CN106872422A - The method of uric acid in quantum dots characterization body fluid - Google Patents
The method of uric acid in quantum dots characterization body fluid Download PDFInfo
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- CN106872422A CN106872422A CN201611265817.7A CN201611265817A CN106872422A CN 106872422 A CN106872422 A CN 106872422A CN 201611265817 A CN201611265817 A CN 201611265817A CN 106872422 A CN106872422 A CN 106872422A
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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"
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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"
- G01N2021/6432—Quenching
Abstract
The present invention is the method for uric acid in quantum dots characterization body fluid, it is related to determine uric acid content using water-soluble CdSe/ZnS quantum dot, using in the presence of HRP, hydrogen peroxide can aoxidize catechol, the oxidation product of formation can make the characteristic that the fluorescence of quantum dot is efficiently quenched, and obtain the uric acid concentration in testing sample.With high sensitivity, small toxicity, specificity is high, accuracy is good the characteristics of.
Description
Technical field
The present invention relates to a kind of method for detecting uric acid in body fluid, and in particular to using uric acid in quantum dots characterization body fluid
Method.
Background technology
Quantum dot is also called fluorescence semiconductor nano particle, and fluorescence can be launched after the irradiation of quantum dot stimulated luminescence, and can
Combined with large biological molecule, with extremely strong biocompatibility.Compared with traditional fluorescent dye, quantum dot has tunable
Launch wavelength, the quantum dot that can possess different emission spectrum to obtain by adjusting its particle diameter.Meanwhile, quantum dot has larger
Stokes shift and symmetric form fluorescence spectrum, can effectively weaken influence of the exciting light to launching light, and then reduce system
System complexity.
In recent years, with the improvement of living standards, the incidence of disease of hyperuricemia and gout rises year by year, and age of onset is carried
Before.The patient of the concurrent hyperuricemia of cardiovascular and cerebrovascular diseases easily induces acute myocardial infarction AMI, palsy, causes case fatality rate to increase.As
The other components of one of component of metabolic syndrome, hyperuricemia and metabolic syndrome are such as:Hypertension, carbohydrate metabolism disturbance, fat
Metabolic disorder, obesity, atherosclerosis etc. are normal and deposit and influence each other.
At present, the method for determining uric acid mainly has enzyme process and the major class of non-enzyme process two.With high performance liquid chromatography, capillary electricity
The non-enzyme process such as swimming method is compared, and the enzyme process based on urate oxidase has specificity high, but because its is cumbersome and used
Expensive reagents, during ascorbic acid oxidase is added to reduce the interference of the reducing substanceses such as ascorbic acid.Therefore still deposit
In many defects.
The content of the invention
The method for we providing uric acid in a kind of quantum dots characterization body fluid, principle is as follows:
Under uric acid enzymatic, oxidation generates allantoin and hydrogen peroxide to uric acid.In the presence of HRP, hydrogen peroxide energy
Enough aoxidize catechol, the oxidation product of formation can be such that the fluorescence of quantum dot is efficiently quenched.Meanwhile, we will using emulsion process
Macromolecular material after hydrophobic modification directly wraps up fat-soluble quantum dot, and the quantum dot of acquisition is more quick to the oxidation product
Sense.And the fluorescent quenching degree of sensor is directly proportional to uric acid concentration in the range of 0.05-10 μm of ol/L, and is capable of detecting when
0.01 μm of ol/L uric acid.It thus provides a kind of uric acid method in detection body fluid, the method compared with prior art, with height
The sensitivity of degree.
Preparation method is as follows:
(1) CdSe/ZnS quantum dots are prepared;
(2) macromolecular material after hydrophobic modification is directly wrapped up by the quantum dot using emulsion process, obtains water-soluble amount
Sub- point;
(3) testing sample and standard sample and the reaction of uricase, HRP and catechol are added into water-soluble quantum dot respectively
After solution, compared after the fluorescent emission peak intensity for determining its 640nm, obtained the uric acid concentration in testing sample.
