CN101750448A - Fluorescence imaging representation method of protein in capillary and microfluidic chip electrophoresis path - Google Patents
Fluorescence imaging representation method of protein in capillary and microfluidic chip electrophoresis path Download PDFInfo
- Publication number
- CN101750448A CN101750448A CN200810240104A CN200810240104A CN101750448A CN 101750448 A CN101750448 A CN 101750448A CN 200810240104 A CN200810240104 A CN 200810240104A CN 200810240104 A CN200810240104 A CN 200810240104A CN 101750448 A CN101750448 A CN 101750448A
- Authority
- CN
- China
- Prior art keywords
- protein
- microfluidic chip
- kapillary
- electrophoresis path
- chip electrophoresis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to a fluorescence imaging representation method of protein in a capillary and a microfluidic chip electrophoresis path, which is used for representing the non-specific adsorption phenomena of the protein in the capillary or the microfluidic chip electrophoresis path and can be used for evaluating the performance of protein coating materials. The method has the principle that fluorescent dye is filled into the capillary or the microfluidic chip electrophoresis path which adsorbs protein or is provided with a protein coating to stand for 5-60 minutes for protein fluorescent dying; the uncombined fluorescent dye is cleaned with solution without fluorescence interference; and finally, a fluorescence microscope is used for imaging observation and image analysis. The operation process is carried out at 4-37 DEG C. The method has the advantages of simple operation and high sensitivity and intuition, is visual and quick on the aspect of non-specific adsorption of protein and representation of protein coating.
Description
Technical field
The present invention relates to fluorescence imaging representation method of protein in a kind of kapillary and the microfluidic chip electrophoresis path, in order to characterize the protein adsorption phenomenon of differential in passage and the performance of protein coat material.
Background technology
Capillary Electrophoresis and micro-fluidic chip technology receive increasing concern as a kind of new technical platform of genomics and proteomics research.Especially aspect gene sequencing and protein analysis, demonstrate unique application advantage especially.
In the Separation of Proteins process, separated protein can produce absorption by electrostatic attraction in quartz capillary or micro-fluidic chip passage, causes the variation of sample concentration variation and capillary surface energy, thereby causes that peak hangover and resolution reduce.The irreversible adsorption of protein, the research that is used for proteomics for Capillary Electrophoresis and micro-fluidic chip technology brings a bottleneck problem.
Yet the absorption of proteins problem is not to be complete adverse factors for concrete research yet.Along with people deepen continuously to the understanding of protein function, protein is used to carry out drug screening, clinical diagnosis and the repercussion study relevant with life science more and more.For this reason, people are fixing in split tunnel protein by the method for physics or chemistry, form the coating of biologically active, are suitable for the new method and the instrument of biochemical and the research of medical treatment ﹠ health aspect so as to development.
To the generation of nonspecific protein adsorption, people are usually by being judged indirect means such as the mensuration of electroosmotic flow and study on the stability thereof.Yet the relation between position, degree and these factors and the separation efficiency of protein adsorption generation does not also have good method to be verified.Similar with it, according to the protein coat that different research purpose makes up, its performance also over-borrowing helps the mensuration of electroosmotic flow and sample separation efficient to investigate, and lacks more definitely, characterization method intuitively.
The method of fluorescence imaging provides feasible experimental considerations for the solution of this difficult problem.The present invention adopts the fluorescent dye of protein, dye to being adsorbed on the protein of differential in passage, behind unconjugated dyestuff flush away, under fluorescent microscope, observe, in order to the sign of carrying out nonspecific proteins matter absorption and the evaluation of protein coat performance.
Summary of the invention
The present invention relates to fluorescence imaging representation method of protein in a kind of kapillary and the microfluidic chip electrophoresis path.This method provides a kind of characterization method visual, simple to operate for the irreversible adsorption phenomenon of protein in research kapillary and the microfluidic chip electrophoresis path.
Another object of the present invention is that the sign of thickness, homogeneity and stability for protein coat or liposome-protein composite coating provides a kind of strong analytical approach.
Protein fluorescence formation method in kapillary and the microfluidic chip electrophoresis path, its concrete technical scheme is: at first, protein is fixing in kapillary and microfluidic chip electrophoresis path, this process both can be to take place in the detachment process of protein, also can be to make up protein coat by physics or chemical method in passage artificially; Then, fill into the protein fluorescence dyestuff in the microchannel and leave standstill 5-60min, carry out protein coating; Then, with the solution or the solvent of no fluorescence interference unconjugated fluorescent dye is cleaned; At last, on fluorescent microscope,, carry out imaging observation and graphical analysis according to the selected observation condition of the excitation wavelength of fluorescent dye.All operating process is carried out under 4-37 ℃ of condition.
