JP2012047457A - Cleaning method of micro fluid device and cleaning liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cleaning method with which a performance of a micro chip can be recovered, and a cleaning liquid.SOLUTION: The present invention relates to a cleaning method of a micro fluid device, including cleaning a fluid channel which is formed in the micro fluid device and has a surface having polymer coating and with which a sample containing nuclear acid and/or protein is brought into contact, by bringing the fluid channel into contact with a cleaning liquid comprised of only an organic solvent having water solubility at least to water of the same volume at 25°C, or a cleaning liquid containing the organic solvent of 50 vol.% or more in a buffer solution. In the cleaning method, the pH of the buffer solution is 8 to 10; the buffer solution further contains a protein denaturant of 3 to 8M; or the pH of the buffer solution is 2 to 4.

Description

The present invention relates to microchip LC and microchip electrophoresis performed in a microfabricated microfluidic device (microchip).
The present invention relates to a microchip cleaning method and a cleaning liquid after use.

  Assays using the characteristic sequences of genes tend to increase year by year, in particular, single or multiplex PCR products, restriction enzyme digests by PCR-RFLP, genotyping such as SNP analysis, PCR reaction condition studies, real time There is a strong demand for simple, rapid and low-cost analysis of reaction solutions after PCR measurement.

In response to this requirement, microchip electrophoresis is becoming a very suitable technique instead of agarose gel electrophoresis. In particular, the separation detection time is as short as 1 to 2 minutes, and it is possible to perform high-precision separation that could not be identified by agarose gel electrophoresis, and it can be displayed and stored as numerical data. It is a feature. Furthermore, if the reaction solution on the PCR plate can be set in the apparatus as it is without being purified or diluted, the operability is greatly improved.
However, since microfabricated microchips (for example, Patent Document 1) are expensive, it is preferable to reuse them rather than disposables in terms of running cost.

  As a cleaning method for reusing the microchip, a method of cleaning the microchip reservoir and the flow path and removing the electrophoresis buffer, the separation medium, the sample, and the like every time the electrophoresis of the sample is completed. (For example, Non-Patent Document 1).

JP-A-2005-214710

Shimadzu Corporation MultiNA Instruction Manual Equipment System

When the amplification product of the PCR plate is directly set in an electrophoresis apparatus and subjected to microchip electrophoresis, the sample contains DNA polymerase, primer DNA, animal / plant-derived target DNA, dNTP, and DNA polymerase. In order to maintain the activity, KCl and MgCl ₂ coexist. In addition, non-ionic surfactants, loading dyes and specific gravity increasing agents premised on the use of agarose gel electrophoresis may be premixed in the PCR reaction buffer for efficient DNA amplification. is there. It is not uncommon for assays that contain restriction enzymes to contain BSA, betaine, and the like. Among these, proteins, basic or hydrophobic compounds contained in samples tend to adsorb on the surface even if the surface of the microchip varies depending on the material such as glass, quartz, resin, etc. When the microchip is repeatedly used, there remains a risk that the separation characteristics change over time. On the other hand, it is possible to reduce the influence on the channel surface by purifying or diluting the crude sample. However, since an operation for the reaction product is added, the convenience is impaired.

Until now, used microchips have been washed with water and reused. However, the recovery of the performance of the microchip may be insufficient due to the adsorption of various components as described above.
Therefore, an object of the present invention is to provide a cleaning method and a cleaning liquid that can recover the performance of a microchip.

The present inventor has found that the object of the present invention can be achieved by using a cleaning liquid based on a polar organic solvent, and has completed the present invention.
The present invention includes the following inventions.

(1)
A flow path formed in a microfluidic device, the flow path having a surface having a polymer film and contacting a sample containing nucleic acid and / or protein,
A microfluidic device for cleaning by bringing it into contact with a cleaning liquid consisting only of an organic solvent having solubility in at least the same volume of water at 25 ° C. or a cleaning liquid containing 50% by volume or more of the organic solvent in a buffer solution Cleaning method.

(2)
The cleaning method according to (1), wherein the pH of the buffer solution is 8 to 10.
(3)
The cleaning method according to (1) or (2), further comprising a protein denaturant of 3 to 8M in the buffer solution.

(4)
The cleaning method according to (1), wherein the pH of the buffer solution is 2 to 4.

