Construction method and application of SERS (Surface Enhanced Raman Scattering) biosensor for detecting vomitoxin

Abstract

The invention provides a construction method and application of an SERS (Surface Enhanced Raman Scattering) biosensor for detecting vomitoxin. The SERS biosensor is used for rapidly detecting the vomitoxin (DON) in real time. Ag is sputtered on the surface of the filter paper through a magnetron sputtering method, so that the surface of the filter paper has a Raman signal enhancement effect. An aptamer (Apt) of vomitoxin is selected as a recognition sensing element to specifically capture DON, Apt is connected to filter paper through an Ag-S bond, Au-4-ATP-cDNA is further modified and connected to the filter paper through complementation of Apt and cDNA, and 4-ATP serves as a signal molecule to provide a Raman signal. When the DON exists, the Apt is combined with the DON and is separated from the cDNA, the Raman signal of the 4-ATP is weakened after the Au-4-ATP-cDNA falls off, and the DON is quantitatively analyzed by detecting the Raman signal change of the 4-ATP. The sensor has high sensitivity, specificity and stability, provides a new idea for on-site rapid detection and real-time monitoring, and has application value and application prospect.

Description

A method for constructing and applying a SERS biosensor for detecting vomitoxin. Technical Field

[0001] This invention relates to the detection of vomitoxin, specifically to a method for constructing and applying a SERS biosensor for detecting vomitoxin. Background Technology

[0002] Vomitoxin (deoxynivalenol, DON) is a common fungal toxin that contaminates grains and foods, primarily affecting cereal crops such as wheat and corn. Residues of DON have also been found in grain products such as bread and biscuits. Animals that consume feed contaminated with DON experience suppressed immunity and become susceptible to disease. Ingestion of food contaminated with DON in humans may cause symptoms of poisoning such as vomiting, diarrhea, and dizziness, and in severe cases, can damage the body's hematopoietic function. DON can also damage the immune system, leading to decreased immunity. Due to the potential health hazards of DON to humans and animals, DON testing is a crucial step in ensuring the safety of grains and foodstuffs.

[0003] Currently, various methods are used for the detection of DON, such as high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC), and electrochemical methods. These traditional detection methods are commonly used for DON detection, but they have many problems. HPLC involves high equipment costs, complex operation, long analysis time, and high consumable costs. ELISA has poor specificity, is difficult to repeat, is cumbersome and time-consuming, and is prone to human error. TLC has cumbersome operation steps, is easily affected by impurities, has limited separation efficiency and capacity, and poor reproducibility. Electrochemical methods have poor stability, the electrode surface is easily contaminated leading to performance degradation, and background interference is significant, affecting the detection sensitivity and signal-to-noise ratio. Therefore, developing a rapid, portable, and highly sensitive method for detecting vomitoxin is of great significance. Summary of the Invention

[0004] This invention addresses the shortcomings of existing methods by designing a SERS technology based on aptamers as recognition elements and flexible filter paper as a substrate, utilizing a handheld Raman spectrometer for the detection of DON. A layer of Ag NPs is uniformly sputtered onto the filter paper surface to obtain a flexible filter paper Raman substrate. Apt (Ag-dependent nucleotide polymorphism) is then incubated on the filter paper substrate, and the Apt is complementary to the cDNA on Au-4-ATP-cDNA. The specific binding of Apt to DON enables quantitative detection of DON. The substrate preparation requires no complex synthetic operations, and the detection method is rapid using a portable Raman spectrometer. Leveraging the advantages of paper-based SERS sensing, a practical technology for the rapid on-site detection of low concentrations of vomitoxin is developed.

