WO2016062231A1 - 凝胶电泳芯片 - Google Patents

凝胶电泳芯片 Download PDF

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Publication number
WO2016062231A1
WO2016062231A1 PCT/CN2015/092242 CN2015092242W WO2016062231A1 WO 2016062231 A1 WO2016062231 A1 WO 2016062231A1 CN 2015092242 W CN2015092242 W CN 2015092242W WO 2016062231 A1 WO2016062231 A1 WO 2016062231A1
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Prior art keywords
gel
gel electrophoresis
electrophoresis chip
strip
chip according
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PCT/CN2015/092242
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English (en)
French (fr)
Inventor
鲍坚斌
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鲍坚斌
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Priority claimed from CN201410563444.6A external-priority patent/CN104359962B/zh
Application filed by 鲍坚斌 filed Critical 鲍坚斌
Priority to US15/520,466 priority Critical patent/US10466200B2/en
Publication of WO2016062231A1 publication Critical patent/WO2016062231A1/zh

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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
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • C07K1/28—Isoelectric focusing
    • C07K1/285—Isoelectric focusing multi dimensional electrophoresis
    • 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
    • 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
    • G01N27/44791—Microapparatus

Definitions

  • the invention relates to the field of protein electrophoresis, in particular to a gel electrophoresis chip.
  • life science research has entered the post-genome era.
  • the main research object of life science turns to protein.
  • the whole genome sequence information is not enough to explain or speculate various life phenomena.
  • Protein is the performer of physiological function, the direct embodiment of life phenomenon, the structure and function of protein. The study will directly clarify the mechanism of change of life under physiological or pathological conditions and before and after drug intervention.
  • proteomic analysis of biological samples in order to determine the type of protein contained in a biological sample or to find a target protein of interest, it is first necessary to separate the proteins in the biological sample.
  • Existing two-dimensional gel electrophoresis analysis can separate proteins in biological samples in two dimensions. This method usually involves first performing protein electrophoresis (first-direction electrophoresis) according to the difference in isoelectric point of the protein. Separation is performed in one dimension, and then polyacrylamide gel electrophoresis (secondary electrophoresis) is performed according to the difference in molecular weight of the protein to separate proteins that have been separated in the first dimension in the second dimension.
  • the protein in the gel is visualized as a protein spot by staining the protein separated on the gel.
  • Different proteins will be located at different locations on the gel due to differences in isoelectric point and molecular weight. For biological samples, there are thousands of protein spots, and even more.
  • the peptide mixture is made into a sample target (ie, a target), and mass spectrometry is performed to obtain mass spectrometry information of the protein, such as peptide mass fingerprint and peptide sequence label.
  • the protein separated by the second-direction gel electrophoresis needs to be subjected to sample preparation including dyeing, gel-cutting, in-gel digestion, extraction of a peptide mixture, and target formation as described above before performing mass spectrometry.
  • sample preparation including dyeing, gel-cutting, in-gel digestion, extraction of a peptide mixture, and target formation as described above before performing mass spectrometry.
  • Each of the protein spots to be detected undergoes a step of gelatinization, in-gel digestion, peptide mixture extraction, and target formation one by one, and such an operation is feasible for a small amount of protein spot detection.
  • Due to the large number of protein spots contained in the biological sample it is very time consuming and laborious to perform sample preparation operations for each protein spot. Even with automatic glue cutters, automatic dezyrists, and automatic spotters, it is still difficult to process all of the protein spots on the gel one by one. This simplifies the second-direction electrophoresis operation and the pre-treatment operation before mass spectrometry
  • Biomicrochip technology offers this possibility, such as microfluidic chip technology, which has been extensively practiced in the field of proteomic analysis: a combination of isoelectric focusing and capillary electrophoresis techniques (AEHerr et al., Anal. Chem., 75, 1180-1187, 2003), a combination of isoelectric focusing and capillary gel electrophoresis techniques (Y. Li et al., Anal. Chem., 76, 742-748, 2004), capillary electrophoresis, fractionation, solidification Integration of phase extraction and electrospray ionization (ESI) techniques (QY Lu, J.-B. Bao, DJ Harrison, 11th Int. Conf. Miniatur. Syst. Chem. Life Sci., p. 44-46, 2007 )Wait.
  • ESI phase extraction and electrospray ionization
  • the object of the present invention is to provide a gel electrophoresis chip, which can perform protein separation, protein digestion, extraction of peptide mixture, and target mass spectrometry sample preparation steps in a biological sample completely in a chip, eliminating the prior art.
  • the step of dyeing and cutting in the sample preparation process, the digestion of all the proteins in the biological sample to be tested, the extraction of the peptide mixture and the time required for the target are digested, extracted and targeted by a protein spot in the prior art. The time is roughly the same. This greatly shortens the time required for mass spectrometry sample preparation and large sample preparation operations of biological samples containing a large amount of protein, enabling large-scale, high-throughput sample preparation and complete biological samples required for proteomics Proteomic analysis.
  • a gel electrophoresis chip comprising the gel electrophoresis chip
  • the second direction is perpendicular to the first direction.
  • the gel electrophoresis chip further comprises a second substrate on the gel strip and the isolation segment in contact with the gel strip and the isolation segment.
  • the gel electrophoresis chip further comprises forming on the same side of each gel strip, respectively A plurality of spacer strips that are in contact with the gel strip and have substantially the same thickness.
  • the gel electrophoresis chip further comprises an isolation strip formed on each side of each gel strip and in contact with the gel strip and having substantially the same thickness, and each of the separation strips is formed with at least one opening at each micro hole.
  • the opening serves as a passage for the current and a passage of a protein or peptide mixture in the gel strip into the microwell.
  • Gel strips in gel electrophoresis chips are used to separate proteins, including but not limited to polyacrylamide gel strips, agar or agarose gel strips, or starch gel strips, with polymerization methods and formulation options and traditional block shapes
  • the gel is the same.
  • the gel strip is used for second-direction separation in two-dimensional gel electrophoresis, the overall width of which preferably matches the length of the solid phase pH gradient strip of the first-direction gel electrophoresis.
  • the overall size of the chip consisting of all of the gel strips, micropores, spacers and spacers can be the same as a conventional block gel.
  • the gel strip has a width of from 1 ⁇ m to 1 cm, more preferably from 10 ⁇ m to 2 mm.
