CN104931711A - Method and device for promoting combination of biological reagents and biomolecules - Google Patents
Method and device for promoting combination of biological reagents and biomolecules Download PDFInfo
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- CN104931711A CN104931711A CN201510358712.5A CN201510358712A CN104931711A CN 104931711 A CN104931711 A CN 104931711A CN 201510358712 A CN201510358712 A CN 201510358712A CN 104931711 A CN104931711 A CN 104931711A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
Abstract
The invention discloses a method and device for promoting combination of biological reagents and biomolecules, and belongs to the field of biological detection. The biomolecules are fixed on a micropore support body, the biological reagents are recycled for multiple times to penetrate the support body through micropores, the biological reagents and the biomolecules are rapidly and effectively combined each time the biological reagents penetrate the support body, and the combination of the biological reagents and the biomolecules is greatly enhanced due to multiple times of repeated penetration. The device for promoting combination of the biological reagents and the biomolecules comprises a column cylinder, a plunger, the micropore support body and the biomolecules fixed on the micropore support body, the plunger moves back and forth in the column cylinder to enable the biological reagents to penetrate the micropore support body repeatedly for multiple times, combination of the biological reagents and the biomolecules is promoted, and compared with an existing detection method, the device has the advantages of convenience, rapidness, flexibility and the like.
Description
Technical field
The invention belongs to field of biological detection, more particularly, relate to a kind of method and apparatus that biological reagent is combined with biomolecule that promotes.
Background technology
Protein is the important composition composition of cell, plays a role in cellular activity process.Protein expression kind in cell and quantity determine its shape and function, and abnormal protein expression can cause disease.A main task of biomedical research is exactly detect the expression pattern of albumen in biological sample.Due to posttranslational modification, there is the protein of specific amino acids primary sequence may occur in different forms in cell.In many cases, only have the albumen of some special shape to participate in certain specific activities of cell directly, valuable information can be provided to the activity and function understanding cell so detect at the albumen of these special shapes of cells.Albumen has various posttranslational modification, as phosphorylation, glycosylation and general elder brotherization.The phosphorylation of serine, methionine or histidine residues is an important mechanism on intracellular signaling.Abnormal protein phosphorylation result in many human diseasess.In the method detecting protein phosphorylation, often used with radioactive isotope metabolic marker cell with antibody mediated immunity detection phosphorylated protein, anti-phosphorylated residue specific antibody, such as PY20 and 4G10, also through being commonly used to detect phosphorylated protein.
Detect protein expression and have application in multiple field, comprise biomedical research, medical diagnosis on disease, find treatment index and drug target and reflect in analysis at toxicity and drug effect.In basic biomedical research, often wish to know which albumen is expressed under special cell or specific condition.By the protein expression of more dissimilar cell, the expression of which albumen and the active character determining a special cells just may be differentiated.In many intracellular signaling passages, some albumen are activated specifically; Detect these by shock protein, such as phosphorylated protein, crucial information can be provided for the principle understanding intracellular signaling passage.Numerous disease can change the expression pattern of albumen, and protein expression abnormal in many cases can be diseases induced.Therefore, the expression pattern of albumen is determined and to contrast normal and paracytic protein expression pattern for understanding the mechanism of disease be useful.The expression detecting some albumen is also widely used in clinical diagnosis.A lot of clinical testing procedure all based on the protein markers detected in sample, as the antibody produced in the antigen of virus and health.In addition, detect protein expression at course of drug development, the such as selection of medicine target application point, the index of toxicology and searching drug response is very useful.
Detection method of protein is a lot, comprises the immunization method based on Ag-Ab specific binding and other nonimmune method.In numerous nonimmune method, mass spectrum is a relatively more conventional method.This technology is by protein molecule is converted into gaseous ion, then utilizes electric field, magnetic field is separated having the protein Ion of extra fine quality with charge ratios (M/Z), collects, determines the M/Z value of ion, carry out Analysis and Identification protein.Mass-spectrometric technique can be used for identifying albumen but be unsuitable for quantitatively detecting expression and some other characteristics of comparison protein, is particularly unsuitable for studying cell inner expression high complexity and the albumen of height mobilism.The immunological method of protein detection also has a lot, has respective principle of work, feature and advantage, shortcoming.The method that research field is commonly used has enzyme linked immunoassay (ELISA), protein immunoblot technology (Western blotting), immunochemistry dyeing etc.Percolation (flow through), sidestream immune chromatography (lateral flow immunoassay) etc. is also often used in disease detection.
