New method for rapidly detecting ochratoxin A based on electrochemical deposition polydopamine coating modified gold electrode

Abstract

The invention discloses a novel method for rapidly detecting ochratoxin A based on an electrochemical deposition polydopamine coating modified gold electrode, and belongs to the technical field of analytical chemistry and biology. The method is based on a polydopamine coating, is combined with an ochratoxin A aptamer with high specificity and high affinity, introduces a DNA-modified nanogold compound as a signal carrier and a signal amplification element, and realizes high-sensitivity detection of ochratoxin A through current change generated by catalyzing H2O2 / TMB by horse radish peroxidase captured on the surface of an electrode.

Description

Technical Field

[0001] The invention belongs to the fields of analytical chemistry and biotechnology, and particularly relates to a new method for rapidly detecting ochratoxin A based on a gold electrode modified with an electrochemically deposited polydopamine coating. Background Art

[0002] Ochratoxins are toxic compounds with structures similar to isocoumarin derivatives, primarily produced by certain Penicillium and Aspergillus fungi. Among them, ochratoxin A (OTA) is the most toxic, second only to aflatoxins. OTA can cause multiple toxic effects in humans, including nephrotoxicity, carcinogenicity, and teratogenicity. OTA is widely distributed, and contamination of traditional Chinese medicines (TCMs) with OTA is a common occurrence. People may ingest OTA through consumption of these contaminated TCMs, and long-term accumulation of OTA can lead to severe pathological reactions. Therefore, the development of highly sensitive, specific, and accurate analytical methods for OTA detection is crucial for the timely detection and disposal of contaminated TCMs, ensuring their quality and safety.

[0003] Polydopamine (PDA), primarily a polymer formed by the oxidation of dopamine (DA) in the presence of an oxidizing agent, is a widely used surface coating material for various interfaces. The earliest reports of PDA as a surface coating material were inspired by mussel adhesive proteins. Dopamine aqueous solutions were dip-coated onto various surfaces. Under alkaline conditions, dopamine self-polymerized on a variety of inorganic and organic materials (including precious metals, semiconductors, and ceramics) to form surface-adherent PDA films. Subsequent studies have demonstrated that PDA can deposit and adhere to virtually any solid substrate, making it a versatile coating material. PDA coatings possess excellent physicochemical properties and biocompatibility, particularly due to the rich functional groups in its chemical structure (such as catechols, amines, and imines). These functional groups can interact with a variety of molecules through hydrogen bonding and chemical reactions (such as Michael additions and Schiff base reactions), providing favorable conditions for secondary surface modification.

[0004] Coating DA on the surface of a material to allow it to self-polymerize is a simple and traditional method for preparing PDA films. However, the coating method for preparing PDA usually takes a long time, and the thickness of the resulting PDA coating is difficult to control and uneven. DA is a redox-active molecule that can be deposited on a conductive substrate by electrochemical methods to obtain a PDA coating. Compared with the coating method, cyclic voltammetry (CV) electrochemical deposition has a higher deposition rate, and the PDA coating thickness is easy to control, resulting in a more uniform PDA coating. Summary of the Invention

