CN104297491A - Protein chromatographic electrophoresis and in-situ chemical imprinting and immune imaging device and method - Google Patents

Abstract

The invention discloses protein chromatographic electrophoresis and an in-situ chemical imprinting and immune imaging device and method. The device comprises chromatographic electrophoresis modules, first solution tanks, a chromatographic electrophoresis separation plate with multiple electrophoresis channels, active chromatographic media and an LED fluorescent light source, wherein two ends of the chromatographic electrophoresis separation plate are respectively connected with the first solution tank and the chromatographic electrophoresis module; the active chromatographic media are contained in each electrophoresis channel and comprise microparticles which are composed of surface-activated ion exchange resin, affinity chromatography fillers and reverse phase chromatography fillers or are non-porous silica microparticles or porous glass microparticles or microparticles made from high molecular materials; and diazirine coupling molecules are dielectrically bonded on the surfaces of the microparticles. A whole process of various chromatographic electrophoresis separation and in-situ chemical imprinting and immune imaging of protein can be finished within two hours. The device and the method disclosed by the invention are high in sensitivity, distinguishability and repeatability while the characteristics of traditional protein chromatography and electrophoresis are maintained.

Description

Protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging apparatus and method

Technical field

The present invention relates to medical science, particularly relate to a kind of protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging apparatus and method.

Background technology

Traditional Western Immuno imaging, or claim Western, the polyacrylamide gel electrophoresis comprising albumen is separated, the transfer trace of albumen, the immune detection relying on antigen antibody interaction and protein immunization imaging analysis four important technologies compositions in gel, is protein analysis method the most frequently used in modern life science and clinic study.Tradition Western analyzes to be needed successively to use multiple instrument and equipment, complex operation step, technical requirement is high, the two day time of usual needs just can complete, and cannot realize robotization, and the result obtained usually because of instrument, reagent, method and operator difference and change.Be made up of four complicated technology because traditional Western analyzes, usually need four kinds of instrument and equipments: protein electrophoresis instrument, albumen transferring film instrument, protein immunoblot instrument and protein immunization imager.The wherein polyacrylamide gel electrophoresis complicated operation of albumen, cannot to be imaged on automatic mode first-class compatible with protein delivery trace, protein immunization.So, go back four complex technologys operations that neither one instrument and equipment can complete traditional Western analysis.Form each individual event instrument and equipment situation that traditional Western analyzes as follows: 1) traditional protein electrophoresis apparatus: namely albumen by size, the character such as electric charge separately.Traditional protein electrophoresis has SDS-PAGE vertical electrophoresis apparatus and isoelectric focusing electrophoresis instrument.Wherein SDS-PAGE vertical electrophoresis apparatus is very general, and isoelectric focusing electrophoresis instrument is with solid phase isoelectric focusing adhesive tape (IPG) electrophoresis system of U.S. Bio-rad Bole and U.S. Thermo thermoelectricity, and the offgel Liquid isoelectric focusing electrophoresis apparatus of Agilent Agilent is main.The critical defect of these two kinds of electrophoresis is all SDS-PAGE vertical electrophoresis apparatus, owing to using polyacrylamide gel medium, all cannot accomplish full-automation; All isoelectric focusing electrophoresises, owing to using IPG adhesive tape, all cannot realize real time imagery.2) namely albumen transferring film instrument is transferred to the albumen in polyacrylamide gel on nitrocellulose filter or PVDF film by its distribution exactly, forms Western blot.Although the transfer liquid that a lot of professional person still continues to use traditional-handwork inhales profit method, electrophoretic transfer is widely used.Electrophoretic transfer must use protein delivery trace instrument.The market of current protein electrophoresis transfer instrument is more chaotic, does not have quality standard.3) protein immunoblot instrument: namely complete antigen antibody interaction on nitrocellulose filter or PVDF film.Owing to will use two kinds of antibody, and will complete protection and the washing of film repeatedly, automation process is more loaded down with trivial details.But process is loaded down with trivial details just, people are very high to the demand of robotization.Over the years, full-automatic protein immunoblot instrument is proposed both at home and abroad respectively.The XD236 automatic westem blot instrument that the B20 type full-automatic Western blotting instrument of such as Britain Bee Robotics, ProfiBlot48, AutoBlot36 of U.S. TECAN and domestic upper Hisoon reach.But due to expensive, it is not very general for using.4) immune imaging instrument: namely nitrocellulose filter or PVDF film developing the color with the interactional albumen of antibody and in situ imaging.According to the difference of antibody labeling method with colour developing principle, immune imaging instrument can be divided into chemiluminescence imaging instrument and phosphorimager.Wherein, with the Odyssey near-infrared laser imager of LICOR, the typhoon Multifunction fluorescent scanner of THERMO and the LAS-4000 fluorescence of FUJI and chemiluminescence imaging instrument for representative.At present, mainly based on import equipment.So, first tradition Western analyzes exists a lot of problem, and traditional Western analyzes to be needed successively to use multiple instrument and equipment, complex operation step, and technical requirement is high, the two day time of usual needs just can complete whole analysis, is unable to catch up with the paces of Development of Modern Science; And the result that traditional Western analyzes usually changes because of the difference of instrument, reagent, method and operator, can not meet the requirement to protein quantification in biomedicine; Four technology ingredients that tradition Western analyzes cannot be compatible on automatic mode, goes back all processes that neither one instrument and equipment can complete traditional Western analysis at present; Finally, Western analytical approach has demand widely clinically, but present operation is all open, and cannot avoid operating personnel and the contact detecting sample, clinical position may be subject to the threat of infectiousness sample.

Be electrophoretic medium with polyacrylamide gel in analyzing relative to traditional Western, Capillary Electrophoresis is is split tunnel with kapillary, take free solution as the trace of albumin separation method of medium.Usually, Capillary Electrophoresis uses elasticity (polyimide) the coating fused quartz kapillary that internal diameter is 25-100 μm, except electrophoretic buffer, does not need solid dielectric.So volume is little, rapid heat dissipation, can bear high electric field (100-1000 V/cm), easily realize robotization.Recently, the full-automatic protein immunization analytical instrument based on Capillary Electrophoresis is come out, and the albumen Western that can complete trace, even tens cells analyzes.The advantage that Capillary Electrophoresis protein immunization is analyzed: 1) efficient, plate number is between 105-107 sheet/m; 2) quick, generally in tens minutes, complete separation; 3) trace, the sample volume needed for sample introduction is for receiving upgrading; 4) robotization is the separation method that current automaticity is higher.The shortcoming of Capillary Electrophoresis protein immunization analysis is 1) because sample size is few, thus preparative capacibility is poor, when particularly needing further mass spectrophotometry, often cannot reach required amount; 2) because capillary diameter is little, make light path too short, detect or image sensitivity lower (as ultraviolet absorption spectroscopy); 3) because electroosmosis is different because of sample composition, the poor reproducibility of Protein Separation; 4) instrument and equipment is expensive, and special kapillary consumables cost is too high, limits applying of it.Under contrast, the advantage that traditional Western analyzes just Capillary Electrophoresis protein immunization analytic system lacks, and the advantage of Capillary Electrophoresis protein immunization analytic system just traditional Western to analyze not available for.So optimal system in conjunction with both advantages, should overcome both shortcomings simultaneously.

