CN106645121A - Two-dimensional liquid-phase colorimetric instrument-free quantitative analysis method - Google Patents
Two-dimensional liquid-phase colorimetric instrument-free quantitative analysis method Download PDFInfo
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- CN106645121A CN106645121A CN201611039046.XA CN201611039046A CN106645121A CN 106645121 A CN106645121 A CN 106645121A CN 201611039046 A CN201611039046 A CN 201611039046A CN 106645121 A CN106645121 A CN 106645121A
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- 239000007791 liquid phase Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229920002472 Starch Polymers 0.000 claims abstract description 10
- 235000019698 starch Nutrition 0.000 claims abstract description 10
- 239000008107 starch Substances 0.000 claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 239000012491 analyte Substances 0.000 claims abstract description 7
- 108010089934 carbohydrase Proteins 0.000 claims description 26
- 239000000976 ink Substances 0.000 claims description 14
- 238000009792 diffusion process Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 229920000856 Amylose Polymers 0.000 claims description 5
- 229920003023 plastic Polymers 0.000 claims description 4
- 230000001419 dependent effect Effects 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- 229920000945 Amylopectin Polymers 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 239000002086 nanomaterial Substances 0.000 claims description 2
- 239000000049 pigment Substances 0.000 claims description 2
- 230000002478 diastatic effect Effects 0.000 abstract description 9
- 238000004458 analytical method Methods 0.000 abstract description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 5
- 239000008103 glucose Substances 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000007246 mechanism Effects 0.000 abstract description 2
- 238000012549 training Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 44
- 238000004737 colorimetric analysis Methods 0.000 description 10
- 239000000523 sample Substances 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 5
- 239000012498 ultrapure water Substances 0.000 description 5
- 239000012496 blank sample Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000007974 sodium acetate buffer Substances 0.000 description 3
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 3
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 2
- 102100022624 Glucoamylase Human genes 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a two-dimensional liquid-phase colorimetric instrument-free quantitative analysis method. The method simultaneously utilizes the reaction that diastatic enzyme catalyzes and hydrolyzes starch molecules with lower water solubility to generate products with good water solubility, such as glucose, and a simple mechanism that color detection reagents with excellent water solubility and water dispersibility can diffuse in different distances in reaction solutions with different water solubility; the diffusing lengths of the color detection reagents in the reaction solutions increase along with the increase of diastatic enzyme activity concentration, so that by visually observing and measuring the diffusing lengths, two-dimensional liquid-phase colorimetric instrument-free quantitative analysis of a target analyte can be realized. The method provided by the invention has a simple detection process, experiments can be developed by a person without professional skill training, quantitative signal reading can be performed without using any instrument equipment, so that the analysis cost is reduced, and the method has broad application prospect.
Description
Technical field
The invention belongs to biochemical analysis and biosensor technique field, and in particular to a kind of Two-dimensional Liquid phase colorimetric is exempted from instrument and determined
Analysis method.
Background technology
Liquid phase colorimetric analysis has simple to operate, cost economy, achievable visually qualitative or half-quantitative detection etc. prominent
Advantage, enjoys for a long time the favor of analysis worker.At present the liquid phase colorimetric analysis of overwhelming majority document report generally makes
With organic dyestuff, zymolyte, noble metal nano particles etc. as colorimetric probe, the concentration of object is converted into into homogeneous reaction molten
The color intensity of liquid is analyzed.Because color intensity is only the one-dimension information of end reaction solution, existing most liquid
Phase colorimetric methods can be collectively referred to as one-dimensional liquid phase colorimetric analysis.Although one-dimensional liquid phase colorimetric analysis has aforementioned many excellent
Point, their a fly in the ointment is essentially consisted in just must can be carried out point by optical instruments such as ultraviolet-visible spectrophotometers
The quantitative determination of analysis thing.
The content of the invention
The purpose of the present invention is the deficiency for most liquid phase colorimetric methods in prior art, there is provided a kind of two
Dimension liquid phase colorimetric exempts from instrumental quantitative analysis method.
