CN109001463A - A kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method - Google Patents
A kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method Download PDFInfo
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- CN109001463A CN109001463A CN201810746656.6A CN201810746656A CN109001463A CN 109001463 A CN109001463 A CN 109001463A CN 201810746656 A CN201810746656 A CN 201810746656A CN 109001463 A CN109001463 A CN 109001463A
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01—MEASURING; TESTING
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
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- 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
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/57446—Specifically defined cancers of stomach or intestine
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Abstract
A kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method, the gastric cancer risk indicator object is pepsinogen Cgene, Pepsinogen II and gastrin 17, test strips include sample pad, gastric cancer risk indicator object antibody magnetism rare-earth fluorescent microballoon label pad, coated film and water absorption pad, and coated film is equipped with three gastric cancer risk indicator object quantitative detection lines and a quality control region C line.Using magnetic field by after the measured matter concentration in sample, using low background, highly sensitive fluorescence immune chromatography method, detection measured matter is carried out, solves the problems, such as that POCT class product sensitivity is low.When can detect blood sample to avoid immune lateral chromatography reagent, for the interference for reducing other substances in blood, tested after being usually diluted sample and caused by the relatively low problem of sensitivity.
Description
Technical field
The invention belongs to biomedicine field, be related to based on use magnetic rare-earth fluorescent microballoon as trace labelling object, stomach
Cancer risk indicator Internet of Things close quantitative testing test paper and preparation method thereof, specifically, a kind of measurement blood (serum, blood plasma, entirely
Blood) in pepsinogen Cgene/Pepsinogen II/gastrin 17 content detection reagent card.
Background technique
Propepsin (PG) is the precursor of pepsin, is divided into two Asias according to its biochemical property and immunogenicity
Group.The immunogenicity of 1-5 component is identical to be known as PG I, is mainly secreted by the chief cell of fundus gland and mucus neck cell, component 6-
7 are known as PG II, in addition to being secreted by the chief cell of fundus gland and mucus neck cell, the mucus of the pyloric gland of cardiac gland and antrum
Neck cell and duodenum upper section can also generate pepsinogen I I.Under normal conditions, there are about 1% PG to penetrate stomach lining hair
Thin blood vessel enters blood circulation, highly stable in blood into sanguimotor PG.Serum PG I and PG II reflect stomach lining
The quantity of body of gland and cell also reflects the secreting function of stomach lining different parts indirectly.
Gastrin is a kind of important gastrointestinal hormone, it is mainly secreted by G cell.G cell is typical open type cell,
It is most with antrum portion, followed by stomach bottom, duodenum and jejunum etc..Serum gastrin 17 is secreted by gastrointestinal tract G cell
Peptide hormone, in conjunction with cholecystokinin receptor after by a series of signal transduction play biological effect, mainly
Gastric acid secretion and nutrition gastrointestinal tract mucosa are participated in, more in recent years research shows that serum G17 is there are also promotion proliferation and inhibits
The effect serum G17 of apoptosis can prompt the functional status of patient's stomach lining, for the screening of disease before gastric cancer and cancer, because
This that is of great significance in the diagnosis of gastrointestinal disease
Current common Serum Pepsinogen I, Pepsinogen II, the detection method of gastrin 17 mainly have: latex enhancing
Immunoturbidimetry, enzyme-linked immunization, Timed resolved fluoroimmunoassay, colloidal gold immunity chromatography etc..Latex enhancing is immune
Turbidimetry, enzyme-linked immunization, Timed resolved fluoroimmunoassay, operating process are complicated, need a large amount of instrument and equipments and specially
Industry person's operation, detection time is longer, and detection sensitivity is high.Colloidal gold immunity chromatography is easy to operate, but sensitivity is not high, nothing
Method accurate quantification.Detection sensitivity and specificity can be improved in pepsinogen Cgene and Pepsinogen II joint-detection, examines identification
Disconnected offer more fully foundation, the two joint-detection, which has, to have complementary advantages and clinical value-added meaning, than individually detecting an item
Mesh is more meaningful.
Summary of the invention
In order to solve the above problem, facilitate pepsinogen Cgene/propepsin in measurement blood (serum, blood plasma, whole blood)
The detection demand of II/gastrin 17 content, the present invention provides a kind of fast and convenient, high sensitivity and the reliable stomaches of testing result
Cancer risk indicator object quantitative detecting method and test paper.
