CN102928468A - Whole blood urea biosensing test paper - Google Patents

Whole blood urea biosensing test paper Download PDF

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CN102928468A
CN102928468A CN2012103386940A CN201210338694A CN102928468A CN 102928468 A CN102928468 A CN 102928468A CN 2012103386940 A CN2012103386940 A CN 2012103386940A CN 201210338694 A CN201210338694 A CN 201210338694A CN 102928468 A CN102928468 A CN 102928468A
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whole blood
blood urea
test paper
reference electrode
sensing test
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CN102928468B (en
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周越
魏晓雨
艾磊
许岳
韩奇
何辉
熊开宇
王瑞元
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Beijing Sport University
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Beijing Sport University
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Abstract

The invention relates to whole blood urea biosensing test paper. The whole blood urea biosensing test paper comprises a substrate, wherein a work electrode, a reference electrode and connecting wires matched with the work electrode and the reference electrode are printed on the substrate; one end of the substrate is printed with an output terminal of the connecting wire to be connected with a test instrument; and reaction layers capable of reacting with urea are printed on the work electrode and the reference electrode. When the whole blood urea biosensing test paper is adopted for testing blood urea, compared with the enzyme kinetics colorimetry method requiring for a semi-automatic or full-automatic instrument in the prior art, testing is more convenient and quick, so that the blood urea index can be more widely applied to sports monitoring or medical analysis, and a simple and quick test method is provided for general people.

