Electrochemical aptamer sensor for rapid detection of saxitoxin and its application

Abstract

The present application relates to a quick detection technique of saxitoxin, and particularly relates to a quick detection electrochemical aptamer sensor of saxitoxin and application thereof. The quick detection electrochemical aptamer sensor of saxitoxin is prepared by the following method: preparing AgNPs@Apt probe; etching AgNPs@Apt probe by potassium ferricyanide: taking 1mM potassium ferricyanide solution and AgNPs@Apt probe to react at 23-28 DEG C for 8-15 min; centrifuging and collecting supernatant after reaction; incubating ferric chloride and supernatant: taking 1mM ferric chloride solution and supernatant to react at 23-28 DEG C for 8-15 min, and obtaining the quick detection electrochemical aptamer sensor of saxitoxin. The advantage lies in that the silver nanoparticle reaction system etched by potassium ferricyanide is used as the source of electrochemical signal, the background interference is reduced, the sensitivity of saxitoxin detection is improved, and the minimum detection limit is 1nM.

Description

Technical Field

[0001] This invention relates to the field of rapid detection technology for salicornin, and more particularly to an electrochemical aptamer sensor for rapid detection of salicornin and its application. Background Technology

[0002] Saxitoxin (STX) is a metabolite produced by marine flagellates and freshwater filamentous cyanobacteria. It is one of the most potent marine toxins and is widely distributed. Due to its high hydrophilicity and thermal stability, STX can be stably present in shellfish and fish, thus entering the food chain. STX harms human health by inhibiting sodium channels in muscle and nerve cells. When people consume food containing STX exceeding safe levels, it primarily damages the nervous system, leading to limb paralysis, headache, fever, respiratory failure, etc. Even a dose of 0.3 mg can cause poisoning and death in adults. Heating and conventional cooking do not destroy STX, and there is no effective antidote. Therefore, it is necessary to strictly control the STX content in seafood to protect human health.

[0003] In recent years, the main methods for detecting STX have included the Mouse Bioassay (MBA), High Performance Liquid Chromatography (HPLC), High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS), and Enzyme-Linked Immunosorbent Assay (ELISA). The AOAC has standardized MBA and recognized it globally as a method for quantitative analysis of STX. However, MBA also has limitations, such as a lack of specificity and sensitivity, inability to identify toxin profiles, and frequent false positive results. Furthermore, due to ethical concerns regarding animal testing, MBA has been phased out. HPLC and HPLC-MS methods offer good sensitivity, specificity, and low detection limits, but require complex sample preparation, specialized operation, and are time-consuming. This makes them difficult to use for rapid on-site detection. ELISA methods offer high sensitivity, cost-effectiveness, speed, and ease of operation. However, ELISA methods have significant limitations; antibody preparation is cumbersome and time-consuming. Therefore, rapid, stable, and sensitive detection of STX is becoming increasingly important in food safety regulation. In recent years, electrochemical biosensors have been extensively studied for STX analysis and detection due to their high sensitivity, fast response speed, and low cost. Compared to electrochemical sensors that rely on a single electrochemical signal change, electrochemical sensors with dual electrochemical signals can effectively reduce interference from electrochemical background signals and have broad application prospects.

[0004] The proportional intensity of dual electrochemical signals is independent of sensor and reagent concentrations. When one substance is the analyte and the other is only used as a reference, proportional processing of the generated dual signals can effectively avoid intrinsic background interference. Proportional electrochemical sensors have received widespread attention in recent years. Currently, there are many analytical methods for proportional dual-signal electrochemical biosensors, but they mainly generate dual electrochemical signals through materials modified on the electrode surface. However, electrode surface modification materials are prone to detachment, leading to unstable electrochemical signals. In most reported studies, the dual electrochemical signals in proportional electrochemical sensors originate from electrode materials prepared using complex processes. However, how to fix two materials with electrochemical signals onto the electrode remains a challenging issue. For example, fixing materials sequentially or simultaneously on a narrow electrode surface is difficult to operate. The content of the two materials on the electrode is difficult to adjust, requiring a time-consuming process and careful operation. To overcome the limitations of previous proportional electrochemical sensors, developing a simple proportional electrochemical sensor remains both challenging and important. Summary of the Invention

[0005] To address the problems of unstable modification of electrochemical electrode materials, leading to poor stability and low sensitivity, this invention provides an electrochemical aptamer sensor for rapid detection of safflower toxin and its application.