In order to reach the purpose of the present invention, technical solution of the present invention is as follows: a kind of gastric cancer risk indicator object quantitative detection examination
Paper, the gastric cancer risk indicator object are pepsinogen Cgene, Pepsinogen II and gastrin 17, the gastric cancer risk indicator object
Quantitative testing test paper includes box body, and the box body includes bottom plate and box cover, and Test paper, the detection examination are equipped in the box body
It is equipped with bottom plate below paper, is connect above Test paper with box cover；The box cover is equipped with through hole area；The through hole area is equipped with well
With result detection window；Detection zone is equipped with above the Test paper, detection zone is corresponding with through hole area；The detection zone includes
Sample pad, gastric cancer risk indicator object antibody magnetism rare-earth fluorescent microballoon label pad, coated film and water absorption pad are set on the coated film
There are three gastric cancer risk indicator object quantitative detection lines and a quality control region C line, the detection line and quality control region C line are arranged in parallel.
Further, the magnetic rare-earth fluorescent microballoon in the magnetic rare-earth fluorescent microballoon label pad is high molecular material package
The sphere diameter of the nanosphere of magnetic rare-earth fluorescent composition, the magnetism rare-earth fluorescent microballoon is 10 ~ 500nm, fluorescent emission wave
Long range is 400 ~ 800nm, and magnetic rare-earth fluorescent microsphere surface modification activities group is amino, carboxyl or sulfydryl.
Further, the coated film both ends respectively with water absorption pad and gastric cancer risk indicator object antibody label pad are mutually overlapping connects
It connects, the coated film is nitrocellulose filter；Sample pad, the sample have been overlayed in gastric cancer risk indicator object antibody label pad
Pad is glass fiber sample pad.
Further, the gastric cancer risk indicator object antibody magnetism rare-earth fluorescent microballoon label pad is that glass fibre or polyester are fine
Dimension.
A kind of gastric cancer risk indicator object quantitative testing test paper preparation method, including gastric cancer risk indicator object antibody label and system
Standby Test paper, the gastric cancer risk indicator object antibody markers step:
1. with the magnetic rare-earth fluorescent microballoon of carboxylic polystyrene microsphere package Eu3+-Fe3O4 compound preparation, partial size
200nm, solid content 1%；
2. taking above-mentioned microballoon 1mL, 7.0 0.02M 3- of 5mL pH (N- Ma Lindai) propane sulfonic acid buffer (MOPS) is added, mixes
Even, 25000rpm is centrifuged 10min, abandons supernatant；5mL MOPS is added, 90W ultrasound, work 2s, and interval 5s is repeated 2 times,
25000rpm is centrifuged 10min, abandons supernatant；
3. 5mL MOPS is added again, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, and completes the cleaning to microballoon；
4. 15mg EDC and 50mg sulfo-NHS is added in microballoon after cleaning, it is vortexed after mixing, 37 DEG C of reaction 15min；
5. 8.6 μ l of 2 mercapto ethanol after the reaction was completed, is added, the activation of carboxyl is terminated；
6. being separately added into pepsinogen Cgene, Pepsinogen II, gastrin 17 corresponds to each 1mg of antibody, it is placed in rotating and culturing case
On, 37 DEG C of 90 ~ 120min of reaction；
7. 100 μ L, 1% PEG20000 of 5% BSA and 100 μ L is then added, unlabelled site is closed, rotation is placed in
Turn on incubator, 37 DEG C of reaction 60min；
8. aforesaid liquid 2000rpm after reaction, is centrifuged 15min, supernatant is abandoned, is added and saves liquid (containing 1% BSA, 10% sugarcane
The pH8.0 0.2M boric acid borax buffer of sugar, 2% trehalose), 5mL, ultrasonic 2s；
9. the microballoon after label is diluted 50 ~ 100 times, every 200 μ L are dispensed, as single part semi-finished product.
Gastric cancer risk indicator Internet of Things close quantitative testing test paper preparation step:
1. with BIODOT three-dimensional metal spraying point film instrument, with 1 μ L/cm parameter, be coated on nitrocellulose filter pepsinogen Cgene,
The corresponding antibody three detections line of Pepsinogen II, gastrin 17 and a sheep anti mouse/goat anti-rabbit igg nature controlling line, 37 DEG C dry
Dry 16 hours, as reaction film.
2. impregnating glass fibre with the 0.1 M PBS solution containing 0.5% Tween-20, then 37 DEG C drying 16 hours, cut out
Cutting 17mm × 300mm is as sample pad.
3. pasting and being assembled in bottom plate sample pad, reaction film and blotting paper (22mm × 300mm) in the form laminated layer by layer
On.
It compared with prior art, include sample pad, gastric cancer risk indicator object the invention has the following beneficial effects: test strips
Antibody magnetism rare-earth fluorescent microballoon label pad, coated film and water absorption pad, it is quantitative that coated film is equipped with three gastric cancer risk indicator objects
Detection line and a quality control region C line utilize low background, highly sensitive using magnetic field by after the measured matter concentration in sample
Fluorescence immune chromatography method carries out detection measured matter, solves the problems, such as that POCT class product sensitivity is low.It can be to avoid immune side
When detecting blood sample to chromatography reagent, for the interference for reducing other substances in blood, carried out after being usually diluted sample
Test and caused by the relatively low problem of sensitivity.
Detailed description of the invention
Fig. 1 is pepsinogen Cgene test curve of the present invention；
Fig. 2 is Pepsinogen II test curve of the present invention；
Fig. 3 is gastrin 17 test curve of the present invention；
Fig. 4 is pepsinogen Cgene measuring method Precision test result of the present invention；
Fig. 5 is Pepsinogen II measuring method Precision test result of the present invention；
Fig. 6 is gastrin 17 measuring method Precision test result of the present invention；
Fig. 7 is structural schematic diagram of the invention；
In figure: 1, bottom plate；2, sample pad；3, gastric cancer risk indicator object antibody magnetism rare-earth fluorescent microballoon label pad；4, coated film；
5, water absorption pad；6, box body；61, box cover；71, well；72, result watch window；8, gastric cancer risk indicator object quantitative detection line；
9, quality control region C line.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The description of specific distinct unless the context otherwise, the present invention in detectable substance title and parameter, quantity both can be single
A form exists, and form that can also be multiple exists, and the present invention is defined not to this.Although step in the present invention with
Label is arranged, but is not used to limit the precedence of step, unless expressly stated the order of step or certain step
Based on rapid execution needs other steps, otherwise the relative rank of step is adjustable.It is appreciated that institute herein
The term "and/or" used one of is related to and covers associated listed item or one or more of any and all possibility
Combination.
A kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method, and the gastric cancer risk indicator object includes stomach
Proproteinase I, Pepsinogen II and gastrin 17, the gastric cancer risk indicator quality testing test paper includes box body, the box body
Including bottom plate and box cover, it is equipped with Test paper in box body, bottom plate is equipped with below Test paper, is connected above Test paper with box cover
It connects；Box cover is equipped with through hole area；The through hole area is equipped with well and result detection window；Detection zone is equipped with above Test paper,
Detection zone is corresponding with through hole area；Detection zone includes sample pad, gastric cancer risk indicator object antibody magnetism rare-earth fluorescent microballoon label
Pad, coated film and water absorption pad, the coated film are equipped with three gastric cancer risk indicator Internet of Things and close quantitative detection line and a Quality Control
Area's C line, the detection line and quality control region C line are arranged in parallel.Magnetic rare-earth fluorescent in magnetic rare-earth fluorescent microballoon label pad is micro-
Ball is the nanosphere of high molecular material coated magnetic rare-earth fluorescent composition, and the sphere diameter of the magnetism rare-earth fluorescent microballoon is
10 ~ 500nm, fluorescence emission wavelengths range are 400 ~ 800nm, and magnetic rare-earth fluorescent microsphere surface modification activities group is ammonia
Base, carboxyl or sulfydryl.Coated film both ends respectively with water absorption pad and pepsinogen Cgene/Pepsinogen II/gastrin 17 antibody mark
The mutually overlapping connection of note pad, has overlayed sample pad in gastric cancer risk indicator object antibody label pad.Sample pad is glass fibre sample
Product pad.Coated film is nitrocellulose filter.Label pad is glass fibre or polyester fiber.
Embodiment, gastric cancer risk indicator Internet of Things close the foundation of quantitative detecting method:
The preparation of 1.1 polystyrene microsphere seeds
1.1.1 it in the styrene and separatory funnel for measuring 100ml, is washed 3 times, is removed in styrene repeatedly with 1 M NaOH
Then impurity is placed in drying basin more than drying for 24 hours then with purifying water washing to neutrality；
1.1.2 argon gas is filled in reaction unit, the oxygen in removing device；
1.1.3 the ethyl alcohol of 100ml is added in reaction unit, 5g PVP is then added, stirs 30min；
1.1.4 40ml washed styrene and 5ml azodiisobutyronitrile (AIBN) is added, warming-in-water is to 45 DEG C, reaction
Then reaction temperature is increased to 75 DEG C by 30min, react 12h；
1.1.5 after reaction, with the above-mentioned reaction solution of screen filtration, centrifugation 3 ~ 5 times then is washed with dehydrated alcohol, then will
Polymer after preliminary removal of impurities makes its natural sedimentation, weighs Sediment weight in 40% ethyl alcohol.