Summary of the invention

Technical matters to be solved by this invention is, overcomes the shortcoming of prior art, provide a kind of can realize full-automatic process protein chromatographic electrophoresis and in-situ chemical trace and immune imaging apparatus and method.

In order to solve above technical matters, the invention provides protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging device, comprise hyperchannel chromatoelectrophoresis module, first solution tank, adopt the hyperchannel chromatoelectrophoresis separating plate of multiple track, active chromatography media and LED fluorescence light source, the liquid-phase outlet of described hyperchannel chromatoelectrophoresis module is connected to one end of hyperchannel chromatoelectrophoresis separating plate, the other end of hyperchannel chromatoelectrophoresis separating plate is connected to the first solution tank, described active chromatography media is placed in the track of chromatoelectrophoresis separating plate, described LED fluorescence light source is for irradiating described hyperchannel chromatoelectrophoresis separating plate, described active chromatography media includes particulate, described particulate is surface active ion exchange resin, affinity chromatography filler, reversed phase chromatography filler or nonporous silica silicon particle or fritted glass particulate or macromolecular material particulate, be situated between altogether on the surface of described particulate and be combined with two a word used for translations and suck class coupling molecule.

Being further defined to of technical solution of the present invention, described hyperchannel chromatoelectrophoresis module is hyperchannel chromatoelectrophoresis handover module, described solution tank is hyperchannel chromatoelectrophoresis handover module, described hyperchannel chromatoelectrophoresis handover module comprises electrophoresis tank, multi-channel loading hole, multi-channel switch and multiple chromatography liquid channel respectively, positive electrode and negative electrode is had respectively in described electrophoresis tank, multi-channel loading hole is connected with electrophoresis tank, multi-channel loading aperture multi-channel switch is connected with track respectively, and described chromatography liquid channel is connected with track respectively.

Being further defined to of technical solution of the present invention, described multi-channel switch is hyperchannel pneumatic switch, a pneumatic switch groove is had in the bottom of described each well, a pneumatic switch film is had in described each pneumatic switch groove, pneumatic switch groove is separated into two passages, described each well is connected with chromatography liquid channel through a passage of pneumatic switch groove, another passage is connected with pneumatic switch gas access, when pneumatic switch film drives from pneumatic switch gas access input malleation or negative pressure, pneumatic switch film opens and closes the path between each well and each chromatography liquid channel respectively.

Being further defined to of technical solution of the present invention, also comprises micro-liquid phase driving pump, and described micro-liquid phase driving pump is placed in porch and the exit of chromatography liquid channel.

Being further defined to of technical solution of the present invention, described particulate to be diameter the be microballoon of 10 microns to 100 microns.

Being further defined to of technical solution of the present invention, described hyperchannel chromatoelectrophoresis separating plate is bonded containing multiple 4-12 mm wide, the glass of 0.1-1 millimeter deep trouth, organic glass or transparent polymer material plate face-to-face by two pieces, form the hyperchannel chromatoelectrophoresis separating tank of multiple 4-12 mm wide, 0.2-2 millimeters deep, two ends have fixing filler to be sealed by swimming lane mouth, form multiple track.

Based on above technical matters, the present invention also provides protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging method, comprise the steps: 1) utilize the active chromatography media in hyperchannel chromatoelectrophoresis separating plate to carry out chromatoelectrophoresis separation, described active chromatography media includes particulate, described particulate is surface active ion exchange resin, affinity chromatography filler, reversed phase chromatography filler or nonporous silica silicon particle or fritted glass particulate or macromolecular material particulate, 2) in described active chromatography media, particulate is its surperficial Jie be altogether combined with the particulate that two a word used for translations suck class coupling molecule, described particulate is penetrated with the illumination of specific wavelength, two a word used for translations activating described chromatoelectrophoresis dielectric surface suck quasi-molecule, two a word used for translations of activation are made to suck the protein generation chemical coupling of quasi-molecule and surrounding, be covalently bound to described microparticle surfaces, protein is made to produce in-situ chemical trace at described microparticle surfaces, 3) first antibody is transported to the in-situ chemical trace protein interaction with generation chemical coupling in hyperchannel chromatoelectrophoresis separating plate, 4) wash unreacted first antibody off, second antibody is transported in hyperchannel chromatoelectrophoresis separating plate and interacts with first antibody, when second antibody is fluorescently-labeled, the surface of second antibody is irradiated with LED fluorescence light source, fluorescence excitation mark is luminous, direct observation or by be positioned at the camera with respective filter directly over hyperchannel chromatoelectrophoresis separating plate or pick-up lens is observed and record protein spring up image, complete protein fluorescence immune imaging, if use enzyme to join the second antibody of mark, the luminous substrate of enzyme connection mark is transported in hyperchannel chromatoelectrophoresis separating tank and joins label with enzyme and to interact and luminous, direct observation or by be positioned at camera directly over hyperchannel chromatoelectrophoresis separating plate or pick-up lens is observed and record protein spring up image, complete the imaging of protein chemistry electrochemiluminescent immunoassay.

Being further defined to of technical solution of the present invention, described step 1 adopts protein chromatographic electrophoresis as claimed in claim 2 and in-situ chemical trace thereof and immune imaging device, concrete steps are as follows: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) open multi-channel switch, path between described multi-channel loading hole and described track is opened, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, protein example in described protein well is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated.

Being further defined to of technical solution of the present invention, described step 1 adopts protein chromatographic electrophoresis as claimed in claim 2 and in-situ chemical trace thereof and immune imaging device, concrete steps are as follows: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) open multi-channel switch, path between described multi-channel loading hole and described track is opened, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, the protein example in described protein well is made to move to the reference position of described hyperchannel chromatoelectrophoresis separating tank in the electric field, 1c) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, meanwhile, by described chromatography liquid channel, required chromatographic flow is inputted hyperchannel chromatoelectrophoresis separating tank mutually, protein example is separated in described chromatoelectrophoresis medium.

Being further defined to of technical solution of the present invention, described step 4) concrete steps are as follows: 4) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, input damping fluid successively by described chromatography liquid channel, one or more can identify the second antibody of the first antibody of some protein, fluorescence or enzyme connection mark.

Based on above technical matters, the present invention also provides multifunctional protein chromatoelectrophoresis and recovery method thereof, protein chromatographic electrophoresis according to claim 2 and in-situ chemical trace thereof and immune imaging device, comprise the steps: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) open multi-channel switch, path between described multi-channel loading hole and described track is opened, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, protein example in described protein well is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated, 1c) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, meanwhile, by described chromatography liquid channel, required chromatographic flow is inputted hyperchannel chromatoelectrophoresis separating tank mutually, make the protein example after separation be exported by the liquid-phase outlet of right hyperchannel chromatoelectrophoresis handover module and be collected respectively with gatherer sequentially.