The ultimate principle of enzyme linked immunoassay is that antigen (or antibody) is attached to surface of solid phase carriers, react by inspection sample (antibody or antigen) and enzyme-labelled antigen (or antibody) by the antigen of different step and surface of solid phase carriers or antibody, by washing, the antigen antibody complex that solid phase carrier is formed and other material are separated again, to be finally combined in enzyme amount on solid phase carrier and sample inspected antibody or antigen amount proportional; After adding enzyme reaction substrate again, substrate is become coloured or luminescent products after enzymatic, carries out qualitative or quantitative test according to its signal power, to understand antibody or antigenic content in tested sample.
Protein immunoblot technology confirms an albumen by antibody-antigene reaction and protein molecular weight two indices.Other detection technique relatively, it has higher sensitivity and specificity, has become one of most important technology in protein research.Immunoblot assay generally includes several step: be first separated by molecular size range by polyacrylamide gel electrophoresis by albumen sample, then move on on blotting membrane by protein transduction; After enforcement is closed, blotting membrane and specific resistance body (primary antibodie) are hatched, and make the destination protein (antigen) on film and an anti-binding.And then add and can resist with two of the tape label of primary antibodie specific binding; Mark on anti-finally by two produces signal, thus can learn the information such as the expression of destination protein in detected biological sample and molecular weight.
The principle of spot immune percolation test (dot immunofiltration assay) is similar to enzyme linked immunoassay, but replaces polystyrene board to make carrier with microporous barrier (as nitrocellulose filter, nylon membrane etc.).During test, the miillpore filter being coated with antigen or antibody is adhered on absorbent material, drips sample, enzyme conjugates, substrate successively, wash respectively, unnecessary sample and enzyme labelled antibody and cleansing solution etc. can diafiltration enter in absorbent material, and last positive sample presents painted spot on film.
Colloidal gold immunochromatographimethod method is simple to operate, quick because detecting, and has been applied in the application of a lot of clinical detection.In this method, capture antigen or antibody are fixed on film with ribbon, and colloid gold label reagent (polyclonal antibody or monoclonal antibody) is adsorbed on pad in advance.After in the sample pad that sample to be checked is added to test strips one end, moved forward by capillary action, react to each other after dissolving the colloid gold label reagent on pad, when moving to the region of fixing capture antigen or antibody again, there is specific binding with it again and be trapped in the bond of thing to be checked and gold marked reagent, be gathered in detection zone, by being observed visually colour developing result.
Protein-chip (dot matrix) technology is a method that simultaneously can detect multiple proteins.Have numerous protein in a protein chip, each albumen is fixed on the ad-hoc location on chip support.Antibody chip is chip with fastest developing speed in protein chip, can be used for analyzing the difference of albumen between sample, determining the character of respective egg white matter.The conventional method using antibody chip is by antibody chip and protein sample incubation, makes the antibody on chip and the albumen in sample (antigen) react, combine, thus they are captured on chip.Afterwards, captured albumen is detected further.A lot of case study shows the important application of antibody chip in biomedicine, as by the expression analyzing different carcinoma cell protein, discloses better cancer cell criteria for classification.
Based on the detection technique of antibody-antigene reaction, as protein immunoblot technology, percolation (flow through) and protein biochip technology, generally all comprise the process that antibody in a solution or antigen are combined with the antigen be fixed on a support or antibody.Generally comprise in method closed (blocking), antibody-antigene combines, cleaning (washing), and the step such as signal generation, detection.These processes generally need dozens of minutes just can complete by several hours.
In actual applications, people wish to shorten the time detecting protein.On the one hand, a lot of application needs to complete at short notice, as clinical real-time test (point-of-care testing).On the other hand, the combination of antibody, antigen is the process of a mobile equilibrium, after antibody, antigenic compound are formed, repeatedly, for a long time cleaning antibody, antigen can be caused to be separated, thus reduction detection sensitivity.Therefore, once after Ag-Ab combination, follow-up detecting step should be completed as early as possible.
The method accelerating the combination of Ag-Ab has multiple.The simplest method is exactly in the process of hatching, constantly rocks solution to increase the effect chance of antibody and antigen.A filter process is under vacuum used in the SNAP i.d. of Mi Libo (Millipore) company
tMon product, help to accelerate antibody-antigene reaction, thus the process of protein immunoblot method was shortened to from several hours be less than 45 minutes.Filtering (filtration) is a method being used for the solid particle in solution to separate from solution.Filtrator can be a solid with micropore, and as filter membrane, solution can pass micropore, and the solid being greater than micropore is intercepted on film.There is the filtrator of multiple different structure, be used in biomedicine to biological solution as calf serum, cell culture fluid filtering or sterilizing.Filter process under vacuum action also with helping a protein or nucleotide is fixed on a film support, also can be used to purify DNA.Such as, 96 orifice plate Bio-Dot of Bio-Rad Company and 48 orifice plate Bio-DotSF microfiltration products are used for the protein in solution or nucleotide to be fixed on film.