The carboxylated of 1.2 polystyrene surfaces is modified
1.2.1 using the resulting polystyrene microsphere of above-mentioned dispersion as seed 1g, dispersed with 0.25% SDS of 10ml；
1.2.2 the hexamethylene of 2 times of seed weights, 35 DEG C of 16 ~ 18h of swelling and then are in the above system added；
1.2.3 it weighs 0.02g benzoyl peroxide, measure 10 μ l ethylene glycol monoemethyl ethers and 10 μ l dimethyl benzene olefin(e) acid ethylene glycol
Ester is added separately in above-mentioned reaction solution, continues to be swollen 12h；
1.2.4 after being swollen, 2.5mg PVA is added, improves reaction temperature to 75 DEG C, the reaction was continued 10 ~ 12h；
1.2.5 it is washed repeatedly with ethyl alcohol 3 times, removes unreacted monomer and oligomer, then 20000rpm centrifugal sedimentation is micro-
Ball.
The preparation of 1.3 magnetism-fluorescent nanometer microsphere
1.3.1 by FeCl2, FeCl3, EuCl3, NaOH solution, example Fe2+:Fe3+:Eu3+:Na+=1:1:2:5 in molar ratio,
Its solution is mixed, is stirred to react, is precipitated, is filtered after being aged 3 ~ 5h, with purifying water washing 2 ~ 3 times, dry, grinding
Eu3+-Fe3O4 compound is obtained afterwards.
1.3.2 the ultrasonic disperse into 20ml isopropanol is added in the polystyrene microsphere for taking 0.1g carboxylated to modify
10min；
1.3.3 Eu3+-Fe3O4 compound 0.5mg is weighed, the ultrasonic disperse 20min into 5ml chloroform is added；
1.3.4 two kinds of liquid in 1.3.2 and 1.3.3 are mixed, 5 ~ 6h is placed in control in vacuum environment, is then being placed in normal pressure 2
~3h；
1.3.5 it by the microballoon after swelling with ethanol washing 3 times, is removed with magnetic field and fails and wrap up the microballoon of magnetic particle, with suitable
Amount is resuspended containing 0.05% sodium azide aqueous solution to get magnetic rare-earth fluorescent microballoon, and 2 ~ 8 DEG C of preservations are forbidden to freeze.
It is prepared by the label of 1.4 antibody
1.4.1 it takes the 200nm of above-mentioned preparation, the microballoon 1ml that solid content is 1%, 7.0 0.02M 3- (N- Ma of 5ml pH is added
Beautiful jade generation) propane sulfonic acid buffer (MOPS), it mixes, 25000rpm is centrifuged 10min, abandons supernatant；
1.4.2 5ml MOPS is added, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, 25000rpm centrifugation 10min, in abandoning
Clearly；
1.4.3 5ml MOPS is added again, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, and completes the cleaning to microballoon；
1.4.4 15mg EDC and 50mg sulfo-NHS is added in microballoon after cleaning, is vortexed after mixing, 37 DEG C of reactions
15min；
1.4.5 after the reaction was completed, 8.6 μ l of 2 mercapto ethanol is added, terminates the activation of carboxyl；
1.4.6 it is separately added into each 1mg(outsourcing of the corresponding antibody of pepsinogen Cgene, Pepsinogen II, gastrin 17), it is placed in
On rotating and culturing case, 37 DEG C of 90 ~ 120min of reaction；
1.4.7 100 μ l, 1% PEG20000 of 5% BSA and 100 μ l is then added, unlabelled site is closed, is set
In on rotating and culturing case, 37 DEG C of reaction 60min；
1.4.8 after reaction, by aforesaid liquid 2000rpm be centrifuged 15min, abandon supernatant, be added save liquid (containing 1% BSA,
The pH8.0 0.2M boric acid borax buffer of 10% sucrose, 2% trehalose), 5ml, ultrasonic 2s；
1.4.9 the microballoon after label is diluted 50 ~ 100 times, every 200 μ l are dispensed, as single part semi-finished product, with stomach cardia
I/Pepsinogen II of proenzyme/gastrin 17 test paper is used cooperatively.