Further, there is provided a kind of method of uric acid in quantum dots characterization body fluid,
(1) with vigorous stirring, by cadmium methide [(CH3)2Cd] and TOPSe (trioctylphosphine phosphorus selenium) be expelled to 300-400 rapidly
DEG C trioctylphosphine oxide (TOPO) (TOPO) in;Then 200-280 DEG C is cooled to rapidly, is then slowly warmed up to 380-320 DEG C;Added
Amount methyl alcohol, by being centrifugally separating to obtain CdSe nano particles;With zinc stearate, hexamethyldisilathiane [(TMS)2S] Zn conducts
Precursor, adds CdSe nano particles, and CdSe/ZnS quantum dots are synthesized in TOPO;
(2) poly- (tert. butylacrylate-ethyl propylene acid esters-methacrylic acid) triblock copolymer is mixed with octylame
It is dissolved in dimethyl sulfoxide, is purified after adding Carbodiimide reaction, obtains Polymer Solution;The CdSe/ZnS that will be prepared
Quantum dot and Polymer Solution are substantially dissolved in chloroform jointly, be added in ultrasonic procedure emulsifying agent Lipoid E-80 and
In distilled water, and continue ultrasound, magnetic agitation then is carried out to the emulsion for obtaining at room temperature again, until chloroform volatilizees completely, very
Sky is dried, that is, obtain water-soluble quantum dot;
(3) a certain amount of water-soluble quantum dot powder is weighed, quantum dot solution is configured to using phosphate buffer solution
It is standby;A certain amount of uricase, HRP and catechol are added in phosphate buffer, after being well mixed, is progressively added with micro syringe
Enter certain density testing sample (human body fluid), react at a certain temperature, quantum dot solution is added after shake well, determine it
The fluorescent emission peak intensity of 640nm;
(4) a certain amount of water-soluble quantum dot powder is weighed, quantum dot solution is configured to using phosphate buffer solution
It is standby;A certain amount of uricase, HRP and catechol are added in phosphate buffer, be well mixed after, with micro syringe respectively by
Step adds the uric acid solution of various concentrations, reacts at a certain temperature, and quantum dot solution is added after shake well, determines it
The fluorescent emission peak intensity of 640nm, builds linear relationship;The testing sample intensity that step (3) is obtained substitutes into formula and calculates it
Uric acid concentration.
Further, there is provided a kind of method of uric acid in quantum dots characterization body fluid,
(1) by 10mg cadmium methides [(CH3)2Cd] and 10mg TOPSe (trioctylphosphine phosphorus selenium) be expelled to what is be stirred vigorously rapidly
In 350 DEG C of trioctylphosphine oxide (TOPO) (TOPO);Then 250 DEG C are cooled to rapidly, are then slowly warmed up to 280 DEG C;Add excessive first
Alcohol, by being centrifugally separating to obtain CdSe nano particles;8mg zinc stearates, 12mg hexamethyldisilathianes are added in TOPO
[(TMS)2S] Zn and CdSe nano particles, micro-wave oven high-temperature heating is added, after after solution natural cooling, filtered with Medium speed filter paper
Insoluble black precipitate is removed, bulky grain is centrifuged off, is collected and is dialysed in supernatant injection bag filter;By the product after dialysis
Thing carries out rotary evaporation in vacuo to solid-like, obtains CdSe/ZnS quantum dots;
(2) by poly- (tert. butylacrylate-ethyl propylene acid esters-methacrylic acid) triblock copolymers of 10mL and 10mL
Octylame mixed dissolution is purified in dimethyl sulfoxide after adding Carbodiimide reaction 6h, obtains Polymer Solution;To prepare
CdSe/ZnS quantum dots and Polymer Solution be substantially dissolved in chloroform jointly, emulsifying agent is added in ultrasonic procedure
In Lipoid E-80 and distilled water, and continue ultrasound, magnetic agitation then is carried out to the emulsion for obtaining at room temperature again, until chlorine
It is imitative to volatilize completely, that is, obtain water-soluble quantum dot;
(3) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to using the phosphate buffer solution of pH=7
For the quantum dot solution of 10 μ g/mL is standby;In the phosphate buffer of pH=7 add containing 0.5mg uricases, 2mg HRP and
The solution of 1mg catechols, after being well mixed, testing sample (human body fluid) is gradually added with micro syringe, in 37 DEG C of reactions, is filled
10mL quantum dot solutions are added after dividing shaking, the fluorescent emission peak intensity of its 640nm is determined;
(4) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to using the phosphate buffer solution of pH=7
For the quantum dot solution of 10 μ g/mL is standby;In the phosphate buffer of pH=7 add containing 0.5mg uricases, 2mg HRP and
The solution of 1mg catechols, after being well mixed, 0.05-10 μm of various concentrations of ol/L is gradually added with micro syringe respectively
Uric acid solution, is reacted at a certain temperature, and quantum dot solution is added after shake well, determines the fluorescent emission peak intensity of its 640nm
Degree, builds linear relationship;The testing sample intensity that step (3) is obtained substitutes into formula and calculates its uric acid concentration.
