CN103760361A - Biological chip for detecting person Hsp90a (heat shock protein 90) and detection method thereof - Google Patents
Biological chip for detecting person Hsp90a (heat shock protein 90) and detection method thereof Download PDFInfo
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- CN103760361A CN103760361A CN201310729381.2A CN201310729381A CN103760361A CN 103760361 A CN103760361 A CN 103760361A CN 201310729381 A CN201310729381 A CN 201310729381A CN 103760361 A CN103760361 A CN 103760361A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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Abstract
The invention discloses a biological chip for detecting person Hsp90a (heat shock protein 90) and a detection method thereof, and relates to a biological chip of the person Hsp90a and the detection method. The biological chip is characterized in that a detection device is composed of a glass piece, a rubber separation piece and metal fixed clamps, wherein the rubber separation piece is arranged above the glass piece, the two sides of the rubber separation piece are respectively inserted into a groove of the metal fixed clamps, and the chip and the rubber separation piece are fixed together. The detection method comprises the following steps of 1, activating a nitrocellulose membrane arranged on the surface of the glass piece; 2, carrying out crosslinking on an antibody protein and biotin so as to increase the bonding strength and sensitivity of an antibody; 3, preparing an antibody chip; 4, detecting the person Hsp90a in a microscale blood specimen by using the antibody chip; and 5, analyzing the data. The method provided by the invention has the advantages that the operation is simple and convenient, meanwhile, multiple samples are detected, the standardization correction is automatically performed, and a signal intensity graph is generated.
Description
Technical field:
The present invention relates to a kind of biochip and detection method of detectable antigens, be specifically related to biochip and the detection method thereof of a kind of human heat shock protein (Hsp90 α).
Background technology:
Biochip refers generally to the microarray hybridization cake core (micro-arrays) that high density is fixed on the biological information molecule (as genetic fragment, DNA fragmentation or polypeptide, protein, glycan molecule, tissue etc.) on mutual supporting dielectric, in array, the sequence of each molecule and position are known, and are pre-set sequence dot matrix.
According to the systemization with biomolecule, biochip can be divided into DNA chip, RNA (RNA (ribonucleic acid)) chip, protein-chip, cell chip and neuron chip.In addition, according to the detection mode of biomolecule, biochip can roughly be divided into two types, and the first is microarray type, and it utilizes capture probe (capturing probe) to explore and is contained in the specific biochemical substances in sample.Particularly, the material that can be used as capture probe (capturing probe) is fixed on the surface of chip, then, after reacting with the biochemical substances of needs analysis, reactionless by detecting and having analyzed, and can obtain the information of biochemical substances.The second is the biochip that utilizes microfluid, microchannel, microchamber and mixing valve are set on chip, to control microfluid, biochemical substances is fixed on after detecting unit, utilize microfluid to make to need the biochemical substances detecting to react with the biochemical substances that is fixed on detecting unit, reactionless to have detected.In the long run, and according to nearest trend toward miniaturization, this is the most active field of research.
Manufacture and utilize biochip need the probe that can react with reactive material technique for fixing, can detect unresponsive detection technique and can process the information processing technology of the information detecting.
Wherein, can detect the general mark that uses ad hoc fashions such as fluorescence, colorimetric and isotope of unresponsive detection technique.This labelling technique is extremely important to improving detection sensitivity.But the problem of existence is that mark may make biomolecule distortion, or lower-molecular substance cannot be labeled.Also the problem of existence is, the sample that labeling process consumption is a large amount of, and need to carry out 2-3 more step, for protein, the marked difference between range protein has increased quantization error.At present, representative mark mode is laser-Induced Fluorescence Detection method.Laser-Induced Fluorescence Detection method is current most popular optical means, wherein, utilizes fluorescent material mark sample, utilizes the fluorescent material of mark to detect and be fixed on to have between the probe on substrate reactionless.Yet the problem that this method also exists is, reactionless in order to have detected, need optical measuring system, this causes needs a lot of time and cost, and this detection method is also in the analytic system based on biochip.
Human heat shock protein 90 α, Hsp90 α, is the important member in heat-shock protein family.External expert's reported first in 1989 gene order of Hsp90 α, confirmed the identity of this albumen.Within 1992, foreign scientist finds, Hsp90 α can be by tumor cell secretion to extracellular, but its Mechanism of Secretion Regulation after this for a long time in and unclear.
