CN104034895A - High-flux three-dimensional micro-fluidic chip immunoassay device and preparation method thereof - Google Patents
High-flux three-dimensional micro-fluidic chip immunoassay device and preparation method thereof Download PDFInfo
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- CN104034895A CN104034895A CN201410260221.2A CN201410260221A CN104034895A CN 104034895 A CN104034895 A CN 104034895A CN 201410260221 A CN201410260221 A CN 201410260221A CN 104034895 A CN104034895 A CN 104034895A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- 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
Abstract
The invention discloses a high-flux three-dimensional micro-fluidic chip immunoassay device and a preparation method of the device. The typical example of the immunoassay device comprises a diversion pipe and a three-dimensional glass core body; the side of the glass core body is modified by a copolymer brush; a detection probe is arranged on the copolymer brush in a way of point print; according to the detection device, the traditional two-dimensional chip is changed into the three-dimensional micro-array chip, the detection flux of the chip is greatly increased, the mass transfer process is promoted, and the combination or reaction rate of a target and probe molecules can be accelerated, so that the detection time is greatly shortened. The preparation method comprises the steps of cleaning and activating the surface of the glass core body, further modifying the polyethylene glycol methyl acrylate (PEGMA)-glycol methyl acrylate (GMA) copolymer brush, arranging the detection probe on the copolymer brush in a way of point print, and putting the product into the diversion pipe to form the three-dimensional micro-fluidic chip immunoassay device. Compared with the existing micro-array immune chip, the immunoassay device is high in detection flux, short in detection time and low in manufacturing cost, and does not need micromachining.
Description
Technical field
The present invention relates to a kind of immunoassay apparatus, particularly high flux three-dimensional microflow control chip immunoassay apparatus of a kind of new ideas and preparation method thereof.
Background technology
Immunoassay is a kind ofly to utilize specific binding between antigen-antibody to react the method that micromolecular compound, biomacromolecule and microorganism etc. are carried out to quantitative and qualitative analysis detection, has been widely used in the fields such as biomedical diagnostic, environmental evaluation, food safety detection.In recent years, microarray and micro-fluidic immuno-chip are because of its sample with reagent dosage is few, analysis throughput is high, be easy to the features such as robotization is widely used in scientific research and daily life.For microarray technology, although have ripe point sample instrument and analysis software on market, under static reactiveness, mass transport process is slower, and on micro-array chip, the antigen-antibody reaction time is longer.For microflow control technique, thus mobile detection system accelerated mass transport process and shortened the reaction time of antibody-antigen, but because micro-processing of chip needs strict condition and expensive instrument, suppressed the development of microflow control technique.
Although have at present researcher by microarray and microflow control technique in conjunction with in order to immune detection, the micro-processing technology that existing apparatus does not still solve microfluidic platforms requires the shortcoming of harshness, somewhat expensive; In addition, existing microarray detects and only in one plane carries out, chip effectively utilize area little, detect flux relatively low.
Summary of the invention
In view of this, the object of this invention is to provide a kind of high flux three-dimensional microflow control chip immunoassay apparatus and preparation method thereof, to improve immune detection flux, to improve detection signal-to-noise ratio and sensitivity and raising testing efficiency.
High flux three-dimensional microflow control chip immunoassay apparatus of the present invention, comprise mozzle and be arranged at the glass core body in mozzle, described glass core body is tubular, the cylindrical or cylinder tube shape of regular prism, regular prism, on the lateral surface of described glass core body, be modified with copolymer brush, on described copolymer brush, point is printed on detector probe.
Further, described glass core body is cuboid.
Further, described detector probe is the monoclonal antibody of target detection thing.
Further, described monoclonal antibody is that anti-human Cea Monoclonal Antibodies is or/and anti-human alpha-fetoprotein monoclonal antibody.
