CN116297779A - Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof - Google Patents

Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof Download PDF

Info

Publication number
CN116297779A
CN116297779A CN202310156273.4A CN202310156273A CN116297779A CN 116297779 A CN116297779 A CN 116297779A CN 202310156273 A CN202310156273 A CN 202310156273A CN 116297779 A CN116297779 A CN 116297779A
Authority
CN
China
Prior art keywords
malachite green
electrode
aptamer
preparation
complementary strand
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310156273.4A
Other languages
Chinese (zh)
Inventor
韩双艳
陈赞林
赵风光
谢苗嘉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Original Assignee
South China University of Technology SCUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT filed Critical South China University of Technology SCUT
Priority to CN202310156273.4A priority Critical patent/CN116297779A/en
Publication of CN116297779A publication Critical patent/CN116297779A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Nanotechnology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Urology & Nephrology (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Composite Materials (AREA)
  • Cell Biology (AREA)
  • Materials Engineering (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Manufacturing & Machinery (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a nucleic acid aptamer electrochemical sensor for detecting malachite green, and a preparation method and application thereof. In the preparation method, the sulfhydrylation complementary strand is combined with malachite green aptamer to form a double-chain structure, and is connected to the nano gold particles through Au-S bond to form a blocking redox probe [ Fe (CN) 6 ] 3‑/4‑ A barrier in contact with the electrode, causing a decrease in current; after adding malachite green, the aptamer and malachite green are specificSex binding, double-stranded structure is destroyed, [ Fe (CN) 6 ] 3‑/4‑ Can be smoothly contacted with the electrode, thereby resulting in current enhancement. The sensor is simple to prepare, low in aptamer price, wide in linear range and low in detection limit, can achieve tens of microamps for current difference values between malachite green with different concentrations, is obvious in difference, can effectively improve detection precision, reduces detection cost and is good in specificity. Can be applied to the accurate, efficient and sensitive detection of malachite green in water.

Description

Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a nucleic acid aptamer electrochemical sensor for detecting malachite green based on a nanocomposite, and a preparation method and application thereof.
Background
Malachite Green (Malachite Green) is an artificially synthesized triphenylmethane dye, has the effects of killing bacteria and parasites, and is also a specific drug for treating saprolegniasis. Malachite green is therefore widely used in commercial aquaculture. In recent years, a great deal of research has shown that malachite green has cytotoxicity, mutagenicity and carcinogenicity, and even a small amount of malachite green can cause damage to the immune system and reproductive system of the human body. In this regard, the European Union regulated that the addition limit of malachite green in fish farming should not exceed 2 μg/kg, and the use of such compounds is more completely prohibited by the Chinese department of agriculture. However, for better profits, individual vendors still illegally use malachite green to increase the survival rate of fish during transportation, and thus it is necessary to develop a method for detecting malachite green in time and rapidly.
Many methods for measuring malachite green have been reported, including high performance liquid chromatography, high performance liquid chromatography-mass spectrometry, immunoassay, surface enhanced raman scattering, molecularly imprinted polymers, and the like. These methods have good accuracy and sensitivity, but often require the use of expensive precision instruments and are operated by trained staff. In recent years, a biosensor technology using a nucleic acid aptamer as a recognition element has become a research hot spot, and the nucleic acid aptamer is an artificial DNA or RNA single-stranded probe which can be folded into different secondary structures so as to be combined with a target substance, and has the advantages of easy synthesis, high chemical stability, simplicity in operation and the like. The electrochemical aptamer sensor is an important aptamer sensor, combines high sensitivity of electrochemical detection and aptamer specificity, and has good application prospect.