[0005] The present invention addresses the shortcomings of the prior art by designing an electrochemical aptamer biosensor for highly sensitive OTA detection using a polydopamine (PDA) coating modified with a highly specific and high-affinity OTA aptamer. DNA-AuNP complexes are introduced as signal carriers and amplification elements. This method provides a novel rapid ochratoxin A detection method based on an electrochemically deposited polydopamine coating modified gold electrode. The method is simple to operate, highly sensitive, and has good selectivity.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A new method for rapid detection of ochratoxin A based on an electrochemically deposited polydopamine-coated gold electrode comprises the following steps: (1) Add 5 mL of 0.2 wt% chloroauric acid aqueous solution and 90 mL of ultrapure water into a brown conical flask, heat to boiling under rapid stirring, then quickly add 5 mL of 1 wt% trisodium citrate aqueous solution, boil and reflux under rapid stirring until the color of the mixed solution turns wine red, continue stirring and boiling for 5 min, then stop heating, cool to room temperature, filter with a 0.22 μm microporous filter membrane, collect the filtrate to obtain AuNP solution; (2) Add TCEP aqueous solution to the Linker to make the final concentrations of Linker and TCEP 100 μM and 0.01 M, respectively. After vortex mixing, activate at 25 ° C for 2 h to obtain an activated Linker solution; 3 μL of the activated Linker solution was mixed with 600 μL of AuNP solution and incubated at 25 ° C for 16 h, and then 50 μL of PB buffer containing 3 M NaCl was slowly added. After vortex mixing, it was aged at 25 ° C for 24 h, centrifuged at 13500 rpm for 30 min, and the precipitate was collected and washed three times with PBS buffer; the washed precipitate was dispersed in 200 μL of PBS buffer containing 1 vol% Tween-20 to obtain a functionalized gold nanoparticle solution; (3) The pretreated gold electrode GE was immersed in PBS buffer containing 5 mM dopamine, and electrochemical polymerization was performed in the potential range of -0.4 V to +1.5 V using the CV method at a scan rate of 20 mV / s for 10 consecutive cycles. The electrode was rinsed with Tris-HCl buffer and dried naturally to obtain a PDA / GE electrode; (4) 10 μL of 1.0 μM amino-modified cDNA aqueous solution was dropped onto the surface of the PDA / GE electrode, incubated at room temperature for 12 h, rinsed the electrode with Tris-HCl buffer and dried with nitrogen to obtain a cDNA-PDA / GE electrode; the cDNA-PDA / GE electrode was immersed in 1 mM ethanolamine aqueous solution, incubated at 25 °C for 1 h, rinsed the electrode with Tris-HCl buffer and dried with nitrogen to obtain an EA-cDNA-PDA / GE electrode; 10 μL of 1.0 μM OTA-specific aptamer solution was dropped onto the surface of the EA-cDNA-PDA / GE electrode, incubated at 25 °C for 1 h, rinsed the electrode with Tris-HCl buffer and dried with nitrogen to obtain an Aptamer-EA-cDNA-PDA / GE electrode; (5) 10 μL of the sample containing ochratoxin A was added to the surface of the Aptamer-EA-cDNA-PDA / GE electrode and incubated at 20°C for 1 h. The electrode was rinsed with Tris-HCl buffer and dried with nitrogen. The electrode was then immersed in a functionalized gold nanoparticle solution and incubated at 30°C for 1 h. The electrode was rinsed with Tris-HCl buffer and dried with nitrogen. The electrode was then immersed in a 2 μg / mL SA-HRP aqueous solution and incubated at room temperature for 30 min. The electrode was rinsed with Tris-HCl buffer and placed in a TMB probe solution containing H2O2 for electrochemical detection.

[0007] Furthermore, the nucleotide sequence of the above-mentioned Linker is: 5'-SH-C6-TTTTTTTGTCCGTGGGTGGCGTAAAGGGAGCATCGGACATA-Biotin-3'.

[0008] Furthermore, the nucleotide sequence of the amino-modified cDNA is: 5'-NH2-C6-TGTCCGATGCTCCCTTTACGCCACCCACGGACA-3'.

[0009] Furthermore, the nucleotide sequence of the above-mentioned OTA-specific aptamer is: 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3'.

[0010] Furthermore, the electrochemical detection method is a chronoamperometry method with the following conditions: initial potential 0.2 V, scan time 100 s, and sampling interval 0.1 s.

[0011] The above-mentioned new method for rapid detection of ochratoxin A based on electrochemically deposited polydopamine coating modified gold electrode is applied in the detection of ochratoxin A.

