carrier in the piranha solution at 40°C for 2 minutes to obtain a hydroxyl surface, then rinsing with distilled water and blowing dry with nitrogen; then, immersing the solid phase carrier in a 10% (v / v) 3-aminopropyltriethoxysilane ethanol solution and performing surface amination at 70°C; then, after washing with water, immersing the amination-modified solid phase carrier in a gold nanoparticle solution and stirring overnight at room temperature to obtain a SERS enhanced nanosubstrate, drying with nitrogen, then sealing and storing at 4°C.

[0019] A ratio biosensor for marine toxin detection is prepared by the above-mentioned ratio biosensor preparation method for marine biological toxin detection.

[0020] The application of a ratio biosensor for marine toxin detection, using the ratio biosensor described above to detect marine toxins, the detection steps are as follows:

[0021] S1, pretreating the to-be-detected substance to obtain a to-be-detected toxin solution;

[0022] S2, detecting the to-be-detected toxin solution by using the ratio biosensor; first, mixing the biomagnetic element FA with the to-be-detected toxin solution, mixing the supernatant with the catalytic reaction solution to generate a catalytic reaction, and dropping the catalytic product on the SERS enhanced nanosubstrate and performing SERS analysis;

[0023] S3, comparing the SERS analysis result with a standard curve to obtain the concentration of the to-be-detected bacteria solution.

[0024] Further, a method for establishing a marine toxin concentration standard curve is provided:

[0025] Prepare marine toxin standard solutions of different concentrations;

[0026] Detect the marine toxin standard solutions of different concentrations by using the ratio biosensor, and establish a standard curve of the marine toxin concentration and the Raman signal characteristic value.

[0027] Further, the S2 inspection step includes: adding the to-be-detected toxin solution into 1mg / mL FA, reacting at 37℃ for 30min; separating the FA using an external magnet, and mixing the obtained supernatant with the catalytic reaction solution, after incubating at room temperature for 5min, immediately adding 3mM hydrochloric acid to terminate the catalytic reaction; finally, 5μL of the catalytic product is dropped on the SERS enhanced nanosubstrate and SERS analysis is performed; the amount ratio of the to-be-detected toxin solution, the FA, and the hydrochloric acid is 50μL: 50μL: 10μL.

[0028] Beneficial effects:

[0029] (1) The present application is based on the affinity of Apt and the target toxin, which triggers the release control of FA Au-cDNA, and the released Au reduces 4-NTP in the catalytic reaction solution to generate a spectral ratio change. Compared with the traditional general ratio strategy which needs the target to directly drive the SERS spectral ratio change of the signal molecule to identify the target, the present application uses an indirect method to realize the triggering of the target by using Apt, thereby effectively overcoming the harsh conditions for specific targets.

[0030] (2) The ratio strategy used in the sensor of the present application can weaken the influence of uneven electromagnetic field enhancement caused by instruments and environmental factors compared with the typical detection method based on single response.

[0031] (3) The use of magnetic nanoparticles in the sensor greatly simplifies the preparation and separation process of the sensor, improves the portability of the detection operation, reduces the cost, and improves the detection efficiency. In addition, compared with antibodies, the use of Apt has the advantages of short synthesis time, low cost, high stability, and strong specificity.

[0032] (4) The released trace amount of AuNPs in the present application can effectively catalyze the reaction of 4-NTP, resulting in a change in the intensity ratio of the characteristic SERS peaks of 4-NTP, and thus realizing the amplification of the SERS signal. This catalytic amplification mechanism endows the sensor with certain sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0034] Figure 1 The construction and principle diagram of the biosensor for detecting okadaic acid (OA) toxin in the embodiments of the present application;

[0035] Figure 2 Scanning electron microscope and transmission electron microscope images of the prepared FA sensor;

[0036] Figure 3 Characterization diagram of gold nano-deposited PDMS in the embodiments;

[0037] Among them, A is the scanning electron microscope image of gold nano-deposited PDMS, and B is the ultraviolet image of PDMS and gold nano-deposited PDMS;

[0038] Figure 4 Ultraviolet and SERS spectra of 4-NTP before and after gold catalysis in the present application;

[0039] Figure 5 SERS intensity standard curve established in the embodiments for detecting different concentrations of okadaic acid;

