CN109765356A - A kind of protein-chip fluorescence detection method - Google Patents
A kind of protein-chip fluorescence detection method Download PDFInfo
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- CN109765356A CN109765356A CN201910046417.4A CN201910046417A CN109765356A CN 109765356 A CN109765356 A CN 109765356A CN 201910046417 A CN201910046417 A CN 201910046417A CN 109765356 A CN109765356 A CN 109765356A
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Abstract
The present invention relates to a kind of protein-chip fluorescence detection methods, comprising: step 1: carrying out PL processing in glass sheet surface；Step 2: ELISA experiment is carried out to the sheet glass for carrying out PL processing；The step 1 includes: that step (1) is successively cleaned by ultrasonic sheet glass using acetone, isopropanol and deionized water respectively；Sheet glass is reacted 2h with after being dried with nitrogen, sheet glass is immersed in be dissolved in the NaOH solution that water concentration is 2.5M by step (2) at room temperature；Step (3) is rinsed sheet glass well using flowing water and is dried with nitrogen, then reacts 2h with glass sheet surface at room temperature with 0.1%PL solution, and the glass carrier that PL is attached with to surface is obtained after cleaning up.Method provided by the invention, it is easy to operate, it is at low cost, the fluorescent detection capabilities of detecting size adjustable section and low concentration protein, operator need to only prepare required PDMS mold first, then be chemically treated glass carrier surface in specific region, the detection zone of required size just can be obtained, capture is uniform.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of protein-chip fluorescence detection method.
Background technique
Protein biochip technology has potential broad prospect of application in field of biomedicine.Protein-chip can be same
When detection biological sample in certain disease or environmental factor damage may relevant all protein content situation, i.e. table
Type fingerprint.Phenotype fingerprint judges that the effect for the treatment of is also of great significance to the process or prediction of monitoring disease.Traditional protein
Chip is usually integrally to carry out special chemical treatment to solid phase carrier, then known protein molecular is fixed thereon capture can be with
Specific binding testing protein to realize biomolecule detection and analysis, be the neck such as antigen and antibody and drug screening
Domain provides strong technical support.However, traditional protein chip solid phase carrier detection zone is big, processing technology is complicated, capture
Unevenly.
Summary of the invention
For above-mentioned problems of the prior art, it can avoid above-mentioned skill occur the purpose of the present invention is to provide one kind
The protein-chip fluorescence detection method of art defect.
In order to achieve the above-mentioned object of the invention, technical solution provided by the invention is as follows:
A kind of protein-chip fluorescence detection method, comprising:
Step 1: PL processing is carried out in glass sheet surface；
Step 2: ELISA experiment is carried out to the sheet glass for carrying out PL processing.
Further, the step 1 includes:
Step (1) is successively cleaned by ultrasonic sheet glass using acetone, isopropanol and deionized water respectively；
Sheet glass is dissolved in the NaOH solution that water concentration is 2.5M with after being dried with nitrogen, sheet glass is immersed in by step (2)
In react 2h at room temperature；
Step (3) is rinsed sheet glass well using flowing water and is dried with nitrogen, then with 0.1%PL solution at room temperature with sheet glass
2h is reacted on surface, and the glass carrier that PL is attached with to surface is obtained after cleaning up.
Further, the step 1 includes:
The PDMS solution of 10:1 proportional arrangement is uniformly mixed by step (1) using glass bar, then vacuumizes 1h except clean gas
Bubble；
The PDMS mixed liquor is slowly poured on clean Si on piece by step (2), and 80 DEG C of heating 1h realize solidification；
PDMS cured block is cut into multiple fritters with knife by step (3), is stamped and is run through in its center with punch on fritter
Round injection port, obtain PDMS mold；
Step (4) uses acetone, and isopropanol and deionized water are successively cleaned by ultrasonic sheet glass and each 5min of PDMS mold；
Step (5) makes glass carrier specific region be directed at fitting with PDMS mold injection port after being dried with nitrogen, then uses fixture
The two is mechanically fixed；
Step (6) picks up PDMS mold come injection port and upper layer on PDMS mold with fixture top plate and lower plywood
Circular hole alignment on plate, fixes fixture with bolt and nut；
Step (7) by the circular hole on top plate to injection port be added 10 μ L 0.1%PL solution, at room temperature with glass
2h is reacted on piece surface, and the glass carrier that PL is attached with to surface is obtained after cleaning up.
