CN210465268U - Pesticide on-site detection device - Google Patents
Pesticide on-site detection device Download PDFInfo
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- CN210465268U CN210465268U CN201921352455.4U CN201921352455U CN210465268U CN 210465268 U CN210465268 U CN 210465268U CN 201921352455 U CN201921352455 U CN 201921352455U CN 210465268 U CN210465268 U CN 210465268U
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Abstract
The utility model discloses a pesticide on-site measuring device belongs to pesticide residue detection area. The pesticide field detection device comprises an injector, the injector comprises an injection port arranged at one end of the injector, and the pesticide field detection device also comprises a reactor connected with the injection port, the reactor comprises an enzyme reaction bin and a color development reaction bin, the color development reaction bin is connected with the injection port, the enzyme reaction bin is far away from the injection port, and the enzyme reaction bin is connected with the color development reaction bin; the device also comprises an adapter cylinder connected with the enzyme reaction bin; a display piece is arranged between the color reaction bin and the enzyme reaction bin, and an enzyme piece is arranged between the enzyme reaction bin and the adapter cylinder. The utility model discloses be applied to the aspect of pesticide residue detects, solved current pesticide on-the-spot detection device because the inhomogeneous testing result who causes of developing is inaccurate, can't realize quantitative analysis, the limited problem of detectivity has that the testing result is accurate reliable, can realize quantitative detection, characteristics that detectivity is high.
Description
Technical Field
The utility model belongs to pesticide residue detection area especially relates to a pesticide on-the-spot detection device.
Background
Pesticides are effective weapons for preventing and controlling crop diseases, pests and weeds in agricultural production, however, the phenomenon of overdose during pesticide application is ubiquitous. Therefore, agricultural and sideline products circulating in the market have pesticide residue problems in different degrees, and become hidden dangers threatening the life and health of people. Thus, the need for on-site detection of pesticide residues is ubiquitous and even shows an increasing trend. The existing pesticide residue detection method mainly comprises gas chromatography, high performance liquid chromatography, mass spectrum, Raman spectrum and the like. Although the methods have high sensitivity and accuracy, the methods depend on large instruments and equipment, have poor mobility, are not suitable for on-site online detection, and are difficult to meet the requirements of preventing and controlling emergency events. The national standard (GB/T5009.199-2003) of rapid detection of residual quantities of organophosphorus and carbamate pesticides in vegetables provides two methods for on-site screening and measuring residual quantities of organophosphorus and carbamate pesticides in vegetables: the rapid test card method (paper method) and the enzyme inhibition rate method (spectrophotometry). Among them, the rapid card method has the advantages of simple operation, no dependence on precise instruments, convenient carrying, intuitive result and the like, and is widely popular. However, this method relies on manual operation to fold the enzyme sheet and then react with the substrate sheet, which is prone to cause errors in manual operation and has certain requirements on the professional knowledge of the operator.
Chinese patent CN 205607868U discloses a rapid detection point card for pesticide residue, which comprises a card holder and a card cover, wherein a plurality of detection holes are arranged on the card holder at intervals, a substrate film (containing substrate indoleacetate) and an enzyme film (containing acetylcholinesterase) are sequentially stacked in the holes from bottom to top, and a sample adding hole corresponding to the detection hole is arranged on the card cover. During detection, a sample is dripped into the detection hole, the acetylcholinesterase on the enzyme membrane is quickly dissolved into a sample liquid to be detected, if the sample liquid does not contain pesticides, the enzyme activity is kept intact, and the sample liquid permeates into the substrate membrane to react with indoleacetic ester, so that the color is green; if the pesticide remains in the sample solution, the enzyme activity is inhibited, the intensity of the color reaction between the enzyme and the substrate is reduced or completely inhibited, and the sample solution is weakly green or colorless.
Chinese patent CN 206594063U discloses a broad-spectrum pesticide residue rapid detection plate, which is provided with a detection hole and a control hole, wherein an enzyme sheet (coated with cholinesterase), a display sheet (coated with dithiodinitrobenzoic acid developer) and a substrate sheet (coated with substrate thioacetyl choline or thiobutyrylcholine) are stacked in sequence from top to bottom in the detection hole. Because the display piece is separated between the enzyme piece and the substrate piece, the storage difficulty caused by direct overlapping of the enzyme and the substrate is avoided. During detection, a sample solution to be detected is firstly contacted with the enzyme sheet, the enzyme permeates into the substrate sheet to decompose the substrate under the driving of the osmosis, an enzyme reaction product is then reacted with the color developing agent, and whether pesticide residues exist or not is judged according to the color depth of yellow color after 8 minutes.
