CN103115919A - Glucose detection test paper - Google Patents
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- CN103115919A CN103115919A CN2012105799539A CN201210579953A CN103115919A CN 103115919 A CN103115919 A CN 103115919A CN 2012105799539 A CN2012105799539 A CN 2012105799539A CN 201210579953 A CN201210579953 A CN 201210579953A CN 103115919 A CN103115919 A CN 103115919A
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Abstract
The invention provides test paper and a detection method for glucose determination. According to the test paper, glucose oxidase and peroxidase are embedded in a cavity of a polymer, namely hollow nanofibers, in an in-situ manner by using a coaxial co-spinning electrospinning technique, and a color developing agent, namely o-dianisidine, is uniformly distributed on shell layer walls of the hollow nanofibers, and thus the glucose detection test paper with a macroscopic non-woven membrane morphology is obtained. When glucose liquid is dripped on the test paper, glucose is oxidized by the glucose oxidase, hydrogen peroxide is produced, and the colorless o-dianisidine is oxidized to a colored product under the catalysis of the peroxidase, so that the test paper can be subjected to color development, and the quantitative detection on the glucose is carried out through determining the color change of the test paper. A glucose detection reagent based on the hollow nanofibers has the outstanding advantages of simplicity in preparation, small enzyme consumption amount, simplicity and convenient in detection operation, and high storage stability and has an application prospect in the quick and accurate determination of the glucose.
Description
Technical field
The present invention relates to a kind of for quantitatively detecting the Test paper of glucose content, comprise for the mensuration test paper of glucose detection and the method for utilizing this test paper to detect, particularly a kind of glucose detection test paper of the immobilized multienzyme system based on the hollow structure nanofiber.
Background technology
Diabetes are global frequently-occurring disease and common disease, and along with the raising of people's living standard and increasing of elderly population, its incidence of disease is obvious ascendant trend.China's incidence of disease has reached 2.5-3%, extremely near the 2-5% of European and American developed countries.Approximately there is patient more than 200,000,000 in the whole world, has become global health care issues, and is seriously threatening the mankind's health, becomes the third-largest dangerous disease that is only second to cardiovascular disease, cancer.Thereby China is not only in diagnosis and the treatment of diabetes, is also the key subjects that whole world medical circle faces.
At present, detecting the main and easy method of glucose content is to utilize blood glucose meter to detect, and the core of blood glucose meter is the test paper that a kind of patient of being applicable to oneself detects blood sugar.The patient can by capillary whole blood as sample drop on blood sugar test paper, then utilize blood glucose meter to carry out measuring content of blood sugar according to different test paper principles.The ultimate principle of blood glucose meter detection in the market is that a kind of glucose oxidase that utilizes comes fast, measures accurately, easily the biology sensor of glucose content as sensitive element.The glucose sensor pick-up unit uses the physical and chemical principle of transducer can be divided into electrochemical sensor, piezoelectric sensor, thermoelectric pickup, acoustic sensor and optical sensor based on it.Wherein, what be most widely used in blood glucose meter is electrochemical sensor and optical sensor, the electrochemical sensor test paper comprises reagent mixture and one or more electrode, and wherein to comprise at least a electron transfer agent be electron mediator and analyte specific biological catalytic proteins such as concrete enzyme to this reagent mixture.Sensors with auxiliary electrode were relies on the transfer of electronics between electron mediator and electrode surface, and plays a role by measuring electrochemical redox reaction.Although the electro-catalysis sensor has long-time stability, high to equipment requirement due to them, device is complicated, have a very large problem to the analyte selectivity is relatively poor.The optical sensor test paper mainly comprises reagent mixture (analyte specific biological catalytic proteins such as concrete enzyme and chromogenic reagent), electrochemical sensor is fairly simple relatively, need not the transfer of electronics between complicated electrode, only the colour developing by special developer realizes the detection to concentration of glucose.
