CN110187106A - A kind of Multilayer film dry plate and its measuring method quantitative determining alpha-amylase activity - Google Patents
A kind of Multilayer film dry plate and its measuring method quantitative determining alpha-amylase activity Download PDFInfo
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- CN110187106A CN110187106A CN201910469655.6A CN201910469655A CN110187106A CN 110187106 A CN110187106 A CN 110187106A CN 201910469655 A CN201910469655 A CN 201910469655A CN 110187106 A CN110187106 A CN 110187106A
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- amylase activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/928—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
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Abstract
The invention discloses the Multilayer film dry plates and its measuring method of a kind of quantitative determination alpha-amylase activity for belonging to medical immunology tested in vitro technical field.The Multilayer film dry plate is to be made of from top to bottom Multifunctional layered, reagent layer and supporting layer, is mainly used for the measurement of serum or urine sample；Wherein Multifunctional layered can quickly and evenly tile sample onto following reagent layer, meanwhile, the white reflection substance in Multifunctional layered containing 95% or more high reflectance；Reagent in reagent layer contains substance required for reaction solution；Supporting layer uses macromolecule polymeric material, plays a part of support and fixation.When sample penetrates into reagent layer by Multifunctional layered, chemically reacted in reagent layer, and generate color change, thus the alpha-amylase activity in quantitatively determining human.The present invention has tool specificity good, strong antijamming capability, it is easy to operate, can quick and precisely obtain being suitable for full-automation analyzer, clinical value is big as a result, be easy to store transport, at low cost.
Description
Technical field
The invention belongs to medical immunology tested in vitro technical field, in particular to a kind of quantitative determination alpha-amylase activity
Multilayer film dry plate and its measuring method.
Background technique
Serum and urine determination of amylase can be used as the diagnosis index of acute pancreatitis, when acute pancreatitis occurs, pancreas
Amylase in gland enters in blood, causes hemodiastase activity is significant to increase, amylase in urine was at 12-24 hours after the onset
It can increase.Certain chronic pancreatic diseases, such as chronic pancreatitis and popular parotitis, serum amylase can also be increased.
Therefore, the diagnosis of the diseases such as acute pancreatitis can be realized by the monitoring to a- amylase activity.
Substrate kind for determination of amylase is various, and the measurement substrate of early stage is starch, due to starch structure not really
It is qualitative, prevent enzymatic reaction from being calculated by chemical examination amount.Occur staining starch method, i.e. dyestuff and insoluble starch again later
It is combined into chromogen substrate, but pigment additional amount is not easy to control in staining starch and this method is using cumbersome, which is of little use.With
Using artificial synthesized substrate determining amylase is formed to the research that deepens continuously of alpha-amylase activity measuring method
Substrate, it can be achieved that stoichiometric relationship calculating.
The method of detection serum and the alpha-amylase activity in urine has very much, mainly there is viscosimetry, mashing system, ratio
Turbid method, enzyme performance rate method etc..The specificity of viscosimetry and turbidimetry, sensitivity are poor.Mashing system is complicated for operation, is unfavorable for clinic
Conventional detection.And enzyme performance rate method is usually due to needing the various large automatic Biochemical Analyzers of adapted, it is expensive, and need
Special start-up, it is complicated for operation, therefore personal family and basic hospital are all bought and are used without condition.
For disadvantages mentioned above, using dry chemical detection method.Dry chemical diagnostic method be will react required for part or
Whole reagents are fixed on the reagent carrier with special construction, avoid to prepare liquid reagent in this way, have operation letter
Single, detection work can be carried out whenever and wherever possible, be polluted the advantages that small, accuracy and precision are higher, thus by testing staff and
The favor of subject.
Summary of the invention
The object of the present invention is to provide a kind of Multilayer film dry plates and its measuring method for quantitative determining alpha-amylase activity；Its
It is characterized in that, the Multilayer film dry plate of the quantitative determination alpha-amylase activity is from top to bottom by Multifunctional layered, reagent layer and branch
Layer composition is supportted, the measurement of serum or urine sample is mainly used for；Wherein Multifunctional layered can be quickly and evenly by sample tiling under
On the reagent layer in face, meanwhile, the white reflection substance in Multifunctional layered containing 95% or more high reflectance；Examination in reagent layer
Agent contains substance required for reaction solution；Supporting layer uses macromolecule polymeric material, plays a part of support and fixation.
Reagent in the reagent layer includes buffer reagent, substrate, activator, preservative and thickener.
The white reflection substance of the high reflectance is barium sulfate, titanium dioxide or barium titanate.
The supporting layer is using transparent plastic made of various macromolecule polymeric materials.
The macromolecule polymeric material is polyethylene, polyvinyl chloride, polyethylene terephthalate.
The Multifunctional layered is by the cellulose acetate of hydrophilic high molecular material, nonionic surface active agent and above-mentioned
Reflective material is constituted.
A kind of alpha-amylase activity measuring method for the Multilayer film dry plate quantitative determining alpha-amylase activity；Its feature exists
In the detecting step of the quantitative determination alpha-amylase activity is:
1) sample to be tested serum or urine are titrated on Multifunctional layered；
2) sample to be tested is quickly and evenly permeated, is tiled onto reagent layer by Multifunctional layered；
3) reagent layer is the region mainly chemically reacted, and sample to be tested is chemically reacted with the reagent in reagent layer
And color change, changed by the depth of color or the optical density of measurement reaction process, thus the α-in quantitatively determining human
Amylase activity；Alpha-amylase catalysis substrate specifically in sample to be tested generates the free chloro- 4- nitrophenol (CNP) of 2-,
I.e. a kind of yellow uitramarine indicator measures dry plate nitre at 405nm with matching used reflective spectrophotometer at 37 DEG C
The generating rate of base phenol carries out data processing, conclusion that you can get it using two point rate assay；Its generating rate and diastatic activity
Property is directly proportional.
Chemical reaction and color change occur for the sample to be tested and the reagent in reagent layer, and to be mainly in reagent include: making
MES buffer is 2-morpholine ethane sulfonic acid buffer, PH 6, concentration 10-100mM；The substrate of use is artificial synthesized more
Sugar derivatives is marked with the chloro- 4- nitrophenol (CNP) of 2-, concentration of substrate 3-30Mm；Activator be calcium chloride, magnesium chloride,
The concentration of middle calcium chloride is 50-130mM, and the concentration of magnesium chloride is 10-60mM；The thickener used is gelatin.
The Multifunctional layered can filter out some macromolecule substances and sample is made quickly and evenly to be diffused into lower interview
In oxidant layer, so that sample be enable sufficiently to react with reagent in reagent layer, develop the color more uniform；Multifunctional layered also acts as reflection simultaneously
The effect of light reduces interference of the coloring matter to reflected light, reduces detection error.
Present invention has the advantages that one kind can reduce measurement interference, it can fast and convenient quantitatively measuring activity of human body alpha-amylase
Multilayer film dry plate technology.This method have it is easy to operate, can quick and precisely obtain result, it is low need blood volume, be easy use and it is small
Type, the advantages that being easy to store transport, is at low cost；Specific good, strong antijamming capability, can resist hemoglobin, anti-bilirubin,
The interference of ascorbic acid is suitable for full-automatic analyzer, there is biggish clinical value；The present invention has rational design, practical
Property is strong, applied widely.
Detailed description of the invention
Fig. 1 is the testing result figure for surveying α-amylasecontent.
Fig. 2 is the reproducible results figure for surveying α-amylasecontent.
Fig. 3 is Multilayer film dry plate structural schematic diagram.
Specific embodiment
The present invention provides a kind of Multilayer film dry plate and its measuring method for quantitative determining alpha-amylase activity；The quantitative survey
The Multilayer film dry plate for determining alpha-amylase activity is to be made of (as shown in Figure 3 Multifunctional layered, reagent layer and supporting layer from top to bottom
Multilayer film dry plate structure), it is mainly used for the measurement of serum or urine sample；In order to make the easier clear reason of the contents of the present invention
Solution, below according to specific implementation case and in conjunction with attached drawing, the present invention is described in further detail, wherein
Analysis is compared below with reference to the Multilayer film dry plate that Fig. 1, Fig. 2 and table survey α-amylasecontent to the present invention.
Concentration of the canonical plotting abscissa of Fig. 1, Fig. 2 for institute's sample, unit U/L；Ordinate is measured response.
Embodiment
Using Multilayer film dry plate as shown in Figure 3；Concrete operation step are as follows:
1) sample to be tested serum or urine are titrated on Multifunctional layered；
2) sample to be tested is quickly and evenly permeated, is tiled onto reagent layer by Multifunctional layered；
3) reagent layer is the region mainly chemically reacted, and sample to be tested is chemically reacted with the reagent in reagent layer
And color change, changed by the depth of color or the optical density of measurement reaction process, wherein table 1 show enzymatic reaction examination
Oxidant layer formula；Table 2 show Multifunctional layered proportion: thus the alpha-amylase activity in quantitatively determining human；Specifically to test sample
The chloro- 4- nitrophenol (CNP) of 2- that alpha-amylase catalysis substrate generation in product dissociates, i.e., a kind of yellow uitramarine indicator,
At 37 DEG C, the generating rate of dry plate nitrophenol at 405nm is measured with matching used reflective spectrophotometer, using two
Point velocity method carries out data processing, conclusion that you can get it；Its generating rate is directly proportional to amylase activity.
1 enzymatic reaction reagent layer formula of table
| Reagent | Concentration |
| MES buffer solution (PH=6) | 10-200mM |
| Substrate | 5-50mM |
| Calcium chloride | 20-220mM |
| Magnesium chloride | 10-100mM |
2 Multifunctional layered of table proportion:
| Reagent | Dosage |
| Barium sulfate, titanium dioxide, barium titanate | 0.1-5g/mL |
| Cellulose acetate | 0.1-5g/mL |
| Acetone | 0.1-5g/mL |
| Nonionic surface active agent | 0.001g-0.01g/mL |
Supporting layer uses transparent plastic sheet, and the applying amount of reagent layer is 10 μ l, and Multifunctional layered amount of reagent is 50 μ l.It will be different
The serum of concentration is added drop-wise on above-mentioned dry plate, at 37 DEG C, continuously monitors reflected light of the dry plate at 405nm with the photometer
The variation of density detects the generating rate of nitrophenol that is, at 405nm, can calculate the concentration of alpha-amylase.Wherein, nonionic
Surfactant refers to unionization in aqueous solution, hydrophilic group mainly by with a certain number of oxygen-containing groups (generally
Ether and hydroxyl) it constitutes.Nonionic surfactant is more superior than ionic surface active agent in some aspects；Because in the solution
It is not ionic condition, so stability is high, the not influence vulnerable to strong electrolyte inorganic salts；Generally use ethylene oxide, second two
Alcohol, glycerol pentaerythrite, anhydrous sorbitol or sucrose.
(1) testing result
As shown in Figure 1,10 μ L various concentration serum standard panels are added dropwise within the scope of 31.25~1000U/L, it is anti-in 37 DEG C
5min is answered, detected at 405nm and records result.The result shows that downward trend is presented in curve with the increase of serum-concentration,
Response is 0.327~0.129.
(2) repeated result
As shown in Fig. 2, the dry plate of preparation is placed 47 days under freezing conditions, the result detected again later.
Claims (9)
1. a kind of Multilayer film dry plate for quantitative determining alpha-amylase activity；It is characterized in that, the quantitative determination alphalise starch enzyme activity
Property Multilayer film dry plate be made of from top to bottom Multifunctional layered, reagent layer and supporting layer, be mainly used for serum or urine sample
Measurement；Wherein Multifunctional layered can quickly and evenly tile sample onto following reagent layer, meanwhile, contain in Multifunctional layered
The white reflection substance of 95% or more high reflectance；Reagent in reagent layer contains substance required for reaction solution；Support
Layer uses macromolecule polymeric material, plays a part of support and fixation.
