CN106872422B - 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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- CN106872422B CN106872422B CN201611265817.7A CN201611265817A CN106872422B CN 106872422 B CN106872422 B CN 106872422B CN 201611265817 A CN201611265817 A CN 201611265817A CN 106872422 B CN106872422 B CN 106872422B
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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 relates to using water-soluble CdSe/ZnS quantum dot measure uric acid content, using in the presence of HRP, hydrogen peroxide can aoxidize catechol, and 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 known as fluorescence semiconductor nano particle, can launch fluorescence after the irradiation of quantum dot stimulated luminescence, and can
To be combined with large biological molecule, there is extremely strong biocompatibility.Compared with traditional fluorescent dye, quantum dot has tunable
Launch wavelength, the quantum dot for possessing different emission spectrum can be obtained by adjusting its particle diameter.Meanwhile quantum dot have it is larger
Stokes shift and symmetric form fluorescence spectrum, can effectively weaken exciting light to launch light influence, 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, age of onset carries
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, determining the method for 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 high specificity, but because its is cumbersome, and it is used
Expensive reagents, during to add ascorbic acid oxidase 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 are as follows：
Uric acid is under uric acid enzymatic, oxidation generation allantoin and hydrogen peroxide.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
High polymer 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 and uric acid concentration are directly proportional in the range of 0.05-10 μm of ol/L, and are 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, this method compared with prior art, have height
The sensitivity of degree.
Preparation method is as follows：
(1) CdSe/ZnS quantum dots are prepared；
(2) high polymer 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 are reacted with uricase, HRP and catechol respectively, adds water-soluble quantum dot
After solution, compared after determining its 640nm fluorescent emission peak intensity, obtain 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 warming up to 380-320 DEG C；Added
Methanol is measured, by being centrifugally separating to obtain CdSe nano particles；With zinc stearate, hexamethyldisilathiane [(TMS)2S] Zn conducts
Precursor, CdSe nano particles are added, 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, obtain 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 of acquisition at room temperature, until chloroform volatilizees completely, very
Sky is dried, that is, obtains 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
640nm fluorescent emission peak intensity；
(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, quantum dot solution is added after shake well, determine it
640nm fluorescent emission peak intensity, build 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 warming 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 solution natural cooling, is filtered with Medium speed filter paper
Insoluble black precipitate is removed, is centrifuged off bulky grain, collects and is dialysed in supernatant injection bag filter；By the production 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 purifies after adding Carbodiimide reaction 6h in dimethyl sulfoxide, obtains Polymer Solution；It will be prepared
CdSe/ZnS quantum dots and Polymer Solution be substantially dissolved in jointly in chloroform, 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 of acquisition at room temperature, until chlorine
Imitative volatilization 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 pH=7 phosphate buffer solution
Quantum dot solution for 10 μ g/mL is standby；In pH=7 phosphate buffer 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, filled
10mL quantum dot solutions are added after dividing shaking, determine its 640nm fluorescent emission peak intensity；
(4) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to using pH=7 phosphate buffer solution
Quantum dot solution for 10 μ g/mL is standby；In pH=7 phosphate buffer add containing 0.5mg uricases, 2mg HRP and
The solution of 1mg catechols, after being well mixed, it is gradually added 0.05-10 μm of ol/L various concentrations respectively with micro syringe
Uric acid solution, quantum dot solution is added after 37 DEG C of reactions, shake well, determines its 640nm fluorescent emission peak intensity, is built
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 high polymer 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, because high polymer material prevents weight in quantum dot well
Metal to external diffusion, toxicity very little.
3rd, it have passed through two-step reaction so that there is no other in testing sample so that the material that quantum dot fluorescence is quenched, therefore
The method of foundation has a specificity of height, and accuracy is good.
Brief description of the drawings
Fig. 1 is the fluorescence spectra that finite concentration UA is added dropwise in the quantum dot solution of embodiment 1, 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.
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 warming 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 solution natural cooling, is filtered with Medium speed filter paper
Insoluble black precipitate is removed, is centrifuged off bulky grain, collects and is dialysed in supernatant injection bag filter；By the production 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 purifies after adding Carbodiimide reaction 6h in dimethyl sulfoxide, obtains Polymer Solution；It will be prepared
CdSe/ZnS quantum dots and Polymer Solution be substantially dissolved in jointly in chloroform, 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 of acquisition at room temperature, until chlorine
Imitative volatilization 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 pH=7 phosphate buffer solution
Quantum dot solution for 10 μ g/mL is standby；In pH=7 phosphate buffer 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, filled
10mL quantum dot solutions are added after dividing shaking, determine its 640nm fluorescent emission peak intensity；
(4) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to using pH=7 phosphate buffer solution
Quantum dot solution for 10 μ g/mL is standby；In pH=7 phosphate buffer add containing 0.5mg uricases, 2mg HRP and
The solution of 1mg catechols, after being well mixed, it is gradually added 0.05-10 μm of ol/L various concentrations respectively with micro syringe
Uric acid solution, quantum dot solution is added after 37 DEG C of reactions, shake well, determines its 640nm fluorescent emission peak intensity, is built
Linear relationship；The fluorescent emission peak intensity for the testing sample that step (3) is obtained substitutes into formula and calculates its uric acid concentration.
In 0.05-10 μm of ol/L, good linear relationship, coefficient correlation 0.9982 is presented in uric acid concentration.
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.
For obtain suitable concentration samples, it is necessary to secondary water dilute 2000 times after determine.Measurement result is shown in Table 1, as a result
Show, the relative deviation of this method is less than 2.23%, and it is good to illustrate that this method has between 97%-103% for the rate of recovery
Preci-sion and accuracy.
Table 1 is loaded recovery experiment
Claims (1)
1. a kind of method of uric acid in quantum dots characterization body fluid, 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 be stirred vigorously 350 DEG C rapidly
In trioctylphosphine oxide (TOPO) TOPO；Then 250 DEG C are cooled to rapidly, are then slowly warming up to 280 DEG C；Add excessive methanol, by from
The isolated CdSe nano particles of the heart；8mg zinc stearates, 12mg hexamethyldisilathianes [(TMS) are added in TOPO2S]Zn
With CdSe nano particles, add micro-wave oven high-temperature heating, after solution natural cooling, filtered off with Medium speed filter paper remove it is insoluble black
Color precipitates, and is centrifuged off bulky grain, collects and is dialysed in supernatant injection bag filter；Product after dialysis is subjected to vacuum rotation
Turn to be evaporated to solid-like, obtain CdSe/ZnS quantum dots；
(2) by poly- (tert. butylacrylate-ethyl propylene acid esters-methacrylic acid) triblock copolymers of 10mL and 10mL octylames
Mixed dissolution purifies after adding Carbodiimide reaction 6h in dimethyl sulfoxide, obtains Polymer Solution；By what is be 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 of acquisition at room temperature, 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, concentration is configured to as 10 using pH=7 phosphate buffer solution
μ g/mL quantum dot solution is standby；Added in pH=7 phosphate buffer containing 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, shake well
10mL quantum dot solutions are added afterwards, determine its 640nm fluorescent emission peak intensity；
(4) a certain amount of water-soluble quantum dot powder is weighed, concentration is configured to as 10 using pH=7 phosphate buffer solution
μ g/mL quantum dot solution is standby；Added in pH=7 phosphate buffer containing 0.5mg uricases, 2mg HRP and 1mg
The solution of tea phenol, after being well mixed, it is gradually added the uric acid of 0.05-10 μm of ol/L various concentrations respectively with micro syringe
Solution, quantum dot solution is added after 37 DEG C of reactions, shake well, determine its 640nm fluorescent emission peak intensity, structure is linear
Relation；The fluorescent emission peak intensity for the testing sample that step (3) is obtained substitutes into formula and calculates its uric acid concentration.
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