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As the most widely used pesticides in modern agriculture, organophosphorus pesticides (OPPs) play an important role
in protecting crops from pests and increasing crop yields.
Unfortunately, due to its misuse worldwide, it causes serious pollution
of water, soil and even agricultural products.
The entry of OPPs into the human body can cause irreversible inhibition of acetylcholinesterase activity, which in turn leads to disorders of the central nervous system and even threatens life
.
Therefore, it is important
to develop a sensitive, fast, reliable and economical method for detecting OPPs.
In contrast, electrochemical sensing detection technology has the advantages of simple operation, rapid detection, high sensitivity and low cost, which is very suitable for simple, fast, sensitive and reliable detection
of OPPs residue.
Yunxia Tian, Yuangen Wu, Han Tao* et al.
of the Key Laboratory of Fermentation Engineering and Biopharmaceuticals of Guizhou Province, School of Brewing and Food Engineering, Guizhou University, constructed plant esterase-based electrochemical biosensors derived from white kidney beans, sensitized the sensing signal by using the excellent physicochemical properties of 1T-WS2@AuNPs nanocomposites, and evaluated
their pesticide detection performance and practical application performance 。 The principle of chlorthion detection is shown in Figure 1, kidney bean esterase (KbE) catalyzes the hydrolysis of substrate 1-naphthyl acetate (1-NA) to produce electrochemical active substance 1-naphthol, when the pesticide thion is present in the system, KbE activity will be inhibited, resulting in a decrease in the production of 1-naphtherol, and the corresponding electrochemical response signal will also decrease, so as to achieve the quantitative detection
of thion killing.
1.
Characterization and analysis of nanomaterials
.
On the one hand, these voids can increase the specific surface area of 1T-WS2 and provide more sites for enzyme attachment.
On the other hand, it also provides a sufficient reaction environment for enzymatic reactions to promote the progress of the reaction
.
Figure 2B shows that AuNPs are homogeneous spherical particles with an average particle size of 13.
5 nm and relatively uniform attachment to the sheet surface
of 1T-WS2.
It can be seen from Figure 2C that 1T-WS2 nanosheets contain tungsten, sulfur, nitrogen and other elements
.
From Figure 2D, it can be seen that compared to 2H-WS2 (software fitting), the peak corresponding to 1T-WS2 is shifted by about 0.
5 eV
to the low binding energy.
Similarly, the high-resolution spectrum of sulfur 2p (Figure 2E) showed a similar trend, and the change of peak binding energy was consistent with the relevant literature reports, indicating that the 1T-WS2 nanosheet prepared in this experiment was dominated by 1T-WS2
nanosheets.
To further determine the presence of the 1T phase, the structure of the 1T-WS2 nanosheet was identified
by Raman spectroscopy.
As shown in Figure 2F, there are two distinct characteristic peaks at 347 cm-1 and 411 cm-1, corresponding to the two molecular vibration modes
in WS2, intraplane (E12g) and interlaminar (A1g).
In addition, several new peaks appeared in the low-frequency region, which were consistently reported in the literature, indicating that 1T-WS2 was successfully prepared, which also corresponds to the characterization results of XPS
.
2.
Analysis of electrochemical characteristics of different modified electrodes
As shown in Figure 3A, a pair of redox peaks
appears in all four electrodes.
Compared with the bare electrode, the oxidation and reduction peak currents on the 1T-WS2/GCE modified electrode are significantly increased.
At the same time, the peak potential difference decreased from 173 mV to 138 mV of the bare electrode, which is attributed to the high conductivity of
1T-WS2.
After compounding with AuNPs, the oxidation and reduction current values of Fe2+/Fe3+ pairs further increased and the peak potential difference decreased to 103 mV, indicating that the 1T-WS2@AuNPs modified electrode had better electrochemical performance, which was related to the good conductivity of AuNPs.
Therefore, the introduction of AuNPs into 1T-WS2 can further improve the conductivity of the electrode, thereby accelerating the transfer
of electrons.
Since KbE and CS are non-conductive biomolecules that hinder the transport of electrons, a decrease in redox current and an increase
in peak potential difference on the CS/KbE/1TWS2@AuNPs/GCE modified electrode were observed.
