JP4213547B2 - Manufacturing method of microreactor - Google Patents

Description

The present invention relates to a method for manufacturing a microreactor in which a fine channel having a width and height of 0.1 to 1500 μm and a liquid chromatograph column are formed between two substrates.

Recently, there is a great need for technology to diagnose and analyze at low cost in the field, such as bedside diagnosis in the vicinity of patients, monitoring of environmental pollutants in the air, water and soil, and food safety inspection. It is getting higher.

For example, if an analysis that previously required an expensive and large device could be replaced by a small portable analyzer, a practitioner could install and use an analyzer that could only be installed in a large hospital. The result can be easily and quickly fed back to the patient. In addition, the home health care environment can be improved by measuring the health index of the elderly by the family of the elderly and managing the health index numerical value at home or by regularly sending it to the hospital and managing it at the hospital. .

If environmental pollutants such as environmental hormones and dioxins can be easily measured without using an expensive and large apparatus, environmental diagnosis can be performed easily and inexpensively. Furthermore, if environmental pollutants can be analyzed on site using a small portable analyzer, a more detailed safe environment can be provided.

In order to perform such a measurement easily, it is necessary to be able to perform analytical detection with high sensitivity using a very small amount of sample. For this purpose, GC-MS, LC-MS, ICP-MS, which are separated by capillary gas chromatography (CGC), capillary liquid chromatography (CLC), induction plasma (ICP), etc., and detected by mass spectrometer (MS) Have been widely used as a method that can be analyzed with a small amount and high sensitivity.

However, since these devices are expensive and large-scale devices, it is very expensive to easily carry or install in a desired location, and can meet the needs of a wide range of medical diagnosis and environmental diagnosis as described above. There wasn't.

As an example of making this as a product form for the purpose of actually carrying the site, several products have been developed with an analysis mechanism such as an electrochemical sensor in the microreactor. A microreactor is a generic term for a microscale fluid transfer means, a reaction part, etc. in a palm-sized chip. For example, the reactor has a width of 0.1 μm to 1500 μm.
Including a fine channel of about m.

As an example of an electrochemical sensor in a microreactor, for example, an ion-selective electrode, that is, an electrode that selectively measures target electrolyte ions by measuring the membrane potential of a polymer membrane formed on the working electrode is mounted. (For example, refer to Patent Document 1).
JP 2000-65791 A

The microreactor uses a microelectrode as an ion selective electrode and realizes a palm-sized microanalysis system by appropriately using a fluid transfer channel having a microlevel width and depth. . Since the system is very small, it can be carried and blood components can be measured without using a large measuring device.

However, in a microreactor, since a trace substance is measured using a trace amount of sample, in order to measure with high sensitivity, it is necessary to concentrate the trace substance to be measured in the sample. At the same time as concentration, it is preferable to separate a substance group that causes a problem as a contaminant.

A liquid chromatograph is generally used as the means for concentration and separation.
It has been difficult to produce a liquid chromatograph column in which a liquid chromatograph packing material is closely and uniformly packed in a microreactor.

That is, as a method for forming a liquid chromatographic column, for example, two substrates on which a concave portion for a liquid chromatographic column and a concave portion for a fine channel are formed are bonded, and a hole is formed in the concave portion for the liquid chromatographic column. Opening and press-fitting the liquid chromatograph filler from this hole, the liquid chromatograph filler was press-fitted into the liquid chromatograph column recess of the substrate on which the liquid chromatographic column recess and the microchannel recess were formed Thereafter, a method of bonding to another substrate is exemplified.

However, the liquid chromatograph column in the microreactor is very small, such as several tens of nl to 10 μl. With these methods, it is difficult to pack the liquid chromatograph packing material in the recess for the liquid chromatograph column closely and uniformly. It was.

In view of the above drawbacks, an object of the present invention is a microreactor having a liquid chromatograph column in which a packing material for liquid chromatograph is closely and uniformly packed, and is very small, easy to detect and highly sensitive. An object of the present invention is to provide a method of manufacturing a microreactor capable of measuring a substance.