As seen from the above analysis, relative to prior art, advantage of the invention is that:
1st, the macromolecular material after hydrophobic modification is directly wrapped up by fat-soluble quantum dot, the quantum of acquisition using emulsion process
Point has high sensitivity.
2nd, compared with commercially available or homemade most of quantum dot, weight in quantum dot is prevented well due to macromolecular material
Metal to external diffusion, toxicity very little.
3rd, have passed through two-step reaction so that there is no other in testing sample so that the material that is quenched of quantum dot fluorescence, therefore
The method of foundation has the specificity of height, and accuracy is good.
Brief description of the drawings
Fig. 1 be embodiment 1 quantum dot solution in be added dropwise over the fluorescence spectra of finite concentration UA, it can be seen that amount
The fluorescence intensity of son point solution has reached peak value at 640nm, and display quantum dot is successfully prepared.
Specific embodiment
Embodiment 1
(1) by 10mg cadmium methides [(CH3)2Cd] and 10mg TOPSe (trioctylphosphine phosphorus selenium) be expelled to what is be stirred vigorously rapidly
In 350 DEG C of trioctylphosphine oxide (TOPO) (TOPO);Then 250 DEG C are cooled to rapidly, are then slowly warmed up to 280 DEG C;Add excessive first
Alcohol, by being centrifugally separating to obtain CdSe nano particles;8mg zinc stearates, 12mg hexamethyldisilathianes are added in TOPO
[(TMS)2S] Zn and CdSe nano particles, micro-wave oven high-temperature heating is added, after after solution natural cooling, filtered with Medium speed filter paper
Insoluble black precipitate is removed, bulky grain is centrifuged off, is collected and is dialysed in supernatant injection bag filter;By the product after dialysis
Thing carries out rotary evaporation in vacuo to solid-like, obtains CdSe/ZnS quantum dots;
(2) by poly- (tert. butylacrylate-ethyl propylene acid esters-methacrylic acid) triblock copolymers of 10mL and 10mL
Octylame mixed dissolution is purified in dimethyl sulfoxide after adding Carbodiimide reaction 6h, obtains Polymer Solution;To prepare
CdSe/ZnS quantum dots and Polymer Solution be substantially dissolved in chloroform jointly, emulsifying agent is added in ultrasonic procedure
In Lipoid E-80 and distilled water, and continue ultrasound, magnetic agitation then is carried out to the emulsion for obtaining at room temperature again, until chlorine
It is imitative to volatilize completely, that is, obtain water-soluble quantum dot;
(3) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to using the phosphate buffer solution of pH=7
For the quantum dot solution of 10 μ g/mL is standby;In the phosphate buffer of pH=7 add containing 0.5mg uricases, 2mg HRP and
The solution of 1mg catechols, after being well mixed, testing sample (human body fluid) is gradually added with micro syringe, in 37 DEG C of reactions, is filled
10mL quantum dot solutions are added after dividing shaking, the fluorescent emission peak intensity of its 640nm is determined;
(4) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to using the phosphate buffer solution of pH=7
For the quantum dot solution of 10 μ g/mL is standby;In the phosphate buffer of pH=7 add containing 0.5mg uricases, 2mg HRP and
The solution of 1mg catechols, after being well mixed, 0.05-10 μm of various concentrations of ol/L is gradually added with micro syringe respectively
Uric acid solution, is reacted at a certain temperature, and quantum dot solution is added after shake well, determines the fluorescent emission peak intensity of its 640nm
Degree, builds linear relationship;The testing sample intensity that step (3) is obtained substitutes into formula and calculates its uric acid concentration.