Enzyme in common glucose detection test paper be all by with enzyme solutions absorption or directly spray the enzyme powder on blank test paper with the enzyme molecule attached in the test paper surface, the enzyme rate is low, complex operation has caused a large amount of wastes but this kind method is carried.In order to guarantee to use enzyme work in the term of validity to reach the requirement of detection sensitivity, often need to roll up the addition of enzyme expensive in reagent, therefore increased to a certain extent the glucose detection cost.Even adhere to so excessive enzyme, the condition of storage of above two kinds of test paper is still harsher, usually needs drying, lucifuge, low temperature and sealing to preserve.Be mainly because of specific enzyme (glucose oxidase and the peroxidase) poor stability in test paper, easily inactivation and some chromogenic reagents are seen what the easy decomposition of light caused.The packing that [CN202414431U] proposed a kind of novel blood sugar test paper improves the stability of blood sugar test paper, this packing comprises the body of establishing a plurality of blister cavity, evenly independently store blood sugar test paper in each described blister cavity, the blister cavity upper opening, the aperture position envelope has aluminium foil.This utility model is provided with a plurality of blister cavity, every blood sugar test paper independently can be put in each blister cavity when producing, and then encloses aluminium foil; During use, corresponding unknown aluminium foil on destruction blister cavity that can be independent, independent taking-up a slice blood sugar test paper does not affect the storage condition of other blood sugar test papers.But the method preparation manipulation more complicated, and further increased again the extra cost of blood sugar test paper.
Various patents all are devoted to provide a kind of more convenient and detection method that cost is low, but still well do not address this problem, and the cost of detection is maintained on very high level.
At present based on the patent of electrochemical glucose sensor mainly by following several:
[CN 101487814A] related to the preparation method that a kind of inclusion is the nano-gold glucose oxidase sensor of electron mediator.This invention is stirring reaction in organic solvent with ferrocene and mercapto-cyclodextrin, adds ethanol to make the reactant precipitate and separate, the absolute ethyl alcohol washing and precipitating, and drying gets yellow inclusion electronic media solid; With HAuCl
4.4H
2O is dissolved in DMF, is dissolved with NaBH4 with another and aforementioned solution mixes rapidly, and stir and react, centrifugation, solid product is used ethanol again: water mixed solvent washing, vacuum drying; Solid product is dissolved in intermediate water, gets glucose oxidase and be dissolved in and form enzyme solutions in intermediate water; Two solution are mixed, mixed liquor is dropped in the platinum disk electrode surperficial, in the saturated vapour of glutaraldehyde, obtain the nano-gold glucose oxidase sensor that inclusion is electron mediator.Although the present invention has solved some defective that the first second generation enzyme sensor exists, preparation process is too loaded down with trivial details, and practicality is too low.
[CN 102621207A] related to a kind of glucose sensor, and the working electrode surface of the glucose sensor of this invention is covered by glucose oxidase and triangle silver nano-grain composite membrane.By the triangle silver nano-grain is used for glucose sensor, the sensitivity that has effectively improved glucose detector makes detectability reach 3-20 μ mol/L.
[CN 101530327A] announced a kind of subcutaneous tissue real-time monitoring and measured glucose sensor and preparation method thereof with the needle-like current type, comprise a needle-like formula reference electrode and at least one needle-like formula working electrode, working electrode comprises conductive layer and enzyme membrane layer, it is characterized in that: described working electrode also comprises macromolecular material theca interna and macromolecular material control diffusion layer, conductive layer, macromolecular material theca interna, enzyme membrane layer and macromolecular material control diffusion layer cover successively from the inside to the outside and form.Method for making according to the wood invention, but glucose sensor good stability, sensitivity are high, the range of linearity of output current and concentration of glucose is extensive, short continuous real-time monitoring of response time, the fastness of enzyme combination, good stability, sensitivity, selectivity and reappearance are high.
[CN 202355415U] utility model patent relates to a kind of Internet of Things blood sugar instrument that can the monitoring human physical exertion.Comprise: the housing of panel, panel is provided with the LCDs of display unit and controls a plurality of keys of controlling of unit, be provided with the human motion energy in housing and consume detecting unit, also be provided with the blood sugar test unit in housing, the blood sugar test unit is provided with the blood sugar test paper detecting position on described housing, the blood sugar test unit with wired or wireless communication unit, display unit, control the unit and be electrically connected to; Control the unit and be provided with energy consumption-blood sugar test switch, energy consumption-blood sugar test switch is provided with energy consumption-blood sugar test conversion operations key on described panel.The utlity model has two monitoring means, be used for energy energy consumption monitoring unit that the monitoring human physical exertion consumes and the monitoring means of monitoring human blood sugar.The utility model can also Monitoring Blood Glucose in energy consumption monitoring, and the data that the monitoring of two aspects obtains are transferred out rapidly by network.