1.5 the preparation of test paper
1.5.1 stomach cardia is coated with respectively on nitrocellulose filter with 1 μ l/cm parameter with BIODOT three-dimensional metal spraying point film instrument
The corresponding antibody three detections line of proenzyme I, Pepsinogen II, gastrin 17 and a sheep anti mouse/goat anti-rabbit igg nature controlling line,
37 DEG C drying 16 hours, as reaction film.
1.5.2 glass fibre is impregnated with the 0.1 M PBS solution containing 0.5% Tween-20, then 37 DEG C of dryings 16 are small
When, cutting 17mm × 300mm is as sample pad.
1.5.3 sample pad, reaction film and blotting paper (22mm × 300mm) are carried out pasting group in the form laminated layer by layer
On PVC bottom plate, after slitting, as pepsinogen Cgene/Pepsinogen II/gastrin 17 measures test paper, is placed in plastic clip
In box, as pepsinogen Cgene/Pepsinogen II/gastrin 17 measures reagent card.
2. the evaluation of pepsinogen Cgene/Pepsinogen II/gastrin 17 detection method
2.1 the range of linearity
2.1.1 at least five kinds of concentration will be diluted to according to a certain percentage close to the high level sample of the range of linearity upper limit respectively, wherein
The sample of low value concentration must be close to the lower limit of the range of linearity；
2.1.2 above-mentioned each concentration samples respectively take 100 μ l, in the label microballoon after being added separately to packing, are vortexed and mix；
2.1.3 the above reaction solution is placed on magnetic field, after 1min, is discarded supernatant, then every 200 μ l PBS(0.02M of addition,
PH7.4)；
2.1.4 magnetic field is removed, is vortexed again for mixing, above each concentration samples are taken into 100 μ l respectively, are added to propepsin
I, Pepsinogen II, gastrin 17 measure in test paper, every concentration retest 3 times；
2.1.5 after reacting 10min, test paper is put into dry type immunofluorescence analysis instrument, T/C value is read, calculates the T/ of each concentration
C mean value, using double-log straight line fitting concentration-response curve, (X-axis is lg (concentration)；Y-axis is lg (T/C)), pepsinogen Cgene
Response curve is y=2.1645x -4.0499, R=0.9919；Pepsinogen II response curve is y=3.1043x-
2.7693 R=0.9903；Gastrin 17 response curve is y=1.1402x -0.3315, R=0.9964.
2.2 minimum detectability
Negative serum is operated by 2.1.2 ~ 2.1.4, retest 20 times, calculates T/C mean value, it is bent to be substituted into 2.1.5 test
In line, the minimum detectability of this method is obtained, wherein pepsinogen Cgene minimum detectability is 70ug/L；Pepsinogen II is most
Low detection limits are 3ug/L；Gastrin 17 minimum detectability is 1pmol/L.
2.3 precision
Calibration object is diluted to high, medium and low three concentration, is operated by 2.1.2 ~ 2.1.4, every concentration retest 10 times.As a result
Shown in Fig. 4-Fig. 6, it can thus be seen that the coefficient of variation of this method test is respectively less than 10%, therefore precision with higher.
This method tests 90 parts of clinical serums, with other product measured value correlations in the market, pepsinogen Cgene result are as follows: y
=0.952x+0.6535, related coefficient 0.972, Pepsinogen II result are as follows: y=0.9321x+0.6015,
Its related coefficient is 0.963, gastrin 17 result are as follows: y=0.9551x+0.6451, related coefficient 0.963.
Synthesis has preferable accurate it is found that this method can detect pepsinogen Cgene/Pepsinogen II/gastrin 17
Degree tests the equal < 10% of the coefficient of variation.Through clinical trial, the present invention, by after the measured matter concentration in sample, is utilized using magnetic field
Low background, highly sensitive fluorescence immune chromatography method, carry out detection measured matter, solve that POCT class product sensitivity is low to ask
Topic.Can to avoid immune lateral chromatography reagent detect blood sample when, for reduce blood in other substances interference, usually by sample
Originally tested after being diluted and caused by the relatively low problem of sensitivity.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (6)
1. a kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method, the gastric cancer risk indicator object include stomach egg
White proenzyme I, Pepsinogen II and gastrin 17, it is characterised in that: the gastric cancer risk indicator Internet of Things close quantitative testing test paper
Including box body, the box body includes bottom plate and box cover, and Test paper is equipped in the box body, is equipped with bottom below the Test paper
Plate, Test paper top are connect with box cover；The box cover is equipped with through hole area；Detection zone, detection are equipped with above the Test paper
Area is corresponding with through hole area；The detection zone includes sample pad, gastric cancer risk indicator object antibody magnetism rare-earth fluorescent microballoon label
Pad, coated film and water absorption pad, the coated film are equipped with three gastric cancer risk indicator object quantitative detection lines and a quality control region C
Line, the detection line and quality control region C line are arranged in parallel.
2. a kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper according to claim 1 and preparation method, feature exist
In: the magnetic rare-earth fluorescent microballoon in the magnetism rare-earth fluorescent microballoon label pad is high molecular material coated magnetic rare-earth fluorescent
The nanosphere of compound, the sphere diameter of the magnetism rare-earth fluorescent microballoon are 10 ~ 500nm, and fluorescence emission wavelengths range is 400
~ 800nm, magnetic rare-earth fluorescent microsphere surface modification activities group is amino, carboxyl or sulfydryl.
3. a kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper according to claim 1 and preparation method, feature exist
In: coated film both ends respectively with water absorption pad and pepsinogen Cgene/Pepsinogen II/gastrin 17 antibody label pad phase
Mutually overlapping connection；The coated film is nitrocellulose filter；Sample has been overlayed above the gastric cancer risk indicator object antibody label pad
Product pad, the sample pad are glass fiber sample pad.
4. a kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper according to claim 3 and preparation method, feature exist
In: the gastric cancer risk indicator object antibody label pad is glass fibre or polyester fiber.
5. a kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method, including gastric cancer risk indicator object antibody label
With prepare Test paper, the gastric cancer risk indicator object antibody markers step:
1. with the magnetic rare-earth fluorescent microballoon of carboxylic polystyrene microsphere package Eu3+-Fe3O4 compound preparation, partial size
200nm, solid content 1%；
2. taking above-mentioned microballoon 1mL, 7.0 0.02M 3- of 5mL pH (N- Ma Lindai) propane sulfonic acid buffer (MOPS) is added, mixes
Even, 25000rpm is centrifuged 10min, abandons supernatant；5mL MOPS is added, 90W ultrasound, work 2s, and interval 5s is repeated 2 times,
25000rpm is centrifuged 10min, abandons supernatant；
3. 5mL MOPS is added again, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, and completes the cleaning to microballoon；
4. 15mg EDC and 50mg sulfo-NHS is added in microballoon after cleaning, it is vortexed after mixing, 37 DEG C of reaction 15min；
5. 8.6 μ l of 2 mercapto ethanol after the reaction was completed, is added, the activation of carboxyl is terminated；
6. being separately added into pepsinogen Cgene, Pepsinogen II, gastrin 17 corresponds to each 1mg of antibody, it is placed in rotating and culturing case
On, 37 DEG C of 90 ~ 120min of reaction；
7. 100 μ L, 1% PEG20000 of 5% BSA and 100 μ L is then added, unlabelled site is closed, rotation is placed in
Turn on incubator, 37 DEG C of reaction 60min；
8. aforesaid liquid 2000rpm after reaction, is centrifuged 15min, supernatant is abandoned, is added and saves liquid (containing 1% BSA, 10% sugarcane
The pH8.0 0.2M boric acid borax buffer of sugar, 2% trehalose), 5mL, ultrasonic 2s；
9. the microballoon after label is diluted 50 ~ 100 times, every 200 μ L are dispensed, as single part semi-finished product, with propepsin
I/Pepsinogen II/gastrin 17 measurement test paper is used cooperatively.
6. a kind of gastric cancer risk indicator Internet of Things according to claim 5 close quantitative testing test paper preparation method, the gastric cancer
Risk indicator Internet of Things close quantitative testing test paper preparation step:
1. with BIODOT three-dimensional metal spraying point film instrument, with 1 μ L/cm parameter, be coated on nitrocellulose filter pepsinogen Cgene,
The corresponding antibody three detections line of Pepsinogen II, gastrin 17 and a sheep anti mouse/goat anti-rabbit igg nature controlling line, 37 DEG C dry
Dry 16 hours, as reaction film；
2. impregnating glass fibre with the 0.1 M PBS solution containing 0.5% Tween-20, then 37 DEG C drying 16 hours, cut
17mm × 300mm is as sample pad；
3. sample pad, reaction film and blotting paper (22mm × 300mm) are pasted and are assembled on bottom plate in the form laminated layer by layer.
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Application publication date: 20181214 |