JP2011039002A - Micro electrophoresis chip and analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chip transferring a protein separated by a micro electrophoresis chip, onto a porous film, and measuring accurately and simply a position on the chip of the detected protein. <P>SOLUTION: In this micro electrophoresis chip 100 provided with: a substrate 107 (glass substrate); and a channel 102 (fine channel) for a specimen sample provided on the substrate 107, recessed parts (a first marker holding part 130 and a second marker holding part 132) for markers showing positions of both terminals of the channel 102 is provided on the substrate 107 separately from the channel 102. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a microelectrophoresis chip and an analysis method.

  After being synthesized, proteins undergo various structural modifications such as phosphorylation and glycosylation. This post-translational modification controls protein activity, intracellular localization, or extracellular secretion, and is greatly involved in the functional expression of the protein. Therefore, simple and rapid detection of post-translational modification is expected to be a useful technique not only in basic research but also in practical aspects such as disease diagnostic marker detection and drug efficacy evaluation. In cells, proteins exist as multiple isoforms having different isoelectric points and molecular weights by being modified. Isoforms are detected by Western blotting using an antibody after being separated by two-dimensional electrophoresis based on the difference in isoelectric point and molecular weight, transferred to a porous membrane, and the like. At this time, two-dimensional electrophoresis and film transfer require about two days in the long case.

  In recent years, with the development of microfabrication technology, research and development related to electrophoresis using microchannels formed on a substrate has been performed, and the time required for electrophoresis has been shortened. In addition, a “two-dimensional” protein analysis technique has been developed by combining isoelectric focusing using such a microelectrophoresis chip and a mass spectrometer.

  Non-Patent Document 1 introduces an example of an analysis technique in which a micro-electrophoresis chip for sample separation and a MALDI (Matrix-Assisted-Laser Desorption Ionization) type mass spectrometer are combined. In this microelectrophoresis chip, a micro flow path is formed by a groove etched by microfabrication on the surface of a glass substrate and a removable PDMS (polydimethylsiloxane) lid covering the micro flow path. The sample containing the protein introduced into the microchannel is separated by electrophoresis and then frozen to preserve the separated state. Next, the PDMS lid is peeled off while the chip is frozen, the sample in the microchannel is freeze-dried, a matrix solution is applied, and the molecular weight is measured by a mass spectrometer. Using this electrophoresis chip, it is possible to perform isoelectric focusing separation of proteins within a short period of several minutes, and then detect the proteins with a mass spectrometer.

Fujita, M .; Hattori, W .; Sano, T .; Baba, M .; Someya, H .; Miyazaki, K .; Kamijo, K .; Takahashi, K .; Kawaura, H .; J. et al. Chromatography A, 1111, pp200-205 (2006).

  However, in the analysis combining the isoelectric focusing by the microelectrophoresis chip described in Non-Patent Document 1 and the mass spectrometer, the physicochemical properties such as the isoelectric point and molecular weight of the protein can be known. It is extremely difficult to identify a specific protein contained in a complex mixture only by this information.

On the other hand, for protein identification, immunological techniques (Western blotting) using antibodies specific to the separated proteins and peptide mass fingerprinting (PMF) using mass spectrometers are generally used. It is done.
Here, in the microelectrophoresis chip described in Non-Patent Document 1, it is possible to directly access the protein in the channel by separating the protein and then peeling off the PDMS lid on the upper side of the channel. However, after the lid was peeled off and freeze-dried, it was separated by performing an immunological detection method involving a reaction operation in a solution on the flow path or an enzyme treatment that is a pretreatment step essential for the PMF method. The protein diffuses and the separated state collapses.

  Therefore, the separation state can be fixed by separating the protein with a microelectrophoresis chip, peeling the PDMS lid on the upper part of the flow path, and transferring the protein in the flow path to a porous film such as a nitrocellulose film. Conceivable. The depth of the flow channel is 10 to several tens of micrometers, and in fact, proteins in the flow channel can be transferred to a porous membrane such as a nitrocellulose membrane in a very short time, compared with the conventional method. It is expected that the time required for electrophoresis and transfer will be greatly shortened.

However, there are problems in transferring a sample from the microelectrophoresis chip disclosed in Non-Patent Document 1 to a porous membrane and applying an immunological detection method or a PMF method. This will be described with reference to FIG. FIG. 1A shows a protein band to be detected separated on the flow channel 102 of the substrate 101 after the lid is peeled off and lyophilized after isoelectric focusing using a microelectrophoresis chip. 103 is schematically represented. FIG. 1B shows a target protein band 105 detected on the nitrocellulose membrane 104. As can be seen from FIG. 1 (B), since there is no information regarding the channel position on the nitrocellulose membrane 104, the distance of the protein band 105 from the end of the channel 102 is unknown. For this reason, it is difficult to accurately grasp the position of the protein band 105.
In the immunological detection method, a protein is usually detected by chemiluminescence using an antibody labeled with peroxidase. The final result is often obtained as a spot on the X-ray film that is exposed to a chemiluminescent signal emitted from the protein of interest. It is difficult to associate the spots on the X-ray film with the position of the band in the flow path.
Further, in immunological detection, it is not easy to distinguish an erroneous signal derived from dirt other than a porous film or a channel on a chip that is often observed. In addition, in order to perform enzymatic digestion of protein on the porous membrane, it is necessary to accurately add the enzyme to the position corresponding to the flow path, and it is necessary to grasp the position of the flow path on the membrane.
As described above, there is a problem as described above in transferring the sample from the microelectrophoresis chip disclosed in Non-Patent Document 1 to the porous membrane and applying the immunological detection method or the PMF method.