[0005] To accomplish the above tasks, the technical solution adopted by this invention is as follows: A method for constructing a SERS biosensor for detecting vomitoxin includes the following steps: Step 1, firstly, filter paper is vapor-deposited to make it hydrophobic, and then an Ag layer is deposited on the surface of the filter paper by magnetron sputtering to obtain a SERS substrate; Step 2, Au NPs are prepared by the trisodium citrate reduction method. 63 μL of 200 mM HAuCl4 is added to a flask, followed by 50 mL of water. After heating and stirring until boiling, 0.5 mL of 3.75% (w / v) trisodium citrate is rapidly added to the solution. Heating and stirring are continued for 30 min, and the solution is cooled to room temperature to obtain a wine-red Au NPs solution, which is stored at 4°C. In a refrigerator at ℃; Step 3, use tris(2-carbonylethyl)phosphohydrochloride (TCEP) to reduce the disulfide bonds (–S–S–) of Apt and cDNA, breaking them into free thiol groups (–SH); Step 4, drop the Apt solution prepared in step 3 onto the Ag layer filter paper substrate from step 1, and incubate the filter paper overnight in a 37℃ constant temperature incubator to obtain filter paper assembled with Apt; Step 5, place the Au prepared in step 2... NPs were incubated with 4-aminothiophenol (4-ATP) to obtain Au-4-ATP, which was then incubated with the cDNA treated in step 3 to obtain Au-4-ATP-cDNA. In step 6, the Au-4-ATP-cDNA solution from step 5 was added to the filter paper containing Apt in step 4 to allow the Apt to hybridize with the cDNA, further assembling the Au-4-ATP-cDNA onto the filter paper. In step 7, a solution containing DON was added to the filter paper substrate from step 6. Since the binding force between Apt and DON is stronger than that between Apt and cDNA, the Au-4-ATP-cDNA originally on the filter paper fell off, thus weakening the 4-ATP signal. In step 8, the changes in the Raman signal of 4-ATP were detected by a handheld Raman spectrometer to achieve quantitative analysis of different concentrations of DON.

[0006] As an improvement, the vapor deposition reagent in step 1 is trichloro(1H,1H,2H,2H-tetrafluoron-octyl)silane (FOS), the vapor deposition conditions are 70 °C, heating for 30 min, and the magnetron sputtering parameters are 60 mA, 15 min.

[0007] As an improvement, the Apt sequence in step 3 is 5'-MB-GCATCACTACAGTCATTACGCATCGTAGGGGGGATCGTTAAGGAAGTGCCCGGAGGCGGTATCGTGTGAAGTGCT-(CH2)6-(SH-SH)-3, with methylene blue (MB) labeled at its 5' end and a hexyl linker (C6) and dithiol (SH-SH) modification at its 3' end. The cDNA sequence is 5'-GGCACTTCCTTAACGATCCC-(CH2)6-(SH-SH)-3', with a hexyl linker (C6) and dithiol (SH-SH) modification at its 3' end. Apt is the aptamer DNA strand of DON, and cDNA is the complementary strand of Apt. The concentration ratio of DNA to TCEP is 1:50.

[0008] As an improvement, the concentration of Apt added in step 4 is 1 μM and the volume is 5 μL, and the filter paper after incubation is washed three times with buffer solution.

[0009] As an improvement, in step 5, the concentration of 4-ATP was 800 nM, the concentration of cDNA was 10 μM, and the Au-4-ATP-cDNA after the reaction was washed three times by centrifugation with distilled water.

[0010] As an improvement, in step 6, the hybridization reaction conditions of Apt and cDNA are 37 °C for 4 h, and the filter paper after the reaction is washed three times with buffer solution.

[0011] As an improvement, in step 7, the DON reaction on filter paper is carried out at 37 °C for 2 h, and the Au-4-ATP-cDNA that has fallen off is washed away with a buffer solution.

[0012] As an improvement, the test height in step 8 is 8 mm, the Raman laser power is 100%, and the integration time is 10 s.

[0013] This invention provides a flexible filter paper SERS substrate with hydrophobic properties, exhibiting excellent Raman enhancement. Based on aptamer recognition, a rapid and sensitive sensing method for DON detection is established by fixing aptamers onto the filter paper substrate and utilizing the aptamer's specific recognition of DON. The filter paper Raman substrate combines the flexibility of filter paper with the enhancement effect of metal nanoparticles, offering advantages such as low cost, simple preparation, and good portability, making it suitable for rapid on-site detection and analysis. The aptamers can bind to the target with high affinity and high specificity, with a short production cycle and low cost. As recognition elements, the aptamers are used to construct highly sensitive and selective biosensors. Through the binding of the aptamer to the target molecule DON and Raman spectroscopy signal amplification technology, rapid and highly sensitive detection of vomitoxin can be achieved. The specific advantages are as follows: 1. The hydrophobic treatment of filter paper can assist SERS detection. During the deposition of Ag, AgNPs on the hydrophobic filter paper will show an aggregation and growth trend, thus forming uniformly distributed Ag NPs. The gaps between Ag NPs provide a large number of "hot spots", which significantly enhances the Raman signal. By controlling the hydrophobic treatment time and the Ag sputtering time, the arrangement of Ag NPs can be controlled, and the filter paper substrate with the best Raman performance can be prepared to achieve highly sensitive detection of the target.