(5)
A microfluidic device cleaning solution comprising only an organic solvent having solubility in at least the same volume of water at 25 ° C. or containing 50% by volume or more of the organic solvent in a buffer solution.
(6)
The cleaning liquid according to claim 5, wherein the buffer has a pH of 8 to 10.
(7)
The cleaning liquid according to (5) or (6), further comprising a protein denaturant of 3 to 8M in the buffer solution.
(8)
The cleaning liquid according to claim 5, wherein the buffer has a pH of 2 to 4.

  ADVANTAGE OF THE INVENTION According to this invention, the cleaning method and cleaning liquid which can recover the performance of a microchip can be provided.

The interaction between the microchip channel surface and the components in the vicinity of the channel is schematically shown. An example of a microchip to be cleaned is shown. It is a comparison of electropherograms using microchips before and after cleaning.

[1. Microfluidic device (microchip)]
In the present invention, the flow path configuration or design of the microfluidic device to be cleaned is not particularly limited as long as it can hold and transport a liquid containing nucleic acids and / or proteins.
The material of the microfluidic device is not particularly limited, such as quartz, glass, and resin. They are preferably highly transparent from the viewpoint of optical properties. In particular, the cleaning method and cleaning liquid of the present invention are effective in the case of a material having a silanol group on the surface.
A channel is formed in the microfluidic device, and the channel is subjected to a surface treatment of a polymer film.

[1-1. Polymer film]
The polymer film may have a hydrophilic group that provides a separation environment for a sample containing nucleic acid or protein, and may protect the surface of the flow channel from interaction with the sample or separation buffer. When performing electrophoretic separation of a sample containing nucleic acid or protein in the flow path, it is also intended to suppress electroosmotic flow. When performing separation by chromatography of a sample containing nucleic acid or protein in the flow path, It can also be a separation carrier or a separation stationary phase.

The polymer film may be formed by adsorption on the channel surface or by a covalent bond.
When the polymer film is formed by covalent bonding to the surface of the flow path, in one embodiment of such a film, one functional group of the bifunctional group is covalently bonded to the surface of the flow path, and the bifunctional group The other functional group is covalently bonded to a polymer that forms an organic film (monomolecular film). Such a polymer film can be formed by bringing a bifunctional compound into contact with the surface of the flow path to covalently bond one functional group to the surface, and then adding a monomer to polymerize with the other functional group. In this case, as a bifunctional compound, in the case of a material having a silanol group on the channel surface, γ-methacryloxypropyltrimethoxysilane, vinyltriacetoxysilane, vinyltri (β-methoxyethoxy) silane, vinyltrichlorosilane, methyl Examples include silane coupling agents such as vinyldichlorosilane. Examples of the monomer include acrylic, acryloyl, methacryl, allyl, vinyl and acrylamide monomers. The monomer may further have a hydrophilic group such as an amino group, a hydroxyl group, or a pyrrole group as a substituent. In particular, in the cleaning method and cleaning liquid of the present invention, when the channel surface has a copolymer polymer film having a pKa of 8 to 10, charging of the polymer film is suppressed, and the components in the channel due to electrostatic interaction are suppressed. It is possible to prevent adsorption and is effective.

  If the polymer film is due to adsorption to the flow path surface, such a film can be formed by applying the polymer or simply mixing the polymer with the separation buffer and contacting the separation buffer with the flow path surface. Can be created by forming a film of the polymer on the surface of the flow path.

  Even in microfluidic devices where liquid chromatography is performed, in addition to a separation carrier in which a silanol group on the channel surface is chemically bonded to a silylating agent having a hydrophobic functional group, it can improve alkali resistance and reduce protein adsorption. Those having a film of a water-soluble polymer for the purpose can be applied. Examples of the water-soluble polymer include methyl cellulose and polyoxyethylene.

[1-2. Flow path]
The microfluidic device only needs to have a channel formed therein, and is not necessarily limited to a small size. The flow path can generally include a main flow path where analysis is performed as the solution moves. For example, an electrophoresis channel including a separation channel in which an electrophoresis step is performed, a liquid feeding channel including a separation carrier in which a liquid chromatography step is performed, and a liquid feeding channel having a surface modification as a separation stationary phase Etc. Furthermore, a sample and / or a mobile phase introduction path leading to the main flow path may be formed. A plurality of each flow path may be formed.