[0040] Among them, A is the Raman signal diagram of the sensor for detecting different concentrations of OA; B is the standard curve established by the ratio of the concentration value of OA and the intensity of the spectral band of the sensor at 1336 and 1077 cm -1 . 1336 1077 DETAILED DESCRIPTION

[0041] ​​Various exemplary embodiments of the present application will now be described in detail, which should be considered in a descriptive sense only and not for purposes of limitation to the present application, as it is understood that the broader aspects of the application will be described in greater detail throughout the present specification.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, it is to be understood that where the application is herein described as comprising or having particular elements and / or features, it is contemplated as being comprised of and / or comprising additional, not mentioned elements and / or features. In other words, it is contemplated that additional elements and / or features not mentioned in the specification can be added to the application without departing from the scope of the application. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, it is to be understood that the use of a singular term, such as, but not limited to "a", "an" and "the" include the plural and vice versa unless the context clearly dictates otherwise. Furthermore, it is to be understood that the description of a particular embodiment is intended to include any and all equivalents unless the context clearly dictates otherwise. Additionally, it is to be understood that the description of a particular embodiment is intended to include any and all alternatives, modifications and variations falling within the scope of the appended claims. It is also to be understood that the use of the term "or" includes any and all combinations of one or more of the associated listed items.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0044] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the description and practice of the application. The description and examples are illustrative only.

[0045] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0046] The "parts" described in the present application are measured by mass unless otherwise specified.

[0047] The present application is described by taking the following biological marine toxins as examples, which are not limited to the following: OA, tetrodotoxin, saxitoxin, and different aptamers are used based on different biological marine toxins, and the corresponding aptamer sequence numbers have been widely studied. The biological sensor is constructed by using the published biological marine toxin aptamer sequence number.

[0048] Example 1

[0049] A preparation method of a ratio biosensor for marine biological toxin detection, comprising:

[0050] Reference Figure 1 and Figure 2Fe3O4nanospheres were prepared by thermal decomposition method. Typically, 1.0802 g of iron(III) chloride hexahydrate and 0.2 g of trisodium citrate were added to 20 mL of ethylene glycol. With thorough stirring, 1.2 g of ammonium acetate was added to the solution and stirring was continued for 30 minutes. The homogeneous precursor was then transferred to a high-pressure vessel and heated at 200 °C for 8 hours. Subsequently, the Fe3O4product was slowly cooled to room temperature and then rinsed with ethanol and water. The synthesized Fe3O4was re-suspended in water and quantified to 1 mg / mL;

[0051] The carboxyl groups on the surface of Fe3O4were initially activated and then reacted with the primary amine of SA to form stable amide bonds to form Fe3O4-SA. 10 mg of prepared Fe3O4, 40 mg of EDC and 20 mg of NHS were added to 20 ml of MES buffer (0.01 M) and reacted for 3 hours under mechanical stirring. Subsequently, the resulting precipitate was separated from the mixture by magnetic separation and washed with water and ethanol three times. Next, 1 mL of 1 mg / mL SA was dispersed in 10 mL of 10 mg / mL carboxyl-activated Fe3O4and incubated at 37 °C for 12 hours. The resulting Fe3O4-SA was thoroughly purified by washing with PBS buffer three times;

[0052] 10 mg of Fe3O4-SA was dispersed in 1 mL of 0.1 M PBS buffer, and then 100 μL of 1 μM Apt solution was added to the PBS buffer solution. After incubation at 37 °C for 8 hours, the reacted Fe3O4-Apt complex was separated and washed with PBS buffer to remove excess aptamer. 50 μL of 1% HAuCl4was dissolved in 10 mL of water and stirred vigorously for about 1 minute. Subsequently, different volumes of 1 mL of 0.1% trisodium citrate solution were quickly added to the boiling solution and continuously refluxed for 30 minutes. Then it was re-dispersed in 10 mL of water for subsequent experiments. At the same time, 200 μL of prepared Au was mixed with 100 μL of 50 nM cDNA PBS buffer solution (0.1 M) and incubated at 37 °C with gentle shaking overnight. After incubation, the Au-cDNA solution was centrifuged to separate the free cDNA, which was then re-dissolved in 5 mL of PBS buffer. In addition, 50 μL of 1 mg / mL Fe3O4-Apt complex was injected into 2 mL of Au-cDNA and mixed at 37 °C for 8 hours to form FA. The prepared FA was washed with PBS buffer three times and re-dispersed in 50 μL of PBS buffer solution for subsequent experiments;

