CN111693595B - Method for evaluating pesticide toxicity based on electrochemical cell sensor - Google Patents
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Abstract
The invention relates to the technical field of biosensing, in particular to a method for detecting pesticide toxicity by using an electrochemical cell sensor; firstly, immersing an AuNDs/ITO electrode in a PLL-Arg solution for incubation, and then airing to obtain an Arg-PLL-AuNDs/ITO electrode; then inoculating HepG2 cells to obtain cell electrodes; then adding thiram solutions with different concentrations; meanwhile, a blank group is set, namely cells are not inoculated after electrode modification; the control group is cell electrodes which are not stimulated by poison; after incubation, the peak currents of three experiments are measured by signals of differential pulse voltammetry, cytotoxicity is calculated according to a formula, and the cytotoxicity is verified by a conventional MTT method. The electrochemical cell sensor prepared by the invention has good conductivity and stability and good electron transfer capacity, simultaneously improves the specific surface area of the electrode, and can realize quick and sensitive toxicity evaluation on pesticides in food.
Description
Technical Field
The invention relates to the technical field of biosensing, in particular to a method for evaluating pesticide toxicity based on an electrochemical cell sensor.
Background
The electrochemical cell sensing technology combines the high sensitivity and the authenticity of cell sensing and the rapidness and the high efficiency of the electrochemical sensing technology, and can evaluate the safety of potential harmful substances and the toxicity thereof in vitro from the cellular level. The electrochemical cell sensor for evaluating toxicity is low in price, high in sensitivity, strong in selectivity, high in response speed and small in size, can be used for long-term monitoring or real-time measurement, can generate a reproducible result, and is an ideal method for detecting harmful substances in food.
At present, related patents construct electrochemical sensors for evaluating cytotoxicity, but the problems exist that the biocompatibility of the surface of an electrode material is poor, cells cannot grow normally on the surface of the electrode material, links such as cell culture, toxicity stimulation, cell state detection and the like are separated, and the real-time and synchronous detection of the cytotoxicity effect of a stimulant cannot be realized. Therefore, the electrochemical sensor which has good biocompatibility and enables cells to grow normally on the surface of the electrode is constructed, and the direct detection of the cytotoxicity of the stimulant is particularly important.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention is directed to solving one of the problems set forth above; the invention aims to construct an electrochemical cell sensor which can synchronously realize cytotoxic stimulation and cell state detection and has high sensitivity.
The invention is realized by the following scheme: preparing an arginine functionalized polylysine solution, preparing dendritic gold nanoparticles with a spike structure on the surface of an ITO conductive glass electrode, modifying the nanoparticles onto the electrode, immersing the electrode into the arginine functionalized polylysine solution, placing the dried electrode in a culture dish, inoculating and culturing cells, changing the shape of the cells after being stimulated by toxic substances or dropping off from the electrode to change an electric signal, and determining by a Differential Pulse Voltammetry (DPV).
The invention provides a method for detecting pesticide toxicity by using an electrochemical cell sensor, which comprises the following steps:
(1) Preparing a dendritic gold nanoparticle/arginine functionalized polylysine modified electrode:
modifying arginine (Arg) on Polylysine (PLL) to obtain PLL-Arg solution; modifying dendritic gold nanoparticles (AuNDs) by an ITO conductive glass electrode through an electrodeposition method to obtain an AuNDs/ITO electrode; immersing the AuNDs/ITO electrode in a PLL-Arg solution for incubation for a period of time, then removing redundant liquid, and drying in the air to obtain an Arg-PLL-AuNDs/ITO electrode, namely the electrochemical cell sensor;
(2) Culturing and fixing cells;
placing the obtained Arg-PLL-AuNDs/ITO electrode in a container, inoculating HepG2 cells, adding a culture medium, and culturing at a certain temperature and in a gas environment for later use;
(3) Detecting the toxicity of the standard substance;
taking the pesticide thiram as an example, weighing thiram standard substances with different qualities, preparing thiram solutions with different concentrations, and respectively adding the thiram solutions into the culture containers containing the electrodes after the culture in the step (1), wherein the thiram solutions and the culture containers for storing the electrodes are in one-to-one correspondence, namely one concentration corresponds to one culture container; continuously placing the mixture in a cell culture box for incubation; after incubation, taking out the cell electrode, washing the cell electrode by PBS, and performing signal measurement of differential pulse voltammetry to obtain differential pulse voltammetry curves of the cell electrode after thiram treatment with different concentrations, wherein the peak current of the cell electrode after thiram treatment is recorded as I toxicant (ii) a Setting a blank group and a control group at the same time; blank set, i.e. peak current without cell inoculation after electrode modification, is marked as I electrode (ii) a The control group is the peak current of the cell electrode without poison stimulation and is marked as I cell (ii) a The cytotoxicity of thiram at different concentrations was further calculated according to the formula: the cytotoxicity calculation formula is as follows:
wherein, I electrode Is the peak current, I, of the non-inoculated cells after electrode modification cell Is the peak current of the cell electrode without poison stimulation, I toxicant Is the peak current of the cell electrode after thiram treatment;
(4) Detection of the actual sample:
firstly, preparing a sample solution, filtering and sterilizing the sample solution through a 0.22-micron filter membrane to be used as an actual sample, respectively adding thiram standard solutions with different concentrations, recording peak currents of electrodes after thiram treatment with different concentrations, and substituting the peak currents into the formula obtained in the step (3) to realize cytotoxicity detection in the sample.