[1-3. Concrete example]
FIG. 2 shows a microchip for electrophoresis as an example of a microfluidic device.
As shown in FIG. 2, the microchip 1 is composed of a pair of transparent substrates (quartz glass or other glass substrate or resin substrate) 11 and 12, and on the surface of one substrate 12, as shown in FIG. The electrophoresis capillary grooves 14 and 15 intersecting each other are formed, and the reservoir 13 is provided as a through hole in the other substrate 11 at a position corresponding to the ends of the grooves 14 and 15 as shown in FIG. The substrates 11 and 12 are overlapped and joined as shown in FIG. 2C, and the capillary grooves 14 and 15 are separated into the separation flow path 15 for electrophoresis separation of the sample and the sample for introducing the sample into the separation flow path. Let it be an introduction flow path 14.
The microchip 1 is basically the one shown in FIGS. 2A to 2C. However, in order to facilitate handling, the microchip 1 is used to apply a voltage as shown in FIG. The electrode terminal may be formed on the chip in advance (described later in the embodiment).

[2. Adsorption of sample-derived components]
The present invention is used for a used microfluidic device. The usage history of the microfluidic device is not particularly limited as long as an electrophoresis buffer containing a separation medium or an intercalator fluorescent dye is enclosed or a sample containing a nucleic acid and / or a protein contacts the flow path surface. Nucleic acids and proteins can be analyzed. At the time of analysis, the sample can come into contact with the channel surface together with the mobile phase or the separation buffer.

[2-1. Adsorption component]
In addition to the nucleic acid (DNA, RNA) as the analysis target, the sample containing the nucleic acid and the mobile phase include, for example, components other than the analysis target, such as nucleic acid components, enzymes, stabilizers, specific gravity increasing agents, fluorescent dyes, Along with salt, it can be included in the buffer. These components can be adsorbed as impurities on the channel surface of the microfluidic device.

More specifically, examples of the enzyme include reverse transcriptase, DNA polymerase, and restriction enzyme.
Examples of nucleic acid components other than those to be analyzed include template DNA, primer DNA, dNTP (dATP, dTTP, dGTP, dCTP, dUTP).
Examples of the stabilizer include glycerol, protein, surfactant, betaine and the like. Examples of the surfactant include Tween 20, Nonidet P-40, Brij-35, Triton X-100 and the like. BSA is mentioned as a protein as a stabilizer.
Examples of fluorescent dyes include bromophenol blue, xylene cyanol, and orange G as loading dyes, and ethidium bromide, SYBR Gold, SYBR Green, SYBR Safe, and GelStar as intercalators.

  The sample containing the nucleic acid can be obtained, for example, as a result of a nucleic acid amplification reaction. Nucleic acid amplification reactions are represented by PCR, but various other reactions are known to those skilled in the art. When PCR is taken as an example, variations include normal PCR, PCR-RFLP, quantitative PCR (real-time PCR), RT-PCR, colony PCR, and all other aspects. Further, the scale of the reaction solution (reaction solution amount) and the amount of amplification product are not particularly limited. The sample containing nucleic acid may be subjected to a purification step and / or a dilution step after the nucleic acid amplification reaction, or may not be subjected to such a step.

[2-2. Assumed mechanism of adsorption]
An example will be described in which electrophoresis separation of DNA molecules is performed in a channel formed in a microfluidic device. When a sample containing DNA molecules and a separation buffer come into contact with the surface of the channel, the DNA molecules basically do not interact with the surface of the channel and bind to a fluorescent dye that can act as an intercalator while negatively charging the DNA. And separated by the sieving effect of the water-soluble polymer in the separation buffer. In this case, various components present in the flow path can be adsorbed by various interactions that can occur between the surface of the flow path and / or between the components as schematically shown in FIG. . FIG. 1 shows a flow path having a silanol group on the surface, and γ-methacryloxypropyltrimethoxysilane is used as a silane coupling agent, and linear polyacrylamide ( When a sample containing DNA is electrophoresed with a separation buffer having a pH of 8.3 using a microfluidic device having a surface of the flow path subjected to a coating treatment of LPA), the surface of the flow path, the sample, and the components containing the separation buffer The interaction is schematically shown.