  The object of the present invention has been made in view of the above circumstances, and in order to transfer a sample separated by a microelectrophoresis chip to a porous membrane and perform analysis by an immunological method or a mass spectrometer, Microelectrophoresis chip that can easily associate the position of the signal derived from the target protein detected on the membrane or X-ray film with the position on the chip channel, analysis of proteins using the microelectrophoresis chip It is to provide a method.

According to the present invention,
A substrate and a flow path for a specimen sample provided on the substrate,
There is provided a microelectrophoresis chip in which concave portions for holding a marker substance indicating positions of both end portions of the flow path are provided on the substrate apart from the flow path.

Moreover, according to the present invention,
Using the microelectrophoresis chip,
Applying a voltage across the flow path to separate the specimen sample in the flow path;
Introducing a marker substance into the recess;
A step of bringing a transfer member into contact with the substrate surface, and transferring the specimen sample in the flow path and the marker substance in the recess to the transfer member;
Detecting the specimen sample transferred to the transfer member,
In the step of detecting the specimen sample, the position data of the specimen sample on the transfer member is associated with the detection data of the specimen sample on the basis of the positions of the marker substances indicating the positions of both ends of the flow path. The analysis method of analyzing the specimen sample is provided.

  According to the present invention, a specimen sample can be accurately detected.

It is the conceptual diagram which shows the electrophoresis using a general microelectrophoresis chip | tip, and the conceptual diagram which shows the nitrocellulose film | membrane after transcription | transfer. It is the conceptual diagram of the electrophoresis using the microchip of this Embodiment, and the conceptual diagram of the nitrocellulose film | membrane after transcription | transfer. It is sectional drawing of the flow-path longitudinal direction of a microelectrophoresis chip. It is a conceptual diagram of transfer from a microelectrophoresis chip to a porous membrane. The rabbit serum introduced into the flow path of the microelectrophoresis chip of the present invention was detected by anti-rabbit IgG antibody after membrane transfer. (A) An image of two-dimensional electrophoresis of HRP. (B) It is a figure which shows the HRP activity detection on the nitrocellulose membrane of a present Example. (A) Two-dimensional electrophoresis image of green fluorescent protein (GFP). (B) It is a figure which shows the migration pattern (fluorescence image) on the microelectrophoresis chip of GFP. (C) It is a figure which shows the result of the immunological detection using the anti- GFP antibody of a present Example. It is a flowchart which shows the analysis method of this Embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

FIG. 2A schematically shows the substrate 107 of the microelectrophoresis chip 100 of the present embodiment. FIG. 3 schematically shows the microelectrophoresis chip 100 of the present embodiment.
The microelectrophoresis chip 100 of the present embodiment will be described.
The microelectrophoresis chip 100 includes a substrate 107 (glass substrate) and a sample sample channel 102 (fine channel) provided on the substrate 107, and a marker substance indicating the positions of both ends of the channel 102. The holding recesses (the first marker holding part 130 and the second marker holding part 132) are provided on the substrate 107 so as to be separated from the flow path 102.
As the specimen sample, a protein (which may be subjected to a predetermined treatment such as SDS treatment), a polymer such as DNA or sugar, or the like can be used. In this embodiment, an example using a protein will be described.
The marker holding units 130 and 132 hold optically detectable marker molecules.
The transfer member to which the protein or marker molecule is transferred is not particularly limited. For example, a porous membrane (protein blotting membrane) can be used. As the porous film, for example, a nitrocellulose film, PVDF (Polyvinylidene difluoride), or the like can be used.

Here, an outline of the present embodiment will be described.
In the present embodiment, when the protein separated by the microelectrophoresis chip 100 is transferred to a porous membrane and analyzed by an immunological method or a mass spectrometer, it is placed on the porous membrane or X-ray film. The position of the signal derived from the target protein to be detected and the position data (or isoelectric point data) on the flow channel 102 can be easily associated with the signal of the marker molecule. Thus, since the position of the flow path 102 on the porous membrane can be grasped by using the markers indicating the positions of both ends of the flow path 102, the protein in the flow path 102 can be accurately detected.
In addition, (i) first detection data by an immunological method can be obtained together with isoelectric point data of the target protein. In addition, (ii) the second detection data can be accurately obtained by the mass analyzer using the position data of the protein. (I) In addition to the analysis of (ii), (iii) since the protein position data and the isoelectric point data match, the first detection data and the second detection data of the protein can be linked and analyzed. . Such analysis can identify the protein.

In the present embodiment, at least one first marker holding portion 130 and second marker holding portion 132 are provided at each of both end portions (first end portion, second end portion) of the flow path 102. (FIG. 2A). Here, the first end is between the flow path 102 and the first reservoir 124, and the second end is between the flow path 102 and the second reservoir 126. In other words, the marker holding units 130 and 132 may be provided so as to indicate the position information of the first end and the second end, respectively.
Therefore, when the protein bands (the first protein band 140 and the second protein band 142) in the channel 102 are transferred, the first protein band 150 and the second protein band 152 are transferred to the nitrocellulose membrane 104. At the same time, the first marker band 120 and the second marker band 122 are transcribed. The position of each band on the nitrocellulose film 104 coincides with the position of the band on the substrate 107 with high accuracy. That is, the relative positional relationship of each band on the substrate 107 is also maintained on the nitrocellulose film 104.
Therefore, the position of the first marker band 120 indicates the position of the first end of the flow path 102, and the position of the second marker band 122 indicates the position of the second end of the flow path 102. (FIG. 2B). Here, in FIG. 2B, a “+” mark indicating the upper left of the chip is written on the nitrocellulose film 104 in order to clearly indicate that the image on the chip surface is inverted.