[0006] The primary objective of this invention is to provide an electrochemical aptamer sensor for rapid detection of safflower toxin, which is prepared by the following method:

[0007] S1. Prepare AgNPs@Apt probes;

[0008] S2. Etching AgNPs@Apt probe with potassium ferricyanide: Take 1mM potassium ferricyanide solution and react with AgNPs@Apt probe at 23-28℃ for 8-15 min; after the reaction, centrifuge at 10000-15000 rpm for 1-3 min and collect the supernatant.

[0009] S3. Incubation with ferric chloride and supernatant: Take 1 mM ferric chloride solution and react with the supernatant collected in step S2 at 23-28℃ for 8-15 min to obtain the electrochemical aptamer sensor for rapid detection of safflower toxin.

[0010] Preferably, the preparation method of the AgNPs@Apt probe includes the following steps:

[0011] S101. Silver nanoparticle solution was prepared by reducing silver nitrate with hydroxylamine hydrochloride.

[0012] S102. Take the silver nanoparticle solution into a centrifuge tube, centrifuge at 8000-12000 rpm for 8-12 min, remove the supernatant, and resuspend in water to the original volume;

[0013] S103. Add the aptamer solution of 1 μM scimitar toxin to 200 μL of silver nanoparticle solution and incubate at 23-28℃ in the dark for 80-100 min.

[0014] S104. After incubation, centrifuge at 10000-15000 rpm for 1-3 min, remove the supernatant, and resuspend in pure water to the original volume to obtain the AgNPs@Apt probe.

[0015] Preferred aptamer nucleotide sequences for salicornin are as follows: 5'-NH2-C6-TTT TTT TGG GGA GTAGGG ACA GGA GGT GG-3';

[0016] The method for preparing the aptamer solution of salicornin includes: taking 2 OD salicornin aptamer, centrifuging at 4000 rpm for 30-60 s, and adding 6.9 mL of 10 mM PBS buffer solution to prepare a 1 μM salicornin aptamer solution.

[0017] Preferably, the volume ratio of ferric chloride solution to supernatant in step S3 is 1:1.

[0018] Preferably, the reaction temperature for steps S2 and S3 is 26°C and the reaction time is 10 min.

[0019] The second objective of this invention is to provide a rapid detection method for safflower toxins, which uses an STX rapid detection electrochemical aptamer sensor and includes the following steps:

[0020] S4. Plot the standard curve for salicornin toxin;

[0021] S5. Take the supernatant of the pretreated sample and add it to 200 μL of Ag NPs@Apt. Incubate for 10–30 min, then add 200 μL of 1 mM potassium ferricyanide and react for 10–15 min. After centrifugation, take 200 μL of the supernatant, then add 200 μL of 1 mM ferric chloride solution, followed by 1 mL of 10 mM PBS (pH = 7.4) solution to obtain the electrolyte.

[0022] S6. Perform electrodeposition pretreatment on the glassy carbon electrode;

[0023] S7. Measure the DPV signal of the electrolyte, analyze the measured DPV data, and determine the concentration of salicornin in the sample by referring to the standard curve of salicornin.

[0024] Preferably, the parameter settings for electrodeposition in step S6 include: initial voltage of -0.3V, running time of 300s, and settling time of 2s; the parameter settings for measuring the DPV signal of the electrolyte in step S7 include: initial voltage of 0V, termination voltage of 0.3V, and settling time of 2s.

[0025] Preferably, the method for preparing the sample extract in step S5 includes: taking the shellfish meat to be tested, adding 0.1M hydrochloric acid solution, boiling and stirring for 4-6 minutes, centrifuging at 5000-8000 rpm for 8-12 minutes at 0-4℃, collecting the supernatant, adjusting the pH to below 4.0 with hydrochloric acid solution; adding 8-10 times the volume of PBS buffer solution, mixing well, and obtaining the sample extract.

[0026] Preferably, the PBS buffer solution has a pH of 7.4.

