JP2010185703A - Sample analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample analysis method which enables the rapid and accurate analysis of the isoform contained in a sample. <P>SOLUTION: The method includes a step of dyeing and capturing the chemical substance in the sample by a phosphor, a step of washing off the non-captured substance in the sample, a step of recovering the captured substance, a step of performing the isoelectric point electrophoresis of the sample after the recovery of the captured substance, and a step of detecting the fluorescence intensity of the sample after the isoelectric point electrophoresis. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a sample analysis method using a fluorescence detection method of a result of electrophoresis of chemical substances, and more particularly to a sample analysis method capable of analyzing a difference in isoform quantitatively and with high reproducibility.

  In recent years, with the development of comprehensive protein analysis technology (proteome analysis technology), there is a lot of movement to measure the expression information of multiple proteins in vivo at once, and to use it for medical, drug discovery and scientific research. .

  Here, the proteome is a coined word that associates a protein with a genome, and represents the entire protein encoded in the genome. Drug discovery is the process of discovering and designing drugs in medicine, biotechnology, and pharmacy.

In particular, it has been reported that some changes in proteins (post-translational modification, processing, etc.) are closely related to life mechanisms, rather than simple protein expression levels.
However, these isoforms (proteins having slightly different structures) cannot be distinguished by immunoassay systems such as ELISA (Enzyme Linked Immuno-Sorbent Assay: Eliza), which are widely used.

  On the other hand, distinction can be made by using a proteome analysis technique such as two-dimensional electrophoresis, but the analysis requires about two days, and there is a problem in processing many samples. As a technique that can solve the above-described problems of two-dimensional electrophoresis, there is a method using a microchip and a mass spectrometer described in Patent Document 1.

Schematic diagrams of the microchip are shown in FIGS.
5A is a plan view of the microchip, FIG. 5B is a partially enlarged view of a wavy line portion of the flow path shown in FIG. 5A, and FIG. FIG. 5B is a sectional view taken along line Vc-Vc in FIG.
A one-dimensional flow path 102 is formed on the glass substrate 101. A pillar structure 104 having a large number of protrusions protruding in a direction perpendicular to the glass substrate 101 is formed in the pillar region 103 indicated by oblique lines in the flow path 102.
Here, the microchip is a very small (several mm square or less) substrate to which a semiconductor microfabrication technology is applied.

Next, a method for analyzing a sample using a microchip will be described with reference to FIGS.
FIGS. 6A to 6E are process diagrams showing a method for manufacturing the microchip shown in FIGS. 5A to 5C.
First, a sample 106 in which a carrier ampholite (amphoteric carrier) or the like is mixed with a reagent necessary for electrophoresis is dropped onto one end (left end in the figure) of the flow path 102 by a pipette 105 (FIG. 6A).
The dropped sample 106 spreads over the entire channel 102 by capillary action. Next, phosphoric acid is dropped onto one end (left end) of the flow path 102 and an aqueous sodium hydroxide solution is dropped onto the other end (right end in this case) (FIG. 6B).
Thereafter, electrodes 108 are introduced into both ends of the channel 102, and a positive voltage is applied to the acid (phosphoric acid) side and a negative voltage is applied to the alkali (sodium hydroxide) side to perform isoelectric focusing (FIG. 6). (C)).
After electrophoresis, the electrode 108 is removed, the sample 106 is dried, and then a matrix 110 such as sinapinic acid is applied to the entire flow path 102 from the needle 109.
Here, the matrix 110 is a substance that promotes ionization of the test sample, which is easily ionized (FIG. 6D).

The applied matrix 110 mixes with the sample 106 in the flow path 102 to form a matrix 110 -sample mixture 111.
Subsequently, the microchip is introduced into the MALDI-TOF type mass spectrometer, and the laser 112 is irradiated to obtain a mass spectrum at the irradiation position.
The MALDI-TOF mass spectrometer is a device that determines the mass of a substance by combining a matrix-assisted laser desorption ionization (MALDI) mechanism with a time-of-flight (TOF) mass spectrometer (MS). Yes, suitable for analysis of biopolymers.

  By performing laser irradiation at each point in the flow path, a mass spectrum that can be placed at each isoelectric point value is obtained. As a result, as in the two-dimensional electrophoresis, protein expression information characterized by two parameters of isoelectric point and molecular weight can be obtained, and isoforms can be easily distinguished. Moreover, the time required for the analysis is 1 to several hours, and the time can be significantly reduced as compared with two-dimensional electrophoresis.