At present based on the patent of optical glucose sensor device mainly by following several:
[CN 101947115A] provides a kind of implantable human blood glucose concentration continuous monitoring based on optical fiber attenuation total reflection, consisted of by double light path, light source is arranged, attenuator, spectroscope, fiber coupling device, optical fiber ATR sensor, measure photo-detector, the reference light detector, data acquisition system (DAS) and computing machine, after light source is decayed through attenuator, divide to serve as reasons through spectroscope and measure two light paths of light and reference light formation, a light path wherein is, measure the gloss fiber coupling device, be coupled into optical fiber ATR sensor by coupling process, the light that sees through through optical fiber ATR sensor receives by measuring photo-detector, another light path is that reference light is directly received by the reference light detector, through measuring signal that photo-detector and reference light detector receive by the data collection system synchronizing collection, be sent to computing machine and carry out analyzing and processing and display measurement result, computing machine also is connected with light source.The present invention adopts in the body direct measuring method, directly measures the concentration of glucose in tissue fluid, has avoided stripped measuring error.
[CN 102469962A] provide a kind of from measuring through time ground optical spectra and the calibrating patterns of biosome, with non-invasive mode through the time ground calculation blood glucose value device for estimating blood sugar level.Possesses the calibrating patterns manufacturing mechanism of making calibrating patterns from a plurality of data groups of a plurality of calibrating patterns or calibrating patterns making of.Consist of as follows: measure person under inspection's biosome spectrum and set benchmark spectrum; Obtain the mensuration spectrum of the timing beyond the said reference spectroscopic assay time and said reference spectrum poor, be difference spectrum; Change the calibrating patterns that uses in above-mentioned calculation according to the variation of above-mentioned difference spectrum.Thus, inferring of blood glucose value can be carried out, especially high the inferring of precision can be carried out through when monitoring time ground.
In addition, also have a lot of patents and document to relate to blood glucose meter.But be to the more apparent little changes of blood glucose meter mostly.
[CN 202421188U] related to a kind of novel blood-sugar detecting instrument, comprise: shell, display, button and test paper mouth that the blood glucose meter original paper is installed, display is arranged on shell upper, button is arranged in the middle part of shell, and the test paper mouth is arranged on outer casing underpart, also comprises bayonet socket blood collecting pen support, support is arranged on shell, the unique difference of this design is fixedly blood collecting pen support is installed on blood-sugar detecting instrument, blood collecting pen can be fixed on blood-sugar detecting instrument, uses easy to carry.
[CN 202421126U] provides a kind of blood glucose meter with water battery, comprise single-chip microcomputer, the input end of described single-chip microcomputer is connected with respectively input circuit and A/D change-over circuit, output terminal is connected with the LCD display unit, the input end of described A/D change-over circuit is connected with the excuse circuit of measuring blood glucose concentration value, and described single-chip microcomputer input end also is connected with water battery and temperature measurement circuit.Characteristics of the present utility model are to obtain to provide by water battery the ability of blood glucose meter work, take full advantage of simultaneously very environmental protection of green energy resource.
[301925966S], [301642379S], [301926004S], [301925968S], [301925636S], [301925711S], [301925740S], [302065336S] philosophy are all delivered patent for the appearance design of blood glucose meter.
For when guaranteeing high detection sensitivity, reduce the use amount, the raising Enzymic stability that detect enzyme in reagent, simplify manufacture craft, reduce cost of manufacture, the present invention designs a kind of new glucose detection test paper, and provides and utilize this test paper to carry out the method for glucose detection.
Summary of the invention
In order to achieve the above object, the present invention is devoted to develop a kind of new detection technique that is used for external quantitative measurement glucose content, mainly comprises for the mensuration test paper of glucose detection and the method for utilizing this test paper to detect.
In order to improve the stability of glucose detection reagent, and detecting step is simplified more, the invention provides a kind of Test paper for glucose detection, and the change color of utilizing this Test paper is carried out the method for glucose detection, it is characterized in that preparing as follows the glucose test paper, carry out the analyzing and testing of glucose:
Glucose detection test paper based on composite reagent A:
1) glucose oxidase, the peroxidase of certain mass is dissolved in the water obtains composite reagent A solution; This composite reagent A solution is fully mixed with glycerine, and wherein the volume ratio of solution and glycerine is 10:90-30:70; With the interior phase solution of this mixed liquor as co-axial electrospinning, join in microsyringe;
2) the ortho-, meta-or p-dianisidine with certain mass is dissolved in organic solvent, and the certain mass high molecular polymer is dissolved in fully dissolving in this organic solvent, the clear solution that obtains joins in another microsyringe as foreign minister's electrospinning liquid of co-axial electrospinning.wherein foreign minister's electrospinning liquid is to be dissolved in N, the percentage by weight of N-dimethyl acetamide (including the ortho-, meta-or p-dianisidine of 5-10mg/ml) is 10～30% polyurethane solutions, be dissolved in N, the percentage by weight of N-dimethyl acetamide (including the ortho-, meta-or p-dianisidine of 5-10mg/ml) is 10～30% Kynoar solution, the percentage by weight that is dissolved in chloroform (including the ortho-, meta-or p-dianisidine of 5-10mg/ml) is 10～30% PLA solution or is dissolved in N, the styrene maleic anhydride copolymer solution of N-dimethyl acetamide (including the ortho-, meta-or p-dianisidine of 5-10mg/ml),
3) inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire;
4) utilize two syringe pumps to control respectively the flow velocity of inside and outside phase electrospinning liquid, carry out coaxial electrostatic spinning, obtain having the polymer nanofiber of hollow structure on dash receiver, wherein be used for the composite reagent A of glucose detection by the cavity of embedding in situ at this hollow Nano fiber in use, the ortho-, meta-or p-dianisidine of developer is evenly distributed in the shell of hollow fiber, thereby prepares the test paper for glucose detection;
5) with above-mentioned steps 4) in the prepared test paper that is used for glucose detection evenly be cut into card punch the diaphragm that diameter is 0.5cm left and right size; Prepare the glucose solution of a series of concentration known, getting 10-30 μ l glucose solution with pipettor drips on the diaphragm that cuts, 30s observes the diaphragm change color behind the left and right, or the optical density instrument reads diaphragm at the reflectivity at 440nm place, draw the working curve between known glucose concentrations and reflectivity, the diaphragm after perhaps developing the color is as standard color comparison card; Drip the glucose solution of 10-30 μ l unknown concentration on the diaphragm that cuts, 30s observes the diaphragm change color behind the left and right, or the optical density instrument reads diaphragm at the reflectivity at 440nm place, can be according to the concentration of glucose in the working curve calculation sample, also can pass through and the standard color comparison card contrast concentration of glucose in the guestimate sample.
Above-mentioned for the test paper for glucose detection as claimed in claim 1, it is characterized in that: above-mentioned steps 1), each component concentration of composite reagent A solution is as follows:
Glucose oxidase: 0.1-2.0g/L
Peroxidase: 0.1-2.0g/L
The phosphate buffer of pH7.0: 50mM
This detection side's ratio juris is as follows: wherein glucose oxidase is gluconic acid with glucose oxidase specifically, and Hydrogen Peroxide; Peroxidase utilizes hydrogen peroxide, colourless ortho-, meta-or p-dianisidine is oxidized to the oxidized form product of rufous.If reaction occurs in solution system, can by detecting solution in the variation of 440nm place light absorption value, come detection reaction speed.In order to improve Enzymic stability, and make to detect and be more prone to operation, the present invention focuses on provides a kind of test paper that utilizes above-mentioned reaction principle to carry out glucose detection, it is characterized in that prepared test paper has the form of non-woven membrane, the glucose oxidase of embedding in situ in hollow Nano fiber in use, peroxidase are in the chamber of hollow Nano fiber in use, keep the free state that disperses, effectively improved enzymic catalytic reaction active.When glucose solution is added drop-wise on test paper, due to occuring, above-mentioned demonstration reaction make test paper become brownish red, and utilize the optical density instrument to measure film at the index of refraction at 440nm place, and take the index of refraction of blank film as reference, the difference DELTA index of refraction (%) of calculating index of refraction.By measuring the Δ index of refraction (%) under different concentration of glucose, with Δ index of refraction (%) ~ log(concentration of glucose) map, can obtain the typical curve of glucose detection.
Because the test paper for the total glucose detection of serum provided by the present invention has the form of non-woven membrane, can also combine with 24 orifice plates or 96 orifice plates, the nano fibrous membrane that is embedded with glucose oxidase, peroxidase and ortho-, meta-or p-dianisidine is laid on the surface of orifice plate, build the Microarray microarray, by dripping successively the solution that contains glucose to be detected in orifice plate, thereby the high flux that can be used for glucose detects.
The test paper that is used for glucose detection that the present invention is prepared, glucose oxidase, peroxidase are by in the cavity of the hollow Nano fiber in use of embedding, the nanometer microenvironment provides special stability mechanism for enzyme and coenzyme, so its stability has obtained significantly improving.Developer is distributed on hollow wall, makes the glucose test paper can quick colour-developing, has greatly reduced detection time, and due to high activity and the high stability of enzyme, makes the consumption of enzyme reduce, thereby can make the testing cost decrease.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and the specific embodiments.
The glucose detection working curve of Fig. 1 embodiment 1 obtains on Unico 2800UV-Vis spectrophotometer.
The described original position of Fig. 2 embodiment 2 has been loaded the hollow polyurethane nanofiber of glucose oxidase, peroxidase and ortho-, meta-or p-dianisidine, namely is used for the stereoscan photograph (sectional drawing) of the test paper of glucose detection.
During the glucose detection detection paper different glucose titer of Fig. 3 embodiment 2, the result that X-Rite 500 optical density instrument detect.
The glucose detection test paper working curve 2 of Fig. 4 embodiment 2 obtains on X-Rite 500 optical density instrument.
Fig. 5 embodiment 2 is described, utilizes in glucose detection detection paper testing sample in the concentration of glucose process, and the test paper color is with the situation of change of concentration of glucose.
Fig. 6 embodiment 2 is described, the storage stability of glucose detection test paper.
Embodiment
The below sets forth embodiments of the present invention with the form of specializing, but the present invention is not restricted in this embodiment.
Embodiment 1 is used for solution morphology reagent and the detection method of glucose detection
The present embodiment is described a kind of mensuration reagent and detection method for total glucose detection, and in reagent, each component concentration is as follows:
Glucose oxidase: 0.01g/L
Peroxidase: 0.01g/L
Ortho-, meta-or p-dianisidine: 0.1mg/mL
The phosphate buffer of pH 7.0: 50mM
Its detection method is to adopt Unico 2800Uv-Vis spectrophotometer to detect, and operates as follows:
Δ
A440(min
-1The working curve of mg)-concentration of glucose is as follows: Δ
A440(min
-1)=0.0205* concentration of glucose/μ mol/L+0.0052; R
2=0.996, detect the range of linearity: 0-20mmol/L.
The detection of concentration of glucose in sample: add 100 μ L glucose oxidases, 100 μ L peroxidase, the 600 ortho-, meta-or p-dianisidines of μ L in detector tube, then add the 200 μ L blood serum sample that contains glucose to be detected, after rapid mixing in the variation that utilizes ultraviolet spectrophotometry at 440nm wavelength place continuous detecting absorbance, calculate reaction rate, quantitatively calculate total concentration of glucose in blood serum sample according to working curve.
Preparation and the detection method of embodiment 2 glucose detection test paper
Be used for the preparation of the test paper of glucose detection: 1mg glucose oxidase, 1mg peroxidase are dissolved in the water of 200 μ L, then with 800 μ L glycerine fully mix, and mixed liquor are joined the injector of 5ml, as the interior phase electrospinning liquid of coaxial electrically spun; Ortho-, meta-or p-dianisidine is dissolved in DMA, is mixed with the solution of 10mg/ml, then the polyurethane of certain mass is dissolved fully that to be made into percentage by weight be 25% solution, as foreign minister's electrospinning liquid, join in the injector of another 5ml; Inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire.Regulating spinning head is 25cm to the distance between dash receiver, and voltage is 17Kv.Utilize syringe pump to control interior phase electrospinning flow velocity and be 0.07ml/h, foreign minister's flow velocity is 0.5ml/h, carries out co-axial electrospinning, obtains the hollow polyurethane nanofiber.The hollow cavity situ of this nanofiber has loaded glucose oxidase and peroxidase, and the ortho-, meta-or p-dianisidine of developer in the shell load, thereby obtains the glucose detection test paper, has the form of non-woven membrane on this glucose test paper macroscopic view.Utilize the form of this test paper of scanning electron microscope observation.
Concentration with glucose detection detection paper glucose, draw the working curve of glucose detection: it is the big or small diaphragm in 0.5cm left and right that the spun hollow-fibre membrane of above-mentioned electricity evenly is cut into diameter with card punch, get the concentration known glucose solution 20 μ l that are dissolved in phosphate buffer, drip on the diaphragm that has cut, observe diaphragm change color or optical density instrument behind about 30s and read 440nm place reflectivity, the test paper color development of the glucose solution by a series of variable concentrations and reflectivity are prepared the standard glucose colorimetric card that develops the color.Thereby can realize directly detecting by an unaided eye the just colour developing test paper of energy guestimate concentration of glucose; Also can by the reflectance readings of optical density instrument, accurately detect concentration of glucose.
By measuring the Δ Reflection under different concentration of glucose, the working curve of drawing Δ Reflection-concentration of glucose is as follows: Δ Reflection=17.57*log (concentration of glucose/mM)-12.83; R
2=0.99, the glucose detection range of linearity: 0-50mM.
Utilize concentration of glucose in glucose detection paper test serum sample: the glucose sample drop that 20 μ L are to be detected is added on Test paper, and after 30s, the optical density instrument to detection paper, detects the reflectivity at 440nm place.According to the working curve of concentration of glucose, calculate the concentration of glucose in the test serum sample, perhaps directly read about concentration of glucose by change color.
The mensuration of the storage stability of glucose detection test paper: the glucose detection test paper of preparing is sealed in fast envelope, be positioned over respectively in 4 ° of C refrigerators and 25 ° of C constant-temperature incubators in, certain interval of time, be the big or small diaphragm in 0.5cm left and right with the even cutting diameter of card punch, get the concentration known glucose solution 20 μ l that are dissolved in phosphate buffer, drip on the diaphragm that has cut, observe diaphragm change color or optical density instrument behind about 30s and read 440nm place reflectivity.By the variation of comparison reflectivity with the test paper resting period, investigate the storage stability of glucose test paper.
Preparation and the detection method of embodiment 3 glucose detection test paper
Be used for the preparation of the test paper of glucose detection: the water that 0.1mg glucose oxidase, 0.5mg peroxidase is dissolved in 100 μ L, then with 900 μ L glycerine fully mix, mixed liquor is joined the injector of 5ml, as the interior phase electrospinning liquid of coaxial electrically spun; Ortho-, meta-or p-dianisidine is dissolved in DMA, is mixed with the solution of 5mg/ml, then the polyurethane of certain mass is dissolved fully that to be made into percentage by weight be 20% solution, as foreign minister's electrospinning liquid, join in the injector of another 5ml; Inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire.Regulating spinning head is 20cm to the distance between dash receiver, and voltage is 15Kv.Utilize syringe pump to control interior phase electrospinning flow velocity and be 0.07ml/h, foreign minister's flow velocity is 0.5ml/h, carries out co-axial electrospinning, obtains the hollow polyurethane nanofiber.The hollow cavity situ of this nanofiber has loaded glucose oxidase and peroxidase, and the ortho-, meta-or p-dianisidine of developer in the shell load, thereby obtains the glucose detection test paper, has the form of non-woven membrane on this glucose test paper macroscopic view.Utilize the form of this test paper of scanning electron microscope observation.
Concentration with glucose detection detection paper glucose, draw the working curve of glucose detection: it is the big or small diaphragm in 0.5cm left and right that the spun hollow-fibre membrane of above-mentioned electricity evenly is cut into diameter with card punch, get the concentration known glucose solution 30 μ l that are dissolved in phosphate buffer, drip on the diaphragm that has cut, observe diaphragm change color or optical density instrument behind about 30s and read 440nm place reflectivity, the test paper color development of the glucose solution by a series of variable concentrations and reflectivity are prepared the standard glucose colorimetric card that develops the color.Thereby can realize directly detecting by an unaided eye the just colour developing test paper of energy guestimate concentration of glucose; Also can by the reflectance readings of optical density instrument, accurately detect concentration of glucose.
Utilize concentration of glucose in glucose detection paper test serum sample: the glucose sample drop that 30 μ L are to be detected is added on Test paper, and after 30s, the optical density instrument to detection paper, detects the reflectivity at 440nm place.According to the working curve of concentration of glucose, calculate the concentration of glucose in the test serum sample, perhaps directly read about concentration of glucose by change color.
Preparation and the detection method of embodiment 4 glucose detection test paper
Be used for the preparation of the test paper of glucose detection: the water that 1.5mg glucose oxidase, 2.0mg peroxidase is dissolved in 200 μ L, then with 800 μ L glycerine fully mix, mixed liquor is joined the injector of 5ml, as the interior phase electrospinning liquid of coaxial electrically spun; Ortho-, meta-or p-dianisidine is dissolved in N, in the N-dimethyl acetamide, be mixed with the solution of 10mg/ml, then the Kynoar of certain mass dissolved fully that to be made into percentage by weight be 25% solution, as foreign minister's electrospinning liquid, join in the injector of another 5ml; Inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire.Regulating spinning head is 20cm to the distance between dash receiver, and voltage is 17Kv.Utilize syringe pump to control interior phase electrospinning flow velocity and be 0.1ml/h, foreign minister's flow velocity is 0.5ml/h, carries out co-axial electrospinning, obtains hollow polyvinylidene fluoride nanometer fiber.The hollow cavity situ of this nanofiber has loaded glucose oxidase and peroxidase, and the ortho-, meta-or p-dianisidine of developer in the shell load, thereby obtains the glucose detection test paper, has the form of non-woven membrane on this glucose test paper macroscopic view.Utilize the form of this test paper of scanning electron microscope observation.
Utilize prepared glucose detection detection paper concentration of glucose, and the method step of the detection of concentration of glucose in testing sample is described with embodiment 2.
Preparation and the detection method of embodiment 5 glucose detection test paper
Be used for the preparation of the test paper of glucose detection: the water that 0.1mg glucose oxidase, 0.1mg peroxidase is dissolved in 300 μ L, then with 700 μ L glycerine fully mix, mixed liquor is joined the injector of 5ml, as the interior phase electrospinning liquid of coaxial electrically spun; Ortho-, meta-or p-dianisidine is dissolved in chloroform, is mixed with the solution of 10mg/ml, then the PLA of certain mass is dissolved fully that to be made into percentage by weight be 25% solution, as foreign minister's electrospinning liquid, join in the injector of another 5ml; Inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire.Regulating spinning head is 15cm to the distance between dash receiver, and voltage is 20Kv.Utilize syringe pump to control interior phase electrospinning flow velocity and be 0.05ml/h, foreign minister's flow velocity is 0.5ml/h, carries out co-axial electrospinning, obtains the hollow polylactic acid nano fiber.The hollow cavity situ of this nanofiber has loaded glucose oxidase and peroxidase, and the ortho-, meta-or p-dianisidine of developer in the shell load, thereby obtains the glucose detection test paper, has the form of non-woven membrane on this glucose test paper macroscopic view.Utilize the form of this test paper of scanning electron microscope observation.
Utilize prepared glucose detection detection paper concentration of glucose, and the method step of the detection of concentration of glucose in testing sample is described with embodiment 2.
Preparation and the detection method of embodiment 6 glucose detection test paper
Be used for the preparation of the test paper of glucose detection: 1mg glucose oxidase, 1mg peroxidase are dissolved in the water of 200 μ L, then with 800 μ L glycerine fully mix, and mixed liquor are joined the injector of 5ml, as the interior phase electrospinning liquid of coaxial electrically spun; Ortho-, meta-or p-dianisidine is dissolved in N, in the N-dimethyl acetamide, be mixed with the solution of 10mg/ml, then the styrene maleic anhydride copolymer of certain mass dissolved fully that to be made into percentage by weight be 20% solution, as foreign minister's electrospinning liquid, join in the injector of another 5ml; Inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire.Regulating spinning head is 25cm to the distance between dash receiver, and voltage is 17Kv.Utilize syringe pump to control interior phase electrospinning flow velocity and be 0.1ml/h, foreign minister's flow velocity is 0.5ml/h, carries out co-axial electrospinning, obtains hollow styrene maleic anhydride copolymer nanofiber.The hollow cavity situ of this nanofiber has loaded glucose oxidase and peroxidase, and the ortho-, meta-or p-dianisidine of developer in the shell load, thereby obtains the glucose detection test paper, has the form of non-woven membrane on this glucose test paper macroscopic view.Utilize the form of this test paper of scanning electron microscope observation.
Utilize prepared glucose detection detection paper concentration of glucose, and the method step of the detection of concentration of glucose in testing sample is described with embodiment 2.
Claims (4)
1. test paper that is used for glucose detection is characterized in that preparation process comprises:
1) glucose oxidase, the peroxidase of certain mass is dissolved in obtains composite reagent A solution in damping fluid; This composite reagent A solution is fully mixed with glycerine, and wherein the volume ratio of solution and glycerine is 10:90-30:70; With the interior phase solution of this mixed liquor as co-axial electrospinning, join in microsyringe;
2) the ortho-, meta-or p-dianisidine with certain mass is dissolved in organic solvent, and the certain mass high molecular polymer is dissolved in fully dissolving in this organic solvent, the clear solution that obtains joins in another microsyringe as foreign minister's electrospinning liquid of co-axial electrospinning.wherein foreign minister's electrospinning liquid is to be dissolved in N, the percentage by weight of N-methylacetamide (including the ortho-, meta-or p-dianisidine of 5-10mg/ml) is 10～30% polyurethane solutions, be dissolved in N, the percentage by weight of N-dimethyl acetamide (including the ortho-, meta-or p-dianisidine of 5-10mg/ml) is 10～30% Kynoar solution, the percentage by weight that is dissolved in chloroform (including the ortho-, meta-or p-dianisidine of 5-10mg/ml) is 10～30% PLA solution or is dissolved in N, the styrene maleic anhydride copolymer solution of N-dimethyl acetamide (including the ortho-, meta-or p-dianisidine of 5-10mg/ml),
3) inside and outside phase electrospinning liquid is connected to respectively the inlet of coaxial electrically spun spinning head by pipeline, the liquid outlet of spinning head is connected with the positive pole of high-voltage power supply, be connected to the negative pole of high-voltage power supply as dash receiver with aluminium-foil paper or stainless (steel) wire;
4) utilize two syringe pumps to control respectively the flow velocity of inside and outside phase electrospinning liquid, carry out coaxial electrostatic spinning, obtain having the polymer nanofiber of hollow structure on dash receiver, wherein be used for the composite reagent A of glucose detection by the cavity of embedding in situ at this hollow Nano fiber in use, the ortho-, meta-or p-dianisidine of developer is evenly distributed in the shell of hollow fiber, thereby prepares the test paper for glucose detection.
2. the test paper for glucose detection as claimed in claim 1 is characterized in that: above-mentioned steps 1), each component concentration of composite reagent A solution is as follows:
Glucose oxidase: 0.1-2.0g/L
Peroxidase: 0.1-2.0g/L
The phosphate buffer of pH 7.0: 50mM.
3. the test paper for glucose detection as claimed in claim 1, it is characterized in that prepared test paper has the form of non-woven membrane, the glucose oxidase of embedding in situ in hollow Nano fiber in use, peroxidase keep the free state that disperses in the chamber of hollow Nano fiber in use.
4. utilize glucose detection test paper claimed in claim 1 to carry out the method for the detection of concentration of glucose, it is characterized in that: with above-mentioned steps 4) in the prepared test paper that is used for glucose detection evenly be cut into card punch the diaphragm that diameter is 0.5cm left and right size; Prepare the glucose solution of a series of concentration known, getting 10-30 μ l glucose solution with pipettor drips on the diaphragm that cuts, 30s observes the diaphragm change color behind the left and right, or the optical density instrument reads diaphragm at the reflectivity at 440nm place, draw the working curve between known glucose concentrations and reflectivity, the diaphragm after perhaps developing the color is as standard color comparison card; Drip the glucose solution of 10-30 μ l unknown concentration on the diaphragm that cuts, 30s observes the diaphragm change color behind the left and right, or the optical density instrument reads diaphragm at the reflectivity at 440nm place, can be according to the concentration of glucose in the working curve calculation sample, also can pass through and the standard color comparison card contrast concentration of glucose in the guestimate sample.
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| CN108120794A (en) * | 2017-12-21 | 2018-06-05 | 深圳市赛亿科技开发有限公司 | A kind of method of concentration of glucose in Intelligent cup and its detection beverage |
| CN108872215A (en) * | 2018-05-10 | 2018-11-23 | 武汉工程大学 | A kind of cellulose base glucose sensor material and preparation method thereof |
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