Multiple document describes the method applying filtration in immunization method.Such as United States Patent (USP) 4,366,241 and 4,632,901 describe and utilize absorbent material, by capillarity solution through a filtering membrane.United States Patent (USP) 5,155,049 describes the method for another solution through film.Utilize similar filter process to have some to limit to strengthen the method combined between biological reagent and biomolecule at present, particularly do not reach maximum combined degree between biological reagent and biomolecule.Therefore, need the method for a research easy economy to realize biological reagent and the maximum combination of biomolecule, improve the sensitivity of biological detection.
In prior art, utilize the combination that similar filter process can realize between biological reagent and biomolecule, in some cases, this method is even superior to other conventional methods.But people do not recognize that existing method does not reach the maximization of molecule combination; Particularly do not find that the method for existing similar filtration can not reach the maximum combined of reagent and molecule usually.The method that biological reagent is combined with biomolecule is promoted in the present invention, significantly can be strengthened effective combination of biological reagent and biomolecule by the pattern of iterative cycles, the result that the result using the method for the invention to carry out testing acquisition obtains than conventional method at least improves five times.
In the method for existing similar filtration, biological reagent is not easy to reclaim.Such as, in spot immune percolation, biological reagent infiltrates in absorbent material, cannot reclaim, and because the biological reagent liquor capacity used is general very little, utilizes the method for vacuum pump to be also difficult to effectively reclaim, is particularly difficult to repeatedly, recycles reagent solution.These difficulties hamper now methodical improvement.In addition, utilize similar filter process to realize the mechanism that combines between biological reagent and biomolecule and unclear.Such as, it is generally acknowledged that the antibody-antigene reaction needed long period combines, but in reality, can realize at short notice combining by filter process, unclear about the mechanism realizing in the short time combining at present, therefore also hamper the improvement of method.
Inventor finds in an experiment, by extending the method for biological reagent and biomolecule binding time, how long no matter extend, all cannot reach higher joint efficiency, such as, biomolecule is fixed on micropore support surface, then hatches with biological reagent, no matter how long, in conjunction with efficiency all do not have the method that repeatedly circulates in the present invention good.Therefore method of the present invention not only ensures that biomolecule can fully contact with biological reagent, but also improve biological reagent effectively in conjunction with concentration.Utilize the method for similar filter process realize biological reagent and biomolecule in conjunction with time, the process through hatching combination is needed before filter operation, use method of the present invention to save and hatch cohesive process, saved the running time, operating efficiency is improved greatly.
3. beneficial effect
Compared to prior art, beneficial effect of the present invention is:
(1) the present invention utilizes a kind of device that biological reagent is combined with biomolecule that promotes, realize biological reagent solution circulation repeatedly through the micropore support being fixed with biomolecule, promote that biomolecule is combined fast, effectively between biological reagent, more quick than existing detection method, sensitive;
(2) the present invention is a kind of promotes the device that biological reagent is combined with biomolecule, comprise post cylinder, plunger and micropore support, micropore support is fixed on one end of post cylinder, the other end that plunger is positioned at post cylinder can do piston movement at post cylinder, in operation, biological reagent solution is placed on by micropore support, in the seal cavity that post cylinder and plunger are formed, micropore support is fixed with biomolecule, plunger does piston movement in post cylinder, biological reagent solution is flowed out through micropore support, collect the biological reagent solution flowed out and repeat aforesaid operations, promote effective combination of biomolecule and biological reagent, the piston movement of plunger realizes, as air pump, vacuum pump, reciprocating pump, peristaltic pump etc. by malleation or negative pressure or mode that both combine,
(3) the present invention is a kind of promotes the device that biological reagent is combined with biomolecule, post cylinder is the hollow structure that one end is closed, plunger is hollow structure, micropore support is positioned at one end of plunger, when plunger does piston movement in post cylinder, biological reagent solution is made to enter in the hollow structure of plunger through micropore support, the second plunger is provided with in the hollow structure of plunger, second plunger can make biological reagent solution again through micropore support by piston movement, repeatable operation like this, promote the combination of biological reagent and biomolecule, have easy and simple to handle, binding time is short, detection sensitivity advantages of higher,
(4) the present invention breaks through traditional biomolecule and biological reagent associated methods, the ingenious abundant combination utilizing the principle of cycling to promote biomolecule and biological reagent, and devise a kind of device of convenient operation based on this, achieve the recycling of biological reagent solution, save the time that a large amount of biomolecule is combined with biological reagent, detection efficiency is significantly improved, and detection sensitivity at least improve five times;
(5) device of the present invention can combinationally use and carry out batch detection, improves detection efficiency; Such as can by device according to 1 × 4,1 × 8,1 × 12 arrangement or according to 2 × 4,4 × 6,8 × 12 aligned transfer such as grade, in addition, can be designed to integrative-structure by multiple post cylinder.