Further study the electrochemical characteristics of different modified electrodes, as shown in Figure 3B, each curve is composed of a semicircle in the high-frequency region and a straight line in the low-frequency region, where the size of the semicircle diameter in the high-frequency region reflects the charge
transfer resistance of the electrode.
The 1T-WS2@AuNPs/GCE modified electrode has the smallest semicircle diameter, that is, the charge transfer resistance is the smallest, indicating that 1T-WS2@AuNPs has excellent electron transfer ability
.
With the further modification and fixation of KbE and CS, the diameter of the semicircle increased significantly, and the electron transfer ability of CS/KbE/1T-WS2@AuNPs/GCE was the worst
.
The above results are consistent with the CV test results and indicate that the CS/KbE/1T-WS2@AuNPs/GCE biosensor has been successfully constructed
.
3.
Analysis of catalytic performance of different modified electrodes
It can be seen from Figure 4 that in the phosphate buffer without 1-NA, the SWV curve of the CS/KbE/1T-WS2@AuNPs/GCE modified electrode is smooth and the peak-free current response is generated.
After the addition of 1-NA, a distinct oxidation peak appears, indicating that this peak is produced by electrochemical oxidation of 1-naphthol generated by KbE hydrolysis of 1-NA on the electrode
.
The above results show that KbE is successfully immobilized on the electrode and maintains its biological activity
.
In addition, compared with CS/KbE/GCE electrodes, the oxidation peak current generated on CS/KbE/1T-WS2/GCE, CS/KbE/AuNPs/GCE, CS/KbE/1T-WS2@AuNPs/GCE modified electrodes increases and decreases
peak potential.
4.
Preparation and optimization of CS/KbE/1T-WS2@AuNPs/GCE modified electrodes
Effect of pH of the base solution
at pH 7.
0.
Therefore, pH 7.
0 is the optimal pH, and phosphate buffer at pH 7.
0 is used for subsequent experiments
.
Effect of 1T-WS2 to AuNPs volume ratio and 1T-WS2@AuNPs load volume
As shown in Figure 5B, the optimal volume ratio of 1T-WS2 to AuNPs is 1:1.
An appropriate amount of nanomaterials modified on the electrode can increase the specific surface area of the electrode and promote electron transfer
between the electrode and the electroactive material.
As shown in Figure 5C, when the load volume of 1T-WS2@AuNPs increases from 6 μL to 12 μL, the peak current increases with the increase of the load volume and reaches a maximum at 12 μL.
As the load volume increases further, the peak current value gradually decreases, possibly because an excessively thick membrane hinders electron transport
.
Therefore, 12 μL was chosen as the optimal loading volume
for the 1T-WS2@AuNPs nanocomposite.
Effect of KbE enzyme activity loaded
As shown in Figure 5D, as the KbE enzyme activity of the load increases, the response current value of the sensor becomes larger, reaching a maximum value at 0.13 U.
The KbE enzyme activity that continues to increase the load decreases slightly, because KbE is a protease and has poor conductivity, and excess enzymes hinder the transfer
of electrons on the sensor surface.
Therefore, 0.
13 U is the optimal enzyme activity
for loading.
Effect of pesticide suppression time
It can be seen from Figure 5E that with the extension of inhibition time, the more enzyme activity decreases, resulting in less 1-naphthol generated, and the inhibition rate increases.When the inhibition time exceeded 15 min, the degree of enzyme inhibition also tended to be stable
due to the saturation of the enzyme active site and the pesticide.
Therefore, 15 min is used as the optimal inhibition time
.
5.
Standard curve of CS/KbE/1T-WS2@AuNPs/GCE sensor to kill thion thiopon
.
It can be seen from Figure 6B that in the range of 0.
1~2 000 μg/L, the inhibition rate has a good linear relationship with the mass concentration (lgC) of thion killing, and the linear standard equation is Y=14.
82lgC+24.
18, the correlation coefficient (R2) is 0.
992, and the detection limit is 0.
04 μg/L (signal-to-noise ratio is 3).
。 Comparing the CS/KbE/1T-WS2@AuNPs/GCE sensor with other reported detection methods (Table 1), the results show that the sensor constructed in this study has a wide linear interval and detection limit.
In addition, it also has the advantages
of simple preparation and low cost.
It shows that it is feasible to replace cholinesterase derived from animals for efficient detection of pesticides by replacing plant esterases, and has the value
of further development and application.