Although the above patent completely abandons the folding or pinching operation mode, and improves the detection accuracy to some extent, it still has the following problems:
(1) uneven color development and influence on result judgment
The color development uniformity is an important basis for accurately judging the detection result. Whether the rapid test card, the finger stall, the elastic sheet clamp or the direct stacking, the aim is to make the enzyme sheet and the display sheet directly contact, and then establish the correlation between the inhibition degree of the pesticide on the enzyme and the color reaction. In the preparation of the display piece, the fixation of the color developing agent is mostly realized by adopting a direct soaking mode, the fixation strength is very weak, the color developing agent can be dissolved when meeting water, at the moment, if a detection mode that an enzyme piece is directly contacted with the display piece is adopted, the distribution of color developing substances on the display piece is inevitably influenced obviously, the uneven color development is finally caused, and the accuracy of a result is influenced;
(2) can only be detected qualitatively, and cannot realize quantitative analysis
Quantitative analysis is of great significance for pesticide residue assessment. The existing rapid test technology can only qualitatively reflect the existence or nonexistence of pesticide residue in a sample by observing the color change of a display piece;
(3) the detection result is not easy to judge, and the detection sensitivity is limited
The color reaction occurring on the conventional display sheet is a color reaction. When unreacted, the developer is typically colorless (e.g., indole acetate, thio-acetylcholine) or has a distinct color from the color product (e.g., 2, 6-difluoroindophenol acetate is red and the enzymatic product indophenol is blue). After reaction with the enzyme reaction product, a colored chromogenic product is formed. Therefore, the more the pesticide residue, the less the amount of the enzyme reaction product is produced, and the lighter the color of the display sheet appears. The color depth of the display piece must be strictly grasped to ensure the detection accuracy. However, the degree of shade of the color is often difficult to observe and difficult to judge, which inevitably affects the sensitivity of detection.
SUMMERY OF THE UTILITY MODEL
The weak point that exists to prior art, the utility model aims to solve the main problem that it is inaccurate to overcome current pesticide on-the-spot detection device because the testing result that the color development is inhomogeneous to cause, can't realize quantitative analysis, the limited problem of detectivity provides a testing result accurate reliable, can realize quantitative determination, the high pesticide on-the-spot detection device of detectivity.
For solving the technical problem, the utility model discloses a technical scheme be:
the utility model provides a pesticide on-site detection device, which comprises an injector, wherein the injector comprises an injection port arranged at one end of the injector, and a reactor connected with the injection port, the reactor comprises an enzyme reaction bin and a color reaction bin, the color reaction bin is connected with the injection port, the enzyme reaction bin is far away from the injection port, and the enzyme reaction bin is connected with the color reaction bin; the device also comprises an adapter cylinder connected with the enzyme reaction bin; a display sheet is arranged between the color reaction bin and the enzyme reaction bin, and an enzyme sheet is arranged between the enzyme reaction bin and the adapter cylinder.
Preferably, the pipette tip is connected with the adapter.
Preferably, the display piece includes a polyester fiber layer and an agarose gel layer including agarose gel and a color developing solution.
Preferably, the enzyme reaction bin comprises a first tubular enzyme reaction bin and a second tubular enzyme reaction bin, and the inner diameter of the second enzyme reaction bin is larger than that of the first enzyme reaction bin;
the color reaction bin comprises a first cylindrical color reaction bin and a second cylindrical color reaction bin, and the inner diameter of the second color reaction bin is larger than that of the first color reaction bin;
the outer diameter of the first enzyme reaction bin is smaller than the inner diameter of the second color reaction bin;
the adapter cylinder is cylindrical, and the outer diameter of the adapter cylinder is smaller than the inner diameter of the second enzyme reaction bin;
the first enzyme reaction bin is partially sleeved in the second color reaction bin, and the adapter tube is partially sleeved in the second enzyme reaction bin.
Preferably, the display sheet is arranged in the second color reaction bin, and the enzyme sheet is arranged in the second enzyme reaction bin.
Preferably, a first silica gel sealing ring is arranged between the display piece and the first enzyme reaction bin, and a second silica gel sealing ring is arranged between the enzyme piece and the adapter cylinder.
Preferably, a first rubber ring is arranged between the first enzyme reaction bin and the second color reaction bin, and a second rubber ring is arranged between the adapter cylinder and the second enzyme reaction bin.
Preferably, the outer diameter of the first enzyme reaction bin is 11mm, the inner diameter of the first enzyme reaction bin is 9mm, and the height of the first enzyme reaction bin is 20 mm; the outer diameter of the second enzyme reaction bin is 14mm, the inner diameter of the second enzyme reaction bin is 12mm, and the height of the second enzyme reaction bin is 15 mm;
the outer diameter of the first color reaction bin is 11mm, the inner diameter of the first color reaction bin is 9mm, and the height of the first color reaction bin is 20 mm; the outer diameter of the second color reaction bin is 14mm, the inner diameter of the second color reaction bin is 12mm, and the height of the second color reaction bin is 15 mm;
the outer diameter of the adapter cylinder is 11mm, the inner diameter of the adapter cylinder is 9mm, and the height of the adapter cylinder is 20 mm;
the display piece is circular, and the diameter of the display piece is 12 mm; the enzyme tablet is round, and the diameter of the enzyme tablet is 12 mm;
the outer diameter of the first rubber ring is 12mm, the inner diameter of the first rubber ring is 11mm, and the height of the first rubber ring is 10 mm; the outer diameter of the second rubber ring is 12mm, the inner diameter of the second rubber ring is 11mm, and the height of the second rubber ring is 10 mm;
the outer diameter of the first silica gel sealing ring is 12mm, and the inner diameter of the first silica gel sealing ring is 10 mm; the external diameter of second silica gel sealing washer is 12mm, and the internal diameter is 10 mm.
Compared with the prior art, the beneficial effects of the utility model reside in that:
1. the utility model provides a pesticide on-site detection device, wherein an enzyme sheet and a display sheet are respectively fixed in different reaction bins, and the enzyme sheet and the display sheet are not in direct contact in the whole detection process, so that the color development is not interfered;
2. the utility model provides a pesticide on-site detection device, a display piece adopts agarose to embed and fix a color developing agent, on one hand, the leakage of the color developing solution can be effectively prevented, and the detection accuracy is ensured; on the other hand, agarose has good light permeability, and realizes quantitative analysis of pesticide residues;
3. the utility model provides a pesticide on-site measuring device adopts the fading reaction, detects according to having or not of colour, changes the observation, and sensitivity obtains improving.