The 200710070381.0 method for rapidly detecting malachite green in aquatic products by using an aptamer adopts an enzyme-linked chromogenic method to detect, and uses single-stranded DNA with a specific sequence as an identification element, but the method needs elution and chromogenic method and may have false positives. The 201410008020.3 patent adopts an electrochemical aptamer sensor to detect malachite green, the target is combined to the surface of an electrode through acting force between the RNA aptamer and the target, and then an antibody corresponding to the target is combined to form a sandwich structure, so that the problem of false positive is avoided to a certain extent, but the RNA aptamer has the problems of high price, instability, difficulty in labeling and the like. Patent 201810625479.6 proposes a method for detecting an aptamer electrochemical biosensor of malachite green, wherein the aptamer is combined with a target object to the surface of an electrode through the specific combination action of the aptamer and the target object, and a sandwich structure is formed by utilizing the combination action of avidin and biotin and horseradish peroxidase. However, the sensor has poor detection capability on low-concentration malachite green, and only can detect malachite green with the concentration of more than 1 mug/L; and the change of the sensing signal is small, and the difference value between the malachite green with the concentration of 1mg/L and the current of the blank sample is only 10 mu A.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary object of the invention is to provide a nucleic acid aptamer electrochemical sensor for detecting malachite green. The sensor increases the current span, has more obvious sensing signal change aiming at malachite green with different concentrations, can effectively improve the detection precision, reduces the detection cost and has good specificity.
The invention further aims at providing a preparation method of the aptamer electrochemical sensor for detecting malachite green. In the preparation method, the sulfhydrylation complementary strand is combined with malachite green aptamer to form a double-chain structure, and is connected to the nano gold particles through Au-S bond to form a blocking redox probe [ Fe (CN) 6 ] 3-/4- A barrier in contact with the electrode, causing a decrease in current; after addition of malachite green, the aptamer specifically binds to malachite green, the double-stranded structure is destroyed, [ Fe (CN) 6 ] 3-/4- Can be smoothly contacted with the electrode, thereby resulting in current enhancement.
A third object of the present invention is to provide the use of the above-described aptamer electrochemical sensor for detecting malachite green.
The aim of the invention is achieved by the following technical scheme:
a nucleic acid aptamer electrochemical sensor for detecting malachite green comprises a modification material, a recognition element and a transduction device. The modification material comprises a multi-wall carbon nano tube, a titanium dioxide coupled composite material and gold nano particles; the recognition element consists of a malachite green aptamer and complementary strands 1 and 2 thereof, wherein the malachite green aptamer preferably has a sequence of 5'-CCATGCGACGGACAGCACGTGTCACCGCGATCAGCC-3' (Chinese patent application No. CN 202210091617.3) and contains 36 bases. According to the two complementary strands (complementary strand 1, complementary strand 2) designed for the malachite green aptamer, complementary strand 1 preferably has a sequence of 5'-TCGCATGGTTTTT-3', contains 13 bases, and is modified at the 3' -end with-SH- (CH) 2 ) 6 -; complementary strand 2 preferably has sequence 5'-TTTTTGGCTGATC-3' and contains 13 bases and is modified at the 5' -end with-SH- (CH) 2 ) 6 -; the transduction device is a common electrochemical workstation (preferably a CHI660E electrochemical workstation), the working electrode is a gold electrode assembled by a modified material and an identification element, the counter electrode is a platinum electrode, and the reference electrode is Ag/AgCl; the electrolyte is potassium ferricyanide solution.
The detection principle is as follows: the composite material and the gold nanoparticles can obviously strengthen the current signal of the sensor and are used for amplifying the current change caused by malachite green. Before adding malachite green, the aptamer DNA molecular layer forms a barrier on the surface of the modified electrode to prevent [ Fe (CN) in the electrolyte 6 ] 3-/4- Reaching the electrode surface, causing the current signal to drop. In the presence of malachite green, the aptamer separates from the complementary DNA and forms a complex with malachite green. Aptamer stripping results in blocking [ Fe (CN) 6 ] 3-/4- Is broken by barrier of [ Fe (CN) 6 ] 3-/4- Can freely contact the surface of the electrode, the current intensity is obviously increased, the degree of increase is positively correlated with the concentration of malachite green, and a current signal is passedThe detection of malachite green can be achieved by the variation of (a).