[0053] The process of preparing gold nano-deposited PDMS is as follows: first, the PDMS film is cut into 0.3 x 0.3 x 0.5 cm (length x width x thickness). Then, Au@PDMS is prepared by electrostatically attaching AuNPs on the aminated PDMS surface. Generally, the PDMS film is first immersed in a Piranha solution (H2O2 / H2SO4 = 1:3, v / v) at 40°C for 2 minutes to obtain a hydroxyl surface, then rinsed with distilled water and blown dry with nitrogen. After that, the PDMS film is immersed in a 10% (v / v) APTES ethanol solution and aminated at 70°C to perform amination. Subsequently, after washing with water, the aminated modified PDMS is immersed in an Au solution and stirred at room temperature overnight. The obtained Au@PDMS is thoroughly rinsed, dried with nitrogen, then sealed and stored at 4°C, as shown in Figure 4

[0054] In combination Figure 1 with the above, the construction and principle diagram of the ratio biosensor for detecting OA toxin as the detection object in this embodiment are shown. For other detection objects, the corresponding aptamer (Apt) and fragment complementary chain (cDNA) need to be selected, so that specific detection of different detection objects can be realized.

[0055] Example 2

[0056] A preparation method of a ratio biosensor for marine biological toxin detection, comprising:

[0057] Referring to Figure 1 and Figure 2 Fe3O4 nanospheres were prepared by a thermal decomposition method. Typically, 1.0802 g of iron(III) chloride hexahydrate and 0.2 g of trisodium citrate were added to 20 mL of ethylene glycol. With thorough stirring, 1.2 g of ammonium acetate was added and stirring was continued for 30 minutes. The homogeneous precursor was then transferred to a polytetrafluoroethylene-lined autoclave and heated at 200°C for 8 hours. Subsequently, the Fe3O4 product was slowly cooled to room temperature and then rinsed with ethanol and water. The synthesized Fe3O4 was resuspended in water and quantified to 1 mg / mL;

[0058] ​The carboxyl groups on the surface of Fe3C>4 were first activated and then reacted with the primary amine of SA to form stable amide bonds, thus forming Fe3C>4-SA. 10 mg of prepared Fe3C>4, 40 mg of EDC and 20 mg of NHS were added to 20 ml of MES buffer (0.01 M) and reacted for 3 hours under mechanical stirring. Subsequently, the resulting precipitate was separated from the mixture by magnetic separation and washed with water and ethanol three times. Subsequently, 1 mL of 1 mg / mL SA was dispersed in 10 mL of 10 mg / mL carboxyl-activated Fe3C>4 and incubated at 37°C for 12 hours. The resulting Fe3C>4-SA was thoroughly purified by washing with PBS buffer three times;

[0059] 10 mg of Fe3C>4-SA was dispersed in 1 mL of 0.1 M PBS buffer, and then 100 μL of 1 μM Apt solution was added to the PBS buffer solution. After incubation at 37°C for 8 hours, the reacted Fe3C>4-Apt complex was separated and washed with PBS buffer to remove excess aptamer. 50 μL of 1% HAuCl4 was dissolved in 10 mL of water and stirred vigorously for about 1 minute. Different volumes of 1 mL of 0.1% trisodium citrate solution were quickly added to the boiling solution and continuously refluxed for 30 minutes. And it was redispersed in 10 mL of water for subsequent experiments. At the same time, 200 μL of prepared Au was mixed with 100 μL of 50 nM cDNA PBS buffer solution (0.1 M), and incubated at 37°C overnight with gentle shaking. After incubation, the Au-cDNA solution was centrifuged to separate the free cDNA, which was then redissolved in 5 mL of PBS buffer. In addition, 50 μL of 1 mg / mL Fe3C>4-Apt complex was injected into 2 mL of Au-cDNA and mixed at 37°C for 8 hours to form FA. The prepared FA was washed with PBS buffer three times and redispersed in 50 μL of PBS buffer solution for subsequent experiments;

[0060] The process of preparing gold nano-deposited PMMA is as follows: PMMA film is cut into 0.3 x 0.3 x 0.5 cm (length x width x thickness). Au@PDMS is prepared by electrostatically attaching AuNPs onto the surface of aminated PMMA. Generally, the PMMA film is first immersed in Piranha solution (H2O2 / H2SO4 = 1:3, v / v) at 40°C for 2 minutes to obtain a hydroxyl surface, then rinsed with distilled water and dried with nitrogen. After that, the PMMA film is immersed in a 10% (v / v) APTES ethanol solution and aminated at 70°C to perform amination. Subsequently, after washing with water, the aminated PDMS is immersed in a gold nanostar solution and stirred at room temperature overnight. The obtained Au@PDMS is thoroughly rinsed, dried with nitrogen, then sealed and stored at 4°C.