The thinking of the present invention：Using carbohydrase can the water-soluble relatively low starch molecule of catalyzing hydrolysis to generate glucose etc. water-soluble
Property good product, it is molten in the water miscible reaction of difference with reference to the colored detection reagent with dispersiveness in excellent water-soluble or water
The simple mechanisms of different distance are can spread in liquid, proposes that a kind of Two-dimensional Liquid phase colorimetric exempts from instrumental quantitative analysis new method." two dimension "
Refer to while by the use of the color intensity of the colored detection reagent in reaction solution as " the first dimension " qualitative information, introduce
The colored length that it spreads in reaction solution is used as " the second dimension " quantitative information.In other words, with existing most liquid phases
Finally obtain in colorimetric methods unlike the homogeneous reaction solution of color, that new method is finally obtained is double-color reverse Ying Rong
The upper and lower of liquid --- solution presents respectively colored and colourless.In reaction solution the length of chrominance section with carbohydrase it is dense
That what is spent increases and increases.Therefore, only spectrophotometry is replaced by using the extremely low ruler of price with visually observing
The optical instruments such as meter simply measure the diffusion length of color solution in reaction solution, or directly read used transparent vessel certainly
The body scale related to the colored diffusion length, you can realize carbohydrase analyte or with the concentration dependent target analysis of carbohydrase
Thing（I.e. with carbohydrase as enzyme labelled probe）Quantitative determination, so as to significantly reduce analysis cost.
Concretely comprise the following steps：
Step one, is sufficiently mixed starch solution with the solution containing carbohydrase in transparent vessel, carries out carbohydrase catalytic water
The reaction of solution starch, is obtained the secondary products reaction solution with good aqueous solubility.
Step 2, in the liquid level position of reaction solution obtained in step one colored detection reagent solution is added, and measures reaction
The diffusion length of colored detection reagent solution in solution, the diffusion length is proportionate with the concentration of carbohydrase in solution, i.e., complete
Two-dimensional Liquid phase colorimetric into target analytes exempts from instrumental quantitative analysis.
The transparent vessel is the one kind in glass material and plastic material, and transparent vessel itself is without scale or in itself
From with a scale, when transparent vessel does not have scale in itself, the colored inspection in transparent vessel outer wall measures reaction solution using ruler
The diffusion length of test agent solution, when transparent vessel itself from it is with a scale when, directly read transparent vessel itself scale and measure instead
Answer the diffusion length of colored detection reagent solution in solution.
The starch is the one kind in the amylose and amylopectin of low aqueous solubility.
The target analytes be carbohydrase itself or with the concentration dependent material of carbohydrase i.e. with carbohydrase as enzyme mark
Note probe.
The colored detection reagent is the color inks with good aqueous solubility, dyestuff, pigment and divides with good water
One kind in the color nano material and micro materials of scattered property.
Compared with existing most liquid phase colorimetric methods, the present invention's has the prominent advantages that：1）Detection process
It is extremely simple, can also carry out experiment without the operating personnel of professional skill training；2）Only with visually observe or simply use price
Cheap ruler replaces the optical instruments such as ultraviolet-visible spectrophotometer to carry out quantifiable signal reading, significantly reduces and is parsed into
This；3）Sugar in various samples can be directly applied in the fields such as clinical diagnosis, medical research, food security, environmental monitoring
Change enzyme analyte or the simple inexpensive Two-dimensional Liquid phase colorimetric using carbohydrase for the object of enzyme labelled probe exempts from instrument quantitative
Detection, has broad application prospects.
Description of the drawings
Fig. 1 is the principle signal that Two-dimensional Liquid phase colorimetric exempts from instrumental quantitative analysis method in Example 1 and Example 2 of the present invention
Figure.Mark in figure：1-1-colourless buffer solution；1-2-carbohydrase；The graduated transparent plastic detection container of 2-outer wall（It is micro-
Amount syringe）；2-1-detection container outer wall scale；2-2-detection container internal piston；3-1-ultra-pure water；3-2-straight chain forms sediment
Powder；The product of 4-saccharification enzymatic hydrolysis starch（Glucose）；5-colored detection reagent（Red ink）；6-mainly contain formyl
Lower floor's solution of amine；7-main containing colored detection reagent（Red ink）Upper solution；8-naked eyes.
Fig. 2 exempts from instrumental quantitative analysis method and detects 0.64 U/ respectively for Two-dimensional Liquid phase colorimetric used in the embodiment of the present invention 1
ML carbohydrase sample solution and blank sample（The not cushioning liquid of analyte-containing）The quarter related to red ink length of flow of gained
The comparison of angle value.
Fig. 3 is that Two-dimensional Liquid phase colorimetric exempts from a series of differences of instrumental quantitative analysis method detection used in the embodiment of the present invention 2
The scale value related to red ink length of flow obtained by the carbohydrase sample solution of active concentration and diastatic activity concentration
Log values（LogC Carbohydrase）Between working curve.
Specific embodiment
Following examples will be further described to the present invention, but not thereby limiting the invention.
Embodiment 1:
Exempt from instrumental quantitative analysis method using Two-dimensional Liquid phase colorimetric and detect 0.64 U/mL carbohydrase sample solution and blank sample respectively
（The not cushioning liquid of analyte-containing）.
As shown in figure 1, the present embodiment is concretely comprised the following steps：Step one, in 50 μ L transparent plastic micro syringes successively
Suck 15 μ L Glucoamylase Solutions（Prepared by the acetic acid-sodium acetate buffer solution of 20 mM, pH 6.0）It is molten with 15 μ L amyloses
Liquid（Prepared by the ultra-pure water heating that resistivity is 18.2 M Ω cm, be cooled to after room temperature and use）, 37 are placed in after being sufficiently mixed
40 min are reacted at DEG C, reaction solution is obtained；Step 2, continues suction and adds 3 μ L red in the liquid level position of above-mentioned reaction solution
Ink solution（It is obtained after the ultra-pure water that ink stock solution resistivity is 18.2 M Ω cm is diluted into 20 times）, red ink solution exists
Flow in reaction solution after 5 min, visually directly read the syringe outer wall surface quarter related to red length of flow in solution
Angle value, that is, the Two-dimensional Liquid phase colorimetric for completing target analytes exempts from instrumental quantitative analysis.
According to identical step, the acetic acid-sodium acetate buffer solution of analysis margin sample, i.e. 20 mM（pH 6.0）, and use
Naked eyes directly read the syringe outer wall surface scale value related to red length of flow in solution.Figure it is seen that with inspection
Survey the scale value obtained by blank sample to compare, detect that the scale value obtained by 0.64 U/mL carbohydrase sample solutions has notable growth.
This is because carbohydrase analyte expeditiously catalyzing hydrolysis low aqueous solubility starch molecule can be generated with good aqueous solubility
Therefore glucose products, the hydrophobicity of whole reaction solution reduces, and causes the water-soluble red ink solution can within the time of setting
With the longer distance that flows.Contrast and experiment in Fig. 2 shows, based on the diastatic activity of Two-dimensional Liquid phase colorimetric method instrument is exempted from
Quantitative determination is practical.
Embodiment 2:
Exempt from saccharification of the instrumental quantitative analysis method detection active concentration range for 0.01 ~ 0.64 U/mL using Two-dimensional Liquid phase colorimetric
Enzyme sample solution.
As shown in figure 1, analyzing concretely comprising the following steps for each carbohydrase sample solution in the present embodiment：In the transparent modelings of 50 μ L
15 μ L Glucoamylase Solutions are sucked successively in material micro syringe（Prepared by the acetic acid-sodium acetate buffer solution of 20 mM, pH
6.0）With 15 μ L amylose solutions（Prepared by the ultra-pure water heating that resistivity is 18.2 M Ω cm, being cooled to after room temperature makes
With）, 40 min are reacted at 37 DEG C are placed in after being sufficiently mixed, reaction solution is obtained；Step 2, in the liquid level position of above-mentioned reaction solution
Put continuation and aspirate 3 μ L red ink solutions of addition（The ultra-pure water that ink stock solution resistivity is 18.2 M Ω cm is diluted into 20 times
After be obtained）, red ink solution flows after 5 min in reaction solution, in visually directly reading syringe outer wall surface and solution
The related scale value of red length of flow.The last and Log by the scale value obtained from all samples to diastatic activity concentration
Value（LogC Carbohydrase）Mapping（Fig. 3）, that is, complete the Two-dimensional Liquid phase colorimetric method quantitative determination of diastatic activity.