Based on above technical matters, the present invention also provides multifunctional protein chromatoelectrophoresis method, protein chromatographic electrophoresis according to claim 3 and in-situ chemical trace thereof and immune imaging device, comprise the steps: 1a) regulate gaseous tension in described pneumatic switch groove, under making described pneumatic switch film be in barotropic state, close the path between described multi-channel loading hole and described chromatography liquid channel, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) regulate gaseous tension in described pneumatic switch groove, under making described pneumatic switch film be in negative pressure state, open the path between described multi-channel loading hole and described chromatography liquid channel, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, protein example in described protein well is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated.

The invention has the beneficial effects as follows: one of innovative point of this patent is exactly unique chromatoelectrophoresis medium and replaces polyacrylamide gel with this chromatoelectrophoresis medium, make protein can complete separation in chromatoelectrophoresis medium on the one hand, the preparation making it be provided with traditional protein electrophoresis and the feature generally used, be that its surface is wrapped in one deck chemical coupling material on the other hand, can activate under the LED light induction of special wavelength, there is coupling reaction in position with albumen, albumen is covalently bound on chromatoelectrophoresis medium.Complete the three-dimensional printing process of original position of traditional protein electrophoresis.These innovative points are all that Capillary Electrophoresis cannot complete, and are the novelty transformations analyzed Capillary Electrophoresis related immune.These are condition easily, conveniently realizes the complete in changing of WESTERN instrument.

The present invention combines traditional liquid chromatography (LC) electrophoresis, chemical quick coupling characteristic effectively, tradition Western analyzes the advantage of three kinds of technology, have developed a kind of novel chromatoelectrophoresis (electrochromatography, electro chromatography, the electrophoresis that finger protein carries out in liquid chromatography (LC) medium, only use in Capillary Electrophoresis at present), conventional Western can be made analysis automated, thus set up full-automatic Western instrument.First, because namely full-automatic Western instrument has preparation and the imaging advantage of traditional Western, have again the robotization of Capillary Electrophoresis and express-analysis function, it is analyzed than traditional Western has more practical and using value widely.Full-automatic Western instrument drastically increases the speed that Western analyzes, and namely saves time, saves manpower again.In addition, do not need senior technician, artificial operate miss can be reduced, promoting than being easier to, reducing the use threshold of Western technology.The biological safety of full-automatic Western instrument: owing to can not avoid the contact of operating personnel and albumen sample in traditional Western analysis, especially when analyzing the clinical samples of the disease that is infectious, operating personnel are subject to potential threat unavoidably.The use of full-automatic Western instrument can protect operating personnel from the threat of the potential infectiousness sample of tool to greatest extent, eliminates biological safety hidden danger.The pathogen that full-automatic Western instrument can be used to detect comprises 1) virus: HIV, HCV, HEV, cytomegalovirus, herpes simplex virus, parvovirus, rubella virus, EB virus etc.; 2) bacterium: conveyor screw, Brucella, helicobacter pylori, syphilis, Yersinia; 3) parasite: toxoplasmosis etc.

The significance of full-automatic Western instrument: full-automatic Western instrument can improve the ability of life science and clinical diagnose, all has huge application prospect in life science, clinical diagnose and disease detection control etc.In clinical diagnosis, the appearance of full-automatic Western instrument will make Western analytical technology be adopted widely.In life science, because Western confirms the requisite standard methods such as protein expression, molecular weight, isoelectric point and protein-interacting, the use of full-automatic Western instrument will greatly improve the speed and quality of publishing an article.Full-automatic Western instrument is applicable to university, medical research unit, hospital laboratory, Center for Disease Control, inspection and quarantine system, centralab etc.So the development of full-automatic Western instrument has very high practicality, significant to the development of modern life science.

Accompanying drawing explanation

Fig. 1 is present protein chromatoelectrophoresis and in-situ chemical trace thereof and immune imaging device cross-sectional schematic;

Fig. 2 is the chromatoelectrophoresis handover module cut-away illustration of present protein chromatoelectrophoresis and in-situ chemical trace and immune imaging device;

Fig. 3 is the chromatoelectrophoresis separating plate schematic perspective view of present protein chromatoelectrophoresis and in-situ chemical trace and immune imaging device;

In figure, 1 chromatoelectrophoresis handover module, 2 chromatoelectrophoresis separating plates, 3 chromatoelectrophoresis media, 4LED fluorescence light source, 5 micro-liquid phase driving pumps, 6 iontophoretic electrodes, 7 electrophoresis tanks, 8 wells, 9 pneumatic switch air intakes, 10 pneumatic switch films, 11 tracks.

Embodiment

embodiment one

One can realize protein chromatographic electrophoresis and in-situ chemical marking medium thereof, comprise chromatography media, described chromatography media is the ion exchange resin of 10-100 micron diameter, various affinity chromatography medium and reversed phase chromatography microballoon, described chromatography media is filled in the track 11 of described hyperchannel chromatoelectrophoresis separating plate 2, when being filled with different chromatography medias in described track 11, add chromatography liquid phase (mobile phase) again, protein will be separated with hydrophobicity etc. according to CHARGE DISTRIBUTION.Microsphere surface is situated between altogether and is combined with two a word used for translations and suckes class coupling molecule.Described microballoon is through sucking compounds coupling with two a word used for translations, form two unique a word used for translations and suck compound parcel microballoon, when penetrating described microballoon with the illumination of specific wavelength, two a word used for translations activating described chromatographic medium surface suck quasi-molecule, two a word used for translations of activation are made to suck the protein generation chemical coupling of quasi-molecule and surrounding, be covalently bound to described microsphere surface, make protein produce in-situ chemical trace at described microsphere surface.

embodiment two

A kind of protein chromatographic electrophoresis and in-situ chemical marking medium thereof, comprise electrophoretic medium, described electrophoretic medium comprises particulate, and the diameter of described particulate is 20-50 micron non-porous silicas microballoon or fritted glass microballoon or other macromolecular material microballoons.Described microballoon is through sucking compounds coupling with two a word used for translations, form two unique a word used for translations and suck compound parcel microballoon, suck compound parcel microballoon with described two a word used for translations and replace conventional polypropylene acrylamide gel as chromatoelectrophoresis medium 3, electrophoretic separation operation can be carried out to protein.Be filled in the track 11 of described hyperchannel chromatoelectrophoresis separating plate 2 with silicon dioxide microsphere or fritted glass microballoon or other macromolecular material microballoon, in addition, when being filled with polysaccharide or ampholyte etc. medium in described track 11, protein is subject to the effect of electrophoretic buffer composition and silicon dioxide microsphere surface molecular in electrophoretic medium, make the swimming of different proteins in described chromatoelectrophoresis medium 3 by its molecular characterization, as the characteristic such as molecular weight and isoelectric point, reach the object that chromatoelectrophoresis is separated.