Description

Whole blood urea bio-sensing test paper
Technical field
The present invention relates to a kind of whole blood urea bio-sensing test paper, belong to the biosensor technique field.
Background technology
The metabolism situation of body protein when blood urine element index can reflect motion, can be used as the sensitive indicator of evaluation amount of exercise and supervision over training, and though therefore be sport training process and after the exercise recovery effect assessment in all be a very important monitoring index.
At present, about the method for measuring the blood urine element condensation method and enzyme coupling rate method etc. are arranged both at home and abroad.Wherein the condensation method precision is relatively poor, and enzyme coupling rate method relatively quick and precisely.These two kinds of methods all need through getting the processes such as blood, centrifugal serum, reagent application of sample, upper machine colorimetric, and the time spent is relatively long.If any scholar's report, the time of a sample of spectrophotometric method test is approximately 40 minutes, and also needing has skilled operating personnel, is unfavorable for the widespread use of blood urine element index, is unfavorable for especially athletic field monitoring.
Enzyme Electrode has the advantages that test speed is fast, specificity is high, highly sensitive, is to simplify test procedure to realize the on-the-spot best approach of using.Sample of Enzyme Electrode test adds and is approximately 15 minutes release time, and blood serum sample do not need other pre-service, and for the sample that painted spectrophotometric method is difficult to measure, Enzyme Electrode is still effective.Traditional Urease electrode mensuration utilizes the urase catalyzing urea, generates NH 3, carry out also can causing the variation of pH value along with reaction, the NH that urea is generated by urase catalysis 3Responded by ammonia gas-sensing electrode, thereby infer the amount that urea according to the amount of ammonia.Can make the complicacy of this method actual sets architectonical also limit it uses widely.
Summary of the invention
For overcoming the defects of prior art, the invention provides a kind of whole blood urea bio-sensing test paper, can carry out quickly and easily the test of whole blood blood urine element, be conducive to widespread use.
The technical solution adopted in the present invention is: a kind of whole blood urea bio-sensing test paper, comprise substrate, be printed on the described substrate working electrode, reference electrode with described working electrode and reference electrode supporting be connected wire, one end of described substrate is printed on for what link to each other with testing tool and is connected wire output end, is printed on the reactant layer that can react with urea on described working electrode and the reference electrode.
The reactant of described reactant layer can comprise α-ketoglutaric acid and NADH.
Preferably, described reactant layer contains α-ketoglutaric acid 7~26.28ug, contains NADH36.64~119.4ug.
Described connection wire and working electrode and reference electrode preferably are made of the carbon coating that is imprinted on described on-chip conductive layer and be imprinted on the described conductive layer, preferably are printed on the Ag/AgCl layer between the carbon coating of described reference electrode and the described reactant layer.
Can paste by double sticky tape paper on described working electrode and the reference electrode and be coated with plastic sheeting, described double sticky tape paper is preferably two, every all laterally sticks on described working electrode and the reference electrode, article two, preferably leave the gap between the described double sticky tape paper, consist of horizontal reaction tank passage.
Described reactant layer can also contain and is useful on the enzyme that the reaction of urea and described reactant is played catalytic action.
The enzyme of described catalytic action can comprise urase, glutamte dehydrogenase and dglutamic oxidase.
Preferably, contain urase 0.12~0.36U in the described enzyme, contain glutamte dehydrogenase 0.24~0.72U, contain dglutamic oxidase 0.24~0.72U.
Described reactant layer can also contain the electronic media material that is used for electronics and transmits when reaction, material and the damping fluid that is used for immobilized enzyme.
Described electronic media material can be the potassium ferricyanide, and described reactant layer preferably contains the potassium ferricyanide 18.19~59.26ug; Described material for immobilized enzyme can be any one of gelatin, fibroin albumen and bovine serum albumin(BSA) and glutaraldehyde cross-linking, and described reactant layer preferably contains the material 20~40ug for immobilized enzyme; Described damping fluid can be imidazole buffer or phosphate buffer, and described reactant layer contains damping fluid and is preferably 40~100ug, and the pH value of described damping fluid is 6.5~7.5.
Beneficial effect of the present invention: when adopting the present invention to test, test paper is inserted the socket of testing tool, form test loop, again droplet of blood is added on the responding layer, reactant is communicated with working electrode and reference electrode, can obtain resistance characteristic or other electrical characteristics between working electrode and the reference electrode by testing tool, because these electrical characteristics are determined with the rear response characteristic that occurs of drop of blood contact by reactant, embody the content of blood urine element in the blood, testing tool carries out can obtaining corresponding blood urine element test result after the computing according to this characteristic thus.Adopt the present invention to carry out the test of blood urine element, under prior art, need the enzyme power colourimetry test of semi-automatic or full-automatic instrument more convenient, can be so that blood urine element index more be widely used in physical culture monitoring or the medical analysis, can or there be the crowd of large test instrument condition that a kind of method of testing of simple and fast is provided for the general population; The design of reaction tank passage of the present invention also makes things convenient for the blood of getting of the different fingers of right-hand man.
Description of drawings
Fig. 1 is structural representation of the present invention;
Fig. 2 is STRUCTURE DECOMPOSITION figure of the present invention.
Embodiment
Referring to Fig. 1 and Fig. 2, the invention provides a kind of whole blood urea bio-sensing test paper, comprise substrate 1, described substrate can adopt PVC sheet material, can adopt on the described substrate silk screen print method be printed on working electrode, reference electrode with described working electrode and reference electrode supporting be connected wire, one end of described substrate is printed on for what link to each other with testing tool and is connected wire output end 2, is printed on the reactant layer 3 that can react with urea on described working electrode and the reference electrode.
The reactant of described reactant layer can comprise α-ketoglutaric acid and NADH, and described reactant layer preferably contains α-ketoglutaric acid 7~26.28ug, as: 7ug, 10ug, 15ug, 20ug or 26.28ug; Described reactant layer preferably contains NADH 36.64~119.4ug, such as 36.64ug, 50ug, 80ug, 100ug or 119.4ug.