The preparation method of high flux three-dimensional microflow control chip immunoassay apparatus of the present invention, comprises the steps:
A. activation is cleaned in the side of glass core body;
B. on the side of the glass core body after activation, modify PEGMA-GMA copolymer brush;
C. on the PEGMA-GMA copolymer brush on glass core body side surface, point prints detector probe;
D. the glass core body that combines detector probe in step c is positioned over and in mozzle, forms three-dimensional microflow control chip immunoassay apparatus.
Further, in described step a, the step of the side of glass core body being cleaned to activation comprises: the 3-aminopropyl triethoxysilane that is first 3% by glass core body volume fraction soaks after 2 hours and cleans up, then puts into vacuum drying chamber, vacuum annealing 2 hours at 110 DEG C.
Further, in described step b, the step of modifying PEGMA-GMA copolymer brush in the side of glass core body comprises:
The glass core body that side is cleaned after activation is put into the mixed solution containing methenyl choloride, triethylamine and 2-bromine isobutyl acylbromide, first in 0 DEG C of ice bath, hatches 20 minutes, under rear placement room temperature, continues to hatch 2 hours; In described mixed solution, in the methenyl choloride mixed solution of every 20mL, the final concentration of triethylamine is 55mM, and the final concentration of 2-bromine isobutyl acylbromide is 50mM; Then glass core body is taken out from mixed solution and put into again polymeric solution after cleaning up and hatch 6 hours, in described polymeric solution, the final volume mark of first alcohol and water is 50%, the ultimate density of copper bromide is 3.35mg/mL, the ultimate density of dipyridine is 4.6mg/mL, the ultimate density of ascorbic acid is 2.38mg/mL, the final volume mark of glycidyl methacrylate is 2%, and the final volume mark of polyethylene glycol methacrylate-styrene polymer is 20%.
Further, in described step c, on the side of glass core body of having modified PEGMA-GMA copolymer brush, comprise in conjunction with the step of detector probe:
Glass core body is rinsed successively with ethanol and deionized water, dry again, then by chip point sample instrument by the detector probe of 100 μ g/mL with microarray form point glass core body four sides on, and at room temperature put into vacuum tank dry 2 hours, finally use Tris buffer solution for cleaning and immersion.
Beneficial effect of the present invention:
1, high flux three-dimensional microflow control chip immunoassay apparatus of the present invention, four sides of its glass core body all have the detector probe of polymer brush and microarray, polymer brush can effectively improve the quantity of sessile antibody and reduce non-specific adsorption, effective usable floor area with respect to existing this immunoassay apparatus of microarray immuno-chip improves greatly, and detection sensitivity is high.
2, high flux three-dimensional microflow control chip immunoassay apparatus of the present invention, it passes through at the external cover mozzle of glass core, detected object can flow under the driving of flow pumps in the microchannel of mozzle, realizes flow detection, thus the reaction time of shortening antibody-antigen that can be very large; Therefore this three-dimensional microflow control chip immunoassay apparatus has not only had microarray and micro-fluidic detection advantage concurrently, and its detection efficiency is now greatly improved for existing microarray and micro-fluidic immuno-chip, be highly suitable for extensive rapid screening, can be used widely at aspects such as disease early screening, Food Safety Analysis, environmental contaminants monitoring.
3, high flux three-dimensional microflow control chip immunoassay apparatus of the present invention, its glass core body and mozzle are simple in structure, and not by special micro-process equipment manufacture, low cost of manufacture.
Brief description of the drawings
Fig. 1 is the perspective view of this high flux three-dimensional microflow control chip immunoassay apparatus;
Fig. 2 is longitudinal cross-sectional schematic of this high flux three-dimensional microflow control chip immunoassay apparatus;
Fig. 3 is this high flux three-dimensional microflow control chip immunoassay apparatus under the circulation condition of static and different in flow rate, antibody-antigen in conjunction with the graph of a relation in reaction time;
Fig. 4 specificity that to be this high flux three-dimensional microflow control chip immunoassay apparatus detect multiple tumor markerses and optionally histogram and fluorescence photo;
Fig. 5 is that this high flux three-dimensional microflow control chip immunoassay apparatus detects to tumor markers AFP and CEA the typical curve and the fluorescence photo that obtain.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment mono-, the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus, comprise mozzle 1 and be arranged at the glass core body 2 in mozzle 1, described glass core body 2 is cuboid, on the side of described glass core body 2, be modified with copolymer brush, on described copolymer brush, point is printed on detector probe.The glass core body 2 of cuboid, printing when detector probe, can be arranged in juxtaposition multiple glass core bodys same time point and print, and can increase work efficiency.Certainly, in different embodiment, described glass core body 2 is gone back but regular prism, the regular prism of other shape are tubular, cylindrical or cylinder tube shape.
The present embodiment high flux three-dimensional microflow control chip immunoassay apparatus, four sides of its glass core body 2 all have the detector probe of polymer brush and microarray, polymer brush can effectively improve the quantity of sessile antibody and reduce non-specific adsorption, effective usable floor area with respect to existing this immunoassay apparatus of microarray immuno-chip improves greatly, and detection sensitivity is high.Further, this high flux three-dimensional microflow control chip immunoassay apparatus, it passes through at glass core body 2 overcoat mozzles 1, detected object can flow under the driving of flow pumps in the microchannel of mozzle 1, and (gap between glass core body 2 and mozzle 1 is little, can make the required detected object consumption less), realize flow detection, thus the reaction time of shortening antibody-antigen that can be very large; Therefore this three-dimensional microflow control chip immunoassay apparatus has not only had microarray and micro-fluidic detection advantage concurrently, and its detection efficiency is now greatly improved for existing microarray and micro-fluidic immuno-chip, be highly suitable for extensive rapid screening, can be used widely at aspects such as disease early screening, Food Safety Analysis, environmental contaminants monitoring.Further, this high flux three-dimensional microflow control chip immunoassay apparatus, its glass core body 2 and mozzle 1 are simple in structure, and not by special micro-process equipment manufacture, low cost of manufacture.
In the present embodiment, described detector probe is the monoclonal antibody of target detection thing.
In the present embodiment, described monoclonal antibody is anti-human Cea Monoclonal Antibodies, and certainly in different embodiment, described monoclonal antibody also can be anti-human alpha-fetoprotein monoclonal antibody.
The preparation method of embodiment bis-, the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus, comprises the steps:
A. activation is cleaned in the side of glass core body;
B. on the side of the glass core body after activation, modify PEGMA-GMA copolymer brush;
C. on the PEGMA-GMA copolymer brush on glass core body side surface, point prints detector probe;
D. the glass core body 2 that combines detector probe in step c is positioned over and in mozzle 1, forms three-dimensional microflow control chip immunoassay apparatus.
In described step a, the step of the side of glass core body being cleaned to activation comprises: 3-aminopropyl triethoxysilane (APTES) the ethanol liquation that is first 3% by glass core body volume fraction soaks 2 hours, then clean up successively with ethanol and deionized water, then dry up with nitrogen, put into again vacuum drying chamber, vacuum annealing 2 hours at 110 DEG C.
In described step b, the step of modifying PEGMA-GMA copolymer brush in the side of glass core body comprises:
The glass core body that side is cleaned after activation is put into the methenyl choloride mixed solution containing triethylamine (TEA) and 2-bromine isobutyl acylbromide (BIB), first in 0 DEG C of ice bath, hatches 20 minutes, under rear placement room temperature, continues to hatch 2 hours; In described mixed solution, in the methenyl choloride mixed solution of every 20mL, the final concentration of triethylamine is 55mM, and the final concentration of 2-bromine isobutyl acylbromide is 50mM; Then glass core body is taken out from mixed solution and put into again polymeric solution after cleaning up and hatch 6 hours, in described polymeric solution, the final volume mark of first alcohol and water is 50%, the ultimate density of copper bromide is 3.35mg/mL, the ultimate density of dipyridine is 4.6mg/mL, the ultimate density of ascorbic acid is 2.38mg/mL, the final volume mark of glycidyl methacrylate (GMA) is 2%, and the final volume mark of polyethylene glycol methacrylate-styrene polymer (PEGMA) is 20%.