In the microreactor manufacturing method of the present invention, two substrates are stacked, a fine channel having a width and height of 0.1 to 1500 μm between the substrates, and one substrate side in the fine channel , For a liquid chromatographic column, a laminate in which a concave portion for a liquid chromatographic column for filling a liquid chromatographic packing material is formed and an opening communicating with the concave portion for a liquid chromatographic column is formed on the other substrate. The fine flow path including the recess is filled with the aqueous medium, the first electrode is placed so as to come into contact with the aqueous medium, and the liquid dispersed filler medium for liquid chromatograph is placed on the opening, and the liquid chromatography is performed. By installing a second electrode in contact with the graphite filler-dispersed aqueous medium and applying a DC voltage between the first electrode and the second electrode, both sides of the recess for the liquid chromatographic column With aqueous media Body forming an electric field between the chromatographic filler dispersed aqueous medium, characterized by filling a filler for liquid chromatography in a recess for a liquid chromatographic column.

The material of the substrate used in the present invention is not particularly limited, and examples thereof include conventionally used inorganic materials such as glass, quartz, and silicon. These inorganic materials are excellent in accuracy, workability, etc. For example, if optical lithography technology widely used in semiconductor microfabrication technology is used, micron-order grooves can be freely formed on glass or silicon substrates. Can do.

However, it is also important that microreactors can be produced in large quantities easily and inexpensively and can be discarded. In such a case, it is not desirable to use glass or silicon whose materials themselves are expensive.

Also, in the medical field, when using glass products, it is obliged to pay an appropriate disposal cost at the time of disposal, and there are other advantages such as lightness and not cracking. The substrate is made of polymer resin because it is possible to form grooves and holes on the surface with very high productivity by performing injection molding and hot press molding using a transfer mold. It is preferable.

The type of the polymer resin is not particularly limited, but a thermoplastic resin is preferable in that it can be easily surface-treated by heating. For example, a polyolefin resin, a polystyrene resin, a polylactic acid resin, Examples thereof include acrylic resins and polycarbonate resins. In the microreactor for analysis in the present invention, fluids with various pHs are often used to adsorb and desorb a substance to be detected in the concentration part, so that a resin having acid resistance and alkali resistance, for example, a polyolefin resin or the like A polyacrylic resin is more preferable.

On the other hand, the thermosetting resin does not have the advantage of being easily plasticized by heating, but it does not have the advantage of being surface-processed, but a precursor liquid mixed with a curing agent or the like is previously introduced into a transfer mold and cured in situ. Thus, it is possible to shape the resin surface.

In this case, since the precursor is liquid, there is an advantage that the shape of the transfer mold is transferred more faithfully. In general, a statically cured resin can be advantageously used because it exhibits a low linear expansion coefficient and a low molding shrinkage ratio. As such a thermosetting resin, an epoxy resin can be advantageously used from the viewpoint of cost and easy handling.

The two substrates are stacked, and a fine channel having a width and height of 0.1 to 1500 μm is interposed between the substrates, and one substrate side in the fine channel is filled with a liquid chromatographic filler. A recess for a liquid chromatograph column is formed, and a first electrode is provided in a fine channel on both sides of the recess for the liquid chromatograph column . The microchannel must be capable of flowing a fluid sample containing a substance to be detected, an eluent used in a liquid chromatograph, and the width and height are designed to be 0.1 to 1500 μm. .

In general, one end of the microchannel is used as an inlet for a fluid sample, and in order from the inlet side, a concentration part (liquid chromatograph column) of a substance to be detected, a detection part for detecting the type or concentration of the substance to be detected, waste liquid A storage unit or the like is connected, and the other end is a waste liquid discharge port or a gas discharge port.

The liquid chromatographic column recess is formed in a fine channel , and after filling the recess with a liquid chromatograph filler, allowing a fluid sample containing a detected substance to pass through, and adsorbing the detected substance, This is a region having a function of passing through the eluent and detaching the substance to be detected for concentration.

