CN104215758A - Instrument-free quantitative analysis method for fast detecting test paper, and application of instrument-free quantitative analysis method - Google Patents
Instrument-free quantitative analysis method for fast detecting test paper, and application of instrument-free quantitative analysis method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses an instrument-free quantitative analysis method for fast detecting test paper, and application of the instrument-free quantitative analysis method. A target analyte or a secondary analyte in a sample can react with a detection reagent to generate insoluble deposits to enhance the hydrophobicity of a reaction area, and the concentration of the analyte is in direct proportion to the enhancement degree of the hydrophobicity of the reaction area; the hydrophobicity of the reaction area decides the volume of a colored hydrophilic reagent solution flowing into a quantification area from the reaction area within set time or in a set distance (such as from the tail end of the reaction area to the quantification area), and the length of colored print in the quantification area is in direct proportion to the volume of the colored reagent solution flowing into the quantification area and is in inverse proportion to the hydrophobicity of the reaction area. Therefore, the concentration of the analyte in the sample is in inverse proportion to the length of the colored print in the quantification area, and the fast quantitative detection of the analyte in the sample can be carried out by measuring the length of the colored print in the quantification area through a ruler with scales. The method can be applied to quantitative detection on target analytes in various liquid samples of urine, blood, serum, saliva, water, etc.
Description
Technical field
The invention belongs to quick detection test paper product development and applied technical field thereof, particularly a kind ofly exempt from instrumental quantitative analysis method and application thereof for quick detection test paper.
Background technology
Quick detection test paper has been widely used in the every aspect of actual life as a kind of analysis tool that is simple, low cost, such as, surveys pregnant test paper, urine detection test paper, pH test paper, nitrite Test paper, the residual Test paper of agriculture etc.But except colloidal gold immune chromatography test and home-use blood glucose instrument, most traditional quick detection test paper can only realize sxemiquantitative or the qualitative analysis of analyzing thing in sample, significantly limit their practical application.In recent years, novel quick detection test paper and relevant quantitative analysis method are constantly in the news.Test paper in the present invention belongs to novel quick detection test paper, to be also referred to as on paper microfluidic analysis device (Microfluidic paper-based analytical device), paper laboratory (Lab-on-paper) on micro-fluidic chip (Paper-based microfluidics) or paper.
The quick detection test paper quantivative approach of current bibliographical information mainly comprises electrochemical assay, fluorometry, chromatmetry, chemoluminescence method, opto-electronic conversion method etc.Although these methods can the quantitative test of quick detection test paper, but involved specialty analysis instrument (such as, electrochemical workstation, fluorescence analyser, chemical analyzer etc.) bulky and expensive, thus analysis cost is significantly increased, operation steps is relatively loaded down with trivial details time-consuming simultaneously, and higher professional skill is required to operating personnel, especially can not meet the real-time test day by day increased and on-the site analysis requirement, be difficult to obtain widespread use in various field.
Summary of the invention
The object of this invention is to provide and a kind ofly exempt from instrumental quantitative analysis method and application thereof for quick detection test paper.
Of the present inventionly exempt from instrumental quantitative analysis side's ratio juris for quick detection test paper: in sample, target analytes or secondary analysis thing can generate insoluble sediment with detection reagent reacting, cause the hydrophobicity of reaction zone to strengthen, analyte concentration and reaction zone hydrophobicity strengthen degree and are directly proportional; The hydrophobicity degree of reaction zone will determine in the time of setting or apart from the volume of (as reaction zone end) in-laws' hydrate colour reagent solution from inflow quantification area, reaction zone, in quantification area, the length of color prints is directly proportional to the color agents liquor capacity flowed into wherein, and is inversely proportional to the hydrophobicity degree of reaction zone.Therefore, in sample, the color prints length of analyte concentration and quantification area is inversely proportional to, and can realize analyzing in sample the Quantitative detection of thing by the length using ruler with a scale to measure color prints in quantification area.
The concrete steps exempting from instrumental quantitative analysis method for quick detection test paper of the present invention are:
(1) making of quick detection test paper: utilize filter paper, chromatographic paper, rice paper or nitrocellulose filter to make quick detection test paper in conjunction with existing test paper method for making, its structure is patterning composite structure or the patterning hydrophilic paper body of hydrophobic material and hydrophilic paper body, at least comprises sample and adds district, for the reaction zone of fixed test reagent and three, quantification area continuous print part.
