CN1769895A - Blood detection microarray and preparation method thereof - Google Patents
Blood detection microarray and preparation method thereof Download PDFInfo
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- CN1769895A CN1769895A CN 200510109371 CN200510109371A CN1769895A CN 1769895 A CN1769895 A CN 1769895A CN 200510109371 CN200510109371 CN 200510109371 CN 200510109371 A CN200510109371 A CN 200510109371A CN 1769895 A CN1769895 A CN 1769895A
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Abstract
The invention discloses a blood detecting micro array and method for making the same. The micro array comprises a base plate and a cover plate; wherein on the base plate being rigidly jointed with porous silica dioxide membrane which is absorbed with detecting protein probe array that can react with blood type, pathogene and antibody; on the top surface of the cover plate being positioned with sample adding hole, micro array reading window; the cover plate covers on the base plate hermetically and there is a 0.1-1.0mm gap between the cover plate and detecting micro array. The inventive method comprises: making base plate, making protein probe micro array and sealing of blood detecting micro array.
Description
Technical field
The present invention relates to a kind of medical and hygiene article, particularly a kind of blood detection microarray and preparation method thereof.
Background technology
Blood group identifies that propagating relevant pathogen detection with blood transfusion is the key link that guarantees transfusion safety.At present, mainly in the blood donation use the blood clotting technology to carry out blood group to identify, use the enzyme-linked immunoassay technology (enzyme linked immunosorbent assay, ELISA) and the gold-marking immunity chromatographic technique relevant pathogen detection of propagation of transfusing blood.In recent years, the research report that adopts biochip technology to detect is also arranged.
The blood clotting technology is the classical way that blood group is identified, realization condition is simple, but its detection architecture is open, and environment is had bigger pollution, and testing crew is constituted exposure hazard.Carrying out ELISA needs repeatedly incubation, washs and carries out absorbance measurement, needs long experimental period (being not less than 120 minutes) and special-purpose experimental facilities, is not suitable for equipping occasion simple, that need report the result fast, as the check of street corner voluntary blood donation.It is also very fast to dependence, the speed of equipment that the colloidal gold immunochromatographimethod product has solved ELISA well, but detecting target antigen to each, the colloidal gold immunochromatographimethod technology all needs two specific antibody, development difficulty is bigger, also be difficult to realize many targets joint-detection, present colloidal gold immunochromatographimethod product all is individual event test strip/card, test item of every increase will increase the one-time detection operation, also be difficult to form unified Quality Control.
Emerging biochip technology, particularly protein chip technology provide a kind of many index tests parallel processing method at present, have overcome the shortcoming of colloidal gold immunochromatographimethod technology effectively.But, at present the protein chip of report mainly uses glass that glutaraldehyde handles etc. as solid support, the solid supports such as glass that glutaraldehyde is handled have better adsorption capability to nucleic acid probe, then adsorptive power is not good to the protein as chip probe, bad with this protein chip homogeneity of making, assay is difficult to guarantee.
Summary of the invention
For overcoming the shortcomings and deficiencies that above-mentioned prior art exists, one of purpose of the present invention is to provide a kind of blood detection microarray, this detects microarray with blood group substance, blood group antigens, pathogen antigen, pathogen antigen and other testing goal thing, be fixed in an orderly manner on the substrates such as the slide that is coated with the porous silica film, plastic sheet, potsherd, form Protein Detection probe microarray, can be used for blood donor, patient are carried out quick blood test, and, avoid contaminated environment owing to adopt enclosed detection architecture.
Another object of the present invention is to provide a kind of method for making of above-mentioned blood detection microarray, the inventive method utilizes that the porous silica film of rigidity combination on the substrate adsorbs, the method for making of fixed test probe, also can be used for making other biochip.
For achieving the above object, a kind of blood detection microarray of the present invention, form at this on-chip cover plate 2 by substrate 1 and buckle closure, the porous silica film 6 that the rigidity combination is arranged on the described substrate 1, be adsorbed with securely on the film can with blood group, the detector probe 3 that pathogen composition and antibody thereof react, described detector probe is arranged as dot matrix in order and forms microarray 4, the end face of described cover plate 2 is provided with well 5 and transparent reading window 7, cover plate 2 buckle closure hermetically has the gap of 0.1-1.0mm between described cover plate 2 and the detection dot matrix on fritted glass substrate 1.