Summary of the invention
1. the problem that will solve
Exist in conjunction with insufficient, that detection sensitivity is not high enough, speed is fast not problem for existing biological reagent and biomolecule, the invention provides a kind of method and apparatus that biological reagent is combined with biomolecule that promotes.In the present invention, biomolecule is fixed on micropore support, and biological reagent solution repeats Multiple through then out micropore through support, each through in process, biological reagent is combined fast, effectively with biomolecule, repeatedly through making the combination of biological reagent and biomolecule greatly strengthen.A kind of in the present invention promotes the device that biological reagent is combined with biomolecule, comprise post cylinder, plunger and micropore support, plunger moves back and forth in post cylinder, impels biological reagent solution repeatedly repeatedly through micropore support, promotes the combination of biological reagent and biomolecule.
2. technical scheme
In order to solve the problem, the technical solution adopted in the present invention is as follows:
Promote to the steps include: the method that biological reagent is combined with biomolecule
(1) biomolecule is fixed on the micropore support with micropore, forms one or more biomolecule point, obtain detection bodies;
(2) biological reagent is passed micropore support by micropore, in the process, biological reagent is combined with biomolecule;
(3) repeat step (2), make biological reagent repeatedly through micropore support.
Preferably, the described micropore support film that is nitrocellulose membrane or nylon membrane or polyacrylamide film or is made up of cellulose nitrate, nylon, polyacrylamide derivative.
Preferably, the cross-sectional area of described micropore support is 10 ~ 1000mm
2.
Preferably, the detection bodies in described step (1) is that multiple biomolecule point is fixed on the biomolecule dot matrix that micropore support is formed.
Preferably, the detection bodies in described step (1) has 2 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
Preferably, the detection bodies in described step (1) has 5 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
Preferably, described detection bodies is that then protein shift through electrophoretic separation and be fixed on the Western blotting film that micropore support is formed.
Preferably, described biomolecule is antibody or described biological reagent is antibody.
Preferably, in described step (2), biological reagent solution passes micropore support with single direction by micropore.
Preferably, in described step (2), biological reagent solution passes micropore support with two-way by micropore.
Preferably, step (2) 2 to 100 times are repeated in described step (3).
Preferably, step (2) 5 to 30 times are repeated in described step (3).
A kind ofly promote the device that biological reagent is combined with biomolecule, comprise detection bodies, post cylinder and plunger, described detection bodies is formed by micropore support and the biomolecule be fixed in the above, described post cylinder is hollow structure, described plunger is arranged in the hollow structure of post cylinder, forms hermetically-sealed construction between plunger and post cylinder.
Preferably, described detection bodies is micropore support is fixed with the biomolecule dot matrix that multiple biomolecule point formed, and have 2 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
Preferably, described detection bodies is micropore support is fixed with the biomolecule dot matrix that multiple biomolecule point formed, and have 5 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
Preferably, described biomolecule is antibody.
Preferably, described detection bodies is that then protein shift through electrophoretic separation and be fixed on the Western blotting film that micropore support is formed.
Preferably, described detection bodies is fixed on one end of post cylinder.
Preferably, described post cylinder is the hollow structure that one end is closed, and described plunger is hollow structure, and detection bodies is fixed on one end of plunger.
Preferably, in the hollow structure of described plunger, be provided with the second plunger, between the second plunger and plunger, form hermetically-sealed construction.
Preferably, the hollow structure of described plunger and post cylinder is elliptical cylinder-shape.
Preferably, the hollow structure of plunger and post cylinder is cubic cylindricality, and corner is with radian.
Preferably, also comprise one or more setting off, the side that setting off described at least one is arranged on detection bodies directly contacts with detection bodies.
Promote the multilevel device that biological reagent is combined with biomolecule, n above-mentioned a kind of device promoting biological reagent to be combined with biomolecule is combined and obtains, wherein n >=2.
Preferably, 2≤n≤384.
Preferably, 2≤n≤96.
Preferably, 4≤n≤96.