Described microballoon is through sucking compounds coupling with two a word used for translations, form two unique a word used for translations and suck compound parcel microballoon, when penetrating described particulate with the illumination of specific wavelength, two a word used for translations activating described chromatographic medium surface suck quasi-molecule, two a word used for translations of activation are made to suck the protein generation chemical coupling of quasi-molecule and surrounding, be covalently bound to described microparticle surfaces, make protein produce in-situ chemical trace at described microparticle surfaces.

embodiment three

As shown in Figure 1 to Figure 2, a kind of protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging device, be commonly called as full-automatic Western instrument, it comprise hyperchannel chromatoelectrophoresis module, hyperchannel chromatoelectrophoresis separating plate 2, be placed in the LED fluorescence light source 4 immediately below hyperchannel chromatoelectrophoresis separating plate 2 and be placed in directly over hyperchannel chromatoelectrophoresis separating plate 2 according to machine machine or pick-up lens.Protein, electrophoretic buffer and chromatographic solution etc. are transported in hyperchannel chromatoelectrophoresis separating plate 2 and carry out separating treatment by hyperchannel chromatoelectrophoresis module in charge, and described hyperchannel chromatoelectrophoresis separating plate 2 holds the electrophoresis chromatography media carrying out being separated.Described LED fluorescence light source 4 can provide the monochromatic source of exciting light chemical reaction and the required wavelength of fluorescence excitation labelled antibody colour developing.Wherein, wherein LED fluorescence light source 4 forms pointolite matrix by the LED of multiple specific wavelength, ensures that two a word used for translations on described chromatoelectrophoresis medium 3 surface are suck quasi-molecule molecule and fully activated.Described photograph or pick-up lens are used for observing and record protein and spring up image.Include particulate in described electrophoresis chromatography media, being situated between altogether on the surface of described particulate is combined with two a word used for translations and suckes class coupling molecule.The diameter of described particulate is the microballoon of 10 microns to 100 microns.Described particulate can be ion exchange resin, affinity chromatography medium, reversed phase chromatographic medium particulate or nonporous silica silicon particle or fritted glass particulate or other macromolecular material microballoon.Hyperchannel chromatoelectrophoresis module comprises two chromatoelectrophoresis handover modules 1, be respectively negative pole chromatoelectrophoresis handover module 1 and positive pole chromatoelectrophoresis handover module 1, negative pole chromatoelectrophoresis handover module 1 and positive pole chromatoelectrophoresis handover module 1 are placed in the left and right sides of track 11 respectively.Chromatoelectrophoresis handover module 1 and described hyperchannel chromatoelectrophoresis separating plate 2 on a plane, wherein at least one chromatoelectrophoresis handover module 1 can slidably reciprocate in plane realize chromatoelectrophoresis handover module 1 and track 11 be separated merging; When two chromatoelectrophoresis handover modules 1 all separate, hyperchannel chromatoelectrophoresis separating plate 2 can freely take out or put back to; When two chromatoelectrophoresis handover modules 1 merge, multiple outlets of chromatoelectrophoresis handover module 1 are alignd with the track 11 of hyperchannel chromatoelectrophoresis separating plate 2 respectively; Chromatoelectrophoresis handover module 1 and hyperchannel chromatoelectrophoresis separating plate 2 binding site have rubber ring or film, to guarantee that track 11 is unimpeded but tight water-tight gentle with the external world.As shown in Figure 3, hyperchannel chromatoelectrophoresis separating plate 2 is multiple tracks 11.Just can check when carrying out sample survey so simultaneously and organize sample more, described track 11 is that upper and lower two blocks of glass with cutting or poly (methyl methacrylate) plate fasten and form, particularly, adopt two pieces and be carved with 0.1-1 millimeters deep, 6-12 mm wide, the glass of 60-120 millimeters long cutting or transparent acrylic material, 0.2-2 millimeters thick is formed after face-to-face bonding, 6-12 mm wide, the track 11 that can hold chromatography electrophoretic medium 3 of 60-120 millimeters long, two ends have fixing filler to be sealed by swimming lane mouth, prevent particulate from flowing out while allowing liquid phase flowing.Described fixing filler can be shaping in the following way: add a small amount of 20 micron silica microballoons and formamide in formed chromatoelectrophoresis swimming lane 11 one end and at high temperature make its coupling solidify, forming the silicon dioxide microsphere cured layer of about 2-5 millimeters thick.Described silicon dioxide microsphere cured layer will allow that liquid passes through, but can stop the outflow of described chromatoelectrophoresis medium 3.Equally also can adopt ion exchange resin or reversed phase chromatography microballoon and formamide and at high temperature make its coupling solidify, forming ion exchange resin or the reversed phase chromatography microballoon cured layer of about 2-5 millimeters thick.As shown in Figure 2, described hyperchannel chromatoelectrophoresis handover module 1 comprises electrophoresis tank 7, multi-channel loading hole 8, hyperchannel pneumatic switch and multiple chromatography liquid channel respectively, positive electrode and negative electrode is had respectively in described electrophoresis tank 7, multi-channel loading hole 8 is connected with electrophoresis tank 7, multi-channel loading hole 8 is connected with chromatography liquid channel respectively through hyperchannel pneumatic switch, and described chromatography liquid channel is connected with track 11 respectively.Micro-liquid phase driving pump 5 is equipped with in entrance or the exit of described multiple chromatography liquid channel.A pneumatic switch groove is had in the bottom of described each well 8, a pneumatic switch film 10 is had in described each pneumatic switch groove, pneumatic switch groove is separated into two passages, described each well 8 is connected with chromatography liquid channel through a passage of pneumatic switch groove, another passage is connected with pneumatic switch air intake 9, pneumatic switch air intake 9 is connected with inlet valve and air outlet valve, when pneumatic switch film 10 inputs malleation or negative pressure driving from pneumatic switch air intake 9, pneumatic switch film 10 opens and closes the path between each well 8 and each chromatography liquid channel respectively.For carrying out electrophoresis and chromatography.

For foregoing invention, its technical characterstic is to carry out protein chromatographic and performance liquid electrophoresis two kinds of operating modes.And described chromatoelectrophoresis modular converter can switch between protein chromatographic and protein electrophorese two kinds of operating modes, reach the object of protein chromatographic and protein electrophorese two kinds operation.For above-mentioned chromatoelectrophoresis handover module 1, when being in electrophoretic, the pneumatic switch air intake 9 of described chromatoelectrophoresis handover module 1 is in negative pressure, pneumatic switch film 10 is in opening, positive and negative iontophoretic electrode 6 can pass through electrophoretic buffer, be connected with the track 11 of pneumatic switch membrane 10 with hyperchannel chromatoelectrophoresis separating plate 2 via well 8, form electrophoresis loop.(or: enter well 8 by the electrophoresis tank 7 cataphoresis electrode 6 of negative pole chromatoelectrophoresis handover module 1 and enter pneumatic switch film 10, enter the track 11 of hyperchannel chromatoelectrophoresis separating plate 2, then enter the positive pole of electrophoresis tank 7 of positive pole chromatoelectrophoresis handover module 1.) when being in chromatography pattern, the pneumatic switch air intake 9 of described chromatoelectrophoresis handover module 1 is in normal pressure usually, pneumatic switch film 10 is in closed condition, now pneumatic switch film 10 turns off, hyperchannel chromatoelectrophoresis separating plate 2 is separated by pneumatic switch film 10 with positive and negative iontophoretic electrode 6 and electrophoretic buffer, electrophoresis loop is interrupted, and micro-liquid phase driving pump 5 is connected with the track 11 of hyperchannel chromatoelectrophoresis separating plate 2 simultaneously, composition liquid chromatography (LC) loop.The solution of needs and reagent can be transported in track 11 by described micro-liquid phase driving pump 5.

