CN204330778U - A kind of alkaline phosphatase detects immunity test strip fast - Google Patents
A kind of alkaline phosphatase detects immunity test strip fast Download PDFInfo
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- CN204330778U CN204330778U CN201420545290.3U CN201420545290U CN204330778U CN 204330778 U CN204330778 U CN 204330778U CN 201420545290 U CN201420545290 U CN 201420545290U CN 204330778 U CN204330778 U CN 204330778U
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Abstract
The utility model relates to alkaline phosphatase and detects immunity test strip fast, comprise base plate and upper strata transparent sealing film, interlacedly successively between described base plate and upper strata transparent sealing film be provided with sample pad, phosphorylation reaction pad, antibody coated film and adsorptive pads, described phosphorylation reaction pad is fixed with the substance that show color combining phosphorylated biomolecule, described antibody coated film is coated with the antibody of anti-described phosphorylated biomolecule or the antibody of anti-described phosphorylated biomolecule dephosphorylation afterproduct, this test strips can the activity of detection of alkaline phosphatase fast, do not need large-scale instrument, testing cost is low, market outlook are good.
Description
Technical field
The utility model belongs to test material field, and particularly a kind of alkaline phosphatase detects immunity test strip fast.
Background technology
Alkaline phosphatase (Alkaline Phosphatase, be called for short ALP) is a kind of embrane-associated protein be extensively present in biosome, and it participates in the physiology courses such as the transfer of phosphate group and metabolism directly.Alkaline phosphatase is present in the nearly all tissue of human body, is mainly used in the diagnosis of disease in the liver and gallbladder and skeletal diseases, especially has great importance for skeletal diseases.Consider from food security angle, alkaline phosphatase is the metabolic product in cow cell, also be the naturally occurring a kind of enzyme in Ruzhong, the pathogen that it exists than Ruzhong the stability of temperature is slightly high, by guaranteeing that to the deactivation of ALP enzyme the pathogenic microorganism of the overwhelming majority is all killed after pasteurization.If ALP shows positive and illustrates that the fresh milk of pasteurize thoroughly or after pasteurization is secondary polluted, so ALP determination of activity is a kind of important sanitary safety index after pasteurization.Therefore European Union and the U.S. evaluate Fresh Milk pasteurize level with ALP is active as a kind of important sanitary safety index in succession.
Present stage ALP Activity determination needs to utilize large-scale instrument to detect by professional, this with regard to limit to portable, fast the alkaline phosphatase in sample to be detected, the application especially in the food security of ox Milk and milk products.Immunochromatographic method (immunochromatography) is a kind of quick diagnosis technology of external rise in recent years, its principle is a certain zone special antibody being first fixed on nitrocellulose filter, after sample is immersed in cellulose nitrate one end of this drying, due to capillarity, sample will move forward along this film, when moving to the region being fixed with antibody, in sample corresponding antigen namely with this antibody generation specific binding, if this region can be made to show certain color with immune colloid gold or Immunoperoxidase Staining, quantitative detection data to biological sample are provided, thus realize specific immunodiagnosis.Compared with routine diagnostic method, this technical Analysis speed is fast, and whole testing process only needs 5-30 minute.Easy and simple to handle, do not need other any instrument, without the need to professional, provide condition for real-time on-site detects.In immuno-chromatographic test paper strip, usually adopt antibody-antigen-antibody to form sandwich or form the detection of competition binding realization to sample.Therefore can only detect the concentration of thing to be checked, and activity analysis can not be provided, therefore on the detection of enzyme material, have certain defect.Therefore, be badly in need of a kind of can the Test paper of detection of alkaline phosphatase, do not need large-scale instrument, simple to operate, can be used in instant detection.
Summary of the invention
In view of this, the purpose of this utility model is to provide a kind of alkaline phosphatase to detect immunity test strip fast, and this test strips can quantitatively detect alkaline phosphatase activities immediately, does not need large-scale instrument, simple to operate.