In 0.05-10 μm of ol/L, uric acid concentration is presented good linear relationship, and coefficient correlation is 0.9982.
Embodiment 2 is loaded recovery experiment
To investigate the feasibility of this method, the content of uric acid in people's urine sample is determined by Standard entertion recovery experiment.
To be determined after obtaining suitable concentration samples, it is necessary to dilute 2000 times with secondary water.Measurement result is shown in Table 1, as a result
Show, the relative deviation of this method is less than 2.23%, and the rate of recovery is between 97%-103%, illustrate that this method has good
Preci-sion and accuracy.
Table 1 is loaded recovery experiment
Claims (3)
1. in a kind of quantum dots characterization body fluid uric acid method, it is characterised in that step is as follows:
(1) CdSe/ZnS quantum dots are prepared;
(2) macromolecular material after hydrophobic modification is directly wrapped up by the quantum dot using emulsion process, obtains water-soluble quantum dot;
(3) testing sample and standard sample and the reaction of uricase, HRP and catechol are added into water-soluble quantum dot solution respectively
Afterwards, compared after the fluorescent emission peak intensity for determining its 640nm, obtained the uric acid concentration in testing sample.
2. in a kind of quantum dots characterization body fluid as claimed in claim 1 uric acid method, it is characterised in that step is as follows:
(1) with vigorous stirring, by cadmium methide [(CH3)2Cd] and TOPSe (trioctylphosphine phosphorus selenium) be expelled to 300-400 DEG C rapidly
In trioctylphosphine oxide (TOPO) (TOPO);Then 200-280 DEG C is cooled to rapidly, is then slowly warmed up to 380-320 DEG C;Add excessive first
Alcohol, by being centrifugally separating to obtain CdSe nano particles;With zinc stearate, hexamethyldisilathiane [(TMS)2S] Zn as precursor,
CdSe nano particles are added, CdSe/ZnS quantum dots are synthesized in TOPO;
(2) by poly- (tert. butylacrylate-ethyl propylene acid esters-methacrylic acid) triblock copolymer and octylame mixed dissolution
In dimethyl sulfoxide, purified after adding Carbodiimide reaction, obtain Polymer Solution;The CdSe/ZnS quantum that will be prepared
Point and Polymer Solution are substantially dissolved in chloroform jointly, and emulsifying agent Lipoid E-80 and distillation are added in ultrasonic procedure
In water, and continue ultrasound, magnetic agitation then is carried out to the emulsion for obtaining at room temperature again, until chloroform volatilizees completely, vacuum is done
It is dry, that is, obtain water-soluble quantum dot;
(3) a certain amount of water-soluble quantum dot powder is weighed, it is standby to be configured to quantum dot solution using phosphate buffer solution;
A certain amount of uricase, HRP and catechol are added in phosphate buffer, after being well mixed, one is gradually added with micro syringe
Determine the testing sample (human body fluid) of concentration, react at a certain temperature, quantum dot solution is added after shake well, determine it
The fluorescent emission peak intensity of 640nm;
(4) a certain amount of water-soluble quantum dot powder is weighed, it is standby to be configured to quantum dot solution using phosphate buffer solution;
A certain amount of uricase, HRP and catechol are added in phosphate buffer, after being well mixed, is progressively added respectively with micro syringe
Enter the uric acid solution of various concentrations, react at a certain temperature, quantum dot solution is added after shake well, determine its 640nm's
Fluorescent emission peak intensity, builds linear relationship;It is dense that the testing sample intensity substitution formula that step (3) is obtained calculates its uric acid
Degree.