As a preferable structure of the recess for the liquid chromatographic column, the height is the same, the flow path width of the fine flow path is gradually increased, and after making the width constant, the flow path width is gradually narrowed to the width of the fine flow path. A substantially hexagonal shape is preferable in plan view.

In addition, fluid samples, eluents, etc., flow out between the liquid chromatographic column recesses and the microchannels so that the liquid chromatographic column packing material does not flow out of the liquid chromatographic column. Although possible, it is preferable to install a filter medium that does not allow the liquid chromatographic packing material to flow out.

As the filter medium, any commonly used material can be used, and examples thereof include glass wool, porous resin beads, and porous glass beads. However, since it is difficult to pack these filter media into the fine flow path, a protrusion having a height and width that can prevent the liquid chromatographic packing material from flowing out at the boundary between the liquid chromatographic column recess and the fine flow path. The part is preferably formed.

The liquid chromatographic filler used in the present invention is not particularly limited as long as it is a fine particle filler generally used in liquid chromatographs. For example, styrene-divinylbenzene copolymer, polymethacrylate resin, poly Examples thereof include fine particles such as hydroxy methacrylate resin, polyvinyl alcohol, silica, and alumina.

Molecules and functional groups for separating and adsorbing specific components by the liquid chromatographic filler may be introduced into the liquid chromatographic filler. Such a compound or functional group is not particularly limited, and has a functional group such as a functional group capable of binding to various ions in the sample, a functional group capable of coordinating binding, a functional group capable of chelate binding, or the like. Compounds such as sulfo group, quaternary ammonium group, octadecyl group, octyl group, butyl group, amino group, trimethyl group, cyanopropyl group, aminopropyl group, nitrophenylethyl group, pyrenylethyl group, diethylaminoethyl group , Sulfopropyl group, carboxyl group, carboxymethyl group, sulfoxyethyl group, orthophosphoric acid group, diethyl (2-hydroxypropyl) aminoethyl group, phenyl group, iminodiacetic acid group, ethylenediamine group, chelate forming group containing sulfur atom ( For example, various mercapto groups, dithiocarbamic acid groups, thiourea groups, etc.) Compound having a group and these functional groups. These may be used alone or 2
More than one type may be used in combination.

As the method for immobilizing the functional group and the compound having these functional groups in the liquid chromatographic filler, any conventionally known method may be employed. For example, the functional group may be included in the liquid chromatographic filler. Examples thereof include a method of directly reacting with a functional group such as a hydroxyl group, a carboxyl group, an aldehyde group, and an amino group, a method of treating an adsorbent carrier with a crosslinking agent such as an acid anhydride and reacting with the crosslinking agent.

The above liquid chromatographic packing materials may be packed alone or in combination of two or more. However, when two or more kinds of substances to be detected are to be measured at a time, functional groups having different functionalities are used. It is preferable to use two or more liquid chromatographic fillers in combination.

The size of the fine particles is not particularly limited, but in order for the measurement sample to efficiently contact the liquid chromatograph filler, the diameter is preferably equal to or less than the minor axis of the microchannel cross section, more preferably,
It is less than half of that.

When an electric field is applied to the dispersion liquid, the fine particles having a charge move with a specific electric mobility due to the charge of the fine particles. Therefore, if a baffle is placed in the middle, it will be collected at a high density on the surface of the baffle.

For example, silica, which is a packing material for liquid chromatography, has silanol groups (—S
iOH), and the silanol group dissociates in an aqueous medium to form a negative ion (-S
iO ⁻ ) and has a surface charge, and moves in the direction of the anode when an electric field is applied.

If the fine particles have no surface charge or are weak, a surfactant such as sodium dodecyl sulfonate, sodium alkylbenzene sulfonate, cetyltrimethylammonium bromide, and an aqueous medium in which a filler for liquid chromatography is dispersed When added to
These surfactants are adsorbed or distributed on the surface of the filler so that the filler has a surface charge or is strengthened.