  • the materials of the isolation segment and the isolation strip are respectively selected from one or more of an inorganic material, an organic material, a polymer material, and a composite material.
  • the polymer material is selected from the group consisting of resin, rubber, and fiber. , plastic, photoresist, adhesive or coating, the material of the separator and the material of the insulation section may be the same or different.
  • the isolated segments used to form the microwell array and the spacers formed on the sides of the gel strip are unable to adsorb or separate proteins, thereby allowing proteins transferred from the gel strips to remain in the microwells. After the protein is separated or the separated protein is digested in the gel, the protein or peptide mixture is transferred from the gel to the microwell by applying a voltage or by applying an extract for further manipulation.
  • the isolation section has a width of from 1 ⁇ m to 5 mm.
  • the spacer has a width of from 1 ⁇ m to 5 mm.
  • each of the micropores has a width of from 1 ⁇ m to 1 cm, more preferably from 10 ⁇ m to 2 mm; and each of the micropores has a length of from 1 ⁇ m to 1 cm, more preferably from 10 ⁇ m to 2 mm.
  • the thickness of the isolation section, the separator and the gel strip are respectively from 1 ⁇ m to 1 cm, more preferably from 10 ⁇ m to 2 mm.
  • the gel electrophoresis chip further comprises a plurality of first blocking blocks respectively located between the gel strips at one end of the first plurality of gel strips.
  • the first stop is used to segment the protein from the solid phase pH gradient strip separated by the first direction electrophoresis to distribute the protein into the second direction gel strip lane.
  • the first stop is triangular.
  • the gel electrophoresis chip further comprises a plurality of second blocking blocks located between the gel strips at the other end of the first plurality of gel strips.
  • the second block is used to ensure a smooth transition of the electric field within each gel strip.
  • the gel electrophoresis chip further comprises terminating and communicating the first plurality of gel strips The first contact area at one end.
  • the gel electrophoresis chip further comprises a second contact region terminating and communicating with the other end of the first plurality of gel strips.
  • the first contact zone is, for example, a concentrated gel for placing, for example, a solid phase pH gradient gel strip and, for example, an electrode buffer strip, the solid phase pH gradient gel strip placement method and conventional electrophoresis.
  • the second contact zone is used to place, for example, an electrode buffer strip.
  • the first contact zone and the second contact zone may be reduced or even not disposed.
  • the materials of the first substrate and the second substrate are selected from one or more of an inorganic insulating material, an organic insulating material, a polymer insulating material and a composite material, preferably a glass piece, a quartz piece, a silicon carbide A sheet, a polymer sheet, or a silicon wafer whose surface is covered with silicon dioxide or other insulating layer.
  • An embodiment of the invention further includes a spare gel zone that can serve as a lane for the protein molecular weight marker and an electrode buffer strip when the protein or peptide mixture is extracted.
  • the gel electrophoresis chip of the invention is suitable for two-dimensional gel electrophoresis except diagonal electrophoresis, and the invention is applicable to ISO-DALT two-dimensional electrophoresis, IPG-DLAT two-dimensional electrophoresis, non-equilibrium pH gradient electrophoresis, non-denaturing BN/SDS- PAGE two-dimensional electrophoresis and CN/SDS-PAGE two-dimensional electrophoresis, etc., the present invention is not limited to the first-direction separation method.
  • microelectromechanical processing technology screen printing technology, 3D printing technology, and photolithography technology.
  • the high precision production ensures the consistency and repeatability of the two-dimensional gel electrophoresis chip, which improves the reproducibility of the two-dimensional gel electrophoresis analysis.
  • the specific operation method is as follows:
  • First-direction electrophoresis (isoelectric focusing electrophoresis) using solid phase pH gradient strip: this step is the same as the first-direction electrophoresis of conventional two-dimensional gel electrophoresis, that is, the protein sample is passed through a conventional solid phase pH gradient strip. Electrospray electrophoresis, sample separation based on the isoelectric point of the protein.
  • the solid phase pH gradient strip can be prepared by itself according to the existing method, or a commercially available product can also be used.
  • step 2) second-direction electrophoresis using a gel electrophoresis chip: the first-phase separated protein sample in the solid phase pH gradient strip of step 1) is subjected to strip balance and then transferred to the gel electrophoresis chip of the present invention.
  • second-direction electrophoresis is performed on the first contact region, and sample separation is performed based on the molecular weight of the protein.
  • In-gel digestion The whole gel electrophoresis chip separated by the second-direction electrophoresis obtained in the step 2) is subjected to in-gel digestion, that is, all the separated proteins in the biological sample on the electrophoresis chip are simultaneously subjected to in-gel digestion.
  • In-gel digestion sample preparation method according to the present invention and single protein spot in the prior art The same method is used for in-gel digestion.
  • the present invention also employs methods such as capillary dropping (dot matrix sample preparation), capillary spray methods, and automated instrument spray methods to apply the desired reagent to the surface of the chip.
  • all the protein spots in the present invention are simultaneously digested in one digestion process, and at the same time, a mixture of peptides corresponding to all the separated proteins is obtained, thereby eliminating the dyeing of the separated proteins and cutting and digesting one by one. step.
  • Extraction of the peptide mixture by adding the peptide extract or applying an extraction voltage to the gel strip, the respective peptide mixture in the gel strip is simultaneously transferred to the corresponding micropores, thereby realizing all the separated The peptide mixture of the protein is simultaneously extracted.
  • the direction in which the extraction voltage is applied is parallel to a second direction in which the second plurality of isolation segments extend.
  • the method of adding the peptide extract is the same as the method of applying the digestion reagent.
  • a protein or peptide mixture is made into a sample target according to different mass spectrometry ionization methods.
  • Methods of mass spectrometry ionization include, but are not limited to, matrix-assisted laser desorption ionization (MALDI), analytical electrospray ionization (DESI), desorption atmospheric photoionization (DAPPI), real-time direct analysis (DART), and the like.
  • MALDI matrix-assisted laser desorption ionization
  • DESI analytical electrospray ionization
  • DAPPI desorption atmospheric photoionization
  • DART real-time direct analysis
  • Ionization and mass spectrometry The peptide mixture or protein in the micropores is ionized, and the ionized sample is subjected to mass spectrometry to obtain mass spectrometry information.
  • mass spectrometry can obtain peptide mass fingerprints or peptide sequence tags, etc., through protein database search, identify the protein species in each microwell or discover new proteins.
  • the ion signal intensity data of all microporous proteins and peptides is a gel electrophoresis map, and the mass spectral data of each microwell is equivalent to one pixel, which is similar to MALDI imaging.
  • the gel electrophoresis chip provided by the present invention can be applied to second-direction electrophoresis of two-dimensional gel electrophoresis, and the conventional second-direction electrophoresis is improved.
  • the gel electrophoresis chip solves the problem of the second-dimensional electrophoresis of the two-dimensional gel electrophoresis and the sample preparation after the second-direction electrophoresis, and realizes the separation of all proteins and the in-gel digestion while separating proteins, without the need of dyeing and gel cutting steps.
  • the gel electrophoresis method according to the present invention can detect a low abundance protein and increase the probability of finding a target protein of interest.
  • Conventional mass spectrometry pretreatment operations require dyeing to show the location of the protein, and any dyeing method has a sensitivity limit and a tendency to protein staining, thus for those with low abundance or low sensitivity to dyes.
  • the protein may not be able to develop color and miss the chance of being detected.
  • the gel electrophoresis method of the present invention the gel electrophoresis chip does not require the above-described operation steps, and all the separated proteins are simultaneously processed, and all the separated proteins can be detected except for the sample which is lower than the sensitivity of the mass spectrometry detection.
  • the gel electrophoresis chip according to the invention is fabricated by microelectromechanical processing, screen printing technology, 3D printing technology, lithography technology, etc., with high precision, good repeatability, wide selection of materials and production parameters, and Separation of proteins into micropores for free use, can be applied to a variety of different mass spectrometry ionization techniques, can obtain digital two-dimensional gel electrophoresis spectra, can improve the reproducibility of two-dimensional gel electrophoresis, can be applied to most two-dimensional gel Electrophoresis systems and more.
  • Fig. 1 is a cross-sectional view showing a gel electrophoresis chip of the present invention.
  • Fig. 2a is a schematic view showing a micropore array region of a gel electrophoresis chip according to Embodiment 1 of the present invention. Dotted lines are used to indicate different areas, not actual ones.
  • Fig. 2b is a partially enlarged view showing the micropores of the gel electrophoresis chip shown in Fig. 2.
  • Fig. 2c shows a cross-sectional view taken along line AA of Fig. 2b.
  • Fig. 2d shows a cross-sectional view taken along line BB of Fig. 2b.
  • Fig. 3a is a partially enlarged view showing the micropores of the gel electrophoresis chip of Example 2 of the present invention.
  • Figure 3b shows a cross-sectional view of Figure 3a.
  • Fig. 4a is a partially enlarged view showing the micropores of the gel electrophoresis chip of Example 3 of the present invention.
  • Figure 4b shows a cross-sectional view of Figure 4a.
  • Fig. 5a is a partially enlarged view showing the micropores of the gel electrophoresis chip of Example 4 of the present invention.
  • Figure 5b shows a cross-sectional view of Figure 5a.
  • 110. a first substrate, 120. a microwell array, 130. a second substrate, 230. a first blocking block, 240. second barrier block, 260. first contact zone, 270. second contact zone, 280. spare gel zone, 290. spare gel zone, 220, 320, 420, 520. gel strip, 210, 310 , 410, 510.
  • Isolation section 311, 411, 412, 511, 512.
  • Isolation zone 250, 350, 450, 550.
  • FIG. 1 and 2 illustrate a gel electrophoresis chip according to Embodiment 1 of the present invention, comprising a first substrate 110, and a plurality of parallel polyacrylamide gel strips extending in a first direction on the first substrate, respectively. 220.
  • the width of the gel strip was 400 ⁇ m and the distance between adjacent gel strips was 1 mm.
  • An epoxy resin segment 210 perpendicular to the gel is formed between adjacent gel strips and has a width of 300 ⁇ m.
  • the gel strip has approximately the same thickness as the epoxy segment, for example about 600 ⁇ m.
  • the gel strip together with the isolation segments form an array 120 of micropores of size 0.6 mm x 0.7 mm, respectively.
  • a second substrate 130 in intimate contact with the gel strip 220 and the isolation segment, such as the epoxy segment 210, on the opposite side of the first substrate 110.
  • a plurality of first blocking blocks 230 are respectively located between the gel strips at one end of the first plurality of gel strips 220.
  • a plurality of second blocking blocks 240 are respectively located between the gel strips at the other ends of the first plurality of gel strips 220.
  • each gel strip gradually increases in width at both ends of the gel strip until it is in communication.
  • the first blocking block and the second blocking block are arranged in a region where the width of the gel strip is gradually increased and have a triangular shape.
  • the gel chip further includes a first contact region 260 formed at one end in the direction in which the gel strip extends to terminate and communicate with each of the gel strips 220.
  • This contact zone is used to place a gel sample that has been separated by electrophoresis in the first direction.
  • the first contact zone has an end face that is perpendicular to the direction in which the gel strip extends.
  • the gel chip further includes a second contact region 270 that terminates at the other end of the gel strip extending direction and communicates with each gel strip.
  • This second contact zone can for example be used to place an electrode buffer strip.
  • the first and second contact regions have a width of, for example, 2 cm.
  • a spare gel zone 280 and a spare gel zone 290 are disposed on the left and right sides of the first plurality of gel strips 220, respectively.
  • the first contact zone 260 is a concentrated gel, which is placed on the cathode buffer strip and the solid phase pH gradient strip during electrophoresis; and the second contact zone 270 is placed in the anode buffer strip during electrophoresis.
  • the first barrier sheet 230 dispenses proteins from the solid phase pH gradient strip into the corresponding gel strips.
  • the second barrier sheet 240 serves to ensure a smooth transition of the electric field within each gel strip.
  • the first contact zone 260 and the second contact zone 270 may not be provided.
  • an extraction voltage is applied to simultaneously transfer all of the protein from the gel strip 220 to the adjacent microwell 250.
  • all of the isolated proteins need to be subjected to in-gel digestion.
  • the gel electrophoresis chip of the invention can simultaneously digest all the proteins in one digestion process, and the extraction method of the digested peptide mixture can be, for example, by applying an extraction voltage, so that all the peptide mixtures are simultaneously from the gel strip. Transfer to the corresponding microwell 250. Alternatively, the peptide mixture can be transferred to the corresponding microwell 250 by adding a peptide extract to the microwell 250. With this extraction method, the peptide mixture in the same gel strip 220 is extracted into the micropores on both sides of the gel strip, i.e., because the same microwell 250 contains the mixture of peptides in the adjacent two gel strips 220.
  • the micropore 250 structure in FIG. 2 has a rectangular or square shape as shown in FIGS. 2b-2d
  • the gel electrophoresis chip has no limitation on the structure of the micropores.
  • any of the microporous structures of Figures 2-5, or other similar structural designs, may be employed.
  • the polyacrylamide sol can be printed first by screen printing. After the sol is completely cured into a gel, the isolated segment of the epoxy resin is printed.
  • the method of screen printing is well known to those skilled in the art and will not be described herein.
  • Gel Electrophoresis Chip This embodiment is modified on the basis of Example 1, and further includes a high-density polyethylene separator 311 disposed on the same side of each gel strip 320 and in contact with the gel strip 320.
  • the separator tape has substantially the same thickness as the gel strip and has a width of 150 ⁇ m, and the formed micropores 350 are as shown in Fig. 3a.
  • the protein components in each gel strip can only be extracted because the spacer is added. Located in the micropores away from the side of the barrier, it is avoided that the peptide mixture in the same gel strip 220 will be extracted into the micropores on both sides of the gel strip, and the same microwell 250 contains two adjacent gel strips. The loss of resolution of the isoelectric focusing electrophoresis caused by the mixture of peptides in 220 improves the resolution of isoelectric focusing electrophoresis.
  • the high-density polyethylene isolation section and the separator tape can be printed by means of 3D printing, and then the polyacrylamide sol can be printed and used after curing.
  • the method of 3D printing is well known to those skilled in the art and will not be described herein.
  • This embodiment is modified on the basis of Embodiment 1, except that the spacer tape 411 and the spacer tape 412 are disposed on both sides of each gel strip 420, respectively.
  • the material of the separator is the photoresist of Example 1, and the thickness is substantially the same as the thickness of the gel strip.
  • the spacer 411 forms two openings at each of the micro holes 450, namely, an opening 451 and an opening 452, and the isolation strip 412 forms an opening, that is, an opening 453, at each of the micro holes 450.
  • openings 451, 452 and 453 serve as current channels, while the openings 453 also serve as an outlet for the protein or peptide mixture when the protein or peptide mixture is extracted.
  • the protein or peptide mixture can be extracted by applying a voltage.
  • two different photoresists can be arranged by photolithography as the isolation segment and the isolation tape, and the gel solution is poured between the isolation bands, and the gel is cured to remove the opening.
  • Photoresist The method of photolithography is well known to those skilled in the art and will not be described herein.
  • This embodiment is modified on the basis of Embodiment 1, except that an isolation strip 511 and an isolation strip 512 are disposed on both sides of each gel strip 520, wherein the spacer strip 511 is formed at each micropore 550.
  • openings 551 and 552 are current channels while the opening 552 also serves as an outlet for the protein or peptide mixture when the protein or peptide mixture is extracted.
  • the protein or peptide mixture can be extracted by applying a voltage.
  • the protein or peptide mixture separated in the gel can be extracted in the micropores by applying a mixture of the extraction voltage or the protein or peptide mixture in the gel. In the middle, the extraction efficiency is improved.
  • Such gel electrophoresis chips have high resolution for proteins with low content in biological samples.

Abstract

一种凝胶电泳芯片，包括第一基片；形成在第一基片上的分别沿第一方向延伸且具有一定宽度的第一多个平行的凝胶条；以及形成在第一基片上，分别位于相邻的凝胶条之间且沿不同于第一方向的第二方向延伸的第二多个平行的隔离段，所述隔离段被布置为与所述凝胶条形成微孔阵列。这种凝胶电泳芯片可在实现传统的蛋白质双向凝胶电泳分离后，高通量地制备适合质谱分析的蛋白质样品，极大地缩短了质谱分析的前处理操作时间，因而适用于生物样品的蛋白质组学分析。

Description

凝胶电泳芯片 技术领域
本发明涉及蛋白质电泳领域，具体地，涉及一种凝胶电泳芯片。
背景技术
随着人类基因组计划的实施和推进，生命科学研究已进入了后基因组时代。在这个时代，生命科学的主要研究对象转向蛋白质，全基因组的序列信息不足以解释或推测各种生命现象，蛋白质是生理功能的执行者，是生命现象的直接体现者，对蛋白质结构和功能的研究将直接阐明生命在生理或病理条件下、药物干预前后的变化机制。
生物样品的蛋白质组学分析中，为了确定生物样品中包含的蛋白质种类或寻找感兴趣的靶蛋白，首先需要将生物样品中的蛋白质进行分离。已有的双向凝胶电泳分析法可以在两个维度对生物样品中的蛋白质进行分离，该方法通常包括先根据蛋白质等电点的差异通过等电聚焦电泳(第一向电泳)使蛋白质在第一维度进行分离，然后再根据蛋白质分子量的差异进行聚丙烯酰胺凝胶电泳(第二向电泳)使已在第一维度分离的蛋白质在第二维度分离。蛋白质经第二向凝胶电泳分离后，通过对凝胶上分离的蛋白质进行染色，使凝胶中的蛋白质以蛋白质斑点的形式显现出来。不同的蛋白质由于等电点和分子量的差异将位于凝胶的不同位置。对于生物样品来说，蛋白质斑点是成千上万的，甚至更多。为了鉴定蛋白质斑点中的蛋白质，需要切割含有蛋白质斑点的凝胶(即切胶)，然后将蛋白质在凝胶内消化(即胶内消化)成肽段混合物，提取肽段混合物，最后对应不同的质谱离子化法，把肽段混合物制成样品靶(即制靶)，进行质谱分析以获得该蛋白质的质谱信息，如肽质量指纹谱和肽序列标签等。因此，经所述第二向凝胶电泳分离后的蛋白质在进行质谱分析前需要经过包括如上所述的染色、切胶、胶内消化、提取肽段混合物和制靶的样品制备。每一个待检测的蛋白质斑点都要逐个地经历切胶、胶内消化、肽段混合物提取和制靶的步骤，对少量蛋白质斑点检测来说，这样的操作是可行的。但由于生物样品中包含的蛋白质斑点数量巨大，对每个蛋白质斑点进行样品制备操作将是非常费时费力的。即便使用自动切胶仪、自动酶解仪和自动点靶仪，要将凝胶上所有的蛋白质斑点逐个进行处理依然很困难。因而简化第二向电泳操作以及电泳后质谱分析前的预处理操作 是生物样品蛋白质组学所急需解决的问题。
生物微芯片技术提供了这种可能性，譬如微流控芯片技术在蛋白质组分析领域已有了大量的实践：等电聚焦和毛细管电泳技术的组合(A.E.Herr et al.,Anal.Chem.,75,1180-1187,2003)，等电聚焦和毛细管凝胶电泳技术的组合(Y.Li et al.,Anal.Chem.,76,742-748,2004)，毛细管电泳、分区分离(fractionation)、固相萃取、和电喷雾离子化(ESI)技术的集成(Q.Y.Lu,J.-B.Bao,D.J.Harrison,11th Int.Conf.Miniatur.Syst.Chem.Life Sci.,p.44-46,2007)等。
然而，目前这些尝试依旧不能满足蛋白质组分析中大量样品快速制备的需要，蛋白质组学需要一种更为实用的微芯片技术。
发明内容
本发明的目的是提供一种凝胶电泳芯片，使生物样品中的蛋白质分离、胶内消化、提取肽段混合物、和制靶等质谱分析样品制备步骤完全在芯片内进行，免去现有技术样品制备过程中染胶和切胶的步骤，使一个待测生物样品中全部蛋白质的消化、肽段混合物提取和制靶所需要的时间与现有技术中一个蛋白质斑点的消化、提取和制靶的时间大致一样。这极大地缩短了包含大量蛋白质的待测生物样品的质谱分析样品制备所需的时间和繁复的制样操作，可实现蛋白质组学所需的大规模、高通量的样品制备，完成生物样品的蛋白质组学分析。
为达到本发明的目的，本发明的技术方案如下：
一种凝胶电泳芯片，该凝胶电泳芯片包括
第一基片；
形成在第一基片上的分别沿第一方向延伸且具有一定宽度的第一多个凝胶条，这些凝胶条是实现蛋白质分离的泳道；
形成在第一基片上，分别位于相邻的凝胶条之间且沿不同于第一方向的第二方向延伸的第二多个隔离段，所述隔离段被布置为与所述凝胶条形成微孔阵列。
优选地，所述第二方向与所述第一方向垂直。
优选地，所述凝胶电泳芯片进一步包括位于所述凝胶条和所述隔离段上与所述凝胶条和所述隔离段接触的第二基片。
优选地，所述凝胶电泳芯片进一步包括分别形成在各凝胶条同一侧、与 凝胶条接触且厚度大致相同的多个隔离带。
优选地，所述凝胶电泳芯片进一步包括分别形成在每一凝胶条两侧、与凝胶条接触且厚度大致相同的隔离带，各隔离带在每一微孔处分别形成有至少一个开口，该开口用作电流的通道和凝胶条中蛋白质或肽段混合物进入所述微孔的通道。
凝胶电泳芯片中的凝胶条用于分离蛋白质，包括但不限于聚丙烯酰胺凝胶条、琼脂或琼脂糖凝胶条、或淀粉凝胶条，其聚合方法和配方选择与传统的块状凝胶相同。凝胶条用来做双向凝胶电泳中的第二向分离，其总体宽度优选与第一向凝胶电泳的固相pH梯度胶条长度相匹配。由所有所述凝胶条、微孔、隔离段和隔离带组成的芯片的整体尺寸可与传统的块状凝胶相同。
优选地，所述凝胶条的宽度为1μm-1cm，更优选10μm-2mm。
优选地，所述隔离段和隔离带的材料分别选自无机材料、有机材料、高分子材料和复合材料中的一种或多种，优选地，所述高分子材料选自树脂、橡胶、纤维、塑料、光刻胶、胶粘剂或涂料，隔离带的材料与隔离段的材料可相同或不同。用于形成微孔阵列的隔离段以及形成在凝胶条侧边的隔离带均不能吸附或分离蛋白质，从而使从凝胶条转移到微孔中的蛋白质保留在微孔中。蛋白质被分离之后或被分离的蛋白质在胶内消化之后，通过加上电压或通过施加提取液，将蛋白质或肽段混合物从凝胶转移到微孔中储存，以待进一步的操作。
优选地，所述隔离段的宽度为1μm-5mm。优选地，所述隔离带的宽度为1μm-5mm。
优选地，所述每个微孔的宽度为1μm-1cm，更优选10μm-2mm；所述每个微孔的长度为1μm-1cm，更优选10μm-2mm。
优选地，所述隔离段、所述隔离带与所述凝胶条的厚度分别为1μm-1cm，更优选10μm-2mm。
优选地，所述凝胶电泳芯片进一步包括在所述第一多个凝胶条一端分别位于凝胶条之间的多个第一阻挡块。该第一挡块用于分割来自经第一向电泳分离的固相pH梯度胶条的蛋白质，以使蛋白质分配到第二向凝胶条泳道中。优选地，所述第一挡块为三角形。
优选地，所述凝胶电泳芯片进一步包括在所述第一多个凝胶条另一端分别位于凝胶条之间的多个第二阻挡块。该第二阻挡块用来保证每个凝胶条内电场的平稳过渡。
优选地，所述凝胶电泳芯片进一步包括端接并连通所述第一多个凝胶条 一端的第一接触区。优选地，所述凝胶电泳芯片进一步包括端接并连通所述第一多个凝胶条另一端的第二接触区。例如对于水平电泳而言，所述第一接触区为例如浓缩胶，用于放置例如固相pH梯度凝胶条和例如电极缓冲液条，所述固相pH梯度凝胶条放置方法与传统电泳相同，第二接触区用来放置例如电极缓冲液条。对于垂直电泳而言，所述第一接触区和所述第二接触区可以缩小、甚至不设置。
优选地，所述第一基片和第二基片的材料选自无机绝缘材料、有机绝缘材料、高分子绝缘材料和复合材料中的一种或多种，优选玻璃片、石英片、碳化硅片、高分子聚合物片、或其表面覆盖了二氧化硅或者其它绝缘层的硅片。
本发明的一个实施例中进一步地包括备用凝胶区，所述备用凝胶区可用作蛋白质分子量标记物的泳道和在提取蛋白质或肽段混合物时放置电极缓冲液条。
本发明所述凝胶电泳芯片适用于除对角线电泳外的双向凝胶电泳，本发明适用于ISO-DALT双向电泳、IPG-DLAT双向电泳、非平衡pH梯度电泳、非变性BN/SDS-PAGE双向电泳和CN/SDS-PAGE双向电泳等等，本发明对第一向分离方法没有限制。
通过微机电加工技术、丝网印刷技术、3D打印技术、光刻技术中的一种或者多种组合技术制成。高精度的制作保证了双向凝胶电泳芯片的一致性和可重复性，进而提高了双向凝胶电泳分析的重现性。
当本发明所述凝胶电泳芯片用于常用的IPG-DLAT双向凝胶电泳时，具体操作方法如下：
1)利用固相pH梯度胶条的第一向电泳(等电聚焦电泳)：该步骤与传统双向凝胶电泳的第一向电泳相同，即将蛋白质样品通过传统的固相pH梯度胶条进行等电聚焦电泳，基于蛋白质的等电点进行样品分离。所述固相pH梯度胶条可以按照现有方法自行制备，也可以采用市售商品。
2)利用凝胶电泳芯片的第二向电泳：将步骤1)的固相pH梯度胶条中经过第一向分离的蛋白质样品经过胶条平衡，然后转移至本发明所述凝胶电泳芯片上例如第一接触区上进行第二向电泳，基于蛋白质分子量的大小进行样品分离。
3)胶内消化：将步骤2)得到的经第二向电泳分离的整块凝胶电泳芯片进行胶內消化，即对电泳芯片上生物样品中的所有被分离的蛋白质同时进行胶内消化。根据本发明的胶内消化制样方法与现有技术中对单一蛋白质斑点 进行胶内消化的方法一样。本发明也采用例如毛细管滴加法(点矩阵样品制备)、毛细管喷雾法、和自动化仪器喷雾法等方法将所需试剂施加到芯片表面。不同于现有方法，本发明中所有的蛋白质斑点在一个消化过程中同时消化，同时得到所有被分离的蛋白质所对应的肽段混合物，免除了对分离的蛋白质进行染色和逐个切胶和消化的步骤。
4)肽段混合物的提取：通过添加肽段提取液或者对凝胶条施加提取电压，将凝胶条中的各肽段混合物同时转移到相应的所述微孔中，从而实现所有被分离的蛋白质的肽段混合物同时提取。所述施加提取电压的方向平行于第二多个隔离段延伸的第二方向。肽段提取液的添加方法与消化试剂的施加方法相同。
5)制备质谱样品靶：根据不同的质谱离子化法把蛋白质或肽段混合物制成样品靶。质谱离子化的方法包括但不限于基质辅助激光解析离子化(MALDI)、解析电喷雾子化(DESI)、脱附常压光离子化(DAPPI)、实时直接分析技术(DART)等。以基质辅助激光解析离子化为例，向所述微孔中添加MALDI基质溶液，所有的蛋白质或肽段混合物和基质同时共结晶，所有所述微孔中的蛋白质或肽段混合物被一次性制成基质辅助激光解析质谱分析的样品，一次性完成所有蛋白质质谱分析的前处理操作。其中基质的添加方法与消化试剂的施加方法相同。
6)离子化及质谱分析：对所述微孔中的肽段混合物或蛋白质进行离子化，离子化后的样品进行质谱分析，得到其质谱信息。如质谱分析可得到肽质量指纹谱或肽序列标签等，通过蛋白质数据库检索，鉴定每个微孔中的蛋白质种类或发现新的蛋白质。将所有微孔的蛋白质和肽段的离子信号强度数据拼起来就是凝胶电泳图谱，每个微孔的质谱数据相当于一个像素，这和MALDI成像技术(MALDI imaging)相似。
本发明的有益效果如下：
1)本发明提供的所述凝胶电泳芯片可适用于双向凝胶电泳的第二向电泳，对传统的第二向电泳进行了改进。凝胶电泳芯片解决了传统的双向凝胶电泳的第二向电泳及第二向电泳后样品制备的难题，实现了在分离蛋白质同时完成所有蛋白质的分离和胶内消化，无须染色和切胶步骤；可同时将所有蛋白质或肽段混合物从所述凝胶条转移到相应的所述微孔中，无须逐个提取；可同时完成所有所述微孔中蛋白质或肽段混合物质谱分析的样品制备操作，无须逐个处理，因而蛋白质质谱分析的样品制备操作的时间极大缩短， 简化了操作，使所有的蛋白质都进行质谱分析成为可能。总之，在传统技术中这些是费时费力、容易出错、容易被污染、难以完全自动化的步骤，根据本发明的电泳芯片实现了蛋白质组分析所要求的高通量和自动化。
2)根据本发明的凝胶电泳方法可以检测到丰度低的蛋白质，提高找到感兴趣靶蛋白质的几率。传统的质谱分析的前处理操作需要染胶来显示蛋白质的位置，而任何一种染胶方法都有灵敏度的限制和蛋白质染色的倾向性，因而对于那些丰度低或含有对染料敏感的氨基酸少的蛋白质，可能不能显色，而错失被检测到的机会。根据本发明的凝胶电泳方法凝胶电泳芯片无需上述操作步骤，对所有被分离的蛋白质同时进行处理，除了低于质谱检测灵敏度的样品，使所有被分离的蛋白质都可以被检测到。
3)根据本发明的凝胶电泳芯片采用微机电加工、丝网印刷技术、3D打印技术、光刻技术等技术制作，精度高、重复性好，制作材料、制作参数的选择范围广，其可以将蛋白质分离到微孔中自由使用，可以适用多种不同的质谱离子化技术，可以得到数字化双向凝胶电泳谱图，可以提高双向凝胶电泳的重现性，可以适用绝大多数双向凝胶电泳系统等等。
所述凝胶电泳芯片的这些特性在生物样品的蛋白质组学分析中具有美好的前景。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出本发明的凝胶电泳芯片剖视图。
图2a示出本发明实施例1的凝胶电泳芯片微孔阵列区的示意图图。虚线用于标示不同的区域，而非实际存在。
图2b示出图2所示凝胶电泳芯片的微孔局部放大图。
图2c示出沿图2b的AA线剖视图。
图2d示出沿图2b的BB线剖视图。
图3a示出本发明实施例2的凝胶电泳芯片的微孔局部放大图。
图3b示出图3a的剖视图。
图4a示出本发明实施例3的凝胶电泳芯片的微孔局部放大图。
图4b示出图4a的剖视图。
图5a示出本发明实施例4的凝胶电泳芯片的微孔局部放大图。
图5b示出图5a的剖视图。
图中110.第一基片，120.微孔阵列，130.第二基片，230.第一阻挡块， 240.第二阻挡块，260.第一接触区，270.第二接触区，280.备用凝胶区，290.备用凝胶区，220，320，420，520.凝胶条，210，310，410，510.隔离段，311，411，412，511，512.隔离带，250，350，450，550.微孔，451，452，453，552，551.开口。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。为清楚且便于理解器件，附图中各部分未按比例绘制。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1：凝胶电泳芯片
图1和图2示出了根据本发明实施例1的凝胶电泳芯片，包括第一基片110，形成在第一基片上分别沿第一方向延伸的多个平行的聚丙烯酰胺凝胶条220。凝胶条的宽度为400μm，相邻凝胶条之间的距离为1mm。相邻凝胶条之间形成有垂直于凝胶的环氧树脂段210，宽度为300μm。凝胶条与环氧树脂段具有大致相同的厚度，例如约600μm。凝胶条与隔离段一起形成了大小分别为0.6mm×0.7mm的微孔的阵列120。根据本发明的一个实施方案，在所述第一基片110相对侧，与所述凝胶条220和所述隔离段例如环氧树脂段210紧密接触的第二基片130。
根据本发明的一个实施方案，在所述第一多个凝胶条220一端分别位于凝胶条之间的多个第一阻挡块230。在所述第一多个凝胶条220另一端分别位于凝胶条之间的多个第二阻挡块240。在该实施方案中，各凝胶条在其两个端部凝胶条的宽度逐渐增加直至连通。第一阻挡块和第二阻挡块被布置在凝胶条宽度逐渐增加的区域并具有三角形的形状。
根据本发明的一个实施方案，凝胶芯片进一步包括在凝胶条延伸方向的一端形成有端接并连通各凝胶条220的第一接触区260。该接触区用于放置已经第一向电泳分离的凝胶样品。优选地，该第一接触区具有垂直于凝胶条延伸方向的端面。根据本发明的一个实施方案，凝胶芯片进一步包括在凝胶条延伸方向的另一端端接并连通各凝胶条的第二接触区270。该第二接触区例如可用于放置电极缓冲液条。第一和第二接触区具有例如2cm的宽度。
根据本发明的一个实施方案，在所述第一多个凝胶条220的左右两侧分别布置备用凝胶区280和备用凝胶区290。
对于水平电泳系统而言，第一接触区260为浓缩胶，电泳时放置于阴极缓冲液条和固相pH梯度胶条；第二接触区270电泳时放置于阳极缓冲液条。第一阻挡片230将来自固相pH梯度胶条的蛋白质分配到相应的凝胶条中。第二阻挡片240用来保证每个凝胶条内电场的平稳过渡。对于垂直电泳系统，第一接触区260和第二接触区270可以不设置。
蛋白质在所述凝胶条220中实现分离后，施加提取电压，使所有蛋白质从所述凝胶条220内同时转移到旁边的所述微孔250中。如需对蛋白质的肽段混合物进行分析，则需要对分离的所有蛋白质进行胶内消化。本发明的凝胶电泳芯片可在一个消化过程中对所有蛋白质同时胶内消化，消化后的肽段混合物的提取方法例如可以是通过施加提取电压，使所有的肽段混合物同时从凝胶条中转移到相应的所述微孔250中。或者，可以通过向所述微孔250中添加肽段提取液，使肽段混合物转移到相应的微孔250中。以这种提取方法同一凝胶条220中的肽段混合物会被提取到凝胶条两边的微孔中，即因同一微孔250中包含相邻两条凝胶条220中的肽段混合物。
需要强调的是，尽管图2中的微孔250结构如图2b-2d所示具有长方形或正方形形状，但是凝胶电泳芯片对微孔的结构没有限制。根据蛋白质组分析的要求不同，可以采用图2-5中任一种微孔结构设计，或其它类似的结构设计。每张凝胶电泳芯片上的凝胶条数、环氧树脂段的数目和微孔的数目同样也没有限制，图2中40×30＝1200个微孔只是一个例子，仅用于示意。
根据该实施例的凝胶电泳芯片，可以通过丝网印刷的方式上先印刷聚丙烯酰胺溶胶。溶胶完全固化为凝胶后，再印刷环氧树脂的隔离段。丝网印刷的方法为本领域技术人员所熟知，在此不再赘述。
实施例2：凝胶电泳芯片
凝胶电泳芯片本实施例是在实施例1的基础上进行修饰，进一步包括布置在各凝胶条320同一侧并与凝胶条320接触的高密度聚乙烯隔离带311。隔离带与凝胶条具有大致相同的厚度，宽度为150μm，所形成的微孔350如图3a所示。
相比于实施例1的凝胶电泳芯片，在使用提取液对凝胶条中的肽段进行提取时，因为增加了隔离带，使每个凝胶条中的蛋白质组分只能被提取到位于远离隔离带一侧的微孔中，避免了因为同一凝胶条220中的肽段混合物会被提取到凝胶条两边的微孔中、同一微孔250中包含相邻两条凝胶条220中的肽段混合物而导致的等电聚焦电泳分辨率的损失，提高了等电聚焦电泳分辨率。
但是这种微孔设计不能通过施加电压的方式提取肽段混合物或蛋白质。
根据该实施例的凝胶电泳芯片，可以通过3D打印的方式上先打印高密度聚乙烯隔离段和隔离带等，再打印聚丙烯酰胺溶胶，固化后即可使用。3D打印的方法为本领域技术人员所熟知，在此不再赘述。
实施例3：凝胶电泳芯片
本实施例是在实施例1的基础上进行修饰，区别在于，在每一凝胶条420两侧分别布置隔离带411和隔离带412。隔离带的材料为实施例1中的光刻胶，厚度与凝胶条厚度大致相同。隔离带411在每一微孔450处形成2个开口，即开口451和开口452，隔离带412在每一微孔450处形成1个开口，即开口453。
其中所述开口451、452和453可作为电流通道，同时所述开口453还作为蛋白质或肽段混合物提取时蛋白质或肽段混合物的出口。
根据该实施例的凝胶电泳芯片，在蛋白质完成第二向分离后，可通过施加电压的方法提取蛋白质或肽段混合物。
根据该实施例的凝胶电泳芯片，可以通过光刻方式上先布置两种不同的光刻胶作为隔离段和隔离带，隔离带之间灌入凝胶溶液，凝胶固化后除去开口处的光刻胶。光刻的方法为本领域技术人员所熟知，在此不再赘述。
实施例4：凝胶电泳芯片
本实施例是在实施例1的基础上进行修饰，区别在于，在每一凝胶条520两侧的布置隔离带511和隔离带512，其中，隔离带511在每一微孔550处形成1个开口，即开口551，隔离带512在每一微孔550处形成1个开口，即开口552。
其中所述开口551和552为电流通道，同时所述开口552还作为蛋白质或肽段混合物提取时蛋白质或肽段混合物的出口。
根据该实施例的凝胶电泳芯片，在蛋白质完成第二向分离后，可通过施加电压的方法提取蛋白质或肽段混合物。
根据实施例3和实施例4的凝胶电泳芯片，通过施加提取电压对凝胶中的蛋白质或肽段混合物，可使分离在凝胶中绝大部分的蛋白质或肽段混合物被提取在微孔中，提高了提取效率。这样的凝胶电泳芯片对于生物样品中含量少的蛋白质具有高的分辨率。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在 上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (21)

  1. 一种凝胶电泳芯片，其特征在于，所述凝胶电泳芯片包括
    第一基片；
    形成在第一基片上的分别沿第一方向延伸且具有一定宽度的第一多个凝胶条；
    形成在第一基片上，分别位于相邻的凝胶条之间且沿不同于第一方向的第二方向延伸的第二多个隔离段，所述隔离段被布置为与所述凝胶条形成微孔阵列。
  2. 根据权利要求1所述的凝胶电泳芯片，其特征在于，所述第二方向与所述第一方向垂直。
  3. 根据权利要求1所述的凝胶电泳芯片，其特征在于，所述凝胶电泳芯片进一步包括位于所述凝胶条和所述隔离段上与所述凝胶条和所述隔离段接触的第二基片。
  4. 根据权利要求1所述的凝胶电泳芯片，其特征在于，所述凝胶电泳芯片进一步包括分别形成在各凝胶条同一侧、与凝胶条接触且厚度大致相同的多个隔离带。
  5. 根据权利要求1所述的凝胶电泳芯片，其特征在于，所述凝胶电泳芯片进一步包括分别形成在每一凝胶条两侧、与凝胶条接触且厚度大致相同的隔离带，各隔离带在每一微孔处分别形成有至少一个开口。
  6. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述凝胶条为用于分离蛋白质的凝胶条，优选聚丙烯酰胺凝胶条、琼脂或琼脂糖凝胶条、或淀粉凝胶条。
  7. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述凝胶条的宽度为1μm-1cm，优选10μm-2mm；所述凝胶条的厚度为1μm-1cm，优选10μm-2mm。
  8. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述隔离段的材料选自无机材料、有机材料、高分子材料和复合材料中的一种或多种；优选地，所述高分子材料选自树脂、橡胶、纤维、塑料、光刻胶、胶粘剂或涂料。
  9. 根据权利要求4或5所述的凝胶电泳芯片，其特征在于：所述隔离带的材料选自无机材料、有机材料、高分子材料和复合材料中的一种或多种；优选地，所述高分子材料选自树脂、橡胶、纤维、塑料、光刻胶、胶粘剂或 涂料，隔离带的材料与隔离段的材料可相同或不同。
  10. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述隔离段的宽度为1μm-5mm；所述隔离段的厚度为1μm-1cm，优选10μm-2mm。
  11. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述每个微孔的宽度为1μm-1cm，优选10μm-2mm，所述每个微孔的长度为1μm-1cm，优选10μm-2mm。
  12. 根据权利要求4或5所述的凝胶电泳芯片，其特征在于，所述隔离带的宽度为1μm-5mm。
  13. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述凝胶电泳芯片进一步包括在所述第一多个凝胶条一端分别位于凝胶条之间的多个第一阻挡块。
  14. 根据权利要求13所述的凝胶电泳芯片，其特征在于：所述凝胶电泳芯片进一步包括在所述第一多个凝胶条另一端分别位于凝胶条之间的多个第二阻挡块。
  15. 根据权利要求1所述的凝胶电泳芯片，其特征在于：所述凝胶电泳芯片进一步包括端接并连通所述第一多个凝胶条一端的第一接触区。
  16. 根据权利要求15所述的凝胶电泳芯片，其特征在于：所述凝胶电泳芯片进一步包括端接并连通所述第一多个凝胶条另一端的第二接触区。
  17. 根据权利要求1所述的一种凝胶电泳芯片，其特征在于：所述第一基片或第二基片的材料包括无机绝缘材料、有机绝缘材料、高分子绝缘材料、复合材料、或组合材料，优选玻璃片、石英片、硅片、碳化硅片或者高分子聚合物片。
  18. 根据权利要求1-17中任一所述的凝胶电泳芯片，其特征在于：所述凝胶电泳芯片通过微机电加工技术、丝网印刷技术、3D打印技术、光刻技术中的一种或者多种组合技术制成。
  19. 权利要求1所述的凝胶电泳芯片的使用方法，其特征在于，该方法包括如下步骤：对待测蛋白质样品利用固相pH梯度胶条进行第一向电泳；利用所述凝胶电泳芯片对经第一向电泳的pH梯度胶条进行第二向电泳；通过添加提取液或者对凝胶条施加提取电压，将凝胶条中的蛋白质转移到相应的微孔中；将各微孔中提取的蛋白质制成样品靶；将所述样品靶进行离子化及质谱分析，得到质谱信息。
  20. 权利要求1所述的凝胶电泳芯片的使用方法，其特征在于，该方法包括如下步骤：对待测蛋白质样品利用固相pH梯度胶条进行第一向电泳； 利用所述凝胶电泳芯片对经第一向电泳的pH梯度胶条进行第二向电泳；将经第二向电泳分离的凝胶电泳芯片进行胶内消化；通过添加肽段提取液或者对凝胶条施加提取电压，将凝胶条中的各肽段混合物转移到相应的微孔中；将各微孔中提取的肽段混合物制成样品靶；将所述样品靶进行离子化及质谱分析，得到质谱信息。
  21. 根据权利要求19或20所述的使用方法，其特征在于，该方法进一步包括将得到的表征各微孔的蛋白质和肽段的离子信号强度的数据拼起来获得数字化双向凝胶电泳图谱。
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