Accompanying drawing explanation
Fig. 1 is biomolecule of the present invention and biological reagent cohesive process schematic diagram;
Fig. 2 is a kind of structural representation promoting the device that biological reagent is combined with biomolecule of the present invention;
Fig. 3 is a kind of structural representation promoting the device that biological reagent is combined with biomolecule of the present invention;
Fig. 4 is a kind of structural representation promoting the device that biological reagent is combined with biomolecule of the present invention;
Fig. 5 is that method more of the present invention and classic method are for detecting an experimental result of antibody;
Fig. 6 is that method more of the present invention and classic method are for detecting an experimental result of antibody;
An experimental result of Fig. 7 to be method more of the present invention and classic method detect in for protein immunoblot protein.
In figure: 1, detection bodies; 2, post cylinder; 3, plunger; 4, hermetically-sealed construction; 5, the second plunger.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.
The invention provides and can promote the method that biological reagent is combined with biomolecule in detection protein and other field of biological molecule.A feature of method uses the process of circulation to make biological reagent solution repeatedly through the micropore support being fixed with biomolecule.Method and apparatus of the present invention can promote the biological reagent in solution and the combination being fixed on the biomolecule on micropore support.Now the term occurred in the present invention is made an explanation:
" biomolecule " this term, with here for the needs being convenient to description and patent right, refers to any molecule relevant with biology, including, but not limited to protein.Biomolecule is often referred to detected material, is fixed on support, can with biological reagent effect, combination.
" biological reagent " this term, with here for the needs being convenient to description and patent right, refers to any material relevant with biology.Biological reagent and biomolecule action, can be used for detecting biomolecule.Biological reagent is including, but not limited to protein, antibody, antigen, polypeptide, DNA, RNA and Small molecular.Biological reagent may reside in solution, and a kind of conventional biological reagent solution is antibody-solutions.
" support " this term, with here for the needs being convenient to description and patent right, refers to for placing the carrier structure with fixing biological molecules.In the present invention, support can be support-" the micropore support " with micropore.Biological reagent solution can by micropore through support.The size of micropore can from 1 nanometer to 1 millimeter, preferably from 10 nanometers to 100 microns.In the ordinary course of things, micro-pore diameter is from 100 nanometers to 10 microns.Micropore support has a variety of, and in the present invention, micropore support can be cellulose nitrate (nitrocellulose), nylon (Nylon) or polyacrylamide (PVDF) film, or the film that their derivants are formed.The micropore support that other material is made, as various glass etc., also in method and apparatus used in the present invention.
Biomolecule is fixed on micropore support and forms detection bodies." detection bodies " this term, with here for the needs being convenient to description and patent right, refers to micropore support and is fixed on the biomolecule on micropore support.In the present invention, detection bodies can be Protein microdot arrays, antibody dot matrix, Western blotting film, or other are with the support of the micropore of protein molecule.
Support is preferably anticipated so that biomolecule can be fixed in the above by with suitable intensity.A kind of method of process support is on solid support, modify one deck polymer compounds, and these polymer compounds interact by non-specific non-covalent bond and biological reagent.Method biomolecule be fixed on support has multiple.Such as, biomolecule can manually o'clock on a nitrocellulose membrane, also can as (Hybridization fingerprinting in genomemapping and sequencing, genome analysis, Voll such as Lehrach, Davies and Tilgham, Eds, Co1d Spring HarborPress, pp.39-81,1990) and Brown (United States Patent (USP): 5807522) etc. described, with the machine point of robotization on support.In the article mentioned above each section any method of describing all may be relevant with the present invention, be incorporated herein by reference at this, as the description to method.
Biomolecule is fixed (Trevan, 1980, Immobilized Enzymes:an introduction andtheir application in biotechnology.Wiley, Chichester) by suction-operated.This absorbability can be non-specific, and hydrophobic or interionic interacts.Typical adsorption material comprises clay, charcoal, hydroxylapatite, and many ion exchange materials such as DEAE-cross-linked glucose.Embedding (entrapment) is the method (Trevan of another kind of fixing biological molecules, 1980, ImmobilizedEnzymes:an introduction and their application in biotechnology.Wiley, Chichester).In theory, the biomolecule be embedded is not combined with medium, is only that their free diffusing is restricted.A kind of embedding medium of frequent use is multi-polyamide gel.
Biomolecule can be fixed on solid support directly or indirectly.Employing is similar to the technology preparing highdensity DNA chip and biomolecule directly can be fixed on to high-density (Shalon et al., Genome Reserch, 1996Jul on support; 6 (7): 639-645).Reagent also can be fixed on support indirectly, and such as, albumin A or G and their mutant can be fixed on support.Antibody is fixed on support by the interaction with albumin A or G.Recombinant protein combines by the acceptor that distinguished sequence and the distinguished sequence therewith on fixing support act on mutually and is fixed.Such as, part (as glutathione or nickel) can first by covalent attachment on support, the recombination fusion protein with distinguished sequence (as GST or 6xHis) is then fixed on by the interaction with part on identical support.Modification distinguished sequence and part can change their affinity thus remove fixing recombinant protein with suitable intensity.