[0012] The technical principle of the present invention is: The present invention utilizes a Schiff base reaction between amino groups and a PDA membrane to immobilize amino-modified cDNA on the surface of a PDA / GE electrode, and uses ethanolamine to block the remaining active sites of the PDA membrane to prevent nonspecific adsorption, thereby forming an EA-cDNA-PDA / GE electrode. The cDNA immobilized on the modified electrode surface can hybridize with an OTA-specific aptamer through base complementary pairing to form a cDNA-Aptamer double-stranded complex. In the absence of OTA, the cDNA immobilized on the electrode surface maintains a stable DNA double-stranded structure with the aptamer. The linker-AuNP complex cannot be captured on the electrode surface due to the lack of binding sites, and can only generate a low signal current under specific potential conditions. In the presence of OTA, the aptamer on the electrode surface can specifically recognize OTA and tends to bind to OTA to form a more stable OTA-aptamer complex, causing the aptamer to be stripped from the cDNA-aptamer double-stranded complex, causing the cDNA to return to a single-stranded state. These single-stranded cDNAs can then undergo base complementary pairing and hybridization with the linker on the DNA-AuNP complex, thereby capturing a large number of linker-AuNP complexes on the electrode surface. Since the 3' end of the linker is modified with biotin, a large amount of SA-HRP can be captured through the interaction of streptavidin-biotin. Finally, the SA-HRP captured on the electrode catalyzes the oxidation of TMB by H2O2 to generate a significant signal current. Therefore, OTA can be detected by recording the signal current changes caused by the presence or absence of the target OTA. The above principle diagram is shown in Figure 1 .

[0013] In this method, aptamers are used as recognition elements, achieving highly specific OTA detection while significantly reducing detection costs. The introduction of a polydopamine functional coating enables efficient cDNA immobilization, effectively improving detection sensitivity. This is further combined with the dual signal amplification mechanism of the DNA-gold nanoparticle composite: on the one hand, the ultra-high surface area of ​​AuNPs provides a large number of loading sites for signal probes; on the other hand, AuNPs possess both excellent electrical conductivity and enzyme-like catalytic activity, synergistically catalyzing the H2O2 / TMB colorimetric reaction, thereby significantly improving detection sensitivity.

[0014] The advantages of the present invention are: 1. The present invention utilizes OTA-specific aptamers as biological recognition elements, ensuring the stability and specificity of the detection method.

[0015] 2. The present invention introduces a polydopamine functional coating that can effectively immobilize biomolecules and improve detection sensitivity and stability.

[0016] 3. In this paper, DNA-AuNP complexes are used as signal carriers and signal amplification elements to design an electrochemical aptamer biosensor for highly sensitive detection of OTA, which can effectively improve the sensitivity and analytical performance of the detection method.

[0017] 4. The present invention achieves highly specific detection of OTA, is easy to operate, has a low detection limit, and has strong anti-interference ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Schematic diagram of OTA electrochemical aptamer sensor based on polydopamine and gold nanoparticles.

[0019] Figure 2 :The electrochemical detection results of the method of the present invention for detecting different concentrations of OTA and the standard working curve established.

[0020] Figure 3 : Specificity verification of the method of the present invention for detecting OTA. DETAILED DESCRIPTION

[0021] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods. However, the following examples are merely examples of the present invention and do not represent the scope of protection of the rights defined by the present invention. The scope of protection of the rights of the present invention shall be subject to the claims.

[0022] Example 1: A new method for rapid detection of ochratoxin A (OTA) based on an electrochemically deposited polydopamine-coated gold electrode comprises the following steps: (1) Preparation of gold nanoparticles (AuNPs): 5 mL of 0.2 wt% chloroauric acid aqueous solution and 90 mL of ultrapure water were added to a 100 mL brown conical flask and heated to boiling under rapid stirring. Then, 5 mL of 1 wt% trisodium citrate aqueous solution was quickly added and heated to reflux under rapid stirring until the color of the mixed solution turned wine red. Stirring and boiling were continued for 5 min, and then heating was stopped. The mixture was cooled to room temperature and filtered with a 0.22 μm microporous filter membrane to remove large-sized AuNPs. The filtrate was collected to obtain the AuNP solution, which was stored in the dark at 4 °C.