A preparation method of a nucleic acid aptamer electrochemical sensor for detecting malachite green comprises the following steps:
(1) Preparation of multi-wall carbon nano tube and titanium dioxide composite material: dissolving the multi-wall carbon nano tube and titanium dioxide in absolute ethyl alcohol, uniformly mixing, and then carrying out ultrasonic treatment until a highly dispersed gray black solution is obtained, thus obtaining a multi-wall carbon nano tube and titanium dioxide composite material;
(2) Polishing and activating treatment of a gold electrode: immersing a gold electrode in a piranha solution (concentrated sulfuric acid: hydrogen peroxide=7:3), washing with ultrapure water, and polishing the surface of the electrode to a mirror surface with alumina powder on a polishing cloth; then respectively placing the gold electrodes in ethanol and ultrapure water for ultrasonic treatment, and drying with nitrogen flow for later use;
(3) Preparation of the identification element: after the malachite green aptamer and the complementary strand 1 and the complementary strand 2 thereof are thawed and diluted to the corresponding concentrations, the complementary strand 1 and the complementary strand 2 with the same volume and concentration and the malachite green aptamer with half concentration of twice volume are taken to be fully and uniformly mixed, and incubated overnight at 2-8 ℃ (preferably 4 ℃) to obtain double-stranded DNA with sulfhydryl groups at two ends, namely the recognition element;
(4) Preparation of modified electrode: and (3) dripping the multi-wall carbon nano tube and titanium dioxide composite material prepared in the step (1) on the surface of the gold electrode activated in the step (2), naturally drying the gold electrode, dripping nano gold particles on the surface of the electrode, naturally drying the gold electrode, dripping the double-stranded DNA prepared in the step (3) on the surface of the electrode, naturally drying the gold electrode, then sealing the non-specific binding sites, naturally drying the gold electrode, sealing the non-specific binding sites, finishing the preparation of the modified electrode, and storing the modified electrode at 2-8 ℃ (preferably 4 ℃) when the modified electrode is not used.
In the step (1), the step of (a),
the ratio of the multi-wall carbon nano tube to the titanium dioxide to the absolute ethyl alcohol is as follows: 40-100 mg: 25-50 mg: 10-25 mL; preferably 100mg:50mg:25mL;
preferably, the mixing time is 3-5 minutes; further 3 minutes;
the ultrasonic treatment time is 40-60 minutes; preferably 60 minutes;
in the step (2), the step of (C),
the soaking time is 5-10 min; preferably for 10min;
the ultrasonic treatment time is 1-2 min; preferably 1min;
in the step (3), the vortex time is 3-5 minutes; preferably 3 minutes;
in the step (4), the step of (c),
the non-specific binding site is blocked by using 0.5 mM-1.5 mM 6-mercapto-1-hexanol; preferably, 1mM 6-mercapto-1-hexanol is used to block the non-specific binding sites;
the step of establishing a standard curve by the sensor comprises the following steps:
1) The modified electrode is used as a working electrode, a platinum electrode is used as a counter electrode, ag/AgCl is used as a reference electrode, a potassium ferricyanide solution is used as an electrolyte, a differential pulse voltammetry is adopted for detection, the scanning potential range is-0.1-0.4V, the amplitude is 0.05V, the pulse time is 0.05ms, and a differential pulse voltammetry spectrogram is recorded; obtaining a blank peak current without malachite green;
2) Dripping a detection base solution containing malachite green standard substance on a working electrode, incubating for 30-60 min, immersing the working electrode in electrolyte, and recording a differential pulse voltammogram of the working electrode; in the concentration range of 10 1 ～10 6 Within ng/L, one concentration was selected every 10-fold, and repeated 3 times.
3) And (3) plotting by taking the logarithmic value lgC of the concentration of the base solution detected by the malachite green standard product as an abscissa and the current value as an ordinate, and establishing a standard curve. According to the standard curve, the concentration of malachite green in different water samples can be detected.
The application of the aptamer electrochemical sensor in detecting malachite green.
Compared with the prior art, the invention has the following advantages and effects:
(1) The linear range of the nucleic acid aptamer electrochemical sensor for detecting malachite green provided by the invention can span 5 orders of magnitude, can detect the malachite green of pmol/L level, and has good detection capability.
(2) The invention solves the problems of high cost, long time consumption, complex detection scheme and need of professional operation of the existing malachite green detection method, and provides a simple, convenient, rapid and efficient preparation method of the malachite green aptamer electrochemical sensor. The sensor is simple to prepare, low in aptamer price, wide in linear range and low in detection limit, and can achieve tens of microamps for current difference between malachite green with different concentrations, and the difference is obvious. Can be applied to the accurate, efficient and sensitive detection of malachite green in water.