Further, Patent Documents 2 and 3 are cited as techniques related to sample analysis.
The invention described in Patent Document 2 is an invention relating to a method for measuring hyaluronic acid, specifically, a reagent containing a hyaluronic acid binding protein (labeled hyaluronic acid binding protein) modified with a labeling substance, and hyaluronic acid It is performed by contacting a sample containing acid, separating hyaluronic acid and labeled hyaluronic acid binding protein, and measuring the labeled substance in the complex or the free labeled hyaluronic acid binding protein. It is.

  According to the invention described in Patent Document 2, hyaluronic acid can be easily measured with high accuracy.

  The invention described in Patent Document 3 relates to a difference detection method using a large number of suitable dyes, specifically, a method for comparing protein composition between at least two different cell samples: a) preparing an extract of a protein obtained from each of said at least two cell samples; (b) a set of compatible luminescent dyes selected from dyes capable of covalently binding to proteins in said protein extract Each dye in the set has (1) a net charge that maintains the overall net charge of the protein at the covalent bond, and any one of the dyes The relative movement of the labeled protein has ionic and pH characteristics that are similar to the relative movement of the protein labeled with another dye in the set; 2) emits luminescence at a wavelength sufficiently different from the emitted luminescence of the remaining dyes in the set, giving a different detectable light signal; (c) each extract of the protein in step (a) Reacting with the different dyes of the set in step (b) to obtain dye-labeled proteins; (d) mixing each of the dye-labeled proteins to form a single mixture of different dye-labeled proteins (E) separating a dye-labeled protein of interest in the mixture; and (f) detecting a difference in luminescence intensity between different dye-labeled proteins of interest by luminescence detection. .

  According to the invention described in US Pat. No. 6,057,049, a simple, relatively fast and reliable method that eliminates the problems associated with gel distortion or column variability and compares and contrasts the protein content of different samples. It is said that it is possible to provide a reliable method.

Japanese Patent No. 4074821 Re-specialized WO2002101389 Special table 2003-506718 gazette

Tim Wehr, Roberto Rodfiguez-Diaz, and Mingde Zhu, "Capillary Electrophoresis of Proteins", from Marcel Dekker Inc. ISBN: 0-8247-0205-0 (Figure 15)

  However, since the microchip disclosed in Patent Document 1 uses mass spectrometry for detection, there is a problem that the quantitative and reproducibility in measuring the expression level of each isoform is poor.

  In addition, in the methods disclosed in Patent Documents 2 and 3, there is a possibility that substances in the sample that have not been captured may remain, and thus there is a possibility that accurate analysis cannot be performed.

  Therefore, an object of the present invention is to provide a sample analysis method capable of quickly and accurately analyzing an isoform contained in a sample.

  The method of the present invention comprises a step of staining and capturing a chemical substance in a sample with a phosphor, a step of washing away the substance in the sample that has not been captured, a step of recovering the captured substance, and a sample after recovery And a step of detecting the fluorescence intensity of the sample after the isoelectric focusing.

  ADVANTAGE OF THE INVENTION According to this invention, provision of the sample analysis method which can perform the exact analysis of a sample is realizable.

(A)-(d) is explanatory drawing for demonstrating the pre-processing for performing sample analysis. (A) is a top view of the microchip used for the sample analysis method concerning this invention, (b) is the elements on larger scale of the pillar area | region of the microchip shown to (a), (c) is It is the IIc-IIc sectional view taken on the line of (b). (A)-(d) is an example of the process figure to which the sample analysis method concerning this invention is applied. (A)-(d) is explanatory drawing for demonstrating the pre-processing of a sample. (A) is the top view of a microchip, (b) is the elements on larger scale of the wavy line part of the flow path shown to (a), (c) is the Vc-Vc line | wire cross section of (b) FIG. (A)-(e) is process drawing which shows the manufacturing method of the microchip shown to Fig.5 (a)-(c).

(Characteristic)
In the present embodiment, the target protein in the document is fluorescently labeled, and then the target is supplemented with an antibody or the like and then washed. Thereafter, the target molecule is recovered, and the recovered liquid is subjected to isoelectric focusing, By observing the converged band with fluorescence, only the isoform captured by the antibody is rapidly and quantitatively measured.

  Here, isoelectric focusing is an electrophoretic technique in which isoelectric focusing (pI) of proteins is used for separation and isoelectric focusing measurement and analysis of the target protein. The charge at the amino acid side chain, amino terminal, and carboxyl terminal constituting the protein changes depending on the pH condition, and the pH value at which the total charge becomes zero is the isoelectric point.