In described step c, on the side of glass core body of having modified PEGMA-GMA copolymer brush, comprise in conjunction with the step of detector probe:
Glass core body is rinsed successively with ethanol and deionized water, dry again, then by chip point sample instrument by the detector probe of 100 μ g/mL with microarray form point glass core body four sides on, and at room temperature put into vacuum tank dry 2 hours, finally use Tris buffer solution for cleaning and immersion; Detector probe described in the present embodiment is mouse-anti people CEA (carcinomebryonic antigen, carcino-embryonic antigen) monoclonal antibody, the epoxy bond of the glycidyl methacrylate in PEGMA-GMA copolymer brush reacts with the amino in monoclonal antibody and forms chemical bond, makes monoclonal antibody covalency be fixed on glass capillary surface.
The concrete steps of the detection tumor markers CEA of the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus are as follows:
To contain respectively 0pg/mL, 0.1pg/mL, 1pg/mL, 10pg/mL, 100pg/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1 μ g/mL, the PBS solution of 10 μ g/mL tumor markers CEA adopts flow pumps to pump into respectively this device, be 15 μ L/min flowing reactive 30 minutes at flow velocity, again the mouse-anti people CEA monoclonal antibody of 1 μ g/mLCy3 mark is pumped into this device 30 minutes, finally TBS solution and deionized water are flow through respectively to this device, after dry, read fluorescence signal with fluorescent scanning instrument, then taking fluorescence intensity as ordinate, the concentration of tumor markers CEA is horizontal ordinate, drawing standard curve, as shown in Figure 5.
The preparation method of embodiment tri-, the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus and the difference of embodiment mono-are: described detector probe is mouse-anti people AFP (alpha-fetoprotein, alpha-fetalprotein) monoclonal antibody, other step of the present embodiment is identical with embodiment bis-, does not repeat them here.
The concrete steps of the detection tumor markers AFP of the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus are as follows:
0pg/mL will be contained respectively, 0.1pg/mL, 1pg/mL, 10pg/mL, 100pg/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1 μ g/mL, the PBS solution of 10 μ g/mL tumor markers APF adopts respectively flow pumps that sample solution is flowed through taking flow velocity as 15 μ L/min, and this installs 30 minutes, again the mouse-anti people AFP polyclonal antibody of 1 μ g/mL Cy3 mark is pumped into this device 30 minutes, finally TBS solution and deionized water are flow through respectively to this device, after dry, read fluorescence signal with fluorescent scanning instrument, then taking fluorescence intensity as ordinate, tumor markers AFP concentration is horizontal ordinate, drawing standard curve, as shown in Figure 5.
The preparation method of embodiment tetra-, the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus and the difference of embodiment mono-are: described detector probe is mouse-anti people AFP (alpha-fetoprotein, alpha-fetal protein) monoclonal antibody and mouse-anti people CEA (carcinomebryonic antigen, carcino-embryonic antigen) monoclonal antibody, other step of the present embodiment is identical with embodiment bis-, does not repeat them here.