When unlatching multi-channel switch, path between described multi-channel loading hole 8 and described track 11 is opened, now under electrophoretic, under the effect of electric field, protein can enter the track 11 of hyperchannel chromatoelectrophoresis separating plate 2 from chromatoelectrophoresis handover module 1 and is separated in chromatoelectrophoresis medium 3.When closedown multi-channel switch, make the path blockade between described multi-channel loading hole 8 and described track 11, now under chromatography pattern, solution and protein enter hyperchannel chromatoelectrophoresis separating plate 2 from chromatoelectrophoresis handover module 1 and carry out chromatography to albumen chromatoelectrophoresis media 3 under the promotion of micro-liquid phase driving pump 5, and can replace liquid phase part.It should be noted that: under chromatography pattern, well 8 does not communicate with track 11, track 11 is entered into by pneumatic switch film 10 in order to make protein, then frequently should open pneumatic switch film 10, simultaneously under the micro-liquid phase driving pump 5 of negative pole or positive pole chromatoelectrophoresis handover module 1 drives, protein is swum to positive pole by negative pole.In order to realize the chromatography of protein, can also adopt alternatively: the reference position namely by electrophoretic, protein being transported to hyperchannel chromatoelectrophoresis separating tank in advance, concrete steps are: regulate gaseous tension in described pneumatic switch groove, under making described pneumatic switch film 10 be in negative pressure state, open the path between described multi-channel loading hole 8 and described chromatography liquid channel, on the hyperchannel chromatoelectrophoresis handover module 1 of two, described left and right, add suitable voltage simultaneously, the protein example in described protein well 8 is made to move to the reference position of described hyperchannel chromatoelectrophoresis separating tank in the electric field.When for chromatography mode of operation, under the driving of micro-liquid phase driving pump 5, the first antibody and second antibody and enzyme with generation in-situ chemical trace protein generation immune imaging is joined the luminous substrate marked and send in hyperchannel chromatoelectrophoresis separating plate 2.

For above-described embodiment, electrophoresis tank 7 can be one also can be multiple, and it also can be connected with track 11 separately in addition, as long as protein is just passable through the conductive path of electrophoresis tank 7.It can arrange accumulator tank on the path of electrophoresis tank 7, the output terminal of protein well 8 is connected with accumulator tank, can ensures completing of electrophoresis process.Described LED fluorescence light source 4 also can be placed in the top of hyperchannel chromatoelectrophoresis separating plate 2, as long as or other position can be irradiated to particulate in separate tank.

The hyperchannel chromatoelectrophoresis separating plate 2 of above-mentioned track 11 is a kind of electrophoretic apparatuss that can use described chromatoelectrophoresis medium 3, its effect to allow described chromatoelectrophoresis medium 3 form physical size as conventional polypropylene acrylamide gel and shape, guarantee that protein effectively can be separated under chromatography and electrophoresis two kinds of patterns, guarantee that protein can complete the operations such as in-situ chemical trace and immune imaging in chromatoelectrophoresis medium 3 simultaneously.

The present invention also comprises a kind of chromatoelectrophoresis medium 3 being used for replacing conventional polypropylene acrylamide gel.The technical requirement of described chromatoelectrophoresis medium 3 comprises 1) replace conventional polypropylene acrylamide gel but still can protein electrophorese separation be carried out, 2) liquid flow velocity of 1-2000 mul/min is allowed, ensure damping fluid and the flowing of solution in chromatoelectrophoresis medium 3,3) there is the chemical reaction characteristic of coupling protein matter, namely react with peripheral protein matter generation chemical coupling under the activation of light or chemical reagent.

embodiment four

Be with embodiment three difference, chromatoelectrophoresis handover module 1 only has one, and it is positioned at the left side of hyperchannel chromatoelectrophoresis separating plate 2, and right side only has the first solution tank, in described first solution tank, have positive electrode.First solution tank is connected with track 11 by chromatography liquid channel, in the electrophoresis tank 7 of described chromatoelectrophoresis handover module 1, have negative electrode, and micro-liquid phase driving pump 5 described is in addition one, and it is positioned at entrance or the outlet of chromatography liquid channel.

embodiment five

A kind of protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging device, comprise hyperchannel chromatoelectrophoresis module, first solution tank, adopt the hyperchannel chromatoelectrophoresis separating plate 2 of multiple track 11, active chromatography media and LED fluorescence light source 4, the liquid-phase outlet of described hyperchannel chromatoelectrophoresis module is connected to one end of hyperchannel chromatoelectrophoresis separating plate 2, the other end of hyperchannel chromatoelectrophoresis separating plate 2 is connected to the first solution tank, described active chromatography media is placed in the track 11 of chromatoelectrophoresis separating plate, described LED fluorescence light source 4 is for irradiating described hyperchannel chromatoelectrophoresis separating plate 2.Described hyperchannel chromatoelectrophoresis module is hyperchannel chromatoelectrophoresis handover module 1, described hyperchannel chromatoelectrophoresis handover module 1 comprises the second solution tank and multi-channel loading hole 8 respectively, described second solution tank is connected with track 11, described multi-channel loading hole 8 is connected with track 11, in first, second solution tank described, have positive electrode and negative electrode respectively.This structure also can realize chromatography pattern and electrophoretic, and under chromatography pattern, the luminous substrate of solution, first antibody and second antibody and enzyme connection mark also can shift to another side from hyperchannel chromatoelectrophoresis separating plate 2 from the second solution tank by gravity.

For above-described embodiment, in fact chromatoelectrophoresis module can adopt various structure, as adopted independently chromatography conveyor module or adopt independently electrophoresis conveyor module, well 8 can adopt separately independently structure to be connected with track 11, and first, second solution tank is then connected with track 11 by another passage.Well 8 also can not independently exist, but directly carries out protein application of sample by solution tank.And these are just simply enumerated, in fact also have a variety of structure that other realizes, these structures should not depart from protection scope of the present invention.

Above-mentioned independently chromatography conveyor module can adopt following structure: described hyperchannel chromatoelectrophoresis module comprises solution tank and multi-channel loading hole 8, and described solution tank is connected with track 11, and described multi-channel loading hole 8 is connected with track 11.This hyperchannel chromatoelectrophoresis module is two, and the one end being positioned at hyperchannel chromatoelectrophoresis separating plate 2, another is positioned at the other end of hyperchannel chromatoelectrophoresis separating plate 2.