[0014] 2. The aptamer Apt has a specific recognition of DON, exhibits good anti-interference properties, and has a strong binding force with DON. The structure after the two bind together is stable, resulting in high accuracy of the detection results.

[0015] 3. Handheld Raman spectrometers offer fast detection speeds, detecting Raman signals within seconds, reducing time costs. Their small size and light weight make them suitable for on-site testing and mobile use. Therefore, they are ideal for on-site testing, enabling rapid acquisition of results. Attached Figure Description

[0016] Figure 1 is a schematic diagram of the DON detection based on a flexible SERS substrate according to the present invention.

[0017] Figure 2 is a diagram showing the feasibility verification of the sensor of the present invention.

[0018] Figure 3 shows the detection results of the sensor of the present invention for different concentrations of DON. Figure A shows the Raman spectra of DON at different concentrations, and Figure B shows the linear correlation curves between different concentrations of DON and the Raman signal.

[0019] Figure 4 shows the specificity of the sensor of the present invention in detecting DON. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the invention. Others skilled in the art can implement the invention in various forms and should not be limited to the embodiments described herein. Embodiment 1

[0021] Fabrication of a flexible filter paper substrate and construction of an Apt-based sensor: The filter paper surface was modified using trichloro(1H,1H,2H,2H-tetrafluorooctyl)silane (FOS), followed by vapor deposition (VPD) treatment for 30 min. An Ag layer was then deposited on the filter paper using magnetron sputtering for 15 min. A flexible SERS substrate with a hydrophobic surface and uniformly deposited Ag layer was successfully prepared. The disulfide bonds of Apt were then activated using TCEP, assembling Apt onto the filter paper surface via Ag-S bonds.

[0022] Au NPs were prepared using the trisodium citrate reduction method. 63 μL of 200 mM HAuCl4 was added to a flask, followed by 50 mL of water. The mixture was heated and stirred until boiling. Then, 0.5 mL of 3.75% (w / v) trisodium citrate was rapidly added, and the mixture was heated and stirred for another 30 min to obtain a wine-red Au NPs solution. Next, 4-ATP was added to the Au NPs solution to a final concentration of 800 nM, and the reaction was carried out at 37 °C for 1 h to obtain Au-4-ATP. The disulfide bonds of cDNA were then reduced using TCEP. The activated cDNA was then added to the Au-4-ATP solution and incubated overnight at 37 °C. Finally, the mixture was centrifuged at 10,000 rpm for 15 min to obtain Au-4-ATP-cDNA.

[0023] Au-4-ATP-cDNA was dropped onto filter paper coated with Apt. The Apt and cDNA reacted at 37 °C for 4 h, successfully assembling Au-4-ATP-cDNA onto the filter paper. The preparation process is shown in Figure 1. Example 2

[0024] The analysis of vomitoxin using a flexible SERS substrate sensor involved adding a solution containing DON to a filter paper substrate of Ag / Apt / Au-4-ATP-cDNA. Because the binding affinity of Apt to DON is stronger than that of Apt to cDNA, the Au-4-ATP-cDNA originally bound to the filter paper surface detached, thus weakening the 4-ATP signal (see Figure 2). For the detection of DON at concentrations ranging from 0-400 ng / mL, the 4-ATP signal was selected at a concentration of 1075 cm⁻¹. -1The Raman signal intensity at the location is fitted with a linear curve to the concentration of DON, showing a good linear relationship, as shown in Figures A and B of Figure 3.

[0025] Other toxins were detected, including ochratoxin A (OTA), T-2 toxin (T-2), zearalenone (ZEN), aflatoxin B1 (AFB1), and fumonisin B1 (FB1), totaling five toxins. Compared with the detection results of DON, the signals of other toxins were significantly higher, almost identical to the blank signal, while only DON showed the lowest signal. This indicates that the sensor has excellent specificity, as shown in Figure 4.