For achieving the above object, the utility model provides following technical scheme:
Alkaline phosphatase detects immunity test strip fast, comprise base plate and upper strata transparent sealing film, interlacedly successively between described base plate and upper strata transparent sealing film be provided with sample pad, phosphorylation reaction pad, antibody coated film and adsorptive pads, the antibody that described phosphorylation reaction pad is fixed with the substance that show color combining phosphorylated biomolecule, described antibody coated film is coated with anti-described phosphorylated biomolecule, the aptamer combined with the phosphorylated biomolecule opposite sex or the antibody resisting described phosphorylated biomolecule dephosphorylation afterproduct.
Preferably, described sample pad, phosphorylation reaction pad, interlaced 2mm between antibody coated film and adsorptive pads.
Preferably, described phosphorylated biomolecule is phosphorylated amino acid, phosphorylated protein, Phosphorylated Peptide or phosphorylated nucleic acids.
Preferred, described phosphorylated amino acid is phosphorylated tyrosine.
Preferably, described substance that show color is gold nano grain, silver nano-grain or quantum dot.
Preferably, the antibody of anti-described phosphorylated biomolecule is the antibody of anti phosphotyrosine; The described aptamer combined with the phosphorylated biomolecule opposite sex is the aptamer with phosphorylated tyrosine specific binding.
Preferably, described upper strata transparent sealing film is overlay.
The beneficial effects of the utility model are: disclosed in the utility model, alkaline phosphatase detects immunity test strip fast, have fast easy, sense cycle is short, the advantage of single part operation, utilize the interaction of alkaline phosphatase and phosphorylated biomolecule, utilize anti-phosphorylated biomolecule antibody to carry out specific recognition to phosphorylated molecules simultaneously, the product generated after acting on substrate to enzyme detects, thus while realizing quantitatively detecting, enzyme functional activity is detected, compensate for Traditional immunochromatographic technology merely to the defect that concentration is investigated, represent direction and the trend of current real-time test technical development.
Accompanying drawing explanation
In order to make the purpose of this utility model, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is structural representation (the 1. sample dropping position of alkaline phosphatase activities immuno-chromatographic test paper strip; 2. the nanogold particle fixed bolster of phosphorylated tyrosine modification; 3. anti phosphotyrosine antibody fixed position (detection zone); 4. thieving paper).
Fig. 2 is the schematic diagram preparing phosphorylated tyrosine marking nano gold grain; (1) halfcystine is combined the nanogold particle obtaining cysteine modified with nm of gold by its sulfydryl; (2) utilize glutaraldehyde by the cysteine cross on phosphorylated tyrosine and nanogold particle surface, obtain the nanogold particle that phosphorylated tyrosine is modified; AuNPs is gold nano grain; AuNPs@Cys-Try-p is the nanogold particle that phosphorylated tyrosine is modified.
Fig. 3 is the characterization result (A: scanning electron microscope (SEM) photograph of the nanogold particle that phosphorylated tyrosine is modified; B: ultraviolet visible absorption spectra; C:Zeta current potential; D. particle size distribution; In B ~ D, a represents the nanogold particle of unmodified; B represents the nanogold particle of cysteine modified; C represents the nanogold particle that phosphorylated tyrosine is modified).
Fig. 4 is the Cleaning Principle figure of the golden alkaline phosphatase activities immuno-chromatographic test paper strip for colour developing spike of phosphorylated tyrosine marking nano.
Fig. 5 is the condition optimizing (A is the condition optimizing result of preparation test strips, and B is the histogram of the condition optimizing result of preparation test strips) of preparation test strips.
Fig. 6 is the experimental result (A is the experimental result that alkaline phosphatase activities immuno-chromatographic test paper strip detects milk alkaline phosphatase content, and B is the typical curve that alkaline phosphatase activities immuno-chromatographic test paper strip detects the experimental result of milk alkaline phosphatase content) that alkaline phosphatase activities immuno-chromatographic test paper strip detects milk alkaline phosphatase content.
Embodiment
Below in conjunction with accompanying drawing, preferred embodiment of the present utility model is described in detail.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
Alkaline phosphatase detects immunity test strip fast, and its structure as shown in Figure 1, comprises base plate and upper strata transparent sealing film, interlacedly successively between described base plate and upper strata transparent sealing film is provided with sample pad, phosphorylation reaction pad, antibody coated film and adsorptive pads.Preferably, sample pad, phosphorylation reaction pad, interlaced 2mm between antibody coated film and adsorptive pads, wherein phosphorylation reaction pad is combined with gold nano grain, gold nano grain is combined with phosphorylated tyrosine, and antibody coated film is coated with the antibody of anti phosphotyrosine or anti-dephosphorylation phosphotyrosine antibody.In the present invention, phosphorylated tyrosine can be replaced with other phosphorylated biomolecule, as phosphorylated protein, Phosphorylated Peptide or phosphorylated nucleic acids etc., antibody on antibody coated film is replaced with the antibody of antibody or the biomolecule dephosphorylation product identifying corresponding phosphorylated biomolecule, gold nano grain also can replace with silver nano-grain or quantum dot simultaneously; Upper strata transparent sealing film is preferably overlay.
Alkaline phosphatase detects the preparation method following (Fig. 2) of immunity test strip fast: the gold nano grain preparation method that phosphorylated tyrosine is modified: first, 2 milliliter of 1% (m/V) HAuCl
4(distilled water dilution) adds in 198 milliliters of distilled waters and mixes, mixed HAuCl
4solution is boiled by oil bath heating, then adds 4 milliliter of 1% (m/V) trisodium citrate aqueous solution, reacts about 30 minutes, HAuCl
4solution becomes red, obtains gold nano grain (AuNPs); Get 10 milliliters of gold nano grain solution and add 10 milliliters of (1 mg/ml-) Cys, stirring reaction 2 hours under room temperature (25 DEG C) condition; Collect mixed liquor in bag filter, dialyse 10 hours in 4 DEG C of aqueous solution, within every 2 hours, change 1 distilled water, obtain the gold nano grain of cysteine modified; Get 5 milliliters of phosphorylated tyrosines (500 μMs) subsequently to mix with the gold nano grain solution of 5mL cysteine modified, then 50 microlitre glutaraldehyde water solution (50% aqueous solution are got, v/v) potpourri of the gold nano grain solution of phosphorylated tyrosine and cysteine modified is slowly added dropwise to, stirring reaction 2 hours under (25 DEG C) condition; Then, under condition of ice bath, drip 50 microlitre 0.1% (w/w) NaBH
4aqueous solution enters potpourri.Finally, remove trip phosphorylated tyrosine and glutaraldehyde by dialysis, and by polyglycol (WM 20000), potpourri is concentrated to 5 milliliters, obtain the gold nano grain that phosphorylated tyrosine is modified.The characterization result of the gold nano grain that phosphorylated tyrosine is modified as shown in Figure 3.Result shows, and the particle diameter of the gold nano grain of the phosphorylated tyrosine modification of acquisition is approximately 21.7nm, and the gold nano grain modified rear ultraviolet-visible absorption spectroscopy red shift in surface is about 10nm, shows that gold nano grain is wrapped up by amino acid gradually in modification; Zeta potential absolute value becomes large in addition, shows that the stability of modified rear gold nano grain is better; Also can find out granularmetric analysis in addition, after modifying, particle diameter is more even, for preparation detects the ideal material of immunity test strip.
The gold nano grain bag that phosphorylated tyrosine is modified is by the preparation of padding: be added drop-wise on glass fibre membrane with the gold nano grain that 10 μ L, 20 μ L, 30 μ L and 40 μ L phosphorylated tyrosines are modified respectively, 25 DEG C of drying for standby; Prepared by anti phosphotyrosine antibody Bao Bei district: 1.5 μ L (concentration is respectively 0.5mg/mL, 1.0mg/mL and 2mg/mL) anti phosphotyrosine monoclonal antibody speckings are to nitrocellulose filter, place after 1 hour for 25 DEG C, nitrocellulose filter is closed 30 minutes with the BSA that massfraction is 2%, dry in room temperature (25 DEG C) again, finally be fixed on PVC base plate by sample pad, phosphorylation reaction pad, antibody coated film and the interlaced 2mm of adsorptive pads, namely surface transparent plastic diaphragm seal is assembled into alkaline phosphatase and detects immunity test strip fast.
Obtained alkaline phosphatase detects phosphorylated tyrosine that immunity test strip utilizes gold nano grain to combine fast and alkaline phosphatase (ALP) reacts, then utilize anti phosphotyrosine antibody (antibody of the aptamer combined with the phosphorylated biomolecule opposite sex or anti-described phosphorylated biomolecule dephosphorylation afterproduct) to be combined concentration and the content of detection of active alkaline phosphatase with phosphorylated tyrosine, its Cleaning Principle as shown in Figure 4.Use the detection of the alkaline phosphatase activities of AuNPs@Cys-Tyr-p based on laterally flowing area in Fig. 4.(A) add sample pad with the sample solution containing alkaline phosphatase, the phosphate group (1) on the phosphorylated tyrosine on nanogold particle is modified in the alkaline phosphatase removal then in sample; Then dephosphorylized nanogold particle migrates to forward anti phosphotyrosine antibody fixed area (2); Last dephosphorylized nanogold particle by anti phosphotyrosine antibody capture, cannot continue to migrate to adsorptive pads forward, therefore generates (3) without visual band at antibody fixed area; If but without alkaline phosphatase or containing sex change alkaline phosphatase in sample solution, phosphorylated tyrosine marking nano gold grain migrates to and secures anti phosphotyrosine antibody regions, and caught by specific antibody, visual band (B) is formed in this region.Institute can pass through visual inspection in this way, analyzes optical density, to realize quantitative test after taking pictures with image software.
Then utilize obtained alkaline phosphatase to detect immunity test strip fast to detect: 35 μ L are detected sample drops and is added in sample pad, liquid move along test strips to adsorptive pads direction, with the alkaline phosphatase of the thermal denaturation negative control thing as detection; After about 10 minutes, utilize the camera on smart mobile phone to take pictures to test strips, all experiments repeat at least three times all independently, and then draw histogram according to colour developing result, result as shown in Figure 5.Result shows, the nanogold particle consumption that 40 microlitre phosphorylated tyrosines are modified and 1.5 microlitre 2mg/ml anti phosphotyrosine antibody test best results.
Utilize the obtained alkaline phosphatase of obtained top condition to detect immunity test strip fast and detect milk alkaline phosphatase content: utilize the milk containing variable concentrations alkaline phosphatase to be added drop-wise in sample pad respectively, liquid moves along test strips to adsorptive pads direction, with the alkaline phosphatase of thermal denaturation as the negative control thing detected; After about 10 minutes, utilize the camera on smart mobile phone to take pictures to test strips, all experiments repeat at least three times all independently, then according to colour developing result drawing standard curve (Fig. 6).Result shows, and is linearly be correlated with within the scope of 1 ~ 100U/L at alkaline phosphatase concentration.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (7)
1. alkaline phosphatase detects immunity test strip fast, it is characterized in that: comprise base plate and upper strata transparent sealing film, interlacedly successively between described base plate and upper strata transparent sealing film be provided with sample pad, phosphorylation reaction pad, antibody coated film and adsorptive pads, the antibody that described phosphorylation reaction pad is fixed with the substance that show color combining phosphorylated biomolecule, described antibody coated film is coated with anti-described phosphorylated biomolecule, the aptamer combined with the phosphorylated biomolecule opposite sex or the antibody resisting described phosphorylated biomolecule dephosphorylation afterproduct.
2. alkaline phosphatase detects immunity test strip fast according to claim 1, it is characterized in that: described sample pad, phosphorylation reaction pad, antibody coated film and adsorptive pads be staggered 2mm successively.
3. according to claim 1 or 2, alkaline phosphatase detects immunity test strip fast, it is characterized in that: described phosphorylated biomolecule is phosphorylated amino acid, phosphorylated protein, Phosphorylated Peptide or phosphorylated nucleic acids.
4. alkaline phosphatase detects immunity test strip fast according to claim 3, it is characterized in that: described phosphorylated amino acid is phosphorylated tyrosine.
5. according to claim 1 or 2, alkaline phosphatase detects immunity test strip fast, it is characterized in that: described substance that show color is gold nano grain, silver nano-grain or quantum dot.
6. according to claim 1 or 2, alkaline phosphatase detects immunity test strip fast, it is characterized in that: the antibody of anti-described phosphorylated biomolecule is the antibody of anti phosphotyrosine, the described aptamer combined with the phosphorylated biomolecule opposite sex is the aptamer with phosphorylated tyrosine specific binding.
7. according to claim 1 or 2, alkaline phosphatase detects immunity test strip fast, it is characterized in that: described upper strata transparent sealing film is overlay.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002282A (en) * | 2015-07-29 | 2015-10-28 | 江苏猎阵生物科技有限公司 | Nucleic acid detecting test strip and preparation method there of and method for detecting nucleic acid |
| CN108375616A (en) * | 2018-02-02 | 2018-08-07 | 云南大学 | A kind of liquid crystal biosensor of detection of alkaline phosphatase and its preparation method and application |
| CN108761058A (en) * | 2018-05-19 | 2018-11-06 | 大连大学 | Paper chip and method for Parallel testing multi-infection disease marker |
| CN109554369A (en) * | 2018-02-02 | 2019-04-02 | 中国科学院化学研究所 | Aptamer is identifying and is combining the application in alkaline phosphatase heterodimer |
| CN109576273A (en) * | 2018-07-03 | 2019-04-05 | 广西医科大学 | A set of tumor-marker molecular nucleic acid aptamers and application based on CRISPR/Cas9 |
| CN109682973A (en) * | 2019-01-02 | 2019-04-26 | 中国科学院化学研究所 | Lesion detection approach and kit based on aptamer |
| CN110354920A (en) * | 2018-03-26 | 2019-10-22 | 首都师范大学 | A kind of enrichment detecting method of paper substrate micro-fluidic chip and the phosphorylated polypeptide based on it |
| CN111856011A (en) * | 2020-07-31 | 2020-10-30 | 郑州大学第一附属医院 | Kit and method for detecting serine hydroxymethyltransferase activity |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002282A (en) * | 2015-07-29 | 2015-10-28 | 江苏猎阵生物科技有限公司 | Nucleic acid detecting test strip and preparation method there of and method for detecting nucleic acid |
| CN108375616A (en) * | 2018-02-02 | 2018-08-07 | 云南大学 | A kind of liquid crystal biosensor of detection of alkaline phosphatase and its preparation method and application |
| CN109554369A (en) * | 2018-02-02 | 2019-04-02 | 中国科学院化学研究所 | Aptamer is identifying and is combining the application in alkaline phosphatase heterodimer |
| CN109554369B (en) * | 2018-02-02 | 2021-11-02 | 中国科学院化学研究所 | Use of aptamers for recognition and binding of alkaline phosphatase heterodimers |
| CN110354920A (en) * | 2018-03-26 | 2019-10-22 | 首都师范大学 | A kind of enrichment detecting method of paper substrate micro-fluidic chip and the phosphorylated polypeptide based on it |
| CN110354920B (en) * | 2018-03-26 | 2021-08-17 | 首都师范大学 | Paper-based micro-fluidic chip and phosphorylated polypeptide enrichment detection method based on same |
| CN108761058A (en) * | 2018-05-19 | 2018-11-06 | 大连大学 | Paper chip and method for Parallel testing multi-infection disease marker |
| CN109576273A (en) * | 2018-07-03 | 2019-04-05 | 广西医科大学 | A set of tumor-marker molecular nucleic acid aptamers and application based on CRISPR/Cas9 |
| CN109682973A (en) * | 2019-01-02 | 2019-04-26 | 中国科学院化学研究所 | Lesion detection approach and kit based on aptamer |
| CN111856011A (en) * | 2020-07-31 | 2020-10-30 | 郑州大学第一附属医院 | Kit and method for detecting serine hydroxymethyltransferase activity |
| CN111856011B (en) * | 2020-07-31 | 2023-06-20 | 郑州大学第一附属医院 | Kit for detecting serine hydroxymethyl transferase activity and detection method |
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