When the electrophoresis condition is, for example, pH 8.3, since free silanol groups remain on the surface of the flow path, SYBR Gold in the buffer can be adsorbed by ionic bonds or a basic component can be adsorbed. Enzymes, nonionic surfactants and BSA that can be included as stabilizers for PCR reactions and the like can be adsorbed by hydrophobic interaction with a silane coupling agent covalently bonded to the channel surface.
Furthermore, linear polyacrylamide (LPA) is considered to have a pKa of 8.0 to 10.0, and is assumed to have a positive charge by washing with buffer or water near pH 8, and DNA molecules, BSA, etc. are electrostatically charged. Can be adsorbed.
Note that the assumed mechanism of adsorption shown in FIG. 1 only illustrates the main interactions. In fact, components other than those shown in FIG. 1 (glycerol, restriction enzymes, etc.) coexist in the flow path. It is thought that various interactions are working.

[3. Cleaning fluid]
[3-1. Polar organic solvent based cleaning solution]
In order to remove these adsorbing components, in the present invention, a cleaning liquid composed of 100% polar organic solvent or a cleaning liquid in which 50% by volume or more, preferably 70% by volume or more polar organic solvent is dissolved in water or a buffer solution. Is brought into contact with the channel surface. If the amount of the polar organic solvent is below the above range, the cleaning effect of the present invention tends to be difficult to obtain.
The polar organic solvent in the present invention has solubility in at least the same volume of water at 25 ° C. Specific examples include alcohol and / or acetonitrile. As the alcohol, methanol, ethanol, isopropyl alcohol and the like are particularly used.
Since such a cleaning liquid can alleviate the hydrophobic interaction, for example, it is possible to remove proteins and stabilizers that can interact with the hydrophobic portion of the silane coupling agent on the surface of the flow path. .

[3-2. Alkaline cleaning solution]
When the cleaning liquid includes a buffer solution, the buffer solution may have a pH of 8 to 10, preferably 8.3 to 8.9, for example.
Buffers include Tris-HCl buffer, Tris-acetate buffer, HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] -ethanesulfonic acid) buffer, MOPS (morpholine propanesulfonic acid) buffer, TAPS (N -[Tris (hydroxymethyl) methyl] -3-aminopropanesulfonic acid) buffer, phosphate buffer and the like can be used.

In an alkaline region such as pH 8 to 10, since the charging of the polymer film on the surface of the flow path can be suppressed, nucleic acid components (for example, DNA) and proteins (for example, BSA) that can have an electrostatic interaction with the flow path surface are removed. Is possible.
When the pH of the buffer solution falls below the above range, the above-described effects tend to be difficult to obtain. When the pH of the buffer solution exceeds the above range, the silane coupling agent tends to hydrolyze and tend to cause irreversible damage to the polymer film.
This cleaning liquid can basically be used for a microfluidic device that has undergone any use history exemplified herein.

[3-3. Denaturant-containing cleaning solution]
The cleaning liquid may further contain a modifying agent. Examples of the denaturing agent include urea and guanidine salts that break the stability of hydrogen bonding. Examples of guanidine salts include guanidine hydrochloride and guanidine isothiocyanate. The concentration of the denaturing agent may be 3-8M in the buffer, preferably 6-8M. Moreover, 3-8M or 6-8M may be sufficient in the liquid mixture of an organic solvent and a buffer solution. When a guanidine salt is used, dithiothreitol as a reducing agent may coexist in addition to the guanidine salt.
Such denaturing agents can alleviate adsorption by losing higher order structure and dissociating into polypeptide chains. For this reason, it is possible to remove proteins (for example, BSA, enzyme) that can be adsorbed on the surface of the flow path by the cleaning liquid containing such a denaturant.
However, if the salt concentration is too high, it is difficult to prepare, and if crystals remain, there is a risk of clogging in the fine channel, so care must be taken.

[3-4. Acid cleaning liquid]
On the other hand, when the cleaning liquid includes a buffer, the buffer may have a pH of 2 to 4, preferably 2.5 to 3.5, for example.
Since the dissociation of silanol groups remaining on the flow path surface is suppressed in the acidic region, the acidic cleaning liquid removes the positively charged fluorescent dyes and basic compounds that can ionically bond to the dissociated silanol groups on the flow path surface. It is possible.
If the pH of the buffer solution is lower than the above range, polyacrylamide may cause hydrolysis of the amide to form a polycarboxylic acid structure, which may affect the separation characteristics (selectivity).