6.
Analysis of repeatability, stability and anti-interference ability of CS/KbE/1T-WS2@AuNPs/GCE sensor
96% and 64.
49%, respectively, which may be that paraquat has a somewhat inhibitory effect on KbE as a highly toxic pesticide, while carbendazim is a carbamate pesticide and also has an inhibitory effect on KbE, which is consistent with
those reported in the literature.
The results show that the CS/KbE/1T-WS2@AuNPs/GCE sensor has good anti-interference ability
.
7.
CS/KbE/1T-WS2@AuNPs/GCE sensor is used for analysis in actual samples
16%~109.
60%, and the relative standard deviation is less than 5%, indicating that the biosensor has good practicability and is expected to be applied to the detection
of OPPs in actual samples.
Conclusion
A 1T-WS2@AuNPs nanocomposite with good electrocatalytic activity was prepared, and a novel electrochemical biosensor was constructed by replacing acetylcholinesterase with KbE, which was used for the simple and efficient detectionof thion killing.
The results of SEM and TEM characterization showed that 1T-WS2@AuNPs nanocomposites had a large specific surface area, which could provide a good microenvironment for enzyme loading, while electrochemical characterization showed that 1T-WS2@AuNPs had good conductivity and synergistic electrocatalytic effect, which could effectively promote electron transport and improve the response sensitivity
of sensors.
Compared with traditional analysis methods, the electrochemical sensor has low cost, simple preparation and high sensitivity, which has potential application value in the detection of OPPs, and provides a certain theoretical and technical basis
for the application of plant esterases in the detection of pesticide residues.
01 Correspondence author profile
Tao Han, Associate Professor, Master Supervisor
, School of Brewing and Food Engineering, Guizhou University.
From 1994 to 1998, he studied at the College of Chemistry and Chemical Engineering of Hunan University and obtained a bachelor's degree.
From 2001 to 2004, he studied at the State Key Laboratory of Biosensing and Metrology of Hunan University and obtained a master's degree.
From 2006 to 2011, he studied at the College of Agriculture, Guizhou University and obtained a Ph.
D.
degree.
He is engaged in teaching and scientific research in Guizhou University, and is currently a master tutor of the School of Brewing and Food Engineering and a researcher of the Guizhou Provincial Key Laboratory of Fermentation Engineering and Biopharmacy
.
His main research direction is new technology
for food quality and safety testing.
At present, his research interests mainly focus on the use of chemical and biological sensing technology, using natural enzymes, artificial enzymes, aptamers, antigens and antibodies as signal recognition molecules, combined with nanotechnology, to screen and establish new laboratory testing or on-site real-time detection methods
for conventional or hazardous ingredients in food.
He has presided over and participated in many projects such as sub-projects
of the National Key R&D Program, National Natural Science Foundation of China, 863 Program Topics, Guizhou Provincial Science and Technology Project, and Guizhou Provincial Science and Technology Department Agricultural Research Project 。 At present, he has published more than 60 papers in domestic and foreign journals (23 papers included in SCI), including Microchimica Acta, Journal of Colloid and Interface Science, Journal of Agricultural and Food Chemistry, Agriculture-Basel, Journal of photochemistry and photobiology A: Chemistry, Analytical Methods, RSC advances, New Journal of Chemistry and other English journals
.
02 First author profile
Tian Yunxia, female, born
in March 1996.
From 2014 to 2018, he studied at the College of Food Science and Engineering, Qingdao Agricultural University and obtained a bachelor's degree; From 2018 to 2021, he studied at the School of Brewing and Food Engineering of Guizhou University and obtained a master's degree
.
His research direction is new technology
for food quality and safety testing.
He has successively won the first-class scholarship of Guizhou University, the third-class scholarship of master's degree, and the outstanding Communist Youth League member of Guizhou University
.
This paper "Construction of white kidney bean esterase electrochemical biosensor based on 1T-WS2@AuNPs composite and its application of thion killing" is from Food Science, Vol.
43, No.
18, 2022, pp.
324-331, authors: Tian Yunxia, Wu Yuangen, Wang Xiao, Ji Chun, Shi Qili, Tao Han
.
DOI:10.
7506/spkx1002-6630-20211005-033
。 Click to view information about
the article.