[0027] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0028] Using the potassium ferricyanide etching silver nanoparticles (AgNPs) reaction system as the electrochemical signal source reduces background interference and improves the sensitivity of STX detection, with a detection limit (LOD) of 1 nM for STX. The proportional electrochemical method of this invention is more suitable as a method for detecting STX. Attached Figure Description

[0029] Figure 1 The following are the characterization results of AgNPs@Apt provided according to embodiments of the present invention: (A) HRTEM image of AgNPs; (B) HRTEM image of AgNPs@Apt; (C) Particle size of AgNPs and AgNPs@Apt; (D) Scanning TEM elemental map of P element in AgNPs@Apt; (E) Scanning TEM elemental map of Ag element in AgNPs@Apt; (F) Zeta potential of AgNPs, Apt and AgNPs@Apt.

[0030] Figure 2 This is a schematic diagram illustrating the principle of the rapid detection method for clam toxins provided in an embodiment of the present invention.

[0031] Figure 3 The results of DPV peak current of (A) STX mixed solution and (B) different C provided by the embodiments of the present invention are as follows: STX Value I AgCl and I Fe The current diagram.

[0032] Figure 4 Different C provided according to embodiments of the present invention STX Value I AgCl and I FeThe linear fit plot.

[0033] Figure 5 This is a standard curve of STX concentration at a DPV peak current of 0.13V to 0.19V provided according to an embodiment of the present invention. Detailed Implementation

[0034] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0036] Example 1

[0037] Preparation of AgNPs@Apt probes; specifically including the following sub-steps:

[0038] S101. Silver nanoparticle (AgNPs) solution was prepared by reducing silver nitrate with hydroxylamine hydrochloride.

[0039] S102. Take 1 mL of silver nanoparticle (AgNPs) solution into a centrifuge tube, centrifuge at 10000 rpm / min for 10 min, remove the supernatant, and resuspend in water to the original volume (1 mL).

[0040] S103. Add the aptamer (Apt) solution of 1 μM scimitar toxin (STX) to 200 μL AgNPs solution and incubate at 26 °C in the dark for 90 min.

[0041] The nucleotide sequence of the aptamer is as follows: 5'-NH2-C6-TTT TTT TGG GGA GTA GGG ACA GGA GGTGG-3';

[0042] The method for preparing the aptamer solution of salicornin includes: taking one centrifuge tube containing 2D salicornin aptamer, centrifuging at 4000 rpm for 30-60 seconds, and then adding 6.9 mL of 10 mM PBS buffer solution (pH=7.4) to prepare a 1 μM salicornin aptamer solution.

[0043] S104. After incubation, centrifuge at 12000 rpm for 2 min, remove the supernatant, and resuspend in pure water to the original volume to obtain the AgNPs@Apt probe.

[0044] The characterization and measurement results of the AgNPs@Apt probe are as follows: Figure 1 .like Figure 1 A and Figure 1 As shown in Figure B, the morphology of relatively monodisperse AgNPs changed after modification with aptamers, with a thin film covering the surface. Elemental analysis of AgNPs@Apt showed that... Figure 1 D and Figure 1 The AgNPs in E contain abundant P and Ag elements on their surface, with the P element likely originating from the aptamer. Next, the particle size and zeta potential of the Ag NPs, aptamers, and Ag NPs@Apt were measured, as shown below. Figure 1 As shown in C and 1F, the results indicate that the aptamer can adsorb onto the AgNPs surface, with the zeta potential varying from -18.4 eV for AgNPs to -13.7 eV for AgNPs@Apt. The particle size of AgNPs@Apt is slightly larger than that of AgNPs. This suggests that aptamer attachment may be one of the reasons for the increased volume. The above characterization demonstrates that the aptamer successfully attaches to AgNPs.

[0045] Example 2

[0046] The preparation method of an electrochemical aptamer sensor for rapid detection of safflower toxins includes the following steps:

[0047] S1. Prepare AgNPs@Apt probes according to the method in Example 1;

[0048] S2. Etching AgNPs@Apt probe with potassium ferricyanide: Take 200 μL of 1 mM potassium ferricyanide solution and react with 200 μL of AgNPs@Apt probe at 26℃ for 10 min; after the reaction, centrifuge at 12000 rpm for 2 min and collect the supernatant;

[0049] S3. Incubation with ferric chloride and supernatant: Take 200 μL of 1 mM ferric chloride solution and 200 μL of the supernatant collected in step S2, and react at 26 °C for 10 min to obtain an electrochemical aptamer sensor for rapid detection of safflower toxin.