The aqueous medium used in the present invention is an electrolyte containing water and may be any medium that can form an electric field by applying a voltage , for example, water alone liquid, water and alcohol such as methanol, ethanol, and the like. The mixed liquid is raised.
In order to improve the electrolyte property of the aqueous medium, a surfactant such as sodium dodecyl sulfonate, sodium alkylbenzene sulfonate, cetyltrimethylammonium bromide may be added to the aqueous medium.

Next, the manufacturing method of the microreactor of this invention is demonstrated with reference to drawings. FIG. 1 is a schematic cross-sectional view showing an example of filling a liquid chromatographic column in a liquid chromatographic column recess.

In the figure, reference numeral 1 denotes an upper substrate, and reference numeral 2 denotes a lower substrate on which protrusions 21 and 22 for preventing outflow of a liquid chromatographic filler are formed. The upper substrate 1 and the lower substrate 2 are laminated, and the liquid chromatographic column recess 3 (between the protrusion 21 and the protrusion 22) and the liquid chromatographic column recess 3 are arranged on the microchannels 4 on both sides (projection 21 in the figure). forming a micro-channel with the right) of the projecting portion 22 in the left side) and the micro channel 41 (FIG. First electrodes 7 and 8 are installed in the micro flow paths 4 and 41, respectively.

The fine channels 4 and 41 and the liquid chromatographic column recess 3 are filled with an aqueous medium 5. An opening 11 is opened in the approximate center of the liquid chromatographic column recess 3 of the upper substrate 1. A liquid dispersion medium 6 for liquid chromatograph is placed on the opening 11, and a second electrode 9 is placed on the liquid dispersion medium 6 for liquid chromatograph.

In the present invention, for applying a voltage between the first electrode 7 and 8 and the second electrode 9. Conductive when applying a pressure, on both sides of the liquid chromatograph column recesses 3 field between the aqueous medium 5 and a liquid chromatograph packing material dispersed aqueous medium 6 is formed, the filler dispersed aqueous medium 6 for liquid chromatograph The liquid chromatographic filler moves with a specific electric mobility due to the charge of the filler particles, and is packed densely from the protrusion 21 and the protrusion 22 side toward the center. The DC voltage to be applied is not particularly limited, but is generally preferably 200 to 2000 V / cm.

A filler for liquid chromatography in densely filled with a liquid chromatographic column recesses 3, in applying a voltage, or varying the dc voltage you applied, cause temporarily reversing the application direction Is preferred.

Varying the dc voltage you applied, the moving speed of the filler for liquid chromatography which has been moved is changed, temporarily reversing the application direction of the voltage, to move in a certain direction, a liquid chromatographic column The liquid chromatograph filler filled in the concave portion 3 is shaken and packed more densely.

In addition, after filling the liquid chromatograph filler having a relatively large diameter so that the liquid chromatograph filler does not flow out from the liquid chromatographic column recess 3 into the fine channels 4 and 41, the diameter is relatively large. Are preferably filled with a small liquid chromatographic filler, and the size of the filler may be three or more.

Further, the width and height of the fine channel are preferably 10 to 200 μm. In this case, the diameter of the liquid chromatographic packing material having a relatively large diameter is 0.5 to 50 μm, and the diameter is relatively small. The diameter of the liquid chromatographic filler is preferably 0.1 to 10 μm.

In the above example, the liquid chromatograph filler dispersed aqueous medium 6 is placed on the upper substrate 1, but the liquid chromatograph filler dispersed aqueous medium 6 may be in liquid contact with the aqueous medium 5, for example, The opening 11 may be connected to a container containing the liquid chromatograph filler-dispersed aqueous medium by a glass tube, a plastic tube or the like, and the second electrode 9 may be installed in the container. In this case, since the sedimentation of the filler can be suppressed by stirring the aqueous medium in the container, the container can be suitably filled.

The first electrodes 7 and 8 may also be in contact with the aqueous medium 5, and may be installed at a position away from the liquid chromatographic column recess 3, for example, at the end of the fine passage.