[0023] (2) Preparation of functionalized gold nanoparticles: The linker was placed in a 1.5 mL microcentrifuge tube and freshly prepared TCEP aqueous solution was added to make the final concentration of linker 100 μM and the final concentration of TCEP 0.01 M. After vortex mixing, the solution was activated at 25°C for 2 h to reduce the disulfide bonds generated by autooxidation in the thiol-modified linker to obtain the activated linker solution. 3 μL of the activated linker solution was mixed with 600 μL of the above-prepared AuNP solution and incubated at 25°C for 16 h to allow the linker to be fixed to the AuNP surface through gold-sulfur bonds to form a linker-AuNP complex. 50 μL of PB buffer (100 mM, pH 7.40) containing 3 M NaCl was then slowly added. After vortex mixing, the solution was aged at 25°C for 24 h. The solution was then centrifuged at 13,500 rpm for 30 min. The precipitate was collected and washed three times with 1× PBS buffer (pH 7.40) to remove unbound linker. The washed precipitate was dispersed into 200 μL of 1× PBS buffer (pH 7.40) containing 1 vol% Tween-20 to obtain a functionalized gold nanoparticle solution, which was stored at 4°C for future use.

[0024] (3) Preparation of polydopamine functionalized gold electrode: A gold electrode (GE) was polished with 1 μm Al2O3 powder until the surface was smooth. The electrode was rinsed with anhydrous ethanol and deionized water, respectively, and then dried with nitrogen to obtain a pretreated GE. The pretreated GE was immersed in 10 mL of 1× PBS buffer (pH 6.50) containing 5 mM dopamine. Electrochemical polymerization was performed using the CV method in the potential range of -0.4 V to +1.5 V at a scan rate of 20 mV / s for 10 consecutive cycles. The electrode was then rinsed with Tris-HCl buffer (0.01 M, pH 7.50) and allowed to dry naturally to obtain a GE electrode with a PDA film functionalized on the surface (i.e., a PDA / GE electrode). The electrode was then covered with a dedicated electrode cap and set aside.

[0025] (4) Construction of OTA electrochemical aptamer sensor: 10 μL of 1.0 μM amino-modified cDNA aqueous solution was added to the surface of the PDA / GE electrode and incubated at room temperature for 12 h. During this period, a special electrode cap was placed to prevent the solution from evaporating. After the incubation, the residual cDNA that was not assembled on the electrode surface was removed by rinsing with Tris-HCl buffer (0.01 M, pH 7.50), and the electrode was blown dry with nitrogen to obtain a cDNA-modified PDA / GE electrode (i.e., cDNA-PDA / GE electrode). The cDNA-PDA / GE electrode was immersed in 1 mM ethanolamine aqueous solution and incubated at 25 °C for 1 h to remove nonspecific adsorption of DNA and block the remaining active sites of the PDA membrane. The electrode was then rinsed with Tris-HCl buffer (0.01 M, pH 7.50) and blown dry with nitrogen to obtain an EA-cDNA-PDA / GE electrode. The OTA-specific aptamer was prepared into a solution using 1× PBS buffer (pH 7.40). Then, 10 μL of a 1.0 μM OTA-specific aptamer solution was dropwise added to the EA-cDNA-PDA / GE surface and incubated at 25°C for 1 h. The electrode was washed with Tris-HCl buffer (0.01 M, pH 7.50) and dried with nitrogen gas to obtain the aptamer-modified working electrode (i.e., Aptamer-EA-cDNA-PDA / GE electrode).

[0026] (5) Electrochemical detection of OTA: The OTA standard solution prepared in methanol was diluted to different concentrations with 1× PBS buffer (pH 7.40), and 10 μL was added dropwise to the surface of the Aptamer-EA-cDNA-PDA / GE electrode. The solution was incubated at 20°C in the dark for 1 h. The electrode was rinsed with Tris-HCl buffer (0.01 M, pH 7.50) and dried with nitrogen. The electrode was then immersed in a functionalized gold nanoparticle solution and incubated at 30°C for 1 h. The electrode was rinsed with Tris-HCl buffer (0.01 M, pH 7.50) and dried with nitrogen. The electrode was then immersed in a 2 μg / mL streptavidin-modified horseradish peroxidase (SA-HRP) aqueous solution and incubated at room temperature for 30 min. The electrode was then rinsed with Tris-HCl buffer (0.01 M, pH 7.50) and dried with nitrogen. 7.50) After thoroughly rinsing to remove unbound SA-HRP from the electrode surface, place the electrode in a H2O2 / TMB substrate solution. Perform electrochemical detection using chronoamperometry with an initial potential of 0.2 V, a scan time of 100 s, and a sampling interval of 0.1 s. (Note: Pure nitrogen needs to be continuously injected into the substrate solution at a low rate to remove oxygen interference.) Finally, quantify the OTA concentration based on the signal current value.

