CN108196048A - A kind of bisphenol-A fluorescence detection test strip, preparation method and application - Google Patents
A kind of bisphenol-A fluorescence detection test strip, preparation method and application Download PDFInfo
- Publication number
- CN108196048A CN108196048A CN201710894094.5A CN201710894094A CN108196048A CN 108196048 A CN108196048 A CN 108196048A CN 201710894094 A CN201710894094 A CN 201710894094A CN 108196048 A CN108196048 A CN 108196048A
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- Prior art keywords
- aptamers
- line
- biotin
- bisphenol
- streptavidin
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Classifications
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention discloses a kind of bisphenol-A fluorescence detection test strip, preparation method and application, bisphenol-A fluorescence detection test strip is included in sample pad, bonding pad, nitrocellulose filter and the absorption pad for pasting overlap joint on liner plate successively；The bonding pad is fluorescent nanometer microsphere aptamers 1. bonding pad, arranged for interval has detection label and reference marker on the nitrocellulose membrane, the detection label includes the detection line 2. printed on nitrocellulose membrane with Streptavidin biotin aptamers, the reference marker includes the line of reference 3. printed on nitrocellulose membrane with Streptavidin biotin aptamers, and the detection line is arranged in parallel with line of reference and detection line is close to sample pad.The present invention uses the principle of competition law, the content of bisphenol-A in sample to be tested is detected by detection line in test strips and the colour developing degree of line of reference, detection is quick, accurate, can complete to detect in 30min, suitable for enterprise or the field quick detection of government regulator.
Description
Technical field
The invention belongs to technical field of fluorescence detection, more particularly, to a kind of bisphenol-A fluorescence detection test strip, preparation method
And application.
Background technology
Bisphenol-A is a kind of environmental endocrine disruptors, due to its specific food safety risk, receives pass in recent years
Note.Bisphenol-A has the function of similar estrogen, and body is absorbed by some approach such as polluted source, food or through skin
Afterwards, synthesis, release and metabolism of body endocrine hormone etc. can be interfered, so as to which organism endocrine be made to lack of proper care, sex premature occurs
Etc. diseases.Bisphenol-A may be sent out closely, in long-time contact process as plastics packed synthesis material with complex matrices food
Raw migration, so as to bring potential food safety hazards.
The measure of bisphenol-A mainly has the detection of the precision instruments such as high performance liquid chromatography, high performance liquid chromatography-tandem mass method
Method, testing cost is higher；Its enzyme-linked immunization is easy to operate, and detection is quick, but biological antibody prepares cumbersome take.
Aptamer is a kind of new bio identification molecule developed in recent years, is obtained by in-vitro screening
One group of oligonucleotide sequence that can be combined with the contour affine, high specific of target molecule such as protein, organic matter or metal ion
(DNA or RNA).Since it has many advantages, such as that molecular weight is smaller, chemically synthesizes, the good extensive pass for receiving researcher of stability
Note.Exploitation provides new approaches based on research of the Rapid detection test strip of aptamer for rapid detection method.
Invention content
First purpose of the present invention is, for the deficiencies in the prior art, provides a kind of bisphenol-A fluoroscopic examination
Test strips.
For this purpose, the above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of bisphenol-A fluorescence detection test strip, the bisphenol-A fluorescence detection test strip, which is included on liner plate to paste successively, to be taken
Sample pad, bonding pad, nitrocellulose filter and the absorption pad connect；1. the bonding pad is combined for fluorescent nanometer microsphere-aptamers
Pad, arranged for interval has detection to mark and reference marker on the nitrocellulose membrane, the detection label include with Streptavidin-
2. detection line that biotin-aptamers are printed on nitrocellulose membrane, the reference marker are included with Streptavidin-biology
3. line of reference that element-aptamers are printed on nitrocellulose membrane, the detection line is arranged in parallel with line of reference and detection line is close
Sample pad.
In order to obtain further technique effect, the present invention can also use technical solution further below：
Preferably, aptamers are 1., 2. aptamers are followed successively by with the nucleotide sequence of aptamers 3.：
Aptamers are 1.：5’-CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA
GCG CAT CAC GGG TTC GCA CCA AAA AAAAAAAAAAAAAAAAAAAAAA-NH2-3’；
Aptamers are 2.：5’-TGG TGC GAA CCC GTG ATG CGC TGG GCC ATA CGC GGA ACG CTA
TCC CAC CTG ACC ACC CAC CGG-Biotin-3’；
Aptamers are 3.：5’-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTT-3’.
Preferably, the bonding pad is：1. fluorescent nanometer microsphere is 1 according to mass ratio with amido modified aptamers:10
It is reacted 1.5 hours at 37 DEG C and obtains fluorescent nanometer microsphere-aptamers 1., 1. fluorescent nanometer microsphere-aptamers are equably applied
It is put on glass fibre membrane, fluorescent nanometer microsphere-aptamers 1. bonding pad is formed after freeze-drying.
Preferably, the detection line is：2. biotin modification aptamers are 1 according to molar ratio with Streptavidin:10
30min is reacted at 37 DEG C and obtains Streptavidin-biotin-aptamers 2., Streptavidin-life is used on nitrocellulose filter
2. cross to obtain comprising the Streptavidin-line of biotin-aptamers 2. object element-aptamers be detection line by solution；
The line of reference is：3. biotin modification aptamers are 1 according to molar ratio with Streptavidin:10 is anti-at 37 DEG C
30min is answered to obtain Streptavidin-biotin-aptamers 3., with Streptavidin-biotin-suitable on nitrocellulose filter
3. cross to obtain comprising the Streptavidin-line of biotin-aptamers 3. ligand be line of reference by solution.
Preferably, the liner plate is that the toughness material not absorbed water is formed, including plastics.
Preferably, the sample pad and/or the material of bonding pad include glass fibre membrane.
Preferably, the absorption pad includes absorbent filter.
Second object of the present invention is, in view of the deficiencies of the prior art, provides a kind of bisphenol-A fluorescence detection test strip
Preparation method.
For this purpose, the above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of preparation method of bisphenol-A fluorescence detection test strip, the preparation method include the following steps：
(1) 1. fluorescent nanometer microsphere and amido modified aptamers are 1 according to mass ratio:10 at 37 DEG C reaction it is 1.5 small
When, obtain fluorescent nanometer microsphere-aptamers 1.；1. fluorescent nanometer microsphere-aptamers are equably applied on glass fibre membrane,
Fluorescent nanometer microsphere-aptamers 1. bonding pad is formed after freeze-drying；
(2) aptamers of biotin modification 2. with the aptamers of biotin modification 3. respectively with Streptavidin according to mole
Than being 1:10 react 30min at 37 DEG C, obtain Streptavidin-biotin-aptamers 2. with Streptavidin-biotin-
Aptamers are 3.；On nitrocellulose membrane with Streptavidin-biotin-aptamers 2. with Streptavidin-biotin-aptamers
3. solution draw two parallel lines respectively, comprising the Streptavidin-line of biotin-aptamers 2. be detection line, include chain
The line of mould Avidin-Biotin-aptamers 3. is line of reference；
(3) on liner plate successively paste overlap joint sample pad, fluorescent nanometer microsphere-aptamers 1. bonding pad, include strepto- parent
It is fine with the detection line of element-biotin-aptamers 2. and the nitric acid comprising the Streptavidin-line of reference of biotin-aptamers 3.
The plain film of dimension and absorption pad；Length of overlapped part between adjacent two paste is 2mm, obtained fluorescence detection test strip drying
It encapsulates and is preserved at 4~25 DEG C.
A further object of the invention is, for the deficiencies in the prior art, provides a kind of bisphenol-A fluoroscopic examination
The application of test strips.
For this purpose, the above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of application of bisphenol-A fluorescence detection test strip, the application of the bisphenol-A fluorescence detection test strip are based on institute above
The bisphenol-A fluorescence detection test strip stated, the application of the bisphenol-A fluorescence detection test strip include：By bisphenol-A fluorescence detection test
The absorption pad end of item is inserted into sample to be detected, and sample solution to be detected does not exceed sample pad highly, sets simultaneously
One group of blank control group, wait for 30s after take out keep flat 20min, detect by an unaided eye testing result in the UV lamp；
If detection line and the line of reference aobvious fluorescence, and detection line fluorescence signal intensity is better than simultaneously on nitrocellulose filter
Or it is suitable with line of reference, then showing to detect content of bisphenol A in sample, less than 10ppb, testing result is feminine gender；
If detection line and the line of reference aobvious fluorescence, and detection line fluorescence signal intensity is shallower than simultaneously on nitrocellulose filter
Line of reference then shows that content of bisphenol A is more than 10ppb in sample, and testing result is the positive；
If the detection line on nitrocellulose filter shows fluorescence, line of reference and do not show fluorescence, show that testing result is invalid.
The present invention provides a kind of bisphenol-A fluorescence detection test strip, preparation method and application, has the advantages that：
(1) present invention uses the principle of competition law, is detected by detection line in test strips and the colour developing degree of line of reference
The content of bisphenol-A in sample to be tested, detection is quick, accurate, can complete to detect in 30min, suitable for enterprise or government
The field quick detection of supervision department；
(2) present invention has the characteristics that specificity is good, of low cost, stability is good, easy to operate, easy to spread.
Description of the drawings
Fig. 1 is a kind of side schematic view of bisphenol-A fluorescence detection test strip provided by the present invention；
Fig. 2 is that the testing result of bisphenol-A fluorescence detection test strip provided by the present invention judges schematic diagram.
Specific embodiment
The present invention is described in further detail with reference to the drawings and specific embodiments.
Embodiment 1：The preparation of bisphenol-A fluorescence detection test strip
(1) 1. fluorescent nanometer microsphere is 1 in mass ratio with amido modified aptamers:10 react 1.5 hours at 37 DEG C,
Generate fluorescent nanometer microsphere-aptamers 1..
1. fluorescent nanometer microsphere-aptamers are uniformly applied on glass fibre membrane, be lyophilized, it is micro- to form fluorescence nano
Ball-aptamers 1. bonding pad.
(2) biotin modification aptamers 2., aptamers 3. respectively with chain and sistomycocin in molar ratio be 1:10 at 37 DEG C
React 30min, generation Streptavidin-biotin-aptamers 2. and Streptavidin-biotin-aptamers 3..
On nitrocellulose filter 5 with Streptavidin-biotin-aptamers 2. with Streptavidin-biotin-adaptation
The solution of body 3. draws two parallel lines respectively, is detection line (T lines) comprising the Streptavidin-line of biotin-aptamers 2.
3, it is line of reference (C lines) 4 comprising the Streptavidin-line of biotin-aptamers 3..
(3) it is fine to paste overlap joint sample pad 1, fluorescent nanometer microsphere-aptamers bonding pad 2, nitric acid successively on plastics lining board 7
The plain film 5 of dimension, absorption pad 6.
(4) length of overlapped part between adjacent two paste is 2mm, and obtained fluorescence detection test strip is dry to encapsulate,
It is preserved under the conditions of 4~25 DEG C.
Aptamers 1., aptamers 2., aptamers 3., biotin-aptamers 2., biotin-aptamers 3., be purchased from Shanghai
Sheng Gong bioengineering Co., Ltd
It is the structure diagram that the present embodiment obtains test strips shown in Fig. 1.Wherein, serial number 1 is sample pad, and length is
18mm；Serial number 2 is fluorescent nanometer microsphere-aptamers 1. bonding pad, length 4mm；Serial number 3 be detection line, width 1mm；Sequence
Numbers 4 be line of reference, width 1mm；Serial number 5 be nitrocellulose filter, length 20mm；Serial number 6 is absorption pad, and length is
18mm；Serial number 7 be plastics lining board, length 60mm.Arrow represents liquid capillarity direction in figure.
Embodiment 2：The application of bisphenol-A fluorescence detection test strip
When being detected using the fluorescence detection test strip of the present invention, test strips arrow end is inserted into sample to be detected
In, do not exceed sample pad, while one group of blank control group is set, 30s is waited for take out and keeps flat 20min, under ultraviolet lamp with the naked eye
Observe testing result.
If detection line and the line of reference aobvious fluorescence, and detection line fluorescence signal intensity is better than simultaneously on nitrocellulose filter
Or it is suitable with line of reference, then showing to detect content of bisphenol A in sample, less than 10ppb, testing result is feminine gender；
If detection line and the line of reference aobvious fluorescence, and detection line fluorescence signal intensity is shallower than simultaneously on nitrocellulose filter
Line of reference then shows that content of bisphenol A is more than 10ppb in sample, and testing result is the positive；
If detection line shows fluorescence, line of reference and do not show fluorescence on nitrocellulose filter, show that testing result is invalid.
Above-mentioned specific embodiment is used for illustrating the present invention, is merely a preferred embodiment of the present invention rather than to this
Invention is limited, and in the protection domain of spirit and claims of the present invention, to any modification of the invention made, is equal
Replace, improve etc., both fall within protection scope of the present invention.
Claims (6)
1. a kind of bisphenol-A fluorescence detection test strip, which is characterized in that the bisphenol-A fluorescence detection test strip is included on liner plate
Sample pad, bonding pad, nitrocellulose filter and the absorption pad of overlap joint are pasted successively；The bonding pad is fluorescent nanometer microsphere-suitable
Ligand 1. bonding pad, arranged for interval has detection to mark and reference marker on the nitrocellulose membrane, the detection label include with
2. detection line that Streptavidin-biotin-aptamers are printed on nitrocellulose membrane, the reference marker are included with strepto- parent
The line of reference 3. printed on nitrocellulose membrane with element-biotin-aptamers, the detection line are arranged in parallel and examine with line of reference
Survey line is close to sample pad.
2. bisphenol-A fluorescence detection test strip according to claim 1, which is characterized in that aptamers 1., aptamers it is 2. and suitable
The nucleotide sequence of ligand 3. is followed successively by：
Aptamers are 1.：5’-CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG
CAT CAC GGG TTC GCA CCA AAA AAAAAAAAAAAAAAAAAAAAAA-NH2-3’；
Aptamers are 2.：5’-TGG TGC GAA CCC GTG ATG CGC TGG GCC ATA CGC GGA ACG CTA TCC
CAC CTG ACC ACC CAC CGG-Biotin-3’；
Aptamers are 3.：5’-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTT-3’.
3. bisphenol-A fluorescence detection test strip according to claim 1, which is characterized in that the bonding pad is：Fluorescence nano
1. microballoon is 1 according to mass ratio with amido modified aptamers:10 reacted at 37 DEG C obtain within 1.5 hours fluorescent nanometer microsphere-
1., by fluorescent nanometer microsphere-aptamers 1. aptamers are equably applied on glass fibre membrane, it is micro- that fluorescence nano is formed after freeze-drying
Ball-aptamers 1. bonding pad.
4. bisphenol-A fluorescence detection test strip according to claim 1, which is characterized in that the detection line is：Biotin is repaiied
2. it is 1 according to molar ratio with Streptavidin to adorn aptamers:10 at 37 DEG C react 30min obtain Streptavidin-biotin-
Aptamers 2., on nitrocellulose filter with Streptavidin-biotin-aptamers 2. solution cross to obtain it is affine comprising strepto-
Element-the line of biotin-aptamers 2. is detection line；
The line of reference is：3. biotin modification aptamers are 1 according to molar ratio with Streptavidin:10 react at 37 DEG C
3. 30min obtains Streptavidin-biotin-aptamers, Streptavidin-biotin-adaptation is used on nitrocellulose filter
3. cross to obtain comprising the Streptavidin-line of biotin-aptamers 3. body be line of reference by solution.
5. a kind of preparation method of bisphenol-A fluorescence detection test strip, which is characterized in that the preparation method includes the following steps：
(1) 1. fluorescent nanometer microsphere and amido modified aptamers are 1 according to mass ratio:10 react 1.5 hours at 37 DEG C, obtain
To fluorescent nanometer microsphere-aptamers 1.；1. fluorescent nanometer microsphere-aptamers are equably applied on glass fibre membrane, be lyophilized
Fluorescent nanometer microsphere-aptamers 1. bonding pad is formed afterwards；
(2) 2. 3. the aptamers of biotin modification are respectively according to molar ratio with Streptavidin with the aptamers of biotin modification
1:10 react 30min at 37 DEG C, obtain Streptavidin-biotin-aptamers 2. with Streptavidin-biotin-adaptation
Body is 3.；On nitrocellulose membrane with Streptavidin-biotin-aptamers 2. with Streptavidin-biotin-aptamers 3.
Solution draws two parallel lines respectively, is detection line comprising the Streptavidin-line of biotin-aptamers 2., includes strepto- parent
It is line of reference with the element-line of biotin-aptamers 3.；
(3) on liner plate successively paste overlap joint sample pad, fluorescent nanometer microsphere-aptamers 1. bonding pad, comprising Streptavidin-
Biotin-aptamers detection line 2. and the nitrocellulose filter for including the Streptavidin-line of reference of biotin-aptamers 3.
And absorption pad；Length of overlapped part between adjacent two paste is 2mm, and obtained fluorescence detection test strip drying encapsulation is simultaneously
It is preserved at 4~25 DEG C.
A kind of 6. application of bisphenol-A fluorescence detection test strip, which is characterized in that the application of the bisphenol-A fluorescence detection test strip
Based on the bisphenol-A fluorescence detection test strip described in any one in claim 1-4, the bisphenol-A fluorescence detection test strip
Using including：The absorption pad end of bisphenol-A fluorescence detection test strip is inserted into sample to be detected, sample solution to be detected
Sample pad is not exceeded highly, while one group of blank control group is set, and is taken out after waiting 30s and is kept flat 20min, used in the UV lamp
Visually observe testing result；
If detection line and line of reference on nitrocellulose filter aobvious fluorescence simultaneously, and detection line fluorescence signal intensity be better than or with
Line of reference is suitable, then showing to detect content of bisphenol A in sample, less than 10ppb, testing result is feminine gender；
If detection line and the line of reference aobvious fluorescence, and detection line fluorescence signal intensity is shallower than reference simultaneously on nitrocellulose filter
Line then shows that content of bisphenol A is more than 10ppb in sample, and testing result is the positive；
If the detection line on nitrocellulose filter shows fluorescence, line of reference and do not show fluorescence, testing result is invalid.
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| CN201710894094.5A CN108196048A (en) | 2017-09-28 | 2017-09-28 | A kind of bisphenol-A fluorescence detection test strip, preparation method and application |
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| CN201710894094.5A CN108196048A (en) | 2017-09-28 | 2017-09-28 | A kind of bisphenol-A fluorescence detection test strip, preparation method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112051398A (en) * | 2020-09-18 | 2020-12-08 | 华侨大学 | Ampicillin and kanamycin joint detection test strip |
| WO2023059283A1 (en) * | 2021-10-08 | 2023-04-13 | Marmara Universitesi Rektorlugu Ozel Kalem Birimi | A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| WO2023059283A1 (en) * | 2021-10-08 | 2023-04-13 | Marmara Universitesi Rektorlugu Ozel Kalem Birimi | A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk |
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