Biomolecule (as antibody) is often fixed on support with the shape of round dot, and biomolecule also can be fixed on support with other shapes.Such as, antibody capable is fixed on support with rectangular shape (0.1 ~ 1 centimetre wide, 0.1 ~ 5 centimeter length).The point of the round dot that biomolecule is formed on support or other shapes is called " biomolecule point ", multiple " the biomolecule points " that support can have a kind of biomolecule formed, also can have multiple " biomolecule point " (each " biomolecule point " is made up of a kind of biomolecule) formed by biomolecule not of the same race, one or more " biomolecule point " forms " biomolecule dot matrix " or " biological molecular chip " on support.Biomolecule is fixed on position fixing in advance on support so that biomolecule to be detected identifies by this specific position.When biomolecule is protein, be exactly " protein array " or " protein-chip " or " Protein microdot arrays ", or " protein chip ".When biological reagent is antibody, be " antibody dot matrix " or " antibody chip ".
In the present invention, can utilize malleation, negative pressure or the combination of the two mode to drive biological reagent solution by micropore through micropore support.The method producing malleation or negative pressure has a lot, such as applies air pump, vacuum pump, reciprocating pump, peristaltic pump etc.
In a kind of method of the present invention, in multiple cyclic process, biological reagent solution passes support micropore with same direction at every turn.That is, biological reagent solution is collected through after support micropore, and then with identical direction through support (as shown in Figure 1, Figure 2 and Figure 3).In another kind of method of the present invention, biological reagent solution in several cycles, passes support micropore with different directions.That is, biological reagent solution through after support micropore, then passes support (Fig. 4 shown device can realize) by micropore.
Embodiment 1
As shown in Figure 2, a kind ofly promote the device that biological reagent is combined with biomolecule, comprise detection bodies 1, post cylinder 2, plunger 3 and hermetically-sealed construction 4, the side of detection bodies 1 is provided with sets off, for detection bodies 1 provides rigid support, set off as filter screen in the present embodiment, be made up, as PTFE film of the material of not having an effect with the reagent in solution; The pore size set off is generally 5 microns to 1 millimeter, and the pore size in the present embodiment is 100 microns; The thickness set off is generally several microns to several centimetres, and preferably from 0.1 millimeter to 10 millimeters, the thickness that sets off of the present embodiment is 0.2 millimeter.Post cylinder 2 is cylindrical hollow configuration, and the hollow structure that plunger 3 is arranged in post cylinder 2 can do piston movement, is provided with hermetically-sealed construction 4 between plunger 3 and post cylinder 2, and detection bodies 1 is fixed with post cylinder 2, is positioned at one end of post cylinder 2.The cavity be made up of detection bodies 1, post cylinder 2 and plunger 3 is for holding biological reagent solution.
Embodiment 2
The present embodiment employs the device being similar to and describing in embodiment 1, but the outside of the inner space of post cylinder 2 and plunger 3 is not right cylinder.In a device optimized, the inner space of post cylinder 2 and the outside of plunger 3 are Elliptic Cylinders.In the device that another is optimized, the inner space of post cylinder 2 and the outside of plunger 3 are rectangular parallelepipeds, and corner, usually with radian, seals to be formed between post cylinder, plunger.Post cylinder, the piston structure of Elliptic Cylinder and rectangular parallelepiped have special advantage in detection protein, particularly detect the biomolecule be fixed on non-circular micropore support.Such as, ProBloot membrane is not generally circular, but square.In some applications, ProBloot membrane is made into strip.Have Elliptic Cylinder and rectangular scapus cylinder, piston structure device can better, use minimum reagent to detect the protein on blotting membrane.
Embodiment 3
The present embodiment employs the device described in embodiment 1.In use, 5ng cardiac muscle troponin I (cTNI) antigen is fixed on formation two the biomolecule points on nitrocellulose filter, the nitrocellulose filter being fixed with cTNI antigen is detection bodies 1, and the micropore cross-sectional area in the present embodiment on nitrocellulose filter is 100 square millimeters.Two kinds of methods are adopted to detect cTNI: traditional method and method of the present invention.In traditional method, detection bodies and cTNI mouse monoclonal antibody solution are in room temperature in conjunction with 2 hours, and sheep anti mouse two anti-binding marked with HRP 1 hour, after adding HRP substrate, chemiluminescence signal G-Box cooled camera obtains.The antibody of same consumption is used to method of the present invention, uses figure tri-shown device, makes antibody-solutions pass nitrocellulose filter 20 times.As shown in Figure 6, the signal (left side) that the method that the signal ratio (the right) of method generation of the present invention is traditional produces is eager to excel at least 5-10 times.Traditional method needs to have come for several hours, and method of the present invention is at every turn through only needing 10 seconds, and whole testing process completed in 20 minutes.