[0027] The nucleotide sequence of the above-mentioned linker is: 5'-SH-C6-TTTTTTTGTCCGTGGGTGGCGTAAAGGGAGCATCGGACATA-Biotin-3'.

[0028] The nucleotide sequence of the amino-modified cDNA is: 5'-NH2-C6-TGTCCGATGCTCCCTTTACGCCACCCACGGACA-3'.

[0029] The nucleotide sequence of the OTA-specific aptamer is: 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3'.

[0030] Example 2: The OTA standard solution prepared in methanol in step (5) of Example 1 was diluted with 1×PBS buffer (pH 7.40) to 0.2 pg / mL, 2 pg / mL, 10 pg / mL, 50 pg / mL, 200 pg / mL, and 1 ng / mL, respectively. The remaining steps were the same as in Example 1, and electrochemical detection of OTA at different concentrations was performed.

[0031] Test results are shown in Figure 2 , it can be seen that with the increase of OTA concentration, the steady-state current I s The value also increases. s and OTA concentration, and a standard curve was established ( Figure 2 ), to achieve quantitative detection of OTA; △I s I is the steady-state current of the sensor before and after combining OTA s The limit of detection (LOD) for OTA was calculated using 3σ / S (where σ represents the standard deviation of the signals obtained from multiple measurements of a blank test solution (1× PBS buffer (pH 7.40)) and S is the slope of the fitted linear equation). The theoretical detection limit for OTA using the method of the present invention was 0.039 pg / mL.

[0032] Example 3: The OTA standard solution prepared in methanol in step (5) of Example 1 was diluted to 0.2 ng / mL with 1×PBS buffer (pH 7.40), and the remaining steps were the same as in Example 1 for electrochemical detection.

[0033] The methanol-prepared OTA standard solution in step (5) of Example 1 was replaced with methanol standard solutions of aflatoxin B1 (AFB1), ochratoxin B (OTB), zearalenone (ZEN), deoxynivalenol (DON) and fumonisin B1 (FB1), respectively, and prepared into 2 ng / mL single-standard working solutions using 1× PBS buffer (pH 7.40). The remaining steps were the same as in Example 1, and electrochemical detection analysis was performed.

[0034] In the mixed detection experiment, a methanol mixed standard solution containing AFB1, OTB, ZEN, DON, FB1, and OTA was used to replace the single standard solution in the original experimental system. The mixed standard solution was diluted with 1× PBS buffer (pH 7.40) to an OTA concentration of 0.2 ng / mL and a concentration of 2 ng / mL for other interfering fungal toxins. The remaining steps were the same as in Example 1 for electrochemical detection.

[0035] Test results are shown in Figure 3 , it can be seen that even if the concentration of interfering fungal toxin is 10 times that of OTA, the generated I s The I values ​​were compared with those generated by the blank test solution (1× PBS buffer (pH 7.40)). s There is no significant change compared with the values, indicating that there is no reaction between these fungal toxins and the OTA aptamer. The cDNA and aptamer on the electrode surface exist stably in a DNA double-stranded structure. The Linker-AuNP complex cannot be captured on the electrode surface due to the lack of binding sites, and can only produce a small background I s In addition, when 0.2 ng / mL of OTA coexists with 2 ng / mL of other mycotoxins, the measured I s The I values ​​were similar to those produced when OTA was present alone at 0.2 ng / mL. s The values ​​were almost the same, indicating that the coexistence of other high concentrations of mycotoxins had an impact on the I s The results showed that the method established in this application has good specificity for OTA detection.

