CN110455759A - A method of Rogor detection kit and detection Rogor concentration based on copper nano particles - Google Patents
A method of Rogor detection kit and detection Rogor concentration based on copper nano particles Download PDFInfo
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- CN110455759A CN110455759A CN201910747364.9A CN201910747364A CN110455759A CN 110455759 A CN110455759 A CN 110455759A CN 201910747364 A CN201910747364 A CN 201910747364A CN 110455759 A CN110455759 A CN 110455759A
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- rogor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
Abstract
A method of Rogor detection kit and application kit detection Rogor concentration based on copper nano particles belong to biosensor technology field.We construct a kind of Portable kit based on hydrogel by being embedded in copper nano particles in agarose aquogel.For accurate quantitative analysis, the high resolution camera of present invention smart phone has recorded the variation of the fluorescence color on Portable kit, its mechanism is when introducing pesticide Rogor in detection architecture, urase catalyzing urea can be inhibited to generate ammonia, when Rogor concentration increases, when introducing pesticide Rogor, the ammonia that pesticide inhibits urase that urase catalyzing urea is caused to generate is reduced, and the amount that ammonia etching copper nano particles generate copper cluster is reduced.Copper cluster has fluorescent effect, the difference for leading to fluorescence intensity according to the pesticide of various concentration amount causes the color change of kit, and corresponding image is then converted into digital information using ImageJ software, image and Rogor log concentration are in a linear relationship, so as to carry out quantitative detection.
Description
Technical field
The invention belongs to biosensor technology fields, and in particular to a kind of Rogor detection reagent based on copper nano particles
The method of box and application kit detection Rogor concentration.
Background technique
Pesticide, by control pest, plays promotion agricultural production and guarantee food storage most important in rural activity
Effect.Typical Representative of the Rogor as organophosphorus pesticide is widely used to prevention and treatment fluke disease.However, wide due to Rogor
It is general using and it is improper processing and caused by pollution by pesticides, it has also become the public pursue agricultural sustainable development most severe challenge it
One.In particular, Rogor can be used as a kind of neurotoxin, inhibit the activity of central nervous system acetylcholinesterase, even if dense
Spend lower, can also inhibit choline to cause nervous centralis transmitting impaired, further damage human health, in addition cause it is fatal after
Fruit.Correct management pesticide, accurate evaluation pollution by pesticides are the primary requirements for ensureing human health, ensureing food quality.Using gas
The goldstandards such as phase chromatography, high performance liquid chromatography, mass spectrum method has carried out high-precision measurement to pesticide residue in agricultural products.This
Although a little technologies have stronger trace analysis ability, the requirement to instrument and equipment is high, time-consuming etc., and seriously to limit it existing
The application of field, especially in case of emergency cannot achieve real-time monitoring.In view of the limitation of traditional detection method, research is fast
Speed, alternative that is easy, selectively detecting Rogor have great importance.
Since cost-effectiveness is high, easy to operate, easy to carry, on the market commercially available colloidal nano test paper for family and
Individual carries out qualitative or sxemiquantitative to pesticide and screens, and the color change of test paper can observe with the naked eye.But these on-site tests
Method cannot identify the slight change of target from signal index, be not suitable for monitoring trace pesticide.In addition, band made of paper lacks
Stability.To meet quick, stable, accurate quantitative analysis needs, consolidate there is an urgent need to other readily identified and efficient identification
Phase carrier.In recent years, agarose aquogel relies on its mechanical stability and flexibility, in sensing, actuator and biomedical neck
Domain causes more and more concerns.No wonder to be, direct signal when by recording using external trigger is converted, and is passed
Sensor is in checkpoint (POC) testing field, to be easier to test.Hydrogel is a kind of up-and-coming analysis of agricultural drugs carrier,
It is also seldom to the research of hydrogel but so far, especially in terms of the on-site test of food samples.Improve poc testing
There are two types of the main paths of energy: enhancing echo signal and enhancing stability.In first method, fluorescent nano material is due to it
Unique physics and chemical characteristic can be used as indicator and carry out intensified response.In the second approach, manufacture has mechanically stable
The hydrogel platform of property may be very effective.
Summary of the invention
The purpose of the invention is to overcome, the testing time existing in the prior art is longer, error is larger, dense to Rogor
The problems such as more demanding, test method is cumbersome and detection sensitivity is lower is spent, is provided a kind of based on copper nano particles detection agriculture
The method of the kit and application kit detection Rogor concentration of medicine Rogor.
This copper nano particles agarose aquogel kit with response characteristic, it is alternative, delicately to Rogor
Carry out accurate measurements.Copper nano particles are pre-loaded in agarose aquogel, it is micro- using commercial 96 holes as signal indicator
Plate makes Portable kit.In the sensor-based system, using ammonia in-situ corrosion technique, convert non-fluorescence copper nanocluster to
Green fluorescence copper nanocluster.Using the concentration of urase catalyzing urea hydrolysis control ammonia, can be used for detecting urease inhibitor.