Further, the step 2 includes:
Step 1) reacts 2h in the cTnI monoclonal antibody solution that 20 μ g/ml are added in the carrier surface at room temperature, realizes
CTnI capture；
5% bovine serum albumen solution, which is added, after step 2) cleaning reacts 1h at room temperature；
Various concentration cTnI solution, which is added, after step 3) cleaning reacts 2h at room temperature；
20 μ g/ml cTnI polyclonal antibody solution are added after step 4) cleaning and react 2h at room temperature；
The rabbit-anti goat IgG solution that 2 μ g/ml fluorescent markers are added after step 5) cleaning reacts 1h at room temperature；
After step 6) cleans up, the corresponding fluorescence intensity of each concentration c TnI is measured under fluorescence microscope, obtains fluorescence
Immune curve, and compared with without chemically treated clean sheet glass.
Further, the fixture includes the top plate and lower plywood of rectangle, the shape and size of top plate and lower plywood
It is all the same, bolt hole there are four opening up, the center of top plate are corresponded on top plate and lower plywood at neighboring
Offer multiple circular holes.
Further, a length of 50mm of fixture top plate, width 40mm, centre offer 9 circular holes, are arranged in 3 rows 3
Column, the distance between capable and row are 6mm, and distance between the column and the column is 10mm, and the distance of long side of the bolt hole apart from plate is
5mm, the distance of the short side apart from plate are 12.5mm.
Further, the fixture is made of glass material, polytetrafluoroethylene material or acrylic material.
Further, in step 3), the cTnI solution concentration range is 1pg/ml-1 μ g/ml.
Protein-chip fluorescence detection method provided by the invention, easy to operate, at low cost, the adjustable section of detecting size
With the fluorescent detection capabilities of low concentration protein, operator need to only prepare required PDMS mold first, then in specific region chemistry
Handle glass carrier surface, so that it may obtain the detection zone of required size, cTnI capture uniformly, can meet well and actually answer
Needs.
Detailed description of the invention
Fig. 1 is the process schematic in glass sheet surface progress PL processing realization cTnI capture of embodiment 1；
Fig. 2 is the mistake handled with PL using the porose PDMS mold in center glass sheet surface specific region in embodiment 2
Journey schematic diagram；
Fig. 3 is the structural schematic diagram of fixture top plate；
Fig. 4 is the structural schematic diagram of fixture lower plywood.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawing and specific implementation
The present invention will be further described for example.It should be appreciated that described herein, specific examples are only used to explain the present invention, and does not have to
It is of the invention in limiting.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of protein-chip fluorescence detection method, comprising:
Step 1: PL processing is carried out in glass sheet surface；Specifically includes the following steps:
Step (1) is successively cleaned by ultrasonic sheet glass 5min using acetone, isopropanol and deionized water respectively；
Step (2) is by sheet glass with after being dried with nitrogen, as shown in Figure 1, sheet glass is immersed in, to be dissolved in water concentration be 2.5M
NaOH solution in react 2h at room temperature；
Step (3) is rinsed sheet glass well using flowing water and is dried with nitrogen, then with 0.1%PL solution at room temperature with sheet glass
2h is reacted on surface, and the glass carrier that PL is attached with to surface is obtained after cleaning up；
Step 2: the sheet glass for carrying out PL processing to the surface carries out ELISA experiment；The process the following steps are included:
CTnI (cTnI) monoclonal antibody solution of 20 μ g/ml is added at room temperature in the carrier surface in step (1)
2h is reacted, realizes cTnI capture；
5% bovine serum albumin (BSA) solution, which is added, after step (2) cleaning reacts 1h at room temperature；
Various concentration (1pg/ml-1 μ g/ml) cTnI solution, which is added, after step (3) cleaning reacts 2h at room temperature；
20 μ g/ml cTnI polyclonal antibody (Capra) solution are added after step (4) cleaning and react 2h at room temperature；
The rabbit-anti goat IgG solution that 2 μ g/ml fluorescent markers are added after step (5) cleaning reacts 1h at room temperature；
After step (6) cleans up, the corresponding fluorescence intensity of each concentration c TnI is measured under fluorescence microscope, obtains fluorescence
Immune curve, and compared with without chemically treated clean sheet glass.
The room temperature mentioned in the present embodiment refers to 22 DEG C~26 DEG C of temperature range.