From the figure 3, it may be seen that with the increase of diastatic activity concentration, corresponding scale value gradually increases.This is because, work as sample
When diastatic activity concentration is larger in this, the amylose of the low aqueous solubility being hydrolyzed also can be more.Now, it is water-soluble good
The quantity of glucose products is also more so that red ink detection reagent solution length of flow wherein（Scale value）Also it is bigger.
Additionally, Fig. 3 shows, using the Log values of the scale value obtained by present invention measurement and diastatic activity concentration in 0.01 ~ 0.64 U/
Good linear relationship is presented in the range of mL.
Claims (1)
1. a kind of Two-dimensional Liquid phase colorimetric exempts from instrumental quantitative analysis method, it is characterised in that concretely comprise the following steps：
Step one, is sufficiently mixed starch solution with the solution containing carbohydrase in transparent vessel, carries out carbohydrase catalytic water
The reaction of solution starch, is obtained the secondary products reaction solution with good aqueous solubility；
Step 2, in the liquid level position of reaction solution obtained in step one colored detection reagent solution is added, and measures reaction solution
The diffusion length of middle colored detection reagent solution, the diffusion length is proportionate with the concentration of carbohydrase in solution, that is, complete mesh
The Two-dimensional Liquid phase colorimetric of mark analyte exempts from instrumental quantitative analysis；
The transparent vessel is the one kind in glass material and plastic material, and transparent vessel itself is without scale or carries in itself
Scale, when transparent vessel does not have scale in itself, the colored detection examination in transparent vessel outer wall measures reaction solution using ruler
The diffusion length of agent solution, when transparent vessel itself from it is with a scale when, directly read transparent vessel itself scale measure reaction it is molten
The diffusion length of colored detection reagent solution in liquid；
The starch is the one kind in the amylose and amylopectin of low aqueous solubility；
The target analytes are carbohydrase itself or visit by enzyme mark of carbohydrase with the concentration dependent material of carbohydrase
Pin；
The colored detection reagent be the color inks with good aqueous solubility, dyestuff, pigment and with good water dispersiveness
Color nano material and micro materials in one kind.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611039046.XA CN106645121B (en) | 2016-11-24 | 2016-11-24 | A kind of Two-dimensional Liquid exempts from instrumental quantitative analysis method compared to color |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611039046.XA CN106645121B (en) | 2016-11-24 | 2016-11-24 | A kind of Two-dimensional Liquid exempts from instrumental quantitative analysis method compared to color |
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| CN106645121A true CN106645121A (en) | 2017-05-10 |
| CN106645121B CN106645121B (en) | 2019-10-11 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201063037Y (en) * | 2007-07-05 | 2008-05-21 | 上海科华生物工程股份有限公司 | Drying chemical test paper for quantitatively measuring activity of human body alpha-amylase |
| CN101676719A (en) * | 2008-09-19 | 2010-03-24 | 河南瑞驰生物科技有限公司 | Method for measuring diastatic enzyme activity using a spectrophotometer |
-
2016
- 2016-11-24 CN CN201611039046.XA patent/CN106645121B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201063037Y (en) * | 2007-07-05 | 2008-05-21 | 上海科华生物工程股份有限公司 | Drying chemical test paper for quantitatively measuring activity of human body alpha-amylase |
| CN101676719A (en) * | 2008-09-19 | 2010-03-24 | 河南瑞驰生物科技有限公司 | Method for measuring diastatic enzyme activity using a spectrophotometer |
Non-Patent Citations (2)
| Title |
|---|
| 夏玉亮: "《直读比色分析技术》", 31 March 1988, 劳动人事出版社 * |
| 曹健等: "《食品酶学》", 28 February 2011, 郑州大学出版社 * |
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Application publication date: 20170510 Assignee: GUILIN VEIRUN MEDICAL TECHNOLOGY Co.,Ltd. Assignor: GUILIN University OF TECHNOLOGY Contract record no.: X2023980046003 Denomination of invention: A Quantitative Analysis Method for Two Dimensional Liquid Chromatographic Immunoassay Granted publication date: 20191011 License type: Common License Record date: 20231108 |
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