(2) in the reaction zone for fixed test reagent, enzyme and substrate is added.
(3) in interpolation district, detection sample is dripped, when sample flows through reaction zone, the enzyme added in target analytes or secondary analysis thing and reaction zone in sample and substrate reactions generate insoluble sediment, the hydrophobicity of reaction zone is caused to strengthen, 3 microlitre to the 20 microlitre water wettability color agents solution dripped subsequently adding district flow through reaction zone and flow into quantification area under capillarity, when reaching the time of setting or flowing through the distance of setting, micropipettor or thieving paper is used to be removed by unnecessary water wettability color agents in interpolation district.
(4) after the water wettability color agents in test paper no longer flows in quantification area, use ruler with a scale to measure the length of color prints in quantification area, namely complete in sample the Quantitative detection analyzing thing.
Described existing test paper method for making is the one in paraffin printer print process, Hand drawing method, mechanical drawing method, photoetch method and laser paper-cut method.
Described enzyme is the one in horseradish peroxidase, glucose oxidase, urate oxidase and alkaline phosphatase.
Described substrate is dimethylbenzidine or tetramethyl benzidine.
Of the present inventionly exempt from for quick detection test paper the quantitative detection that instrumental quantitative analysis method is applied to target analytes in the various liquid samples such as urine, blood, serum, saliva, water.
Method of the present invention is without any need for additional professional instrument, and only use cheap ruler with a scale to get final product the quantitative detection of Realization analysis thing, analysis cost is cheap.Meanwhile, quantitative analysis process is simple, fast, operating personnel are almost without the need to any professional skill training.In addition, the method directly can extend to existing quick detection test paper platform, thus monitors at disease care diagnostic and field screening, site environment, the numerous areas such as food security has boundless commercial applications prospect.
Accompanying drawing explanation
Fig. 1 has the test paper of patterning composite structure that circular sample adds district, circular reaction zone and rectangle quantification area.
Mark in figure: 1-is paraffin; 2-is filter paper; 3-is that sample adds district; 4-is reaction zone; 5-is quantification area.
Fig. 2 detects the test paper (left side) of variable concentrations hydrogen peroxide and the calibration curve between contact length and concentration of hydrogen peroxide (right side) in the embodiment of the present invention.
Fig. 3 detects the test paper (left side) of different glucose and the calibration curve between contact length and concentration of glucose (right side) in the embodiment of the present invention.
Embodiment
embodiment:
The present embodiment exempts from instrumental quantitative analysis for variable concentrations hydrogen peroxide sample, and concrete steps are:
(1) according to the schematic diagram of Fig. 1, laser paper-cut legal system circular sample of having got everything ready is adopted to add district, the test paper of the patterning composite structure of circular reaction zone and rectangle quantification area, first semi-refined solid paraffin is made into the paraffin thin slice that thickness is about 1mm, subsequently it is lain against its surface, and then utilize laser engraving instrument under the parameter (engraving electric current and carving speed are respectively 6mA and 20mm/s) of setting, to export the figure designed in advance to paraffin sheet surface, to be heated melting by the paraffin fractions of laser engraving, and absorbed by the filter paper of relevant position, and then hydrophobic distance is formed after condensation in filter paper, finally obtain that there is sample and add district, the patterning compound test paper of reaction zone and quantification area.
(2) the first hydrophilic region being used for the reaction zone of fixed test reagent at test paper drips 0.4 μ L concentration is 3 of 2 mg/mL, 3', the ethanolic solution of 5,5'-tetramethyl benzidine, dry under room temperature, continue in 3 of drying, 3', 5,5'-tetramethyl benzidine surface drips the horseradish peroxidase solution that 0.4 μ L concentration is 1mg/mL, and (10mM phosphate buffered solution is prepared, pH is 7), dry under room temperature.