[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A novel method for rapid detection of ochratoxin A based on an electrochemically deposited polydopamine-coated gold electrode, characterized by: The following steps are involved: (1) Add 5 mL of 0.2 wt% chloroauric acid aqueous solution and 90 mL of ultrapure water into a brown conical flask, heat to boiling under rapid stirring, then quickly add 5 mL of 1 wt% trisodium citrate aqueous solution, boil and reflux under rapid stirring until the color of the mixed solution turns wine red, continue stirring and boiling for 5 min, then stop heating, cool to room temperature, filter with a 0.22 μm microporous filter membrane, collect the filtrate to obtain AuNP solution; (2) Add TCEP aqueous solution to the Linker to make the final concentrations of Linker and TCEP 100 μM and 0.01 M, respectively. After vortex mixing, activate at 25 ° C for 2 h to obtain an activated Linker solution; 3 μL of the activated Linker solution was mixed with 600 μL of AuNP solution and incubated at 25 ° C for 16 h, and then 50 μL of PB buffer containing 3 M NaCl was slowly added. After vortex mixing, it was aged at 25 ° C for 24 h, centrifuged at 13500 rpm for 30 min, and the precipitate was collected and washed three times with PBS buffer; the washed precipitate was dispersed in 200 μL of PBS buffer containing 1 vol% Tween-20 to obtain a functionalized gold nanoparticle solution; (3) The pretreated gold electrode GE was immersed in PBS buffer containing 5 mM dopamine, and electrochemical polymerization was performed in the potential range of -0.4 V to +1.5 V using the CV method at a scan rate of 20 mV / s for 10 consecutive cycles. The electrode was rinsed with Tris-HCl buffer and dried naturally to obtain a PDA / GE electrode; (4) 10 μL of 1.0 μM amino-modified cDNA aqueous solution was dropped onto the surface of the PDA / GE electrode, incubated at room temperature for 12 h, rinsed the electrode with Tris-HCl buffer and dried with nitrogen to obtain a cDNA-PDA / GE electrode; the cDNA-PDA / GE electrode was immersed in 1 mM ethanolamine aqueous solution, incubated at 25 °C for 1 h, rinsed the electrode with Tris-HCl buffer and dried with nitrogen to obtain an EA-cDNA-PDA / GE electrode; 10 μL of 1.0 μM OTA-specific aptamer solution was dropped onto the surface of the EA-cDNA-PDA / GE electrode, incubated at 25 °C for 1 h, rinsed the electrode with Tris-HCl buffer and dried with nitrogen to obtain an Aptamer-EA-cDNA-PDA / GE electrode; (5) 10 μL of the sample containing ochratoxin A was added to the surface of the Aptamer-EA-cDNA-PDA / GE electrode and incubated at 20°C for 1 h. The electrode was rinsed with Tris-HCl buffer and dried with nitrogen. The electrode was then immersed in a functionalized gold nanoparticle solution and incubated at 30°C for 1 h. The electrode was rinsed with Tris-HCl buffer and dried with nitrogen. The electrode was then immersed in a 2 μg / mL SA-HRP aqueous solution and incubated at room temperature for 30 min. The electrode was rinsed with Tris-HCl buffer and placed in a TMB probe solution containing H2O2 for electrochemical detection.

2. The novel method for rapid detection of ochratoxin A based on an electrochemically deposited polydopamine-coated gold electrode according to claim 1, characterized in that: The nucleotide sequence of the linker is: 5'-SH-C6-TTTTTTTGTCCGTGGGTGGCGTAAAGGGAGCATCGGACATA-Biotin-3'.

3. The novel method for rapid detection of ochratoxin A based on an electrochemically deposited polydopamine-coated gold electrode according to claim 1, characterized in that: The nucleotide sequence of the amino-modified cDNA is: 5'-NH2-C6-TGTCCGATGCTCCCTTTACGCCACCCACGGACA-3'.

4. The novel method for rapid detection of ochratoxin A based on an electrochemically deposited polydopamine-coated gold electrode according to claim 1, characterized in that: The nucleotide sequence of the OTA-specific aptamer is: 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3'.

5. The novel method for rapid detection of ochratoxin A based on an electrochemically deposited polydopamine-coated gold electrode according to claim 1, characterized in that: The electrochemical detection method is chronoamperometry, with the following conditions: initial potential 0.2 V, scan time 100 s, and sampling interval 0.1 s.

6. Use of the novel method for rapid detection of ochratoxin A based on electrochemically deposited polydopamine coating modified gold electrode as claimed in claim 1 in detecting ochratoxin A.

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