(First embodiment)
Next, a first embodiment of the present invention will be described in detail with reference to FIGS. 1 (a) to (d), FIGS. 2 (a) to (c), and FIGS. 3 (a) to (d). To do.
FIGS. 1A to 1D are explanatory diagrams for explaining preprocessing for performing sample analysis.
A fluorescent labeling reagent is added to the sample containing the target molecule 201, and the target molecule 201 is labeled with the phosphor 203. However, in this case, not only the target molecule 201 but also the non-target molecule 202 is labeled simultaneously (FIG. 1 (a)).

Next, the antibody 204 against the target molecule 201 is fixed to a support such as glass, and the sample solution obtained in FIG. Only the target molecule 201 is captured by the antibody 204 by the antigen-antibody reaction.
Here, the antibody is a general term for immunoglobulins that react specifically with an antigen, and is composed of two heavy chains and two light chains, and has a Y-shape. An antibody is a basic glycoprotein that plays a central role in immune responses, and has a neutralizing action, an opsonin effect, and a complement activating action (FIG. 1 (b)).

  After that, when the sample solution is discarded and replaced with the buffer solution, only the target molecule 204 captured by the antibody 204 remains (FIG. 1C).

  Next, by replacing with an acidic buffer solution, the bond between the target molecule 202 and the antibody 204 is cut, and the target molecule 204 is recovered (FIG. 1 (d)).

Carrier amphorite or the like is mixed with the solution containing the collected target molecules 204, and isoelectric focusing is performed. As a device for performing isoelectric focusing, a microchip as shown in FIGS. 2A to 2C can be used as an example.
2A is a plan view of a microchip used in the sample analysis method according to the present invention, and FIG. 2B is a partially enlarged view of the pillar region of the microchip shown in FIG. 2A. FIG. 2C is a cross-sectional view taken along the line IIc-IIc in FIG.
A linear channel 102 is formed on the glass substrate 101. The channel 102 is formed by processing the surface of the glass substrate 101 into a recess. A pillar region 103 is formed in a part of the channel 102. The pillar region 103 is composed of an array of pillar structures 104 made of cylindrical protrusions.

Next, the sample analysis procedure will be described with reference to FIGS.
3A to 3D are examples of process diagrams to which the sample analysis method according to the present invention is applied.
A sample 106 mixed with a reagent necessary for electrophoresis is dropped onto one end (the left end in the figure) of the channel 102 with a pipette 105. Since the pillar region 103 (see FIG. 2A) is formed in the channel 102, and the surface area is large and the hydrophilicity is increased, the dropped sample 106 spreads over the entire channel 102 by capillary action (see FIG. FIG. 3 (a)).

  Next, phosphoric acid is dropped onto one end (left end) of the flow path 102, and an aqueous nanothorium hydroxide solution is dropped onto the other end (right end) (FIG. 3B).

  Thereafter, a pair of electrodes 108 are respectively introduced into both ends of the channel 102, and a positive voltage is applied to the acid side (left end) and a negative voltage is applied to the alkali side (right end) to perform isoelectric focusing (FIG. 3). (C)).

  After electrophoresis, the electrode 108 is removed, the sample 106 is dried, and then the microchip is placed on a scanner (not shown) to measure fluorescence. Since the target molecule 201 (see FIGS. 1A to 1D) is labeled with the phosphor 203, the amount of the target molecule 201 can be easily measured by monitoring the fluorescence intensity. In addition, 113 shows a band and 114 shows fluorescence (FIG.3 (d)).

(Microchip manufacturing method)
Next, a manufacturing method of the microchip in the first embodiment will be described.
First, for example, a resist for photolithography is applied to a glass substrate having a thickness of about 0.5 mm, light irradiation is performed using a semiconductor exposure apparatus such as a stepper, and a resist pattern is obtained after development. In this case, the width of the flow path 102 is 0.1 to 5 mm, and the length of the flow path 102 is 10 to 100 mm. The diameter of the pillar structure 104 is 0.1 to 100 μm, and the distance between the nearest pillar structures is about 0.1 to 100 μm.

Next, using the resist as a mask, a groove having a depth of 0.1 to 100 μm is formed on the surface of the glass substrate 101 by reactive ion etching using CF ₄ (tetrafluoromethane) gas, and the resist is removed by ashing. To do.
Here, the ashing process is a process of decomposing and removing a resist on the wafer in a gas phase.

  As described above, according to the present embodiment, when isoelectric points of isoforms are different, they are spatially separated by electric point electrophoresis, so that isoforms can be distinguished by performing fluorescence observation. The electrophoresis time is as fast as several minutes to several tens of minutes, and the detection by fluorescence is excellent in reproducibility and quantification as compared with MS.

  Examples of electrophoresis include agarose gel electrophoresis, pulse field electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, and two-dimensional electrophoresis.

A microchip was manufactured by the above manufacturing method.
The width, length, and depth of the channel 102 are, for example, 20 mm, 40 mm, and 10 μm, respectively. The pillar structure 104 sounds from a rectangular convex structure when viewed from above, and has a length along the flow path 102 of about 10 μm and a length perpendicular to the flow path 102 of about 5 μm. The distance between the pillar structures in the direction along the flow path 102 is about 10 μm, and the distance between the pillar structures in the direction perpendicular to the flow path 102 is about 5 μm.

As a sample 106, 50 μg of transthyretin (transthyretin: molecular weight: about 15 kDa (g / mol), manufactured by Sigma) was dissolved in 1 ml of water and then labeled with 1 nmol of Cy3 fluorescent reagent. Thereafter, magnetic beads on which an anti-transthyretin antibody was immobilized were introduced and incubated (cultured) at room temperature for about 1 hour. Thereafter, the magnetic beads were washed with PBS (phosphate buffered saline), and transthyretin was collected using formic acid at pH = 2. After recovery, the recovered solution was neutralized with ammonium hydroxide.
Instead of PBS, another buffer for pH adjustment (for example, TRIS: Tris (HOCH ₂ ) ₃ CNH ₂ ) may be used.

After 15 microliters of the collected sample solution was mixed with 1 microliter of carrier ampholite and 30 microliters of water, an amount of 1 microliter was introduced into the microchip.
After the introduction, a phosphoric acid aqueous solution with pH = 2 was introduced on the anode side, and a sodium hydroxide aqueous solution with pH = 12 was introduced on the cathode side.
Next, isoelectric focusing was performed by applying a voltage of 3 kV between the electrodes for 10 minutes. After electrophoresis, the sample on the microchip was air-dried and placed on the fluorescence scanner device together with the chip, and the fluorescence distribution was measured. Three distinct fluorescent peaks were observed around pI = 6.8, confirming the separation of transthyretin isoforms by electrophoresis. Further, the fluorescence peak intensity between the channels was within ± 10% of the variation between the microchips, and a linear relationship with the amount of introduced transthyretin was obtained. In view of signal variation of several tens of percent or more when mass spectrometry is used, detection by fluorescence is superior to mass spectrometry in reproducibility and quantitativeness. Moreover, the time required from the fluorescence labeling to the fluorescence detection was about 4 hours.

  The above-described embodiments and examples show preferred examples of the present invention, and the present invention is not limited thereto, and various modifications can be made without departing from the scope of the invention. .

For example, the chemical substance is captured after the chemical substance in the sample is stained with a phosphor.
Moreover, after capturing the chemical substance in the sample, it is stained with a phosphor, and the residual phosphor after staining is removed.

In addition, capture of chemical substances may be performed by an antibody, aptamer, receptor, or molecular imprinting method.
Here, an aptamer is a synthetic DNA / RNA molecule having the ability to specifically bind to a target substance.
In addition, an acid, an alkali, a denaturing agent, a protease, or DNase can be used for collecting the captured substance.
Here, protease is a general term for enzymes that catalyze the hydrolysis of peptide bonds (—CO—NH—), and is divided into proteinases and peptidases. Proteinase is an enzyme that hydrolyzes peptide bonds of protein molecules, and is also called endoptidase. When it acts on a high molecular weight protein, rapid depolymerization occurs, and the protein becomes peptone. Peptidase is an enzyme that hydrolyzes the peptide bond at the amino terminus or carboxy terminus of the peptide chain, and is also called an exopeptidase, which sequentially releases amino acids from the end of the peptide chain.

DNase (Deoxyribonuclease, DNase) is a DNA that is a nucleic acid, and has two nucleotides (base-deoxyribose-phosphate) having a phosphate group at the 5 ′ end of deoxyribose (dinucleotide). Or it is an enzyme that produces several (oligonucleotides).
Deoxyribonuclease is a general term for a group of enzymes that cleave the phosphodiester bond of a phosphodiester nucleic acid of deoxyribonucleic acid and decompose it into oligonucleotides or mononucleotides.

  In addition, isoelectric focusing may be performed in a channel formed in a capillary.

