CN106124759B - A kind of silaenafil colloidal gold test - Google Patents
A kind of silaenafil colloidal gold test Download PDFInfo
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- CN106124759B CN106124759B CN201610703381.9A CN201610703381A CN106124759B CN 106124759 B CN106124759 B CN 106124759B CN 201610703381 A CN201610703381 A CN 201610703381A CN 106124759 B CN106124759 B CN 106124759B
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- gold
- pad
- preparation
- colloidal gold
- silaenafil
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention discloses a kind of silaenafil colloidal gold tests and preparation method thereof, colloid gold test paper detection is sensitive, accuracy is high, specificity is good, and result is easy to observe the rapid screening, it can be achieved that batch samples, substantially reduces testing cost, workload is reduced, there is practical significance in Food and drug administration.
Description
Technical field
The invention discloses a kind of colloidal gold test and preparation method thereof, more particularly to a kind of silaenafil colloidal gold
Test paper and preparation method thereof.
Background technology
The PDE-5 inhibitor such as silaenafil belongs to prescription medicine, there is specific indication, contraindication and side effect, certain diseases
People cannot take.If patient takes in without knowing it, serious adverse reaction is easily caused, death is even resulted in.By
In immense success of the PDE-5 types inhibitor in terms for the treatment of ED, and the price of the drug is very expensive, more due to its synthesis side
Method is relatively easy so that the case where PDE-5 type inhibitor is illegally added in tonifying kidney and strengthening yang class Chinese patent drug and health food occurs simultaneously
As a kind of universal phenomenon.
Currently, whether adulterating the detection method side of being mainly the following of silaenafil in Chinese patent drug and health food
Method：Thin-layered chromatography, high performance liquid chromatography, tablets by HPLC-MS and physics and chemistry screening method.Former three is both needed to
By complicated extraction process and Instrumental Analysis is borrowed, it is more demanding to the professional technique of testing staff, be not suitable for basic staff
Execute-in-place.Although and physics and chemistry screening method operating procedure it is less but operation require it is stringenter, sensitivity is low and is susceptible to mistake
Sentence phenomenon.Against the above deficiency, the present invention provides a kind of easy to use, fast and convenient, detection health foods of high sensitivity
Or the colloidal-gold detecting-card of silaenafil is illegally added in Chinese patent drug.
Invention content
It is fast and convenient the purpose of the present invention is to provide a kind of easy to use, the detection health food of high sensitivity or in
The quick screening method and its detection used card of silaenafil are illegally added in patent medicine.
The present invention provides a kind of preparation method of silaenafil colloidal gold test, specific preparation process is：
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 25~35nm；
(2) preparation of golden labelled antibody：The colloidal gold solution prepared is adjusted into PH to 6~6.8, then with distilled water or
PBS dilutes labelled antibody to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, is kept stirring 10-
15min, be added BSA to quality final concentration of 0.2~0.5%, close exposed gold particle, continue stir 20-30min, 4 DEG C,
4000r/min centrifuges 30min, is resuspended and is washed using 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass is impregnated using the phosphate buffer that pH is 6.2~7, molar concentration is 0.2M
Fiber sample pad, is dried overnight, and cutting is spare；In buffer solution, contain the final concentration of 0.1~1%BSA of quality, 0.05~0.2%
Tween-80,0.1~1% polyvinylpyrrolidone；
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
Technical scheme of the present invention provide it is a kind of preparing colloid gold test paper and preparation method thereof, the colloid gold test paper detection
Sensitive, accuracy is high, is as a result easy to observe the rapid screening, it can be achieved that batch samples, substantially reduces testing cost, reduces
Workload has practical significance in Food and drug administration.
Specific implementation mode
With reference to embodiment, the claim of the present invention is described in further detail, but do not constituted pair
Any restrictions of the present invention, the modification of anyone limited number of time made within the scope of the invention as claimed, still the present invention's
In claims.
The preparation (1) of 1 silaenafil colloidal gold test of embodiment
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 30nm；
(2) preparation of golden labelled antibody：It is dilute to 6.8, then with distilled water or PBS that the colloidal gold solution prepared is adjusted into PH
Labelled antibody is released to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, 10-15min is kept stirring, is added
BSA to final concentration of 0.2%, closes exposed gold particle, continues to stir 20-30min, 4 DEG C, 4000r/min centrifuges 30min, profit
It is resuspended and is washed with 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare, and quality final concentration of 1% is contained in buffer solution
Tween-80；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibers are impregnated using the phosphate buffer that pH is 7.0, molar concentration is 0.2M
Sample pad is tieed up, is dried overnight, cutting is spare；In buffer solution, gather containing the final concentration of 1%BSA of quality, 0.2% Tween-80,1%
Vinylpyrrolidone.
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
The preparation (2) of 2 silaenafil colloidal gold test of embodiment
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 35nm；
(2) preparation of golden labelled antibody：It is dilute to 6.3, then with distilled water or PBS that the colloidal gold solution prepared is adjusted into PH
Labelled antibody is released to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, 10-15min is kept stirring, is added
BSA to final concentration of 0.2%, closes exposed gold particle, continues to stir 20-30min, 4 DEG C, 4000r/min centrifuges 30min, profit
It is resuspended and is washed with 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare, final concentration of containing quality in buffer solution
0.05% Tween-80；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibers are impregnated using the phosphate buffer that pH is 6.8, molar concentration is 0.2M
Sample pad is tieed up, is dried overnight, cutting is spare；In buffer solution, containing the final concentration of 0.5%BSA of quality, 0.1% Tween-80,
0.5% polyvinylpyrrolidone.
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
The preparation (3) of 3 silaenafil colloidal gold test of embodiment
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 25nm；
(2) preparation of golden labelled antibody：It is dilute to 6.4, then with distilled water or PBS that the colloidal gold solution prepared is adjusted into PH
Labelled antibody is released to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, 10-15min is kept stirring, is added
BSA to final concentration of 0.5%, closes exposed gold particle, continues to stir 20-30min, 4 DEG C, 4000r/min centrifuges 30min, profit
It is resuspended and is washed with 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare, final concentration of containing quality in buffer solution
0.1% Tween-80；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibers are impregnated using the phosphate buffer that pH is 6.5, molar concentration is 0.2M
Sample pad is tieed up, is dried overnight, cutting is spare；In buffer solution, containing the final concentration of 0.2%BSA of quality, 0.05% Tween-80,
0.2% polyvinylpyrrolidone.
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
The preparation (4) of 4 silaenafil colloidal gold test of embodiment
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 25nm；
(2) preparation of golden labelled antibody：The colloidal gold solution prepared is adjusted PH to dilute to 6, then with distilled water or PBS
Then labelled antibody is added dropwise in the colloidal gold solution of stirring to a concentration of 1mg/ml, be kept stirring 10-15min, is added
BSA to final concentration of 0.4%, closes exposed gold particle, continues to stir 20-30min, 4 DEG C, 4000r/min centrifuges 30min, profit
It is resuspended and is washed with 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare, final concentration of containing quality in buffer solution
0.2% Tween-80；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibers are impregnated using the phosphate buffer that pH is 6.2, molar concentration is 0.2M
Sample pad is tieed up, is dried overnight, cutting is spare；In buffer solution, containing the final concentration of 0.1%BSA of quality, 0.1% Tween-80,
0.4% polyvinylpyrrolidone.
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
The preparation (5) of 5 silaenafil colloidal gold test of embodiment
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 30nm；
(2) preparation of golden labelled antibody：It is dilute to 6.8, then with distilled water or PBS that the colloidal gold solution prepared is adjusted into PH
Labelled antibody is released to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, 10-15min is kept stirring, is added
BSA to final concentration of 0.5%, closes exposed gold particle, continues to stir 20-30min, 4 DEG C, 4000r/min centrifuges 30min, profit
It is resuspended and is washed with 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare, final concentration of containing quality in buffer solution
0.5% Tween-80；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibers are impregnated using the phosphate buffer that pH is 7.0, molar concentration is 0.2M
Sample pad is tieed up, is dried overnight, cutting is spare；In buffer solution, containing the final concentration of 0.4%BSA of quality, 0.2% Tween-80,
0.1% polyvinylpyrrolidone.
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
The preparation (6) of 6 silaenafil colloidal gold test of embodiment
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is equal that particle is fired using magnetic force heating stirrer
The colloid gold particle that one diameter is about 30nm；
(2) preparation of golden labelled antibody：It is dilute to 7.5, then with distilled water or PBS that the colloidal gold solution prepared is adjusted into PH
Labelled antibody is released to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, 10-15min is kept stirring, is added
BSA to final concentration of 0.2%, closes exposed gold particle, continues to stir 20-30min, 4 DEG C, 4000r/min centrifuges 30min, profit
It is resuspended and is washed with 0.2M phosphate buffers, then centrifuged, is repeated 2 times, concentration is spare, and quality final concentration of 1% is contained in buffer solution
Tween-80；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark knot handled well using spray film instrument
It closes on object pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose using film instrument stroke film is drawn
Film detection zone and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibers are impregnated using the phosphate buffer that pH is 7.5, molar concentration is 0.2M
Sample pad is tieed up, is dried overnight, cutting is spare；In buffer solution, gather containing the final concentration of 1%BSA of quality, 0.2% Tween-80,1%
Vinylpyrrolidone.
(6) assembling of test paper：It is adhered to support by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Bottom plate over-assemble test paper, and cut, it packs.
The detection range of 7 silaenafil colloidal gold test of embodiment
The colloid gold test paper that the preparation method of Examples 1 to 6 is prepared, detectable concentration is the west of 100 μ g/ml respectively
Ground that non-, Vardenafil, Acctildenafil, Tadalafei.Testing result is as follows, wherein+indicate that testing result is the positive ,-indicate
Testing result is feminine gender：
The experimental result of colloidal gold detection
The sensitivity of 8 silaenafil colloidal gold test of embodiment detection
Colloidal gold colloidal gold detection test paper strip prepared by the embodiment of embodiment 1 is detected into its sensitivity：Silaenafil is dissolved,
It is prepared into the solution of 0.5mg/mL, every 10 times of gradient dilutions detect the detection sensitivity of colloidal gold test.Testing result table
It is bright, according to colloidal gold colloidal gold detection test paper strip prepared by the embodiment of embodiment 1, a concentration of 0.5ppb that minimum detectable range measures.
Claims (2)
1. a kind of preparation method of silaenafil colloidal gold test, it is characterised in that include the following steps：
(1) preparation of colloid gold particle：Using trisodium citrate reduction method, it is uniform that particle is fired using magnetic force heating stirrer
The colloid gold particle that diameter is about 25~35nm；
(2) preparation of golden labelled antibody：It is dilute to 6~6.8, then with distilled water or PBS that the colloidal gold solution prepared is adjusted into PH
Labelled antibody is released to a concentration of 1mg/ml, is then added dropwise in the colloidal gold solution of stirring, 10-15min is kept stirring, is added
BSA closes exposed gold particle to quality final concentration of 0.2~0.5%, continues to stir 20-30min, 4 DEG C, 4000r/min is centrifuged
30min is resuspended using 0.2M phosphate buffers and is washed, then centrifuged, and is repeated 2 times, and concentration is spare；
(3) preparation of gold mark conjugate pad：The labelled antibody of gold label is sprayed on to the gold mark conjugate handled well using spray film instrument
On pad and vacuum freeze drying is spare；
(4) it is coated with nitrocellulose filter：Antibody and antiantibody will be captured and be coated in nitrocellulose filter inspection using film instrument stroke film is drawn
Survey area and quality control region, drying for standby；
(5) preparation of sample treatment pad：Glass fibre is impregnated using the phosphate buffer that pH is 6.2~7, molar concentration is 0.2M
Sample pad is dried overnight, and cutting is spare；In buffer solution, spat containing the final concentration of 0.1~1%BSA of quality, 0.05~0.2%
Warm -80,0.1~1% polyvinylpyrrolidone；
(6) assembling of test paper：It is adhered to support baseboard by sample pad, gold mark conjugate pad, nitrocellulose filter, water absorption pad sequence
Over-assemble test paper, and cut, it packs；
In buffer solution in the gold labelled antibody preparation, the Tween-80 containing 0.05~1% mass final concentration.
2. silaenafil colloidal gold test prepared by preparation method according to claim 1, including PVC board and PVC
Sample pad, gold-marking binding pad, coated film and the water absorption pad composition being connected successively on plate, it is characterised in that：The gold mark combines
Pad is adsorbs the glass fibre of silaenafil monoclonal antibody-colloidal gold composite and IgG- colloidal gold composites, in coated film
On captured successively with silaenafil antibody solution printing linear recessive detection line T lines, printed with antiantibody IgG solution
Linear recessiveness nature controlling line C line, two lines are arranged in parallel.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610703381.9A CN106124759B (en) | 2016-08-22 | 2016-08-22 | A kind of silaenafil colloidal gold test |
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| CN201610703381.9A CN106124759B (en) | 2016-08-22 | 2016-08-22 | A kind of silaenafil colloidal gold test |
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| CN106124759B true CN106124759B (en) | 2018-07-27 |
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| CN107300617A (en) * | 2017-07-17 | 2017-10-27 | 广东志道医药科技有限公司 | The inhibitor colloidal gold immuno-chromatography test paper strips of PDE 5 and its preparation and application |
| CN107727862A (en) * | 2017-11-06 | 2018-02-23 | 北京农学院 | A kind of Ribavirin test strip |
| CN108548925A (en) * | 2018-03-28 | 2018-09-18 | 韶关学院 | Ternary system Immune competition method detects the quantum dot immune chromatography detection card and detection method of silaenafil similar drug |
| CN112881679A (en) * | 2021-01-19 | 2021-06-01 | 南昌大学 | Preparation method of test strip for simultaneously detecting two water-soluble mycotoxins |
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| US20050031544A1 (en) * | 2003-08-07 | 2005-02-10 | Njemanze Philip Chidi | Receptor mediated nanoscale copolymer assemblies for diagnostic imaging and therapeutic management of hyperlipidemia and infectious diseases |
| CN101620226B (en) * | 2009-08-07 | 2013-08-28 | 四川迈克生物科技股份有限公司 | Quick test kit of schistosomiasis and preparation method thereof |
| CN101914155A (en) * | 2010-08-09 | 2010-12-15 | 江苏省南通药品检验所 | Technique for artificially synthesizing antigen of Sildenafil and derivative thereof |
| CN102174474B (en) * | 2011-01-11 | 2013-01-09 | 南通市伊士生物技术有限责任公司 | Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil |
| CN104407130B (en) * | 2013-11-21 | 2016-08-17 | 王海艳 | Detection capripox virus colloidal gold strip and preparation method thereof |
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