Drawings
Fig. 1A is a front view of a pesticide field detection device provided in an embodiment of the present invention;
fig. 1B is a cross-sectional view of a pesticide field detection device provided by the embodiment of the present invention;
fig. 1C is a schematic view of a structure of a part of the on-site pesticide detection device provided in the embodiment of the present invention;
fig. 2 is an absorption spectrum measured by comparing the display sheet and the sample liquid display sheet after the detection of the pesticide on-site detection device provided by the embodiment of the utility model;
in the above figures: 1. an injector; 2. an injection port; 3. a color development reaction bin; 31. a first color reaction bin; 32. a second color reaction bin; 4. a display sheet; 5. an enzyme reaction bin; 51. a first enzyme reaction bin; 52. A second enzyme reaction bin; 6. an enzyme tablet; 7. a transfer cylinder; 8. a pipette tip; 9. a first silica gel sealing ring; 10. a first rubber ring; 11. a second silica gel sealing ring; 12. a second rubber ring; 13. a first rubber plug; 14. and a second rubber plug.
Detailed Description
The present invention is specifically described below by way of exemplary embodiments. It should be understood, however, that elements, structures and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.
In the description of the present invention, it should be understood that the terms "upper", "lower", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientation or positional relationships shown in fig. 1, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be construed as limiting the present invention.
The terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.
In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "connected" and "connected" are to be interpreted broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood in specific cases to those skilled in the art.
As shown in fig. 1A, fig. 1B and fig. 1C, the utility model provides a pesticide on-site detection device, including injector 1, injector 1 includes injection port 2 that locates injector 1 one end, still includes the reactor that links to each other with injection port 2, and the reactor includes enzyme reaction storehouse 5 and color development reaction storehouse 3, and color development reaction storehouse 3 links to each other with injection port 2, and enzyme reaction storehouse 5 keeps away from injection port 2, and enzyme reaction storehouse 5 links to each other with color development reaction storehouse 3; the device also comprises an adapter cylinder 7 connected with the enzyme reaction bin 5; a display sheet 4 is arranged between the color reaction bin 3 and the enzyme reaction bin 5, and an enzyme sheet 6 is arranged between the enzyme reaction bin 5 and the adapter cylinder 7. The color development uniformity is an important basis for accurately judging the detection result. Whether the rapid test card, the finger stall, the elastic sheet clamp or the direct stacking, the aim is to make the enzyme sheet 6 and the display sheet 4 directly contact, and then the inhibition degree of the pesticide to the enzyme and the color reaction are correlated. In the preparation of the display sheet 4, the fixation of the color developing agent is mostly realized by adopting a direct soaking mode, the fixation strength is very weak, the color developing agent can be dissolved when meeting water, at the moment, if a detection mode that the enzyme sheet 6 is in direct contact with the display sheet 4 is adopted, the distribution of the color developing substance on the display sheet 4 is certainly influenced, the color development unevenness is finally caused, and the accuracy of the result is influenced. The utility model provides a detection device is fixed in different reaction storehouses respectively with enzyme piece 6 and display piece 4, uses this device, combines syringe 1's piston "to push away-draw" and the permeability of diaphragm substrate, can control solution and flow between different reaction storehouses, contact enzyme reaction and color reaction. In the whole detection process, the enzyme sheet 6 and the display sheet 4 are not in direct contact, and the color development is not interfered. Therefore, use the utility model provides a detection result that detection device finally obtained is the color development even, is convenient for accurately judge pesticide residue's level. The injector 1 further comprises an injection cavity, a piston sleeved in the injection cavity, a piston head arranged in the injection cavity, and a push rod.
In order to facilitate the solution suction, a pipette tip 8 connected with the adapter cylinder 7 is also included.
In order to prevent the color developing solution from leaking into the reaction solution, the display sheet 4 includes a polyester fiber layer and an agarose gel layer including agarose gel and the color developing solution. The existing rapid test technology can only qualitatively reflect the existence or nonexistence of pesticide residue in a sample by observing the color change of the display piece 4. The display piece 4 of the utility model adopts agarose to embed and fix the color developing agent, on one hand, the leakage of the color developing solution can be effectively prevented, and the detection accuracy is ensured; agarose, on the other hand, has good light permeability. The utility model provides two reading modes in the final result reading, firstly, the qualitative judgment and semi-quantitative measurement of the pesticide residue in the sample can be obtained by comparing the standard colorimetric card directly through the mode of visual observation; in addition, the gel sheet of the display sheet 4 can be carefully peeled off from the polyester fiber film, folded in half from the middle, placed in a constant cuvette containing 1.6mL of water, and then combined with an ultraviolet-visible spectrophotometer to implement quantitative analysis by applying Lambert-Beer law.
In order to facilitate the assembly of the device, the enzyme reaction chamber 5 comprises a first enzyme reaction chamber 51 and a second enzyme reaction chamber 52, wherein the inner diameter of the second enzyme reaction chamber 52 is larger than that of the first enzyme reaction chamber 51;
the color reaction bin 3 comprises a first color reaction bin 31 and a second color reaction bin 32, wherein the first color reaction bin 31 and the second color reaction bin 32 are cylindrical, and the inner diameter of the second color reaction bin 32 is larger than that of the first color reaction bin 31;
the outer diameter of the first enzyme reaction bin 51 is smaller than the inner diameter of the second color reaction bin 32;
the adapter cylinder 7 is cylindrical, and the outer diameter of the adapter cylinder 7 is smaller than the inner diameter of the second enzyme reaction bin 52;
the first enzyme reaction chamber 51 is partially sleeved in the second color reaction chamber 32, and the adapter tube 7 is partially sleeved in the second enzyme reaction chamber 52.