Drawings
FIG. 1 is a schematic diagram of the assembly flow and detection principle of an aptamer electrochemical sensor for detecting malachite green.
FIG. 2 is a graph showing the current response of the electrode after modification of the multiwall carbon nanotube and titanium dioxide composite with the nanogold.
FIG. 3 is a graph of differential voltammetric pulse profile of a nucleic acid aptamer electrochemical sensor for detecting malachite green at different concentrations; wherein the concentration of the alpha- & gt g malachite green is 0 and 10 in sequence 1 ，10 2 ，10 3 ，10 4 ，10 5 And 10 6 ng/L。
FIG. 4 is a standard curve of a nucleic acid aptamer electrochemical sensor for detecting malachite green.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
Example 1: sensor detection for malachite green at 1mg/L
The method comprises the following steps ofAn aptamer electrochemical sensor for detecting malachite green comprises a modification material, a recognition element and a transduction device. The assembly flow and the detection principle of the aptamer electrochemical sensor for detecting malachite green are schematically shown in fig. 1. The modification material comprises a multi-wall carbon nano tube, a titanium dioxide coupled composite material and gold nano particles; the recognition element consists of three single-stranded DNA, which are malachite green aptamer, and the sequence is 5'-CCATGCGACGGACAGCACGTGTCACCGCGATCAGCC-3', and contains 36 bases. Two complementary strands of malachite green aptamer, complementary strand 1 of 5'-TCGCATGGTTTTT-3' and complementary strand 2 of 5'-TTTTTGGCTGATC-3', each containing 13 bases, are modified at the 3 'and 5' ends by-SH- (CH) respectively 2 ) 6 -; the transduction device is a commonly used CHI660E electrochemical workstation, the working electrode is a gold electrode assembled by a modified material and an identification element, the counter electrode is a platinum electrode, and the reference electrode is Ag/AgCl; the electrolyte is potassium ferricyanide solution.
The preparation method of the aptamer electrochemical sensor for detecting malachite green specifically comprises the following steps and parameters:
(1) Preparation of multi-wall carbon nano tube and titanium dioxide composite material: dissolving 100mg of multi-wall carbon nano tube and 50mg of titanium dioxide in 25mL of absolute ethyl alcohol, placing the mixture on a vortex mixer, uniformly mixing for 3 minutes, and then performing ultrasonic treatment for 60 minutes until a highly dispersed gray black solution is obtained, which shows that the multi-wall carbon nano tube and titanium dioxide composite material is obtained, and the MWCNTs-TiO is recorded 2 。
(2) Polishing and activating treatment of a gold electrode: immersing a gold electrode in a piranha solution (concentrated sulfuric acid: hydrogen peroxide=7:3) for 10min, cleaning with ultrapure water, drying with nitrogen flow, placing above a polishing cloth, taking 1.0 mu m alumina powder, placing the electrode surface in the alumina powder, circling and grinding for 2min until the electrode surface is polished to a mirror surface. Then the gold electrodes are respectively placed in ethanol and ultrapure water for ultrasonic treatment for 1min, the treatment is carried out for three times, and the nitrogen flow is used for drying. Scanning for 20 circles by cyclic voltammetry at a scanning speed of 0.1V/s and a potential range of-0.4-0.8V; after flushing with ultrapure water, drying with nitrogen flow for later use;
(3) Preparation of the identification element: after thawing the aptamer and its complementary strand, diluting to the corresponding concentration with phosphate buffer, 1mL of 8. Mu.M complementary strand 1, 1mL of 8. Mu.M complementary strand 2 and 2mL of 4. Mu.M malachite green aptamer were vortexed for 3 minutes to mix them thoroughly, and incubated overnight at 4℃to obtain a double-stranded DNA with thiol groups at both ends at a concentration of 2. Mu.M, i.e., a recognition element.