  In addition, after staining with a phosphor having a different color development wavelength for each sample, the sample is mixed, and the fluorescence intensity for each color development is detected.

(Second Embodiment)
Next, a second embodiment of the present invention will be described in detail with reference to FIGS.
4A to 4D are explanatory views for explaining sample pretreatment.
An antibody 204 against the target molecule 201 is fixed to a support such as glass, a sample containing the target molecule 201 is introduced, and only the target molecule is captured by the antibody 204 by an antigen-antibody reaction (FIG. 4A).

Thereafter, when the sample solution is discarded and replaced with a buffer solution, only the target molecule 201 captured by the antibody 204 remains.
Next, by substituting with an acidic buffer solution, the bond between the target molecule 201 and the antibody 204 is cut, and the target molecule 201 is recovered (FIG. 4B).

  After that, a fluorescent labeling reagent is added, and the target molecule 201 is labeled with the phosphor 203 (FIG. 4C).

  Next, the phosphor 203 that has not contributed to the binding of the target 201 is removed using a separation device such as a spin column (FIG. 4D).

  Thereafter, a reagent such as carrier ampholite is mixed, but this step and the subsequent steps are the same as those in the first embodiment.

  Since this embodiment requires a phosphor removal step, there is room for improvement with respect to the first embodiment in terms of work complexity, increase in work time, and sample loss.

(Third embodiment)
Next, a third embodiment of the present invention will be described.
In the first embodiment, formic acid is used for recovery of transthyretin, but urea is used in this embodiment.
For example, by using an aqueous urea solution having a concentration of 7 mol / ml, transthyretin can be released from the antibody and recovered. Since the aqueous urea solution is neutral, there is no need for neutralization after recovery as in the case of formic acid.

(Fourth embodiment)
Next, a fourth embodiment of the present invention will be described.
First, a sample (sample 1) in which 50 micrograms of transthyretin was dissolved in 1 milliliter of water and a sample in which 25 micrograms were dissolved (sample 2) were prepared.

  Next, the sample 1 was labeled with the fluorescent compound Cy3, the sample 2 was labeled with the fluorescent compound Cy5, and then both were mixed. Thereafter, immunoprecipitation and recovery were performed according to the procedure of the first example. This was introduced into a microchip and subjected to isoelectric focusing and air drying. In detection, detection according to the wavelength of the fluorescent compound Cy3 and detection according to the wavelength of the fluorescent compound Cy5 were performed. The fluorescence amount of the fluorescent compound Cy5 was half of the fluorescence amount of the fluorescent compound Cy3, and a result reflecting the amount of transthyretin in the sample was obtained. At this time, the intensity ratio variation between the fluorescent compound Cy3 and the fluorescent compound Cy5 was within 5% between the chips.

(effect)
The effect of the present embodiment is that the expression level of each isoform contained in the sample can be detected at high speed while ensuring quantitativeness and reproducibility.

  As an application example of the present invention, analysis of isoforms contained in a sample can be mentioned. In particular, it can be applied to the medical field where rapid and accurate isoform detection is important.

DESCRIPTION OF SYMBOLS 101 Glass substrate 102 Flow path 103 Pillar area | region 104 Pillar structure 105 Pipette 106 Sample 107 Electrode liquid 108 Electrode 109 Needle 110 Matrix 111 Matrix-sample mixture 112 Laser 113 Band 114 Fluorescence 201 Target molecule 202 Non-target molecule 203 Phosphor 204 Antibody

Claims (7)

Staining and capturing a chemical substance in a sample with a phosphor;
Washing away the material in the sample that was not captured;
Collecting the captured material; and
A step of performing isoelectric focusing of the sample after collection;
And a step of detecting the fluorescence intensity of the sample after the isoelectric focusing.   The sample analysis method according to claim 1, wherein the chemical substance is captured after the chemical substance in the sample is stained with the phosphor.   The sample analysis method according to claim 1, wherein the chemical substance in the sample is captured and then stained with the phosphor, and the residual phosphor after staining is removed.   The sample analysis method according to claim 1, wherein the chemical substance is captured by an antibody, aptamer, receptor, or molecular imprinting method.   The sample analysis method according to claim 1, wherein an acid, an alkali, a denaturant, a protease, or DNase is used for collecting the captured substance.   The sample analysis method according to claim 1, wherein the isoelectric focusing is performed in a flow path formed on a capillary or a microchip.   The sample according to any one of claims 1 to 3, wherein the sample is mixed after being stained with phosphors having different color development wavelengths for each sample, and the fluorescence intensity is detected for each color development. analysis method.

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