In order to ensure the stability of the display sheet 4 and the enzyme sheet 6, the display sheet 4 is arranged in the second color reaction chamber 32, and the enzyme sheet 6 is arranged in the second enzyme reaction chamber 52.
In order to improve the sealing performance, a first silica gel sealing ring 9 is arranged between the display piece 4 and the first enzyme reaction bin 51, and a second silica gel sealing ring 11 is arranged between the enzyme piece 6 and the adapter cylinder 7.
Further, a first rubber ring 10 is arranged between the first enzyme reaction bin 51 and the second color reaction bin 32, and a second rubber ring 12 is arranged between the adapter cylinder 7 and the second enzyme reaction bin 52.
In order to improve the detection effect of the detection device, the outer diameter of the first enzyme reaction chamber 51 is 11mm, the inner diameter is 9mm, and the height is 20 mm; the outer diameter of the second enzyme reaction bin 52 is 14mm, the inner diameter is 12mm, and the height is 15 mm;
the outer diameter of the first color reaction bin 31 is 11mm, the inner diameter is 9mm, and the height is 20 mm; the outer diameter of the second color reaction bin 32 is 14mm, the inner diameter is 12mm, and the height is 15 mm;
the outer diameter of the adapter cylinder 7 is 11mm, the inner diameter is 9mm, and the height is 20 mm;
the display piece 4 is circular, and the diameter of the display piece 4 is 12 mm; the enzyme tablet 6 is round, and the diameter of the enzyme tablet 6 is 12 mm;
the outer diameter of the first rubber ring 10 is 12mm, the inner diameter is 11mm, and the height is 10 mm; the outer diameter of the second rubber ring 12 is 12mm, the inner diameter is 11mm, and the height is 10 mm;
the outer diameter of the first silica gel sealing ring 9 is 12mm, and the inner diameter is 10 mm; the outer diameter of the second silica gel sealing ring 11 is 12mm, and the inner diameter is 10 mm.
In a preferred embodiment, the display sheet 4 is prepared by the following method:
taking a polyester fiber wafer, placing a cylindrical mold on the polyester fiber wafer, pouring a mixed solution of agarose gel liquid and color development liquid into the cylindrical mold, cooling and solidifying the mixed solution to obtain an agarose gel layer, taking down the cylindrical mold, and punching a hole in the center of the agarose gel layer by using a puncher to obtain a display piece 4;
the outer diameter of the cylindrical die is smaller than the diameter of the polyester fiber wafer; the volume ratio of the agarose gel liquid to the color development liquid is 1:1-1: 2. To ensure the thickness of the agarose gel layer, the height of the cylindrical mold was 3 mm. The size of the display piece 4 is adjusted according to the sizes of the enzyme reaction bin 5 and the color reaction bin 3, and the leakage is prevented on the premise that the display piece 4 is fully contacted with the solution.
In a preferred embodiment, the color developing solution is obtained by the following method:
adding hydrochloric acid solution and FeTMPyP4Uniformly mixing the solution, the TMB solution and the hydrogen peroxide solution, reacting at 37 ℃ for 30-70min, changing the mixed solution from colorless to dark blue, and performing ultrasonic treatment at room temperature for 2-5min to obtain a color developing solution. The color development principle of the color development liquid is as follows: tetra (N-methyl-4-pyridine) iron porphyrin (FeTMPyP)4) Having peroxidase mimic enzyme activity, and being capable of catalyzing hydrogen peroxide to generate hydroxyl radicals under acidic conditions so as to catalyze 3,3 ', 5, 5' -Tetramethylbenzidine (TMB)Oxidation reaction to produce blue oxidized TMB. The plant esterase can catalyze the hydrolysis of the acetate-1-naphthyl ester serving as a substrate to generate the 1-naphthol. 1-naphthol has reducing property, and can reduce blue oxidized TMB to colorless TMB. Therefore, the activity of the plant esterase can be determined from the degree of reduction of oxidized TMB. Research shows that the pesticide (especially organophosphorus pesticide and carbamate) can inhibit the activity of plant esterase with high sensitivity, and the residue degree of the pesticide is in negative correlation with the activity of plant esterase. Therefore, the reduced degree of the oxidized TMB can indirectly reflect the residual degree of the pesticide in the sample, i.e., the more the pesticide residue, the smaller the reduced degree of the oxidized TMB, and the darker the color thereof.
In a preferred embodiment, the concentration of the hydrochloric acid solution is 0.001-0.05mol/L, FeTMPyP4The concentration of the solution is 10-20 mu mol/L, the concentration of the TMB solution is 5-10mmol/L, and the concentration of the hydrogen peroxide solution is 5-10 mmol/L; hydrochloric acid solution, FeTMPyP4The volume ratio of the solution, the TMB solution and the hydrogen peroxide solution is 1:1:6: 4. In this example, the hydrochloric acid solution and FeTMPyP were specifically defined4The concentration and volume ratio of the solution, the TMB solution and the hydrogen peroxide solution are that the prepared developing solution has better developing effect and higher sensitivity under the proportion. The concentration of the hydrochloric acid solution may be 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, or any range thereof, FeTMPyP4The solution may also have a concentration of 12, 14, 16, 18, and any range therein, the TMB solution may also have a concentration of 6, 7, 8, 9, and any range therein, and the hydrogen peroxide solution may also have a concentration of 6, 7, 8, 9, and any range therein.