Further, the specific preparation process of the PLL-Arg solution in the step (1) is as follows: PLL (25 mg) was mixed with amino-protected arginine (Fmoc-Arg (Pbf) -OH,0.12 mmol) and N, N-diisopropylethylamine (DIPEA, 0.40 mmol), which was dissolved in anhydrous N, N-dimethylformamide (DMF, 25 mL); stirring the obtained mixture under nitrogen for 10-15min, adding O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.12 mmol) and 1-hydroxybenzotriazole (HOBt, 0.12 mmol), and reacting at room temperature for 48h; thereafter, the solvent was removed by distillation under reduced pressure; the residue was dissolved in chloroform/methanol (v/v = 5:1) and purified by column chromatography (silica gel, chloroform/methanol = 10; to deprotect PLL-Fmoc-Arg (Pbf), 90mg of purified PLL-Fmoc-Arg (Pbf) was dissolved in 20mL mixed piperidine/DMF (v/v = 2:8) solution and stirred at room temperature for 1h, then the solvent was removed under reduced pressure and diethyl ether was added to wash the residue; dry in vacuo for 6h, then dissolve the residue in 20mL of a mixed trifluoroacetic acid (TFA)/water (v/v = 95) solution and continue stirring at room temperature for 2h; the resulting mixture was concentrated and dropped into cold ether, and the precipitate was collected by centrifugation and then dissolved in 20mL of sterilized deionized water to adjust the pH to 7.4 to 8.0 to obtain a PLL-Arg solution.
Further, the step (1) of depositing AuNDs on the ITO conductive glass electrode comprises the following specific steps: the three-electrode system is composed of an ITO conductive glass electrode as a working electrode, a platinum wire auxiliary electrode as a counter electrode and a silver/silver chloride (3 mol/L potassium chloride) electrode as a reference electrode; before AuNDs is deposited, cutting an ITO conductive glass electrode into the size of 2cm multiplied by 1cm multiplied by 1.1mm, sequentially soaking in acetone and ethanol for ultrasonic cleaning for 15min, and then washing with deionized water for several times to remove the acetone or the ethanol; soaking the three-electrode system in an electrolyte solution containing 10mmol/L chloroauric acid and 0.25mol/L sulfuric acid, applying a voltage of-0.5 to-2V to the working electrode, and depositing for 50-450s.
Further, the electrode in the step (1) is soaked in the PLL-Arg solution for incubation for 5-15min.
Further, in the step (2), the final concentration of the HepG2 cell in the culture medium is 10 5 cells/mL。
Further, in the step (2), the culture medium is a DMEM medium containing fetal bovine serum with a volume concentration of 8 to 13%.
Further, in the step (2), the incubation temperature and gas atmosphere are 37 ℃ and 5% by weight of CO, respectively 2 (ii) a The culture time is 20-30h.
Further, in step (3), the concentration of thiram is 0-125 μmol/L, and 3 parallel samples are prepared at each concentration.
Further, in the step (3), the incubation time of the cells is 3-24h.
Further, in the step (3), the signal measurement conditions of the differential pulse voltammetry are as follows: CHI600E electrochemical workstation with platinum wire auxiliary electrode as counter electrode and silver/silver chloride (3 mol/L potassium chloride) electrode as reference electrode, containing 1mmol/L Fe (CN) 6 3-/4- And 1.0mol/L potassium chloride, pulse period: 0.5s, scan range: 0 to 0.5V, amplitude: 0.05V.
Compared with other electrochemical cell sensors, the electrochemical cell sensor prepared by the invention has the following advantages when used for toxicity detection:
(1) The dendritic gold nanoparticle and arginine functionalized polylysine modified electrode is constructed for the first time, and the electrode has good cell compatibility and signal sensitivity.