[0050] Example 3

[0051] The rapid detection method for salicornin uses the electrochemical aptamer sensor for rapid detection of salicornin prepared in Example 2, and specifically includes the following steps:

[0052] S4. Plot the standard curve for salicornin toxins, specifically including:

[0053] S401. Mix the prepared AgNPs@Apt probe with STX standard solution and incubate at 26℃ for 30 min; then add 200 μL of 1 mM potassium ferricyanide solution and react at 26℃ for 10 min. After centrifugation, take 200 μL of the supernatant and add 200 μL of 1 mM ferric chloride solution and react at 26℃ for 10 min.

[0054] S402. Then add 1 mL of 10 mM PBS buffer solution (pH = 7.4) to prepare the electrolyte;

[0055] S403. Perform electrodeposition pretreatment on the glassy carbon electrode. The main parameters are: initial voltage -0.3V, running time 300s, and settling time 2s.

[0056] The S404 electrochemical differential pulse voltammetry (DPV) was used to determine the DPV signal. The main parameters were: initial voltage of 0V, termination voltage of 0.3V, and settling time of 2s. The measured DPV data were analyzed, and the standard curve of STX was plotted.

[0057] S5. Take 10 μL of the pretreated sample extract, add 200 μL of the electrochemical aptamer sensor for rapid detection of safflower toxin, incubate at 26 °C for 10 min, and then add 1 mL of 10 mM PBS buffer solution (pH = 7.4) to prepare the electrolyte.

[0058] Preparation method of sample extract: Take 10g of sample (shellfish meat), add 70mL of 0.1M hydrochloric acid solution, boil and stir for 5min, centrifuge at 6000rpm for 10min at 4℃, collect the supernatant, and adjust the pH to below 4.0 with hydrochloric acid solution; take 100μL of extract, add 900μL of PBS buffer solution (pH=7.4), mix well to obtain sample extract.

[0059] S6. Perform electrodeposition pretreatment on the glassy carbon electrode. The main parameters are: initial voltage -0.3V, running time 300s, and settling time 2s.

[0060] S7. Measure the DPV signal of the electrochemical electrolyte. The main parameters are: initial voltage 0V, termination voltage 0.3V, and settling time 2s. Analyze the measured DPV data and determine the concentration of STX in the sample by referring to the standard curve of STX.

[0061] Principle: When STX is absent, K3Fe(CN)6 solution can etch AgNPs@Apt, causing some AgNPs to dissociate into Ag. + And it reduces K3Fe(CN)6 to K4Fe(CN)6. After the addition of FeCl3, Fe...3+ and Fe(CN)6 4- Combined to form Fe[Fe(CN)6] - Cl - With Ag + AgCl was generated, and a low-ratio electrochemical signal was detected by differential pulse voltammetry (DPV). However, upon the addition of STX, the aptamers on the surface of AgNPs detached due to STX binding, exposing the surface sites of AgNPs. The etching reaction of AgNPs@Apt by K3Fe(CN)6 was accelerated, exhibiting a high-ratio electrochemical signal as observed by DPV. Therefore, with increasing STX concentration, the signal intensity of the electrochemical signal ratio continuously increases. A schematic diagram of the principle is shown below. Figure 2 As shown.

[0062] Example 4

[0063] The standard curve of the concentration of the salicornin standard solution and the peak current of DPV includes:

[0064] 1. Prepare STX mixed solutions of different concentrations (0.04, 0.06, 0.08, 0.10, 0.15, 0.20 μM) and determine the different C values. STX Value I AgCl and I Fe The peak current of DPV, the results are as follows Figure 3 As shown. From Figure 3 As can be seen from A, with the increase of STX concentration, at 0.13V ( Figure 2 A in a) and 0.19V ( Figure 3 At point b) in A, a peak current is observed. At both peak points, the peak current of AgCl increases significantly, while the peak current of Fe decreases slightly. Figure 3 As can be seen from B, there is a good linear relationship in the range of 0.04 to 0.15 μM.

[0065] 2. Perform linear regression analysis on the peak currents of 0.13V and 0.19V respectively. The fitted curves of the linear regression analysis are shown below. Figure 4 As shown, by comparing the I of different concentrations of STX AgCl and I Fe When analyzing the electrochemical response signals, a poor fit was found between the two (R0). 2 =0.9436, R 2 =0.9418), the fitting effect is poor and needs improvement. Figure 5 In the study, the peak current ratio showed a good linear relationship with the STX concentration in the range of 0.04 μM to 0.15 μM, and the regression equation was Y = -8.0 + 233.7X(R). 2=0.9956). The detection limit (LOD) of STX was calculated to be 1 nM (S / N = 3). The electrochemical aptamer sensor of the present invention was compared with other electrochemical biosensing strategies, and the results showed that the electrochemical aptamer sensor of the present invention has high sensitivity and good stability.