[4. Cleaning method]
The cleaning method is not particularly limited as long as the cleaning liquid can be brought into contact with the channel surface. The flow path may be filled with a cleaning solution using the same method as that for introducing a sample, a mobile phase, or a separation buffer into the microfluidic device. For example, a method in which a constant volume of cleaning liquid is continuously passed through the device flow path by a pumping means such as a pump, or after the cleaning liquid is pumped by the means and the flow path is filled with the cleaning liquid, the device is And a method of immersing in the cleaning liquid for a predetermined time.

The cleaning time is not particularly limited, but in order to effectively remove the components adsorbed on the surface of the flow path, the above-described liquid passing method is preferably 0.1 to 4 hours at a flow rate of 20 to 500 μL / min, for example. If it is the above-mentioned method to immerse at 50 microliters / minute for 1-2 hours, it will take 2 to 48 hours, for example, Preferably it takes about 6 to 18 hours.
The temperature at which cleaning is performed is not particularly limited, and can be performed at room temperature (for example, 20 ° C. ± 15 ° C.).

  Depending on the composition of the sample and the mobile phase or separation buffer, the components remaining on the channel surface and the amount thereof may vary. Therefore, the number and frequency of cleaning (that is, how many times the microfluidic device is used after being analyzed) It is not limited as to whether it is appropriate. For example, cleaning can be performed periodically every time it is used tens to hundreds of times (for example, 20 times to 500 times).

  More specifically, for example, in the case of a normal nucleic acid amplification reaction (PCR reaction scale: 50 uL, amplification product amount: about 5 ng / μL), after regeneration by washing with water and using it about 100 to 500 times, the present invention Can be cleaned. In addition, when the amount of amplified product is large (50 ng / μL or more), especially when the amount of polymerase having a strong adsorption tendency is large, or when the concentration of stabilizer and specific gravity increasing agent is high, analysis is performed for each analysis or several tens of analyzes ( For example, the cleaning according to the present invention can be performed every 20 to 50 analysis).

An automatic cleaning function that periodically performs cleaning at a predetermined number of times and frequency may be incorporated in the apparatus and processed according to a time schedule. Periodic cleaning makes it possible to maintain the maximum performance of the microfluidic device.
The cleaning liquid can be selected according to the component considered to be adsorbed. At this time, the selection can be made based on the description of 3-1 to 3-4 described above.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples. In the following, the amount indicated by% for representing the composition of the cleaning liquid indicates a volume percentage.

1. Microfluidic device (microchip)
135 microchips (total number) whose performance deteriorated due to repeated use of electrophoresis analysis of samples containing nucleic acids were used as test samples. Specifically, this microchip is schematically shown in FIG. FIG. 2D shows a plan view of the microchip 1 used. The microchip 1 is composed of a quartz substrate, and the reservoir 13 is also a port for applying a voltage to the flow paths 14 and 15. Ports # 1 and # 2 are ports located at both ends of the sample introduction channel 14, and ports # 3 and # 4 are ports located at both ends of the separation channel 15. In order to apply a voltage to each port, electrode patterns 21 to 24 formed on the surface of the chip 1 are formed so as to extend from the respective ports to the side end portions of the microchip 1, and the high voltage power supply unit for electrophoresis To be connected to. The inner walls of the flow channels 14 and 15 have LPA films on their surfaces via γ-methacryloxypropyltrimethoxysilane as a silane coupling agent.

Regarding the prepared use history of 135 microchips, the types of analysis provided with the microchips were analysis of samples including nucleic acids out of 135, and were classified into the following five categories.
i) Analysis of normal PCR product amount (by reaction system not containing BSA),
ii) Analysis of normal PCR product amount (by reaction system containing BSA),
iii) Analysis of the product (unpurified and undiluted) obtained by PCR-RFLP,
iv) Analysis of large amount of PCR product (by reaction system containing excess polymerase)
v) Analysis using excess amount of SYBR Gold intercalator

  The amount of PCR products is usually about 5 ng / μL, and the amount of large-scale PCR products (for example, by multiplex PCR) is 50 ng / μL or more. Excess polymerase is an amount of polymerase that constructs the PCR system necessary to generate the large amount of PCE product. An excessive amount of intercalator is, for example, an intercalator having a concentration higher than 10,000-fold dilution.

  Randomly selected from 123 microchips, 29 with cleaning liquid A (the composition of the cleaning liquid will be described later), 25 with cleaning liquid B, 47 with cleaning liquid C, and 22 with cleaning liquid Cleaned with Liquid D. However, 5 out of 22 cleaned with the cleaning liquid D were those whose performance was not recovered by cleaning with the cleaning liquid C.

  The analysis with the remaining 12 microchips out of 135 is an electrophoresis analysis of a purified DNA ladder for electrophoresis analysis of nucleic acids, or an electrophoresis analysis of a standard material for electrophoresis analysis of nucleic acids. . These 12 pieces were cleaned with the cleaning liquid E.

2. Cleaning liquids Cleaning liquids A to E having the following compositions were prepared.
A: Methanol 100%
B: Ethanol 100%
C: 10% TAPS-NaOH buffer (pH 8.9) 30% containing 70% ethanol and 1 mM EDTA
D: 10% TAPS-NaOH buffer (pH 8.9) 30% containing 70% ethanol and 1 mM EDTA and 8M guanidine-HCl
E: Acetonitrile 70% and 20 mM phosphate (Na) buffer (pH 2.5) 30% containing 0.1 M NaClO ₄

3. Flow path cleaning procedure Cleaning with the cleaning liquid A was performed by a liquid passing method. Cleaning by the flow-through method uses a liquid pump (liquid chromatography pump, syringe pump, etc.) to deliver liquid from port # 4 to ports # 1 to 3 at a constant flow rate of 50 μL / min for 80 minutes. Performed according to the procedure to be performed.

Cleaning with the cleaning liquids B to E was performed by an immersion method. Cleaning by the dipping method was performed according to the following procedure.
(1) The cleaning solution was injected by 0.2 mL from the port # 4 of the chip with a 1 mM disposable syringe.
(2) The cleaning liquid overflowing the ports # 1 to # 3 was not sucked as it was, and the chip was immersed in the cleaning liquid placed in a falcon tube (10 mL or 50 mL).
(3) The mixture was allowed to stand for 18 to 24 hours.
(4) The chip was removed from the cleaning solution, and 0.2 mL or more of milli-Q water was injected from port # 4 with a 1 mL disposable syringe. The overflowing water was removed by suction, and the tip surface was wiped off with a becot. (If salt remains on the chip surface and Bencot does not slide on the quartz surface, the surface was lightly wiped again with water.)
(5) Set the chip on the chip frame and clean the chip on MultiNA.

4). Performance Evaluation Using the Shimadzu Microchip Electrophoresis Device MultiNA, the performance of the chips in groups (a) to (b) under the analysis conditions of DNA-1000 on-chip mode and TE buffer blank analysis (6 analyzes / chip) Evaluation was performed.

Details of the analysis conditions are as follows.
Reagent kit used: DNA-1000 manufactured by Shimadzu (applicable DNA size range is 100bp to 1000bp)
Analysis mode: On-chip mixing mode (analysis mode in which sample and internal standard marker are automatically mixed on the chip)
Sample used for performance evaluation: TE buffer (Blank analysis. Only two types of internal standard markers (low molecular side internal standard marker, polymer side internal standard marker) are detected.)
Number of analysis: 24 analysis (6 analysis / chip) (The system can set up to 4 chips, and created a schedule to analyze 4 chips alternately 6 analysis per chip alternately)

5. Cleaning effect Table 1 shows the cleaning effect of each cleaning liquid in terms of recovery rate (%) (that is, the ratio of the number of chips whose performance has been recovered to the number of chips subjected to cleaning) and the average number of theoretical plates of the polymer-side internal standard peak (av .) And migration time (average value).
The definition of performance recovery of microchips is that the number of theoretical plates on the polymer side internal standard (hereinafter also referred to as UM) peak is 80,000 or more, and the UM migration time (that is, the time from the start of separation until the UM peak is detected) ) Was 123 seconds or less. In the state of maintaining the initial performance in which no deterioration of the chip is observed, the number of theoretical plates of the UM peak in the above performance evaluation conditions is 80,000 or more, and the migration time is 113 ± 10 seconds.

  FIG. 3 shows a comparison of electropherograms before and after cleaning. FIG. 3 shows the case where the cleaning liquid C is used among those described in Table 1 above. The four pherograms correspond to four microchips. The first peak shows the internal standard marker on the low molecule side, and the second peak shows the internal standard marker on the polymer side. Before cleaning, the second peak spreads, and the migration time is slow and the variation between chips is large. On the other hand, after cleaning, the peak spread is improved and the migration time is fast.