In Portable kit, inhibits urease activity by target spot of Rogor, further suppress hydrolysis, become with fluorescence signal
Change, to determine the amount of pesticide Rogor according to the variation of fluorescence signal.For accurate quantitative analysis, we use the high score of smart phone
Fluorescence color variation of the resolution cameras record on Portable kit, mechanism of the present invention is in detection architecture
When introducing pesticide Rogor, urase catalyzing urea can be inhibited to generate ammonia, when Rogor concentration increases, when introducing pesticide Rogor, pesticide
The ammonia for inhibiting urase that urase catalyzing urea is caused to generate is reduced, and the amount that ammonia etches copper nano particles generation copper cluster is reduced.Copper cluster tool
There is fluorescent effect, the difference of fluorescence intensity is caused according to the pesticide of various concentration amount, causes the color change of kit, it is then sharp
Corresponding image is converted into digital information with ImageJ software, image and Rogor log concentration are in a linear relationship, so as to
Carry out quantitative analysis.It is worth noting that, the kit based on hydrogel is combined with smart phone reader, successfully answer
With skilled execute-in-place platform, the accurate content for detecting Rogor acid in pear juice, as a result satisfactory, analytic process is convenient.By
In its great convenience and high stability, the availability of field portable kit will be such that frequent screening and monitoring becomes can
Can, to ensure food safety and ensureing that human health provides new approach.The kit can be easy, quickly, with sensitivity
Detect pesticide Rogor.
The purpose of the present invention can be achieved through the following technical solutions:
(1) ascorbic acid (AA) is used as preparing the reducing agent of copper nano particles and capping agent.By 0.8~1.2mL, 80~
100mmol L-1Cu (NO3)2Aqueous solution is added drop-wise to 8~10mL, 80~100mmol L with vigorous stirring-1Ascorbic acid
In aqueous solution, obtained mixture is stirred at room temperature 50~70 minutes, static 10~20 hours, it is molten to obtain copper nano particles
Liquid；
(2) at 100 DEG C, the agarose of 5~10mg is dissolved in the centrifuge tube equipped with 0.3~0.4mL water, fine jade is obtained
Lipolysaccharide hydrogel；The copper nano particles solution and 200~250 μ L, 20~40mmol that will be generated in 800~900 μ L steps (1)
L-1Sodium hydrate aqueous solution is added in above-mentioned agarose aquogel at 100 DEG C；50~60 DEG C are dropped to temperature of reaction system
When, stirring is uniformly mixed reactant, obtains agarose mixed solution；Then the agarose for being 150~200 μ L by 10 parts of volumes
Mixed solution is respectively added in 10 holes of 96 hole elisa Plates, etc. temperature drop to room temperature, the agarose in 96 hole elisa Plates is mixed
Solution natural coagulation is closed to store at room temperature to obtain copper nano particles kit；
It (3) is respectively 0.001,0.005,0.01,0.02,0.05,0.1,0.5,1,2 and 5mgL by 20~30 μ L, concentration-1
Rogor aqueous solution be added to 45~55 μ L, 9~11 U mL-1Inhibit 25~35 minutes in urase aqueous solution, then again by 100
~150 μ L, 9~11mmol L-1Urea is added in the mixed liquor of Rogor and urase, is mixed 10~20 minutes；Finally, by 10 parts
Isometric urea, Rogor and urase mixed solution are added to the Sheng that step (2) obtains with agarose mixed solution in step (2)
Have in the corresponding aperture of 96 hole elisa Plates of agarose mixed solution, reacts 10~15 minutes；Under 365nm ultraviolet irradiation,
The color of agarose aquogel in 10 holes is imported into ImageJ program by the picture that smart phone is shot, clicks Analyze
The image information of kit is converted to form and aspect parameter i.e. intensity by → Gels → Plot lanes → Analyze → Measure；
It is fitted to obtain linear standard curve by origin software again, wherein abscissa is the logarithm of Rogor concentration, and ordinate is reagent
Intensity corresponding to box color；
Urea, Rogor and urase mixed solution are added in copper nano particles kit, mixed solution touches 96 holes
The chromogenic reaction of the agarose aquogel solidified in plate, kit is triggered.Under the irradiation of ultraviolet lamp, can be observed with pleasure
The color of the increase of fruit concentration, kit is gradually become colorless by green, and change of gradient is presented.