Described connection wire and working electrode and reference electrode preferably are made of the carbon coating 6 that is imprinted on described on-chip conductive layer 5 and be imprinted on the described conductive layer.The material of described conductive layer can be conductive silver paste printing ink, and the material of described carbon coating can be carbon printing ink, can be printed on Ag/AgCl layer 4 between the carbon coating of described reference electrode and the described reactant layer.
Described reactant layer can also contain and is useful on the enzyme that the reaction of urea and described reactant is played catalytic action, and the enzyme of described catalytic action can comprise urase, glutamte dehydrogenase and dglutamic oxidase.Preferably contain urase 0.12~0.36U in the described enzyme, as: 0.12U, 0.15U, 0.2U, 0.25U, 0.3U or 0.36U; Preferably contain glutamte dehydrogenase 0.24~0.72U in the described enzyme, as: 0.24U, 0.3U, 0.5U, 0.6U or 0.72U; Preferably contain dglutamic oxidase 0.24~0.72U in the described enzyme, as: 0.24U, 0.3U, 0.5U, 0.6U or 0.72U.
Described reactant layer can also contain the electronic media material that is used for electronics and transmits when reaction, material and the damping fluid that is used for immobilized enzyme.Described electronic media material can be the potassium ferricyanide, and described reactant layer preferably contains the potassium ferricyanide 18.19~59.26ug, as: 18.19ug, 20ug, 30ug, 50ug or 59.26ug; Described material for immobilized enzyme can be any one of gelatin, fibroin albumen and bovine serum albumin(BSA) and glutaraldehyde cross-linking, and described reactant layer preferably contains the material 20~40ug for immobilized enzyme, as: 20ug, 25ug, 30ug, 35ug or 40ug; Described damping fluid can be imidazole buffer or phosphate buffer, and described reactant layer preferably contains damping fluid 40~100ug, as: 40ug, 60ug, 80ug, 90ug or 100ug, the pH value of described damping fluid is 6.5~7.5.
Can paste by double sticky tape paper 7 on described working electrode and the reference electrode and be coated with plastic sheeting 8, described double sticky tape paper is preferably two, every all laterally sticks on described working electrode and the reference electrode, article two, leave the gap between the described double sticky tape paper, consist of horizontal reaction tank passage 9, be convenient to guide blood to be measured fully to contact with described responding layer by siphonage, this kind design, in the time of need to testing, the opening that only needs human body to be got any end of contact reaction pond, blood position passage gets final product, rely on the syphonic effect of reaction tank passage blood can be sucked described reaction tank passage, the opening at described reaction tank passage two ends is applicable to the different finger of right-hand man and gets blood.Described plastic sheeting can prevent that also described working electrode and reference electrode from leaking in air cruelly, impacts test result.
The end that described substrate arranges described working electrode and reference electrode can be provided with outward extending handheld terminal 10, the length of described handheld terminal is preferably 10~18mm, the design of described handheld terminal, be conducive to taking of test paper, when having avoided taking test paper conversion zone polluted and affect the test effect.
Embodiment 1:
Connect wire conductive layer, working electrode conductive layer and reference electrode conductive layer at the PVC substrate with the conductive silver paste ink printing with method for printing screen, be connected with the reference electrode conductive layer with the carbon printing ink printing and connect wire carbon coating, working electrode carbon coating and reference electrode carbon coating at connection wire conductive layer, working electrode conductive layer, potpourri with silver paste printing ink and AgCl is printed on formation Ag/AgCl layer on the reference electrode carbon coating again, and the blending ratio of silver paste printing ink and AgCl is 10/1 (W/W).On working electrode and reference electrode, laterally paste two double sticky tape paper, leave the gap between two double sticky tape paper, consist of the reaction tank passage.The mixed liquor 5ul that will contain α-ketoglutaric acid 26.28ug, NADH119.4ug, urase 0.36U, glutamte dehydrogenase 0.72U, dglutamic oxidase 0.72U, potassium ferricyanide 59.26ug, gelatin 20ug, pH value and be 7 imidazole buffer 40ug drips on the working electrode of reaction tank passage, forms responding layer under the room temperature of drying.Article two, cover the whole piece plastic sheeting on the double sticky tape paper, cooperate the reaction tank that consists of the bilateral opening with the reaction tank passage.The end that the PVC substrate is provided with working electrode and reference electrode is provided with the blank end of the 12mm that stretches out as handheld terminal.
Embodiment 2:
Connect wire conductive layer, working electrode conductive layer and reference electrode conductive layer at the PVC substrate with the conductive silver paste ink printing with method for printing screen, be connected with the reference electrode conductive layer with the carbon printing ink printing and connect wire carbon coating, working electrode carbon coating and reference electrode carbon coating at connection wire conductive layer, working electrode conductive layer, potpourri with silver paste printing ink and AgCl is printed on formation Ag/AgCl layer on the reference electrode carbon coating again, and the blending ratio of silver paste printing ink and AgCl is 10/1 (W/W).On working electrode and reference electrode, laterally paste two double sticky tape paper, leave the gap between two double sticky tape paper, consist of the reaction tank passage.The mixed liquor 5ul that will contain α-ketoglutaric acid 26.28ug, NADH119.4ug, urase 0.36U, glutamte dehydrogenase 0.72U, dglutamic oxidase 0.72U, potassium ferricyanide 59.26ug, fibroin albumen 40ug, pH value and be 7 phosphate buffer 100ug drips on the working electrode of reaction tank passage, forms responding layer under the room temperature of drying.Article two, cover the whole piece plastic sheeting on the double sticky tape paper, cooperate the reaction tank that consists of the bilateral opening with the reaction tank passage.The end that the PVC substrate is provided with working electrode and reference electrode is provided with the blank end of the 12mm that stretches out as handheld terminal.
Biochemical reaction process of the present invention is as follows:
Figure BSA00000777273800071
Figure BSA00000777273800072
Figure BSA00000777273800073
In the time of need to testing, test paper is provided with the socket of the end insertion testing tool of described connection wire output end, connection wire output end of described whole blood urea bio-sensing test paper is connected with corresponding testing tool input terminal, form test loop, again droplet of blood is added on the responding layer, reactant is communicated with working electrode and reference electrode, can obtain resistance characteristic or other electrical characteristics between working electrode and the reference electrode by testing tool, because these electrical characteristics are determined with the rear response characteristic that occurs of drop of blood contact by reactant, embody the content of blood urine element in the blood, testing tool carries out can obtaining corresponding blood urine element test result after the computing according to this characteristic thus, and be presented on the display screen, therefore can be from direct read test data on the testing tool.