(a) (Evaluation result of cleaning with cleaning liquid A)
Performance recovered with 15 out of 29 chips. In addition, the UM migration time improved to 118.1 seconds on average from the delay tendency of 123.0 seconds on average before cleaning.
(b) (Evaluation result of cleaning with cleaning liquid B)
Performance recovered with 11 chips out of 25 chips. Moreover, the migration time of UM was improved from an average of 122.1 seconds before cleaning to an average of 118.0 seconds.
From the results of (a) and (b), a recovery rate of about 50% was obtained by cleaning with 100% polar organic solvent. From this, it is inferred that the components adsorbed on the channel surface were removed mainly by hydrophobic interaction.

(c) (Evaluation result of cleaning with cleaning liquid C)
Performance recovered with 42 chips out of 47 chips. Also, the UM migration time was improved from 121.2 seconds to 117.0 seconds on average, and a 91% recovery rate was obtained. From this, it can be inferred that in addition to removal of the adsorbed component by the hydrophobic interaction with the polar organic solvent, the adsorbed component by the electrostatic interaction can also be effectively removed by changing the pH to alkali.
(d) (Evaluation result of cleaning with cleaning liquid D)
Performance recovered with 16 of 22 chips. The migration time of UM was 118.1 seconds compared with an average of 119.4 seconds, and no significant difference was observed. The 22 chips contain 5 chips that were not recovered by the cleaning liquid C, and all of these were recovered by the cleaning liquid D, so that the protein components (for example, BSA and restriction enzymes) contained in the sample components were denatured. As a result of dissociation into the polypeptide chain by the agent, it is presumed that the cleaning effect was improved.

(e) (Evaluation result of cleaning with cleaning liquid E)
Performance recovered with 6 out of 12 chips. The UM run time was also reduced from an average of 114.0 seconds to 108.2 seconds. The 12 chips cleaned with the cleaning solution E do not have analysis history of samples containing nucleic acids such as PCR products and PCR-RFLP products as described above. As a result, for microchips where adsorption of SYBR Gold contained in the separation buffer seems to be the main cause of performance degradation, a certain effect on treatment (pH 2.5) that suppresses ion binding to silanol groups It is guessed that was seen.

  From the above, it was shown that the organic solvent-based cleaning liquid removes the adsorbed component derived from the sample, and has a certain effect on the recovery of the chip whose performance is temporarily deteriorated.

  Further, the number of specimens was increased to 427, and the cleaning method of the present invention was similarly performed. Table 2 shows an outline of the cleaning method of the microchip according to the present invention. B to E are used as the cleaning liquid, and the usage history of the cleaned microchip is the microchip subjected to the analysis in the above categories i) to v). The cleaning effects of cleaning liquids B to E were summarized for each microchip usage history. “Remarkable effect” means that the recovery rate is 50% or more, “Effective” means that the recovery rate is 25 to 50%, and “No significant effect” means In this case, the recovery rate is 25% or less. A person skilled in the art can select an appropriate cleaning liquid composition based on the microchip usage history based on Table 2.

Claims (8)

A flow path formed in a microfluidic device, the flow path having a surface having a polymer film and contacting a sample containing nucleic acid and / or protein,
A microfluidic device for cleaning by bringing it into contact with a cleaning liquid consisting only of an organic solvent having solubility in at least the same volume of water at 25 ° C. or a cleaning liquid containing 50% by volume or more of the organic solvent in a buffer solution Cleaning method.   The cleaning method according to claim 1, wherein the buffer solution has a pH of 8 to 10.   The cleaning method according to claim 1 or 2, further comprising a protein denaturant of 3 to 8M in the buffer solution.   The cleaning method according to claim 1, wherein the buffer solution has a pH of 2 to 4.   A microfluidic device cleaning solution comprising only an organic solvent having solubility in at least the same volume of water at 25 ° C. or containing 50% by volume or more of the organic solvent in a buffer solution.   The cleaning liquid according to claim 5, wherein the buffer has a pH of 8 to 10.   The cleaning liquid according to claim 5 or 6, further comprising a protein denaturing agent of 3 to 8M in the buffer.   The cleaning liquid according to claim 5, wherein the buffer has a pH of 2 to 4.