(3) hydrogen peroxide exempt from instrumental quantitative analysis.The hydrogen peroxide sample solution (10mM phosphate buffered solution is prepared, and pH is 7) that 1 μ L contains variable concentrations is dripped in sample interpolation district.When sample solution flows through reaction zone from interpolation district under capillarity, horseradish peroxidase in reaction zone can hydrogen peroxide in catalysis sample and 3,3', and 5,5'-tetramethyl benzidine reacts, and the insoluble sediment generated thus causes the hydrophobicity of reaction zone to strengthen.Add after district and reaction zone solution drying until sample, continue to drip 4 μ L water wettability blue inks in interpolation district.When this ink solution just flows to reaction zone end, micropipettor or thieving paper is used to be removed by unnecessary ink in interpolation district.
After (4) 2 minutes, after the blue agents in test paper no longer flows in quantification area, the length using the ruler of band grade scale to measure color prints in quantification area is respectively 3.8mm, 5.9 mm, 8.7 mm, 10.7 mm, 11.8 mm.Analysis of drawing obtains the calibration curve (right see Fig. 2) between contact length and concentration of hydrogen peroxide.
The present embodiment exempts from instrumental quantitative analysis for different glucose sample, and specific implementation process is as follows:
(1) method identical in embodiment 1 is adopted to make phase diagram patterning composite structure test paper.Specific implementation process is with example 1.
(2) the first hydrophilic region being used for the reaction zone of fixed test reagent at test paper drips the ethanolic solution that 0.4 μ L concentration is 3,3', 5, the 5'-tetramethyl benzidines of 2 mg/mL, dry under room temperature.Continue to drip the horseradish peroxidase solution (10 mM phosphate buffered solution preparations, pH is 7) that 0.4 μ L concentration is 1 mg/mL on 3,3', 5,5'-tetramethyl benzidine surfaces of drying, dry under room temperature.Continue to drip the glucose oxidase solution (10 mM phosphate buffered solution preparations, pH is 7) that 1 μ L concentration is 0.01 mg/mL on horseradish peroxidase surface, dry under room temperature.
(3) hydrogen peroxide exempt from instrumental quantitative analysis.The glucose sample solution (10 mM phosphate buffered solution preparations, pH is 7) that 1 μ L contains variable concentrations is dripped in sample interpolation district.When sample solution flows through reaction zone from interpolation district under capillarity, glucose oxidase in reaction zone is by catalysis glucose Hydrogen Peroxide, and horseradish peroxidase can catalyzing hydrogen peroxide and 3,3', 5, the reaction of 5'-tetramethyl benzidine generates insoluble sediment, and then causes the hydrophobicity of reaction zone to strengthen.Add after district and reaction zone solution drying until sample, continue to drip 4 μ L water wettability blue inks in interpolation district.When this ink solution just flows to reaction zone end, micropipettor or thieving paper is used to be removed by unnecessary ink in interpolation district.
After (4) 1.5 minutes, after the blue agents in test paper no longer flows in quantification area, the ruler of band grade scale is used to measure the length of color prints in quantification area.In sample, the concentration of glucose and the color prints length of quantification area are inversely proportional to.(right see Fig. 3).
Claims (2)
1. exempt from an instrumental quantitative analysis method for quick detection test paper, it is characterized in that concrete steps are:
(1) making of quick detection test paper: utilize filter paper, chromatographic paper, rice paper or nitrocellulose filter to make quick detection test paper in conjunction with existing test paper method for making, its structure is patterning composite structure or the patterning hydrophilic paper body of hydrophobic material and hydrophilic paper body, at least comprises sample and adds district, for the reaction zone of fixed test reagent and three, quantification area continuous print part;
(2) in the reaction zone for fixed test reagent, enzyme and substrate is added;
(3) in interpolation district, detection sample is dripped, when sample flows through reaction zone, the enzyme added in target analytes or secondary analysis thing and reaction zone in sample and substrate reactions generate insoluble sediment, the hydrophobicity of reaction zone is caused to strengthen, 3 microlitre to the 20 microlitre water wettability color agents solution dripped subsequently adding district flow through reaction zone and flow into quantification area under capillarity, when reaching the time of setting or flowing through the distance of setting, micropipettor or thieving paper is used to be removed by unnecessary water wettability color agents in interpolation district;
(4) after the water wettability color agents in test paper no longer flows in quantification area, use ruler with a scale to measure the length of color prints in quantification area, namely complete in sample the Quantitative detection analyzing thing;
Described existing test paper method for making is the one in paraffin printer print process, Hand drawing method, mechanical drawing method, photoetch method and laser paper-cut method;
Described enzyme is the one in horseradish peroxidase, glucose oxidase, urate oxidase and alkaline phosphatase;
Described substrate is dimethylbenzidine or tetramethyl benzidine.