Embodiment 4
As shown in Figure 3, a kind ofly promote the device that biological reagent is combined with biomolecule, comprise detection bodies 1, post cylinder 2, plunger 3 and hermetically-sealed construction 4, post cylinder 2 is cylindrical hollow configuration, the hollow structure that plunger 3 is arranged in post cylinder 2 can do piston movement, be provided with hermetically-sealed construction 4 between plunger 3 and post cylinder 2, detection bodies 1 is fixed on one end of plunger 3.The cavity be made up of detection bodies 1, post cylinder 2 and plunger 3 is for holding biological reagent solution.
Embodiment 5
The present embodiment employs the device executed and describe in example 4.The mouse monoclonal antibody (Fig. 5 A and 5B) that 5 kinds of about 1ng are different or rabbit many anti-(Fig. 5 C and 5D) are fixed on the detection bodies being formed with five biomolecule points on nitrocellulose filter; These detection bodies or be combined (Fig. 5 A and 5C) with sheep anti mouse/goat-anti rabbit that HRP marks by traditional method, or use method of the present invention, use the device executed and describe in example 4, be combined (Fig. 5 B and 5D) with sheep anti mouse/goat-anti rabbit that HRP marks.As shown in the figure, the signal (5A and 5C) that the method that the signal ratio (5B and 5D) of method generation of the present invention is traditional produces is eager to excel several times.Traditional method needs come for 1-2 hour, and method of the present invention completed in 10 minutes.
Embodiment 6
As shown in Figure 4, a kind ofly promote the device that biological reagent is combined with biomolecule, comprise detection bodies 1, post cylinder 2, plunger 3 and hermetically-sealed construction 4, the both sides of detection bodies 1 are provided with sets off as detection bodies 1 provides rigid support, sets off as filter screen in the present embodiment, be made up of the material of not having an effect with the reagent in solution, film as surface hydrophobicity process (refers to United States Patent (USP) 5,792,425 and 5,141,639); Set off the effect can playing buffer zone, ensure that solution is uniformly distributed on whole support.The size generally setting off micropore sets off greatly than the micropore of support, and the size setting off micropore is generally 5 microns to 1 millimeter, and the pore size in the present embodiment is 100 microns; The thickness set off is generally several microns to several centimetres, and preferably from 0.1 millimeter to 10 millimeters, the thickness that sets off of the present embodiment is 0.2 millimeter.Post cylinder 2 is cylindrical hollow configuration, the hollow structure that plunger 3 is arranged in post cylinder 2 can do piston movement, hermetically-sealed construction 4 is provided with between plunger 3 and post cylinder 2, plunger 3 is also hollow structure, detection bodies 1 is fixed on one end of plunger 3, be provided with in the hollow structure of plunger 3 between second plunger 5, second plunger 5 and plunger 3 and be provided with hermetically-sealed construction, the second plunger 5 can do piston movement in the hollow structure of plunger 3.The cavity be made up of detection bodies 1, post cylinder 2 and plunger 3 and the cavity that is made up of detection bodies 1, plunger 3 and the second plunger 5 are for holding biological reagent solution.
Embodiment 7
In the present embodiment, first use gel electrophoresis to be separated by SDS/PAGE by the protein sample (serum) containing PCT, then the protein delivery after separation is formed a ProBloot membrane to pvdf membrane.Using ProBloot membrane as micropore support.Use method of the present invention, cohesive process as shown in Figure 1, makes antibody-solutions under vacuum, through blotting membrane.In the process, antibody is effectively combined with antigen.This process is automatically repeated repeatedly to reach the maximum combined (Fig. 7, right) of antibody-antigene.The antibody-antigene binding signal obtained by traditional method relatively very weak (Fig. 7, left).Because this cyclic process is very quick, antibody antigen reaction required time can be reduced.Cleaning process is also complete with this similar procedure, effectively can remove the antibody of non-specific binding.Use antibody-antigene circular response technology antibody-antigene cohesive process can be shortened to a few minutes from several hours, thus greatly accelerate the speed of protein detection.Antibody-antigene circular response technology ensure that the maximum combined of antibody-antigene, thus improves the sensitivity of detection.