Wherein, fluorescent dye is meant and can the organic fluorescent dye that covalently or non-covalently effect also combines takes place with protein, include but are not limited to: fluorescein, fluorescein isothiocynate (FITC), rhodamine B, tetramethyl rhodamine (TMR), SYPROOrange, SYPRO Red, SYPRO Ruby, acridine red, cy3 and cy5.
Method involved in the present invention aspect the sign of the non-specific adsorption of protein and protein coat, has simple to operate, highly sensitive, visual pattern and advantage fast.
Description of drawings
Fig. 1 is the shows fluorescent microscopy images of quartz capillary sample introduction end adsorbed proteins.
Fig. 2 is the ordinary optical microscope figure (1) and the shows fluorescent microscopy images (2) of polymethyl methacrylate micro flow control chip electrophoresis path adsorbed proteins.
Fig. 3 is the shows fluorescent microscopy images of low-density lipoprotein coating (1), human serum albumins coating (2), Vtg coating (3) and no coatings capillary pipe (4).
Embodiment
The fluorescence imaging analysis of non-specific adsorption protein on embodiment 1 quartz capillary
Getting an internal diameter is fused quartz kapillary (the length overall 31cm of 50 μ m, effective length 21cm), insert Beckman MDQ capillary electrophoresis system, at+15kY voltage, 20mM phosphate buffer (pH 7.4), pressure sample introduction (0.5psi, 5s) under the condition, continuous 5 operation the separating of 0.2mg/mL protein biased sample (the former A of lysozyme, cytochrome c, ribonuclease A and Chymetin).After operation is finished, kapillary is continued with 20mM phosphate buffer (pH 7.4) flushing 10min.
Measure the SYPRO Orange fluorescent dye of 1.0 μ L, with 20mM phosphate buffer (pH 7.4) dilution and the mixing of 5mL.This solution filling is gone in the kapillary, leave standstill 15-30min under the room temperature, dye.After dyeing is finished, with 20mM phosphate buffer (pH 7.4) and each 10min of deionized water rinsing.Kapillary is taken out from instrument, slowly feed high pure nitrogen 1-2min.With quartzy cutter kapillary on average is cut to 3 sections, is labeled as sample introduction end, center section and outflow end respectively, and scrape off surperficial polyimide clad.At last, observe image CCD and digital camera collection with Zeiss AxioSkop 40 type fluorescent microscopes.
Find that according to the fluoroscopic image interpretation of result protein mainly occurs in sample introduction end (seeing accompanying drawing 1) in the absorption of capillary wall.The fluorescence imaging analysis of non-specific adsorption protein on embodiment 2 micro-fluidic chips
Get the micro-fluidic chip of a slice polymethylmethacrylate (PMMA) matrix, it is of a size of: long 50mm, wide 30mm, thickness 3mm, and be carved with that 35mm is long, the split tunnel of the wide about 130 μ m of substrate, under field intensity 200V/cm, damping fluid 40mM phosphate (pH 7.2) condition, continuous 5 operation the separating of 0.2mg/mL protein biased sample (the former A of lysozyme, cytochrome c, ribonuclease A and Chymetin).After operation was finished, the microchannel continued to use SYPRO Orange fluorescent dye (diluting 5000 times) dyeing 15min then with 40mM phosphate buffer (pH 7.2) flushing 10min.After dyeing is finished,, observe image CCD and digital camera collection at last with Zeiss AxioSkop40 type fluorescent microscope with 20mM phosphate buffer (pH 7.4) and each 10min of deionized water rinsing.
According to the fluoroscopic image result as can be seen, on the micro-fluidic chip of PMMA matrix, in sample introduction end (being right-angled intersection place) passage, the absorption of proteins situation is serious (seeing accompanying drawing 2) relatively, illustrates that the PMMA material has stronger non-specific adsorption effect to protein.
The fluorescence imaging analysis of embodiment 3 low-density lipoproteins (LDL) capillary coating
The capillary coating of low-density lipoprotein (LDL) adopts the chemical bonding preparation: the first kapillary that is 50 μ m to an internal diameter with the methanol solution of 10% aminopropyl trimethoxysilane carries out silylation modification, introduces amino reactive group; Use 5% glutaraldehyde (in the 20mM phosphate buffer, pH 7.4) to handle kapillary 2h then; LDL solution (in the 20mM phosphate buffer, pH 7.4) with 1mg/mL pours in the kapillary again, and 20 ℃ are reacted 2h down; Last sodium cyanoborohydride solution (pH 6.0) reduction of using 0.5mg/mL again, and with 20mM phosphate buffer (pH 7.4) and each 10min of deionized water rinsing kapillary.
