JP2005224110A - Method for detecting polynucleotide or oligonucleotide and micro fluid device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly detecting the presence or absence or amount of a polynucleotide or an oligonucleotide having a target base sequence in a specimen solution with good accuracy, especially a method for detecting SNP (single nucleotide polymorphism) or single nucleotide mutation. <P>SOLUTION: The method for detecting the polynucleotide or oligonucleotide comprises bringing the specimen solution containing the polynucleotide or oligonucleotide which is the object of detection into contact with each probe-immobilizing part of a micro fluid device having a porous layer where a probe is immobilized on the surface in each capillary-like channel, then washing the probe-immobilizing part, removing components of the specimen solution unbound to the probe and subsequently measuring the fluorescence intensity of the probe-immobilizing part. In the method, the transmittance of light at 670 nm wavelength in a site for measuring the fluorescence intensity of the porous layer where the probe is immobilized is 10-60% and the measurement of the fluorescence intensity is made at ≥650 nm wavelength. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a polynucleotide or oligonucleotide analysis method for detecting a polynucleotide or oligonucleotide having a specific base sequence, and relates to a microfluidic device having a porous layer in which a probe is immobilized on the inner surface of a capillary channel The present invention relates to an analysis method using. The present invention also relates to a microfluidic device used for analysis of polynucleotides or oligonucleotides.

  The test polynucleotide in the sample solution has a specific base sequence for detection of the polynucleotide (hereinafter sometimes referred to as “target base sequence”), does not have a target base sequence, or is a target base A DNA chip method is known as an analysis method for detecting a polynucleotide having a specific base sequence such as the concentration or amount of a polynucleotide having a sequence (Non-patent Document 1). In the DNA chip method, a probe that specifically binds to a polynucleotide or oligonucleotide to be detected is immobilized on a solid phase, and the polynucleotide or oligonucleotide in a sample solution is selectively bound (hybridized) thereto. Since it is possible to detect various sequences at a time and is easy to operate, it is most frequently used among DNA analysis methods.

  However, when a polynucleotide with a small difference in base sequence, such as a single base polymorphism (SNP) or single base mutation analysis of a polynucleotide, that is different from the target base sequence by only one base is selectively detected. In other cases, for example, a polynucleotide slightly different from the target base sequence by one base, for example, would also be hybridized (hybrid formation having a mismatch) with the probe, so that it was difficult to detect. In order to avoid this difficulty and sufficiently increase the difference in the amount of binding, incubation for several hours or more is required, leading to a long analysis time (see Non-Patent Document 1).

  On the other hand, according to the study by the present inventors, the hybridization temperature is increased, for example, not less than the melting temperature (Tm) of the hybrid of the probe and its complementary polynucleotide or oligonucleotide, and not more than “Tm + 10 ° C.”. However, the binding speed was low, the amount of saturated bonds was reduced, and the sensitivity was lowered, and the reliability of the analysis was also lowered.

  Patent Document 1 discloses a method for detecting a mutant allele from a mixed wild-type and mutant allele sample using a carrier carrying a probe having a base sequence complementary to a wild-type allele. Has been. In this method, a mixed allele sample of a wild type and a mutant type is passed through a packed flow channel in which an allele having a base sequence complementary to the wild type allele is fixed, and the wild type allele is selectively flowed by hybridization. In this method, the mutant allele is concentrated by allowing the mutant allele to flow out without being adsorbed.

  However, since this method is a method of separating a wild type allele and a mutant allele by hybridizing a wild type allele with an allele having a base sequence complementary to the gene, In order to realize sufficiently high selectivity so that a single base mutation can be detected, a long incubation time is required as in the DNA chip method.

  On the other hand, an immunodiagnostic drug (immunoassay) is known in which a pseudoantigen is immobilized on, for example, a polyamide porous membrane having a thickness of 100 to 200 μm. The use of a porous membrane as the stationary phase of the pseudoantigen increases the amount of immobilization and enables highly sensitive detection of the color former-labeling substance. However, when measuring the fluorescence of a fluorescent labeling substance, if a porous membrane is used for the fixed layer, the background of the fluorescence increases remarkably and the S / N ratio decreases, resulting in lower accuracy of determination. It was.

  In addition, as described in Patent Document 2 by the present inventors, an attempt has been made to immobilize probes such as antigens and antibodies on a thin porous layer formed in a substrate shape instead of the porous film. However, in the case of detecting by fluorescence intensity, the background was not sufficiently suppressed and the reliability was insufficient. Moreover, since the scattered light of the excitation light increases and this is superimposed on the fluorescence and the fluorescence background increases, the ratio of the fluorescence intensity of the binding portion and the non-binding portion of the polynucleotide (A) is reduced, and detection is performed. There has been a problem that the reliability of the system has decreased.

Norio Ohno, “Newton”, July 1999, p. 60-61 JP-T-2001-514504 JP 2000-002705 A

  The problem to be solved by the present invention is to provide a method for quickly and accurately detecting the presence or amount or presence or absence of a polynucleotide or oligonucleotide having a target base sequence in a sample solution. It is to provide a method for detecting type and single nucleotide mutations. Moreover, it is providing the microfluidic device used for this measurement.

  That is, the present invention detects on the probe fixing part of a microfluidic device having a porous layer on which a probe that specifically binds to a polynucleotide or oligonucleotide having a target base sequence is immobilized on the inner surface of a capillary channel. After contacting the sample solution containing the target polynucleotide or oligonucleotide, and then washing the probe fixing part to remove the sample solution component that did not bind to the probe, the detection control polynucleotide or oligonucleotide In the polynucleotide or oligonucleotide detection method for measuring the fluorescence intensity of the probe fixing part based on binding to a probe, light having a wavelength of 670 nm at a site for measuring the fluorescence intensity of the porous layer on which the probe is immobilized is measured. The transmittance is 10 to 60%, and Measurement of light intensity is to provide a method of detecting a polynucleotide or oligonucleotide is a fluorescent intensity measured at a wavelength of above 650 nm.

  Furthermore, the present invention provides a microfluidic device having a porous layer in which a probe that specifically binds to a polynucleotide or oligonucleotide to be detected is immobilized on the inner surface of a capillary channel, the porous layer comprising: A microfluid having a thickness of 1 to 20 μm, a light transmittance of 10 to 60% in a state filled with water, and formed in a part of the circumference in the flow path cross section A device is provided.

  In the present invention, the above-mentioned problem has been solved by setting the light transmittance at a specific wavelength in the porous layer to a specific value and measuring the fluorescence at the specific wavelength. That is, the light transmittance of the porous layer is such that the value at 670 nm in the state where the pores of the porous layer are filled with water is 10 to 60%, preferably 15 to 50%. Even when a porous layer is used, the ratio of the fluorescence intensity and fluorescence background derived from the sample increases (that is, the S / N ratio increases), and the polynucleotide or oligonucleotide has high accuracy and high reliability. Detection is possible.

  The method for analyzing a polynucleotide of the present invention comprises a porous layer in which the amount of immobilized probes can be increased and the transmittance of light at 670 nm is 10 to 60% in the state where the pores of the porous layer are filled with water. The base sequence of the test polynucleotide or test oligonucleotide is slightly different from the standard base sequence by measuring fluorescence of 650 nm or more generated by the binding between the probe and the polynucleotide. Can be detected in a short time and with high reliability, and in particular, a base sequence that differs from the standard base sequence by only one base can be suitably analyzed.

  Furthermore, according to the method of the present invention, since the sample solution is brought into contact with a fixed probe in a minute region in the capillary channel, even a small amount of sample can be analyzed accurately without drying. In addition, multiple samples and multiple items can be measured at the same time by fixing multiple probes to different areas in one flow path, or by fixing them in multiple flow paths. , The sample amount is not excessive.

Hereafter, the principal part of this invention is demonstrated in detail.
[Target base sequence]
The target base sequence refers to a specific base sequence for detection purposes, and a base sequence portion characteristic of the polynucleotide for detection is used as a target base sequence. In addition, when the purpose of analysis is to discriminate single nucleotide differences between polynucleotides, for example, in single nucleotide polymorphism (SNP) or mutation, it is a wild type (normal type) polynucleotide or a mutant type (abnormal type). In determining whether the polynucleotide is a polynucleotide, a base sequence portion including a portion (hot spot) in which a mutation occurs is set as a target base sequence. In this case, the target base sequence may be a wild-type base sequence or a mutant base sequence, but a probe described later using the wild-type base sequence as a target base sequence and a mutant type It is preferable to use both probes having the base sequence as a target base sequence because of high reliability.

[Polynucleotide]
A polynucleotide or oligonucleotide to be detected, which is contained in a sample solution to be subjected to the analysis method of the present invention (hereinafter, the polynucleotide or oligonucleotide is referred to as a test polynucleotide. In addition, the complicated explanation is avoided. Therefore, unless otherwise distinguished, “polynucleotide” as used in the present specification includes “oligonucleotide” as well as DNA, RNA, and other modified polynucleotides. obtain. The DNA can be, for example, total DNA, cDNA, a DNA fragment cleaved with a restriction enzyme, a DNA fragment amplified by polymerase chain reaction (PCR), or the like. Among these, a DNA fragment amplified by polymerase chain reaction (PCR) or the like is particularly useful and preferable.

  The length of the polynucleotide is arbitrary except that it is not less than the number of bases of the target base sequence, preferably 10 to 3000 bases, more preferably 15 to 1000, and most preferably 20 to 500. This range is preferable because the selectivity can be improved and the analysis time can be shortened. When prepared by PCR (polymerase chain reaction) or restriction enzymes, the number of bases of these polynucleotides can be set by selecting primers and restriction enzymes.

  The test polynucleotide contained in the sample solution subjected to the analysis method of the present invention is a polynucleotide having a target base sequence (hereinafter, the polynucleotide is referred to as “polynucleotide (A)”) alone, a target base. A polynucleotide having no sequence (hereinafter, the polynucleotide is referred to as a polynucleotide (B)) alone or a mixture thereof may be used. In addition, it may be single-stranded or double-stranded, and being double-stranded means that a biological sample can be directly applied to a polynucleotide product amplified by PCR (polymerase chain reaction) or the like. Since it can be used as a test polynucleotide, it is particularly useful and preferred. Moreover, you may contain several types of polynucleotide (A) and polynucleotide (B), respectively. In the present invention, in particular, when the ratio of the polynucleotide (B) to the sum of the polynucleotide (A) and the polynucleotide (B) is approximately 0, 1/2, or 1, that is, a single nucleotide In the case of the determination of the homo-wild type, the hetero-type, and the homo-mutant type, the determination reliability becomes high, which is preferable. Of course, as a result of the analysis, it may be determined that neither the polynucleotide (A) nor the polynucleotide (B) is contained in the sample solution. In the present invention, these detection methods are collectively referred to as polynucleotide detection methods.

  Here, the polynucleotide (B) may be a polynucleotide having no target base sequence, or may have a base sequence slightly different from the target base sequence, for example, a base sequence different by one base. In the case of slight differences, the different places in the sequence may be due to substitutions, deletions or insertions. The place where the sequence is different may be at the end or inside of the target base sequence. In the present invention, when the base sequence is different from the target base sequence by one base, the effect is particularly exhibited as compared with the conventional method.

  Examples of polynucleotides having a nucleotide sequence that is completely different from or substantially different from the target nucleotide sequence include bacterial and viral DNAs that are pathogens. Examples of the polynucleotide having a base sequence different from the target base sequence by one base include a single nucleotide polymorphism (SNP) and a mutant gene. The polynucleotide (A) and the polynucleotide (B) are each single-stranded, but may be either a sense strand or an antisense strand.

  Further, it is necessary that the test polynucleotide generates fluorescence when bound to the probe. In order to generate such fluorescence, a polynucleotide labeled with a fluorescent substance may be used, or a fluorescent intercalator may be used in the sample solution. In the present invention, even if a porous layer capable of increasing the amount of probe immobilized is used as the probe immobilization part by using a fluorescent wavelength of 650 nm or more, the fluorescence background does not increase so much. Highly reliable analysis can be performed.

[probe]
A probe that specifically binds to a polynucleotide having a target base sequence, that is, a probe having a base sequence substantially complementary to the target base sequence is used. Here, “substantially complementary” means a base sequence portion having a base sequence complementary to the target base sequence or having a relatively small number of sequences different from the base sequence complementary to the target base sequence. It means having a base sequence complementary to most of the target base sequence. [Hereinafter, “complementary (to the target base sequence)” may be expressed as “perfect match (to the target base sequence)”, and “[the base sequence complementary to the target base sequence] "N (where N is a natural number) base sequence is different" may be expressed as "to generate a mismatch (of target base sequence and N base)". Such a probe may be a polynucleotide ( Hybridizes to A) more strongly than a polynucleotide that produces more mismatches. When the probe causes a mismatch with respect to the target nucleotide sequence, the portion causing the mismatch may be any of substitution, insertion, and deletion. However, substitution is an effect of introducing the mismatch. Is preferable.

  It is also preferred that the probe has a base sequence that causes a small number of mismatches with respect to the target base sequence. The number of mismatches depends on the length of the target base sequence, and it is preferable to increase the number of mismatches as the target base sequence length increases. However, 1-5 are preferred, 1-4 are more preferred, and 2-3 are most preferred. By making the probe a base sequence that causes a small number of mismatches to the target base sequence, the difference in the amount of hybridization between the polynucleotide (A) and the probe of the polynucleotide (B) can be increased, Increased analysis reliability.

  Probes that can be used include, for example, oligo DNA, oligo RNA, oligo DNA or oligo RNA in which the sugar moiety or phosphate group moiety is modified or substituted, and any other compound. Such probes can be synthesized and are commercially available for microarrays and the like.

  The probe may have an irrelevant base sequence portion outside the end of the target base sequence portion, whether or not it has a portion that causes a mismatch with the target base sequence. In this case, only the target base portion and the substantially complementary portion contribute to the hybridization. The length of the irrelevant part in the probe is arbitrary, but is preferably as short as possible, and it may be zero, that is, only the target base sequence part may cause the unknown base sequence part of the test polynucleotide to hybridize by chance. It is preferable from the viewpoint of improving the reliability of analysis.

  The probe may have a portion that is not a base sequence. For example, a functional group such as an amino group for immobilizing the probe on the solid phase when solid-phase synthesizing, an alkylene group or a polyethylene glycol group serving as a spacer between the base sequence portion and the immobilizing functional group, etc. Can do.

  The probe used in the present invention may be a single-stranded probe or a double-stranded probe, and can be selected according to the purpose, but is preferably a single-stranded probe. When the test polynucleotide is double-stranded, it is also preferable to fix both probes having base sequences complementary to each other in independent regions. In that case, it is preferable that the two regions have a mutual distance that allows sufficient diffusion mixing, for example, 100 μm or less.

[Microfluidic device, channel]
The shape of the microfluidic device used in the present invention is arbitrary as long as the fluorescence of a porous layer (hereinafter referred to as “probe fixing portion”) to which the probe is fixed can be observed from outside the microfluidic device. The shape may be a sheet shape (including a sheet shape, a film shape, a ribbon shape, etc.), a tube shape, a rod shape, a lump shape, or the like, but a plate shape is preferable because it is easy to manufacture and use.

  A capillary channel (hereinafter may be simply referred to as [channel]) is formed in the microfluidic device, and may be arbitrarily provided that it is formed at a position where the fluorescence of the probe fixing portion can be observed from the outside. However, it is preferable that the plate-like microfluidic device is formed substantially parallel to the surface on the flat side because fluorescence observation is easy. The microfluidic device includes a plate-shaped member having a groove and a member serving as a plate-shaped lid fixed to a groove forming surface of the member having the groove, and the flow path is a member serving as the groove and the lid. It is also preferable that these are formed. Further, the member having the groove may be a member in which a plate-like member having a defect portion penetrating the front and back of the member is laminated and fixed to another member, and the defect portion may form a groove.

  The channel amplifies a polynucleotide such as PCR (polymerase chain reaction) in one microfluidic device, any other mechanism, for example, a mechanism for pretreatment such as polynucleotide extraction, concentration, and purification. It is also preferable that these processes and the analysis of the present invention be carried out in succession. It is also preferable to perform multi-sample simultaneous measurement using a microfluidic device having a plurality of channels.

  The members constituting the microfluidic device are arbitrary as long as they are transparent so that excitation light irradiation and fluorescence observation are possible. Examples of materials that can be preferably used include crystals such as glass and quartz, and organic polymers (including organic-containing inorganic polymers such as polydimethylsiloxane). Of course, as long as the flow path wall facing the probe fixing portion is formed of the above-described material, the other portions may be opaque.

The cross-sectional shape of the flow path is arbitrary, and may be, for example, a rectangle, a slit, a triangle, a trapezoid, a circle, an ellipse, etc., but a rectangle, a trapezoid, or a semicircle is preferable because it is easy to manufacture and measure. . The cross-sectional area of the flow path is preferably 10 μm ² to 10 mm ² , more preferably 30 μm ^{2 to 1} mm ² , and most preferably 100 μm ^{2 to} 0.3 mm ² μm. By making the flow path cross-sectional area larger than 10 μm ² , it is possible to prevent an increase in pressure loss, facilitate the flow of the sample solution through the flow path, and improve the fluorescence detection sensitivity. By making the flow path diameter smaller than 10 mm ² , even a small amount of sample solution can be analyzed.

  The distance from the surface of the porous layer to which the probe is fixed to the facing surface of the inner wall of the flow channel is preferably 1 to 300 μm, more preferably 3 to 100 μm, and most preferably 5 to 50 μm. By setting this range, the test polynucleotide in the sample solution has a high contact probability with the fixed probe, and the time for the hybridization step can be shortened.

[Probe fixing part]
A porous layer is formed by being fixed to the inner surface of the flow path, and a probe is fixed to the pore surface of the porous layer (hereinafter, the porous layer portion where the probe is fixed is referred to as “probe fixing portion”). Called). The porous layer is formed on the inner surface of the channel in a shape that does not block the channel cross section. Even if the porous body is formed in a shape that closes the cross section of the flow path, the fluid such as the sample solution flows through the pores, but the pressure loss increases, so the pressure resistance of the microfluidic device needs to be increased. In addition, a liquid feeding means capable of feeding a high pressure is required. The porous layer may be formed on the entire circumference in the cross section of the flow path, but is preferably formed on a part of the circumference and not on the facing surface. Thereby, since the fluorescence of the surface of the porous layer can be measured through the flow path, the fluorescence background is reduced, the detection sensitivity is improved, and the quantitative reliability is improved. At this time, the capillary channel is provided inside the plate-like or film-like member substantially parallel to the plane-side surface thereof, and the probe fixing portion is seen from the plane-side surface of the member. In addition, the inner surface facing the channel is preferable because excitation and fluorescence measurement are easy. Further, the microfluidic device includes a plate-like member having a groove and a member that becomes a plate-like lid fixed to a groove forming surface of the member having the groove, and the flow path includes the groove and the lid. It is preferable that the porous layer is formed on the bottom surface of the groove or the member serving as the lid because the microfluidic device can be easily manufactured.

By setting the solid phase for immobilizing the probe as the pore surface of the porous layer, the surface area increases, the density of immobilizing the probe per apparent area increases, and the detection sensitivity increases. In addition, since the detection is possible at a stage where a small amount, for example, a few tenths, is hybridized compared to the saturation amount of hybridization, the hybridization time can be shortened.
The microfluidic device may have a porous layer portion to which the probe is not fixed in addition to the probe fixing portion, and such a portion is also preferably used as a fluorescence background measurement portion. .

  However, when the fluorescence of the probe fixing part formed by the porous layer is measured, the scattered light of the excitation light that is not completely cut increases compared to the case where the probe is fixed on a smooth solid surface, which overlaps with the fluorescence. The fluorescence background (fluorescence intensity measurement value when there is no fluorescent light-emitting substance to be measured) is increased. For this reason, the ratio of the fluorescence intensity of the binding part and the non-binding part of the polynucleotide (A) becomes small, and there has been a problem that the reliability of detection is lowered.

  In the present invention, this problem is solved by setting the light transmittance of the porous layer to a specific value. That is, the transmittance of light having a wavelength of 670 nm at the site for measuring the fluorescence intensity of the porous layer on which the probe is immobilized is 10 to 60% in a state where the pores of the porous layer are filled with water, Is 15-50%. The transmittance can be measured directly through the channel using a microdensitometer. Alternatively, a measurement model in which a porous layer is formed in a large area on a substrate may be measured using a normal spectrophotometer. By setting the light transmittance to the above lower limit or more, the ratio of the fluorescence intensity derived from the sample and the background of the fluorescence increases (that is, the S / N ratio increases), and the accuracy and reliability of the measurement increase. Measurement time can be shortened. In addition, by setting the amount to be equal to or less than the above upper limit, it is possible to sufficiently increase the probe fixing amount while reducing the deterioration of the S / N ratio. Detection sensitivity increases and analysis time can be shortened.

  The light transmittance is measured in a state where the probe is not fixed when the probe fixed to the porous layer has an absorption band at the fluorescence measurement wavelength. If this is not possible, the measurement shall be made at the shortest wavelength without absorption on the long wavelength side of the absorption band. The reason why the light transmittance is measured in a state where the pores of the porous layer are filled with water is that the transmittance is remarkably lowered in the dry state and the measurement error increases to a degree that cannot be ignored. Incidentally, when the light transmittance at 670 nm with the pores filled with water is 10 to 60%, the fluorescence measurement in the dry state and the fluorescence measurement with the water (or buffer solution) filled The S / N ratio is not so different.

  The method for setting the light transmittance in the above range is arbitrary, for example, a method in which the thickness of the porous layer is 1 to 20 μm, preferably 2 to 10 μm, the average pore diameter of the porous layer is less than 0.1 μm, or 10 μm. The method described above, the method of narrowing the pore size distribution of the porous layer to reduce the pore size portion of 0.2 to 3 μm, and the method of making the pore shape of the porous layer into a cylindrical shape facing the fluorescence measurement direction A method of using a material having a low refractive index, preferably a material having a refractive index of 1.3 to 1.49, as the material constituting the porous layer can be employed. Of course, it is also preferable to carry out these simultaneously. Among these, the method of adjusting the thickness of the porous layer and the method of forming the porous layer with a low refractive index material are preferable.

  In the method of setting the thickness of the porous layer to a specific value, it is possible to form a porous layer that is so thin that it cannot be self-supported by fixing the porous layer to the inner wall of the flow path. By reducing the thickness, the scattering intensity of the excitation light can be lowered and the fluorescence background can be lowered in the fluorescence measurement, so that the measurement reliability is improved. In addition, in the hybridization step and the step of washing and removing components that did not bind to the probe after the hybridization step, a predetermined treatment can be performed in a short time, thereby speeding up the analysis.

  The porous layer has a peak in the range of 0.03 to 3 μm, for example, the pore size distribution measured by the mercury intrusion method, so that the amount of immobilized probe is increased and the polynucleotide of the test polynucleotide is hybridized. This is preferable because the speed can be increased. On the other hand, for example, the surface of a glass or polystyrene surface that is roughened by sandblasting or the like to form irregularities has an insufficient surface area increase, and the effect of increasing the amount of probe fixation is small. On the surface having such irregularities, the pore size distribution does not have a clear peak in the range.

  When the porous layer is formed of a material having a low refractive index, when the light transmittance of the porous layer is within the above range, the allowable range of the pore diameter and thickness of the porous layer is widened. Therefore, the amount of the probe immobilized can be increased, and the pore diameter and thickness can be selected so as to increase the hybridization rate of the test polynucleotide, and the production is facilitated. By setting the refractive index to 1.3 or more, it becomes easy to select a material having preferable physical properties such as transparency, hardness, and hydrophilicity. By setting the refractive index to 1.49 or less, sufficient S / N ratio is improved. Can be planned.

  Examples of materials having a refractive index in the above range include glass such as quartz glass, crystals such as fluorite, fluorine-based materials such as (meth) acrylic resin, polyvinyl acetate, silicon resin, and polytetrafluoroethylene. Organic polymers such as resins can be mentioned. Of these, a (meth) acrylic resin is most preferred because it is easy to form a porous layer, easily adjust hydrophilicity, and easily fix the probe. The (meth) acrylic resin may be a thermoplastic resin or a thermosetting resin. Among these, an ultraviolet curable resin is preferable because it is easy to form a flow path and a porous layer and to easily fix the probe.

  A plurality of the porous layers may be formed on the inner surface of the flow path, and a probe may be fixed to each porous layer portion. By forming the porous layer as a plurality of regions independent of each other, different types of probes can be easily fixed without being contaminated with each other. At this time, depending on the purpose of the analysis, different probes are fixed to the probe fixing portions formed as a plurality of regions in one flow path, or separately to the probe fixing portions in the plurality of flow paths formed in parallel. The probe may be fixed to the surface. Moreover, it is also preferable to provide a probe fixing part which is composed of a background measurement part, a negative control measurement part, and a positive control measurement part.

  The method of fixing the probe used in the present invention to the probe fixing part is arbitrary, but it is necessary that the fixed probe is not substantially detached from the solid phase at the hybridization temperature. Examples of such an immobilization method include binding via a protein such as biotin-avidin and immobilization by covalent bond. However, since the bond is strong and the probe is unlikely to be detached from the solid phase, covalent immobilization is possible. Preferably there is.

[Detection method]
In the detection method of the present invention, first, a sample solution is caused to flow through a flow path, and is brought into contact with a porous layer on which the probe is fixed, and a test polynucleotide contained in the sample solution is bound to the fixed probe by hybridization. (Hereinafter sometimes referred to as “hybridization step”). This step is performed under conditions where the polynucleotide (A) selectively hybridizes with the probe.

  The temperature of the hybridization step is arbitrary as long as the probe and the nucleotide (A) can form a hybrid, but the analysis discriminates the difference of one base in the nucleotide sequence of the polynucleotide or oligonucleotide. In this case, the temperature is preferably not lower than the melting temperature (Tm) of the hybrid of the probe and the nucleotide (A) and not higher than [Tm + 10 ° C.]. When the target base sequence has a preferred length of 20 to 25 bases and no mismatch is introduced into the probe, the temperature of the hybridization step is preferably 75 to 100 ° C.

  On the other hand, in the conventional DNA chip method, the hybridization step is generally performed in the range of [melting temperature of the hybrid of the probe and the nucleotide (A) (Tm) −35 ° C.] to [the Tm−15 ° C.], For example, it is performed at 50 to 70 ° C.

  The analysis method of the present invention can use such a high hybridization temperature that could not be used in the prior art. In the present invention, since the amount of probe binding can be greatly increased by using a porous layer in the probe fixing part, a small amount of polynucleotide (for example, about 1/30 of the saturation binding amount) This is because the bond of A) can be detected with sufficient sensitivity, and a temperature with a low saturation bond amount but high selectivity can be employed. In addition, performing the hybridization step at a high temperature as described above could not be performed because the conventional slide glass-type DNA chip method would dry, but hybridization in the flow path can be performed without any problem. .

  By performing the hybridization step in the above temperature range, the ratio of the binding amount of the polynucleotide (A) and the polynucleotide (B) becomes small as in the conventional method when the difference in the one base is analyzed in a short time. In addition, the amount of binding of the polynucleotide (A) does not decrease and the sensitivity does not decrease. For example, when the analysis is for discriminating a difference in base sequence of 3 or more bases, the analysis time can be further shortened by adopting a temperature lower than the above range.

  The melting temperature (Tm) depends on the number of bases in the target base sequence, the GC content, the number of mismatches introduced into the probe, the composition of the solvent, and the like. For example, “Gene Engineering Keyword Book” (page 355 “Modification”, Ogata) It can be measured by the method described in Nobuo, Hiroshi Nojima, 1996, Yodosha). A simple method for calculating the melting temperature is described, for example, in “Bio-Experiment Illustrated, (3) Really PCR” (Hiroki Nakayama, 2003, Shujunsha).

  In the hybridization step, the contact time of the sample solution with the polynucleotide fixing part is arbitrary, but it is preferably a short time within a detectable range with sufficient sensitivity. From this viewpoint, 1 to 10 minutes is preferable. Preferably, 2 to 6 minutes is more preferable.

  Any method can be used to bring the sample solution into contact with the fixed portion of the probe, and the sample solution can be contacted in the flow path in one direction or in a reciprocating manner or in a retained state. This is preferable because the contact time can be shortened.

  When the polynucleotide in the sample solution is a double-stranded polynucleotide, prior to the hybridization step, the sample solution is heated and subjected to the hybridization step in a state in which the test polynucleotide is nautical miles; Alternatively, it is preferable that the sample solution is heated and dissociated in contact with the probe, and then held at the hybridization step temperature for a predetermined time.

[Washing process]
After the hybridization step, a washing solution is flowed through the flow path to remove the sample solution component that has not bound to the probe (this is referred to as a washing step). The washing solution is optional, but it must be one that does not excessively remove the polynucleotide (A) bound to the probe. Examples of such liquids include those known as liquids that strongly denature polynucleotides, such as formamide and sodium hydroxide solution. Whether or not the washing solution excessively removes the polynucleotide (A) bound to the probe can be confirmed by experiments. The reference liquid at that time is the solvent of the sample solution. The washing solution that can be preferably used is water or a buffer solution in addition to the solvent of the sample solution. Of course, the washing liquid must be one that does not interfere with detection. For example, it is necessary that it does not emit fluorescence at the fluorescence measurement wavelength and does not have strong absorption at the fluorescence excitation wavelength.

[Detection process]
After the washing step, fluorescence emitted when a polynucleotide or oligonucleotide is bound to the probe is measured, and the presence or absence of binding or the amount of binding is measured from the intensity (this is referred to as a detection step). That is, the fluorescence intensity varies depending on the amount of polynucleotide or oligonucleotide bound to the probe fixing part. Of course, when a plurality of fluorescent dyes are used as the labeling fluorescent dye, it can be determined from the color or spectrum of the fluorescence, but this is also a modification of the measurement of the fluorescence intensity at a specific wavelength.

  As long as the fluorescence is measured by measuring the fluorescence intensity of the probe fixing part and measuring the binding amount of the polynucleotide bound to the site, the target fluorescent light-emitting substance and the measuring method are arbitrary. . For example, a method of using a fluorescently labeled polynucleotide or oligonucleotide as the test polynucleotide, a method of measuring fluorescence of the polynucleotide or oligonucleotide itself, a double strand of a hybrid formed by the polynucleotide or oligonucleotide and a probe Examples include a method of adding a fluorescent intercalator that intercalates to change the fluorescence emission yield or change the fluorescence spectrum. Among these, the use of a fluorescently labeled polynucleotide is preferable because of high measurement sensitivity and measurement accuracy. Subsequently, the use of a fluorescent intercalator that intercalates into double strands and increases the fluorescence emission yield is preferred.

  The measurement of the fluorescence intensity can be measured from any position of the porous layer of the probe fixing part, but is measured from the surface side of the porous layer in contact with the sample solution through the flow path. However, high sensitivity is preferable. The measurement of the fluorescence intensity may be measurement of a plurality of probe fixing parts, and in that case, the plurality of probe fixing parts may be measured simultaneously or sequentially by scanning. Using the fluorescence intensity of the probe-fixed part that is not in contact with the sample solution as a background, subtract it from the fluorescence intensity of the measurement part, or bring the sample solution into contact with the porous part where the probe is not fixed, and perform the hybridization process. It is also preferable to subtract the intensity from the fluorescence intensity of the measurement part as a negative control.

  EXAMPLES Hereinafter, although this invention is demonstrated in more detail using an Example and a comparative example, this invention is not limited to the range of a following example. In this example, “part” represents “part by mass”, and “%” regarding the mixing ratio represents “% by mass”.

(Irradiation with ultraviolet lamp 1)
Using a UE031-353CHC type UV irradiation device manufactured by Eye Graphics Co., Ltd. using a 3000 W metal halide lamp as a light source, ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² were irradiated in a nitrogen atmosphere at room temperature unless otherwise specified.

(Irradiation with ultraviolet lamp 2)
Using a light source unit for multi-light 250W series exposure apparatus manufactured by USHIO INC., Which uses a 250W high-pressure mercury lamp as a light source, ultraviolet light with an ultraviolet intensity at 365 nm of 50 mW / cm ² is used in a nitrogen atmosphere at room temperature unless otherwise specified. Irradiated.

(Fluorescence intensity measurement method)
Measurement was performed using a plate reader FLA-3000G manufactured by Fuji Photo Film Co., Ltd. When Dy5 was used as the labeling dye, the excitation wavelength was 633 nm (laser excitation), and the detection wavelength was 675 nm or more (with a cut filter having a cutoff wavelength of 675 nm). When FITC was used as the labeling dye, the measurement was carried out with an excitation wavelength of 480 nm (laser excitation) and a detection wavelength of 520 nm or more (by a cut filter with a cutoff wavelength of 520 nm).

(Example 1)
[Preparation of film-forming solution (Y1)]
As energy beam polymerizable compound (b), trifunctional urethane acrylate oligomer “Unidic V-4263” (Dainippon Ink & Chemicals, Inc.) having an average molecular weight of 2000, 72 parts, dicyclopentanyl diacrylate “R-684” 18 parts (manufactured by Nippon Kayaku Co., Ltd.), 10 parts glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), 180 parts of methyl decanoate (manufactured by Wako Pure Chemical Industries, Ltd.) as a poor solvent (R), volatile As a good solvent, 10 parts of acetone and 3 parts of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as an ultraviolet polymerization initiator were uniformly mixed to prepare a film-forming solution (Y1).

[Preparation of Composition (X1)]
70 parts of trifunctional urethane acrylate oligomer “Unidic V-4263” (manufactured by Dainippon Ink & Chemicals, Inc.) having an average molecular weight of 2000 as energy beam polymerizable compound (a), hexanediol diacrylate “New Frontier HDDA” (first 30 parts by Kogyo Seiyaku Co., Ltd., 3 parts by weight of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (Ciba Geigy) as a photopolymerization initiator, and 2,4-diphenyl-4-methyl-1- A composition (X1) was prepared by mixing 0.5 parts of pentene (manufactured by Kanto Chemical Co., Inc.).

[Preparation of composition for lid]
80 parts of a trifunctional urethane acrylate oligomer “Unidic V-4263” (Dainippon Ink Chemical Industries, Ltd.) having an average molecular weight of 2000 as an energy ray polymerizable compound, hexanediol diacrylate “New Frontier HDDA” (Daiichi Kogyo Seiyaku Co., Ltd.) 20 parts of a company) and 2 parts of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a photopolymerization initiator were mixed to prepare a composition for a lid.

[Target base sequence, probe, polynucleotide (A), polynucleotide (B)]
[Target base sequence]
The base sequence of 25 bases shown in Table 1 was used as the target base sequence.

[Probe (PR0)]
A 25-base long oligo DNA (manufactured by Espec Oligo Service Co., Ltd.) having a base sequence complementary to the target base sequence amino-modified at the 5 ′ end was used as a probe (PR0) (Table 1).

[Probe (PR1)]
An oligo DNA (manufactured by Espec Oligo Service Co., Ltd.) similar to the probe (PR0) except that the 13th guanine in the target base sequence was replaced with adenine was used as the probe (PR1) (Table 1).

[Test polynucleotide (F0)]
The 5 ′ end is modified with a fluorescent substance “Cy5”, and a 25-base long oligo DNA (manufactured by Espec Oligo Service Co., Ltd.) having a base sequence portion complementary to the probe (PR0) is used as a test polynucleotide (F0). (Table 1). The oligo DNA (F0) is a polynucleotide (A) having a base sequence complementary to the probe (PR0), and is a polynucleotide (B) that causes a single base mismatch to the probe (PR1). .

[Test polynucleotide (F1)]
A 5'-end oligo DNA (manufactured by Espec Oligo Service Co., Ltd.) having a base sequence portion complementary to the probe (PR1) is modified with a fluorescent substance “Cy5” at the 5 ′ end, and a test polynucleotide (F1) (Table 1). The oligo DNA (F1) is a polynucleotide (B) that causes a single base mismatch with respect to the probe (PR0), and is a polynucleotide (A) having a complementary base sequence with respect to the probe (PR1). .

[Test polynucleotide (F0 ′)]
Except that the labeled fluorescent substance was “FITC”, the same oligo DNA (manufactured by Espec Oligo Service Co., Ltd.) as the test polynucleotide (F0) was used as the test polynucleotide (F0 ′) (Table 1).

[Test polynucleotide (F1 ′)]
Except that the labeled fluorescent substance was “FITC”, the same oligo DNA (manufactured by Espec Oligo Service Co., Ltd.) as the test polynucleotide (F1) was used as the test polynucleotide (F1 ′) (Table 1).

[Production of microfluidic devices]
[Formation of a member having a groove and a fixing portion]
A 1 mm-thick acrylic plate is used as the support (1), and a film-forming solution (Y1) is applied onto the support (1) using a spin coater, and ultraviolet rays are applied to the film-forming solution (Y1). The film-forming solution (Y1) is cured by irradiating ultraviolet rays from the lamp 1 for 40 seconds, and at the same time the phases are separated, and the poor solvent (R) is washed and removed with n-hexane to form a white porous layer (2). Formed.

  A composition (X1) is applied onto the porous layer (2) using a spin coater, the composition (X1) is filled in the pores of the porous layer (2), and passed through a photomask. The portion other than the portion to be formed as the fixing portion (5) is irradiated with ultraviolet rays by the ultraviolet lamp 2 for 120 seconds to cure the composition (X1) in the irradiated portion and fill the pores in the non-irradiated portion. The uncured composition (X1) is removed with ethanol, and the porous fixing part (5a), (5b), (5c) [hereinafter, these three members are collectively referred to as the fixing part (5)]. Formed on a support. That is, the porous layer (2) other than the fixing part (5) is transparent because the pores are filled with the cured product of the composition (X1).

  The composition (X1) is applied on the porous layer (2) subjected to the above-described treatment using a spin coater, and the ultraviolet ray by the ultraviolet lamp 2 is applied to a portion other than the portion to be the groove (6) through a photomask. Irradiation is performed for 120 seconds to form a coating film (3) in which the irradiated part is semi-cured, and the uncured composition (X1) in the non-irradiated part is removed with ethanol to fix the fixed parts (5a) and (5b), respectively. ), (5c) were exposed on the bottom, and a groove (6) branched into three grooves (6a), (6b), (6c) was formed.

[Probe fixation]
A 5% aqueous solution of 5% polyallylamine (molecular weight: 15000, manufactured by Nittobo Co., Ltd.) is placed on the entire groove (6) prepared in the above step 2, and a cover brass (not shown) is placed on the groove (6). After allowing to stand for a period of time and reacting some of the amino groups in the polyallylamine with the epoxy groups in the porous layer, it was washed with running water for 15 minutes to introduce the amino groups into the fixing part (5). .

  The member having the fixing part (5) into which the amino group was introduced was placed in a 5% glutaraldehyde (Wako Pure Chemical Industries, Ltd.) aqueous solution and allowed to stand at 50 ° C. for 2 hours. After reacting the amino group with one aldehyde group in glutaraldehyde, the aldehyde group was introduced into the fixing part (5) by washing with running water for 10 minutes.

  Next, 2 μL of a 50 μM aqueous solution of the probe (PR0) amino-modified at the 5 ′ end is placed on the fixing part (5a), which is one of the fixing parts (5), and the probe (5b) Distribute 2 μL of 50 μM aqueous solution of (PR1), protect it with a masking tape (not shown) so as not to move to other parts, put a cover glass (not shown), and humidity 100% at 50 ° C. After standing for 15 hours, the terminal amino group of the DNA was reacted with the aldehyde group of the porous layer. Thereafter, the member is immersed in a 0.2% aqueous solution of sodium tetrahydroborate, subjected to a reduction reaction for 5 minutes, then rinsed with a 0.2 × SSC / 0.1% SDS solution, and then 0.2% After rinsing with × SSC, further rinsing with distilled water, and air drying, DNA (PR0) as a probe was fixed to the fixing part (5a), and DNA (PR1) as a probe was fixed to the fixing part (5b). The probe is not fixed to the fixing part (5c). (Here, 0.2 × SSC is 0.03 NaCl, 3 mM sodium citrate aqueous solution, and 0.1% SDS is 0.1% sodium dodecyl sulfate aqueous solution.)

[Fixing the lid]
The composition for a lid was coated on a biaxially stretched polypropylene film (manufactured by Nimura Chemical Co., Ltd.) (not shown) having a thickness of 30 μm on one side subjected to corona discharge treatment using a bar coater having a coating thickness of 150 μm. . The uncured coating film (not shown) is irradiated with ultraviolet rays for 2 seconds by an ultraviolet lamp 1 to form a semi-cured coating film (not shown) of the composition, and the groove (6) of the member produced above A resin layer (4) which is laminated on the forming surface is formed as a lid, and the ultraviolet lamp 1 is irradiated with ultraviolet rays for 40 seconds to completely cure all the layers including the resin layer (4). A microfluidic device was manufactured using 6) as a flow path (6).

  Thereafter, holes (7a), (7b), (7c) having a diameter of 0.7 mm that pass through the support (1) and the porous layer (2) at both ends of the channel and communicate with the channel (6). , (8) is formed using a drill, and a polyvinyl chloride pipe having an inner diameter of 3 mm and a height of 5 mm is bonded to the holes (7a), (7b), (7c), and (8), respectively. Side openings (9a), (9b), (9c), and outlet side openings (10) were formed to produce a microfluidic device.

[Measurement of characteristics]
[Dimensions of each part]
The dimensions of each part of the manufactured microfluidic device were as follows. The outer shape is 75 mm x 25 mm x about 1.2 mm, and the flow path (6) is branched into three (6a), (6b), and (6c) near the end on the outlet side. The thickness (depth) is about 100 μm and the length is about 50 mm. The thickness of the resin layer (4) serving as a lid was about 100 μm. The fixing portions (5a), (5b), and (5c) are provided in the central portions of the flow paths (6a), (6b), and (6c), respectively, and each has a width of 500 μm and a length in the flow path direction of about It was 3 mm.

(Porous layer)
When the surface of the porous layer (2) was observed with a scanning electron microscope, pores having an average pore diameter of about 0.7 μm were formed as gaps between aggregated particles having a diameter of about 0.5 μm. Moreover, the thickness of the porous layer (2) was about 6 μm.
In the middle of the manufacturing process, when the porous layer (2) was formed on the support (1), the porous layer (2) was filled with distilled water, and the light transmittance at a wavelength of 670 μm was measured. The rate was about 28%.

  Further, as a resin model for forming the porous layer (2), a component obtained by removing the phase separation agent from the film-forming solution (Y1), that is, “Unidic V-4263” (72 parts, “R-684” 18 Part, 10 parts of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and 3 parts of “Irgacure 184” were prepared, and this was a biaxially stretched polypropylene having a thickness of 30 μm and subjected to corona discharge treatment on one side. A film (made by Nimura Chemical Co., Ltd.) (not shown) is coated using a bar coater, cured by irradiating it with ultraviolet lamp 60 for 60 seconds, and then peeled off. The refractive index of this film measured with an Abbe refractometer was 1.48.

[Preparation of sample solution]
1 μL of a 500 μM solution of oligo DNA (F0) labeled with a fluorescent substance “Cy5” having a base sequence portion complementary to the probe (PR0) and 499 μL of a 0.2 × SSC solution are mixed uniformly to obtain a concentration. A 1 μM DNA solution was prepared. Further, this DNA solution was mixed with 50 μL and 0.2 × SSC solution 50 μL to obtain a measurement sample solution (SF0) having a concentration of 0.5 μM.
In addition, a sample solution for measurement (SF1) was prepared in the same manner as described above except that an oligo DNA (F1) having a base sequence portion complementary to the probe (PR1) was used instead of the oligo DNA (F0). did.

[Measurement of sample solution (SF0)]
A heater block (not shown) was brought into contact with the lid (4) side of the flow path type microfluidic device (D) to adjust the microfluidic device (D) to 85 ° C.
Next, 20 μL each of the sample solution for measurement (SF0) is put into the inlet side openings (9a) and (9b) of the microfluidic device (D), and 20 μL of the buffer solution is put into the inlet side opening (9c). From the outlet side opening (10) of the fluidic device (D1), the sample solution (SF0) in the inlet openings (9a) and (9b) and the buffer solution in (9c) are all discharged using a micro syringe pump. Suction was performed at a rate of 15 μL / min until it moved to the side opening (10). Next, the microfluidic device (D) is naturally cooled to room temperature, and the inside of the flow paths (6a), (6b), (6c) is washed with 20 μL of 0.2 × SSC solution. X SSC solution was used for fluorescence measurement while the flow paths (6a), (6b), and (6c) were filled.

  The fluorescence measurement was performed using a fixed part (5a) [probe (PR0) fixed part], a fixed part (5b) [probe (PR1) fixed part], and a fixed part (5c) [probe ratio fixed part. [Background]. The measurement results are summarized in Table 1. As can be seen from Table 1, the probe (PR0) immobilization part and the probe (PR1) immobilization, although the contact time of the hybridization step is about 5 minutes, which is very short compared with several hours of the conventional method. The fluorescence intensity ratio of the part is as large as about 9.7 (ratio of the value obtained by subtracting the background), and the value of the background [fixed part (5c)] is sufficiently small, and a highly reliable result was obtained. .

[Measurement of specimen solution (SF1)]
The measurement was performed in the same manner as in the case of “Measurement of sample solution (SF0)” except that the sample solution (SF1) was used as the sample solution. The results are shown in Table 1.
As can be seen from Table 1, the fluorescence intensity ratio between the probe (PR0) immobilization part and the probe (PR1) immobilization part is about 2 even though the contact time of the hybridization step is as short as about 5 minutes. 7 (ratio of values obtained by subtracting the background) and the background was sufficiently small, and a highly reliable result was obtained.

[Comparative example]
The same experiment as in Example 1 was performed, except that “FITC” was used instead of “Cy5” as the fluorescent labeling dye for DNA, and that the excitation wavelength and the measurement wavelength were the wavelengths for the FITC. The results are summarized in Table 2.
In this case, the difference in fluorescence intensity between the polynucleotide (A) and the polynucleotide (B) is very small, and the background [fixed part (5c)] value is high, indicating that the reliability is poor.

It is a schematic diagram of the channel for chromatography of the present invention used for the example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Support body 2 ... Porous layer 3 ... Coating film, resin layer 4 ... Resin layer, member used as a lid | cover 5, 5a, 5b, 5c ... Fixed part 6, 6a, 6b , 6c ... groove, flow path 7a, 7b, 7c, 8 ... hole 9a, 9b, 9c ... entrance side opening 10 ... exit side opening

Claims (12)

A polynucleotide to be detected or a polynucleotide to be detected on a probe fixing portion of a microfluidic device having a porous layer on which a probe that specifically binds to a polynucleotide or oligonucleotide having a target base sequence is immobilized on the inner surface of a capillary channel After contacting the sample solution containing the oligonucleotide, and then washing the probe fixing part to remove the sample solution component that did not bind to the probe, the detection control polynucleotide or oligonucleotide was bound to the probe. In the polynucleotide or oligonucleotide detection method for measuring the fluorescence intensity of the probe immobilization part based on the above, the transmittance of light having a wavelength of 670 nm at the site for measuring the fluorescence intensity of the porous layer on which the probe is immobilized is 10 ~ 60% and measurement of the fluorescence intensity Detection method of a polynucleotide or oligonucleotide is a fluorescent intensity measured at a wavelength of above 650 nm. The method for detecting a polynucleotide or oligonucleotide according to claim 1, wherein the detection of the polynucleotide or oligonucleotide selectively detects a difference of one base in the base sequence. The temperature at which the sample solution is brought into contact with the probe fixing part is not less than the melting temperature of the hybrid of the probe and a polynucleotide or oligonucleotide that specifically binds to the probe and not more than [the melting temperature + 10 ° C.]. Item 3. A method for detecting a polynucleotide or oligonucleotide according to Item 1 or 2. The method for detecting a polynucleotide or oligonucleotide according to claim 1 or 2, wherein a temperature at which the sample solution is brought into contact with the probe fixing part is in the range of 75 to 100 ° C. The method for detecting a polynucleotide or oligonucleotide according to any one of claims 1 to 4, wherein the time for contacting the sample solution and the probe is in the range of 1 to 10 minutes. The method for detecting a nucleotide according to claim 1, wherein the porous layer has a thickness of 1 to 20 μm. The method for detecting a polynucleotide or oligonucleotide according to any one of claims 1 to 6, wherein the porous layer is formed of a material having a refractive index in the range of 1.3 to 1.49. The capillary channel is provided in a plate-like or film-like member approximately parallel to the plane-side surface thereof, and the porous layer to which the probe is fixed is provided on the plane-side surface of the member. It is an inner surface facing the flow channel as viewed from the above, and the fluorescence is measured from the surface side of the porous layer through the flow channel from the surface side surface direction of the member. The method for detecting a nucleotide according to any one of the above. A microfluidic device having a porous layer in which a probe that specifically binds to a polynucleotide or oligonucleotide to be detected is immobilized on the inner surface of a capillary channel, wherein the porous layer has a thickness of 1 A microfluidic device having a transmittance of 10 to 60% of light having a wavelength of 670 nm in a state of ˜20 μm and filled with water, and formed in a part of the circumference in the flow path cross section. The microfluidic device according to claim 9, wherein the porous layer is formed of a material having a refractive index in the range of 1.3 to 1.49. The microfluidic device according to claim 9 or 10, wherein the porous layer is formed of a (meth) acrylic resin. The microfluidic device includes a plate-like or film-like member having a groove, and a member that becomes a plate-like or film-like lid fixed to the groove forming surface of the member having the groove, and the flow path is formed of the groove and The microfluidic device according to any one of claims 9 to 11, wherein the microfluidic device is formed by a member serving as a lid, and the porous layer is formed on a bottom surface of the groove or a member serving as the lid.

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