In a preferred embodiment, the enzyme tablet 6 is prepared by the following method:
soaking the polyester fiber wafer by adopting a phosphate buffer solution, and naturally airing to obtain a pretreated polyester fiber wafer;
and uniformly mixing the phosphate buffer solution, the plant esterase enzyme solution, the bovine serum albumin solution and the glutaraldehyde solution to obtain a mixed solution, dropwise adding the mixed solution to the pretreated polyester fiber wafer, and naturally drying to obtain an enzyme sheet 6. The polyester fiber wafer is soaked by adopting a phosphate buffer solution, so that the activity of the plant esterase is kept, wherein the pH value of the phosphate buffer solution is 7.2-7.4, and the polyester fiber wafer is dried in the shade and in a ventilation manner. The bovine serum albumin in the enzyme sheet 6 plays a role of providing a protein environment beneficial to the stability of the plant esterase, and the glutaraldehyde serves as a crosslinking agent of the protein in the enzyme sheet 6 so that the plant esterase is fixed on the membrane sheet in a crosslinking state. In addition, the size of the enzyme tablet 6 is adjusted according to the sizes of the enzyme reaction bin 5 and the adapter cylinder 7, and the leakage is prevented on the premise of ensuring that the enzyme tablet 6 is fully contacted with the solution.
In a preferred embodiment, the concentration of the phosphate buffer solution is 0.01-0.03mol/L, the concentration of the bovine serum albumin solution is 0.5-1%, and the concentration of the glutaraldehyde solution is 0.05-0.1%;
the volume ratio of the phosphate buffer solution to the plant esterase enzyme solution to the bovine serum albumin solution to the glutaraldehyde solution is 16.5:25:7.5: 1;
the plant esterase enzyme solution is prepared by the following method:
mixing flour and distilled water according to the mass: after being uniformly mixed according to the volume ratio of 1:4-1:6, the mixture is frozen and centrifuged at the rotating speed of 4000-.
In this embodiment, the concentrations of the phosphate buffer, the bovine serum albumin solution and the glutaraldehyde solution are specifically limited, the concentration of the phosphate buffer may be 0.02mol/L, the concentration of the bovine serum albumin solution may be 0.6%, 0.7%, 0.8%, 0.9% and any value within the range, and the concentration of the glutaraldehyde solution may be 0.06%, 0.07%, 0.08%, 0.09% and any value within the range.
The detection method for detecting the pesticide on site by using the pesticide on-site detection device comprises the following steps:
a pesticide field detection device is adopted to absorb the sample solution to an enzyme reaction bin 5, the sample solution is fully contacted with an enzyme sheet 6, and the reaction is carried out for 1 to 3 hours at room temperature; continuously sucking the acetic acid-1-naphthyl ester substrate solution to an enzyme reaction bin 5, and reacting for 20-40min at room temperature;
inverting the pesticide field detection device, pulling a push rod of the injector 1 to transfer all the solution in the enzyme reaction bin 5 to the color reaction bin 3, fully contacting the solution with the display piece 4, and reacting for 20-40min at room temperature;
the display piece 4 is taken out, and the detection result is displayed and analyzed.
According to the detection method provided by the embodiment, the enzyme sheet 6 and the display sheet 4 are respectively positioned in different reaction bins in the detection process, the enzyme sheet and the display sheet are not in direct contact, the color development process is not interfered, the color development is more uniform, and the accuracy of the detection result is improved.
In a preferred embodiment, the displaying and analyzing of the detection result includes at least one of qualitative analysis, semi-quantitative analysis or quantitative analysis;
the qualitative analysis comprises the steps of comparing the colors of a display piece 4 obtained by detecting the sample solution with the colors of a display piece 4 obtained by detecting water by taking water as a control group, thereby qualitatively judging whether the pesticide exists or not;
the semi-quantitative analysis comprises the steps of manufacturing a standard colorimetric card, detecting the color of a display piece 4 obtained by comparing a standard colorimetric card with a sample solution, and roughly judging the content of the pesticide;
the quantitative detection comprises the steps of making a standard curve, measuring the absorbance of the agarose gel layer in the display piece 4 at the wavelength of 609nm by adopting an ultraviolet-visible spectrophotometer to detect the sample solution, and obtaining the residual concentration of the pesticide by utilizing the standard curve.
Wherein, the qualitative analysis process comprises the following steps:
1) set up control experiment
And (3) detecting the water by using a pesticide field detection device by using the water as a sample, wherein the obtained display piece 4 is colorless, which indicates that the contrast setting is successful.
2) Qualitative analysis
The color of the display piece 4 in the sample detection is carefully observed for the display piece 4 obtained after the comparison sample solution is detected. If the display piece 4 is colorless as same as the control display piece 4, indicating that no enzyme activity inhibitor exists in the sample; if the display piece 4 is blue, the enzyme activity inhibitor (including organophosphorus pesticides and carbamate pesticides) exists in the sample no matter how the degree is, and accordingly, qualitative judgment can be made on whether the sample contains organophosphorus and carbamate pesticides.