Further, the porous silica film 6 on the described substrate 1, energy non-specific adsorption proteantigen, antibody;
Further, described microarray assay probe 3 is selected among blood group A material, B material, blood group A antibody, B antibody, D antibody, anti-HBsAg, HCV recombinant antigen, HCV antibody, HIVp24, HIVgp36, HIVp24 antibody, HIVgp36 antibody, TP recombinant antigen, the goat anti-human igg one or more and is separately fixed on the porous silica film, forms orderly array and arranges;
Further, the end face of described cover plate 2 is provided with bar code 8, and this bar code identification has microarray assay content and probe arrangement mode; Further, described substrate 1 is slide, plastic sheet or potsherd.A kind of method for making of blood detection microarray is characterized in that may further comprise the steps:
A makes the substrate that the porous silica film covers
Get common microslide, transparent plastic sheet or potsherd, the clean surface is standby according to a conventional method; And microslide covers 1-20% Na on this
2SiO
3100ul/cm
25-60min in the 0.05-0.5mol/L hydrochloric acid solution is immersed in dry rapidly back; Deionized water soaks 3min above 3 times; Under 180 ℃-300 ℃ condition, react 10-60min, form the meagre rete of porous silica of one deck 10-500nm on this slide surface, promptly obtain the microarray substrate;
B makes the probe microarray
With detector probe, positive control and negative control, be separately fixed on the microarray substrate of making, form orderly microarray, then with bSA or polyglycol sealing, drying;
C encapsulates blood detection microarray
With cover plate hermetically buckle closure on the substrate that has the detector probe microarray, cover plate and detect the gap that keeps 0.1-1.0mm between the microarray;
Further, the detector probe among the step b is with in blood group A material, B material, blood group A antibody, B antibody, D antibody, anti-HBsAg, HCV recombinant antigen, HCV antibody, HIVp24, HIVgp36, HIVp24 antibody, HIVgp36 antibody, the TP recombinant antigen one or more;
Further, can stick the bar code of this microarray assay content of sign and probe arrangement mode on the cover plate among the step c.
Compared with prior art, blood detection microarray of the present invention, utilize of the strong suction-operated of porous silica film to protein, fixedly the antigen of any kind, antibody are as the detector probe of microarray, and this strong suction-operated is relevant with thickness, the aperture of porous silica layer, the thickness of porous silica layer, aperture make that easily by chemical substance concentration, reaction time and the controlling reaction temperature of its reaction of formation the differences between batches of the microarray substrate that this method is made are little; Owing to adopt enclosed detection architecture, avoided contaminated environment simultaneously.Adopt preparation method of the present invention, utilize fritted glass substrate adsorption technology, purpose antigen that needs are detected or the corresponding antibody of antibody or antigen are fixed on to form on the glass substrate of special facture and detect microarray, can be used for making the microarray protein chip that comprises the arbitrary protein probe, technology is simple, and cost is low.
Description of drawings
Fig. 1 is the structural representation of blood detection microarray of the present invention;
Fig. 2 is the structural representation of blood detection microarray substrate of the present invention.
Embodiment
The present invention will be further described below in conjunction with the drawings and the specific embodiments.
As shown in Figure 1 and Figure 2, blood detection microarray of the present invention, comprise by substrate and buckle closure and forming at this on-chip cover plate, on the described substrate 1 the detection microarray is arranged, by firmly being adsorbed on the orderly dot matrix 3 that on-chip albumen probe is formed, the end face of described cover plate 2 is provided with well 4, microarray and reads window 5, cover plate 2 hermetically buckle closure on substrate 1.The testing staff can also can use chip scanner to read the sheet judged result by the change color of the 5 observation albumen probe dot matrix of the reading window on cover plate 2 end faces.
Embodiment 1
The making of visual blood detection microarray and detection
A makes the glass substrate that the porous silica film covers
Get common microslide, the clean surface is standby according to a conventional method; And microslide covers 1%Na on this
2SiO
3100ul/cm
25min in the 0.05mol/L hydrochloric acid solution is immersed in dry rapidly back; Deionized water soaks 3min above 3 times; Under 180 ℃ condition, react 10min, form the meagre rete of porous silica of one deck 10-500nm on this slide surface, promptly obtain the microarray substrate;
B makes microarray albumen probe
With anti-A antibody, anti-B antibody, anti-HBsAg, HCV recombinant antigen, HIV P24, HIV gp36 and syphilis recombinant antigen as detector probe, with the goat anti-human igg as positive control, with fetal bovine serum albumin as negative control, be separately fixed on the microarray substrate of making, form 3 * 3 microarray, each spot diameter 1mm in the array, interval 2mm is then with bSA sealing, drying;
C encapsulates blood detection microarray
With cover plate hermetically buckle closure on the substrate that has the detector probe microarray, promptly make blood detection microarray;
D blood testing and interpretation as a result
Detecting operation: get 20ul blood donor blood, be dissolved among the 0.1mol/L pH7.4PBS that 180ul contains 1%SDS and 0.1-5mmol/L gold mark Clq, hemolysate is joined microarray surface, behind the 5min, with the 0.1mol/L pH7.4PBS washing microarray of 1%SDS.Interpretation as a result: goat anti-human igg site colour generation shows that testing result is effective, detects once more otherwise should change detection arrays; Anti-A site colour generation and anti-B site be colour generation not, shows that the blood donor is the A type; Anti-B site colour generation and anti-A site be colour generation not, shows that the blood donor is a Type B; Anti-A site colour generation and anti-B site colour generation show that the blood donor is the AB type; Anti-A site and anti-B site be colour generation not, shows that the blood donor is the O type; Blood group A material site colour generation shows that the blood donor may be Type B or O type; Blood group B material site colour generation shows that the blood donor may be A type or O type; Blood group A material, B material site be colour generation all, and the blood donor is the O type; Blood group A material, B material site be colour generation not, and the blood donor is the AB type; Anti-D site colour generation shows blood donor Rh (D) positive, and colour generation is not negative; The anti-HBsAg colour generation shows the blood donor HBsAg positive; HCVAb antiantibody site colour generation shows the blood donor HCVAb positive; HIV P24/gp36 chimeric antigen site colour generation shows blood donor HIV antibody positive; Anti-HIV p24 site colour generation shows blood donor HIV-1 virus-positive; Anti-HIV gp36 site colour generation shows blood donor HIV virus-positive; Anti-HCMV site colour generation shows blood donor HCMV virus-positive; Anti-EBV site colour generation shows blood donor HCMV virus-positive; Syphilis antiantibody site colour generation shows blood donor's syphilis antibody positive.
Embodiment 2
Automatic blood detects microarray and makes and detect
A makes the transparent plastic substrates that the porous silica film covers
Get the normal transparent plastic sheet, the clean surface is standby according to a conventional method; And microslide covers 130%Na on this
2SiO
3100ul/cm
230min in the 0.3mol/L hydrochloric acid solution is immersed in dry rapidly back; Deionized water soaks 3min above 3 times; Under 240 ℃ condition, react 40min, form the meagre rete of porous silica of one deck 10-500nm on this slide surface, promptly obtain the microarray substrate;
B makes the probe microarray
With blood group A material, B material, blood group A antibody, B antibody, D antibody, anti-HBsAg, HCV recombinant antigen, HCV antibody, HIVp24, HIVgp36, HIVp24 antibody, HIVgp36 antibody, TP recombinant antigen as detector probe, with the goat anti-human igg as positive control, with fetal bovine serum albumin as negative control, be separately fixed on the microarray substrate of making, form 5 * 3 microarray, each spot diameter 0.2mm in the array, interval 0.5mm is then with bSA sealing, drying;
C encapsulates blood detection microarray
With cover plate hermetically buckle closure on the substrate that has the detector probe microarray, stamp or stick the bar code of the information of this microarray assay content of sign, probe arrangement mode at the barcode size or text field of cover plate, promptly make blood detection microarray;
D blood testing and result judge
Detecting operation: get 20ul blood donor blood, be dissolved among the 0.1mol/L pH7.4PBS that 180ul contains 1%SDS and 0.1-5mmol/L gold mark Clq, hemolysate joined microarray surface, behind the 5min, 0.1mol/L pH7.4PBS washing microarray with 1%SDS will detect microarray.Interpretation as a result: microarray scanner scans detecting microarray, from reading the crossing pattern that window reads microarray, read information such as microarray assay content and probe arrangement mode from the bar code district, and call embedded software in view of the above testing result is judged, testing result can be printed also can deposit computing machine in.
The making of visual blood detection microarray and detection
A makes the glass substrate that the porous silica film covers
Get potsherd, the clean surface is standby according to a conventional method; And potsherd covers 20%Na on this
2SiO
3100ul/cm
260min in the 0.5mol/L hydrochloric acid solution is immersed in dry rapidly back; Deionized water soaks 3min above 3 times; Under 300 ℃ condition, react 60min, form the meagre rete of porous silica of one deck 10-500nm on this potsherd surface, promptly obtain the microarray substrate;
B makes microarray albumen probe;
With anti-A antibody, anti-B antibody, anti-HBsAg, HCV recombinant antigen, HIV P24, HIV gp36 and syphilis recombinant antigen as detector probe, with the goat anti-human igg as positive control, with fetal bovine serum albumin as negative control, be separately fixed on the microarray substrate of making, form 3 * 3 microarray, each spot diameter 1mm in the array, interval 2mm is then with bSA sealing, drying;
C encapsulates blood detection microarray
With cover plate hermetically buckle closure on the substrate that has the detector probe microarray, promptly make blood detection microarray;
D blood testing and interpretation as a result
Detecting operation: get 20ul blood donor blood, be dissolved among the 0.1mol/L pH7.4PBS that 180ul contains 1%SDS and 0.1-5mmol/L gold mark Clq, hemolysate is joined microarray surface, behind the 5min, with the 0.1mol/L pH7.4PBS washing microarray of 1%SDS.Interpretation as a result: goat anti-human igg site colour generation shows that testing result is effective, detects once more otherwise should change detection arrays; Anti-A site colour generation and anti-B site be colour generation not, shows that the blood donor is the A type; Anti-B site colour generation and anti-A site be colour generation not, shows that the blood donor is a Type B; Anti-A site colour generation and anti-B site colour generation show that the blood donor is the AB type; Anti-A site and anti-B site be colour generation not, shows that the blood donor is the O type; Blood group A material site colour generation shows that the blood donor may be Type B or O type; Blood group B material site colour generation shows that the blood donor may be A type or O type; Blood group A material, B material site be colour generation all, and the blood donor is the O type; Blood group A material, B material site be colour generation not, and the blood donor is the AB type; Anti-D site colour generation shows blood donor Rh (D) positive, and colour generation is not negative; The anti-HBsAg colour generation shows the blood donor HBsAg positive; HCVAb antiantibody site colour generation shows the blood donor HCVAb positive; HIV P24/gp36 chimeric antigen site colour generation shows blood donor HIV antibody positive; Anti-HIV p24 site colour generation shows blood donor HIV-1 virus-positive; Anti-HIV gp36 site colour generation shows blood donor HIV virus-positive; Anti-HCMV site colour generation shows blood donor HCMV virus-positive; Anti-EBV site colour generation shows blood donor HCMV virus-positive; Syphilis antiantibody site colour generation shows blood donor's syphilis antibody positive.
Claims (8)
1, a kind of blood detection microarray, form at this on-chip cover plate (2) by substrate (1) and buckle closure, it is characterized in that: the porous silica film (6) that the rigidity combination is arranged on the described substrate (1), be adsorbed with securely on the film can with blood group, the detector probe that pathogen composition and antibody thereof react (3), described detector probe is arranged as dot matrix in order and forms microarray (4), the end face of described cover plate (2) is provided with well (5) and transparent reading window (7), cover plate (2) buckle closure hermetically has the gap of 0.1-1.0mm between described cover plate (2) and the detection dot matrix on porous substrates (1).
2, a kind of blood detection microarray according to claim 1 is characterized in that: the porous silica film (6) on the described substrate (1), energy non-specific adsorption proteantigen, antibody.
3, a kind of blood detection microarray according to claim 1, it is characterized in that: described microarray assay probe (3) is selected among blood group A material, B material, blood group A antibody, B antibody, D antibody, anti-HBsAg, HCV recombinant antigen, HCV antibody, HIVp24, HIVgp36, HIVp24 antibody, HIVgp36 antibody, TP recombinant antigen, the goat anti-human igg one or more and is separately fixed on the porous silica film, forms orderly array and arranges.
4, a kind of blood detection microarray according to claim 1 is characterized in that: the end face of described cover plate (2) is provided with bar code (8), and this bar code identification has microarray assay content and probe arrangement mode.
5, a kind of blood detection microarray according to claim 1 is characterized in that: described substrate (1) is slide, plastic sheet or potsherd.
6, a kind of method for making of blood detection microarray box is characterized in that may further comprise the steps:
A makes the substrate that the porous silica film covers
Get common microslide, plastic sheet or potsherd, the clean surface is standby according to a conventional method; And microslide covers 1-20%Na on this
2SiO
3100ul/cm
25-60min in the 0.05-0.5mol/L hydrochloric acid solution is immersed in dry rapidly back; Deionized water soaks 3min above 3 times; Under 180 ℃-300 ℃ condition, react 10-60min, form the meagre rete of porous silica of one deck 10-500nm on this slide surface, promptly obtain the microarray substrate;
B makes the probe microarray
With detector probe, positive control and negative control, be separately fixed on the microarray substrate of making, form orderly microarray, then with bSA or polyglycol sealing, drying;
C encapsulates blood detection microarray
With cover plate hermetically buckle closure on the substrate that has the detector probe microarray, cover plate and detect the gap that keeps 0.1-1.0mm between the microarray.
7, the method for making of blood detection microarray according to claim 6 is characterized in that: the detector probe among the step b is for in blood group A material, B material, blood group A antibody, B antibody, D antibody, anti-HBsAg, HCV recombinant antigen, HCV antibody, HIVp24, HIVgp36, HIVp24 antibody, HIVgp36 antibody, the TP recombinant antigen one or more.
8, the method for making of blood detection microarray according to claim 6 is characterized in that: the bar code that can stick this microarray assay content of sign and probe arrangement mode on the cover plate among the step c.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104714034A (en) * | 2013-12-17 | 2015-06-17 | 天津德祥生物技术有限公司 | Blood type detection method based on membrane structure |
| CN104769429A (en) * | 2012-06-11 | 2015-07-08 | Abo血型诊断公司 | In vitro diagnosis device and uses thereof |
| CN104950114A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Screening method for serum (plasma) antibody based on membrane structure and preparation method of screening and detecting kit |
| CN104950115A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Reverse typing detecting method for human ABO blood type based on membrane structure |
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| US5770416A (en) * | 1989-05-26 | 1998-06-23 | Upfront Chromatography A/S | Permeable hollow particles having an outer shell of mechanically rigid porous material |
| CN1048695C (en) * | 1994-07-02 | 2000-01-26 | 雷慧绪 | Method for producing silicon dioxide powder for electronic industry |
| US5951295A (en) * | 1996-02-08 | 1999-09-14 | Materials Evolution And Development Usa, Inc. | Ceramic fused fiber enhanced dental materials |
| US6037186A (en) * | 1997-07-16 | 2000-03-14 | Stimpson; Don | Parallel production of high density arrays |
| AU2001249437A1 (en) * | 2000-03-24 | 2001-10-08 | Lyles, Mark B | Diagnostic devices containing porous material |
| US7011841B2 (en) * | 2000-03-24 | 2006-03-14 | Mark B. Lyles | High density porous materials |
| CN1138145C (en) * | 2001-04-27 | 2004-02-11 | 上海晶泰生物技术有限公司 | Multiple-sample microarray biochip |
| CN1217003C (en) * | 2003-02-20 | 2005-08-31 | 北京博奥生物芯片有限责任公司 | Micro array reaction apparatus and uses thereof |
| CN1289904C (en) * | 2003-08-01 | 2006-12-13 | 博奥生物有限公司 | Micro array reaction unit and its use |
| CN1641335A (en) * | 2004-01-18 | 2005-07-20 | 陈尚松 | Contactless liquid sample collecting and packaging system |
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2005
- 2005-10-17 CN CNB2005101093714A patent/CN100425990C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104769429A (en) * | 2012-06-11 | 2015-07-08 | Abo血型诊断公司 | In vitro diagnosis device and uses thereof |
| CN104769429B (en) * | 2012-06-11 | 2018-02-16 | 迪亚加斯特公司 | In-vitro diagnosis device and its use |
| CN104714034A (en) * | 2013-12-17 | 2015-06-17 | 天津德祥生物技术有限公司 | Blood type detection method based on membrane structure |
| CN104950114A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Screening method for serum (plasma) antibody based on membrane structure and preparation method of screening and detecting kit |
| CN104950115A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Reverse typing detecting method for human ABO blood type based on membrane structure |
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