Embodiment 2
A kind of protein-chip fluorescence detection method, comprising:
Step 1: glass sheet surface specific region is handled with PL using the porose PDMS mold in center；PL, that is, poly- L- relies
Propylhomoserin, the abbreviation of poly-l-lysine；Specifically includes the following steps:
Step (1) is mixed the PDMS solution (PDMS, that is, dimethyl silicone polymer) of 10:1 proportional arrangement using glass bar equal
It is even, then 1h is vacuumized except clean bubble；
The PDMS mixed liquor is slowly poured on clean Si on piece by step (2), and 80 DEG C of heating 1h realize solidification；
The PDMS cured block is cut into the small cured block of 2cm*2cm one by one using pocket knife by step (3), is taken wherein 3 pieces small
Cured block runs through the injection port of small cured block by the circle that punch beats 1mm, 2mm and 3mm diameter respectively in its center, obtains
Three PDMS molds；As shown in Figure 2；
Step (4) uses acetone, and isopropanol and deionized water are successively cleaned by ultrasonic sheet glass and each 5min of PDMS mold；
Step (5) makes glass carrier specific region be directed at fitting with PDMS mold injection port after being dried with nitrogen, then uses fixture
The two is mechanically fixed；
As shown in Figure 3 and Figure 4, the fixture includes the top plate and lower plywood of rectangle, the shape of top plate and lower plywood
It is all the same with size, it is corresponding at the neighboring on top plate and lower plywood to open up there are four bolt hole, in top plate
Heart position offers multiple circular holes, and the quantity and diameter of circular hole are set according to actual needs, and the present embodiment is 9 circles
Hole.In the present embodiment, a length of 50mm of fixture top plate, width 40mm, intermediate 9 circular holes are arranged in 3 rows 3 column, capable and row
The distance between be 6mm, distance between the column and the column is 10mm, and the distance of long side of the bolt hole apart from plate is 5mm, apart from plate
The distance of short side is 12.5mm, these sizes can be set as other numerical value according to the needs of practical application, such as circular hole is straight
Diameter can be 1mm, 2mm or 3mm etc..
The fixture can use following different materials: the first for glass fixture, advantage be cleaning it is transparent, can at any time into
Row optical detection；Second is polytetrafluoroethylene (PTFE) (polytetrafluoroethylene, PTFE), and advantage is high temperature high voltage resistant,
And bio-compatibility is excellent, hardly reacts with any reagent；The third is acrylic, advantage be it is low in cost, processing is simple.
Step (6) picks up PDMS mold come injection port and upper layer on PDMS mold with fixture top plate and lower plywood
Circular hole alignment on plate, fixes fixture with bolt and nut；
Step (7) by the circular hole on top plate to injection port be added 10 μ L 0.1%PL solution, at room temperature with glass
2h is reacted on piece surface, surface certain bits are obtained after cleaning up is equipped with a border circular areas (diameter 1mm, 2mm or 3mm) being attached with PL
Glass carrier, which is located at the center of the glass carrier.
Step 2: the sheet glass for carrying out PL processing to the surface carries out ELISA experiment；The process the following steps are included:
CTnI (cTnI) monoclonal antibody solution of 20 μ g/ml is added in the sheet glass carrier surface for step (1)
2h is reacted at room temperature, realizes cTnI capture；
5% bovine serum albumin (BSA) solution, which is added, after step (2) cleaning reacts 1h at room temperature；
Various concentration (1pg/ml-1 μ g/ml) cTnI solution, which is added, after step (3) cleaning reacts 2h at room temperature；
20 μ g/ml cTnI polyclonal antibody (Capra) solution are added after step (4) cleaning and react 2h at room temperature；
The rabbit-anti goat IgG solution that 2 μ g/ml fluorescent markers are added after step (5) cleaning reacts 1h at room temperature；
Step (6) measures the corresponding fluorescence intensity of each concentration c TnI under fluorescence microscope after cleaning up, obtain fluorescence
Immune curve, and compared with without chemically treated clean sheet glass.
The room temperature mentioned in the present embodiment refers to 22 DEG C~26 DEG C of temperature range.
Embodiment 2 can be realized in carrier surface to be treated solid simply and at low cost by the fixed PDMS mold of fixture
The chemical treatment of phase carrier-specific area (100 micron of -1 cm size size, specific shape).
Protein-chip fluorescence detection method provided by the invention, easy to operate, at low cost, the adjustable section of detecting size
With the fluorescent detection capabilities of low concentration protein, operator need to only prepare required PDMS mold first, then in specific region chemistry
Handle glass carrier surface, so that it may obtain the detection zone of required size, cTnI capture uniformly, can meet well and actually answer
Needs.