  The shape of the marker holding portions 130 and 132 is not particularly limited, but can be a rectangle or a triangle in plan view of the substrate 107. When the marker holding portions 130 and 132 are rectangular, the marker bands 120 and 122 are also rectangular, and the longitudinal direction indicates a position corresponding to the end of the flow path 102 (FIG. 2B). When the marker holding portions 130 and 132 are triangular, the marker bands 120 and 122 are also substantially triangular, and the extension line of the apex of the triangle indicates the position corresponding to the end of the channel 102.

  Moreover, the marker holding | maintenance parts 130 and 132 may further be provided in the position which opposes on both sides of the flow path 102. That is, the first end portion and the second end portion may be provided with a pair of first marker holding portions 130 and a pair of second marker holding portions 132, respectively, with the flow channel 102 interposed therebetween. . Accordingly, the straight line connecting the pair of marker holding portions 130 and 132 positioned opposite to each other indicates the positions of both end portions of the channel 102 (FIG. 2A). Thereby, as shown in FIG. 2B, the straight line connecting the pair of marker bands 120 and 122 positioned opposite to each other indicates the positions of both end portions of the channel 102. For this reason, on the nitrocellulose membrane 104, the position corresponding to the end of the channel 102 can be grasped more accurately.

Furthermore, as shown in FIG. 2A, a pair of marker holding portions 108 (concave portions) are provided on the substrate 107 in the extending direction of the flow channel 102, particularly in the extending direction of the central line of the flow channel 102. Also good.
As shown in FIG. 2B, the pair of marker bands 121 corresponding to the marker holding portion 108 can accurately grasp the trajectory of the flow path 102 on the nitrocellulose membrane 104.
2B, on the nitrocellulose membrane 104, the extension lines of the pair of first marker bands 120, the extension lines of the pair of second marker bands 122, and the pair of marker bands 121 The extension lines are arranged so as to be orthogonal. The intersection of the extension lines indicates the end of the channel 102. Thereby, the position of the both ends of the flow path 102 on the nitrocellulose membrane 104 and its trajectory can be determined more accurately.
In addition, a marker holding portion may be disposed so as to surround the flow path 102 (may be surrounded continuously or discontinuously). At this time, the region surrounded by the marker band on the nitrocellulose membrane 104 indicates the shape of both ends and the orbit of the channel 102.

  As shown in FIG. 2, the flow path 102 communicates with the first reservoir 124 and the second reservoir 126 at both ends, respectively. A liquid containing a specimen sample is introduced into the channel 102. In the channel 102, the components (first protein band 140, second protein band 142) in the sample are separated by isoelectric point separation. At this time, the flow path 102 includes an isoelectric point separation region where a pH gradient is formed, a pair of electrodes (not shown) for applying an electric field to the isoelectric point separation region, and a specimen sample and a sample in the isoelectric point separation region. And a sample introduction part (reservoir 124, 126) for introducing a liquid containing an electrode solution. When an electric field is applied between the reservoirs 124 and 126, the entire flow path 102 can be used as an isoelectric point separation region.

As described above, in the present embodiment, the region from the first end to the second end indicates the isoelectric point separation region of the flow path 102. Therefore, in the nitrocellulose membrane 104, the region from the position of the first marker band 120 (corresponding to the first end portion) to the second marker band 122 (corresponding to the second end portion) is the same as above. It corresponds to the electric point separation region.
As described above, the isoelectric point separation region information in the flow channel 102 appears accurately in the nitrocellulose membrane 104 by the position information of the marker bands 120 and 122.
That is, according to the present embodiment, the isoelectric points of the first protein band 150 and the second protein band 152 can be grasped even in the nitrocellulose membrane 104. Therefore, according to the present embodiment, the isoelectric point data (position data) of each protein band, which is the same as the isoelectric point data obtained in the flow channel 102, can be acquired also in the nitrocellulose membrane 104.

The shape of the flow path 102 may be, for example, an I shape, a Y shape, a U shape, or the like when viewed from the substrate 107 direction. The cross-sectional shape perpendicular to the extending direction of the flow path 102 may be, for example, a rectangular shape, a square shape, a trapezoidal shape, a triangular shape, or the like. In addition, a plurality of flow paths 102 may be formed on the substrate 107.
Moreover, in the flow path 102, a flow path width can be 0.4-2 mm, for example, and a flow path length can be 30-50 mm, for example.

  The material of the substrate 107 is not particularly limited, and for example, glass such as quartz, silicone resin such as polydimethylsiloxane (PDMS), and silicon may be used. In this embodiment, a glass substrate is used as the substrate 107.