Above-mentioned independently electrophoresis conveyor module can adopt following structure: described hyperchannel chromatoelectrophoresis module comprises solution tank and multi-channel loading hole 8, and described solution tank is connected with well 8, and described multi-channel loading hole 8 is connected with track 11.This hyperchannel chromatoelectrophoresis module is two, and the one end being positioned at hyperchannel chromatoelectrophoresis separating plate 2, another is positioned at the other end of hyperchannel chromatoelectrophoresis separating plate 2.The solution tank of one end and the other end of being arranged in hyperchannel chromatoelectrophoresis separating plate 2 has negative electrode and positive electrode respectively.

embodiment six

Be with embodiment three difference: described hyperchannel chromatoelectrophoresis module is hyperchannel chromatoelectrophoresis handover module 1, described hyperchannel chromatoelectrophoresis handover module 1 comprises electrophoresis tank 7, multi-channel loading hole 8, multi-channel switch and multiple chromatography liquid channel respectively, positive electrode or negative electrode is had respectively in described electrophoresis tank 7, multi-channel loading hole 8 is connected with electrophoresis tank 7, multi-channel loading hole 8 is connected with track 11 respectively through multi-channel switch, and described chromatography liquid channel is connected with track 11 respectively.Described multi-channel switch can be various forms of switch, as mechanical switch etc.

embodiment seven

A kind of protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging method, comprise the steps: 1) utilize the active chromatography media in hyperchannel chromatoelectrophoresis separating plate 2 to carry out electrophoresis chromatography, described active chromatography media includes particulate, described particulate is surface active ion exchange resin, affinity chromatography filler, reversed phase chromatography filler or nonporous silica silicon particle or fritted glass particulate or macromolecular material particulate, when for nonporous silica silicon particle or fritted glass particulate or macromolecular material particulate, described step 1 adopts protein chromatographic electrophoresis as described in embodiment six and in-situ chemical trace thereof and immune imaging device, the concrete steps of described step 1) are as follows: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole 8 and described track 11, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well 8, electrophoretic buffer is added in electrophoresis tank 7, 1b) open multi-channel switch, path between described multi-channel loading hole 8 and described track 11 is opened, on the hyperchannel chromatoelectrophoresis handover module 1 of two, described left and right, add suitable voltage simultaneously, protein example in described protein well 8 is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated.2) in described active chromatography media, particulate is its surperficial Jie be altogether combined with the particulate that two a word used for translations suck class coupling molecule, described particulate is penetrated with the illumination of specific wavelength, two a word used for translations activating described chromatoelectrophoresis medium 3 surface suck quasi-molecule, two a word used for translations of activation are made to suck the protein generation chemical coupling of quasi-molecule and surrounding, be covalently bound to described microparticle surfaces, make protein produce in-situ chemical trace at described microparticle surfaces.3) after completing in-situ chemical trace, chromatoelectrophoresis handover module 1 is switched to chromatography pattern, by micro-liquid phase driving pump 5 first antibody is transported in hyperchannel chromatoelectrophoresis separating plate 2 with the in-situ chemical trace protein interaction that chemical coupling occurs; 4) wash unreacted first antibody off, by micro-liquid phase driving pump 5 second antibody is transported in hyperchannel chromatoelectrophoresis separating plate 2 and interacts with first antibody; When second antibody is fluorescently-labeled, the surface of second antibody is irradiated with LED fluorescence light source 4, fluorescence excitation mark is luminous, direct observation or by be positioned at the camera with respective filter directly over hyperchannel chromatoelectrophoresis separating plate 2 or pick-up lens is observed and record protein spring up image, complete protein fluorescence immune imaging.If use enzyme to join the second antibody of mark, if second antibody is peroxidase mark, by micro-liquid phase driving pump 5 luminous substrate of peroxidase is transported in hyperchannel chromatoelectrophoresis separating tank and interacts with peroxidase and luminous, direct observation or by be positioned at camera directly over hyperchannel chromatoelectrophoresis separating plate 2 or pick-up lens is observed and record protein spring up image, complete the imaging of protein chemistry electrochemiluminescent immunoassay.Described step 4) concrete steps are as follows: 4) close multi-channel switch, make the path blockade between described multi-channel loading hole 8 and described track 11, in described hyperchannel chromatoelectrophoresis separating tank, input damping fluid successively by described chromatography liquid channel, one or more can identify the second antibody of the first antibody of some protein, fluorescence or enzyme connection mark.

Present invention also offers and a kind of the method for Real-Time Monitoring is carried out to fluorescent marker protein in described chromatoelectrophoresis medium 3.When namely chromatoelectrophoresis being carried out to fluorescent marker protein, by opening LED fluorescence light source 4, exciting fluorescent marker protein in chromatography track 11, by naked eyes or camera installation, protein being sprung up and carries out Real Time Observation and imaging.

When adopting ampholyte or polysaccharide to carry out electrophoretic separation, the method of operating of the present embodiment is as follows: in the electrophoresis tank 7 of two chromatoelectrophoresis handover modules 1, add appropriate electrophoretic buffer, by micro-liquid phase driving pump 5 electrophoretic buffer, the fluid pipeline in chromatoelectrophoresis handover module 1 and hyperchannel chromatoelectrophoresis separating plate 2 is rinsed, with containing ampholyte or polysaccharide, hyperchannel chromatoelectrophoresis separating plate 2 is balanced by micro-liquid phase driving pump 5.

When using ampholyte (ampholyte) to fill the track 11 of hyperchannel chromatoelectrophoresis separating plate 2, protein can be undertaken being separated (isoelectric focusing) by isoelectric point; If now do not contain silicon dioxide microsphere in chromatoelectrophoresis medium 3, then in-situ chemical trace and immune imaging cannot be carried out.

When using the electrophoretic buffer containing polysaccharide to fill the track 11 of hyperchannel chromatoelectrophoresis separating plate 2, protein can be separated by molecular size range.If now do not contain silicon dioxide microsphere in chromatoelectrophoresis medium 3, then in-situ chemical trace and immune imaging cannot be carried out.

Protein example is joined in the well 8 of chromatoelectrophoresis handover module 1, be switched to electrophoretic by chromatoelectrophoresis handover module 1 and start electrophoresis, under the effect of electric field, protein will enter hyperchannel chromatoelectrophoresis separating plate 2 from well 8 and will be separated chromatoelectrophoresis medium 3.

Above-mentioned protein example also can change fluorescently-labeled protein into.

After bromophenol blue indicator goes to suitable position in hyperchannel chromatoelectrophoresis separating plate 2, open described LED fluorescence light source 4, two a word used for translations on activation chromatography electrophoretic medium 3 surface suck class coupling molecule, bring out the free radical coupling reaction with peripheral protein matter, complete the in-situ chemical trace that protein springs up image.

Be switched to chromatography pattern by chromatoelectrophoresis handover module 1, first antibody be transported to protein interaction with coupling in chromatoelectrophoresis medium 3 by micro-liquid phase driving pump 5 by the input port of micro-liquid phase driving pump 5.

Under chromatography pattern, wash unreacted first antibody off by micro-liquid phase driving pump 5, then second antibody is transported in chromatoelectrophoresis medium 3 and interacts with first antibody.