3. in a kind of quantum dots characterization body fluid as claimed in claim 1 or 2 uric acid method, it is characterised in that step is as follows:
(1) by 10mg cadmium methides [(CH3)2Cd] and 10mg TOPSe (trioctylphosphine phosphorus selenium) be expelled to 350 DEG C for being stirred vigorously rapidly
Trioctylphosphine oxide (TOPO) (TOPO) in;Then 250 DEG C are cooled to rapidly, are then slowly warmed up to 280 DEG C;Excessive methanol is added, is led to
Cross and be centrifugally separating to obtain CdSe nano particles;8mg zinc stearates, 12mg hexamethyldisilathianes [(TMS) are added in TOPO2S] Zn and CdSe nano particles, micro-wave oven high-temperature heating is added, after after solution natural cooling, removal is filtered not with Medium speed filter paper
Dissolubility black precipitate, is centrifuged off bulky grain, collects and is dialysed in supernatant injection bag filter;Product after dialysis is carried out
Rotary evaporation in vacuo obtains CdSe/ZnS quantum dots to solid-like;
(2) by poly- (tert. butylacrylate-ethyl propylene acid esters-methacrylic acid) triblock copolymers of 10mL and 10mL octylames
Mixed dissolution is purified in dimethyl sulfoxide after adding Carbodiimide reaction 6h, obtains Polymer Solution;By what is prepared
CdSe/ZnS quantum dots and Polymer Solution are substantially dissolved in chloroform jointly, and emulsifying agent Lipoid is added in ultrasonic procedure
In E-80 and distilled water, and continue ultrasound, magnetic agitation then is carried out to the emulsion for obtaining at room temperature again, until chloroform is waved completely
Hair, that is, obtain water-soluble quantum dot;
(3) a certain amount of water-soluble quantum dot powder is weighed, it is 10 to be configured to concentration using the phosphate buffer solution of pH=7
The quantum dot solution of μ g/mL is standby;Added in the phosphate buffer of pH=7 and contain 0.5mg uricases, 2mg HRP and 1mg
The solution of tea phenol, after being well mixed, testing sample (human body fluid) is gradually added with micro syringe, in 37 DEG C of reactions, is fully shaken
10mL quantum dot solutions are added after shaking, the fluorescent emission peak intensity of its 640nm is determined;
(4) a certain amount of water-soluble quantum dot powder is weighed, it is 10 to be configured to concentration using the phosphate buffer solution of pH=7
The quantum dot solution of μ g/mL is standby;Added in the phosphate buffer of pH=7 and contain 0.5mg uricases, 2mg HRP and 1mg
The solution of tea phenol, after being well mixed, the uric acid for being gradually added the 0.05-10 μm of various concentrations of ol/L respectively with micro syringe is molten
Liquid, is reacted at a certain temperature, and quantum dot solution is added after shake well, determines the fluorescent emission peak intensity of its 640nm, is built
Linear relationship;The testing sample intensity that step (3) is obtained substitutes into formula and calculates its uric acid concentration.
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Cited By (4)
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CN108896750A (en) * | 2018-05-11 | 2018-11-27 | 江苏大学 | A kind of preparation method and purposes of BSA-Au/Ag NCs/OPD/HRP proportional-type fluorescent optical sensor |
CN111999275A (en) * | 2020-08-25 | 2020-11-27 | 中南民族大学 | Method for rapidly and quantitatively measuring pH value and/or uric acid |
CN117849015A (en) * | 2024-03-04 | 2024-04-09 | 臻萃(江苏)酶科技发展有限公司 | Uric acid detection method |
CN117849015B (en) * | 2024-03-04 | 2024-05-10 | 臻萃(江苏)酶科技发展有限公司 | Uric acid detection method |
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Granted publication date: 20180403 Termination date: 20191230 |