[0061] Example 3

[0062] Based on the method disclosed in the above Examples 1, 2, and 3, a ratio biosensor for marine toxin detection can be prepared, which is used to detect OA in this example. The specific steps are as follows:

[0063] In order to achieve specific and quantitative detection, it is necessary to establish a standard curve of the concentration of okadaic acid toxin OA, and the process is as follows:

[0064] Prepare standard solutions of okadaic acid toxin OA with different concentrations; use the ratio biosensor to detect the standard solutions of okadaic acid toxin OA with different concentrations, and establish a standard curve of the concentration of okadaic acid toxin OA and color, Raman signal characteristic value. For example, 50 μL of okadaic acid toxin standard solution is added to 50 μL of 1 mg / mL FA, and reacted at 37°C for 30 min. The FA is separated using an external magnet, and 30 μL of the supernatant is mixed with a uniform solution containing 200 μL of 10 mM NaBH4 and 20 μL of 1 mM 4-NTP. After incubation at room temperature for 5 minutes, 10 μL of 3 mM hydrochloric acid is immediately added to terminate the catalytic reaction. Finally, 5 μL of the catalytic product is dropped on the SERS enhanced nanosubstrate and subjected to SERS analysis; and finally a standard curve of the concentration of okadaic acid toxin OA and Raman signal characteristic value is established, as shown in Figure 5 .

[0065] Based on the standard curve of the concentration of okadaic acid toxin OA, the ratio biosensor of the present application is used to detect okadaic acid toxin OA in the test object, and the process is as follows:

[0066] The test object is pretreated to obtain a test toxin solution. First, the biomagnetic element FA is mixed with the test toxin solution, the supernatant is mixed with a catalytic reaction solution to undergo a catalytic reaction, the catalytic product is dropped on the SERS enhanced nanosubstrate and subjected to SERS analysis; the SERS analysis result is compared with the established standard curve to obtain the concentration of the test bacteria solution.

[0067] Example 4

[0068] This example takes tetrodotoxin as an example to explain. First, a standard curve of the concentration of tetrodotoxin is established, and the process is as follows:

[0069] Prepare standard solutions of tetrodotoxin at different concentrations; use the ratiometric biosensor to detect the standard solutions of tetrodotoxin at different concentrations, and establish a standard curve of the concentration of tetrodotoxin versus color and Raman signal characteristic values. For example, 50 μL of a standard solution of tetrodotoxin is added to 50 μL of 1 mg / mL FA, and reacted at 37°C for 30 min. The FA is separated using an external magnet, 30 μL of the supernatant obtained is mixed with a uniform solution containing 200 μL of 10 mM NaBH4 and 20 μL of 1 mM 4-NTP, and incubated at room temperature for 5 min, and then 10 μL of 3 mM hydrochloric acid is immediately added to terminate the catalytic reaction. Finally, 5 μL of the catalytic product is dropped on a SERS-enhanced nanosubstrate and subjected to SERS analysis; and finally a standard curve of the concentration of tetrodotoxin versus Raman signal characteristic values is established.

[0070] Based on the concentration standard curve of tetrodotoxin, the ratiometric biosensor of the application is used to detect tetrodotoxin in a test sample, and the process is as follows:

[0071] The test sample is pretreated to obtain a test toxin solution, the biomagnetic element FA is first mixed with the test toxin solution, the supernatant is mixed with a catalytic reaction solution to undergo a catalytic reaction, the catalytic product is dropped on a SERS-enhanced nanosubstrate and subjected to SERS analysis; the SERS analysis result is compared with the established standard curve to obtain the concentration of the test bacterial solution.

