CN103278643A - Preparation method of microchip for microprotein detection - Google Patents
Preparation method of microchip for microprotein detection Download PDFInfo
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- CN103278643A CN103278643A CN2013101827197A CN201310182719A CN103278643A CN 103278643 A CN103278643 A CN 103278643A CN 2013101827197 A CN2013101827197 A CN 2013101827197A CN 201310182719 A CN201310182719 A CN 201310182719A CN 103278643 A CN103278643 A CN 103278643A
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Abstract
The invention relates to a preparation method of a microchip for microprotein detection. The preparation method comprises the following steps of: respectively exposing and developing a silicon wafer taken as a substrate material and SU-8 photoresist taken as a masking layer so as to manufacture a mold A and a mold B; mixing PDMS (Polydimethylsiloxane) with a curing agent, respectively pouring the mixture into a mold A and a mold B, heating and curing; respectively stripping A-PDMS and B-PDMS; punching the A-PDMS, gluing a processed glass sheet to the punched A-PDMS; and stripping away the A-PDMS, aligning and gluing the glass sheet on which an antibody is fixed with the B-PDMS, and gluing another punched glass sheet on the other surface of the B-PDMS, thus obtaining the microchip. The microchip can be used for integrating the separation and the detection of blood plasma in whole blood and detecting multiple targets, has the characteristics of specificity, rapidness and high sensitivity and can be expected to be applied to the clinical diagnosis and detection of multiple proteins in trace amounts of whole blood.
Description
Technical field
The invention belongs to the detection range of separating plasma and protein, particularly a kind of preparation method of the microchip that detects for trace of albumin.
Background technology
Whole blood is made up of compositions such as liquid blood plasma, red blood cell, leucocyte, blood platelets.In modern medicine detects, because there are very big interference in haemocyte or protoheme to spectral analysis, all at first blood plasma is separated from blood sample usually, be used for biochemistry or immunodiagnosis analysis then.At present, centrifuge method and filtration method are the clinical middle the most general methods of using.Centrifuge method is to utilize the principle of inertial centrifugal force and material sedimentation coefficient to reach separating effect, and filtration method is the separation that realizes blood plasma by filter membrane or solid support thing.But all there is certain weak point in these two kinds of methods, and bulky, the complicated operation of the former separation equipment, latter's filter membrane pore volume are easily stopped up by haemocyte and cause separation efficiency to reduce and sample contamination.Therefore, how effectively blood plasma being separated from whole blood and organically become one with detection is the focus of studying.
In recent years, micro-total analysis system because of have microminiaturization, integrated and intelligentized characteristics enjoy the researcher to pay close attention to, especially fast, the required sample size of analysis speed is several microlitres or lower, so micro-total analysis system will provide better detection platform for biomedical, analytical chemistry.Microflow control technique is the core of micro-total analysis system, and the microchip size adopts the MEMS technology to produce the integrated multifunction chips of components and parts such as channel network, reaction mixing pit, detecting device at base materials such as silicon chip, glass or plastics in a centimetre magnitude.Therefore, realize that at microchip the separation of micro-example is the focus that everybody pays close attention to detecting always.
Summary of the invention
Technical matters to be solved by this invention provides a kind of preparation method of the microchip that detects for trace of albumin, this microchip connects as one separation, the detection of blood plasma in the whole blood, can be once at the detection of a plurality of targets, have special, quick and high-sensitive characteristics, and do not need complicated instrument and the experiment condition of enzyme reaction, be expected to be applied to diagnosis and the detection of multiple protein in the clinical middle micro whole blood.
The preparation method of a kind of microchip that detects for trace of albumin of the present invention comprises:
Be base material with the silicon chip, with the SU-8 photoresist as mask layer, expose respectively, develop the mould A that makes ankyrin with separates detection mould B; After PDMS and hardening agent (Sylgard184curing agent) mixed with weight ratio 5～10:1, be cast in respectively on mould A and the mould B, be heating and curing; Peel off the PDMS structure B-PDMS on PDMS structure A-PDMS, the mould B on the mould A respectively; After A-PDMS beats sample holes and sample outlet hole, fit with treated glass sheet, be used for fixing a plurality of antibody; After A-PDMS taken off, will fixedly there be the glass sheet of antibody to aim at applying with B-PDMS, and gets the another side that another glass sheet through punching is fitted in B-PDMS, constitute the sandwich structure of glass-PDMS-glass, namely get microchip.