Since the structure of the manufacturing method of the microreactor of the invention of claim 1 is as described above,
The liquid chromatograph column of the obtained microreactor is packed tightly and uniformly with a liquid chromatographic filler. Therefore, the obtained microreactor is very small and can measure a substance to be detected easily and with high sensitivity.

Since the structure of the manufacturing method of the microreactor of the invention according to claim 2 is as described above,
The liquid chromatograph column of the obtained microreactor is packed more tightly and uniformly with the liquid chromatographic packing material, so that the substance to be detected can be measured with higher sensitivity.

Since the structure of the manufacturing method of the microreactor of the invention according to claims 3 to 5 is as described above, the liquid chromatograph column is packed in the liquid chromatograph column without flowing out into the fine flow path. A microreactor in which the agent is packed tightly and uniformly is obtained.

Since the structure of the manufacturing method of the microreactor of the invention according to claim 6 is as described above,
The obtained liquid chromatograph column of the microreactor is packed with two or more kinds of liquid chromatograph packing materials having different functional groups, and two or more kinds of different substances to be detected can be measured at a time.

  Next, examples of the present invention will be described, but the present invention is not limited to the following examples.

Example 1
In the microreactor shown in FIG. 1, the cross-sectional shape of the microchannels 4 and 41 is square, the length of one side is 200 μm, the cross-sectional shape of the recess 3 for the liquid chromatograph column is rectangular, and the height is 200 μm. The width was 1000 μm. Further, the protrusions 21 and 22 are provided at substantially the center of the fine channels 4 and 41, and have a height of 190 μm and a width of 50 μm.

The microchannels 4 and 41 and the liquid chromatographic column recess 3 of the microreactor were filled with an aqueous medium consisting of 20 wt% distilled water and 80 wt% ethanol as the aqueous medium 5.

As a filler-dispersed aqueous medium 6 for liquid chromatography, 1 part by weight of silica powder having a particle diameter of 20 μm and a sulfone group fixed on the surface was dispersed in 100 parts by weight of an aqueous medium composed of 20% by weight of distilled water and 80% by weight of ethanol. A filler-dispersed aqueous medium was placed on the upper substrate 1.

When the second electrode 9 is used as an anode and the first electrodes 7 and 8 are used as a cathode and a DC voltage of 10 KV (voltage gradient 500 V / cm) is applied for 5 hours, the liquid chromatographic column recess 3 is filled with silica. A microreactor with a liquid chromatograph column was obtained.

(Example 2)
A microreactor was obtained in the same manner as in Example 1 except that the electric field application method was changed as follows.

First, the second electrode 9 is used as an anode, the first electrodes 7 and 8 are used as a cathode, a DC voltage of 10 KV (voltage gradient 500 V / cm) is applied for 1 hour, and then the second electrode 9 is used as a cathode . A DC voltage of 10 KV (voltage gradient of 500 V / cm) was applied for 10 seconds using 1 electrode 7 and 8 as an anode. When this 1-hour application to 10-second application cycle was carried out five times, a microreactor having a liquid chromatograph column in which the liquid chromatograph column recess 3 was filled with silica was obtained.

(Example 3)
The microreactors 4 and 41 of the microreactor used in Example 1 and the liquid chromatographic column recess 3 were filled with an aqueous medium consisting of 30% by weight of distilled water and 70% by weight of methanol as the aqueous medium 5.

An aqueous medium 100 comprising 1 part by weight of silica powder having a particle diameter of 15 μm, 29.9% by weight of distilled water, 70% by weight of ethanol, and 0.1% by weight of sodium alkylbenzenesulfonate as the filler-dispersed aqueous medium 6 for liquid chromatography. The filler-dispersed aqueous medium dispersed in parts by weight was placed on the upper substrate 1.