[0072] Example 5

[0073] This example is illustrated by taking saxitoxin as an example. First, a concentration standard curve of saxitoxin is established, and the process is as follows:

[0074] Prepare standard solutions of saxitoxin at different concentrations; use the ratiometric biosensor to detect the standard solutions of saxitoxin at different concentrations, and establish a standard curve of the concentration of saxitoxin versus color and Raman signal characteristic values. For example, 50 μL of a standard solution of saxitoxin is added to 50 μL of 1 mg / mL FA, and reacted at 37°C for 30 min. The FA is separated using an external magnet, 30 μL of the supernatant obtained is mixed with a uniform solution containing 200 μL of 10 mM NaBH4 and 20 μL of 1 mM 4-NTP, and incubated at room temperature for 5 min, and then 10 μL of 3 mM hydrochloric acid is immediately added to terminate the catalytic reaction. Finally, 5 μL of the catalytic product is dropped on a SERS-enhanced nanosubstrate and subjected to SERS analysis. Finally, a standard curve of the concentration of saxitoxin versus Raman signal characteristic values is established.

[0075] Based on the concentration standard curve of saxitoxin, the ratiometric biosensor of the application is used to detect saxitoxin in a test sample, and the process is as follows:

[0076] The test substance is pretreated to obtain a test toxin solution, the biomagnetic element FA is first mixed with the test toxin solution, the supernatant is mixed with a catalytic reaction solution to generate a catalytic reaction, the catalytic product is dropped on a SERS enhanced nano substrate and SERS analysis is performed; the SERS analysis result is compared with the established standard curve to obtain the concentration of the test bacteria solution.

[0077] The above examples are only used for illustrating the design idea and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the present application and to implement it, and the protection scope of the present application is not limited to the above examples. Therefore, any equivalent changes or modifications made according to the disclosed principles and design ideas of the present application are within the protection scope of the present application.

Claims

1. A method for the preparation of a ratiometric biosensor for marine biotoxin detection, characterized by, The ratio biosensor comprises a biomagnetic element FA with specific detection, a catalytic reaction solution and a SERS enhanced nanosubstrate; The preparation method of the biomagnetic element FA is as follows: synthesizing carboxylated magnetic nanoparticles Fe3O4-COOH, then sequentially combining the same with streptavidin SA and 5'-end biotin-labeled aptamer Apt to form a Fe3O4-Apt complex; grafting a fragment complementary chain cDNA of the aptamer to a surface of gold nanoparticles AuNPs to form an Au-cDNA complex; hybridizing the Au-cDNA with the Fe3O4-Apt through a base pair complementary bond to form the biomagnetic element FA; The preparation method of the catalytic reaction solution is as follows: mixing 4-nitrothiophenol 4-NTP and sodium borohydride to obtain the catalytic reaction solution; The preparation method of the SERS enhanced nanosubstrate is as follows: depositing gold nanoparticles on a surface of a solid phase carrier to form the SERS enhanced nanosubstrate; The synthesis steps of the biomagnetic element FA include: 1) synthesizing the carboxylated magnetic nanoparticles through a thermal decomposition method, mixing ferric chloride hexahydrate, trisodium citrate and ethylene glycol and fully stirring; then, adding ammonium acetate and continuously stirring for 25-35 minutes; subsequently, transferring the uniform precursors into a polytetrafluoroethylene-lined autoclave and heating at 200 DEG C for 8 hours; after completion, slowly cooling the Fe3O4 product to room temperature and rinsing with ethanol and water; the mass ratio of the ferric chloride hexahydrate, the trisodium citrate, the ethylene glycol and the ammonium acetate is 1.0802:0.2:20:1.2 g; 2) synthesizing the Fe3O4-Apt, which is as follows: adding the prepared Fe3O4, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysulfosuccinimide into a 0.01M MES buffer and mechanically stirring for 3 hours; then, separating the carboxyl-activated Fe3O4 from the mixture through magnetic separation and washing with water and ethanol for three times; then, dispersing streptavidin SA into the carboxyl-activated Fe3O4 and incubating at 37 DEG C for 12 hours; thoroughly purifying the obtained Fe3O4-SA by washing with a PBS buffer for three times; subsequently, adding an Apt solution of 0.8-1.2 μM, incubating at 37 DEG C for 6-10 hours, separating the reacted Fe3O4-Apt complex through magnetic separation and washing with a PBS buffer to remove the excess aptamer; the ratio of the Fe3O4, the EDC, the NHS, the MES, the SA and the Apt is 0.01 g:0.04 g:0.02 g:20 g:0.1 g:100 μL; 3) The synthesis of Au-cDNA step is: synthesis of AuNPs with different sizes by trisodium citrate reduction method; dissolve 1% gold chloride tetrahydrate in water and stir vigorously for about 1 minute; then, add 1% trisodium citrate solution to the boiling solution quickly and continue to reflux for 30 minutes; mix the prepared AuNPs with 50 nM cDNA and shake gently at 37°C overnight to obtain Au-cDNA complex; the amount ratio of gold chloride tetrahydrate, water, trisodium citrate solution and cDNA is 50 μL: 10 mL: 0.5-1 mL: 5 mL; 4) The FA synthesis step includes: inject 50 μL, 0.8-1.2 mg / mL Fe3O4-Apt into 2 mL Au-cDNA and mix at 37°C for 8 hours to form FA.