It is wide that described A-PDMS is of a size of 100 μ m height * 100 μ m.
Described B-PDMS comprises separating plasma pipeline, cell deposition pipeline and waste liquid pool, wherein the separating plasma pipeline is of a size of 15 μ m * 100 μ m * 23mm, the cell deposition pipeline is of a size of 100 μ m * 500 μ m * 20mm, and waste liquid pool is of a size of 100 μ m * 1.6mm * 9mm.
The described temperature that is heating and curing is 90 ℃, and the time of curing is 1 hour.
Described treated glass sheet is the aldehyde group modified glass sheet of process.
The temperature of described fixing a plurality of antibody is 37 ℃, and the time is 2 hours; Wherein, a plurality of antibody specifics are BSA, CEA, CyFRA21-1 and IgG antibody.
Trace of albumin during described trace of albumin detects is antigen or antibody.
The described microchip that obtains cooperates nano gold mark antibody to be applied to the separation of blood plasma in the whole blood and the detection of protein; Concrete steps comprise as follows:
After nano gold mark monoclonal two anti-NP and the sample mix, the injection port that adds microchip, haemocyte deposition during by separated region, blood plasma and NP flow into surveyed area simultaneously, form NP-antigen-primary antibodie sandwich structure with monoclonal primary antibodie fixing on the chip, thereby cause signal to amplify, obtain a result by the nm of gold colour developing.
The preparation method of described nano gold mark antibody comprises:
(1) configuration concentration is 1mM HAuCl
4Solution and concentration are the citric acid three sodium solution of 38.8mM, with HAuCl
4Solution is heated to 130 ℃, adds the 25ml citric acid three sodium solution in the time of 20 minutes, continues stirring until solution and is cooled to room temperature, namely forms claret solution; With 0.22 μ m cellulose nitrate nylon membrane filtering solution, can obtain evengranular 13nm nano-Au solution, 4 ℃ of preservations are standby;
(2) get the above-mentioned nano-Au solution of 500 μ l, regulating pH with sal tartari is 8.2, adds the two anti-of 30 μ l, places the 0.1M NaCl and the 0.01M PB that repeatedly add 4 μ l after 1～2 hour, 4 ℃ of preservations.
Nano-Au solution concentration in the step (2) is 12.2nM, and two anti-concentration are 0.1mg/ml.
The synoptic diagram of microstructure as shown in Figure 2 in the micro-fluid chip of the present invention (abbreviation microchip).Utilize the making of the photoetching process realization microstructure of standard, prepared required microchip with glass sheet and the reversible sealing-in of microstructure.This microchip connects as one separation and the protein detection of blood plasma in the whole blood.The entire chip size is 75mm * 25mm, is made up of separated region and two parts of surveyed area.
The present invention is based on nano-probe and micro-fluidic network combination, separation, reaction and the detected set of sample are become one, the deposition separated plasma in microchannel by haemocyte, and fixing target is combined on microchip, labelled antibody on nanogold particle, by the amplification step by step of signal, reach separation and protein detection to blood plasma in the micro whole blood.
Beneficial effect
Microchip of the present invention connects as one separation, the detection of blood plasma in the whole blood, can be once at the detection of a plurality of targets, have special, quick and high-sensitive characteristics, and do not need complicated instrument and the experiment condition of enzyme reaction, be expected to be applied to diagnosis and the detection of multiple protein in the clinical middle micro whole blood.
Description of drawings
Fig. 1 is the preparation of microchip of the present invention and the process chart that carries out the detection of separating plasma and protein;
Fig. 2 is the structural representation of microchip of the present invention;
Fig. 3 is the testing result of embodiment 1;
Fig. 4 is the testing result of embodiment 2.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
The separating plasma of healthy people's whole blood and the detection of albumen
Adopt microfluid system to detect to separating plasma in healthy people's whole blood and to albumen, concrete steps are as follows:
(1) preparation of micro-fluid chip and proteopexy
1, silicon chip is base material, and as mask layer, exposure, the mould A that makes ankyrin that develops detect mould B with separating with the SU-8 photoresist;
2, with PDMS, hardening agent (Sylgard184curing agent) with the 10:1(weight ratio) mix, be cast in respectively on mould A and the mould B except pour into a mould behind the bubble, 90 ℃ were heating and curing 1 hour, the making microstructure;
3, peel off PDMS piece (A-PDMS) on the mould A, with perforating needle punching sample holes and sample outlet hole, fit together with aldehyde group modified glass sheet is reversible then;
4, add 4 kinds of different primary antibodie solution (BSA, CEA, CyFRA21-1 and IgG antibody, BSA is as negative control, IgG is positive control) successively from the injection port of step 3, fix 2 hours at 37 ℃; Add PBS damping fluid (containing 1%BSA) flushing, dry naturally; Take the A-PDMS structure off, then the glass sheet that antibody is fixedly arranged is aimed at punching (sample holes and waste liquid pool).
5, peel off PDMS piece (B-PDMS) on the mould B, with 4 in the glass sheet of ankyrin aim at applying;
6, get the another side (no picture surface) that common glass sheet is fitted in B-PDMS then, form the sandwich structure of a glass sheet-PDMS-glass sheet, finish the making of chip and fixing of albumen.
(2) preparation of nm of gold and antibody labeling
1, the preparation of nano-Au solution
Configuration concentration is 1mM HAuCl
4Solution and concentration are the citric acid three sodium solution of 38.8mM.With HAuCl
4Solution is heated to 130 ℃, guarantees to stir in the heating process fully, and the citric acid three sodium solution that rapid adding newly prepares in the time of 20 minutes (is noted insulation, do not allowed HAuCl in this process
4Solution cooling) 25ml continues stirring until solution and is cooled to room temperature, namely forms claret solution.With 0.22 μ m cellulose nitrate nylon membrane filtering solution, can obtain evengranular 13nm nano-Au solution, 4 ℃ of preservations are standby.
2, nano gold mark antibody (NP)
Get the collaurum of 500 μ l, regulating pH with sal tartari is near 8.2, adds the two anti-of 30 μ l, places the 0.1M NaCL, the 0.01M PB(that add 4 μ l after 1 hour successively and adds three times), make solution keep certain salinity, 4 ℃ of preservations.
The NP probe is used for catching micro-antigen to be detected, because antigen-antibody reaction forms the NP-antigenic compound.
(3) detect and interpretation as a result
1, the PDMS piece that will take out negative pressure is placed on the sample outlet hole of the above-mentioned sandwich microchip for preparing.
2,10 μ l are fresh healthy people's whole blood (not containing CEA and CyFRA21-1 antigen through identifying) mixes with 0.5 μ l NP probe, get 3 μ l whole blood-NP mixed solutions and be added in the injection port of chip, blood flow is gone in the microchannel under suction function, the haemocyte natural subsidence is in pipeline, blood plasma-NP mixed liquor separates the back and flows into surveyed area, the protein fixing with chip surface has formed sandwich structure (being NP-antigen-albumen microchip), hatches 30 minutes for 37 ℃.
3, the 10mM PBS phosphate that adds 3 μ l at outlet, pH7.4 cleans three times.Unreacted NP probe, blood plasma etc. are washed off, and waste liquid flows out from injection port.
4, add 3 μ l nm of gold staining reagents, observations behind the 10min at outlet.Only fixedly black appears in the zone of IgG antibody, and all the other all do not have the black appearance, and this increment of presentation of results does not originally contain CEA and CyFRA21-1 antigen (see figure 3).
Embodiment 2
The separating plasma of clinical whole blood and the detection of albumen
(1) preparation of micro-fluid chip
Identical with (one) among the embodiment 1.
(2) preparation of nm of gold and antibody labeling
Identical with (two) among the embodiment 1.
(3) detect and interpretation as a result
1, the PDMS piece that will take out negative pressure is placed on the sample outlet hole of the above-mentioned sandwich microchip for preparing.
2,10 μ l are fresh lung cancer whole blood (containing CEA and CyFRA21-1 antigen through identifying the inside), 0.5 μ lNP probe mix, get 3 μ l whole blood-NP mixed solutions and be added in the injection port of chip, blood flow is gone in the microchannel under suction function, the haemocyte natural subsidence is in pipeline, blood plasma/NP mixed liquor separates the back and flows into surveyed area, the protein fixing with chip surface has formed sandwich structure (being NP-antigen-albumen microchip), hatches 30 minutes for 37 ℃.