(2) The invention constructs a high-performance modified interface, is beneficial to fixing and maintaining the activity of cells, and the cells can grow on the surface of the electrode, thereby realizing the direct detection of the cytotoxicity of the stimulus and being easy for the toxicity evaluation of the pesticide.
(3) The dendritic gold nanoparticle modified interface prepared by the method has good conductivity and stability and good electron transfer capacity, improves the specific surface area of the electrode, and provides more active sites for subsequent material modification and cell attachment, thereby amplifying signals; after the polylysine is subjected to arginine functionalization, ammonium ions are increased, so that the surface of the electrode is provided with more abundant polycations, cell surface glycoprotein (negative) is easily adsorbed, a modified interface has good biocompatibility and higher electrical activity, the electrochemical performance of the surface of the electrode is further improved, and quick and sensitive toxicity evaluation of pesticides in food can be realized.
Drawings
FIG. 1 is a flow chart of the construction of an electrochemical cell sensor.
FIG. 2 is an SEM image of AuNDs (AuNDs/ITO electrode) with modified electrode surface.
FIG. 3 is a diagram of electrochemical voltammetry characterization of a cell sensor modification process, wherein (a) is ITO, (b) is AuNDs/ITO, (c) is Arg-PLL-AuNDs/ITO, and (d) is cell/Arg-PLL-AgNDs/ITO.
FIG. 4 is a graph showing the growth of cells on the electrode in three days as measured by the MTT method.
The solid line in fig. 5 is the differential pulse voltammetry curves obtained after the cell electrode is exposed to 0, 1, 10, 25, 50, 75, 100 and 125 μmol/L thiram respectively from bottom to top, and the dotted line is the differential pulse voltammetry curve of the cell after the electrode modification.
FIG. 6 is a standard curve of the toxicity of thiram obtained by the electrochemical cell sensor designed in example 1.
Detailed Description
In order to more clearly illustrate the content of the present invention, the present invention will be further described with reference to specific examples, and it is apparent that the described examples are only a part of the examples of the present invention and should not be construed as all the examples of the present invention.
Example 1
(1) Preparation of dendritic gold nanoparticle/arginine functionalized polylysine modified electrode
Preparation of PLL-Arg solution: PLL (25 mg) was mixed with amino-protected arginine (Fmoc-Arg (Pbf) -OH,0.12 mmol) and N, N-diisopropylethylamine (DIPEA, 0.40 mmol), which was dissolved in anhydrous N, N-dimethylformamide (DMF, 25 mL); after the resulting mixture was stirred for 10min under a nitrogen atmosphere, O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.12 mmol) and 1-hydroxybenzotriazole (HOBt, 0.12 mmol) were added and reacted at room temperature for 48h. Thereafter, the solvent was removed by distillation under reduced pressure. The residue was dissolved in chloroform/methanol (v/v = 5:1) and purified by column chromatography (silica gel, chloroform/methanol = 20; to deprotect PLL-Fmoc-Arg (Pbf), 90mg of purified PLL-Fmoc-Arg (Pbf) was dissolved in 20mL mixed piperidine/DMF (v/v = 2:8) solution and stirred at room temperature for 1h. Thereafter, the solvent was removed under reduced pressure and ether was added to wash the residue, dried under vacuum for 6h, then the residue was dissolved in 20mL of a mixed trifluoroacetic acid (TFA)/water (v/v = 95) solution and stirred at room temperature for 2h, the resulting mixture was concentrated and dropped into cold ether, the precipitate was collected by centrifugation, and then it was dissolved in 20mL of sterilized deionized water to adjust its pH to 7.5 to obtain PLL-Arg solution, diluted 100-fold for use;
preparing an AuNDs/ITO electrode: the three-electrode system is composed of an ITO conductive glass electrode as a working electrode, a platinum wire auxiliary electrode as a counter electrode and a silver/silver chloride (3 mol/L potassium chloride) electrode as a reference electrode; before AuNDs are deposited, an ITO conductive glass electrode is cut into the size of 2cm multiplied by 1cm multiplied by 1.1mm, the ITO conductive glass electrode is sequentially soaked in acetone and ethanol for ultrasonic cleaning for 15min, deionized water is used for washing for several times to remove the acetone or the ethanol, a three-electrode system is soaked in an electrolyte solution containing 10mmol/L chloroauric acid and 0.25mol/L sulfuric acid, a voltage of-1.5V is applied to a working electrode, and the deposition time is 300s;
preparing an Arg-PLL-AuNDs/ITO electrode: immersing the AuNDs/ITO electrode in PLL-Arg solution for incubation for 10min, then sucking excess liquid and airing for standby application, namely the electrochemical cell sensor;
FIG. 2 is an SEM image of AuNDs modified on the surface of an electrode, the deposited gold nanoparticles present a dendritic microstructure, and the nanosheets consist of a trunk with a length of several microns and primary, secondary and tertiary branches, indicating that AuNDs are successfully modified on the electrode;
(2) The modified electrode is placed in a culture dish with the diameter of 35mm, each dish is 3mL of DMEM culture medium containing fetal bovine serum with the volume concentration of 8-13%, and the concentration of the inoculated HepG2 cells in the culture medium is 10 5 cells/mL at 37 ℃ C. And 5% CO 2 Culturing for 24 hours in an incubator;
FIG. 3 is an electrochemical characterization of the cell sensor modification process, with the peak currents of curves (a) (b) (c) increasing in order, indicating that AuNDs can increase the electron transfer performance of the electrode, and Arg-PLL can further increase the electron transfer performance of the electrode; the peak current of curve (d) is greatly reduced compared to curve (c), indicating that the cells are well immobilized on the electrodes and thus hinder electron transport;
(3) MTT method for detecting survival and growth of cells on electrode
After cells are inoculated on the surface of an electrode, the cells are digested from the surface of the electrode by pancreatin every 12 hours, the cells are diluted by a culture medium according to a certain proportion to prepare cell suspension, a 96-well plate is added, 200 mu L of each well is formed, and 6 multiple wells are arranged on the cell suspension prepared by each electrode; 5% of CO 2 Incubating at 37 ℃ until the cells adhere to the wall; adding 20 mu L of MTT solution (0.5%) into each well, and continuing culturing for 4h; terminating the culture, absorbing the culture solution in the wells, adding 150 μ L of dimethyl sulfoxide into each well, placing on a shaking table, shaking at low speed for 10min, detecting the absorbance (OD value) of each well at 490nm with an enzyme linked immunosorbent assay (ELISA), and taking the average value of 6 wells;
FIG. 4 is a graph showing the growth curve of the cells on the electrode measured by MTT method, which shows that the cells grow slowly in 0-24h, grow fastest in 24-48h, and grow slowly in 48-72h, reflecting that the cells can continuously proliferate on the electrode and can maintain better activity.
(4) Detection of Standard toxicity
Weighing thiram standard substances with different qualities, preparing thiram solutions with different concentrations, and respectively adding the thiram solutions into the culture dish for placing the electrodes, wherein the final concentration of thiram is 0 and 110, 25, 50, 75, 100 and 125 mu mol/L, and placing the mixture in a cell culture box for incubation for 12 hours. After the cell electrode was taken out and washed with PBS, differential pulse voltammetry was performed to measure the signal using CHI600E electrochemical workstation, platinum wire auxiliary electrode as counter electrode, silver/silver chloride (3 mol/L potassium chloride) electrode as reference electrode, and a solution containing 1mmol/L Fe (CN) 6 3-/4- And 1.0mol/L potassium chloride, pulse period: 0.5s, scan range: 0 to 0.5V, amplitude: 0.05V;
the solid line in fig. 5 is the differential pulse voltammetry curve obtained after the cell electrode is exposed to different concentrations of thiram, and the dotted line is the differential pulse voltammetry curve of the cell after the electrode modification.
(5) Analysis of cytotoxicity
And (4) recording peak currents of the electrodes after the thiram with different concentrations is processed according to the differential pulse voltammetry curve obtained in the step (4), and further calculating the cytotoxicity of the thiram with different concentrations according to a formula:
wherein, I electrode Is the peak current, I, of the non-inoculated cells after electrode modification cell Is the peak current of the cell electrode without poison stimulation, I toxicant Is the peak current of the cell electrode after thiram treatment.
Substituting the peak current data in fig. 5 into the above formula to calculate cytotoxicity, and drawing a standard curve by taking the thiram concentration as an abscissa and the cytotoxicity as an ordinate to obtain the standard curve of the thiram cytotoxicity of fig. 6, wherein the standard curve is y =0.47203x +11.76909. Cytotoxicity increased linearly with the concentration of thiram in the range of 1 to 125. Mu. Mol/L (R) 2 ＝0.99799)；
(6) Detection of actual samples
Selecting apple juice as sample liquid; apple juice was sterilized by filtration through a 0.22 μm filter, and as actual samples, thiram standard solutions of different concentrations were added, respectively, and toxicity tests were performed using the prepared electrochemical cell sensor, and compared with the results of the conventional MTT method, and the results are shown in table 1. The results of the two methods have no obvious difference, and the accuracy and the reliability of the method are verified.
TABLE 1 toxicity of thiram in the actual samples measured by the method and MTT method
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (8)
1. A method for evaluating pesticide toxicity based on an electrochemical cell sensor is characterized by comprising the following steps:
(1) Firstly, preparing and obtaining an arginine Arg functionalized polylysine PLL solution; then modifying the dendritic gold nanoparticles by an ITO conductive glass electrode through an electrodeposition method to obtain an AuNDs/ITO electrode; immersing the AuNDs/ITO electrode in an arginine Arg functionalized polylysine PLL solution, incubating for a period of time, removing redundant liquid, and airing to obtain an Arg-PLL-AuNDs/ITO electrode;
(2) Culturing and fixing cells: placing the obtained Arg-PLL-AuNDs/ITO electrode in a container, inoculating HepG2 cells, adding a culture medium, and culturing at a certain temperature and in a gas environment for later use;
(3) Weighing thiram standard substances with different qualities, preparing thiram solutions with different concentrations, and adding the thiram solutions into the culture containers containing the electrodes after the culture in the step (2), wherein the thiram solutions and the culture containers for storing the electrodes are in a one-to-one correspondence relationship, namely one concentration corresponds to one culture container; continuously placing the mixture in a cell culture box for incubation; after incubation, the cell electrodes were taken out and washed with PBS,performing signal measurement of differential pulse voltammetry to obtain differential pulse voltammetry curves of the cell electrode after being treated by thiram with different concentrations, and recording the peak current of the cell electrode after being treated by thiram as I toxicant (ii) a Simultaneously setting a blank group and a control group, wherein the blank group is the peak current of the non-inoculated cells after electrode modification and is marked as I electrode (ii) a The peak current of the cell electrode in the control group without poison stimulation is marked as I cell (ii) a And further calculating the cytotoxicity of thiram at different concentrations according to a formula: the cytotoxicity calculation formula is as follows:
wherein, I electrode Is the peak current, I, of the non-inoculated cells after electrode modification cell Is the peak current of the cell electrode without being stimulated by poison, I toxicant Is the peak current of the cell electrode after thiram treatment;
(4) Detection of the actual sample: and (3) filtering and sterilizing the prepared sample solution through a 0.22-micrometer filter membrane to be used as an actual sample, respectively adding thiram standard solutions with different concentrations, recording peak currents of the electrodes after the thiram standard solutions with different concentrations are processed, and substituting the peak currents into the calculation formula of the cytotoxicity in the step (3) to realize the detection of the cytotoxicity in the sample.
2. The method for evaluating the toxicity of pesticides based on electrochemical cell sensor according to claim 1, wherein the electrode of step (1) is immersed in arginine Arg functionalized polylysine PLL solution for 5-15min.
3. The method for evaluating the toxicity of pesticides based on electrochemical cell sensor according to claim 1, wherein in the step (2), the final concentration of the HepG2 cell in the culture medium is 10 5 cells/mL。
4. The method for evaluating the toxicity of agricultural chemicals based on the electrochemical cell sensor of claim 1, wherein in the step (2), the culture medium is a DMEM culture medium containing fetal bovine serum with a volume concentration of 8-13%.
5. The method for evaluating toxicity of agricultural chemicals based on electrochemical cell sensor as claimed in claim 1, wherein in the step (2), the certain temperature and gas atmosphere are 37 ℃, 5% of CO 2 (ii) a The culture time is 20-30h.
6. The method for evaluating toxicity of agricultural chemicals based on electrochemical cell sensor as claimed in claim 1, wherein in step (3), the concentration of thiram is 0-125 μmol/L, and each concentration is used to prepare 3 parallel samples.
7. The method for evaluating the toxicity of pesticides based on electrochemical cell sensor according to claim 1, wherein in the step (3), the cell incubation time is 3-24h.
8. The method for evaluating the toxicity of pesticides based on electrochemical cell sensor according to claim 1, wherein in the step (3), the signal measurement conditions of the differential pulse voltammetry are as follows: CHI600E electrochemical workstation with platinum wire auxiliary electrode as counter electrode and silver/silver chloride electrode as reference electrode, in a solution containing 1mmol/L Fe (CN) 6 3-/4- And 1.0mol/L potassium chloride, pulse period: 0.5s, scan range: 0 to 0.5V, amplitude: 0.05V.
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