Being firmly established at present of this brand-new tumor markers of Hsp90 α, opened the molecular difference of Hsp90 α in extracellular hsp 90 α and cell simultaneously, further illustrate secreting type Hsp90 α and can promote tumor invasion and transfer, and its content and malignancy positive correlation in blood.These discoveries have proved in blood that Hsp90 α is as the good potential quality of tumor markers.Hsp90 α tumor markers has the characteristic of wide spectrum, can be used for the clinical detection of a plurality of knurl kinds such as liver cancer, breast cancer, colorectal cancer, can reflect real-time and accurately result for the treatment of.Hsp90 α, as tumor markers, checks more convenient and quicker, and cost also reduces greatly.Only need get one and bleed, can detect by this kit the content of Hsp90 α in blood plasma.Although some tumor patient does not show related symptoms, but its Hsp90 alpha content may raise, if first detect Hsp90 α level in blood, carry out again testing in depth testing, just may be diagnosed as tumour, this can make patient receive treatment early, thereby greatly improves cure rate or extend life cycle.
In addition, in patient's blood plasma, the height of Hsp90 alpha content has good correspondence with change of illness state, therefore can reflect in real time, more exactly result for the treatment of, for doctor formulates and adjust in time therapeutic scheme, provides reference.
Summary of the invention:
The biochip and the detection method thereof that the object of this invention is to provide a kind of detection human heat shock protein (Hsp90 α), each film piece can detect 80 blood samples simultaneously, utilize rubber shim and slide geometrical clamp, can on a chip, there be 16 film pieces, easy and simple to handle, save sample and time, can automatically carry out standardization correction, and generate the chart of each autoantibody signal intensity.
The present invention is by the following technical solutions: its pick-up unit is comprised of slide, rubber shim and metal clip, and rubber shim is arranged on slide top, and insert respectively in the groove of metal clip both sides, and chip and rubber shim are fixed together.
Detection method: 1, the activation of surface of glass slide nitrocellulose filter: slide is immersed in certain density affinity cellulose solution, room temperature is placed 30min, after taking out, with PBS solution, wash once, centrifugal drying, puts 4 ℃ of refrigerators standby: 2, antibody protein and biotin are crosslinked: the bond strength and the flexibility ratio that increase antibody; 3, Dispersal risk chip: be 1mg/mL by antibody dilution, with injecting type micro-array point sample instrument by antibody point on the NC of surface of glass slide film, on every slide, be divided into 16 film pieces, each film piece contains 80 antibody and 8 reference proteins, during point sample, humidity remains on 75%, after point sample, room temperature is placed 2h, then vacuum packaging: 4, utilize antibody chip to detect human heat shock protein (Hsp90 α) in micro blood sample; 5, data analysis: analyze with corresponding analysis software.
The described about 1mm of rubber shim thickness, size is consistent with slide, has 16 holes on shim, and the position in hole and aperture are suitable with antibody spot sample district, and rubber shim is covered to chip surface, and antibody spot sample district is positioned at the center position in each hole.
The monoclonal antibody of the selected human heat shock protein of the present invention (Hsp90 α) oneself provides for this laboratory, by protein cross technology, antibody and biotin (Biotin) are connected, then utilize microarray technology, with micro-array point sample instrument, biotinylated antibody is fixed on the slide carrier that has been coated with Avidin (Streptavidin).Every slide (25mm * 73mm) is divided into 16 film pieces, and each film piece contains 80 antibody and 8 reference proteins.Therefore, every chip all contains 16 microarraies that comprised 80 antibody and 8 kinds of reference proteins, and each film piece can detect 80 samples simultaneously.Pick-up unit is the rubber separate spacers and two geometrical clamps that have 16 holes, and rubber separate spacers and slide are in the same size, and on the position in its hole and size and slide, the position in 16 albumen point sample districts meets.During detection, first rubber shim is positioned over to surface of glass slide, with geometrical clamp, slide and rubber shim are fixed together, after 1uL sample is mixed with 50uL sample diluent, add in the hole of rubber shim and react, after 60min, abandon reactant liquor, add 100uL cleansing solution, vibration washing 3 times, each 5min.Then the goat anti-human igg antibody's (dilution in 1: 1000) who adds affinity element and the fluorescein Cy5 mark of fluorescein Cy3 mark, room temperature reaction 60min, suck reactant liquor, add cleansing solution to wash 3 times, remove geometrical clamp and separate spacers, slide is immersed to rinsing 30s in PBS damping fluid, and low-speed centrifugal (500rpm) 3min makes slide dry.Use laser scanner scans slide, scanning wavelength is 535nm (Cy3) and 632nm (Cy5).Image Genepix6.0 software analysis, draws the fluorescence intensity of each point Cy3 and Cy5, and take Cy3 and calculate the correction signal intensity of each point as internal reference, then uses analysis software, calculates the mean intensity of each antigen.
The present invention can be easy and simple to handle, save sample and time, can automatically carry out standardization correction, and generate the chart of counter sample signal intensity.
Accompanying drawing explanation:
Fig. 1 is the structural representation of slide in the present invention;
Fig. 2 is the structural representation of slide after processing in the present invention;
Fig. 3 is the structural representation of rubber shim in the present invention;
Fig. 4 is the structural representation of metal clip in the present invention;
Fig. 5 is the assembly structure schematic diagram of pick-up unit of the present invention.
Embodiment:
With reference to Fig. 1-5, this embodiment is by the following technical solutions: its pick-up unit is comprised of slide 1, rubber shim 2 and metal clip 3, rubber shim 2 is arranged on slide 1 top, insert respectively in the groove of metal clip 3 both sides, and slide 1 and rubber shim 2 are fixed together.
Its detection method is:
1, the activation of surface of glass slide nitrocellulose filter: slide is immersed in certain density affinity element (Streptavidin) solution, and room temperature is placed 30min, washes once after taking out with PBS solution, and centrifugal drying, puts 4 ℃ of refrigerators standby.
2, antibody protein and biotin are crosslinked: the bond strength and the flexibility ratio that increase antibody.
3, the preparation of antibody chip: be 1mg/mL by antibody dilution, and proceed in 384 orifice plates.With injecting type micro-array point sample instrument, antigen is printed on the NC film of surface of glass slide.16 film pieces of some system on every slide, each film piece contains 80 antigens and 8 reference proteins, and each albumen repeats twice on each film piece.During point sample, humidity remains on 75%.After completing, room temperature is placed 2h, and then vacuum packaging, can preserve 2 years for 4 ℃.
4, utilize antibody chip to detect human heat shock protein (Hsp90 α) in micro blood sample: during detection, first rubber shim is positioned over to surface of glass slide, with geometrical clamp, slide and rubber shim are fixed together, after 1uL sample is mixed with 50uL sample diluent, add in the hole of rubber shim and react, after 60min, abandon reactant liquor, add 100uL cleansing solution, vibration washing 3 times, each 5min.Then add the affinity element of fluorescein Cy3 mark and the goat-anti people 1gG antibody of fluorescein Cy5 mark (dilution in 1: 1000), room temperature reaction 60min, suck reactant liquor, add cleansing solution to wash 3 times, remove geometrical clamp and separate spacers, slide is immersed to rinsing 30s in PBS damping fluid, and low-speed centrifugal (500rpm) 3min makes slide dry.Use laser scanner scans slide, scanning wavelength is 535nm (Cy3) and 632nm (Cy5).Image Genepix6.0 software analysis, draws the fluorescence intensity of each point Cy3 and Cy5, and take Cy3 and calculate the correction signal intensity of each point as internal reference.
5, data analysis: analyze with corresponding analysis software.
The monoclonal antibody of the selected human heat shock protein of the present invention (Hsp90 α) oneself provides for this laboratory, by protein cross technology, antibody and biotin (Biotin) are connected, then utilize microarray technology, with micro-array point sample instrument, biotinylated antibody is fixed on the slide carrier that has been coated with Avidin (Streptavidin).Every slide (25mm * 73mm) is divided into 16 film pieces, and each film piece contains 80 antibody and 8 reference proteins.Therefore, every chip all contains 16 microarraies that comprised 80 antibody and 8 kinds of reference proteins, and each film piece can detect 80 samples simultaneously.Pick-up unit is the rubber separate spacers and two geometrical clamps that have 16 holes, and rubber separate spacers and slide are in the same size, and on the position in its hole and size and slide, the position in 16 albumen point sample districts meets.During detection, first rubber shim is positioned over to surface of glass slide, with geometrical clamp, slide and rubber shim are fixed together, after 1uL sample is mixed with 50uL sample diluent, add in the hole of rubber shim and react, after 60min, abandon reactant liquor, add 100uL cleansing solution, vibration washing 3 times, each 5min.Then the goat anti-human igg antibody's (dilution in 1: 1000) who adds affinity element and the fluorescein Cy5 mark of fluorescein Cy3 mark, room temperature reaction 60min, suck reactant liquor, add cleansing solution to wash 3 times, remove geometrical clamp and separate spacers, slide is immersed to rinsing 30s in PBS damping fluid, and low-speed centrifugal (500rpm) 3min makes slide dry.Use laser scanner scans slide, scanning wavelength is 535nm (Cy3) and 632nm (Cy5).Image Genepix6.0 software analysis, draws the fluorescence intensity of each point Cy3 and Cy5, and take Cy3 and calculate the correction signal intensity of each point as internal reference, then uses analysis software, calculates the mean intensity of each antigen.
The present invention can be easy and simple to handle, save sample and time, can automatically carry out standardization correction, and generate the chart of counter sample signal intensity.
Claims (3)
1. biochip and a detection method thereof that detects human heat shock protein (Hsp90 α), the pick-up unit that it is characterized in that it is comprised of slide, rubber shim and metal clip, rubber shim is arranged on slide top, insert respectively in the groove of metal clip both sides, and slide and rubber shim are fixed together.
2. biochip and the detection method thereof of a kind of detection human heat shock protein according to claim 1 (Hsp90 α), the detection method that it is characterized in that it is: 1, the activation of surface of glass slide nitrocellulose filter: slide is immersed in certain density affinity cellulose solution, room temperature is placed 30min, after taking out, with PBS solution, wash once, centrifugal drying, puts 4 ℃ of refrigerators standby; 2, antibody protein and biotin are crosslinked: the bond strength and the flexibility ratio that increase antibody; 3, Dispersal risk chip: be 1mg/mL by antibody dilution, with injecting type micro-array point sample instrument by antibody point on the NC of surface of glass slide film, on every slide, be divided into 16 film pieces, each film piece contains 80 antibody and 8 reference proteins, each albumen repeats point sample twice on each film piece, during point sample, humidity remains on 75%, and after point sample, room temperature is placed 2h, then vacuum packaging; 4, utilize antibody chip to detect human heat shock protein (Hsp90 α) in micro blood sample; 5, data analysis: analyze with corresponding analysis software.
3. biochip and the detection method thereof of a kind of detection human heat shock protein according to claim 1 (Hsp90 α), it is characterized in that the described about 1mm of rubber shim thickness, size is consistent with slide, on shim, there are 16 holes, the position in hole and aperture are suitable with antibody spot sample district, rubber shim is covered to chip surface, and antibody spot sample district is positioned at the center position in each hole.
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| CN201310729381.2A CN103760361A (en) | 2013-12-14 | 2013-12-14 | Biological chip for detecting person Hsp90a (heat shock protein 90) and detection method thereof |
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| CN201310729381.2A CN103760361A (en) | 2013-12-14 | 2013-12-14 | Biological chip for detecting person Hsp90a (heat shock protein 90) and detection method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104155436A (en) * | 2014-07-28 | 2014-11-19 | 王泰朕 | Biological chip for urine analysis |
| CN105572353A (en) * | 2014-10-17 | 2016-05-11 | 广州瑞博奥生物科技有限公司 | Antibody chip reagent kit for detecting hepatoma marker |
| CN106248957A (en) * | 2016-07-15 | 2016-12-21 | 陶少强 | A kind of biochip for detecting HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 |
-
2013
- 2013-12-14 CN CN201310729381.2A patent/CN103760361A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104155436A (en) * | 2014-07-28 | 2014-11-19 | 王泰朕 | Biological chip for urine analysis |
| CN105572353A (en) * | 2014-10-17 | 2016-05-11 | 广州瑞博奥生物科技有限公司 | Antibody chip reagent kit for detecting hepatoma marker |
| CN105572353B (en) * | 2014-10-17 | 2018-10-30 | 广州瑞博奥生物科技有限公司 | A kind of antibody chip kit for detecting liver cancer marker |
| CN106248957A (en) * | 2016-07-15 | 2016-12-21 | 陶少强 | A kind of biochip for detecting HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 |
| CN106248957B (en) * | 2016-07-15 | 2018-07-03 | 安徽国科生物科技有限公司 | A kind of biochip for being used to detect human heat shock protein |
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