JP2007093374A - Molecular analysis method using microchannel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of eliminating the need for preliminary preparatory work on a liquid containing specimen molecules to be analyzed and directly analyzing the liquid containing specimen molecules having various variable factors such as concentration, viscosity, and the presence of impurities. <P>SOLUTION: Both a laminar flow of a liquid containing probe molecules and a laminar flow of an adjustment liquid are fed in a microchannel in parallel with each other, and a laminar flow of the liquid containing the specimen molecules is fed in between both laminar flows in such a way as to be sandwiched. Changes in the amount of the probe molecules diffusing into the laminar flow of the liquid containing the specimen molecules or the laminar flow of the adjustment liquid or the amount of remaining probe molecules in the laminar flow of the liquid containing the probe molecules are detected and analyzed to qualitatively or quantitatively analyze the specimen molecules. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for qualitative or quantitative analysis of a specific molecule using a microfluidic system.

In the research in the genetic field, the sequencing of the human genome is almost complete, and based on the results, the identification and functional analysis of genes related to gene expression, mutation, single nucleotide polymorphism, etc., and the structure and function of the protein associated therewith The center of research is shifting to the analysis.
On the other hand, technology development is being promoted to make use of these series of research results for medical and welfare purposes.

By the way, one of the techniques developed in connection with such research is to use the interaction between a sample molecule such as a biomolecule and a molecule that forms a complex (hereinafter referred to as a probe molecule). There is a way to detect.
In this method, a complex formed by a specific interaction between a probe molecule immobilized on a solid phase carrier and a biomolecule is detected by a signal emitted from a fluorescent substance or an electrochemically active substance that has been labeled in advance. Thus, the presence of biomolecules is qualitatively detected and quantified.

  As a method for detecting a biological substance at this time, for example, a method using an electrochemical reaction (see Non-Patent Document 1), a method using a crystal resonator (see Non-Patent Document 2), surface plasmon, etc. A method using resonance (see Non-Patent Document 3) and the like are known.

  These all have a nucleic acid fragment or protein immobilized on the gold surface as a probe molecule, and a biological substance or its related substance that has a specific interaction with it is bound, and the electrochemical response generated at that time, a quartz crystal This is to detect and analyze the change in the frequency of light and the change in the refractive index due to surface plasmon.

  However, in order to perform quantification with high accuracy in these methods, it is necessary to strictly control the probe molecules immobilized on the solid phase carrier, but the efficiency of immobilizing the probe molecules on the solid phase carrier is inadequate. Due to the uniformity, low reproducibility, complexity of the substance diffusion behavior at the solid-liquid interface, and lack of skill of the operator when working with the analyte molecules, it was inevitable that the limit was generated.

  In addition, a method performed without immobilizing any probe molecule or analyte molecule on a solid phase carrier, for example, a method using a polymerase chain (PCR) reaction (see Non-Patent Document 4), a method using a molecular beacon (Non-Patent Document) 5) is known.

  However, since a method using a PCR reaction is a reaction in which the PCR reaction itself is exponentially amplified, it is theoretically difficult to perform highly accurate quantification. In addition, a method using a molecular beacon is one in which a fluorescent site and a quenching site are introduced at both ends of the probe DNA, but before forming a complex with a test molecule, it is bent to take a self-complementary sequence and the fluorescence is quenched. However, after the formation of the complex, it is a method of analysis utilizing the property of emitting light by the probe molecule. In order to carry out this method, there are restrictions on the sequence design of the probe DNA, DNA and other However, it can be applied only to the analysis of nucleic acids.

  In order to overcome these drawbacks in the conventional method, the present inventors previously used a method of performing qualitative and quantitative analysis of a sample molecule using a complex of a probe molecule and a sample molecule, that is, a probe molecule-containing solution. When a sample molecule-containing solution is flowed through a microchannel, both of them form a laminar flow and do not mix with each other, so the difference in diffusivity is detected by the strength of selective interaction between the two. Thus, there has been proposed a method for analyzing a sample molecule such as a biomolecule with high accuracy without being immobilized on a solid phase carrier (see Patent Document 1).

  However, in this method, there remains a problem that the sample molecule-containing solution to be analyzed requires prior work such as purification, concentration, and controlled preparation of the physical properties of the solution.

JP 2004-53417 A (Claims and others) “Anal. Chem.”, 2000, Vol. 72, p. 1334-1341 “Journal of the American Chemical Society” (J. Am. Chem. Soc.), 1992, 114, p. 8299-8300 Nagata and Handa, “Real-time analysis experiment of biological material interaction”, Springer Fairlark Tokyo, 1998 “Procedures of the National Academy of Sciences of the United States of America” (Proc. Natl. Acad. Sci. USA), 1997, Vol. 94, p. 10756 “Anal. Chem.”, 2000, Vol. 72, p. 747A-753A

  Under such circumstances, the present invention eliminates the need for the preliminary preparation of the analyte molecule-containing liquid to be analyzed, and directly applies the analyte-containing liquid having various variation factors such as concentration, viscosity, and the presence of impurities. It was made for the purpose of providing a method that can be analyzed.

  In the analysis method using a microchannel, the present inventors do not need a preparatory work of a specimen-containing liquid and directly analyze a specimen-containing liquid having various properties such as concentration, viscosity, density, and impurities. As a result of diligent research to develop a method, a probe molecule-containing liquid and a separately prepared adjustment liquid are fed into a microchannel while forming a laminar flow, and the laminar flow of the two liquids By supplying the sample molecule-containing liquid so as to be sandwiched between the two, it has various properties while maintaining the simple operability and high accuracy of the analytical method with only the minimum required amount of sample-containing liquid. The inventors have found that the specimen-containing liquid can be directly analyzed, and have reached the present invention based on this finding.

  That is, the present invention sends the probe molecule-containing liquid laminar flow and the adjustment liquid laminar flow in parallel to the microchannel, and sends the sample molecule-containing liquid laminar flow between the two laminar flows. A sample molecule characterized by detecting and analyzing a change in the amount of probe molecules diffusing into the sample molecule-containing liquid laminar flow or the regulated liquid laminar flow or the amount of remaining probe molecules in the probe molecule-containing liquid laminar flow A qualitative or quantitative analysis method is provided.

  In the method of the present invention, the probe molecule-containing liquid and the adjustment liquid are fed into the microchannel while forming a laminar flow, and the sample molecule-containing liquid is sent as a laminar flow so as to be sandwiched between the two laminar flows. As a result, the diffusion degree of the complex formed by the specific interaction between the probe molecule and the analyte molecule into each laminar flow changes depending on the amount and strength of the complexation. This is detected by a signal emitted from the probe molecule, and the sample is analyzed by analyzing the result.

In the conventional method, considering the properties such as viscosity and density of the sample molecule-containing liquid, if the preparation work is not performed in advance, this sample molecule will sink to the lower part of the microchannel due to the action of gravity. The phenomenon occurred and the correct analysis could not be performed.
On the other hand, by feeding the probe molecule-containing liquid and the adjustment liquid so as to be sandwiched by the method of the present invention, it is possible to perform a correct analysis without adjusting properties such as viscosity and density. .

The microchannel used in the method of the present invention needs to be provided on an inert material substrate. This inert material is a material that does not react with probe molecules, analyte molecules, the solvent used, and the resulting complex. For example, glass, quartz, or silica, Si / SiO ₂ , magnesia. , Zirconia, alumina, apatite, silicon nitride, and ceramics such as oxides, carbides, nitrides, borides, and silicides of metals such as titanium, aluminum, yttrium, and tungsten.
In addition, metals and plastics can be used as long as they satisfy the above requirements. As the shape of this base, a plate-like body is common, but if desired, an arc-like body, a sphere, a grain or the like can be used.

  These materials are appropriately selected according to the means to be selected, the type of analyte and probe molecules, and the solvent. However, when detecting by optical means, at least at the detection site, sufficient for the wavelength of light to be used. It is necessary to use a material that exhibits transparency.

The microchannel in the present invention needs to have a size and shape in a range in which a laminar flow is formed in terms of fluid engineering. That is, it is engraved on the inert material substrate with a width of 10 to 500 μm, preferably 50 to 400 μm, and a depth of 10 to 500 μm, preferably 50 to 400 μm. The length of the microchannel is not particularly limited and is appropriately selected in consideration of the residence time. Specifically, although it depends on the size of the inert material used, it is usually selected in the range of 100 to 300 mm.
In addition, the size of the introduction flow channel before the plurality of liquids fed into the flow channel join may be the same or different. In particular, it is advantageous that the channel for the analyte molecule-containing liquid is narrower than others. The flow path after the merge does not always need to be a fixed flow path. For example, by providing a tapered flow path structure, the reaction can be promoted depending on the type of reaction.

  Such a micro flow path is engraved on a substrate by mechanical means using a machine tool such as a micro drill, for example, or after forming a groove by a photolithographic technique used for manufacturing a semiconductor integrated circuit, etc. It can be manufactured by bonding another substrate. Alternatively, it can also be produced by transferring a substrate on which a microchannel is engraved in this way as a mold to another substrate.

  The fluid flowing through such an extremely thin flow path flows while forming a laminar flow without being mixed even if it is a soluble solvent. And such an ultra-fine flow path has the characteristic that the diffusion distance of a substance is short.

Next, in the method of the present invention, three kinds of fluids, that is, a specimen molecule-containing liquid, a probe molecule-containing liquid, and an adjustment liquid are used for feeding into the microchannel and forming a laminar flow. The sample molecule-containing liquid is a solution in which a sample to be analyzed is dissolved or suspended, and water, a physiological buffer solution, or an organic solvent such as ethanol or propanol is used as the carrier medium.
The probe molecule-containing liquid is a molecule that forms a complex with the above-described analyte molecule, that is, a liquid in which the probe molecule is dissolved or suspended, and the support medium is an example of the support medium for the sample molecule-containing liquid. The same is used.

  On the other hand, the adjustment liquid for forming the third laminar flow of the method of the present invention is a liquid for adjusting the diffusivity, as long as it does not contain analyte molecules, probe molecules, or molecules that react with them. Although there is no particular limitation, pure water or a buffer solution is usually used. However, it is also possible to easily adjust the diffusivity by adding a salt, acid, base or other substance.

  The method of the present invention has the advantage that the analyte-containing liquid can be fed into the microchannel at a high concentration, and the complex formation reaction is promoted near the interface with the probe molecule-containing liquid laminar flow.

  In addition, the laminar flow formation by feeding into the microchannel in the method of the present invention is not limited to the three-layer flow format of the probe molecule-containing liquid, the adjustment liquid, and the specimen-containing liquid. A multilayer flow format by adding the probe molecule-containing liquid, the adjustment liquid, and the specimen molecule-containing liquid may be used. Furthermore, the order in which the layers of the large number of liquids are arranged is not limited, and can be selected as appropriate depending on the purpose of the analysis.

  The method of forming the multi-layer flow can be formed in the vertical direction in addition to the method of forming the multi-layer flow simply in the horizontal direction, and how to arrange the multi-layer flow is appropriately selected according to the purpose of the analysis.

  In the method of the present invention, the feeding speeds of the probe molecule-containing liquid, the adjustment liquid, and the specimen-containing liquid may be the same or different. This can be appropriately selected depending on the purpose of analysis, but it is particularly advantageous to feed the sample-containing solution at a low flow rate with respect to the probe molecule-containing solution and the adjustment solution.

  In this way, by feeding the specimen-containing liquid at a slow flow rate, the necessary amount of liquid necessary for analysis can be reduced.

  In general, at the interface of multiple laminar flows that flow through a microchannel, the solute diffuses naturally toward the other liquid if it is soluble in the solvent, but there is a special gap between the probe molecule and the analyte molecule. When there is such an interaction, a complex is generated only in the vicinity of the interface, and the generated complex can be moved and diffused in a desired place and direction by fluid operation.

  If this phenomenon is used, the signal emitted from the functional group introduced into the probe molecule, the specific characteristics of the probe molecule itself (absorption of light of a specific wavelength, etc.), or the formed complex can be selectively used. Since it is possible to detect signals and characteristics emitted by the substance to be bound, it is possible to know the amount of extra diffusion added to the natural diffusion, and the sample can be analyzed based on the amount.

  Therefore, the method of the present invention can be generally used in the case where a specific interaction occurs between the probe molecule and the analyte molecule. That is, when a nucleic acid fragment is used as a probe, a nucleic acid fragment having a specific sequence can be detected and analyzed. In addition, when an antigen or antibody is used as a probe, a specific antibody or antigen can be detected. When a protein is used as a probe, a specific protein, enzyme, nucleic acid, or the like can be detected, and it has protease inhibitory activity. If such various peptides are used as probes, it is possible to detect a specific enzyme and evaluate its activity. Furthermore, if various sugars are used as probes, nucleic acids and proteins that specifically recognize them can be detected and quantified. If various cells are used as probes, living bodies such as various natural or artificial drugs and environmental substances can be detected. Can be screened for effects.

  In the method of the present invention, the present invention is not limited to these, and any combination can be used for detecting any compound if a combination that causes a specific interaction between the probe molecule and the analyte molecule is selected. In the above example, a single molecule in the chemical sense is used, but other than this, it can be applied to cells and other substances in general.

  Furthermore, in the method of the present invention, the skill of the operator is not required in the series of operations unlike the conventional method, and the uncertainty of the analysis result due to the difference in the skill level of the operator can be eliminated. . The conventional method requires an experimental operation for forming a complex of a probe molecule and an analyte molecule, such as a hybridization operation in detecting a nucleic acid fragment, and is caused by a difference in skill level of workers in this experimental operation. Uncertainty of the analysis result has been a problem, but in the method of the present invention, since the liquid is simply poured into the flow path with a syringe or the like, the skill level is irrelevant, and if an instrument such as a syringe pump is used, Furthermore, the difference in the skill level of the operator can be eliminated.

  The time required for detection in the method of the present invention corresponds to the time required for the liquid sent to the micro flow path to flow through the flow path, but the time required to flow through an extremely thin flow path having a size of about several hundred μm. Even if the amount of liquid fed is at least short, the time required for detection in the method of the present invention is naturally shortened. This time depends on the size of the microchannel to be used, but is usually within a few seconds to a few tens of seconds.

  The method of the present invention performs qualitative or quantitative analysis of a sample molecule based on a change in the diffusion degree of a complex formed between the sample molecule and the probe molecule in a laminar flow. In other words, when there is no affinity between the analyte molecule and the probe molecule, both simply detect the diffusivity between the laminar flows based on the normal mixing behavior, but there is no affinity between them. If so, a complex is formed, which can be moved and diffused to the desired location and orientation by fluid manipulation and detected as an increased diffusivity. Therefore, by comparing such a difference in diffusivity, it is possible to quantitatively know the action of the analyte molecule on the probe molecule.

  And since the above detection is easy to measure, usually the amount of probe molecules diffusing to the specimen molecule-containing liquid or adjustment liquid side is measured directly, or the amount of probe molecules on the probe molecule liquid side is measured. Is done. This measurement is performed by detecting a characteristic signal attached to the probe molecule with a sensor, and in particular, using a fluorescent molecule imparted with fluorescence by introducing a fluorescent functional group as the probe molecule, its fluorescence It is advantageous to determine the change in the diffusivity of the complex formed by tracking the strength of the complex.

  In addition, using a probe molecule into which an electrochemically active functional group has been introduced, a method for determining the change in the amount of current, and measuring the absorption intensity of the probe molecule for specific ultraviolet light, visible light, or infrared light Methods are also available. In this way, the analyte molecule can be analyzed without chemical modification for obtaining a signal specific to the probe molecule.

  Prior to measurement of the diffusivity of the complex, a calibration curve can be created for a known amount, and then the detection result can be compared with this to quantitatively analyze the concentration of the analyte molecule.

  The above is a case where the amount of the probe molecule diffused to the sample molecule-containing liquid side is measured. If desired, the complex of the sample molecule and the probe molecule diffused to the probe molecule-containing liquid side is measured to obtain the sample molecule. The amount of can also be detected. Thus, the detection means in the method of the present invention is not particularly limited, and any means may be used as long as the diffusion state of the complex of the probe molecule and the analyte molecule is known.

  Next, in the method of the present invention, in order to send each liquid into the microchannel, for example, a syringe can be connected and manually performed, but the liquid feeding speed and the feeding speed can be increased by mechanical means such as a syringe pump. It is advantageous to carry out while controlling the liquid pressure and the like.

For example, the specimen molecule-containing liquid, the probe molecule-containing liquid, and the adjustment liquid are simultaneously fed into the microchannel, and after passing through the channel for a certain distance, the detection operation is performed. Here, at least one kind of specimen molecule-containing liquid and probe molecule-containing liquid are required, but a plurality of pieces of information can be obtained simultaneously by flowing a plurality of liquids simultaneously.
For example, when the detection at this time is performed by a fluorescence method, the detection site is irradiated with light from a laser beam or other excitation light source, and the intensity of the fluorescence emitted therefrom is measured. The stronger the fluorescence emitted from the specimen-containing liquid side, the greater the amount of probe molecules present, that is, the intensity and amount of interaction between the probe molecules and the specimen. In this way, it is possible to know the presence / absence and the amount of the target sample.

  Next, a specific description will be given by taking as an example a case where detection of DNA having a predetermined sequence is performed using a probe molecule into which a fluorescent functional group is introduced. That is, the sample DNA fragment solution and the fluorescent functional group-introduced DNA fragment solution as a probe are fed into the microchannel. After flowing through the channel for a certain distance, the sample solution side is irradiated with excitation light such as laser light. If there is no or weak fluorescence emitted therefrom, it is judged that there is no sequence complementarity between the probe DNA fragment and the sample DNA fragment. Conversely, if the fluorescence detected there is strong, it is determined that there is sequence complementarity between the probe DNA fragment and the sample DNA fragment.

  In this case, before examining the unknown sample, it is possible to analyze the fluorescence intensity and the degree of complementarity of the known sample more accurately, and at the same time to quantify the amount of the DNA fragment having the complementary sequence. Can do.

  In this case, instead of DNA as a probe molecule, a DNA-binding peptide (a peptide having a structure similar to DNA and having a higher sequence recognition ability than DNA) or LNA (the 2 ′ and 5 ′ positions of the ribose ring of DNA) Can be used to perform analysis with higher accuracy. Also, unlike DNA, PNA is soluble in organic solvents other than water. By using the sample as an aqueous solution and the probe PNA dissolved in an organic solvent, the diffusion of the sample to the probe solution side is suppressed. It is possible to perform analysis with higher accuracy.

  According to the present invention, the probe-containing liquid, the specimen-containing liquid, and the adjustment liquid are simply fed into the microchannel as a laminar flow without being fixed to the solid phase carrier, and the probe molecule and the specimen molecule are combined at a predetermined position. Qualitative and quantitative analysis of specimens can be performed with high accuracy simply by measuring the diffusivity that reflects the concentration of the body. In addition, since it is performed by a simple operation, errors in analysis results due to differences in the skill level of workers can be suppressed to a minimum, and there are no restrictions on the molecular species of the sample that can be applied. There is an advantage that it can be applied to a wide range without requiring work.

  Next, the best mode for carrying out the present invention will be described by way of examples, but the present invention is not limited to these examples.

Example As shown in FIG. 1, a micro flow path was engraved on a silicon wafer by a dry etching method, and this was used as a first master mold, and a polydimethylsiloxane resin was poured and hardened to form a second master mold. That is, in this state, the portion that becomes the microchannel has a convex shape. Further, a polydimethylsiloxane resin was poured into the second matrix and hardened to produce a microchannel. A target polydimethylsiloxane resin microchannel engraved microreactor was fabricated by affixing another polydimethylsiloxane resin plate thereon. This is a plate having a size of 70 mm in width and 30 mm in length, and the size of the engraved microchannel is 200 μm in depth and 300 μm in width.

  Next, analysis was performed using peptide diffusion (PNA) having a structure of FO- (N-terminal) -ATGCACGCGC-Lys- (C-terminal) as a probe molecule. Here, F represents a fluorescent substance, fluorescein, O represents ethylene glycol as a linker, and Lys represents lysine, which is an amino acid for ensuring water solubility.

  As a specimen sample, a PCR product having 195 base pairs was used. Two cases (MT1) of wild type (WT) having a sequence complementary to the probe molecule near the center of the DNA strand, and a mutant type in which one of the bases is mutated from guanine (G) to adenine (A) And MT2) were prepared in total.

  The feeding into the microchannel was performed using a syringe pump from the a, b, and c positions shown in FIG. The liquid feeding speed was 40 μl / min for the probe molecule-containing liquid and the adjustment liquid, and 1 μl / min for the sample molecule-containing liquid. In this example, pure water was used as the adjustment liquid. The probe molecule-containing solution was an aqueous solution having a concentration of 200 fmol / μl.

  Next, fluorescence is generated by irradiating light of 488 nm emitted from an argon gas laser to the probe molecule-containing liquid flow channel side and the adjustment liquid flow channel side at the position A in FIG. 1, and the intensity ratio (adjustment liquid flow channel side) / Probe molecule-containing liquid channel side). As described above, the evaluation is performed in order to correct the influence of the analysis value due to the fluctuation of the laser intensity or the machine setting. The results are shown in FIG. 2 as a bar graph. This graph is an average value of the above ratios measured 10 times, and the range of standard deviation is indicated by error bars. Only WT having a base sequence complementary to the probe PNA fragment obtained a larger value than the other two mutant sequence samples. The amount of PCR product sample required for this analysis was about 2 to 3 μl. From this result, it is understood that those having a special interaction can be analyzed. The measurement result was about 2% in terms of the coefficient of variation, and showed extremely high reproducibility.

  The present invention is suitable for analysis of various biological materials because it can easily qualitatively and quantitatively analyze a micro sample such as DNA or LNA.

The top view which shows an example of the microchannel used by the method of this invention. The bar graph which shows the result of an Example.

Claims (6)

  The probe molecule-containing liquid laminar flow and the adjustment liquid laminar flow are sent in parallel to the microchannel, and the sample molecule-containing liquid laminar flow is sandwiched between the two laminar flows. A method for qualitative or quantitative analysis of analyte molecules, characterized by detecting and analyzing a change in the amount of probe molecules diffusing into a flow or adjusted liquid laminar flow or the amount of remaining probe molecules in a probe molecule-containing liquid laminar flow.   The qualitative or quantitative analysis method according to claim 1, wherein the flow rates of the probe molecule-containing liquid laminar flow and the adjustment liquid laminar flow are made larger than the flow rates of the sample molecule-containing liquid laminar flow.   The qualitative or quantitative analysis method according to claim 1 or 2, wherein a change in the amount of the complex formed between the probe molecule and the analyte molecule is detected by a change in the amount of the probe molecule.   The qualitative or quantitative analysis method according to claim 1, 2 or 3, wherein the probe molecule has fluorescence.   The qualitative or quantitative analysis method according to claim 3, wherein the concentration of the analyte molecule is determined by comparing the diffusion degree of the complex with a calibration curve formed in advance. 6. The qualitative or quantitative analysis method according to claim 1, wherein the sample molecule is a DNA fragment having a specific sequence.

Images