CN-121978322-A - Method for detecting mycotoxin by using carbon nano tube field effect transistor biosensor based on nucleic acid conformational change

CN121978322ACN 121978322 ACN121978322 ACN 121978322ACN-121978322-A

Abstract

The invention provides a method for detecting mycotoxin by using a carbon nano tube field effect transistor biosensor based on nucleic acid conformational change, which adopts a CNT-FET biosensor provided with an ion shielding gold film layer above a channel, anchors a modified nucleic acid aptamer on the surface of the gold film layer through gold sulfide bonds to construct a sensing interface, wherein the modified nucleic acid aptamer consists of an original nucleic acid aptamer sequence of target mycotoxin and an oligo-thymidine nucleotide fragment directionally inserted into the 5' end of the original nucleic acid aptamer sequence, and when in detection, mycotoxin in a sample to be detected is specifically combined with the modified nucleic acid aptamer to trigger the secondary conformational change of the mycotoxin approaching the gold film layer, the CNT-FET biosensor converts an electric field effect caused by conformational displacement into a signal of change of channel carrier concentration, and quantitative detection is realized according to the linear relation between the signal and the concentration of mycotoxin. According to the method, the nucleic acid aptamer is directionally modified, so that the electrical response of the low-charge/neutral mycotoxin combined with the sensor is enhanced, and the detection sensitivity is greatly improved.

Inventors

LIU XIAOFENG
YU HONGXIANG
TONG JIAJUN
LIU YIWEI
Cao Juexian

Assignees

湘潭大学

Dates

Publication Date: 20260505
Application Date: 20260209

Claims (10)

1. A method for detecting mycotoxins by a carbon nanotube field effect transistor biosensor based on conformational changes of nucleic acids, comprising the steps of: A carbon nano tube field effect transistor (CNT-FET) biosensor is adopted, a gold film layer with shielding effect is arranged above a channel of the carbon nano tube field effect transistor biosensor, and a modified nucleic acid aptamer is anchored on the surface of the gold film layer through Jin Liujian to construct a sensing interface, wherein the modified nucleic acid aptamer comprises an original nucleic acid aptamer sequence of target mycotoxin and an oligo-thymidine nucleotide fragment directionally inserted into the 5' -end of the original nucleic acid aptamer sequence, and the oligo-thymidine nucleotide fragment enables the modified nucleic acid aptamer to form a preset space conformation in a non-target state; Dropwise adding a buffer solution to the sensing interface, eliminating interference of ions in the buffer solution on sensing signals by utilizing the ion shielding effect of a gold film layer, and simultaneously measuring an initial electrical transfer characteristic curve by the CNT-FET biosensor to serve as a detection baseline; Dripping a sample to be detected to the sensing interface to enable mycotoxin in the sample to be detected to be specifically combined with the modified nucleic acid aptamer, wherein the combination triggers the modified nucleic acid aptamer to undergo secondary conformational change approaching to the surface of the gold film layer; and measuring transfer characteristic curve change after specific binding by the CNT-FET biosensor, converting an electric field effect caused by conformational displacement of the modified aptamer into a change signal of carrier concentration in a channel of the CNT-FET biosensor, and quantitatively detecting mycotoxin in a sample to be detected according to a linear proportional relation between the change signal and the mycotoxin concentration.
2. The method for detecting mycotoxin by using a carbon nanotube field effect transistor biosensor based on a conformational change of nucleic acid according to claim 1, wherein the length of the oligo-thymidine nucleotide fragment is 5 nt-20 nt.
3. The method for detecting mycotoxins of a carbon nanotube field effect transistor biosensor based on conformational change of nucleic acids according to claim 1, wherein the predetermined spatial conformation is such that the modified nucleic acid aptamer appears away from or parallel to the gold membrane surface in a non-target state.
4. The method for detecting mycotoxins based on a carbon nanotube field effect transistor biosensor of claim 1, wherein the free ends of the oligothymidine fragments form gold-sulfur bonds with the gold membrane layer through sulfhydryl groups.
5. The method for detecting mycotoxins according to claim 1, wherein the 3' end of the modified aptamer probe is free, and the modified aptamer is in an extended state away from the surface of the gold membrane by flexible support of the oligonucleotide fragment when the target mycotoxin is not bound.
6. The method for detecting mycotoxin by using a carbon nanotube field effect transistor biosensor based on a conformational change of nucleic acid according to claim 1, wherein the thickness of the gold film layer is 15 nm-30 nm.
7. The method for detecting mycotoxins by a carbon nanotube field effect transistor biosensor based on conformational change of nucleic acid according to claim 1, wherein the CNT-FET biosensor comprises, in order from bottom to top: The substrate layer is a silicon wafer with a carbon nano tube layer pre-deposited; the source electrode and the drain electrode are symmetrically arranged above the carbon nano tube layer, and the carbon nano tube between the source electrode and the drain electrode forms a channel; The gate dielectric layer covers the channel, the source electrode and the drain electrode; the gold film layer covers the gate dielectric layer above the channel; the passivation layer covers the gate dielectric layer above the source electrode and the drain electrode and defines a sensing window exposing the gold film layer; the biosensing layer is composed of modified nucleic acid aptamer, and gold sulfide bonds are formed by the modified nucleic acid aptamer and the gold film layer exposed by the sensing window, so that the anchoring of the modified nucleic acid aptamer is realized.
8. The method for detecting mycotoxins in a carbon nanotube field effect transistor biosensor based on conformational change of nucleic acids according to any of claims 1-6, wherein the mycotoxins comprise gliotoxins.
9. The method for detecting mycotoxins of claim 8, wherein the method comprises the steps of: the modified nucleic acid aptamer is characterized in that the sequence of the modified nucleic acid aptamer is HS-TTTTTTTTCATGCGTCAGCATGGAGGGGACCT.
10. The method for detecting mycotoxins of claim 1, wherein the method has a lower limit of detection of gliotoxin of 3.9 fg/mL.

Description

Method for detecting mycotoxin by using carbon nano tube field effect transistor biosensor based on nucleic acid conformational change Technical Field The invention relates to the technical field of biosensors, in particular to a method for detecting mycotoxin by using a carbon nano tube field effect transistor biosensor based on nucleic acid conformational change. Background The biotoxin is a natural chemical substance with toxicity or pathogenicity produced by microorganisms, plants or animals, is mostly secondary metabolites of organisms, has the characteristics of strong toxicity, trace existence and wide distribution range, and is different from artificial synthetic chemical toxins, the biotoxin is derived from natural biological processes, is easy to transfer through food chains or pollute food and environment under specific conditions, and brings potential threat to food safety and ecological environment. Among them, mycotoxins small molecules with low charge or neutrality are the types with prominent harm in biotoxin, the trace detection difficulty of the molecules is high due to the charge characteristics of the molecules, and the gliotoxin is a typical representative of the mycotoxins as a high-toxicity metabolite produced by aspergillus fumigatus, and can invade human bodies and cause damage through various ways such as food contact, environmental exposure and the like. Therefore, a high-sensitivity detection method of low-charge-quantity or electrically neutral mycotoxins micromolecules is established, the efficient screening of the toxins in food detection and the early identification and intervention of trace target toxins in the environment are realized, and the method has important practical significance for preventing and controlling the toxic action of the mycotoxins and guaranteeing the food safety and the human health. The detection method for the gliotoxin mainly comprises a high-efficiency liquid phase method, a colorimetric method, an ELISA method and the like, but the existing methods have obvious technical defects that the detection limit of the high-efficiency liquid phase method only reaches micrograms per milliliter level, the pretreatment of a sample is complex, the detection flow is highly dependent on professional operators and precise matched instruments, the detection limit of the biological probe-based colorimetric method can be increased to nanograms per milliliter level, but the defects of narrow detection linear range and long detection time exist, and the ELISA method also has the problems of high antibody preparation cost and easy interference of detection specificity. Meanwhile, the method is difficult to meet the timeliness requirements of practical application scenes such as toxin enrichment detection in a field environment and emergency rapid detection in the food safety field, and popularization of basic detection scenes cannot be realized due to the problems of high operation threshold, severe detection conditions and the like. In view of the above technical drawbacks, field Effect Transistor (FET) based biosensors have been developed in the field of biological detection due to unique electrical detection mechanisms, and carbon nanotube field effect transistor (CNT-FET) biosensors have been developed particularly. The CNT-FET is a field effect transistor which takes a carbon nanotube as a channel material, has higher response speed, excellent electrical performance and higher detection sensitivity, has the detection principle of realizing the detection of a biomarker based on the electrical performance change of a semiconductor material, can carry out trace detection on a target molecule with extremely low concentration, has short response time, is generally in the level of seconds to minutes, is far superior to the traditional immunoassay or chromatographic analysis method, meets the instant detection requirement, has good integration capability, can be combined with microfluidic technology, wearable equipment and the like, realizes the in-situ real-time detection of the target molecule, and has good application prospect in the detection of food and environmental biotoxin. The biological probe is used as a core recognition element of the biosensor, is a key for realizing accurate recognition of detection substances, and common biological probes comprise an antigen-antibody system, enzyme, molecularly imprinted polymer, nucleic acid aptamer and the like. The type selection and the structural design of the biological probe directly determine the detection sensitivity, the signal to noise ratio and other core performances of the biological sensor, and the nucleic acid aptamer has the advantages of high binding sensitivity, high response speed, adaptation to the recognition of small molecular targets and the like aiming at the recognition application of the small molecular targets such as biotoxins, and becomes a preferable recognition element in the detection field