Claims (10)

1. whole blood urea bio-sensing test paper, comprise substrate, be printed on the described substrate working electrode, reference electrode with described working electrode and reference electrode supporting be connected wire, one end of described substrate is printed on for what link to each other with testing tool and is connected wire output end, it is characterized in that being printed on described working electrode and the reference electrode reactant layer that can react with urea.
2. whole blood urea bio-sensing test paper as claimed in claim 1 is characterized in that the reactant of described reactant layer comprises α-ketoglutaric acid and NADH.
3. whole blood urea bio-sensing test paper as claimed in claim 2 is characterized in that described reactant layer contains α-ketoglutaric acid 7~26.28ug, contains NADH36.64~119.4ug.
4. whole blood urea bio-sensing test paper as claimed in claim 3, it is characterized in that described connection wire and working electrode and reference electrode are made of the carbon coating that is imprinted on described on-chip conductive layer and be imprinted on the described conductive layer, are printed on the Ag/AgCl layer between the carbon coating of described reference electrode and the described reactant layer.
5. whole blood urea bio-sensing test paper as claimed in claim 4, it is characterized in that being coated with plastic sheeting by the stickup of double sticky tape paper on described working electrode and the reference electrode, described double sticky tape paper is two, every all laterally sticks on described working electrode and the reference electrode, article two, leave the gap between the described double sticky tape paper, consist of horizontal reaction tank passage.
6. such as claim 1,2,3,4 or 5 described whole blood urea bio-sensing test paper, it is characterized in that described reactant layer also contains to be useful on the enzyme that the reaction of urea and described reactant is played catalytic action.
7. whole blood urea bio-sensing test paper as claimed in claim 6 is characterized in that the enzyme of described catalytic action comprises urase, glutamte dehydrogenase and dglutamic oxidase.
8. whole blood urea bio-sensing test paper as claimed in claim 7 is characterized in that containing urase 0.12~0.36U in the described enzyme, contains glutamte dehydrogenase 0.24~0.72U, contains dglutamic oxidase 0.24~0.72U.
9. whole blood urea bio-sensing test paper as claimed in claim 8 is characterized in that described reactant layer also contains the electronic media material that is used for electronics and transmits when reaction, material and the damping fluid that is used for immobilized enzyme.
10. whole blood urea bio-sensing test paper as claimed in claim 9 is characterized in that described electronic media material is the potassium ferricyanide, and described reactant layer contains the potassium ferricyanide 18.19~59.26ug; Described material for immobilized enzyme is any one of gelatin, fibroin albumen and bovine serum albumin(BSA) and glutaraldehyde cross-linking, and described reactant layer contains the material 20~40ug for immobilized enzyme; Described damping fluid is imidazole buffer or phosphate buffer, and described reactant layer contains damping fluid 40~100ug, and the pH value of described damping fluid is 6.5~7.5.
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CN104198421A (en) * 2014-08-14 2014-12-10 上海睿康生物科技有限公司 Detection kit for measuring content of urea without interference of endogenous ammonia in serum
CN105807042A (en) * 2014-12-29 2016-07-27 张红 Biomarker outer driving immune lateral chromatography device and method
CN109946353A (en) * 2018-11-08 2019-06-28 利多(香港)有限公司 Potential type biosensor and detection method
CN109946352A (en) * 2018-11-08 2019-06-28 利多(香港)有限公司 Reaction reagent and its application for potential type biosensor
CN116144731A (en) * 2023-04-07 2023-05-23 南京晶捷生物科技有限公司 Reactive enzyme liquid, test paper and device system for electrochemical urea nitrogen detection

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104198421A (en) * 2014-08-14 2014-12-10 上海睿康生物科技有限公司 Detection kit for measuring content of urea without interference of endogenous ammonia in serum
CN105807042A (en) * 2014-12-29 2016-07-27 张红 Biomarker outer driving immune lateral chromatography device and method
CN109946353A (en) * 2018-11-08 2019-06-28 利多(香港)有限公司 Potential type biosensor and detection method
CN109946352A (en) * 2018-11-08 2019-06-28 利多(香港)有限公司 Reaction reagent and its application for potential type biosensor
CN116144731A (en) * 2023-04-07 2023-05-23 南京晶捷生物科技有限公司 Reactive enzyme liquid, test paper and device system for electrochemical urea nitrogen detection

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