(4) the unknown Rogor aqueous solution of 20~30 μ L, concentration is added to 45~55 μ L, 9~11 U mL-1Urase is water-soluble
Inhibit in liquid 25~35 minutes, then again by 100~150 μ L, 9~11mmol L-1Urea is added to the mixing of Rogor and urase
In liquid, mix 10~20 minutes；Finally, urea that will be isometric with agarose mixed solution in step (2), Rogor and urase are mixed
It closes solution to be added in the corresponding aperture of 96 hole elisa Plates for filling agarose mixed solution that step (2) obtains, reaction 10~15
Minute；Under 365nm ultraviolet irradiation, the picture after the color of agarose aquogel is shot by smart phone is imported
ImageJ program clicks Analyze → Gels → Plot lanes → Analyze → Measure, by the image information of kit
Be converted to form and aspect parameter i.e. intensity；In the standard curve that intensity data substitution step (3) is obtained again, so that Rogor be calculated
The concentration of aqueous solution.
The invention has the characteristics that:
(1) the Rogor detection kit prepared by the present invention based on copper nano particles is active high, easily prepared etc. excellent
Point can carry out qualitative and quantitative analysis to Rogor using its fluorescent characteristic.
(2) kit applied by the present invention has easy building process, easily operated, fast response time, at low cost etc.
Advantage.The detection method of the Rogor concentration based on copper nano particles be used to construct kit sensor simultaneously, realize to Rogor
Carry out the rapid detection in scene, moreover it is possible to the picture importing ImageJ program after being shot by smart phone, click Analyze →
It is i.e. strong to be converted to form and aspect parameter by Gels → Plot lanes → Analyze → Measure for the image information of kit
To realize detection.
The present invention is based on the Rogor detection kit of copper nano particles in building portable minisize diagnostic device to realize i.e.
When examine (POCT) in terms of have huge potentiality.
Detailed description of the invention
Fig. 1: obtained standard curve is fitted to Rogor quantitative detection based on copper nano particles kit.
Specific embodiment
Embodiment 1: the synthesis of copper nano particles
Ascorbic acid (AA) is used as preparing the reducing agent of copper nano particles and capping agent.By the Cu (NO of 1.0mL3)2It is water-soluble
Liquid (100mmol L–1) it is added drop-wise to 10mL aqueous ascorbic acid (100mmol L with vigorous stirring-1) in.It is anti-by what is obtained
It answers mixture to be stirred at room temperature 60 minutes, static 12 hours, obtains copper nano particles solution.
Embodiment 2: the production of kit
At 100 DEG C, 7.5g agarose is dissolved in the centrifuge tube equipped with 0.375mL water, agarose aquogel is obtained；
The copper nano particles solution and 225 μ L sodium hydrate aqueous solution (30mmol L that will be generated in 900 μ L steps (1)-1) be added on
It states to obtain in agarose aquogel, when temperature of reaction system being waited to drop to 50 DEG C, is stirred for being uniformly mixed reactant, obtains agar
Sugared mixed solution.Then the agarose mixed solution that 10 parts of volumes are respectively 200 μ L is respectively added to the 10 of 96 hole elisa Plates
In a hole, etc. temperature drop to room temperature, the agarose solution natural coagulation in 96 hole elisa Plates has obtained copper nano particles reagent
Box stores at room temperature.
Embodiment 3: application of the kit to the detection of Rogor content
It is respectively 0.001,0.005,0.01,0.02,0.05,0.1,0.5,1,2 and 5mgL by 25 μ L, concentration-1Rogor
Aqueous solution is added to 50 μ L, 10U mL-1Inhibit 30 minutes in urase aqueous solution, then again by 150 μ L, 10mmol L-1Urea adds
Enter into the mixed liquor of Rogor and urase, mixes 15 minutes；Finally, by the bodies such as 10 parts and agarose mixed solution in embodiment 2
Long-pending 200 μ L urea, Rogor and urase mixed solution is added to the 96 hole enzymes for filling agarose mixed solution that embodiment 2 obtains
In the corresponding aperture of target, react 15 minutes；Under 365nm ultraviolet irradiation, by the color of agarose aquogel in 10 holes
The picture importing ImageJ program shot by smart phone, click Analyze → Gels → Plot lanes → Analyze →
The image information of kit is converted to form and aspect parameter i.e. intensity by Measure；It is fitted by origin software and is linearly marked again
Directrix curve, wherein abscissa is the logarithm of Rogor concentration, and ordinate is intensity corresponding to kit color.
When Rogor concentration is 1mgL-1When, under the irradiation of ultraviolet lamp, kit is in green fluorescence and can be within a certain period of time
Keep colour stable.
Under the irradiation of ultraviolet lamp, it with the naked eye can be observed increase with Rogor concentration, the color of kit is from green
Color gradually arrives colourless and change of gradient is presented.