2. The method of claim 1, wherein the ratio biosensor for detecting marine toxin is prepared by the steps of: The volume ratio of NTP and sodium borohydride is 200:20 μL, and the concentration ratio is 10:1 mM.

3. The method for preparing a ratiometric biosensor for detecting marine biological toxins according to claim 1, characterized in that, 4-NTP was reduced to 4-aminobenzenethiol in the presence of sodium borohydride as a hydrogen source under the catalysis of gold nanoparticles. During the reduction process, the intensity of the SERS spectrum of 4-NTP at 1336 cm –1 was significantly reduced, while the intensity at 1077 cm –1 was essentially unchanged.

4. The method of claim 1, wherein the ratio biosensor for detecting marine toxin is prepared by the steps of: The solid phase carrier of the SERS enhanced nanosubstrate includes but is not limited to silicon wafer, glass, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyethylene (PE) or transparent tape.

5. The method for preparing a ratiometric biosensor for detecting marine biotoxins according to claim 4, characterized in that, The step of depositing gold nanoparticles on the surface of the solid phase carrier includes: immersing the solid phase carrier in the piranha solution at 40°C for 2 minutes to obtain a hydroxyl surface, then rinsing with distilled water and blowing dry with nitrogen; then, immersing the solid phase carrier in a 10% volume concentration of 3-aminopropyl triethoxysilane ethanol solution and performing surface amination at 70°C; then, after washing with water, immersing the aminated solid phase carrier in a gold nanoparticle solution and stirring overnight at room temperature to obtain a SERS enhanced nanosubstrate, drying with nitrogen, then sealing and storing at 4°C.

6. A ratiometric biosensor for marine toxin detection, characterized in that, The ratio biosensor for detecting marine toxins is prepared by the method of claim 1.

7. Use of a ratiometric biosensor for marine biotoxin detection, characterized in that, The ratio biosensor for detecting marine toxins is used to detect marine toxins, and the detection steps are as follows: S1, pretreating the test substance to obtain a test toxin solution; S2, detecting the test toxin solution by using the ratio biosensor; first, mixing the biomagnetic element FA with the test toxin solution, mixing the supernatant with the catalytic reaction solution to occur catalytic reaction, and dropping the catalytic product on the SERS enhanced nanosubstrate and performing SERS analysis; S3, comparing the SERS analysis result with the standard curve to obtain the concentration of the test bacteria solution.

8. Use of a ratiometric biosensor for detection of marine toxins according to claim 7, characterized in that, Method for establishing marine toxin concentration standard curve: Prepare marine toxin standard solutions with different concentrations; Use the ratio biosensor to detect marine toxin standard solutions with different concentrations to establish the standard curve of marine toxin concentration and color, Raman signal characteristic value. Method for establishing marine toxin concentration standard curve: Prepare marine toxin standard solutions with different concentrations; Use the ratio biosensor to detect marine toxin standard solutions with different concentrations to establish the standard curve of marine toxin concentration and color, Raman signal characteristic value.

9. Use of a ratiometric biosensor for detection of marine toxins according to claim 8, characterized in that, The S2 checking step comprises: adding the to-be-tested toxin solution into 1 mg / mL FA, reacting for 30 min at 37°C; separating the FA using an external magnet, and mixing the obtained supernatant with a catalytic reaction solution; after incubation at room temperature for 5 min, 3 mM hydrochloric acid is immediately added to terminate the catalytic reaction; finally, 5 μL of the catalytic product is dropped on a SERS-enhanced nano substrate and subjected to SERS analysis; the amount ratio of the to-be-tested toxin solution, the FA and the hydrochloric acid is 50 μL: 50 μL: 10 μL.

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