If second antibody is fluorescently-labeled, described LED fluorescence light source 4 can be opened, fluorescence excitation mark is luminous, and observed by the photograph directly over hyperchannel chromatoelectrophoresis separating plate 2 or pick-up lens and record protein spring up image, complete protein chromatographic electrophoresis and in-situ chemical trace thereof and fluorescence immunoassay imaging.

If second antibody is peroxidase mark, by micro-liquid phase driving pump 5 luminous substrate of peroxidase is transported in chromatoelectrophoresis medium 3 and interacts with peroxidase and luminous, spring up image by camera installation observing protein, complete protein chromatographic electrophoresis and in-situ chemical trace thereof and chemiluminescence immunoassay imaging.

When adopting ion-exchange or anti-phase affinity chromatography to carry out chromatography, the method for operating of the present embodiment is as follows:

If use ion-exchange or anti-phase affinity chromatography etc., when making spent ion exchange resin substituted layer analyse electrophoretic medium 3, protein will be separated according to CHARGE DISTRIBUTION; When using reversed phase chromatographic medium (C8, C18 etc.) to replace chromatography electrophoretic medium 3, protein will be separated according to hydrophobicity; Protein example is joined after in the well 8 of chromatoelectrophoresis handover module 1 and be switched to chromatography pattern, under the driving of the micro-liquid phase driving pump 5 of positive pole chromatoelectrophoresis handover module 1, protein and chromatographic solution entered hyperchannel chromatoelectrophoresis separating plate 2 from well 8 and be separated chromatoelectrophoresis medium 3.It should be noted that: under chromatography pattern, well 8 does not communicate with track 11, track 11 is entered into by pneumatic switch film 10 in order to make protein, then frequently should open pneumatic switch film 10, simultaneously under the micro-liquid phase driving pump 5 of negative pole or positive pole chromatoelectrophoresis handover module 1 drives, protein is swum to positive pole by negative pole.Also by electrophoretic, protein can be transported to the reference position of hyperchannel chromatoelectrophoresis separating tank in addition in advance.

Other step is with identical when adopting ampholyte or polysaccharide to carry out electrophoretic separation.

Above-mentioned protein also can be fluorescently-labeled protein.

Because protein is fluorescently-labeled, can open described LED fluorescence light source 4, fluorescence excitation mark is luminous, and observed by the photograph directly over hyperchannel chromatoelectrophoresis separating plate 2 or pick-up lens and record protein spring up image.

embodiment eight

Step 1 is: described step 1 concrete steps are as follows: 1a) close multi-channel switch with embodiment seven difference, make the path blockade between described multi-channel loading hole 8 and described track 11, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well 8, electrophoretic buffer is added in electrophoresis tank 7, 1b) open multi-channel switch, path between described multi-channel loading hole 8 and described track 11 is opened, on the hyperchannel chromatoelectrophoresis handover module 1 of two, described left and right, add suitable voltage simultaneously, the protein example in described protein well 8 is made to move to the reference position of described hyperchannel chromatoelectrophoresis separating tank in the electric field, 1c) close multi-channel switch, make the path blockade between described multi-channel loading hole 8 and described track 11, meanwhile, by described chromatography liquid channel, required chromatographic flow is inputted hyperchannel chromatoelectrophoresis separating tank mutually, protein example is separated in described chromatoelectrophoresis medium 3.

embodiment nine

The present embodiment provides a kind of novel protein chromatoelectrophoresis and retracting device thereof, can be undertaken being separated and the protein after separation can being reclaimed by isoelectric point, molecular weight or other molecular characterization to protein, namely add auto partial sampler, described auto partial sampler is 96-orifice plate part gatherer.Other parts are identical with embodiment three.Auto partial sampler described in the present embodiment is placed in the downstream of positive pole chromatoelectrophoresis handover module 1, after the micro-liquid phase driving pump 5 of positive pole chromatoelectrophoresis handover module 1.By the micro-liquid phase driving pump 5 of positive pole chromatoelectrophoresis handover module 1, the protein in the track 11 of hyperchannel chromatoelectrophoresis separating plate 2 slowly can be pumped, be collected by 96 components by auto partial sampler.Wherein 96-orifice plate auto partial sampler can be existing auto partial sampler on market.

The method of operating of the present embodiment is roughly as follows:

Performance liquid electrophoretic separation: the first step, under the effect of electric field, fluorescent marker protein will enter hyperchannel chromatoelectrophoresis separating plate 2 from well 8 and will be separated chromatoelectrophoresis medium 3.Second step, opens described LED fluorescence light source 4, and fluorescence excitation mark is luminous, and springs up image by the photograph directly over hyperchannel chromatoelectrophoresis separating plate 2 or pick-up lens record protein.3rd step, is switched to chromatography pattern, is collected respectively by the fluorescent marker protein after separation in hyperchannel chromatoelectrophoresis separating plate 2 under the driving of the micro-liquid phase driving pump 5 of positive pole chromatoelectrophoresis handover module 1 by 96-orifice plate part gatherer by component.

Protein chromatographic is separated: the first step, under the driving of the micro-liquid phase driving pump 5 of positive pole chromatoelectrophoresis handover module 1, protein will enter hyperchannel chromatoelectrophoresis separating plate 2 from well 8 and will be separated chromatoelectrophoresis medium 3.Second step, opens described LED fluorescence light source 4, and fluorescence excitation mark is luminous, and springs up image by the photograph directly over hyperchannel chromatoelectrophoresis separating plate 2 or pick-up lens record protein.3rd step, is collected the note protein after separation in hyperchannel chromatoelectrophoresis separating plate 2 respectively by component by 96-orifice plate part gatherer under the driving of the micro-liquid phase driving pump 5 of positive pole chromatoelectrophoresis handover module 1.

In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of application claims.

Claims (12)

1. protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging device, it is characterized in that: comprise hyperchannel chromatoelectrophoresis module, first solution tank, adopt the hyperchannel chromatoelectrophoresis separating plate of multiple track, active chromatography media and LED fluorescence light source, the liquid-phase outlet of described hyperchannel chromatoelectrophoresis module is connected to one end of hyperchannel chromatoelectrophoresis separating plate, the other end of hyperchannel chromatoelectrophoresis separating plate is connected to the first solution tank, described active chromatography media is placed in the track of chromatoelectrophoresis separating plate, described LED fluorescence light source is for irradiating described hyperchannel chromatoelectrophoresis separating plate, described active chromatography media includes particulate, described particulate is surface active ion exchange resin, affinity chromatography filler, reversed phase chromatography filler or nonporous silica silicon particle or fritted glass particulate or macromolecular material particulate, be situated between altogether on the surface of described particulate and be combined with two a word used for translations and suck class coupling molecule.

2. protein chromatographic electrophoresis according to claim 1 and in-situ chemical trace thereof and immune imaging device, it is characterized in that: described hyperchannel chromatoelectrophoresis module is hyperchannel chromatoelectrophoresis handover module, described first solution tank is hyperchannel chromatoelectrophoresis handover module, described hyperchannel chromatoelectrophoresis handover module comprises electrophoresis tank respectively, multi-channel loading hole, multi-channel switch and multiple chromatography liquid channel, positive electrode and negative electrode is had respectively in described electrophoresis tank, multi-channel loading hole is connected with electrophoresis tank, multi-channel loading aperture multi-channel switch is connected with track respectively, described chromatography liquid channel is connected with track respectively.