Claims (2)
1. a kind of method of the Rogor detection kit detection Rogor concentration based on copper nano particles, its step are as follows:
(1) by 0.8~1.2mL, 80~100mmolL-1Cu (NO3)2Aqueous solution is added drop-wise to 8~10mL, 80 with vigorous stirring
~100mmol L-1Aqueous ascorbic acid in, obtained mixture is stirred at room temperature 50~70 minutes, static 10~
20 hours, obtain copper nano particles solution；
(2) at 100 DEG C, the agarose of 5~10mg is dissolved in the centrifuge tube equipped with 0.3~0.4mL water, agarose is obtained
Hydrogel；The copper nano particles solution and 200~250 μ L, 20~40mmol L that will be generated in 800~900 μ L steps (1)-1Hydrogen
Aqueous solution of sodium oxide is added in above-mentioned agarose aquogel at 100 DEG C；When temperature of reaction system drops to 50~60 DEG C, stir
Mixing is uniformly mixed reactant, obtains agarose mixed solution；Then the agarose that 10 parts of volumes are 150~200 μ L is mixed
Solution is respectively added in 10 holes of 96 hole elisa Plates, etc. temperature drop to room temperature, the mixing of agarose in 96 hole elisa Plates is molten
Liquid natural coagulation stores at room temperature to obtain copper nano particles kit；
It (3) is respectively 0.001,0.005,0.01,0.02,0.05,0.1,0.5,1,2 and 5mgL by 20~30 μ L, concentration-1Pleasure
Fruit aqueous solution is added to 45~55 μ L, 9~11U mL-1Inhibit 25~35 minutes in urase aqueous solution, then again by 100~150 μ
L, 9~11mmolL-1Urea is added in the mixed liquor of Rogor and urase, is mixed 10~20 minutes；Finally, by 10 parts and step
(2) what the isometric urea of agarose mixed solution, Rogor and urase mixed solution were added to that step (2) obtains in fills agar
In the corresponding aperture of 96 hole elisa Plates of sugared mixed solution, react 10~15 minutes；Under 365nm ultraviolet irradiation, by 10
The color of agarose aquogel imports ImageJ program by the picture that smart phone is shot in hole, clicks Analyze → Gels
The image information of kit is converted to form and aspect parameter i.e. intensity by → Plot lanes → Analyze → Measure；Pass through again
Origin software is fitted to obtain linear standard curve, and wherein abscissa is the logarithm of Rogor concentration, and ordinate is kit color
Corresponding intensity；
(4) the unknown Rogor aqueous solution of 20~30 μ L, concentration is added to 45~55 μ L, 9~11U mL-1Press down in urase aqueous solution
System 25~35 minutes, then again by 100~150 μ L, 9~11mmolL-1Urea is added in the mixed liquor of Rogor and urase, is mixed
It closes 10~20 minutes；Finally, urea, Rogor and urase mixed solution that will be isometric with agarose mixed solution in step (2)
It is added in the corresponding aperture of 96 hole elisa Plates for filling agarose mixed solution that step (2) obtains, reacts 10~15 minutes；In
Under 365nm ultraviolet irradiation, the picture after the color of agarose aquogel is shot by smart phone imports ImageJ journey
Sequence clicks Analyze → Gels → Plot lanes → Analyze → Measure, the image information of kit is converted to color
Phase parameter, that is, intensity；In the standard curve that intensity data substitution step (3) is obtained again, so that Rogor aqueous solution be calculated
Concentration.
2. a kind of Rogor detection kit based on copper nano particles, it is characterised in that: it is to be prepared by following steps,
(1) by 0.8~1.2mL, 80~100mmolL-1Cu (NO3)2Aqueous solution is added drop-wise to 8~10mL, 80 with vigorous stirring
~100mmol L-1Aqueous ascorbic acid in, obtained mixture is stirred at room temperature 50~70 minutes, static 10~
20 hours, obtain copper nano particles solution；
(2) at 100 DEG C, the agarose of 5~10mg is dissolved in the centrifuge tube equipped with 0.3~0.4mL water, agarose is obtained
Hydrogel；The copper nano particles solution and 200~250 μ L, 20~40mmol L that will be generated in 800~900 μ L steps (1)-1Hydrogen
Aqueous solution of sodium oxide is added in above-mentioned agarose aquogel at 100 DEG C；When temperature of reaction system drops to 50~60 DEG C, stir
Mixing is uniformly mixed reactant, obtains agarose mixed solution；Then the agarose that 10 parts of volumes are 150~200 μ L is mixed
Solution is respectively added in 10 holes of 96 hole elisa Plates, etc. temperature drop to room temperature, the mixing of agarose in 96 hole elisa Plates is molten
Liquid natural coagulation stores at room temperature to obtain copper nano particles kit.
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| CN114371158A (en) * | 2022-01-29 | 2022-04-19 | 吉林大学 | Preparation method of hydrogel kit for monitoring degradation of organophosphorus pesticide |
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