(4) Preparation of modified electrode: and (3) dripping 2 mu L of the composite material prepared in the step (1) on the surface of the gold electrode after the activation in the step (2), naturally drying, dripping 2 mu L of nano gold particles (AuNPs) on the surface of the electrode, naturally drying, dripping 2 mu L of the double-stranded DNA prepared in the step (3) on the surface of the electrode, naturally drying, then taking 2 mu L of 1mM 6-mercapto-1-hexanol (MCH) to seal a non-specific binding site, naturally drying and then sealing the 6-mercapto-1-hexanol to finish the preparation of the modified electrode. Wherein, gold electrode is used as blank control, MWCNTs @ TiO is dripped 2 The gold electrode of the composite material without the nano gold particles was used as an experimental control, and the electrode current response change chart is shown in fig. 2. As can be seen from FIG. 2, when the surface of the gold electrode is modified with MWCNTs @ TiO 2 After the composite material, the current peak response increased from 58 μA to 172 μA; when the nano gold particles are modified continuously, the current peak response is increased from 172 mu A to 220 mu A, which shows that the modified material is favorable for enhancing electric signal conduction so as to amplify detection sensitivity.
(5) And (3) detecting by using a differential pulse voltammetry with a scanning potential range of-0.1-0.4V, an amplitude of 0.05V and a pulse time of 0.05ms, and recording a differential pulse voltammetry spectrogram (see figure 3) to obtain a blank peak current without malachite green, wherein the differential pulse voltammetry is used as a working electrode, the platinum electrode is used as a counter electrode, the Ag/AgCl is used as a reference electrode, and the potassium ferricyanide solution is used as an electrolyte.
(6) And 2 mu L of detection base solution containing malachite green standard substances is dripped on the working electrode, the working electrode is immersed into electrolyte after incubation for 30min, and a differential pulse voltammogram of the working electrode is recorded. In the concentration range of 10 1 ～10 6 Within ng/L, one concentration was selected every 10-fold, and repeated 3 times. The differential pulse voltammogram is shown in figure 3,indicating that over a range, the current peak response is proportional to the malachite green concentration.
(7) The logarithmic lgC of the concentration of the base liquid detected by the malachite green standard is plotted on the abscissa, and the current value is plotted on the ordinate, so as to establish a standard curve, as shown in FIG. 4. At 10 1 ～10 6 In the ng/L concentration range, the current value and the logarithmic value of the malachite green standard concentration show good linear relation (R 2 = 0.99816), the linear equation is y=51.0585+3.8892x, and the detection limit is 8.68pg/mL. According to the standard curve, the concentration of malachite green in different water samples can be detected.
(8) 2 mu L of detection base solution with the concentration of malachite green of 1mg/L is dripped on a working electrode, the working electrode is immersed into electrolyte after incubation for 30min, a differential pulse voltammogram of the working electrode is recorded, and the peak current is about 74 mu A and the peak current difference between a blank sample without malachite green is 27 mu A.
Example 2: sensor detection for malachite green at a concentration of 10ng/L
A sensor was prepared with reference to example 1, with a malachite green concentration of 10ng/L;
after the working electrode is prepared, 2 mu L of detection base solution with the concentration of malachite green of 10ng/L is dripped on the working electrode, the working electrode is immersed into electrolyte after incubation for 30min, a differential pulse voltammogram of the working electrode is recorded, and the peak current is about 55 mu A, and the peak current difference between the blank sample without malachite green is 8 mu A.
Example 3: sensor stability detection
The sensor was prepared with reference to example 1, in which the working electrode was stored at 4 ℃ for 7 days.
After the working electrode is prepared, 2 mu L of malachite green detection base solution with the concentration of 100 mu g/L is dripped on the working electrode, the working electrode is immersed into the electrolyte after incubation for 30min, and a differential pulse voltammogram of the working electrode is recorded, so that the peak current is about 63 mu A, namely the detection efficiency of the sensor stored for 7 days at 4 ℃ can still reach more than 90%.
Example 4: sensor-specific detection
The sensor was prepared according to reference example 1 with a glyphosate concentration of 5mg/L and a tricyclazole concentration of 5mg/L;
after the working electrode is prepared, 2 mu L of detection base solution with the concentration of 5mg/L of glyphosate and 2 mu L of detection base solution with the concentration of 5mg/L of tricyclazole are respectively dripped on different working electrodes, the working electrodes are immersed into electrolyte after incubation for 30min, and differential pulse voltammograms of the working electrodes are recorded. It is known that the peak current of the working electrode for dropping glyphosate was about 55. Mu.A and that of the working electrode for dropping tricyclazole was about 58. Mu.A. The peak current of the differential pulse voltammetry spectrum of the sensor for detecting 100 mug/L malachite green is about 70 mugA, even if the concentration is 50 times different, the current difference still exceeds 12 mugA, and the difference is obvious, so that the sensor has good specificity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. An aptamer electrochemical sensor for detecting malachite green, which is characterized in that: comprises a finishing material, an identification element and a transduction device;
the modification material comprises a multi-wall carbon nano tube, a titanium dioxide coupled composite material and gold nano particles;
the recognition element consists of a malachite green aptamer, a complementary strand 1 and a complementary strand 2, wherein the sequence of the malachite green aptamer is 5'-CCATGCGACGGACAGCACGTGTCACCGCGATCAGCC-3', the sequence of the complementary strand 1 is 5'-TCGCATGGTTTTT-3', and the 3' -end is modified with-SH- (CH) 2 ) 6 -; complementary strand 2 has a sequence 5'-TTTTTGGCTGATC-3' and is modified at the 5' -end with-SH- (CH) 2 ) 6 -；
The transduction device is a commonly used electrochemical workstation.
2. The method for preparing the aptamer electrochemical sensor for detecting malachite green according to claim 1, comprising the following steps:
(1) Preparation of multi-wall carbon nano tube and titanium dioxide composite material: dissolving the multi-wall carbon nano tube and titanium dioxide in absolute ethyl alcohol, uniformly mixing, and then carrying out ultrasonic treatment until a highly dispersed gray black solution is obtained, thus obtaining a multi-wall carbon nano tube and titanium dioxide composite material;
(2) Polishing and activating treatment of a gold electrode: immersing a gold electrode in a piranha solution, cleaning the gold electrode by using ultrapure water, and polishing the surface of the electrode to a mirror surface by using alumina powder on polishing cloth; then respectively placing the gold electrodes in ethanol and ultrapure water for ultrasonic treatment, and drying with nitrogen flow for later use;
(3) Preparation of the identification element: after the malachite green aptamer and the complementary strand 1 and the complementary strand 2 thereof are thawed and diluted to the corresponding concentrations, the complementary strand 1 and the complementary strand 2 with the same volume and concentration and the malachite green aptamer with half concentration of twice volume are taken to be fully and uniformly mixed, and incubated overnight at the temperature of 2-8 ℃ to obtain double-chain DNA with mercapto groups at two ends, namely the recognition element;
(4) Preparation of modified electrode: and (3) dripping the multiwall carbon nanotube and titanium dioxide composite material prepared in the step (1) on the surface of the gold electrode activated in the step (2), naturally drying the gold electrode, dripping nano gold particles on the surface of the electrode, naturally drying the gold electrode, dripping the double-stranded DNA prepared in the step (3) on the surface of the electrode, naturally drying the gold electrode, sealing the non-specific binding sites, and finishing the preparation of the modified electrode for detection.
3. The preparation method according to claim 2, characterized in that:
and (3) when the modified electrode in the step (4) is not used, storing at 2-8 ℃.
4. The preparation method according to claim 2, characterized in that:
in the step (1), the ratio of the multi-wall carbon nano tube to the titanium dioxide to the absolute ethyl alcohol is as follows: 40-100 mg: 25-50 mg: 10-25 mL.
5. The method of manufacturing according to claim 4, wherein:
in the step (1), the ratio of the multi-wall carbon nano tube to the titanium dioxide to the absolute ethyl alcohol is as follows: 100mg:50mg:25mL.
6. The method according to any one of claims 2 to 5, wherein:
in the step (1), the mixing time is 3-5 minutes;
in the step (1), the ultrasonic treatment time is 40-60 minutes;
in the step (2), the soaking time is 5-10 min;
in the step (2), the ultrasonic treatment time is 1-2 min;
in the step (3), the time of the vortex is 3-5 minutes.
7. The method according to any one of claims 2 to 5, wherein:
in the step (4), the non-specific binding site is blocked by using 0.5 mM-1.5 mM 6-mercapto-1-hexanol.
8. The method of manufacturing according to claim 7, wherein:
in step (4), the non-specific binding site is blocked with 1mM 6-mercapto-1-hexanol.