[0026] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a SERS biosensor for detecting vomitoxin, characterized in that, The process includes the following steps: Step 1: First, the filter paper is vapor-deposited to make it hydrophobic, and then an Ag layer is deposited on the filter paper surface by magnetron sputtering to obtain a SERS substrate; Step 2: Au NPs are prepared using the trisodium citrate reduction method. 63 μL of 200 mM HAuCl4 is added to a flask, followed by 50 mL of water. After heating and stirring to boiling, 0.5 mL of 3.75% (w / v) trisodium citrate is quickly added to the solution. Heating and stirring continues for 30 min, and the solution is cooled to room temperature to obtain a wine-red Au NPs solution, which is then stored at 4 °C; Step 3: The disulfide bonds (–S–S–) of Apt and cDNA are reduced using tris(2-carbonylethyl)phosphohydrochloride (TCEP) to break them into free thiol groups (–SH); Step 4: The Apt solution prepared in Step 3 is dropped onto the Ag layer filter paper substrate from Step 1, and the filter paper is placed at 37 °C. Step 5: Incubate the Au NPs prepared in Step 2 with 4-aminobenzylthiophenol (4-ATP) to obtain Au-4-ATP, and then incubate with the cDNA treated in Step 3 to obtain the complex Au-4-ATP-cDNA; Step 6: Add the Au-4-ATP-cDNA solution from Step 5 to the filter paper with Apt attached in Step 4 to allow Apt and cDNA to hybridize and further assemble the Au-4-ATP-cDNA onto the filter paper; Step 7: Add a solution containing DON to the filter paper substrate from Step 6. Since the binding force between Apt and DON is stronger than that between Apt and cDNA, the Au-4-ATP-cDNA originally bound to the filter paper falls off, thus weakening the 4-ATP signal; Step 8: Quantitative analysis of different concentrations of DON is achieved by detecting the Raman signal changes of 4-ATP using a handheld Raman spectrometer.

2. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, The vapor deposition reagent in step 1 is trichloro(1H,1H,2H,2H-tridecylfluoron-octyl)silane (FOS), and the vapor deposition conditions are 70 °C, heated for 30 min, and the magnetron sputtering parameters are 60 mA, 15 min.

3. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, In step 3, the Apt sequence is 5'-MB-GCATCACTACAGTCATTACGCATCGTAGGGGGGATCGTTAAGGAAGTGCCCGGAGGCGGTATCGTGTGAAGTGCT-(CH2)6-(SH-SH)-3', and the cDNA sequence is 5'-GGCACTTCCTTAACGATCCC-(CH2)6-(SH-SH)-3'. Apt is the aptamer DNA strand of DON, and cDNA is the complementary strand of Apt. The concentration ratio of DNA to TCEP is 1:

50.

4. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, In step 4, the concentration of Apt added was 1 μM and the volume was 5 μL. The filter paper after incubation was washed three times with buffer solution.

5. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, In step 5, the concentration of 4-ATP was 800 nM and the concentration of cDNA was 10 μM. The Au-4-ATP-cDNA after the reaction was washed three times by centrifugation with distilled water.

6. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, In step 6, the hybridization reaction conditions for Apt and cDNA are 37 °C for 4 h, and the filter paper after the reaction is washed three times with buffer solution.

7. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, In step 7, DON was reacted on filter paper at 37 °C for 2 h, and the Au-4-ATP-cDNA that had fallen off was washed away with a buffer solution.

8. The method for constructing a SERS biosensor for detecting vomitoxin according to claim 1, characterized in that, In step 8, the test height is 8 mm, the Raman laser power is 100%, and the integration time is 10 s.

9. The application of the biosensor obtained by the construction method according to any one of claims 1-7 in the detection of vomitoxin, characterized in that, Ag was sputtered onto the surface of filter paper using magnetron sputtering to enhance the Raman signal. An aptamer for vomitoxin (Apt) was selected as the recognition sensor element to specifically capture doxycycline (DON). Apt was attached to the filter paper via an Ag-S bond. Au-4-ATP-cDNA was further modified and attached to the filter paper through complementarity between Apt and cDNA. 4-ATP provided the Raman signal as a signaling molecule. In the presence of DON, Apt bound to DON and detached from the cDNA. The detachment of Au-4-ATP-cDNA led to a weakening of the 4-ATP Raman signal. The changes in the 4-ATP Raman signal were used to quantitatively analyze DON.

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