The semi-quantitative analysis process comprises the following steps:
1) production of standard colorimetric card
The plant esterase enzyme solutions were diluted with water to give dilutions of various doses (0%, 20%, 25%, 29%, 33%, 40%, 67%, 100%). Uniformly mixing 0.03mol/L phosphate buffer solution (pH7.2-7.4), plant esterase diluent, 0.5% bovine serum albumin solution and 0.05% glutaraldehyde solution according to the volume ratio of 16.5:25:7.5:1, taking 25 mu L of the mixture, dropwise adding the mixture onto a polyester fiber wafer, and placing the wafer in a shade for natural drying to obtain a plurality of enzyme sheets 6 with different enzyme dosages; these enzyme sheets 6 having different enzyme dosages were mounted in different detection apparatuses, respectively, and the detection process was performed according to the detection method of the above-described embodiment. The obtained display sheet 4 was subjected to color information collection with a digital camera (nikon D7000) and printed on a sheet of a4 paper (100 g of tara) by a printer (hewlett packard 377dw) to form a standard color chart.
2) Semi-quantitative analysis
Comparing the display piece 4 obtained in the sample detection with a standard colorimetric card, the content of the uninhibited enzyme can be roughly judged, and the inhibition condition of the enzyme activity can be further obtained.
The quantitative analysis process comprises the following steps:
1) drawing of standard curve
The plant esterase enzyme solution was diluted with water to give 20%, 25%, 29%, 33%, 40% dilutions. Uniformly mixing 0.03mol/L phosphate buffer solution (pH7.2-7.4), plant esterase diluent, 0.5% bovine serum albumin solution and 0.05% glutaraldehyde solution according to the volume ratio of 16.5:25:7.5:1, taking 25 mu L of the mixture, dropwise adding the mixture onto a polyester fiber wafer, and placing the wafer in a shade for natural drying to obtain a plurality of enzyme sheets 6 with different enzyme dosages; the enzymes are dosed in different amountsThe sheets 6 were respectively mounted in different detection devices, the detection process was carried out according to the detection method of the above example to obtain the display sheet 4, the gel sheet in the display sheet 4 was carefully peeled off from the polyester fiber film, folded in half from the center, placed in a constant cuvette containing 1.6mL of water, the absorption spectrum in the range of 750nm was measured with a conventional UV-Vis spectrophotometer, and the absorbance at 609nm (A) was recorded609). With A609The ordinate represents the dosage of the plant esterase, the abscissa represents the dosage of the plant esterase, the graph is drawn, and finally the standard curve can be obtained through linear fitting.
2) Quantitative analysis
For the display piece 4 obtained in the sample test, the gel piece was carefully peeled off from the polyester fiber film, folded in half in the middle, placed in a constant cell containing 1.6mL of water, and measured for A609. A is to be609The value is put into a standard curve linear fitting formula, so that the content of the uninhibited enzyme can be calculated, and the condition of the pesticide residue in the sample can be evaluated.
In order to describe the pesticide field detection device provided by the embodiment of the present invention in more detail, the following description will be made with reference to specific embodiments.
Examples
(1) Assembly of detection device
Step 1: preparation of enzyme tablet 6
① preparation of plant esterase
After uniformly mixing commercially available flour and distilled water according to the proportion of 1:5(W/V), freezing and centrifuging at the rotating speed of 6000r/min, and collecting supernatant, namely the plant esterase enzyme solution.
② activation treatment of polyester fiber film
The polyester fiber film was cut into a circular piece having a diameter of 2.5cm by a circular punch. Then, immersing the wafer into 0.02mol/L phosphate buffer solution (pH7.2-7.4), standing at 4 ℃ for 2 hours, and naturally airing in a cool and ventilated place for later use.
③ preparation of enzyme tablet 6
Uniformly mixing 0.02mol/L phosphate buffer solution (pH7.2-7.4), plant esterase enzyme solution, 0.5% bovine serum albumin solution and 0.05% glutaraldehyde solution according to the proportion of 16.5:25:7.5:1(V/V), taking 25 mu L of the mixture, dropwise adding the mixture onto a polyester fiber wafer, and placing the wafer in a shade for natural drying to obtain an enzyme sheet 6. And placing the enzyme tablets 6 in a self-sealing bag, sealing, and storing at a low temperature of-20 ℃ for later use.
Step 2: preparation of display sheet 4
① preparation of color developing solution
Uniformly mixing 0.02mol/L hydrochloric acid solution, 15 mu mol/L FeTMPyP4 solution, 8mmol/L TMB solution and 8mmol/L hydrogen peroxide solution according to the ratio of 1:1:6:4(V/V), placing in a constant temperature box at 37 ℃ for reaction for 50min, changing the mixed solution from colorless to dark blue, and performing ultrasound at room temperature for 3min to obtain a color developing solution.
② preparation of display sheet 4
Firstly, a polyester fiber wafer with the diameter of 2.5cm is taken, and a cylindrical mold with the diameter of 2cm and the height of 3mm is placed on the polyester fiber wafer. Then, agarose gel liquid with the concentration of 45mmol/L and the temperature of 65 ℃ is uniformly mixed with developing solution according to the proportion of 1:2(V/V), and then poured into a cylindrical mold. And after the gel is cooled and solidified, taking down the mold. A circular hole having a diameter of 3mm was punched in the center of the gel by a punch to obtain a display piece 4. The display piece 4 is soaked in water and stored at 4 ℃ for later use.
