CN108445069A - Isoelectric focusing electrophoresis chip with microarray immobilization pH gradient column and method - Google Patents
Isoelectric focusing electrophoresis chip with microarray immobilization pH gradient column and method Download PDFInfo
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- CN108445069A CN108445069A CN201810554632.0A CN201810554632A CN108445069A CN 108445069 A CN108445069 A CN 108445069A CN 201810554632 A CN201810554632 A CN 201810554632A CN 108445069 A CN108445069 A CN 108445069A
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- 238000002493 microarray Methods 0.000 title claims abstract description 89
- 238000001962 electrophoresis Methods 0.000 title claims abstract description 54
- 238000001155 isoelectric focusing Methods 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 9
- 239000003292 glue Substances 0.000 claims abstract description 15
- 230000005540 biological transmission Effects 0.000 claims abstract description 12
- 238000004458 analytical method Methods 0.000 claims abstract description 10
- 230000003287 optical effect Effects 0.000 claims abstract description 9
- 230000005484 gravity Effects 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims description 6
- 238000009738 saturating Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 11
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000004064 recycling Methods 0.000 abstract description 5
- 230000004907 flux Effects 0.000 abstract description 3
- 238000001531 micro-dissection Methods 0.000 abstract description 2
- 238000003384 imaging method Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 230000005611 electricity Effects 0.000 description 4
- 239000002390 adhesive tape Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005684 electric field Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 230000017525 heat dissipation Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44795—Isoelectric focusing
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Abstract
A kind of isoelectric focusing electrophoresis chip and method with microarray immobilization pH gradient column, including：Microarray holder, microelectrophoresis bath, electrode and the microarray optical transmission window from top to bottom set gradually, wherein：The bottom of microarray holder is equipped with several microarray microtrabeculaes pair of array arrangement, and the end of each microarray microtrabeculae pair is equipped with minisize pH gradient column.Minisize pH gradient column includes：Minisize pH gradient glue positioned at microarray frame bottom and miniature support film, pH gradient glue are convenient for microarray pH gradient column sample sample introduction, minisize pH gradient column to be come into full contact with electrode to form electrophoresis circuit by microarray holder and its own gravity downward.The operation for effectively preventing Traditional IP G focusing electrophoresis complexity improves IEF separation analysis loading speed, accelerates focusing speed, especially being capable of direct-on-line detection.Meanwhile compared with capillary/chip focusing electrophoresis technology, pH gradient column of the invention have stability is good, flux is high, focus protein band can micro-dissections recycling etc. special advantages.
Description
Technical field
It is specifically a kind of to carry microarray immobilization pH gradient the present invention relates to a kind of technology of field of biological detection
The isoelectric focusing electrophoresis chip and method of column (IPG).
Background technology
Isoelectric focusing electrophoresis (isoelectric focusing, IEF) is to generate continuous pH gradient in the electric field, loading
Protein, which is realized according to its isoelectric point (isoelectric point, pI) difference in IEF electric fields, focuses separation analysis.It is common
It is to be passed through based on immobilization pH gradient adhesive tape (immobilized pH gradient, IPG strip) isoelectric focusing electrophoresis
Prefabricated pH gradient is formed by being chemically bonded Immobiline on 70~240mm long substrates, and then passes through focusing electrophoresis
Realize the separation analysis of polypeptide.This long substrate isoelectric-focusing electrophoresis has many advantages, such as the good high resolution of stability,
But it is clearly present following drawback：Manual albumen sample sample introduction is cumbersome (10 multistep), and loading hydration time length (is more than 10 hours), gathers
Burnt speed slow (5-10 hours) and interminable proteopexy dyeing imaging analysis (being more than 7 hours) etc..
Invention content
For the problem present on, the present invention proposes a kind of Isoelectric Focusing with microarray immobilization pH gradient column
The drawbacks of swimming chip and method, having abandoned traditional immobilization pH gradient focusing electrophoresis and existing capillary/chip focusing electrophoresis, knot
The focusing electrophoresis technological merit for having closed the two has not only well solved four disadvantages of traditional immobilization pH gradient focusing electrophoresis
End, and the technical issues of preferably solve above-mentioned capillary/chip focusing technology three are not resolved always：Low pass
Amount, sample are difficult to recycle, and isoelectric focusing caused by natural gradient is unstable.