3. protein chromatographic electrophoresis according to claim 2 and in-situ chemical trace thereof and immune imaging device, it is characterized in that: described multi-channel switch is hyperchannel pneumatic switch, a pneumatic switch groove is had in the bottom of described each well, a pneumatic switch film is had in described each pneumatic switch groove, pneumatic switch groove is separated into two passages, described each well is connected with chromatography liquid channel through a passage of pneumatic switch groove, another passage is connected with pneumatic switch gas access, when pneumatic switch film drives from pneumatic switch gas access input malleation or negative pressure, pneumatic switch film opens and closes the path between each well and each chromatography liquid channel respectively.

4. the protein chromatographic electrophoresis according to Claims 2 or 3 and in-situ chemical trace thereof and immune imaging device, it is characterized in that: also comprise micro-liquid phase driving pump, described micro-liquid phase driving pump is placed in porch and/or the exit of chromatography liquid channel.

5. protein chromatographic electrophoresis according to claim 1 and 2 and in-situ chemical trace thereof and immune imaging device, is characterized in that: described particulate to be diameter the be microballoon of 10 microns to 100 microns.

6. protein chromatographic electrophoresis according to claim 1 and 2 and in-situ chemical trace thereof and immune imaging device, it is characterized in that: described hyperchannel chromatoelectrophoresis separating plate is bonded containing multiple 4-12 mm wide, the glass of 0.1-1 millimeter deep trouth, organic glass or transparent polymer material plate face-to-face by two pieces, form the hyperchannel chromatoelectrophoresis separating tank of multiple 4-12 mm wide, 0.2-2 millimeters deep, two ends have fixing filler to be sealed by swimming lane mouth, form multiple track.

7. protein chromatographic electrophoresis and in-situ chemical trace thereof and immune imaging method, it is characterized in that: comprise the steps: 1) utilize the active chromatography media in hyperchannel chromatoelectrophoresis separating plate to carry out chromatoelectrophoresis separation, described active chromatography media includes particulate, described particulate is surface active ion exchange resin, affinity chromatography filler, reversed phase chromatography filler or nonporous silica silicon particle or fritted glass particulate or macromolecular material particulate, 2) in described active chromatography media, particulate is its surperficial Jie be altogether combined with the particulate that two a word used for translations suck class coupling molecule, described particulate is penetrated with the illumination of specific wavelength, two a word used for translations activating described chromatoelectrophoresis dielectric surface suck quasi-molecule, two a word used for translations of activation are made to suck the protein generation chemical coupling of quasi-molecule and surrounding, be covalently bound to described microparticle surfaces, protein is made to produce in-situ chemical trace at described microparticle surfaces, 3) first antibody is transported to the in-situ chemical trace protein interaction with generation chemical coupling in hyperchannel chromatoelectrophoresis separating plate, 4) wash unreacted first antibody off, second antibody is transported in hyperchannel chromatoelectrophoresis separating plate and interacts with first antibody, when second antibody is fluorescently-labeled, the surface of second antibody is irradiated with LED fluorescence light source, fluorescence excitation mark is luminous, direct observation or by be positioned at the camera with respective filter directly over hyperchannel chromatoelectrophoresis separating plate or pick-up lens is observed and record protein spring up image, complete protein fluorescence immune imaging, if use enzyme to join the second antibody of mark, the luminous substrate of enzyme connection mark is transported in hyperchannel chromatoelectrophoresis separating tank and joins label with enzyme and to interact and luminous, direct observation or by be positioned at camera directly over hyperchannel chromatoelectrophoresis separating plate or pick-up lens is observed and record protein spring up image, complete the imaging of protein chemistry electrochemiluminescent immunoassay.

8. protein chromatographic electrophoresis according to claim 7 and in-situ chemical trace thereof and immune imaging method, it is characterized in that: described step 1 adopts protein chromatographic electrophoresis as claimed in claim 2 and in-situ chemical trace thereof and immune imaging device, concrete steps are as follows: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) open multi-channel switch, path between described multi-channel loading hole and described track is opened, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, protein example in described protein well is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated.

9. protein chromatographic electrophoresis according to claim 7 and in-situ chemical trace thereof and immune imaging method, it is characterized in that: described step 1 adopts protein chromatographic electrophoresis as claimed in claim 2 and in-situ chemical trace thereof and immune imaging device, concrete steps are as follows: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) open multi-channel switch, path between described multi-channel loading hole and described track is opened, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, the protein example in described protein well is made to move to the reference position of described hyperchannel chromatoelectrophoresis separating tank in the electric field, 1c) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, meanwhile, by described chromatography liquid channel, required chromatographic flow is inputted hyperchannel chromatoelectrophoresis separating tank mutually, protein example is separated in described chromatoelectrophoresis medium.

10. according to the protein chromatographic electrophoresis in claim 7 to 9 described in any one claim and in-situ chemical trace thereof and immune imaging method, it is characterized in that: described step 4) concrete steps are as follows: 4) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, input damping fluid successively by described chromatography liquid channel, one or more can identify the second antibody of the first antibody of some protein, fluorescence or enzyme connection mark.

11. multifunctional protein chromatoelectrophoresis and recovery methods thereof, it is characterized in that: protein chromatographic electrophoresis according to claim 2 and in-situ chemical trace thereof and immune imaging device, comprise the steps: 1a) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) open multi-channel switch, path between described multi-channel loading hole and described track is opened, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, protein example in described protein well is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated, 1c) close multi-channel switch, make the path blockade between described multi-channel loading hole and described track, meanwhile, by described chromatography liquid channel, required chromatographic flow is inputted hyperchannel chromatoelectrophoresis separating tank mutually, make the protein example after separation be exported by the liquid-phase outlet of right hyperchannel chromatoelectrophoresis handover module and be collected respectively with gatherer sequentially.

12. multifunctional protein chromatoelectrophoresis methods, it is characterized in that: protein chromatographic electrophoresis according to claim 3 and in-situ chemical trace thereof and immune imaging device, comprise the steps: 1a) regulate gaseous tension in described pneumatic switch groove, under making described pneumatic switch film be in barotropic state, close the path between described multi-channel loading hole and described chromatography liquid channel, in described hyperchannel chromatoelectrophoresis separating tank, required chromatoelectrophoresis damping fluid is added by described chromatography liquid channel, the protein example needing chromatographic analysis is added in described protein well, electrophoretic buffer is added in electrophoresis tank, 1b) regulate gaseous tension in described pneumatic switch groove, under making described pneumatic switch film be in negative pressure state, open the path between described multi-channel loading hole and described chromatography liquid channel, on the hyperchannel chromatoelectrophoresis handover module of two, described left and right, add suitable voltage simultaneously, protein example in described protein well is moved in described hyperchannel chromatoelectrophoresis separating tank be in the electric field separated.