Measure the SYPRO Orange fluorescent dye of 1.0 μ L, with 20mM phosphate buffer (pH 7.4) dilution and the mixing of 5mL.This solution is filled in the kapillary of above-mentioned low-density lipoprotein coating, 15min is left standstill under the room temperature in the sealing two ends.Use 20mM phosphate buffer (pH 7.4) and each 10min of deionized water rinsing kapillary again.Observe image CCD and digital camera collection at last with Zeiss AxioSkop40 type fluorescent microscope.Fluorescence imaging the results are shown in accompanying drawing 3 (1).
Experimental result shows that the method for fluorescence imaging can be used for the sign of performances such as protein coat thickness, homogeneity.
Take by weighing 2.0mg human serum albumins (HSA), fully be dissolved in the 20mM phosphate buffer (pH 7.4) of 1mL, mixing makes the human serum albumins coating solution.Get a quartz capillary (internal diameter 50 μ m), use NaOH, secondary water, 1M hydrochloric acid, the secondary water treatment 15-30min of 1M successively, high pure nitrogen dries up.The filling of above-mentioned HSA solution in kapillary, is sealed two ends with silicon rubber, under room temperature, leave standstill 2h.Repeat above-mentioned filling and leave standstill operation 1 time.To manage interior solution with 20mM phosphate buffer (pH 7.4) and wash away, and continue to clean 10min, make human serum albumins dress coating.
Measure the fluorescein isothiocynate (FITC) of 1.0mg, with 20mM phosphate buffer (pH 7.4) dilution and the mixing of 5mL.This solution is filled in the kapillary of above-mentioned human serum albumins coating, the sealing two ends, 30min dyes under the room temperature.Use 20mM phosphate buffer (pH 7.4) and each 10min of deionized water rinsing kapillary again.Observe image CCD and digital camera collection at last with Zeiss AxioSkop40 type fluorescent microscope.Fluorescence photo is seen accompanying drawing 3 (2).
Experimental result shows, different types of fluorescent dye only need select suitable optical filter that excitation wavelength is modulated, and all can carry out the fluorescence imaging analysis.
The fluorescence imaging analysis of embodiment 5 Vtgs (Vtg) capillary coating
The making of Vtg (Vtg) coatings capillary pipe and sign and embodiment 3 described methods are similar, only need to change the low-density lipoprotein among the embodiment 3 into Vtg, simultaneously capillary inner diameter are changed into 100 μ m.Its fluorescence photo is seen accompanying drawing 3 (3).
By the fluorescence imaging result as can be seen, fluorescence intensity can reflect information such as coating thickness, degree of uniformity and stability, but does not significantly distinguish for the kind of protein.
Claims (5)
1. fluorescence imaging representation method of protein in kapillary and the microfluidic chip electrophoresis path is characterized in that, adopts fluorescent dye that the protein in kapillary and the microfluidic chip electrophoresis path is dyeed, and uses fluorescent microscope to carry out imaging analysis.
2. kapillary according to claim 1 and microfluidic chip electrophoresis path fluorescence imaging representation method of protein is characterized in that, are used for the absorption behavior of profiling protein matter on capillary tube inner wall and microfluidic chip electrophoresis path.
3. fluorescence imaging representation method of protein in kapillary according to claim 1 and the microfluidic chip electrophoresis path, it is characterized in that, be used for characterizing thickness, homogeneity and the stability of kapillary and microfluidic chip electrophoresis path protein coat, and can be used for observing the interaction between protein and biomacromolecule.
4. fluorescence imaging representation method of protein in kapillary according to claim 1 and the microfluidic chip electrophoresis path, it is characterized in that, concrete technical scheme is: protein is fixing in kapillary or microfluidic chip electrophoresis path, this process both can be to take place in the detachment process of protein, also can be the protein coat that makes up in passage by physics or chemical method artificially; Then, fill into fluorescent dye in the microchannel and leave standstill 5-60min, carry out protein coating; Then, solution or the solvent with no fluorescence interference washes 10-30min with unconjugated fluorescent dye; At last, on fluorescent microscope,, carry out imaging analysis according to the selected observation condition of the excitation wavelength of fluorescent dye.
5. method according to claim 4 is characterized in that, all operates under the 4-37 ℃ of condition to carry out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810240104A CN101750448A (en) | 2008-12-18 | 2008-12-18 | Fluorescence imaging representation method of protein in capillary and microfluidic chip electrophoresis path |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810240104A CN101750448A (en) | 2008-12-18 | 2008-12-18 | Fluorescence imaging representation method of protein in capillary and microfluidic chip electrophoresis path |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101750448A true CN101750448A (en) | 2010-06-23 |
Family
ID=42477744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810240104A Pending CN101750448A (en) | 2008-12-18 | 2008-12-18 | Fluorescence imaging representation method of protein in capillary and microfluidic chip electrophoresis path |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101750448A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091385A (en) * | 2013-02-05 | 2013-05-08 | 中央民族大学 | Capillary enzyme array micro-reactor and its application |
| CN106607107A (en) * | 2015-10-21 | 2017-05-03 | 中国科学院大连化学物理研究所 | Three-dimensional structure characterization method of microfluidic chip |
| CN112368077A (en) * | 2018-12-07 | 2021-02-12 | 元素生物科学公司 | Flow cell device and use thereof |
-
2008
- 2008-12-18 CN CN200810240104A patent/CN101750448A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091385A (en) * | 2013-02-05 | 2013-05-08 | 中央民族大学 | Capillary enzyme array micro-reactor and its application |
| CN103091385B (en) * | 2013-02-05 | 2015-07-08 | 中央民族大学 | Capillary enzyme array micro-reactor and its application |
| CN106607107A (en) * | 2015-10-21 | 2017-05-03 | 中国科学院大连化学物理研究所 | Three-dimensional structure characterization method of microfluidic chip |
| CN112368077A (en) * | 2018-12-07 | 2021-02-12 | 元素生物科学公司 | Flow cell device and use thereof |
| US11426732B2 (en) | 2018-12-07 | 2022-08-30 | Element Biosciences, Inc. | Flow cell device and use thereof |
| CN112368077B (en) * | 2018-12-07 | 2023-11-24 | 元素生物科学公司 | Flow cell device and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101287406B1 (en) | A method for identification of a substance | |
| US9400277B2 (en) | Methods and devices for analyte detection | |
| US20190331692A1 (en) | Fluidic separation and detection | |
| Miller et al. | A digital microfluidic approach to heterogeneous immunoassays | |
| EP2916128B1 (en) | Method and device for analyte detection | |
| US20090301885A1 (en) | Electrophoresis extraction device | |
| JP2004042012A (en) | Separation apparatus, analysis system, separating method, and method of manufacturing the apparatus | |
| JP2003527606A5 (en) | ||
| WO2008053822A1 (en) | Method of detecting specific bond reaction of molecule by single molecule fluorometry | |
| CA2819237A1 (en) | Protein renaturation microfluidic devices and methods of making and using the same | |
| JP2007510935A (en) | Multi-dimensional electrophoresis device | |
| US7465381B2 (en) | Electrokinetic molecular separation in nanoscale fluidic channels | |
| CN101750448A (en) | Fluorescence imaging representation method of protein in capillary and microfluidic chip electrophoresis path | |
| Esene et al. | 3D printed microfluidic devices for integrated solid-phase extraction and microchip electrophoresis of preterm birth biomarkers | |
| WO2017213074A1 (en) | Fluid device, system, and method of detecting specimen substance | |
| CA2895275A1 (en) | Lipid membrane enveloped particles with membrane proteins | |
| JP2001165840A (en) | Method and apparatus for analyzing gene | |
| US20160153978A1 (en) | Automated chemical analysis apparatus with integrated microfluidic microchip | |
| KR100757348B1 (en) | Microfluidic device including superporous agarose immuno-beads and immunoassay using the same | |
| Riordan | Developing Microfluidic Devices for the Optimization of Nanodiscs to Measure Membrane Protein Structure and Function | |
| D'onofrio et al. | Controlling and measuring local composition and properties in lipid bilayer membranes | |
| Sandy | New Instrumentation and Methods for Analyzing Biological Compounds for Drug Discovery and Discovering Novel Disease Markers | |
| Teerapanich | Fluorescence-based nanofluidic biosensor platform for real-time measurement of protein binding kinetics | |
| Morani | Novel electrokinetic and analytical approaches for isolation and detection of extracellular vesicles | |
| Murukeshan et al. | Diagnostic sensing of specific protein in breast cancer cells using hollow-core photonic crystal fiber |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100623 |