As shown in FIG. 3, the microelectrophoresis chip 100 of the present embodiment further includes a PDMS lid 110 and a glass lid 111 on a substrate 107.
In the microelectrophoresis chip 100 of the present embodiment, the lids (PDMS lid 110, glass lid 111) may be made of a plate-like member. Thereby, the upper part of the flow path 102 can be reliably coat | covered. Moreover, in the microelectrophoresis chip 100, the upper surface of the flow path 102 (microspace) can be covered with a removable lid (PDMS lid 110, glass lid 111) during use. For this reason, it is possible to perform a process such as electrophoresis on the specimen sample while suppressing evaporation and drying of the contents (specimen sample and electrolyte) in the flow path 102, leakage of the contents, or invasion of foreign substances. . In addition, after use, when removing the lid from the surface of the substrate 107, the lid can be removed in a direction perpendicular to the surface of the substrate 107. For this reason, it is possible to reliably prevent the specimen sample in the flow channel 102 from moving to the gap between the substrate 107 and the lid by capillary action and causing contamination of the contents. Further, the lid can be removed and the upper portion of the flow channel 102 can be opened while stably holding the sample sample in the flow channel 102, so that mixing of the sample sample in the flow channel 102 can be suppressed. . For example, according to the microelectrophoresis chip 100 of the present embodiment, mixing of components that are spatially separated in one channel 102 by electrophoresis or the like can be suppressed.
Further, after use, the flow path 102 can be opened, so that it is possible to directly operate the contents present in an arbitrary region in the flow path 102 section. Therefore, a quick and reliable operation is possible.

  Here, as means for pressing the lid (PDMS lid 110 and glass lid 111) on the surface of the substrate 107, for example, a screw, a hydraulic device, a hydraulic device, or a pneumatic device can be employed. Accordingly, the substrate 107 and the lid can be detachably fixed in accordance with the purpose of use of the microelectrophoresis chip 100.

In the microelectrophoresis chip 100, the inside (bottom surface and side surface) of the flow path 102 and the marker holding portions 108, 130, and 132 can be hydrophilic. Accordingly, even when the flow channel 102 and the marker holding units 108, 130, and 132 are fine, (i) liquid (specimen sample) is supplied to the flow channel 102, and (ii) marker molecules are applied to the marker holding units 108, 130, and 132. Can be more reliably introduced. For example, the surface of the substrate 107 can be made hydrophilic by ozone ashing or coating.
Further, regions other than the channel 102 and the marker holding units 108, 130, 132, that is, the surface of the substrate 107 in the vicinity of the channel 102 and the marker holding units 108, 130, 132 (recesses) can be made hydrophobic. Thereby, it is possible to prevent (i) liquid (specimen sample) from overflowing from the flow path 102 and (ii) marker molecules from the marker holding portions 108, 130, 132 to the upper surface of the substrate 107. For example, the surface of the substrate 107 can be subjected to water repellent treatment by coating a water repellent material such as Teflon (registered trademark).

  In addition, as marker molecules (marker substances), alkaline phosphatase, peroxidase, fluorescent substance, phosphorescent substance, Coomassie blue, and proteins labeled or stained with alkaline phosphatase, peroxidase, fluorescent substance, phosphorescent substance or Coomassie blue, etc. should be used. Can do.

A method for manufacturing the microelectrophoresis chip of this embodiment will be described. For example, as a microelectrophoresis chip, a microchannel chip described in Japanese Patent Application No. 2005-513937 and a chip appropriately modified according to experimental conditions can be adopted.
In the present embodiment, first, an optical mask corresponding to the shape of the flow path 102 is prepared. Then, an optical resist is applied on the substrate 107 by a spin coating method. Subsequently, using a mask prepared in advance, the optical resist is optically exposed to a flow pattern and developed, thereby producing a resist pattern. Then, using the obtained pattern as a mask, the flow path 102 and the respective liquid reservoirs (the first reservoir 124 and the second reservoir 126) communicating therewith are formed by dry etching or wet etching of the substrate 107.
As a specific example of the manufacturing method, first, an electron beam drawing resist is applied to a glass substrate having a thickness of about 0.5 mm, and electron beam irradiation is performed using an electron beam drawing apparatus. After the development, for example, a resist pattern having a channel width of 0.4 to 2 mm, a channel length of 30 to 50 mm, and a long side of 1 to 2 mm and a short side of 0.5 to 1 mm is obtained as a positioning marker holder. The center line in the longitudinal direction of the marker holding part is made to coincide with the extension of the flow path center line in the holding part 108 and to the end of the flow path in the holding part 109. The depth of the channel is several μm to several tens of μm, and a columnar separation structure having a cylinder diameter of 50 to 1000 nm and a distance between the nearest cylinders of about 50 to 1000 nm is installed on the bottom surface of the channel. An appropriate distance between the columns is several μm to several tens of μm. Next, using the resist as a mask, grooves are formed on the surface of the glass substrate by reactive ion etching using CF ₄ gas or the like, and the resist is removed by ashing.

Next, the analysis method of the present embodiment will be described. The analysis method of the present embodiment relates to protein analysis, particularly to protein identification.
FIG. 8 shows a flowchart of the analysis method of the present embodiment.
The analysis method of the present embodiment includes a step of using the microelectrophoresis chip 100 of the present embodiment and applying a voltage across the flow channel 102 to separate the specimen sample (protein) in the flow channel 102. (S100), a step of introducing marker molecules into the recesses (marker holding portions 108, 130, 132), and a transfer member (nitrocellulose film 104) in contact with the surface of the substrate 107, A step of transferring the marker molecule in the recess to the transfer member (S102), and a step of detecting the specimen sample transferred to the transfer member (S104).
In the analysis method of the present embodiment, the sample sample position data on the transfer member and the sample sample detection data are associated with each other by using the positions of the marker molecules indicating the positions of both ends of the flow path 102 as a reference. Analyze the sample.

  In the analysis method of the present embodiment, the components of the specimen sample (protein mixture) to be analyzed are separated in the flow path 102 (S100). Specifically, in the flow channel 102, electrophoretic separation is performed with appropriate physical or chemical properties such as size and isoelectric point. At this time, after preparing a sample solution for electrophoresis and introducing it into the channel 102, an electrode (for example, platinum) is inserted into each of the first reservoir 124 and the second reservoir 126, and a voltage is applied between the electrodes. Then, the separation operation is performed. Further, instead of the method of separating by applying voltage, a separation method of applying pressure may be used.

Here, the solution for electrophoresis is appropriately selected according to the use, but may be a solution for isoelectric focusing. Isoelectric focusing is one of electrophoresis methods for separating an amphoteric carrier according to an isoelectric point (pI) specific to the amphoteric carrier such as a protein that is a component in a sample. pI refers to the pH at which the positive and negative charges of the components in the sample are exactly equal. The components in the sample have a charge corresponding to the pH of the solution in which they are dissolved, and electrophorese to each pI region. When each pi is reached, the electrophoretic mobility of the components in the sample becomes zero, and the electrophoresis is terminated. By this phenomenon, the components in the sample can be concentrated and separated for each pI. In addition to the components in the sample to be separated, the solution for isoelectric focusing includes amphoteric carriers for forming a pH gradient. In order to create a pH gradient in the flow channel 102, for example, a solution for isoelectric focusing is introduced into the flow channel 102, and then an acid solution is introduced into the first reservoir 124 and an alkaline solution is introduced into the second reservoir 126. And a voltage may be applied between the electrodes arranged in the reservoir.
By isoelectric focusing, the specimen sample can be accurately separated into protein bands (per pI). In addition, the position where the protein band appears by isoelectric focusing is highly reproducible.

  Subsequently, the marker molecule is introduced into the marker holding unit 108, 130, 132. The introduction method is not particularly limited, but can be introduced using, for example, a dropper. The introduction time is not particularly limited, and may be before the specimen sample is separated.

  Subsequently, after the separation, the separated sample is dried by a method such as freeze-drying, and fixed to the separated position in the flow path 102. By these procedures, an analytical sample such as protein is dried and fixed on the microelectrophoresis chip 100.

Subsequently, the specimen sample (first protein band 140, second protein band 142) in the channel 102 and the marker molecules in the marker holding units 108, 130, 132 are transferred to the nitrocellulose membrane 104 (S102). ).
In order to transfer, the substrate 107 and the nitrocellulose film 104 may be brought into contact with each other. For example, a pressure can be applied between the substrate 107 and the nitrocellulose film 104 for transfer. Specifically, as shown in FIG. 4, a nitrocellulose film 104 that has been subjected to a predetermined treatment is brought into close contact with the upper surface of the substrate 107, and an acrylic plate 113 and a weight of about 500 g (weight 114) are placed on the upper portion of the film through a filter paper 112. ) Can be transferred to the nitrocellulose membrane 104 (the first protein band 140 and the second protein band 142). As other transfer means, voltage or air pressure (suction force) can also be used.
As described above, the nitrocellulose film 104 to which the protein band and the marker band are transferred can be obtained.

Subsequently, the protein indicated by the band in the nitrocellulose film 104 obtained in the above step is analyzed (S104).
Thereafter, the protein (first protein band 150, second protein band 152) and marker molecule (first marker band 120, second marker band 122, marker band) are obtained by a method suitable for the target protein and marker molecule. 121) are detected (FIG. 2B). At this time, the protein and the marker molecule can be detected simultaneously.

Hereinafter, each detection method will be described in detail.
In the immunological method (Western blotting method) according to the present embodiment, detection is performed by reacting an antibody with a target protein. By combining the excellent resolution of electrophoresis and the high specificity of the antigen-antibody reaction, the target protein can be detected from a plurality of protein bands (the first protein band 150 and the second protein band 152).
For the detection of protein, an antibody or a fluorescent sample may be used. In the case of using an antibody, a primary antibody is reacted with the target protein, and a secondary antibody labeled with, for example, AP (Alkaline phosphatase) or HRP (Horse radish peroxidase) is reacted with this primary antibody, and coloring by enzyme activity is performed. And detect chemiluminescence. When a fluorescent sample is used, exposure to the X-ray film is detected, or a scanner capable of detecting the fluorescent sample is used. These detection methods facilitate the detection of trace amounts of protein. In addition, the technique using a fluorescent sample has good sensitivity and excellent quantitativeness.

  Thus, the target protein contained in a trace amount in the protein solution of the specimen sample can be clearly detected by utilizing an antigen-antibody reaction or the like with high specificity. Thereby, it can be determined whether the protein present in the band (spot) on the nitrocellulose film 104 is the target protein, and the protein can be identified for each band.

The effect of this Embodiment is demonstrated.
In the present embodiment, a protein with high antibody specificity can be detected by the presence or absence of a spot. Further, as described above, the position of the signal derived from the target protein detected on the porous membrane or the X-ray film and the isoelectric point data on the flow channel 102 are easily associated with the signal of the marker molecule. Can do. Therefore, the isoelectric point of the protein spot (band) can be determined from the position information of the marker bands 120 and 122. As described above, in the present embodiment, both the first detection data (presence / absence of spot) by the immunological method and the isoelectric point data of the spot indicated by the target protein are obtained on the nitrocellulose membrane 104. be able to. For this reason, since it can detect from two viewpoints in one process, protein (detection object) can be detected correctly. Two proteins can identify this protein.

  In the present embodiment, position data corresponding to the flow path 102 can be accurately grasped on the nitrocellulose membrane 104 based on the position information of the marker bands 120 and 122. Therefore, if the spot on the X-ray film is outside the range of the position data, it can be easily determined that the spot is not a target protein band in the channel 102. Thus, it can be easily understood whether or not the spot is a band of the target protein. Therefore, the protein can be accurately detected by preventing misidentification of the band (signal) of the target protein.

Moreover, in mass spectrometry according to the present embodiment, a protein can be identified by collating a peptide mass fingerprint, which is mass spectrometry data of a protein enzyme digest, with a protein database. A protein band (spot) on the nitrocellulose membrane 104 is subjected to an enzyme treatment, and mass fragment data, that is, a peptide mass fingerprint, is obtained by mass spectrometry of the obtained fragment peptide mixture by MALDI method. At this time, after peptide fragmentation, a matrix which is an ionization accelerator is added and mass spectrometry is performed. A matrix suitable for the protein band on the nitrocellulose membrane 104 can be added using a predetermined method such as a spray method or a dropping method.
In the enzyme treatment, a protein is cleaved at a specific site in the amino acid sequence using a proteolytic enzyme (such as trypsin). For this reason, the mass peak data (second detection data) obtained in the present embodiment is protein-specific data.

In the present embodiment, position data corresponding to the flow path 102 can be accurately grasped on the nitrocellulose film 104 based on the position information of the marker bands 120 and 122. Therefore, the enzyme can be accurately added at a position corresponding to the flow path 102 on the nitrocellulose membrane 104. As a result, operational errors are reduced and reproducibility is increased, so that proteins can be detected accurately.
Moreover, since the position (position corresponding to the flow path 102) which performs mass spectrometry is known on the nitrocellulose membrane 104, protein can be detected accurately.

In the present embodiment, the second detection data (peptide mass fingerprint) by the mass analyzer and the position data from both ends of the protein flow channel 102 are known. This position data corresponds to the isoelectric point data. Therefore, based on the protein position data obtained from the position information of the marker bands 120 and 122, the first detection data (the type of antibody to which the protein specifically reacts) and the second detection data (mass data) are obtained. Can be tied.
As described above, in this embodiment, since basic protein data (position data, particularly isoelectric point data) and a plurality of types of detection data can be associated with each other, each detection data is integrated to more accurately identify the protein. Can be identified.

  In addition, in the detection of protein-specific activity according to the present embodiment, a physiologically active substance (an antibody, a fluorescent substance, a staining substance, etc.) is added to the protein band on the nitrocellulose membrane 104 to measure absorbance and luminescence. Etc. Thereby, the activity specific to the desired protein, particularly the enzyme activity can be detected.

  In the present embodiment, the same effect as that obtained in the case of the immunological method can be obtained. Moreover, the third detection data (enzyme activity) can be further integrated with basic protein data (position data, particularly isoelectric point data) by detecting the intrinsic activity of the protein. Thereby, protein can be identified more correctly.

As described above, in the present embodiment, the positioning marker molecule added to the concave portion of the positioning marker holding portion maintains the relative positional relationship between the microchannel and the protein separated on the channel, and the porous membrane Since it is transferred to the top, the position of the protein signal detected on the porous membrane or the X-ray film in the flow path can be easily and accurately measured.
In the present embodiment, proteins separated by electrophoresis in a fine channel formed on a glass substrate are transferred to a porous film such as a nitrocellulose film and detected by an immunological method in a subsequent process. The present invention relates to a method, and particularly provides a chip capable of specifying a signal derived from a protein and measuring a position on the chip quickly, accurately and simply in detecting a protein transferred onto a porous membrane. Can do.
In the present embodiment, a specific protein in a biological sample can be quickly and specifically separated and detected using a microelectrophoresis chip, and protein expression analysis, marker protein detection for disease and drug efficacy determination Alternatively, it can be applied to detection of specific proteins in agricultural products and foods.

Example 1
The microelectrophoresis chip 100 used in this embodiment will be described. As the microelectrophoresis chip 100, a microchannel chip described in Japanese Patent Application No. 2005-513937 (WO 2005/026742 pamphlet) or a chip appropriately modified according to experimental conditions can be employed. A microchip was produced in accordance with the above manufacturing method. The channel 102 is linear, the channel length is 35 mm, and the width and depth are 2 mm and 10 μm, respectively. The flow channel 102 is composed of an array of elliptical pillar-shaped separation structures. The major axis of the pillar is 30 μm, the minor axis is 10 μm, and the interval between the pillars is 10 μm. Further, the depth of the channel 102 and the positioning marker holders 108, 130, and 132 was 10 μm. The PDMS lid 110 was brought into close contact with the substrate 107 to form the flow path 102, and a glass lid 111 was further placed on the top in order to strengthen the PDMS lid 110.

After transferring to the nitrocellulose membrane 104, the rabbit serum IgG was detected using an anti-rabbit IgG antibody to verify whether the protein sample could be transferred. PBS (Phosphate Buffered Saline, containing 8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ in 1 liter of pure water, pH 7.4) 1 μl of rabbit serum diluted to 100 was injected into the first reservoir 124, aspirated from the opposite second reservoir 126, and introduced into the channel 102. After freezing the chip, the lid was peeled off, and 0.2 μl of rabbit serum diluted with PBS to 1/100 was added to the marker holders 108, 130, 132, and then freeze-dried. The nitrocellulose membrane 104 is washed in advance in a solution containing 30% methanol, 25 mM Tris-HCl, 192 mM glycine for 10 minutes, further washed with pure water for 5 minutes, and then excess water is removed with Kimwipe (registered trademark) or the like. Then, no moisture remained on the film surface. The wet nitrocellulose membrane 104 pre-treated as described above is brought into close contact with the top surface of the chip, and two to three dried filter papers 112 are placed on the top, on which an acrylic plate 113 and a heavy object of about 500 g (weight) 114), the proteins (the first protein band 140 and the second protein band 142) were transferred to the nitrocellulose membrane 104 (FIG. 4). The transfer can also be performed by applying an appropriate pressure using an instrument such as a roller. Blocking was performed by incubating the nitrocellulose membrane 104 in Block Ace (registered trademark, Dainippon Pharmaceutical) at room temperature for 30 minutes. A peroxidase (HRP) -labeled anti-rabbit IgG antibody (MPBiomedicals) diluted 3000-fold with PBS containing 3% bovine serum albumin (Sigma) is applied over the entire nitrocellulose membrane 104 (0.2-0.0.2). 4 ml / 1 cm ² nitrocellulose membrane) and incubating for 2 hours at room temperature while preventing drying in a humidity-controlled chamber. Excess antibody was washed while shaking the nitrocellulose membrane 104 in TBS-T buffer (20 mM Tris-HCl, pH 7.4, 9 g / L NaCl, 0.05% Tween-20) for 15 minutes. After further washing twice, the nitrocellulose membrane 104 was reacted with a chemiluminescent substrate (Enhanced Chemiluminescence detection reagent, GE Healthcare) for 1 minute. The nitrocellulose membrane 104 was wrapped and adhered to an X-ray film (Hyper Film ECL, GE Healthcare) to detect light generated by the chemiluminescence reaction.

  As shown in FIG. 5, the rabbit IgG was detected uniformly throughout the shape of the channel 102 (in FIG. 5, the solid line indicates the position of the positioning marker of the channel on the nitrocellulose membrane). ). In addition, the channel end coincided with the positioning marker (first marker band 120, second marker band 122). Thus, in this Embodiment, it turned out that the position of the both ends of the flow path 102 on the nitrocellulose membrane 104 can be correctly grasped | ascertained by the position of a marker band.

(Example 2)
(Detection of isoelectric point of protein)
The solution was prepared as follows. cIEF gel (8.25 μl), carrier ampholite (pH 4.0-6.0, 0.25 μl), fluorescent marker (0.75 μl mixture of pI 4.0, 4.5 and 5.5, Fluka), HRP Water was added to 1 μl (Sigma, 5.6 μg / ml) to a total volume of 12.5 μl. The anode electrode solution is prepared by mixing 10 μl of a mixture of 5 μl of 1M phosphoric acid solution and 50 μl of cIEF gel and 90 μl of cIEF gel. A 30 mM aqueous sodium hydroxide solution was used as the cathode electrode solution.

  Using a micropipette, 1 μl of the sample solution was injected into the anode reservoir and introduced into the flow path by capillary action and suction by a vacuum pump from the cathode side. After injecting 8 μl of each electrode solution into the cathode and anode reservoir, the total amount was recovered to remove excess sample solution and to wash the reservoir. 7 μl of electrode solution was injected into each reservoir. Isoelectric focusing was performed by applying 1500 V to both electrodes for 1 minute and then applying 3000 V for 12 minutes. The temperature of the chip during electrophoresis was set to 10 ° C. using a Peltier element. After completion of the electrophoresis, the chip temperature was quickly lowered to −30 ° C., and the solution in the flow path was frozen. After freezing, the lid was peeled off, 0.2 μl of HRP (5.6 μg / ml) was added to the positioning marker holding part, and freeze-drying was performed. The PDMS lid was removed and lyophilization was performed at -20 ° C. After lyophilization, the position of the fluorescent marker was detected using a fluorescent microscope. The sample was transferred to a nitrocellulose membrane by the method described in Example 1. The transferred HRP was detected in the same manner as in Example 1 using the Enhanced Chemiluminescence detection reagent.

FIG. 6A shows an image of two-dimensional electrophoresis of HRP (in FIG. 6, the solid line shows the position of the positioning marker of the flow path on the nitrocellulose membrane).
According to Sigma, the HRP preparation used in Example 2 is said to contain at least 5 isoforms with different pI. This HRP sample was analyzed by ordinary two-dimensional electrophoresis. Specifically, HRP (2 μg) was separated by isoelectric focusing at pH 3-10 in the first dimension and SDS-PAGE (12.5% polyacrylamide) in the second dimension, and then visualized by silver staining. At least 5 spots of HRP were observed in the pH range of 5.3 to 6.7 (FIG. 6A).

  This HRP sample was subjected to isoelectric focusing with the microelectrophoresis chip of the present embodiment, transferred to nitrocellulose, and then detected by observing the activity on the membrane. As a result, four bands were found as shown in FIG. These bands were found to be in the region of pI 5.4 to 5.9 based on the position of the fluorescent marker on the channel and the position of the positioning marker.

Example 3
(Isoelectric focusing and immunological detection of green fluorescent protein)
The sample solution was prepared as follows. cIEF gel (8.25 μl, Beckman), carrier ampholite (pH 4.0-6.0, 0.25 μl, Beckman), fluorescent marker (0.75 μl of a mixture of pI4.0, 4.5 and 5.5, Fluka), green fluorescent protein (GFP, Becton Dickinson, 0.25 μg / μl) 0.5 μl was added with water to a total volume of 12.5 μl. The anode and cathode electrode solutions were prepared in the same manner as in Example 2.

  Injection of the sample solution into the flow path, injection of the electrode solution, and isoelectric focusing were performed in the same manner as in Example 2. After the electrophoresis was completed, the chip was quickly lowered to -30 ° C. to freeze the solution in the flow path. After freezing, the lid of PDMS was peeled off, 0.2 μl of GFP (0.25 μg / μl) was dropped on the positioning marker holder, and freeze-dried at −20 ° C. After lyophilization, the position of the fluorescent marker was detected using a fluorescent microscope.

FIG. 7 (A) shows a two-dimensional electrophoresis image of green fluorescent protein (GFP) (in FIG. 7, the solid line indicates the position of the positioning marker of the flow path on the nitrocellulose membrane).
The Becton Dickinson GFP used in Example 3 is reported to contain at least two isoforms with different pI. As shown in FIG. 7A, GFP (1.5 μg) was separated by isoelectric focusing at pH 4-7 in the first dimension and SDS-PAGE (12.5% polyacrylamide) in the second dimension. Later, it was visualized by silver staining. Three spots were seen in the range of pH 4.9-5.2.

FIG. 7B shows a migration pattern (fluorescence image) on a GFP microelectrophoresis chip.
Isoelectric focusing was performed using a microelectrophoresis chip, and after freeze-drying, a fluorescence image of GFP on the chip was obtained with an image scanner (Typhoon 9400, GE Healthcare). That is, GFP (0.125 μg) was subjected to isoelectric point separation using a carrier ampholite having a pH of 4 to 6, and after lyophilization, a separation pattern of GFP was observed on a chip using a fluorescent scanner. As shown in FIG. 7B, although the pI value is a little low, 4.1 to 4.2, in addition to one major band, there are two minor bands as in the result of normal two-dimensional electrophoresis. Admitted. Therefore, although the isoelectric points were slightly different, three GFP bands were confirmed as in normal two-dimensional electrophoresis.

FIG. 7C shows the result of immunological detection using an anti-GFP antibody.
It transferred to the nitrocellulose film | membrane by the method of Example 1, and blocked. Anti-GFP antibody (Zymed, diluted 750 times with PBS containing 3% bovine serum albumin) was applied to a nitrocellulose membrane in the same manner as in Example 1, and incubated at room temperature for 2 hours while preventing drying in the chamber. After washing in the same manner as in Example 1, an HRP-labeled anti-mouse IgG antibody (MP Biomedicals) diluted 3000 times with PBS containing 3% bovine serum albumin (Sigma) was applied, followed by incubation and washing as before. Further, after reacting with the chemiluminescent substrate as before, a signal was detected with an X-ray film (FIG. 7C). The pI of these bands was the same pI value obtained from the fluorescence scanner from comparison with the positioning marker and the fluorescent marker.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

DESCRIPTION OF SYMBOLS 100 Microelectrophoresis chip 101 Substrate 102 Channel 103 Protein band 104 Nitrocellulose membrane 105 Protein band 106 Reservoir 107 Substrate 108 Marker holding part 110 PDMS lid 111 Glass lid 112 Filter paper 113 Acrylic plate 114 Weight 120 First marker band 121 Marker band 122 2nd marker band 124 1st reservoir 126 2nd reservoir 130 1st marker holding part 132 2nd marker holding part 140 1st protein band 142 2nd protein band 150 1st protein band 152 1st 2 protein bands

Claims (13)

A substrate and a flow path for a specimen sample provided on the substrate,
A microelectrophoresis chip, wherein a recess for holding a marker substance indicating positions of both end portions of the flow path is provided on the substrate apart from the flow path.   The microelectrophoresis chip according to claim 1, wherein a pair of the concave portions are provided so as to face each other with the flow channel interposed therebetween.   The microelectrophoresis chip according to claim 1, wherein the concave portion is provided in an extending direction of the flow path.   The microelectrophoresis chip according to claim 1, wherein the inside of the recess is hydrophilic.   The microelectrophoresis chip according to claim 1, wherein the substrate surface in the vicinity of the recess is hydrophobic. Using the microelectrophoresis chip according to any one of claims 1 to 5,
Applying a voltage across the flow path to separate the specimen sample in the flow path;
Introducing a marker substance into the recess;
A step of bringing a transfer member into contact with the substrate surface, and transferring the sample sample in the flow path and the marker substance in the recess to the transfer member;
Detecting the specimen sample transferred to the transfer member,
In the step of detecting the specimen sample, the position data of the specimen sample on the transfer member is associated with the detection data of the specimen sample with reference to the positions of the marker substances indicating the positions of both ends of the flow path. And analyzing the specimen sample.   The analysis method according to claim 6, wherein the specimen sample is analyzed by integrating the position data of the specimen sample and a plurality of types of the detection data.   The analysis method according to claim 6 or 7, wherein the detecting step is a step of detecting the specimen sample by an immunological method using an antibody.   The analysis method according to claim 6 or 7, wherein the detecting step is a step of detecting the specimen sample by a mass spectrometry method.   The analysis method according to claim 6 or 7, wherein the detecting step detects an activity specific to the specimen sample.   The analysis method according to claim 6, wherein the detecting step is performed simultaneously with the step of detecting the position of the marker substance.   12. The analysis according to claim 11, wherein in the step of detecting the position of the marker substance, the position of the specimen sample in the flow path is determined on the transfer member based on the detected position of the marker substance. Method.   The marker substance is at least one selected from the group consisting of alkaline phosphatase, peroxidase, fluorescent substance, phosphorescent substance, Coomassie blue, and protein labeled or stained with alkaline phosphatase, peroxidase, fluorescent substance, phosphorescent substance or Coomassie blue. The analysis method according to claim 6, comprising: