Homogeneous anode photoelectrochemical aflatoxin detection method based on bismuth tungstate
阅读说明：本技术 一种基于钨酸铋的均相阳极光电化学检测黄曲霉毒素的方法 (Homogeneous anode photoelectrochemical aflatoxin detection method based on bismuth tungstate ) 是由 王光丽 刘田利 孙冬雪 顾萌萌 于 2019-11-08 设计创作，主要内容包括：本发明属于分析检测领域,涉及一种基于钨酸铋的均相阳极光电化学检测黄曲霉毒素的方法。采用钨酸铋修饰的ITO电极作为光电阳极,信号分子(亚甲基蓝或硫磺素T)能够作为电子供体增大阳极光电流。在均相溶液中,结合黄曲霉毒素与适配体的识别反应所介导的滚环扩增(RCA)反应与信号分子特异性嵌入G-四链体的特性,构建了信号“增强型”检测平台。该发明对黄曲霉毒素的检测灵敏度很高,线性范围为0.01-10000pg/mL,检测限低至2.6fg/mL。与传统方法相比,本发明所提出的方法成本低,操作简便(无需标记及生物分子的电极固定),试剂用量小,实用性强,有望成为检测黄曲霉毒素的高效方法之一。(The invention belongs to the field of analysis and detection, and relates to a homogeneous anode photoelectrochemical detection method of aflatoxin based on bismuth tungstate. An ITO electrode modified by bismuth tungstate is used as a photoelectric anode, and a signal molecule (methylene blue or thioflavine T) can be used as an electron donor to increase anode photocurrent. In homogeneous solution, a signal 'enhanced' detection platform is constructed by combining the Rolling Circle Amplification (RCA) reaction mediated by the recognition reaction of the aflatoxin and the aptamer and the characteristic that signal molecules are specifically embedded into a G-quadruplex. The detection sensitivity of the kit to aflatoxin is high, the linear range is 0.01-10000pg/mL, and the detection limit is as low as 2.6 fg/mL. Compared with the traditional method, the method provided by the invention has the advantages of low cost, simple and convenient operation (no need of labeling and electrode fixation of biomolecules), small reagent dosage and strong practicability, and is expected to become one of efficient methods for detecting aflatoxin.)
1. A homogeneous anode photoelectrochemical detection method of aflatoxin based on bismuth tungstate is characterized in that:
a、Bi2WO6the preparation of (1): mixing 20mL of bismuth nitrate solution and 20mL of tungstate solution, stirring for 20 minutes, transferring into an autoclave, and heating for a certain time at a certain temperature; naturally cooling to room temperature, alternately washing the product with absolute ethyl alcohol and distilled water, and drying in a vacuum drying oven overnight to obtain a powdery product;
b、Bi2WO6preparing a modified ITO electrode: weighing the prepared Bi2WO6Preparing 1mg/mL suspension of the powder; 30 μ L of Bi was applied by dropping on the surface of the ITO electrode which had been previously cleaned2WO6Drying the suspension at 40 ℃ for 2 hours for later use;
c. biological recognition and signal amplification reaction: mixing the aptamer probe with aflatoxin solutions with different concentrations in Tris-HCl buffer with pH of 7.5, and incubating with shaking at 37 ℃ for 1 hour; add padlock probe and contain 10mM MgCl2At a pH of 7.5, for 1 hour at 37 ℃; then, T4 DNA ligase was added and 10mM MgCl was added20.5mM MATP, 7.540mM Tris-HCl buffer, incubated at 22 ℃ for 1 hour; followed by the addition of phi29 DNA polymerase and 10mM MgCl2、50mM KCl、5mM (NH4)2SO4At a pH of 7.540mM, Tris-HCl buffer solution and deoxyribonucleoside triphosphates, and carrying out an amplification reaction at 30 ℃ for 2 hours; finally 5 is multiplied by 10-6Adding a signal molecule of M into the solution, and reacting for 30min at 37 ℃;
d. measurement of photocurrent: by current-time technique, adding Bi2WO6And (c) placing the modified ITO electrode serving as a working electrode in the reaction solution in the step (c), and measuring the photocurrent at a voltage of 0.2V relative to an Ag/AgCl reference electrode.
2. A method as claimed in claim 1A homogeneous phase photoelectrochemical detection method of aflatoxin based on bismuth tungstate is characterized in that Bi is prepared2WO6The mass concentration ratio of the bismuth nitrate solution to the tungstate solution is 2:1, the tungstate is one of ammonium tungstate or sodium tungstate, the reaction temperature is 160-180 ℃, and the reaction time is 10-16 hours.
3. The homogeneous photoelectrochemical detection method of aflatoxins based on bismuth tungstate as claimed in claim 1, wherein the aptamer base sequence adopted in the biological recognition and signal amplification reaction is 5'-TGC ACG TGT TGT CTCTCT GTG TCT CGT GCT TTT TT-3'; the padlock probe base sequence is 5'-AAC AGA GAG ACA CCC ACCCAC CCA CCC ACA CAC AGA GCA CG-3'.
4. The homogeneous photoelectrochemical detection method of aflatoxins based on bismuth tungstate according to claim 1, wherein the signal molecule used in the biological recognition and signal amplification reaction is one of methylene blue or thioflavine T.
Technical Field
The invention relates to an analysis and detection technology, and belongs to the technical field of analysis and detection.
Background
The state of the art and the problems that exist are described closest to the present invention.
Aflatoxins are produced primarily by aspergillus flavus and aspergillus parasiticus [ reverbei M, riceli a, ZjalicS, Fabbri a, Fanelli c.appl.microbiol.biotechnol.2010,87: 899-. The international agency for research on cancer (IARC) has classified aflatoxins as a
Photoelectrochemical (PEC) analysis is a novel analytical method established on the basis of direct or indirect interaction between a photoactive material under photoexcitation and an analyte. By virtue of its low background, high sensitivity, and inexpensive instrumentation, PEC analysis has evolved rapidly in recent years and is widely used in many fields.
The performance of PEC analysis is strongly related to the signal molecules present in the photoelectrode and solution. The bismuth-based compound is composed of (Bi)2O2)2+The layered structure formed by the host layer and other guest ion layers is beneficial to the migration of photo-generated electrons between layers, effectively reduces the recombination of the photo-generated electrons and holes and further improves the utilization efficiency of photo-generated carriers. Therefore, the present invention employs Bi2WO6As photoelectric activityThe material is used for preparing a photoelectrode, and the exploration finds that a signal molecule serving as an electron donor can obviously promote a photocurrent signal thereof through the action of a photogenerated hole. The invention constructs a novel PEC (molecular enhanced Raman scattering) aflatoxin detection method by combining a target aflatoxin aptamer-mediated Rolling Circle Amplification (RCA) reaction and the characteristic that a signal molecule is specifically embedded into a G-quadruplex. The method is convenient and rapid to use, does not need the fixation of biomolecules on the surface of an electrode and the marking of a biological probe, and has high sensitivity and good selectivity.
Disclosure of Invention
The technical problem is as follows: the technical problem to be solved by the invention is to achieve the aim.
Most of the photoelectrochemical immunosensors or aptamer sensors for aflatoxin detection reported at present need high-cost antibodies or labeled nucleic acid probes, and have the defects of high cost, complex operation steps, low sensitivity (because the recognition efficiency of surface-immobilized biomolecules is reduced), and the like. The invention aims to provide a novel anode photoelectrochemical detection method which is low in cost, convenient, rapid and sensitive so as to realize the detection of aflatoxin.
A homogeneous anode photoelectrochemical method for detecting aflatoxin is provided based on the current increasing effect of signal molecules on a semiconductor material bismuth tungstate and the embedding effect of the signal molecules on nucleic acid molecules. The rolling circle amplification reaction mediated by the recognition reaction of the aflatoxin and the aptamer and the characteristic that the signal molecule is specifically embedded into the G-quadruplex are combined, the concentration of the signal molecule in the solution is changed, the photocurrent value is further changed, and a signal 'enhanced' detection method is constructed.
The technical scheme is as follows: the invention discloses a complete technical means and a method.
The object of the invention can be achieved by the following technical measures:
the invention provides a homogeneous anode photoelectrochemical method for detecting aflatoxin, which is realized by the following technical measures:
a、Bi2WO6the preparation of (1): mixing 20mL of bismuth nitrate solution and 20mL of tungstate solution, stirring for 20 minutes, transferring into an autoclave,heating at a certain temperature for a certain time; naturally cooling to room temperature, alternately washing the product with absolute ethyl alcohol and distilled water, and drying in a vacuum drying oven overnight to obtain a powdery product;
b、Bi2WO6preparing a modified ITO electrode: weighing the prepared Bi2WO6Preparing 1mg/mL suspension of the powder; 30 μ L of Bi was applied by dropping on the surface of the ITO electrode which had been previously cleaned2WO6Drying the suspension at 40 ℃ for 2 hours for later use;
c. biological recognition and signal amplification reaction: mixing the aptamer probe with aflatoxin solutions with different concentrations in Tris-HCl buffer with pH of 7.5, and incubating with shaking at 37 ℃ for 1 hour; add padlock probe and contain 10mM MgCl2At a pH of 7.5, for 1 hour at 37 ℃; then, T4 DNA ligase was added and 10mM MgCl was added20.5mM MATP, 7.540mM Tris-HCl buffer, incubated at 22 ℃ for 1 hour; followed by the addition of phi29 DNA polymerase and 10mM MgCl2、50mM KCl、5.0mM(NH4)2SO47.540mM Tris-HCl buffer solution and deoxyribonucleoside triphosphate, and carrying out an amplification reaction at 30 ℃ for 2 hours; finally 5 is multiplied by 10-6Adding a signal molecule of M into the solution, and reacting for 30min at 37 ℃;
d. measurement of photocurrent: by current-time technique, adding Bi2WO6And (c) placing the modified ITO electrode serving as a working electrode in the reaction solution in the step (c), and measuring the photocurrent at a voltage of 0.2V relative to an Ag/AgCl reference electrode.
The object of the invention can also be achieved by the following technical measures:
preparation of Bi2WO6The mass concentration ratio of the bismuth nitrate solution to the tungstate solution is 2:1, the tungstate is ammonium tungstate or sodium tungstate, the reaction temperature in the autoclave is 160-180 ℃, and the reaction time is 10-16 hours; the aptamer base sequence adopted in the biological recognition and signal amplification reaction is 5'-TGC ACG TGT TGT CTC TCT GTG TCT CGTGCTTTT TT-3'; the base sequence of the padlock probe is 5'-AAC AGA GAG ACA CCC ACC CAC CCA CCCACACACAGAGCACG-3'; the signal molecule used is one of Methylene Blue (MB) or thioflavine T (ThT). When no target aflatoxin exists, the aptamer can be combined with the padlock probe to cause constant-temperature rolling circle amplification, so that the nucleic acid is exponentially increased in a short time, and the high-efficiency amplification of signals is realized. The amplification reaction finally produces a single-stranded DNA rich in G bases, at K+Under the action, the formed G-quadruplex can effectively embed free signal molecules in the solution, so that a photocurrent signal different from the free signal molecules is generated, and the detection purpose is achieved.
Has the advantages that: the advantages brought by the invention are the indexes achieved.
The homogeneous anode photoelectrochemical biosensor for detecting aflatoxin does not need to be marked by a probe on one hand, and on the other hand, biological reaction is carried out in a homogeneous solution (complex processes of fixing biomolecules on the surface of an electrode and repeatedly washing the electrode are not needed), and moreover, efficient signal amplification is obtained by utilizing rolling circle amplification reaction, so that the homogeneous anode photoelectrochemical biosensor has the advantages of low cost, simplicity and convenience in operation, strong practicability, high sensitivity and the like.
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Description of the meanings indicated in the figures
Detailed Description
Illustrating according to what is contained in the claims
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