2., as claimed in claim 1 for the application of exempting from instrumental quantitative analysis method of quick detection test paper, it is characterized in that this instrumental quantitative analysis method of exempting from for quick detection test paper is applied to the quantitative detection of target analytes in urine, blood, serum, saliva and water liquid sample.
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Cited By (9)
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| CN104931489A (en) * | 2015-05-17 | 2015-09-23 | 桂林理工大学 | Quantitative analysis method for measuring rapid test strip on basis of iodine-starch variable-color distance |
| CN105044070A (en) * | 2015-08-18 | 2015-11-11 | 上海微银生物技术有限公司 | Tear glucose detection device |
| CN106404758A (en) * | 2015-07-28 | 2017-02-15 | 珠海和凡医药股份有限公司 | Test paper for detecting uric acid content range in urine |
| CN106526201A (en) * | 2016-11-10 | 2017-03-22 | 陕西师范大学 | Method for detecting antigens in qualitative and semi-quantitative mode based on paper chip immunoreaction distance |
| CN106596532A (en) * | 2016-11-24 | 2017-04-26 | 桂林理工大学 | Simple and low cost method for detecting activity of alkaline phosphatase |
| CN108535472A (en) * | 2018-02-27 | 2018-09-14 | 上海艾瑞德生物科技有限公司 | A kind of detection strip significantly improving lateral flow immunochromatography |
| CN109856124A (en) * | 2018-12-13 | 2019-06-07 | 迪瑞医疗科技股份有限公司 | A kind of hydrogen peroxide detection drying chemical reagent paper and preparation method thereof |
| CN110672863A (en) * | 2019-09-29 | 2020-01-10 | 桂林理工大学 | Instrument-free quantitative detection method for divalent lead ions |
| CN113984753A (en) * | 2021-11-04 | 2022-01-28 | 长春美泰仪器有限公司 | Formaldehyde detection test paper, preparation method thereof and formaldehyde detection system |
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Cited By (15)
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| CN104931489A (en) * | 2015-05-17 | 2015-09-23 | 桂林理工大学 | Quantitative analysis method for measuring rapid test strip on basis of iodine-starch variable-color distance |
| CN106404758A (en) * | 2015-07-28 | 2017-02-15 | 珠海和凡医药股份有限公司 | Test paper for detecting uric acid content range in urine |
| CN105044070A (en) * | 2015-08-18 | 2015-11-11 | 上海微银生物技术有限公司 | Tear glucose detection device |
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| CN106596532A (en) * | 2016-11-24 | 2017-04-26 | 桂林理工大学 | Simple and low cost method for detecting activity of alkaline phosphatase |
| CN106596532B (en) * | 2016-11-24 | 2019-07-23 | 桂林理工大学 | A kind of detection method of simple alkaline phosphatase activities |
| CN108535472A (en) * | 2018-02-27 | 2018-09-14 | 上海艾瑞德生物科技有限公司 | A kind of detection strip significantly improving lateral flow immunochromatography |
| CN108535472B (en) * | 2018-02-27 | 2021-09-24 | 上海艾瑞德生物科技有限公司 | Detection test strip capable of remarkably improving lateral flow immunochromatography |
| CN109856124A (en) * | 2018-12-13 | 2019-06-07 | 迪瑞医疗科技股份有限公司 | A kind of hydrogen peroxide detection drying chemical reagent paper and preparation method thereof |
| CN110672863A (en) * | 2019-09-29 | 2020-01-10 | 桂林理工大学 | Instrument-free quantitative detection method for divalent lead ions |
| CN113984753A (en) * | 2021-11-04 | 2022-01-28 | 长春美泰仪器有限公司 | Formaldehyde detection test paper, preparation method thereof and formaldehyde detection system |
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