[0066] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0067] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An electrochemical aptamer sensor for rapid detection of clam toxins, characterized in that, Prepared by the following method: S1. Prepare AgNPs@Apt probes; S2. Etching AgNPs@Apt probe with potassium ferricyanide: Take 1mM potassium ferricyanide solution and react with AgNPs@Apt probe at 23-28℃ for 8-15 min; after the reaction, centrifuge at 10000-15000 rpm for 1-3 min and collect the supernatant. S3. Incubation with ferric chloride and supernatant: Take 1 mM ferric chloride solution and react with the supernatant collected in step S2 at 23-28℃ for 8-15 min to obtain an electrochemical aptamer sensor for rapid detection of safflower toxin.

2. The electrochemical aptamer sensor for rapid detection of clam toxins according to claim 1, characterized in that: The preparation method of the AgNPs@Apt probe includes the following steps: S101. Silver nanoparticle solution was prepared by reducing silver nitrate with hydroxylamine hydrochloride. S102. Take the silver nanoparticle solution into a centrifuge tube, centrifuge at 8000-12000 rpm for 8-12 min, remove the supernatant, and resuspend in water to the original volume; S103. Add the aptamer solution of 1 μM scimitar toxin to 200 μL of silver nanoparticle solution and incubate at 23-28℃ in the dark for 80-100 min. S104. After incubation, centrifuge at 10000-15000 rpm for 1-3 min, remove the supernatant, and resuspend in pure water to the original volume to obtain the AgNPs@Apt probe.

3. The electrochemical aptamer sensor for rapid detection of safflower toxin according to claim 2, characterized in that: The aptamer nucleotide sequence of the sarcotoxin is as follows: 5'-NH2-C6-TTT TTT TGG GGA GTA GGG ACA GGAGGT GG-3'; The method for preparing the aptamer solution of the sarcotoxin includes: taking 2D of sarcotoxin aptamer, centrifuging at 4000rpm for 30-60s, and adding 6.9mL of 10mM PBS buffer solution to prepare a 1μM sarcotoxin aptamer solution.

4. The electrochemical aptamer sensor for rapid detection of safflower toxin according to claim 3, characterized in that: In step S3, the volume ratio of ferric chloride solution to supernatant is 1:

1.

5. The electrochemical aptamer sensor for rapid detection of clam toxins according to claim 4, characterized in that: The reaction temperature for steps S2 and S3 is 26°C, and the reaction time is 10 min.

6. A rapid detection method for salicornin, comprising using the electrochemical aptamer sensor for rapid detection of salicornin as described in claim 1, characterized in that, Includes the following steps: S4. Plot the standard curve for salicornin toxin; S5. Take the pretreated sample extract, add 15 to 25 times the volume of the electrochemical aptamer sensor for rapid detection of shifang clams, incubate at 23 to 28°C for 8 to 15 minutes, and then add 10 mM PBS buffer solution to prepare the electrolyte. S6. Perform electrodeposition pretreatment on the glassy carbon electrode; S7. Measure the DPV signal of the electrolyte, analyze the measured DPV data, and determine the concentration of salicornin in the sample by referring to the standard curve of salicornin.

7. The rapid detection method for clam toxins according to claim 6, characterized in that: The parameter settings for electrodeposition in step S6 include: initial voltage of -0.3V, running time of 300s, and settling time of 2s; the parameter settings for measuring the DPV signal of the electrochemical electrolyte in step S7 include: initial voltage of 0V, termination voltage of 0.3V, and settling time of 2s.

8. The rapid detection method for clam toxins according to claim 7, characterized in that: The preparation method of the sample extract in step S5 includes: taking the shellfish meat to be tested, adding 0.1M hydrochloric acid solution, boiling and stirring for 4-6 minutes, centrifuging at 5000-8000 rpm for 8-12 minutes at 0-4℃, collecting the supernatant, adjusting the pH to below 4.0 with hydrochloric acid solution; adding 8-10 times the volume of PBS buffer solution, mixing well, and obtaining the sample extract.

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