9. The method according to any one of claims 2 to 5, wherein:
1) Detecting by differential pulse voltammetry with a scanning potential range of-0.1-0.4V, an amplitude of 0.05V and a pulse time of 0.05ms by using the modified electrode prepared in the step (3) as a working electrode, a platinum electrode as a counter electrode, ag/AgCl as a reference electrode and potassium ferricyanide solution as an electrolyte; obtaining a blank peak current without malachite green;
2) Dripping a detection base solution containing malachite green standard substance on a working electrode, incubating for 30-60 min, immersing the working electrode in electrolyte, and recording a differential pulse voltammogram of the working electrode; in the concentration range of 10 1 ～10 6 Within ng/L, selecting a concentration every 10 times, repeating for 3 times;
3) And (3) plotting by taking the logarithmic value lgC of the concentration of the base solution detected by the malachite green standard product as an abscissa and the current value as an ordinate, and establishing a standard curve.
10. Use of the aptamer electrochemical sensor of claim 1 for detecting malachite green.
CN202310156273.4A 2023-02-23 2023-02-23 Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof Pending CN116297779A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310156273.4A CN116297779A (en) 2023-02-23 2023-02-23 Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310156273.4A CN116297779A (en) 2023-02-23 2023-02-23 Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN116297779A true CN116297779A (en) 2023-06-23

Family

ID=86829862

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310156273.4A Pending CN116297779A (en) 2023-02-23 2023-02-23 Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN116297779A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116908265A (en) * 2023-09-11 2023-10-20 常州先趋医疗科技有限公司 Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116908265A (en) * 2023-09-11 2023-10-20 常州先趋医疗科技有限公司 Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids
CN116908265B (en) * 2023-09-11 2023-12-12 常州先趋医疗科技有限公司 Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids

Similar Documents

Publication Publication Date Title
Chen et al. Electrochemical sensing and biosensing based on square wave voltammetry
Zamfir et al. Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection
Thévenot et al. Electrochemical biosensors: recommended definitions and classification
CN110618185B (en) Ratiometric electrochemical detection method of ochratoxin A
CN111175364B (en) Preparation method of ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A
Ciszewski et al. Electrochemical detection of nitric oxide using polymer modified electrodes
Tao et al. Fabrication of an electrochemical sensor based on spiropyran for sensitive and selective detection of fluoride ion
Uludag et al. Design and characterisation of a thin-film electrode array with shared reference/counter electrodes for electrochemical detection
Qiang et al. A new potentiometric immunosensor for determination of α-fetoprotein based on improved gelatin–silver complex film
Yuan et al. A label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of Japanese B encephalitis vaccine
Pisoschi Biosensors as bio-based materials in chemical analysis: a review
Nodoushan et al. Electrochemical detection of aflatoxin B1: an aptasensor prepared using graphene oxide and gold nanowires
Zuo et al. A sensitive ratiometric electrochemiluminescence biosensor for hypoxanthine detection by in situ generation and consumption of coreactants
CN116297779A (en) Aptamer electrochemical sensor for detecting malachite green and preparation method and application thereof
Aboul-Kasim Anodic adsorptive voltammetric determination of the vitamin B1 (thiamine)
Liu et al. A novel electrochemical immunosensor for ochratoxin A with hapten immobilization on thionine/gold nanoparticle modified glassy carbon electrode
Zhou et al. An amperometric immunosensor based on an electrochemically pretreated carbon–paraffin electrode for complement III (C3) assay
CN113588752B (en) Preparation method and application of electrochemiluminescence aptamer sensor
Zheng et al. A simple electrochemical aptasensor for saxitoxin detection
Zhang et al. Potentiometric immunosensor based on antiserum of Japanese B encephalitis immobilized in nano-Au/polymerized o-phenylenediamine film
CN111307907B (en) Reproducible electrochemical sensor and method for detecting aflatoxin
Shankaran et al. Determination of sulfur dioxide based on a silver dispersed functional self-assembled electrochemical sensor
Xiao et al. Highly enhanced electrochemiluminescence based on pseudo triple-enzyme cascade catalysis and in situ generation of co-reactant for thrombin detection
Mongra et al. Review study on electrochemical-based biosensors
Kurita et al. Differential measurement with a microfluidic device for the highly selective continuous measurement of histamine released from rat basophilic leukemia cells (RBL-2H3)

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination