Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for rapidly detecting imidacloprid in water.
The technical scheme for solving the technical problems is as follows: a method for rapidly detecting imidacloprid in water comprises the following steps:
step 1: preparation of imidacloprid biosensing element
Carrying out surface hydroxylation treatment on the biosensing element to obtain a surface hydroxylated biosensing element;
carrying out surface alkylation treatment on the surface hydroxylated biosensor to obtain a surface alkylated biosensor;
connecting the surface alkylated biosensor element with the bifunctional cross-linking agent to obtain a biosensor element connected with the bifunctional cross-linking agent;
fixing the imidacloprid coating antigen on the reaction site of the biological sensing element connected with the bifunctional cross-linking agent to obtain the biological sensing element fixed with the imidacloprid coating antigen;
sealing unreacted sites of the biological sensing element fixed with the imidacloprid coating antigen to obtain the imidacloprid biological sensing element;
step 2: preparation of fluorescence-labeled Imidacloprid antibody
Performing primary dialysis on the imidacloprid antibody to obtain an imidacloprid antibody subjected to primary dialysis;
reacting the imidacloprid antibody subjected to preliminary dialysis with a fluorescent dye to obtain a reaction solution;
carrying out secondary dialysis on the reaction solution to obtain a fluorescence-labeled imidacloprid antibody;
and step 3: establishing a standard curve
In the evanescent wave fluorescence biosensor, PBS buffer solution is used as a blank control, mixed with the fluorescence-labeled imidacloprid antibody obtained in the step 2, pre-reacted, reacted on the surface of the imidacloprid biosensor element obtained in the step 1, and a blank fluorescence signal value is read;
mixing the imidacloprid standard working solution with the concentration gradient with the fluorescence-labeled imidacloprid antibody obtained in the step 2, carrying out pre-reaction, carrying out reaction on the surface of the imidacloprid biosensor obtained in the step 1, and reading a fluorescence signal value of the imidacloprid standard working solution;
taking the ratio of the fluorescence signal value of the imidacloprid standard working solution to the blank fluorescence signal value as a vertical coordinate, and taking the concentration of the imidacloprid standard working solution as a horizontal coordinate to obtain a standard curve;
and 4, step 4: detection of imidacloprid content in water sample to be detected
And (3) mixing a water sample to be detected with the fluorescence-labeled imidacloprid antibody obtained in the step (2), carrying out pre-reaction, then carrying out reaction on the surface of the imidacloprid biosensor element obtained in the step (1), reading a fluorescence signal of the water sample to be detected, carrying out normalization treatment on the fluorescence signal and the blank fluorescence signal value obtained in the step (3), and substituting the fluorescence signal and the blank fluorescence signal value into the standard curve obtained in the step (3) to obtain the content of the imidacloprid in the water sample to be detected.
The invention discloses an explanation of a principle of a method for rapidly detecting imidacloprid in water, which comprises the following steps:
in step 1 of the invention, the surface of the biosensor element contains a large amount of silicon dioxide components, and cannot be directly connected with the imidacloprid coating antigen. Therefore, the surface hydroxylation treatment is firstly carried out on the biosensor element to generate silicon hydroxyl, then the surface alkylation treatment is carried out, then one side functional group (4-maleimide butyrate) of the bifunctional cross-linking agent is modified on the surface of the surface alkylated biosensor element, then the other side functional group (N-hydroxysuccinimide ester) of the bifunctional cross-linking agent is reacted with the amino group on the imidacloprid coating antigen, and finally the imidacloprid coating antigen is fixed on the surface of the biosensor element.
In the step 2 of the invention, the imidacloprid antibody is fluorescently labeled, so that the imidacloprid antibody can be conveniently detected in an evanescent wave fluorescence biosensor.
In step 3 of the invention, the evanescent wave fluorescence biosensor is based on the laser-induced fluorescence biosensing principle and adopts an indirect competitive biological detection mode for detection. When the light wave is transmitted in the medium in a total reflection mode, evanescent waves can be generated on an interface. The imidacloprid coating antigen fixed on the surface of the biosensor is combined with the residual fluorescence-labeled imidacloprid antibody after the pre-reaction. Fluorescence is generated by fluorescent molecules under the excitation of evanescent waves, a fluorescence signal is collected by an optical fiber and transmitted to a signal acquisition unit, and the imidacloprid detection is realized according to the size of the fluorescence signal.
In conclusion, the invention utilizes the principle of laser-induced fluorescence, utilizes light with certain wavelength to excite the fluorescent molecules carried by the imidacloprid antibody bound on the surface of the biosensor element, monitors the intensity of the fluorescent signal in real time and realizes the real-time monitoring of the imidacloprid.
The method for rapidly detecting imidacloprid in water has the advantages that:
1. the detection method disclosed by the invention can realize real-time monitoring of imidacloprid in water, and has the advantages of high sensitivity, short test time, high detection efficiency and the like.
2. The detection method of the invention can realize repeated use by using the biological sensing element, thereby greatly saving the detection cost.
3. The detection method has good repeatability and reproducibility, strong anti-interference capability and simple operation, and is suitable for large-scale popularization and application.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in step 1, the biosensor is a biochip or an optical fiber sensor.
The adoption of the further beneficial effects is as follows: both the two biosensing elements can realize the technical scheme of the invention.
Furthermore, the biochip is a K9 glass sheet, and the optical fiber sensor is a quartz optical fiber.
Further, in step 1, the method for surface hydroxylation treatment is as follows: and (3) immersing the biosensor into Piraha solution, heating and reacting at 100-120 ℃ for 60min, then washing with ultrapure water until the pH value of the washing liquid is 7, and blow-drying with nitrogen at room temperature to obtain the surface hydroxylated biosensor.
The adoption of the further beneficial effects is as follows: by adopting the method, the surface hydroxylation of the biosensing element can be realized.
Further, the Piraha solution is prepared from concentrated H2SO4And H2O2Is prepared by evenly mixing according to the volume ratio of 3: 1.
The further beneficial effects of the adoption are as follows: can remove impurities on the surface of the biosensing element and can also hydroxylate the surface of the biosensing element.
Further, in step 1, the surface alkylation treatment method is as follows: and (3) immersing the surface hydroxylated biosensor into an anhydrous toluene solution of a 3-mercaptopropyltrimethoxysilane solution, and reacting for 2h at the temperature of 20-25 ℃ to obtain the surface alkylated biosensor.
The adoption of the further beneficial effects is as follows: by the method, the surface alkylation of the biosensing element can be realized.
3-mercaptopropyltrimethoxysilane, 3-trimethylsilylpropanethiol, MTS for short, with the molecular formula C6H16O3SSi, CAS number 4420-74-0.
Furthermore, in the anhydrous toluene solution of 3-mercaptopropyltrimethoxysilane, the volume fraction of 3-mercaptopropyltrimethoxysilane is 2%.
The further beneficial effects of the adoption are as follows: the anhydrous toluene solution of the 3-mercaptopropyltrimethoxysilane with the parameters can form a single-layer effective alkylation coating layer on the surface of the biosensor element with surface hydroxylation.
Further, in step 1, the method for connecting the bifunctional crosslinking agent is as follows: cleaning the surface alkylated biosensor with anhydrous toluene for 3-5 times, drying the surface alkylated biosensor with nitrogen under the condition of dust-free air, then putting the surface alkylated biosensor into an N-succinimidyl-4-maleimide butyrate solution with the concentration of 2mmol/L, reacting for 1h, taking out the surface alkylated biosensor, washing the surface alkylated biosensor with anhydrous ethanol for 3 times, then washing the surface alkylated biosensor with high-purity water, and drying the surface alkylated biosensor with nitrogen to obtain the biosensor connected with the bifunctional crosslinking agent.
The adoption of the further beneficial effects is as follows: with the above method, a bifunctional crosslinking agent can be attached to the surface-alkylated biosensor element. The thiol group on the biosensor after alkylation reacts with the maleimide group to form a stable thioether bond, thereby introducing N-succinimidyl-4-maleimidobutyrate to the biosensor.
N-succinimidyl-4-maleimidobutyrate, having the name of 2,5-Dioxopyrrolidin-1-yl 4- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) butanoate, abbreviated as GMBS (formula C)12H12N2O6CAS number 80307-12-6.
N-succinimidyl-4-maleimidobutyrate is an amine-to-sulfhydryl crosslinker containing NHS ester and maleimide reactive groups located at opposite ends of a short spacer arm (7.3 angstroms).
Further, in step 1, the specific method for immobilizing the imidacloprid coating antigen is as follows: and (3) dripping 5-10 mu L of 1-4 mg/mL imidacloprid coated antigen solution onto the reaction sites of the biological sensing element connected with the bifunctional cross-linking agent, standing at 4 ℃ for 12 hours, completely absorbing the redundant imidacloprid coated antigen solution, washing with ultrapure water, and blow-drying with nitrogen to obtain the biological sensing element fixed with the imidacloprid coated antigen.
The adoption of the further beneficial effects is as follows: by adopting the method, the immobilization of the imidacloprid coating antigen can be realized. And (3) placing the obtained biological sensing element fixed with the imidacloprid coating antigen into a culture dish, and storing at 4 ℃ for later use. The imidacloprid-coated antigen solution can be purchased commercially, for example, from Shandong LvDu Biotech, Inc.
Further, in step 1, the specific method for blocking is as follows: and (3) sealing unreacted sites of the biological sensing element fixed with the imidacloprid coated antigen for 60min at room temperature by adopting 2mg/mL bovine serum albumin, then washing the unreacted sites with ultrapure water, and drying the unreacted sites with nitrogen to obtain the imidacloprid biological sensing element.
The adoption of the further beneficial effects is as follows: by adopting the method, the closure of unreacted sites on the biological sensing element fixed with the imidacloprid coating antigen can be realized. And placing the obtained imidacloprid biosensing element into a culture dish, and storing at 4 ℃ for later use.
Bovine Serum Albumin (BSA), which is called Bovine Serum Albumin in English name, is abbreviated as BSA in English and has a CAS number of 9048-46-8.
Further, in step 2, the method of the preliminary dialysis is: putting the imidacloprid antibody into a mixed solution of sodium carbonate and sodium bicarbonate with the pH value of 9.0-9.3 and the concentration of 0.1mol/L according to the volume ratio of 1:9 in a dialysis bag, and dialyzing for 2h to obtain the imidacloprid antibody after primary dialysis.
The adoption of the further beneficial effects is as follows: by adopting the method, the preliminary dialysis of the imidacloprid antibody can be realized. Wherein the minimum molecular weight cut-off of the dialysis bag is 3500, the flattening width is 34mmol/L, 2 sections are prepared, and the dialysis bag is boiled in advance three times.
Further, in step 2, the specific method for reacting the imidacloprid antibody after the preliminary dialysis with the fluorescent dye is as follows: and (3) reacting the imidacloprid antibody subjected to preliminary dialysis with a fluorescent dye Cy5.5-N-hydroxysuccinimide ester at the molar ratio of 1:15 at 37 ℃ for 2 hours to obtain a reaction solution.
The adoption of the further beneficial effects is as follows: by the above method, a reaction solution can be obtained.
Further, in step 2, the specific method of the secondary dialysis is as follows: and (3) placing the reaction solution into PBS buffer solution with the mass percent of 0.01% sodium azide and the concentration of 10mmol/L, dialyzing for 4h, taking out, placing the reaction solution into PBS buffer solution with the mass percent of 0.01% sodium azide and the concentration of 10mmol/L, dialyzing for 12h, and taking out to obtain the fluorescence-labeled imidacloprid antibody.
The adoption of the further beneficial effects is as follows: by adopting the method, the secondary dialysis of the reaction solution can be realized, so that the fluorescence-labeled imidacloprid antibody can be obtained.
Further, in the step 2, the labeling ratio of the fluorescence-labeled imidacloprid antibody is 1.5-4.
The adoption of the further beneficial effects is as follows: if the label ratio is too low or too high, the fluorescence signal is weak and cannot be used for experimental procedures. The invention finds that the labeling ratio is 1.5-4 properly through research.
Furthermore, the method for calculating the mark ratio is as follows: diluting the imidacloprid antibody after secondary dialysis by 3-5 times with PBS buffer solution with concentration of 10mmol/L, and detecting OD with ultraviolet spectrophotometer280nmAnd OD678nmThe absorbance of the compound (A) is substituted by formula 1, the labeling ratio is calculated,
[Cy5.5]=OD678nm/250000
[Ab]=(OD280nm–0.18×OD678nm)/170000
[ D/P ] ═ Cy5.5]/[ Ab ] (formula 1)
Wherein [ Cy5.5] is the concentration of Cy5.5-N-hydroxysuccinimide ester as fluorescent dye, [ Ab ] is the concentration of imidacloprid antibody, and [ D/P ] is the labeling ratio.
The adoption of the further beneficial effects is as follows: by the above method, the mark ratio can be calculated. The imidacloprid antibody after the secondary dialysis needs to be diluted by 3-5 times by using PBS buffer solution with the concentration of 10mmol/L, so that the test signal can be read or more accurately.
The imidacloprid antibody and the fluorescent dye respectively have characteristic absorption peaks at 280nm and 678nm, the imidacloprid antibody marked by fluorescence is scanned by ultraviolet-visible full spectrum, the concentration value of the imidacloprid antibody and the concentration value of the fluorescent dye Cy5.5-N-hydroxysuccinimide ester are quantified according to Lambert beer law by utilizing the characteristic absorption peaks at corresponding positions, and the marking ratio of the antibody and the marker is calculated.
The molar extinction coefficient of the fluorescent dye Cy5.5-N-hydroxysuccinimide ester at 678nm is250000, the molar extinction coefficient of the imidacloprid antibody at 280nm was 170000. Since the fluorescent dye Cy5.5-N-hydroxysuccinimide ester also has certain absorption at 280nm, and the absorbance value is about 18% of that at 678nm, the absorbance value needs to be revised when the imidacloprid antibody concentration is calculated, namely [ Cy5.5]]=OD678nm/250000,[Ab]=(OD280nm–0.18×OD678nm)/170000。
Further, in the step 3, the concentration of the PBS buffer solution is 10mmol/L, and the volume ratio of the PBS buffer solution to the fluorescence-labeled imidacloprid antibody is 1 (3-6); the volume ratio of the imidacloprid standard working solution to the fluorescence-labeled imidacloprid antibody is 1 (3-6).
Further, in the step 3, the concentration gradient of the imidacloprid standard working solution is 0-400 mug/L.
The adoption of the further beneficial effects is as follows: the concentration gradient of the standard working solution adopts the range, and the possible concentration range of the imidacloprid in the water sample to be detected can be covered. The specific preparation method comprises the following steps: taking an imidacloprid standard substance, and diluting the imidacloprid standard substance by using PBS buffer solution with the concentration of 10 mmol/L. Imidacloprid standards are commercially available, e.g., from Olympic Biotechnology, Inc. of the Beijing century at a concentration of 1000 mg/L.
Further, in step 3, the pre-reaction time is 300s, and the reaction time is 500 s.
The adoption of the further beneficial effects is as follows: tests show that when the pre-reaction time and the reaction time are respectively less than 300s and 500s, the signal value is always reduced along with the increase of the time; after that, the signal values reach equilibrium, so the pre-reaction time should be set to 300s and the reaction time should be 500 s.
Further, in the step 4, the volume ratio of the water sample to be detected to the fluorescence-labeled imidacloprid antibody is 1 (3-6).
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Examples
The method for rapidly detecting imidacloprid in water comprises the following steps:
step 1: preparation of imidacloprid biosensing element
Adding concentrated H2SO4And H2O2And uniformly mixing the components according to the volume ratio of 3:1 to prepare a Piraha solution.
And (3) soaking a K9 glass sheet into Piraha solution, heating and reacting at 110 ℃ for 60min, then washing with ultrapure water until the pH value of the washing liquid is 7, and drying with nitrogen at room temperature to obtain the biosensor with the hydroxylated surface.
Preparing an anhydrous toluene solution of 3-mercaptopropyltrimethoxysilane in an argon environment. In the anhydrous toluene solution of 3-mercaptopropyltrimethoxysilane, the volume fraction of 3-mercaptopropyltrimethoxysilane is 2%.
And (3) immersing the surface hydroxylated biosensor into an anhydrous toluene solution of a 3-mercaptopropyltrimethoxysilane solution, and reacting for 2h at 22 ℃ to obtain the surface alkylated biosensor.
Cleaning the surface alkylated biosensor element with anhydrous toluene for 3 times, drying the surface alkylated biosensor element with nitrogen under the dustless air condition, then putting the surface alkylated biosensor element into an N-succinimidyl-4-maleimide butyrate solution with the concentration of 2mmol/L, taking out the surface alkylated biosensor element after reacting for 1h, firstly washing the surface alkylated biosensor element with anhydrous ethanol for 3 times, then washing the surface alkylated biosensor element with high-purity water, and then drying the surface alkylated biosensor element with nitrogen to obtain the biosensor element connected with the bifunctional cross-linking agent.
And (3) dropwise adding 5 mu L of 4mg/mL imidacloprid coated antigen solution onto the reaction sites of the biological sensing element connected with the bifunctional cross-linking agent, standing at 4 ℃ for 12 hours, completely sucking the redundant imidacloprid coated antigen solution, washing with ultrapure water, and blow-drying with nitrogen to obtain the biological sensing element fixed with the imidacloprid coated antigen. Placing into a culture dish, and storing at 4 ℃ for later use.
And (3) sealing unreacted sites of the biological sensing element fixed with the imidacloprid coated antigen for 60min at room temperature by adopting 2mg/mL bovine serum albumin, then washing the unreacted sites with ultrapure water, and drying the unreacted sites with nitrogen to obtain the imidacloprid biological sensing element. Placing into a culture dish, and storing at 4 ℃ for later use.
Step 2: preparation of fluorescence-labeled Imidacloprid antibody
Putting the imidacloprid antibody into a mixed solution of sodium carbonate and sodium bicarbonate with the pH value of 9.0 and the concentration of 0.1mol/L according to the volume ratio of 1:9 in a dialysis bag, and dialyzing for 2h to obtain the imidacloprid antibody after primary dialysis.
Reacting the imidacloprid antibody subjected to preliminary dialysis and a fluorescent dye Cy5.5-N-hydroxysuccinimide ester at the molar ratio of 1:10 and 1:15 for 2 hours at 37 ℃ to obtain a reaction solution.
And (3) placing the reaction solution into PBS buffer solution with the mass percent of 0.01% sodium azide and the concentration of 10mmol/L, dialyzing for 4h, taking out, placing the reaction solution into PBS buffer solution with the mass percent of 0.01% sodium azide and the concentration of 10mmol/L, dialyzing for 12h, and taking out to obtain the fluorescence-labeled imidacloprid antibody.
Calculating the labeling ratio of the fluorescence-labeled imidacloprid antibody: diluting the imidacloprid antibody after secondary dialysis by 3 times with PBS buffer solution with concentration of 10mmol/L, and detecting OD with ultraviolet spectrophotometer280nmAnd OD678nmThe absorbance of (A) was, as shown in FIG. 1, OD when the molar ratio of imidacloprid antibody to fluorescent dye was 1:10678nm=0.98,OD280nm0.617; when imidacloprid is used as insecticideOD at a molar ratio of 1:15 of body to fluorescent dye678nm=1.16,OD280nm0.605. Substitution of formula 1 resulted in labeling ratios of 1.51 and 1.99, respectively.
[Cy5.5]=OD678nm/250000
[Ab]=(OD280nm–0.18×OD678nm)/170000
[ D/P ] ═ Cy5.5]/[ Ab ] (formula 1)
Wherein [ Cy5.5] is the concentration of Cy5.5-N-hydroxysuccinimide ester as fluorescent dye, [ Ab ] is the concentration of imidacloprid antibody, and [ D/P ] is the labeling ratio.
And step 3: and (3) a computer-on detection step:
the evanescent wave fluorescence biosensor is divided into four parts, wherein a standard solution reagent is mixed and pre-reacted, liquid and a chip are reacted after pre-reaction, a reaction tank is flushed and a signal is measured, and the chip is activated.
The method comprises the following specific operation steps: pre-reacting 1.6mL of solution to be detected and 200 mu L of fluorescence labeling imidacloprid antibody in a reactor, setting the pre-reaction time to be 100s-500s, then entering the surface of a chip through an automatic sample injection system, reacting the imidacloprid antibody containing binding sites with imidacloprid coating antigen fixed on the surface of an imidacloprid biosensor element, and setting the reaction time to be 200s-700 s. After unbound imidacloprid antibody is washed away by PBS buffer solution with the concentration of 10mmol/L, evanescent waves on the surface of the imidacloprid biosensor can excite Cy5.5-N-hydroxysuccinimide ester serving as a fluorescent dye to generate fluorescence, and the content of the imidacloprid in a sample to be detected can be quantitatively analyzed through the size of a fluorescence signal. And (3) after each reaction is finished, regenerating by using SDS eluent with the mass concentration of 0.5%, wherein the regeneration time is set to be 200-1200 s, and the SDS eluent can be used for next detection.
And 4, step 4: establishing a standard curve
And (3) taking PBS buffer solution with the concentration of 10mmol/L as a blank control, mixing the blank control with the fluorescence-labeled imidacloprid antibody obtained in the step (2) according to the volume ratio of 1:4, carrying out pre-reaction, carrying out reaction on the surface of the imidacloprid biosensor obtained in the step (1), and reading a blank fluorescence signal value.
And (3) taking an imidacloprid standard substance with the concentration of 1000mg/L, and diluting the imidacloprid standard substance into an imidacloprid standard working solution with the concentration gradient of 0 mu g/L-400 mu g/L by using PBS buffer solution with the concentration of 10 mmol/L. Mixing the imidacloprid standard working solution with the concentration gradient with the fluorescence-labeled imidacloprid antibody obtained in the step 2 according to the volume ratio of 1:4, carrying out pre-reaction, then carrying out reaction on the surface of the imidacloprid biosensor obtained in the step 1, and reading the fluorescence signal value of the imidacloprid standard working solution; the labeling ratio of the fluorescence-labeled imidacloprid antibody used was 1.99, and the concentration was 6. mu.g/mL.
And taking the ratio of the fluorescence signal value of the imidacloprid standard working solution to the blank fluorescence signal value as a vertical coordinate, and taking the concentration of the imidacloprid standard working solution as a horizontal coordinate to obtain a standard curve, as shown in figure 2. The standard curve corresponds to a unary linear equation of
y 0.2707+ (0.9322-0.2707)/(1+ (x/2.8425). times. 1.3244) (formula 2)
In the formula, y is the ratio of the fluorescence signal value of the imidacloprid standard working solution to the blank fluorescence signal value, and x is the concentration of the imidacloprid standard working solution. The correlation coefficient is 0.995, the detection range is 0.998. mu.g/L-8.096. mu.g/L, and the detection limit is 0.0247. mu.g/L.
And 5: reaction parameter optimization
And testing and optimizing the pre-reaction, the reaction time and the elution time by setting a series of time gradients. As can be seen from FIGS. 3 and 4, when the pre-reaction time and the reaction time are less than 300s and 500s, respectively, the signal value is always decreased as the time increases; then the signal value reaches the balance, so the pre-reaction time is set to be 300s, and the reaction time is set to be 500 s; as can be seen from FIG. 5, when the elution time is less than 800s, the signal value decreases with the increase of the elution time; when the reaction value was 800s, the signal value reached equilibrium, so the elution time was set to 800 s.
Step 6: cross reaction rate test
In order to detect the specificity of the imidacloprid antibody, carbendazim and dibutyl phthalate are selected to determine the cross reaction rate, the 50% inhibition concentration of the imidacloprid antibody is respectively obtained through the standard curves of the two substances, and the cross reaction rate of the imidacloprid antibody to other antibiotics is calculated by using a formula 3. The smaller the cross-reactivity, the better the detection specificity of this imidacloprid antibody for imidacloprid, and the assay was repeated 3 times with the results averaged as shown in table 1.
Cross-reactivity (%). ratio (concentration causing 50% inhibition of imidacloprid/other antibiotic substance causing 50% inhibition) × 100% (formula 3)
TABLE 1 specificity of Imidacloprid antibodies
Name of drug
|
Imidacloprid
|
Carbendazim
|
Dibutyl phthalate
|
Cross reaction Rate (%)
|
100
|
<0.1
|
<0.1
|
IC50Value (μ g/L)
|
2.84
|
>1000
|
>1000 |
Experiments show that the imidacloprid antibody has good specificity on imidacloprid, namely the imidacloprid antibody can detect the content of imidacloprid in a water sample.
And 7: detection of imidacloprid content in water sample to be detected
Respectively adding imidacloprid into tap water without imidacloprid and effluent water of a sewage treatment plant until the final concentration of the imidacloprid is 6.4 mu g/L, and calculating the accuracy respectively by making 5 replicates for each concentration.
And (3) mixing the tap water sample with the fluorescence-labeled imidacloprid antibody obtained in the step (2) according to the volume ratio of 1:4, carrying out pre-reaction, carrying out reaction on the surface of the imidacloprid biosensor obtained in the step (1), wherein the fluorescence signal value of the tap water sample is 2567.9, the blank fluorescence signal value obtained in the step (4) is 5836.335, the blank fluorescence signal value is 1, the normalized signal value is the actual sample fluorescence signal value/blank fluorescence signal value, and the normalized result obtained by the tap water sample is 2567.98/5836.335, and calculating to obtain 0.44. The normalized signal value is the y value in the standard curve obtained in step 3. The value of x in the standard curve is calculated by using the value, namely the concentration of the imidacloprid in the sample is 6.36 mu g/mL.
The calculation mode of the effluent water sample of the sewage treatment plant is the same as that of the tap water sample. The results are shown in Table 2.
TABLE 2 actual water sample labeling test results
The result shows that the adding accuracy of the tap water and the effluent sample of the sewage treatment plant is 70-120%, and the variation coefficient is less than 15%, which indicates that the test result of the embodiment is reliable. In addition, the detection time of the embodiment of the invention is shorter and is 0.5 h.
Comparative example
Respectively adding imidacloprid into tap water without imidacloprid and effluent water of a sewage treatment plant until the final concentration of the imidacloprid is 6.4 mu g/L, and calculating the accuracy respectively by making 5 replicates for each concentration.
And (3) detecting by adopting an indirect competitive immune enzyme-linked method. The concentration of the imidacloprid antibody used was 0.09. mu.g/mL, and the concentration of the coating antigen was 1. mu.g/mL. The detection range is as follows: 0.498 to 16.836 mu g/L. Detection limit: 0.025. mu.g/L.
The detection range and detection limit of the indirect competitive immune enzyme-linked reaction of the comparative example are similar to those of the embodiment of the invention, but the detection time is longer and is at least 6 h. Compared with the detection time of the embodiment of the invention, the detection time is increased by 0.5 h.
Therefore, the detection method disclosed by the invention can realize real-time monitoring of imidacloprid in water, and has the advantages of high sensitivity, short test time, high detection efficiency and the like.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.