Claims (25)
1. promote the method that biological reagent is combined with biomolecule, its step comprises:
(1) biomolecule is fixed on the micropore support with micropore, forms one or more biomolecule point, obtain detection bodies;
(2) biological reagent is passed micropore support by micropore, in the process, biological reagent is combined with biomolecule;
(3) repeat step (2), make biological reagent repeatedly through micropore support.
2. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: the film that described micropore support is nitrocellulose membrane or nylon membrane or polyacrylamide film or is made up of cellulose nitrate, nylon, polyacrylamide derivative.
3. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: described detection bodies is that multiple biomolecule point is fixed on the biomolecule dot matrix that micropore support is formed.
4. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 3, it is characterized in that: described detection bodies has 2 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
5. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 3, it is characterized in that: described detection bodies has 5 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
6. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: described detection bodies is that then protein shift through electrophoretic separation and be fixed on the Western blotting film that micropore support is formed.
7. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: described biomolecule is antibody or described biological reagent is antibody.
8. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: in described step (2), biological reagent solution passes micropore support with single direction by micropore.
9. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: in described step (2), biological reagent solution passes micropore support with two-way by micropore.
10. a kind of method that biological reagent is combined with biomolecule that promotes according to claim 1, is characterized in that: repeat step (2) 2 to 100 times in described step (3).
11. a kind of methods that biological reagent is combined with biomolecule that promote according to claim 10, is characterized in that: repeat step (2) 5 to 30 times in described step (3).
12. 1 kinds promote the device that biological reagent is combined with biomolecule, it is characterized in that: comprise detection bodies (1), post cylinder (2) and plunger (3), described detection bodies is formed by micropore support and the biomolecule be fixed in the above, described post cylinder (2) is hollow structure, described plunger (3) is arranged in the hollow structure of post cylinder (2), forms hermetically-sealed construction (4) between plunger (3) and post cylinder (2).
13. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 12, it is characterized in that: the biomolecule dot matrix that described detection bodies (1) is formed for micropore support being fixed with multiple biomolecule point, have 2 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
14. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 12, it is characterized in that: the biomolecule dot matrix that described detection bodies (1) is formed for micropore support being fixed with multiple biomolecule point, have 5 biomolecule points at least, each biomolecule point to be fixed on micropore support at least one predetermined position.
15. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 12, is characterized in that: described biomolecule is antibody.
16. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 12, is characterized in that: described detection bodies is that then protein shift through electrophoretic separation and be fixed on the Western blotting film that micropore support is formed.
17. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 12, is characterized in that: described detection bodies (1) is fixed on one end of post cylinder (2).
18. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 12, it is characterized in that: the hollow structure that described post cylinder (2) is closed for one end, described plunger (3) is hollow structure, and detection bodies (1) is fixed on one end of plunger (3).
19. a kind of devices that biological reagent is combined with biomolecule that promote according to claim 18, it is characterized in that: in the hollow structure of described plunger (3), be provided with the second plunger (5), between the second plunger (5) and plunger (3), form hermetically-sealed construction.
20. promote according to a kind of in claim 12 ~ 19 described in any one the device that biological reagent is combined with biomolecule, it is characterized in that: the hollow structure of described plunger (3) and post cylinder (2) is elliptical cylinder-shape.
21. promote according to a kind of in claim 12 ~ 19 described in any one the device that biological reagent is combined with biomolecule, it is characterized in that: the hollow structure of plunger (3) and post cylinder (2) is cubic cylindricality, and corner is with radian.
22. promote according to a kind of in claim 12 ~ 19 described in any one the device that biological reagent is combined with biomolecule, it is characterized in that: also comprise one or more setting off, the side that setting off described at least one is arranged on detection bodies (1) directly contacts with detection bodies (1).
23. 1 kinds promote to it is characterized in that the multilevel device that biological reagent is combined with biomolecule: combined by a kind of device promoting biological reagent to be combined with biomolecule in a n claim 12 ~ 22 described in any one and obtain, wherein n >=2.
24. a kind of multilevel devices that biological reagent is combined with biomolecule that promote according to claim 23, is characterized in that: 2≤n≤384.
25. a kind of multilevel devices that biological reagent is combined with biomolecule that promote according to claim 24, is characterized in that: 4≤n≤96.
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| CN109929735A (en) * | 2019-04-25 | 2019-06-25 | 湖南工业大学 | A kind of plunger type detection of nucleic acids integration cartridge and its detection method |
| WO2021169089A1 (en) * | 2020-02-25 | 2021-09-02 | 中山大学 | Microfluidic chip-based rapid immunoassay method |
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