Alumina membrane nanopore gate-based bimodal sensor and applications thereof

Abstract

The application relates to the application of photoelectrochemical detection combined with colorimetric detection in the field of biosensing technology, and discloses a bimodal sensor based on alumina membrane nanopore gating and application thereof. The bimodal sensor comprises an electrochemical workstation, a photoelectric detection area and a colorimetric detection area. The electrochemical workstation is in working connection with the photoelectric detection area. The photoelectric detection area and the colorimetric detection area are separated by an alumina membrane nanopore gating which is modified with nanogold and aptamer in a pore and is in a closed state. After a to-be-detected substance is combined with the aptamer, the alumina membrane nanopore gating is opened. The opening degree and range of the alumina membrane nanopore gating are related to the amount of the to-be-detected substance. Then, the photoelectric detection area and the colorimetric detection area detect the size of the photoelectric current and the strength of the colorimetric intensity in the detection area respectively, and the concentration of the to-be-detected substance is obtained. The application greatly improves the accuracy of the result and reduces the detection false positive under a single sensing mode.

Description

Technical Field

[0001] This invention relates to the application of combining photoelectric detection and colorimetric detection in the field of biosensing technology, and particularly to a dual-mode sensor based on alumina film nanopore gating and its application. Specifically, it relates to a photoelectrochemical and visual colorimetric dual-mode sensor based on alumina film nanopore gating, its construction method, and its application in detecting T-2 toxin. Background Technology

[0002] Photoelectrochemical (PEC) sensing analysis is an analytical method based on the principle of photoelectrochemistry, which outputs information about the analyte in the form of an electrical signal in the presence of a light source. Because of the difference between the input energy and the output signal, it has many advantages, such as low background signal, high sensitivity, simple instrumentation, and ease of operation. Photoactive materials, as the medium for the conversion between light energy, electrical energy, and chemical energy in the PEC process, have always received considerable attention. CdS is a semiconductor photoelectrochemical material with a suitable band gap. Furthermore, nucleic acid molecules or some small molecules are closely related to human diseases or food safety, making the development of corresponding sensitive and convenient photoelectrochemical sensing platforms of great significance. Visual colorimetry is a common chemical analysis method that uses the color intensity of a solution to determine its concentration. This method is simple and easy to perform, requiring no complex instruments. The principle of visual colorimetry is based on the fundamental principle of colorimetry, namely that the color intensity of a solution is directly proportional to its concentration. In visual colorimetry experiments, a set of standard solutions is first prepared, and then the test solution is compared with the standard solutions by colorimetry. The concentration of the test solution is determined by comparing the color intensity of the two solutions. The advantage of visual colorimetry is its simplicity and ease of use, requiring no complex instruments and equipment, and providing quick results. However, it has some disadvantages, such as being susceptible to errors due to factors like light and color differences.

[0003] Nanoporous materials refer to porous materials with significant surface effects and a certain porosity, exhibiting a pore size of 1–100 nm. Due to the controllable size of pores at the atomic, molecular, and nanoscale, they can distinguish and interact with molecules and clusters. They also possess advantages such as good stability, durability, good adsorption kinetics, high selectivity, and high adsorption capacity, leading to their widespread application in biosensors, drug delivery, gas separation, energy storage and fuel cell technology, nanocatalysis, and photonics. Porous alumina (AAO) membranes, as a type of nanoporous material, offer structural stability, easily controllable pore size, good biocompatibility, low preparation cost, and reusability, making them suitable for applications in biosensing, DNA sensors, drug delivery, and electrochemical sensors.

[0004] More than 100 mycotoxins have been isolated and identified to date. T-2 toxin, a toxic metabolite produced by *Fusarium cladosporum*, is widely distributed in cereal products and animal feed, and is stable and difficult to eliminate. Long-term exposure to humans and animals can cause serious damage to body tissues, disorders, slowed growth, and even death. Current methods for detecting T-2 toxin include thin-layer chromatography, high-performance liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay (ELISA), and electrochemical analysis. While these methods are accurate, they are time-consuming, costly, and complex, limiting their practical application. Therefore, developing a simple, rapid, and low-cost analytical method is essential. Summary of the Invention

[0005] Objective of the Invention: To address the problems existing in the prior art, this invention provides a dual-mode sensor based on alumina film nanopore gating and its application. The dual-mode sensor provided by this invention integrates advantages such as simple operation, high sensitivity, good selectivity, low cost, and dual detection. When applied to the detection of T-2 toxin, it greatly improves the accuracy of the results and significantly reduces false positives that may occur in single-sensor mode.

[0006] Technical Solution: This invention provides a dual-mode sensor based on alumina film nanopore gating, comprising an electrochemical workstation, a photoelectric detection region, and a colorimetric detection region. The electrochemical workstation is operatively connected to the photoelectric detection region. The photoelectric detection region and the colorimetric detection region are separated by an alumina film nanopore gating system with gold nanoparticles and aptamers modified within the pores and in a closed state. After the analyte binds to the aptamer, the alumina film nanopore gating system opens, and the degree and range of opening are related to the amount of the analyte. The photocurrent magnitude and colorimetric intensity within the detection region are then detected by the photoelectric detection region and the colorimetric detection region, respectively, to obtain the concentration of the analyte.

[0007] Furthermore, the electrochemical workstation is connected to the photoelectric detection area via a working electrode, a reference electrode, and a counter electrode: if the modifying material on the working electrode is an anodic photoelectric semiconductor material, then the photoelectric detection area contains a reducing reagent, and the colorimetric detection area contains a system that changes color with an oxidizing reagent; If the modifying material on the working electrode is a cathode photoelectric semiconductor material, then the photoelectric detection area contains an oxidizing reagent, and the colorimetric detection area contains a system that changes color with a reducing reagent.

[0008] Furthermore, the working electrode is a CdS / ITO modified electrode; the specific preparation method of the CdS / ITO modified electrode is as follows: CdS quantum dots were drop-coated onto the washed and dried ITO surface, and then a naphthol solution was added to form a uniform thin film to fix the material. After air drying, a CdS / ITO modified electrode was obtained.

[0009] Preferably, the reference electrode is an Ag / AgCl reference electrode; the counter electrode is a Pt counter electrode.

[0010] Furthermore, the photoelectric detection area contains a PBS solution containing ascorbic acid; the colorimetric detection area contains a NaAc-HAc solution containing TMB, H2O2 and a catalyst.

[0011] Furthermore, the PBS solution has a pH of 7.4 and a concentration of 0.1 mol / L; the concentration of ascorbic acid in the PBS solution is 1-10 mmol / L. The NaAc-HAc solution has a pH of 3.5 and a concentration of 0.1 mol / L; the NaAc-HAc solution contains 10-50 mmol / L H2O2, 1-5 mg / mL TMB, and 0.1-0.5 mg / mL catalyst.

[0012] Furthermore, the specific preparation method of the alumina film nanopore gating is as follows: Au NPs solution was mixed evenly with n-hexane, and anhydrous ethanol was added dropwise. After separation, Au NPs aggregated at the liquid-liquid interface to form an Au NPs membrane. An AAO membrane was placed on the Au NPs membrane, allowing the Au NPs to automatically adsorb onto the surface of the AAO membrane. The membrane was then heated and dried to form an Au-AAO nanomembrane. A T-2 toxin aptamer solution was prepared using PBS as a solvent and added dropwise to the Au-AAO nanomembrane. After reaction, unadsorbed aptamers were removed by rinsing with PBS solution. MCH solution was then added dropwise to block non-specific active sites, yielding an alumina nanoporous gated apt / Au-AAO membrane.

[0013] Furthermore, the volume ratio of the Au NPs solution, n-hexane, and anhydrous ethanol is 10:5:8; The PBS solution has a pH of 6.0-8.0 and a concentration of 0.1-1 mol / L; The volume of the MCH solution is 10-50 μL, and the concentration is 0.5-10 mmol / L.

[0014] Furthermore, the volume of the T-2 toxin aptamer solution is 20 μL, and the concentration is 0-10. 6μmol / L; The T-2 toxin aptamer is a thiol-containing sequence, with the sequence: 5'-GTAT ATCA AGCA TCGC GTGT TTAC ACAT GCGA GAGGTGAA-SH-3'.

[0015] Furthermore, the specific conditions for the reaction are: reaction temperature 4-6℃, reaction time 12h.

[0016] The present invention also provides an application of the dual-mode sensor as described in any of the preceding claims in the detection of T-2 toxin.

[0017] Theoretical Explanation: In the dual-mode sensor constructed in this invention, the alumina film nanopore gating system modifies the nanopores of the alumina film with gold nanoparticles and aptamers. The volume of the gold nanoparticles and the extension of the aptamers partially block the nanochannels on the alumina film, inhibiting the mass transfer capacity of the alumina film nanochannels and keeping the sensor in a "closed" state. When a analyte is present, it binds to the aptamer, causing the aptamer to curl and fold, opening part of the nanochannels and switching the sensor gating system from a "closed" to an "open" state. The degree and range of opening of the alumina film nanochannels are related to the amount of analyte: the greater the amount of analyte, the larger the opening range of the alumina film nanochannels, resulting in greater flux of reactants in both the photoelectric detection area and the colorimetric detection area, leading to a larger change in photocurrent and a more significant change in colorimetric intensity. Conversely, the smaller the amount of analyte, the smaller the opening range of the alumina film nanochannels, resulting in less flux of reactants in both the photoelectric detection area and the colorimetric detection area, leading to a smaller change in photocurrent and a weaker change in colorimetric intensity. Reactants in both the photoelectric detection region and the colorimetric detection region can spontaneously undergo redox reactions, and their concentrations determine the magnitude of the photocurrent and the intensity of the colorimetric color, respectively. By measuring the magnitude of the photocurrent and the intensity of the colorimetric color, the concentration of toxins can be determined.

[0018] Beneficial Effects: Compared with existing technologies, the dual-mode sensor based on alumina film nanopore gating designed in this invention spatially separates the detection signal from the analyte, reducing sensor assembly steps and effectively improving detection accuracy. The detection signal is more controlled by the nanopores than the signal itself, contributing to improved detection sensitivity. Simultaneous detection of the same analyte using dual signals, with mutual verification between the two signals, reduces false positives and false negatives that may occur in single-signal mode compared to single-signal detection. This invention combines the advantages of simple operation, high sensitivity, good selectivity, low cost, and dual detection, facilitating the development of inexpensive functional diagnostic kits. Attached Figure Description

[0019] Figure 1This is a device diagram of the dual-mode sensor based on alumina membrane nanopore gating according to the present invention; wherein, 1, dual-mode sensor; 2, photoelectric detection area; 3, colorimetric detection area; 4, CdS; 5, TMB; 6, AA; 7, nanozyme catalyst; 8, working electrode; 9, counter electrode; 10, reference electrode; 11, alumina membrane nanopore gating; 12, analyte; 13, wire; 14, electrochemical workstation; Figure 2 This is a diagram illustrating the detection mechanism of the dual-mode sensor based on alumina film nanopore gating constructed in this invention. Figure 3 The Au-AAO membrane nanochannel gating diagram is shown in the dual-mode sensor based on alumina membrane nanopore gating constructed in this invention. Figure 4 The X-ray diffraction pattern of Au NPs in Implementation Method 1; Figure 5 The UV-Vis absorption spectrum of Au NPs in Implementation Method 1; Figure 6 This is a scanning electron microscope image of the AAO membrane in Implementation Method 1; Figure 7 This is a scanning electron microscope image of the Au-AAO nanofilm in Embodiment 1; Figure 8 The graph shows the linear relationship between the lg value and the CdS photoelectric sensor current value for different T-2 toxin concentrations. Figure 9 Color changes of TMB at different T-2 toxin concentrations; Figure 10 The UV absorbance of TMB; Figure 11 The linear relationship between the lg value and the CdS photoelectric response current value for different T-2 toxin concentrations; Figure 12 The linear relationship between the lg value and the TMB UV absorption peak for different T-2 toxin concentrations is shown. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the embodiments.