After applying a 6 KV DC voltage (voltage gradient 300 V / cm) for 1 hour using the second electrode 9 as the anode and the first electrodes 7 and 8 as the cathode, the filler dispersed aqueous medium 6 for liquid chromatography is 1 part by weight of silica powder having a particle diameter of 7 μm and having a sulfone group fixed on the surface thereof was dispersed in 100 parts by weight of an aqueous medium comprising 29.9% by weight of distilled water, 70% by weight of ethanol and 0.1% by weight of sodium alkylbenzenesulfonate. A microreactor having a liquid chromatograph column in which two types of silica are packed in the concave portion 3 for a liquid chromatographic column when a DC voltage of 10 KV (voltage gradient 500 V / cm) is applied for 5 hours by replacing with a filler-dispersed aqueous medium. was gotten.

Physical property measurement Using the obtained microreactor, an ion exchange experiment was conducted by the following method.
(Regeneration process)
5 ml of 0.001N nitric acid was passed through the obtained microreactor at a flow rate of 0.2 ml / min to regenerate the sulfone group on the particle surface into a hydrogen type.

(Adsorption process)
Next, 1 ml of a 1 ppm divalent lead ion solution was passed through the microreactor at a flow rate of 0.2 ml / min.
(Desorption process)
Finally, 1 ml of 0.001N nitric acid was passed through the microreactor at a flow rate of 0.2 ml / min. At this time, the liquid passing through the reactor was collected and used as a test solution for analysis.

(analysis)
The amount of divalent lead ions in the analytical test solution was measured using an atomic absorption device (Perkin Elmer, ZL-40).
00 type). The recovery rate was calculated from the amount of lead ions in the test solution and the amount of lead ions used in the adsorption process, and the results are shown in Table 1.

The recovery rate was obtained from the following formula.
Recovery rate (%) = [Amount of lead ion in analytical solution (μg)] × 100 / [Amount of lead ion in adsorption process (μg) = 1 μg]

It is a schematic cross section which shows an example which fills the recessed part for liquid chromatograph columns with the filler for liquid chromatographs.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Upper board | substrate 11 Opening part 2 Lower board | substrate 3 Recessed part for liquid chromatograph columns 4, 41 Fine flow path 5 Aqueous medium 6 Filler dispersion aqueous medium for liquid chromatographs 7, 8 First electrode
9 Second electrode

Claims (6)

Two substrates are stacked, and a fine channel having a width and height of 0.1 to 1500 μm is filled between the substrates, and one substrate side in the fine channel is filled with a liquid chromatographic filler. An aqueous medium is applied to the microchannel including the liquid chromatographic column concave portion of the laminate in which the concave portion for the liquid chromatographic column is formed, and an opening communicating with the concave portion for the liquid chromatographic column is formed on the other substrate. The first electrode is placed so as to be filled and in contact with the aqueous medium, the liquid dispersion for liquid chromatograph dispersion is placed on the opening, and the liquid dispersion for liquid chromatography is in contact with the filler. The second electrode is installed as described above, and a direct current voltage is applied between the first electrode and the second electrode, so that the aqueous medium on both sides of the recess for the liquid chromatograph column and the packing material for the liquid chromatograph With dispersed aqueous media Forming an electric field between method of producing a micro-reactor, characterized by filling a filler for liquid chromatography in a recess for a liquid chromatographic column. The process according to claim 1, wherein the microreactor, characterized in that conductive during application of pressure, temporarily changing the DC voltage applied to voltage, or electric and temporarily reversing the direction of application of pressure.   3. The method for producing a microreactor according to claim 1, wherein the liquid chromatograph packing material having a relatively large diameter is filled, and then the liquid chromatographic packing material having a relatively small diameter is packed.   The diameter of the packing material for liquid chromatography having a relatively large diameter is 0.5 to 50 μm, and the diameter of the packing material for liquid chromatography having a relatively small diameter is 0.1 to 10 μm. The method for producing a microreactor according to claim 3.   5. A projection for preventing the liquid chromatographic packing material from flowing out is formed near the boundary between the liquid chromatographic column recess and the fine channel. A method for producing the microreactor as described.   The method for producing a microreactor according to any one of claims 1 to 5, wherein the liquid chromatographic filler comprises two or more liquid chromatographic fillers having different functional functional groups. .

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