The present invention is achieved by the following technical solutions：
The present invention relates to a kind of isoelectric focusing electrophoresis chips with microarray immobilization pH gradient column, including：By upper
Under the microarray holder, microelectrophoresis bath, wire electrode and the microarray optical transmission window that set gradually, wherein：Array arrangement it is several
The end of a microarray microtrabeculae pair, each microarray microtrabeculae pair is equipped with minisize pH gradient column, and minisize pH gradient column passes through microarray
Holder and its own gravity come into full contact with electrode to form electrophoresis circuit.
The minisize pH gradient column includes：Minisize pH gradient glue positioned at microarray frame bottom and miniature support film,
Wherein：PH gradient glue is convenient for microarray pH gradient column sample sample introduction downward.
The minisize pH gradient column has positive-negative polarity, and its positive and negative anodes polarity is consistent with microarray holder polarity.
The microarray optical transmission window is embedded in microelectrophoresis bath bottom by seal frame, and microarray optical transmission window with
The arragement direction of microarray pH gradient column is consistent.
The microarray optical transmission window includes transparent panel and microarray photomask.
The electrode includes：Anode electrode silk and cathode electrode silk.
The array arrangement refers to：Single display pH gradient pole unit is formed by 12~24 minisize pH gradient columns, 1~
8 single display pH gradient pole units constitute microarray pH gradient column battle array, and 12~192 minisize pH gradient columns may be implemented simultaneously
Aquation sample introduction and the different test samples of up to 192 kinds of progress.
The present invention relates to a kind of electrophoresis methods based on said chip, are directly added different samples simultaneously with volley of rifle fire pipettor
When being added to the enterprising traveling sample of different microarray pH gradient columns, then by minisize pH gradient column both ends jointed anode wire electrode and cathode
Wire electrode is focused electrophoretic separation analysis.
Technique effect
Compared with Traditional IP G focusing electrophoresis technologies, the technology of the present invention effect includes：The first, the operating procedures such as aquation sample introduction
It substantially reduces.Traditional IPG focusing electrophoresis needs the operation of 10 steps or more that could complete the operations such as sample introduction, and the present invention only row of need
Microarray pH gradient column is transferred to miniature electrophoresis tank and operates the assembling for completing entire chip after rifle sample introduction and loading, realizes IEF
Operation.Therefore, experimental procedure reduces 5 times.The second, aquation sample introduction speed of the present invention is significantly promoted.When traditional IPG sample introductions
Between at least 10 hours, and the present invention only needs to complete within 30 minutes albumen sample aquation sample introduction, and sample introduction speed improves 20 times or more.
The main reason is that the specific surface area of minisize pH gradient column promotes 7-26 times compared with Traditional IP G adhesive tape.The focusing electricity of third, the present invention
Swimming speed is greatly improved.Traditional IPG focusing electrophoresis times need 5-10 hours, and focal time of the present invention only needs 4
Minute, focusing speed improves 75-150 times.The main reason is that the more existing traditional IPG adhesive tape contracting of the pH gradient column of the present invention
7-24 times short, the corresponding focusing electrophoresis time reduces 7-24 times.And because high-specific surface area causes rapid heat dissipation, the present invention can
The electric field strength of 2-4 times of Traditional IP G focusing electrophoresis is withstood greater than, the corresponding focusing electrophoresis time reduces 2-4 times.4th, it and passes
System IPG focusing electrophoresis is compared, and detection speed of the present invention promotes 210-420 times.It needs to be up to 7 after the completion of Traditional IP G focusing electrophoresis
Hour or more proteopexy, dyeing and decoloration and scanning analysis, and the present invention only needs 2 minutes 24 channels can be completed
The online dynamic imaging of albumen sample is analyzed.Therefore, focusing electrophoresis post-processing work is not only greatly simplified, but also detection speed carries
It is 210-420 times high.
Compared with tradition is based on capillary/chip I EF technologies, the technology of the present invention effect includes：The first, stabilization of the invention
Property is greatly improved.Capillary/chip I EF is the electrophoretic techniques based on natural pH gradient, there is pH gradient drift etc. always
Caused by it is unstable；It is basicly stable to thereby result in the pH gradient within 15 clocks, is more than this time pH gradient, that is, unstable.And
The pH gradient of the present invention can continue to keep at least 24 hours or more stablizing, therefore stability at least promotes 80 times or more.The second,
The focusing electrophoresis flux of the present invention is obviously improved.Conventional capillary/chip I EF tests for single channel, tests and analyzes one every time
The time of a protein sample, detection is 10-15 minutes.And the present invention can carry out the same of up to 12-192 albumen sample simultaneously
Focusing electrophoresis separation analysis is walked, analysis time is 6 minutes.Therefore, flux of the invention promotes 24-384 times.Third, the present invention
Solves the problems, such as the trace of albumin band extraction recycling that capillary/chip I EF technologies are difficult to realize.Capillary/chip I EF technologies
The extraction recycling that cannot achieve albumen after focusing can not carry out subsequent mass spectrum and western blotting identifications.And this hair
It is bright to solve the problems, such as this respect (Fig. 3) well.
Description of the drawings
Fig. 1 is schematic structural view of the invention；
Fig. 2 is that the isoelectric focusing electrophoresis chip performance of microarray pH gradient column of the present invention is tested
The isoelectric focusing electrophoresis chip of Fig. 3 microarray pH gradient columns of the present invention focuses protein band micro-dissections recycling imaging
Figure
Fig. 4 is the isoelectric focusing electrophoresis chip schematic diagram with one group of microarray pH gradient column；
Fig. 5 is the isoelectric focusing electrophoresis chip schematic diagram with two groups of microarray pH gradient columns；
Fig. 6 is the isoelectric focusing electrophoresis chip schematic diagram with three groups of microarray pH gradient columns；
Fig. 7 is the isoelectric focusing electrophoresis chip schematic diagram with four groups of microarray pH gradient columns；
In figure：It is 1 microarray pH gradient column, 1.1 minisize pH gradient glues, 1.2 miniature support films, 2 microarray holders, 2.1 micro-
Array microtrabeculae to, 3 optical transmission windows, 3.1 microarray photomasks, 4 microelectrophoresis baths, 5 cathode electrode silks, 6 anode electrode silks, 7 negative electricity
Extreme mouth, 8 positive electrode ports, 9 micro sealing frames, 10 light-transmitting plates.
Specific implementation mode
As shown in Figure 1, the present embodiment includes：Microarray holder 2 with microarray pH gradient column 1 carries negative electrode
The microelectrophoresis bath 4 of port 7 and positive electrode port 8, cathode electrode silk 5, anode electrode silk 6, light-transmitting plate 10, microarray optical transmission window
3 and micro sealing frame 9.
The microarray pH gradient column 1 includes：The minisize pH gradient column of several array arrangements, every minisize pH gradient column
Including：Miniature support film 1.2 and the minisize pH gradient glue being set on the microarray microtrabeculae pair 2.1 of 2 bottom of microarray holder
1.1, wherein：Its positive and negative anodes polarity is consistent with 2 polarity of microarray holder.
There are microarray microtrabeculae pair 2.1, the miniature support film 1.2 of every minisize pH gradient column on the microarray holder 2
It is adhered on the microtrabeculae pair 2.1 of microarray holder 2, pH gradient glue-line 1.1 is convenient for microarray pH gradient column 1 to carry out sample downward
Sample introduction；The distance of adjacent minisize pH gradient column by microarray holder 2 adjacent microtrabeculae pair 2.1 determine, and with microarray holder 2
Polarity it is consistent.
Since microarray holder 2 has certain weight, it is attached on microarray pH gradient column 1 by gravity pressure；On the one hand
Microarray microtrabeculae pair 2.1 is supported, on the other hand ensures that minisize pH gradient glue 1.1 comes into full contact with wire electrode, using positive electrode
Port 8 and negative electrode port 7 finally connect electrophoresis power, form loop.
The microarray optical transmission window 3 includes：Transparent panel 10 and microarray photomask 3.1, and be fitted by seal frame 9
In 4 bottom of microelectrophoresis bath, arrange unanimously with microarray pH gradient column 1.
Microarray groove is carved in the 4 intermediate both sides of microelectrophoresis bath, consistent with microarray pH gradient 1 array arrangement of column；
Anode electrode silk 6 and cathode electrode silk 5 are built in microelectrophoresis bath bottom, and electrophoresis polarity keeps one with microarray pH gradient column 1
It causes.
The pH gradient refers to：The pH gradient range of each minisize pH gradient column in minisize pH gradient column 1 meets：
PH range=pH X~Y, wherein：0.3pH units≤pH Y-pH X≤12pH units,
2≤pH of pH X≤pH 11.7,2.3≤pH of pH Y≤pH 12.
The minisize pH gradient column length is 5mm~50mm, preferred 10~30mm.
The minisize pH gradient column width is 0.3mm~2.0mm, preferred 0.7~1.5mm.
The minisize pH gradient column thickness is 0.1mm~2.0mm, preferred 0.6~1.5mm.
The dry glue layer thickness of the minisize pH gradient column is 0.03~0.3mm, preferred 0.1~0.2mm.
The microarray pH gradient column battle array is formed by 3~960 minisize pH gradient column array arrangements, preferably by 12~
96 minisize pH gradient column array arrangements are formed, as shown in Figure 4 to 7.
Single group microarray pH gradient column chip is carried out at the same time equal electricity for 1~12 kind of different proteins sample in the present embodiment
Focusing electrophoresis (Fig. 4)；Two groups of microarray pH gradient column chips are carried out at the same time for up to 24 kinds of different proteins samples in Fig. 5
Electric focusing；Three groups of microarray pH gradient column chips are carried out at the same time equal electricity for up to 36 kinds of different proteins samples in Fig. 6
Focusing electrophoresis；Four groups of microarray pH gradient column chips the voltolisation such as are carried out at the same time for up to 48 kinds of different proteins samples in Fig. 7
Burnt electrophoresis efficiently solves a systems such as loading time caused by sample feeding in isoelectronic focusing electrophoretic device is long and complicated for operation
Row problem especially realizes the separation analysis of high-throughput quickly pH gradient focusing electrophoresis.
A kind of isoelectric-focusing electrophoresis chip of the microarray pH gradient column, Fig. 2 be using albumen marker into
The isoelectric focusing electrophoresis chip performance test (Fig. 2) of row microarray pH gradient column, experiment condition are：800 volts of voltage, electric current 1200
Microampere, 240 microlitres of loading total amount, loading time 30 minutes, focal time 4 minutes, imaging time 2 minutes.Experiment is shown
Superior performance：30 minutes sample sample introductions, 4 minutes electrophoretic focusing time carry out 24micro-columns imagings for 2 minutes and move
State is analyzed and the operation of simplicity, admirably solves the problems, such as four of classical IPG IEF.Meanwhile microarray Isoelectric Focusing
Chip of swimming has a high stability, high-throughput and protein band cutting recycling can be focused on microarray electrophoresis chip, very
Solve the problems, such as three of capillary/chip focusing technology well.
Above-mentioned specific implementation can by those skilled in the art under the premise of without departing substantially from the principle of the invention and objective with difference
Mode carry out local directed complete set to it, protection scope of the present invention is subject to claims and not by above-mentioned specific implementation institute
Limit, each implementation within its scope is by the constraint of the present invention.
Claims (8)
1. a kind of isoelectric focusing electrophoresis chip with microarray immobilization pH gradient column, which is characterized in that including：From top to bottom
Microarray holder, microelectrophoresis bath, microelectrophoresis bath, electrode and the microarray optical transmission window set gradually, wherein：Microarray holder
Bottom be equipped with several microarray microtrabeculaes pair of array arrangement, the end of each microarray microtrabeculae pair is equipped with miniature pH gradient
Column；Minisize pH gradient column includes：Minisize pH gradient glue positioned at microarray frame bottom and miniature support film, pH gradient glue is downward
Convenient for microarray pH gradient column sample sample introduction, minisize pH gradient column is come into full contact with by microarray holder and its own gravity with electrode
To form electrophoresis circuit.
2. isoelectric focusing electrophoresis chip according to claim 1, characterized in that the minisize pH gradient column includes：Position
Minisize pH gradient glue in end and miniature support film, wherein：PH gradient glue is convenient for microarray pH gradient glue sample sample introduction downward.
3. isoelectric focusing electrophoresis chip according to claim 1, characterized in that the minisize pH gradient glue has positive and negative
Polarity, and its positive and negative anodes polarity is consistent with microarray holder polarity.
4. isoelectric focusing electrophoresis chip according to claim 1, characterized in that the narrow window of microarray light transmission passes through close
Envelope frame is embedded in microelectrophoresis bath bottom, and the narrow window of microarray light transmission is consistent with the arragement direction of microarray pH gradient glue.
5. isoelectric focusing electrophoresis chip according to claim 1, characterized in that the narrow window of microarray light transmission includes saturating
Isotropic disk and microarray photomask.
6. isoelectric focusing electrophoresis chip according to claim 1, characterized in that the array arrangement refers to：By 12~
24 minisize pH gradient columns form single display pH gradient pole unit, and 1~8 single display pH gradient pole unit constitutes microarray
PH gradient column battle array, 12~192 minisize pH gradient columns may be implemented, and aquation sample introduction and the different samples of up to 192 kinds of progress are surveyed simultaneously
Examination.
7. isoelectric focusing electrophoresis chip according to claim 1, characterized in that the pH gradient, i.e., miniature pH gradient
The pH gradient range of each minisize pH gradient column in column meets：PH range=pH X~Y, wherein：0.3pH units≤pH
Y-pH X≤12pH units, 2≤pH of pH X≤pH 11.7,2.3≤pH of pH Y≤pH 12.
8. a kind of electrophoresis method based on chip described in any of the above-described claim, which is characterized in that directly use volley of rifle fire pipettor
When different samples to be added to the enterprising traveling sample of different microarray pH gradient columns simultaneously, then by minisize pH gradient column jointed anode
Wire electrode and cathode electrode silk are focused electrophoretic separation analysis.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111505094A (en) * | 2020-04-28 | 2020-08-07 | 上海交通大学 | Food feed species tracing identification method based on isoelectric focusing electrophoresis |
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| CA2364115A1 (en) * | 2001-11-27 | 2003-05-27 | Royce Technologies Llc, A Nevada Corporation | Method of two-dimensional gel electrophoresis for protein display |
| US20060049051A1 (en) * | 2004-09-03 | 2006-03-09 | Combisep, Inc. | Microfabricated chip and method of use |
| CN102120754A (en) * | 2010-11-30 | 2011-07-13 | 上海交通大学 | Miniature capillary array device for isoelectric focusing electrophoresis |
| CN103235026A (en) * | 2013-04-02 | 2013-08-07 | 上海交通大学 | Protein isoelectric focusing method and apparatus |
| CN208334265U (en) * | 2018-06-01 | 2019-01-04 | 上海交通大学 | Isoelectric focusing electrophoresis chip with microarray immobilization pH gradient column |
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2018
- 2018-06-01 CN CN201810554632.0A patent/CN108445069B/en active Active
Patent Citations (5)
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|---|---|---|---|---|
| CA2364115A1 (en) * | 2001-11-27 | 2003-05-27 | Royce Technologies Llc, A Nevada Corporation | Method of two-dimensional gel electrophoresis for protein display |
| US20060049051A1 (en) * | 2004-09-03 | 2006-03-09 | Combisep, Inc. | Microfabricated chip and method of use |
| CN102120754A (en) * | 2010-11-30 | 2011-07-13 | 上海交通大学 | Miniature capillary array device for isoelectric focusing electrophoresis |
| CN103235026A (en) * | 2013-04-02 | 2013-08-07 | 上海交通大学 | Protein isoelectric focusing method and apparatus |
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| CN111505094A (en) * | 2020-04-28 | 2020-08-07 | 上海交通大学 | Food feed species tracing identification method based on isoelectric focusing electrophoresis |
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