And step 3: assembly of detection device
As shown in fig. 1A-1C, the color reaction chamber 3 is inverted, and the display plate 4 and the first silica gel sealing ring 9 are sequentially placed; after the upper end of the enzyme reaction bin 5 is sleeved with the first rubber ring 10, the color reaction bin 3 is inserted, and the display piece 4 is firmly fixed in the color reaction bin 3. Then, sequentially putting the enzyme sheet 6 and the second silica gel sealing ring 11 into the enzyme reaction bin 5; after one end of the adapter tube 7 is sleeved with the second rubber ring 12, the enzyme reaction bin 5 is inserted, and the enzyme sheet 6 is firmly fixed in the enzyme reaction bin 5. And finally, inserting a second rubber plug 14 into the other end of the adapter cylinder 7, connecting a pipette tip 8, connecting the syringe 1 and the color reaction chamber 3 through a first rubber plug 13, and finishing the assembly of the detection device.
(2) The specific steps of sample detection are as follows:
step 1: enzyme tablet 6 reaction
1mL of the sample solution was aspirated by the syringe 1 in the detection apparatus, and the sample solution was brought into sufficient contact with the enzyme sheet 6 and reacted at room temperature for 2 hours. Then, 500. mu.L of 0.03mol/L acetic acid-1-naphthyl ester substrate solution (the solvent is a mixed solution of ethanol and water in a ratio of 1:1 (V/V)) was continuously sucked and reacted at room temperature for 30 min.
Step 2: display sheet 4 reaction
And (3) inverting the device, continuously pulling a push rod of the injector 1, completely transferring the solution in the enzyme reaction bin 5 into the color reaction bin 3, and reacting for 30min at room temperature. The enzyme reaction bin 5 and the color reaction bin 3 are disassembled, the color reaction bin 3 is opened, and the display piece 4 is taken out.
And step 3: set up control experiment
With water as the sample, repeating steps 1 and 2, the resulting display piece 4 was colorless, indicating that the control setting was successful.
And 4, step 4: qualitative judgment
The color of the display sheet 4 in the sample test was carefully observed in comparison with the control display sheet 4. If the display piece 4 is colorless as same as the control display piece 4, indicating that no enzyme activity inhibitor exists in the sample; if a blue color is present on display chip 4, it indicates, to whatever extent, that an enzyme activity inhibitor (including organophosphorous pesticides and carbamate pesticides) is present in the sample. Therefore, qualitative judgment can be made on whether the sample contains organophosphorus pesticides and carbamate pesticides.
And 5: semi-quantitative analysis
① Standard colorimetric card
The plant esterase enzyme solutions were diluted with water to give dilutions of various doses (0%, 20%, 25%, 29%, 33%, 40%, 67%, 100%). And then uniformly mixing 0.03mol/L phosphate buffer solution (pH7.2-7.4), plant esterase diluent, 0.5% bovine serum albumin solution and 0.05% glutaraldehyde solution according to the volume ratio of 16.5:25:7.5:1, taking 25 mu L of the mixture, dropwise adding the mixture onto a polyester fiber wafer, and placing the wafer in a shade for natural drying to obtain a plurality of enzyme sheets 6 with different enzyme dosages.
These enzyme sheets 6 having different enzyme dosages were set in different detection apparatuses, and the detection process was performed according to step 1 and step 2, respectively, using water as a sample. The obtained display sheet 4 was subjected to color information collection with a digital camera (nikon D7000) and printed on a sheet of a4 paper (100 g of tara) by a printer (hewlett packard 377dw) to form a standard color chart.
② semi-quantitative analysis
Comparing the display piece 4 obtained in the sample detection with a standard colorimetric card, the content of the uninhibited enzyme can be roughly judged, and the inhibition condition of the enzyme activity can be further obtained.
Step 6: quantitative analysis
① drawing standard curve
From the display pieces 4 obtained when the standard colorimetric card was produced in step 4, the display pieces 4 corresponding to the enzyme pieces 6 having the enzyme doses of 20%, 25%, 29%, 33%, and 40% were selected, respectively. Carefully peeling the gel sheet in the display sheet 4 from the polyester fiber film, folding the gel sheet in the middle, placing the gel sheet in a constant cuvette containing 1.6mL of water, measuring the absorption spectrum in the range of 500-750nm by using a conventional ultraviolet-visible spectrophotometer, and recording the absorbance (A) at 609nm609). With A609The ordinate represents the dosage of the plant esterase, the abscissa represents the dosage of the plant esterase, the graph is drawn, and finally the standard curve can be obtained through linear fitting.
② quantitative analysis
For the display piece 4 obtained in the sample test, the gel piece was carefully peeled off from the polyester fiber film, folded in half in the middle, placed in a constant cell containing 1.6mL of water, and measured for A609. A is to be609The value is put into a standard curve linear fitting formula, so that the content of the uninhibited enzyme can be calculated, and the condition of the pesticide residue in the sample can be evaluated.
(3) Analysis of sample detection results
The device and the method for detecting the pesticide on site are applied, and the common organophosphorus pesticide dichlorvos is used as a detection object for testing. The result shows that the utility model discloses detection device and method can detect the dichlorvos that concentration is as low as 0.15 mug/kg, and sensitivity is higher than current on-the-spot detection technique far away.