3, the 10mM PBS phosphate that adds 3 μ l at outlet, PH7.4 cleans three times.Unreacted NP probe, blood plasma etc. are washed off, and waste liquid flows out from injection port.
4, add 3 μ l nm of gold staining reagents, observations behind the 10min at outlet.Fixedly black appears in the zone of IgG antibody, CEA antibody and CyFRA21-1, and BSA does not have black and occurs, this increment of presentation of results CEA and CyFRA21-1 antigen test positive (see figure 4) originally.
Claims (10)
1. preparation method who is used for the microchip that trace of albumin detects comprises:
Be base material with the silicon chip, with the SU-8 photoresist as mask layer, expose respectively, develop the mould A that makes ankyrin with separates detection mould B; After PDMS and hardening agent mixed with weight ratio 5～10:1, be cast in respectively on mould A and the mould B, be heating and curing; Peel off the PDMS structure B-PDMS on PDMS structure A-PDMS, the mould B on the mould A respectively; After A-PDMS beats sample holes and sample outlet hole, fit with treated glass sheet, be used for fixing a plurality of antibody; After A-PDMS taken off, will fixedly there be the glass sheet of antibody to aim at applying with B-PDMS, and gets the another side that another glass sheet through punching is fitted in B-PDMS, constitute the sandwich structure of glass-PDMS-glass, namely get microchip.
2. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1, it is characterized in that: it is wide that described A-PDMS is of a size of 100 μ m height * 100 μ m.
3. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1, it is characterized in that: described B-PDMS comprises separating plasma pipeline, cell deposition pipeline and waste liquid pool, wherein the separating plasma pipeline is of a size of 15 μ m * 100 μ m * 23mm, the cell deposition pipeline is of a size of 100 μ m * 500 μ m * 20mm, and waste liquid pool is of a size of 100 μ m * 1.6mm * 9mm.
4. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1, it is characterized in that: the described temperature that is heating and curing is 90 ℃, the time of curing is 1 hour.
5. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1 is characterized in that: described treated glass sheet is for through aldehyde group modified glass sheet.
6. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1, it is characterized in that: the temperature of described fixing a plurality of antibody is 37 ℃, and the time is 2 hours; Wherein, a plurality of antibody specifics are BSA, CEA, CyFRA21-1 and IgG antibody.
7. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1, it is characterized in that: the trace of albumin during described trace of albumin detects is antigen or antibody.
8. the preparation method of a kind of microchip that detects for trace of albumin according to claim 1, it is characterized in that: the described microchip that obtains cooperates nano gold mark antibody to be applied to the separation of blood plasma in the whole blood and the detection of protein; Concrete steps comprise as follows:
After nano gold mark monoclonal two anti-NP and the sample mix, the injection port that adds microchip, haemocyte deposition during by separated region, blood plasma and NP flow into surveyed area simultaneously, form NP-antigen-primary antibodie structure with monoclonal primary antibodie fixing on the chip, thereby cause signal to amplify, obtain a result by the nm of gold colour developing.
9. the preparation method of a kind of microchip that detects for trace of albumin according to claim 8, it is characterized in that: the preparation method of described nano gold mark antibody comprises:
(1) configuration concentration is 1mM HAuCl
4Solution and concentration are the citric acid three sodium solution of 38.8mM, with HAuCl
4Solution is heated to 130 ℃, adds the 25ml citric acid three sodium solution in the time of 20 minutes, continues stirring until solution and is cooled to room temperature, namely forms claret solution; With 0.22 μ m cellulose nitrate nylon membrane filtering solution, can obtain evengranular 13nm nano-Au solution, 4 ℃ of preservations are standby;
(2) get the above-mentioned nano-Au solution of 500 μ l, regulating pH with sal tartari is 8.2, adds the two anti-of 30 μ l, places the 0.1M NaCl and the 0.01M PB that repeatedly add 4 μ l after 1～2 hour, 4 ℃ of preservations.
10. the preparation method of a kind of microchip that detects for trace of albumin according to claim 9, it is characterized in that: the nano-Au solution concentration in the described step (2) is 12.2nM, two anti-concentration are 0.1mg/ml.
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