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (8)
1. a kind of protein-chip fluorescence detection method characterized by comprising
Step 1: PL processing is carried out in glass sheet surface；
Step 2: ELISA experiment is carried out to the sheet glass for carrying out PL processing.
2. protein fluorescence detection method according to claim 1, which is characterized in that the step 1 includes:
Step (1) is successively cleaned by ultrasonic sheet glass using acetone, isopropanol and deionized water respectively；
Sheet glass is dissolved in room in the NaOH solution that water concentration is 2.5M with after being dried with nitrogen, sheet glass is immersed in by step (2)
Temperature is lower to react 2h；
Step (3) is rinsed sheet glass well using flowing water and is dried with nitrogen, then with 0.1%PL solution at room temperature with glass sheet surface
2h is reacted, the glass carrier for being attached with PL to surface is obtained after cleaning up.
3. protein fluorescence detection method according to claim 1, which is characterized in that the step 1 includes:
The PDMS solution of 10:1 proportional arrangement is uniformly mixed by step (1) using glass bar, then vacuumizes 1h except clean bubble；
The PDMS mixed liquor is slowly poured on clean Si on piece by step (2), and 80 DEG C of heating 1h realize solidification；
PDMS cured block is cut into multiple fritters with knife by step (3), stamps perforative circle in its center with punch on fritter
Shape injection port obtains PDMS mold；
Step (4) uses acetone, and isopropanol and deionized water are successively cleaned by ultrasonic sheet glass and each 5min of PDMS mold；
Step (5) makes glass carrier specific region be directed at fitting with PDMS mold injection port after being dried with nitrogen, then with fixture by two
Person is mechanically fixed；
Step (6) picks up PDMS mold come on the injection port and top plate on PDMS mold with fixture top plate and lower plywood
A circular hole alignment, fix fixture with bolt and nut；
Step (7) by the circular hole on top plate to injection port be added 10 μ L 0.1%PL solution, at room temperature with sheet glass table
2h is reacted in face, and the glass carrier that PL is attached with to surface is obtained after cleaning up.
4. protein fluorescence detection method according to claim 1-3, which is characterized in that the step 2 packet
It includes:
Step 1) reacts 2h in the cTnI monoclonal antibody solution that 20 μ g/ml are added in the carrier surface at room temperature, realizes that cTnI is caught
It obtains；
5% bovine serum albumen solution, which is added, after step 2) cleaning reacts 1h at room temperature；
Various concentration cTnI solution, which is added, after step 3) cleaning reacts 2h at room temperature；
20 μ g/ml cTnI polyclonal antibody solution are added after step 4) cleaning and react 2h at room temperature；
The rabbit-anti goat IgG solution that 2 μ g/ml fluorescent markers are added after step 5) cleaning reacts 1h at room temperature；
After step 6) cleans up, the corresponding fluorescence intensity of each concentration c TnI is measured under fluorescence microscope, obtains fluorescence immunoassay
Curve, and compared with without chemically treated clean sheet glass.
5. protein fluorescence detection method according to claim 1 to 3, which is characterized in that the fixture includes the upper of rectangle
The shape and size of laminate and lower plywood, top plate and lower plywood are all the same, on top plate and lower plywood at neighboring
Bolt hole there are four opening up is corresponded to, the center of top plate offers multiple circular holes.
6. protein fluorescence detection method described in -5 according to claim 1, which is characterized in that fixture top plate it is a length of
50mm, width 40mm, centre offer 9 circular holes, are arranged in 3 rows 3 column, and the distance between capable and row is 6mm, between the column and the column
Distance be 10mm, the distance of long side of the bolt hole apart from plate is 5mm, and the distance of the short side apart from plate is 12.5mm.
7. protein fluorescence detection method according to claim 1 to 3, which is characterized in that the fixture be glass material,
Polytetrafluoroethylene material or acrylic material are made.
8. protein fluorescence detection method according to claim 1 to 3, which is characterized in that in step 3), cTnI solution
Concentration range is 1pg/ml-1 μ g/ml.
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| JP3368981B2 (en) * | 1994-04-15 | 2003-01-20 | 石川島播磨重工業株式会社 | Restraint jig |
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| CN101681866A (en) * | 2008-02-22 | 2010-03-24 | 泰尼克斯有限公司 | Wafer chucking apparatus for plasma process |
| US20140030755A1 (en) * | 2011-03-04 | 2014-01-30 | University College Cardiff Consultants Limited | Microplate for correlative microscopy |
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