The experimental steps are as follows:
1mL of a 0.15. mu.g/kg DDVP solution was sucked into the enzyme reaction chamber 5 by the syringe 1 of the detection apparatus, and the solution was brought into sufficient contact with the enzyme chip 6 and reacted at room temperature for 1 hour. Continuously sucking 500 mu L of 0.05mol/L acetic acid-1-naphthyl ester substrate solution, and reacting for 30min at room temperature. Then, the device is inverted, the push rod of the injector 1 is continuously pulled, all the solution in the enzyme reaction chamber 5 is transferred into the color reaction chamber 3, and the reaction is carried out for 30min at room temperature. The enzyme reaction bin 5 and the color reaction bin 3 are disassembled, the color reaction bin 3 is opened, and the display piece 4 is taken out. The color change was observed compared to the control display piece 4. Carefully peeling the gel sheet from the polyester fiber film, folding the gel sheet in the middle, placing the gel sheet in a constant cuvette containing 1.6mL of water, measuring the absorption spectrum within the range of 500-750nm, and recording A609。
The experimental results are as follows:
as shown in fig. 2, when the color of the display sheet 4 is observed, the display sheet 4 appears faint blue, as compared with the control display sheet 4. Further comparing the absorption spectra measured in the control display piece 4 and the display piece 4 after the detection of 0.15. mu.g/kg of dichlorvos, the absorption spectrum of the control display piece 4 does not have an absorption peak at 609nm and thus appears colorless, as shown in the following figure. And the detected display piece 4 has an obvious absorption peak at 609nm, which is consistent with weak blue.
Claims (8)
1. The utility model provides a field detection device of pesticide, includes syringe (1), syringe (1) is including locating injection port (2) of syringe (1) one end, its characterized in that: the device is characterized by further comprising a reactor connected with the injection port (2), wherein the reactor comprises an enzyme reaction bin (5) and a color reaction bin (3), the color reaction bin (3) is connected with the injection port (2), the enzyme reaction bin (5) is far away from the injection port (2), and the enzyme reaction bin (5) is connected with the color reaction bin (3); the device also comprises an adapter cylinder (7) connected with the enzyme reaction bin (5); a display sheet (4) is arranged between the color reaction bin (3) and the enzyme reaction bin (5), and an enzyme sheet (6) is arranged between the enzyme reaction bin (5) and the adapter cylinder (7).
2. The on-site pesticide detection device according to claim 1, characterized in that: the pipette tip (8) is connected with the adapter cylinder (7).
3. The on-site pesticide detection device according to claim 1, characterized in that: the display sheet (4) comprises a polyester fiber layer and an agarose gel layer, wherein the agarose gel layer comprises agarose gel and a color developing solution.
4. The on-site pesticide detection device according to claim 1, characterized in that: the enzyme reaction bin (5) comprises a first tubular enzyme reaction bin (51) and a second tubular enzyme reaction bin (52), and the inner diameter of the second enzyme reaction bin (52) is larger than that of the first enzyme reaction bin (51);
the color reaction bin (3) comprises a first cylindrical color reaction bin (31) and a second cylindrical color reaction bin (32), and the inner diameter of the second color reaction bin (32) is larger than that of the first color reaction bin (31);
the outer diameter of the first enzyme reaction bin (51) is smaller than the inner diameter of the second color reaction bin (32);
the adapter cylinder (7) is cylindrical, and the outer diameter of the adapter cylinder (7) is smaller than the inner diameter of the second enzyme reaction bin (52);
the first enzyme reaction bin (51) is partially sleeved in the second color reaction bin (32), and the adapter cylinder (7) is partially sleeved in the second enzyme reaction bin (52).
5. The on-site pesticide detection device according to claim 4, characterized in that: the display sheet (4) is arranged in the second chromogenic reaction chamber (32), and the enzyme sheet (6) is arranged in the second enzyme reaction chamber (52).
6. The on-site pesticide detection device according to claim 5, characterized in that: the display piece (4) with be equipped with first silica gel sealing washer (9) between first enzyme reaction storehouse (51), enzyme piece (6) with be equipped with second silica gel sealing washer (11) between a section of thick bamboo (7).
7. The on-site pesticide detection device as set forth in claim 6, wherein: a first rubber ring (10) is arranged between the first enzyme reaction bin (51) and the second color reaction bin (32), and a second rubber ring (12) is arranged between the adapter cylinder (7) and the second enzyme reaction bin (52).
8. The on-site pesticide detection device as set forth in claim 7, wherein: the outer diameter of the first enzyme reaction bin (51) is 11mm, the inner diameter of the first enzyme reaction bin is 9mm, and the height of the first enzyme reaction bin is 20 mm; the outer diameter of the second enzyme reaction bin (52) is 14mm, the inner diameter is 12mm, and the height is 15 mm;
the outer diameter of the first color reaction bin (31) is 11mm, the inner diameter of the first color reaction bin is 9mm, and the height of the first color reaction bin is 20 mm; the outer diameter of the second color reaction bin (32) is 14mm, the inner diameter is 12mm, and the height is 15 mm;
the outer diameter of the adapter cylinder (7) is 11mm, the inner diameter is 9mm, and the height is 20 mm;
the display piece (4) is circular, and the diameter of the display piece (4) is 12 mm; the enzyme sheet (6) is round, and the diameter of the enzyme sheet (6) is 12 mm;
the outer diameter of the first rubber ring (10) is 12mm, the inner diameter of the first rubber ring is 11mm, and the height of the first rubber ring is 10 mm; the outer diameter of the second rubber ring (12) is 12mm, the inner diameter of the second rubber ring is 11mm, and the height of the second rubber ring is 10 mm;
the outer diameter of the first silica gel sealing ring (9) is 12mm, and the inner diameter of the first silica gel sealing ring is 10 mm; the outer diameter of the second silica gel sealing ring (11) is 12mm, and the inner diameter is 10 mm.
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