The concrete steps that the present embodiment high flux three-dimensional microflow control chip immunoassay apparatus detects polycomponent tumor markers are simultaneously as follows:
Adopt flow pumps by flow through taking flow velocity as 15 μ L/min this device 30 minutes of the PBS mixed solution that contains 100ng/mL tumor markers APF and CEA or serum, again the PBS mixed solution of the mouse-anti people CEA monoclonal antibody of the mouse-anti people AFP monoclonal antibody of 1 μ g/mL Cy3 mark and 1 μ g/mL Cy3 mark is pumped into this device 30 minutes, finally TBS solution and deionized water are flow through respectively to this device, after dry, read fluorescence signal with fluorescent scanning instrument, then taking fluorescence intensity as ordinate, tumor markers AFP and CEA are horizontal ordinate, draw histogram, as shown in Figure 4.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (8)
1. a high flux three-dimensional microflow control chip immunoassay apparatus, it is characterized in that: comprise mozzle and be arranged at the glass core body in mozzle, described glass core body is tubular, the cylindrical or tubular of regular prism, regular prism, on the lateral surface of described glass core body, be modified with copolymer brush, on described copolymer brush, point is printed on detector probe.
2. high flux three-dimensional microflow control chip immunoassay apparatus according to claim 1, is characterized in that: described glass core body is cuboid.
3. high flux three-dimensional microflow control chip immunoassay apparatus according to claim 1, is characterized in that: described detector probe is the monoclonal antibody of target detection thing.
4. high flux three-dimensional microflow control chip immunoassay apparatus according to claim 3, is characterized in that: described monoclonal antibody is that anti-human Cea Monoclonal Antibodies is or/and anti-human alpha-fetoprotein monoclonal antibody.
5. a preparation method for arbitrary described high flux three-dimensional microflow control chip immunoassay apparatus in claim 1-4, is characterized in that: comprise the steps:
A. activation is cleaned in the side of glass core body;
B. on the side of the glass core body after activation, modify PEGMA-GMA copolymer brush;
C. on the PEGMA-GMA copolymer brush on glass core body side surface, point prints detector probe;
D. the glass core body that combines detector probe in step c is positioned over and in mozzle, forms three-dimensional microflow control chip immunoassay apparatus.
6. the preparation method of high flux three-dimensional microflow control chip immunoassay apparatus according to claim 5, it is characterized in that: in described step a, the step of the side of glass core body being cleaned to activation comprises: the 3-aminopropyl triethoxysilane that is first 3% by glass core body volume fraction soaks after 2 hours and cleans up, put into again vacuum drying chamber, vacuum annealing 2 hours at 110 DEG C.
7. the preparation method of high flux three-dimensional microflow control chip immunoassay apparatus according to claim 5, is characterized in that: in described step b, the step of modifying PEGMA-GMA copolymer brush in the side of glass core body comprises:
The glass core body that side is cleaned after activation is put into the mixed solution containing methenyl choloride, triethylamine and 2-bromine isobutyl acylbromide, first in 0 DEG C of ice bath, hatches 20 minutes, under rear placement room temperature, continues to hatch 2 hours; In described mixed solution, in the methenyl choloride mixed solution of every 20mL, the final concentration of triethylamine is 55mM, and the final concentration of 2-bromine isobutyl acylbromide is 50mM; Then glass core body is taken out from mixed solution and put into again polymeric solution after cleaning up and hatch 6 hours, in described polymeric solution, the final volume mark of first alcohol and water is 50%, the ultimate density of copper bromide is 3.35mg/mL, the ultimate density of dipyridine is 4.6mg/mL, the ultimate density of ascorbic acid is 2.38mg/mL, the final volume mark of glycidyl methacrylate is 2%, and the final volume mark of polyethylene glycol methacrylate-styrene polymer is 20%.
8. the preparation method of high flux three-dimensional microflow control chip immunoassay apparatus according to claim 5, is characterized in that: in described step c, on the side of glass core body of having modified PEGMA-GMA copolymer brush, comprise in conjunction with the step of detector probe:
Glass core body is rinsed successively with ethanol and deionized water, dry again, then by chip point sample instrument by the detector probe of 100 μ g/mL with microarray form point glass core body four sides on, and at room temperature put into vacuum tank dry 2 hours, finally use Tris buffer solution for cleaning and immersion.
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