[0021] Implementation method 1: This embodiment provides a dual-mode sensor based on alumina film nanopore gating (such as...). Figure 1The system includes an electrochemical workstation 14, a photoelectric detection area 2, and a colorimetric detection area 3. The electrochemical workstation 14 is connected to the photoelectric detection area 2. The photoelectric detection area 2 and the colorimetric detection area 4 are separated by an alumina film nanopore gate 11, which is modified with gold nanoparticles and aptamers and is in a closed state. After the analyte binds to the aptamer, the alumina film nanopore gate 11 opens. The degree and range of opening are related to the amount of the analyte. Then, the photoelectric detection area 2 and the colorimetric detection area 3 are used to detect the magnitude of the photocurrent and the intensity of the colorimetric color within the detection area, respectively, to obtain the concentration of the analyte.

[0022] The electrochemical workstation 14 is connected to the photoelectric detection region 2 via a CdS / ITO modified electrode, an Ag / AgCl reference electrode, and a Pt counter electrode.

[0023] The photoelectric detection area 2 contains a PBS solution containing ascorbic acid; the pH of the PBS solution is 7.4 and the concentration is 0.1 mol / L; the concentration of ascorbic acid in the PBS solution is 5.17 mmol / L.

[0024] Colorimetric detection area 3 contains a NaAc-HAc solution containing TMB, H2O2, and Fe3O4@PDA@ZIF-67 nanozyme catalyst; the pH of the NaAc-HAc solution is 3.5, and the concentration is 0.1 mol / L; in the NaAc-HAc solution, the concentration of H2O2 is 0.0216 M, the concentration of TMB is 2 mg / ml, the concentration of Fe3O4@PDA@ZIF-67 nanozyme catalyst is 0.225 mg / ml, and the volume ratio of H2O2, TMB, and Fe3O4@PDA@ZIF-67 nanozyme catalyst is 0.6∶3∶4.

[0025] The specific preparation method of the above-mentioned CdS / ITO modified electrode is as follows: (1) Measure 50 mL of 0.01 M CdCl2 solution and 250 µL of TGA and add them to a 100 mL three-necked flask. Stir magnetically for 30 min under nitrogen atmosphere. During this period, adjust the pH of the mixture to 11 with 1 M NaOH solution. Then, add 5.5 mL of 0.1 M Na2S solution and heat under reflux at 110 °C for 4 h under nitrogen atmosphere. The synthesized CdS quantum dots are diluted with deionized water and stored in a refrigerator at 4 °C for later use.

[0026] (2) The ITO glass was ultrasonically washed three times for 20 minutes in anhydrous ethanol and deionized water and dried. The insulating tape was punched with a punch and then adhered to the conductive surface of the ITO glass. CdS quantum dots were dropped onto the ITO surface and then naphthol solution was added to form a uniform thin film fixing material. The material was then air-dried to obtain the CdS / ITO modified electrode.

[0027] The specific preparation method of the above Fe3O4@PDA@ZIF-67 nanozyme catalyst is as follows: (1) Fe3O4 nanoparticles dispersed in water were synthesized by hydrothermal method. FeCl3·6H2O and sodium citrate were dissolved in ethylene glycol, and anhydrous sodium acetate was dissolved in ethylene glycol. After both were completely dissolved, they were mixed and transferred to a reaction vessel and reacted at 200℃ for 10 h. After natural cooling, the mixture was washed with ethanol and water and then freeze-dried for collection. (2) The synthesized Fe3O4 was ultrasonically dispersed in sterile water, and dopamine hydrochloride and tris(hydroxymethyl)aminomethane were added. After mechanical stirring for 2 hours, the mixture was magnetically separated, washed with sterile water, and vacuum dried to obtain Fe3O4@PDA. (3) Fe3O4@PDA was dissolved in a methanol solution containing cobalt nitrate to obtain solution A. 2-methylimidazole was dissolved in methanol to obtain solution B. Solution A was quickly added to solution B and mechanically stirred for 0.5 h. After standing at room temperature for 24 h, magnetic separation was performed. The solution was washed with methanol, dried under vacuum, and then carbonized at 800 °C under N2 atmosphere for 2 h to finally obtain Fe3O4@PDA@ZIF-67.

[0028] The specific preparation method of the above-mentioned alumina film nanopore gate is as follows: (1) Place 10 mL of Au NPs solution in a beaker, then add 5 mL of n-hexane and mix well. Next, add 8 mL of anhydrous ethanol. After separation, Au NPs can be seen agglomerating at the liquid-liquid interface. After removing the n-hexane, the Au NPs self-assemble and stabilize into a compact film at the gas-liquid interface. Carefully place the AAO film onto the surface of the solution in the beaker. The Au NPs will automatically adsorb onto the surface of the AAO film. After heating and drying, an Au-AAO nanofilm is formed. Figure 3 That is, Au-AAO nanopore gating; (2) A 2 μmol / L T-2 toxin aptamer solution was prepared using 0.1 mol / L PBS (pH 7.4) as the solvent. The T-2 aptamer sequence was: 5'-GTAT ATCA AGCA TCGC GTGT TTAC ACAT GCGA GAGG TGAA-SH-3'. 20 μL of the T-2 toxin aptamer solution was added dropwise to Au-AAO and reacted at 4°C for 12 h. The mixture was then rinsed multiple times with PBS buffer (pH 7.4) to remove unadsorbed aptamers. 20 μL of 1 mmol / L MCH solution was then added dropwise to block non-specific active sites, resulting in an alumina membrane nanoporous gated apt / Au-AAO.

[0029] This embodiment also provides a method for constructing the above-mentioned dual-mode sensor based on alumina film nanopore gating: 20 μL of T-2 toxin solutions of different concentrations were dropped onto an alumina membrane nanopore-gated apt / Au-AAO and incubated at 37 °C for 45 min to obtain T-2 / apt / Au-AAO. The photodetector region and the colorimetric detection region were separated by the T-2 / apt / Au-AAO. The photodetector region contained a PBS solution containing ascorbic acid (AA), and the colorimetric detection region contained a NaAc-HAc solution containing TMB, H2O2, and Fe3O4@PDA@ZIF-67 nanozyme catalyst. Using a three-electrode system, an electrochemical workstation was connected to the photodetector region via a CdS / ITO modified electrode, a Pt counter electrode, and an Ag / AgCl reference electrode. The photoresponse intensity current value of the CdS / ITO modified electrode and the UV absorption peak of TMB were detected, thus constructing a dual-mode sensor based on alumina membrane nanopore gating.

[0030] Implementation Method 2: This embodiment provides an application of a dual-mode sensor based on alumina film nanopore gating in the detection of T-2 toxin, the specific steps of which are as follows: S1. A dual-mode sensor based on alumina membrane nanopore gating was constructed according to Embodiment 1. 20 μL of T-2 toxin solutions of different concentrations were dropped onto the alumina membrane nanopore gating apt / Au-AAO and incubated at 37°C for 45 min to prepare T-2 / apt / Au-AAO. Different concentrations of T-2 toxin loaded on the apt / Au-AAO membrane (resulting in different membrane channel opening ranges) resulted in different amounts of AA and H2O2 passing through the apt / Au-AAO membrane within the same time period, causing changes in the photodetector region's light response and a lighter color in the colorimetric detection region. Figure 9 A three-electrode system was connected to the Chi660e electrochemical workstation for detection. The photoresponse intensity of the CdS / ITO modified electrode was used as the output signal to plot a standard curve A between the CdS / ITO photoresponse intensity current value and the T-2 toxin concentration. Figure 11 The UV absorption peak of TMB versus the standard curve of T-2 toxin concentration ( ) and TMB standard curve ( ) Figure 12 ); S2. Using the same method as in Example 1, a dual-mode sensor based on alumina film nanopore gating was constructed from the T-2 toxin solution to be detected. The photoresponse intensity current value of the CdS / ITO modified electrode and the ultraviolet absorption peak of TMB were detected. Substituting the results into standard curves A and B, the concentration of the T-2 toxin solution to be detected could be obtained. Figure 11-12 As shown, the T-2 virus exhibits a very good linear range between 1 ng / mL and 106 ng / mL.

[0031] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A dual-mode sensor based on alumina film nanopore gating, characterized in that: It includes an electrochemical workstation (14), a photoelectric detection area (2) and a colorimetric detection area (3). The electrochemical workstation (14) is connected to the photoelectric detection area (2). The photoelectric detection area (2) and the colorimetric detection area (3) are separated by an alumina film nanopore gate (11) with nano-gold and aptamers modified in the pores and in a closed state. After the analyte binds to the aptamer, the nanopore gate (11) of the alumina film opens. The degree and range of opening are related to the amount of the analyte. Then, the photocurrent magnitude and colorimetric intensity within the detection area are detected by the photoelectric detection area (2) and the colorimetric detection area (3), respectively, to obtain the concentration of the analyte. The electrochemical workstation (14) is connected to the photoelectric detection area (2) via the working electrode (8), the reference electrode (10), and the counter electrode (9): if the modification material on the working electrode (8) is an anodic photoelectric semiconductor material, then the photoelectric detection area (2) contains a reducing agent, and the colorimetric detection area (3) contains a system that changes color with an oxidizing agent; if the modification material on the working electrode (8) is a cathodic photoelectric semiconductor material, then the photoelectric detection area (2) contains an oxidizing agent, and the colorimetric detection area (3) contains a system that changes color with a reducing agent. The working electrode (8) is a CdS / ITO modified electrode; the specific preparation method of the CdS / ITO modified electrode is as follows: CdS quantum dots are drop-coated onto the washed and dried ITO surface, and then naphthol solution is added to form a uniform thin film fixing material, which is then air-dried to obtain the CdS / ITO modified electrode. The photoelectric detection area (2) contains a PBS solution containing ascorbic acid; the colorimetric detection area (3) contains a NaAc-HAc solution containing TMB, H2O2 and a catalyst; the PBS solution has a pH of 7.4 and a concentration of 0.1 mol / L; the ascorbic acid concentration in the PBS solution is 1-10 mmol / L; the NaAc-HAc solution has a pH of 3.5 and a concentration of 0.1 mol / L; the H2O2 concentration in the NaAc-HAc solution is 10-50 mmol / L, the TMB concentration is 1-5 mg / mL, and the catalyst concentration is 0.1-0.5 mg / mL.

2. The dual-mode sensor based on alumina film nanopore gating according to claim 1, characterized in that: The specific preparation method of the alumina film nanopore gate (11) is as follows: Au NPs solution was mixed evenly with n-hexane, and anhydrous ethanol was added dropwise. After separation, Au NPs aggregated at the liquid-liquid interface to form an Au NPs membrane. An AAO membrane was placed on the Au NPs membrane, allowing the Au NPs to automatically adsorb onto the surface of the AAO membrane. The membrane was then heated and dried to form an Au-AAO nanomembrane. A T-2 toxin aptamer solution was prepared using PBS as a solvent and added dropwise to the Au-AAO nanomembrane. After reaction, unadsorbed aptamers were removed by rinsing with PBS solution. MCH solution was then added dropwise to block non-specific active sites, yielding an alumina nanoporous gated apt / Au-AAO membrane.

3. The dual-mode sensor based on alumina film nanopore gating according to claim 2, characterized in that: The volume ratio of the AuNPs solution, n-hexane, and anhydrous ethanol is 10:5:

8. The PBS solution has a pH of 6.0-8.0 and a concentration of 0.1 mol / L-1 mol / L; The volume of the MCH solution is 10-50 μL, and the concentration is 0.5-10 mmol / L.

4. The dual-mode sensor based on alumina film nanopore gating according to claim 2, characterized in that: The volume of the T-2 toxin aptamer solution was 20 μL, and the concentration was 0-10. 6 μmol / L; The T-2 toxin aptamer is a thiol-containing sequence: 5'-GTAT ATCA AGCA TCGC GTGT TTAC ACAT GCGA GAGG TGAA-SH-3'.

5. The dual-mode sensor based on alumina film nanopore gating according to claim 2, characterized in that: The specific conditions for the reaction are: reaction temperature 4-6 ℃, reaction time 12 h.

6. The application of a dual-mode sensor as described in any one of claims 1-5 in the detection of T-2 toxin.

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