2. quantitative determining the Multilayer film dry plate of alpha-amylase activity according to claim 1；It is characterized in that, the reagent layer
In reagent include buffer reagent, substrate, activator, preservative and thickener.
3. quantitative determining the Multilayer film dry plate of alpha-amylase activity according to claim 1；It is characterized in that, the high reflection
The white reflection substance of coefficient is barium sulfate, titanium dioxide or barium titanate.
4. quantitative determining the Multilayer film dry plate of alpha-amylase activity according to claim 1；It is characterized in that, the supporting layer
Using transparent plastic made of various macromolecule polymeric materials.
5. quantitative determining the Multilayer film dry plate of alpha-amylase activity according to claim 4；It is characterized in that, the macromolecule
Polymeric material is polyethylene, polyvinyl chloride, polyethylene terephthalate.
6. quantitative determining the Multilayer film dry plate of alpha-amylase activity according to claim 4；It is characterized in that, described multi-functional
Layer is made of the cellulose acetate of hydrophilic high molecular material, nonionic surface active agent and above-mentioned reflective material.
7. quantitative determining the alpha-amylase activity measurement side of the Multilayer film dry plate of alpha-amylase activity described in a kind of claim 1
Method；It is characterized in that, the detecting step of the quantitative determination alpha-amylase activity is:
1) sample to be tested serum or urine are titrated on Multifunctional layered；
2) sample to be tested is quickly and evenly permeated, is tiled onto reagent layer by Multifunctional layered；
3) reagent layer is the region mainly chemically reacted, and chemical reaction and face occur for the reagent in sample to be tested and reagent layer
Color change is changed by the depth of color or the optical density of measurement reaction process, thus the alphalise starch in quantitatively determining human
Enzymatic activity；The chloro- 4- nitrophenol (CNP) of 2- that alpha-amylase catalysis substrate generation specifically in sample to be tested dissociates, i.e., one
Kind yellow uitramarine indicator measures dry plate nitrobenzene at 405nm with matching used reflective spectrophotometer at 37 DEG C
The generating rate of phenol carries out data processing, conclusion that you can get it using two point rate assay；Its generating rate and amylase activity at
Direct ratio.
8. quantitative determining the alpha-amylase activity measurement side of the Multilayer film dry plate of alpha-amylase activity according to claim 7
Method；It is characterized in that, chemical reaction occurs for the reagent in the sample to be tested and reagent layer and color change is mainly in reagent
Include: the MES buffer used is 2-morpholine ethane sulfonic acid buffer, PH 6, concentration 10-100mM；The substrate of use is people
Work synthesizes the chloro- 4- nitrophenol (CNP) of polysaccharide derivates 2-, concentration of substrate 3-30Mm；Activator be calcium chloride, magnesium chloride,
Wherein the concentration of calcium chloride is 50-130mM, and the concentration of magnesium chloride is 10-60mM；The thickener used is gelatin.
9. quantitative determining the alpha-amylase activity measurement side of the Multilayer film dry plate of alpha-amylase activity according to claim 7
Method；It is characterized in that, the Multifunctional layered can filter out some macromolecule substances and keep sample quickly and evenly multi-functional
Into following reagent layer, so that sample be enable sufficiently to react with reagent in reagent layer, develop the color more uniform；While Multifunctional layered is also
Play the role of reflected light, reduces interference of the coloring matter to reflected light, reduce detection error.
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Cited By (2)
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| CN110794128A (en) * | 2019-10-31 | 2020-02-14 | 无锡锦帛诚医疗器械科技有限公司 | Dry chemical method multi-layer membrane reagent for in vitro biochemical diagnosis |
| CN111879591A (en) * | 2020-09-01 | 2020-11-03 | 中科嘉睿(天津)医疗科技发展有限公司 | Dry chemical standard reagent tablet and preparation method thereof |
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