KR20100077991A - Apparatus for separating sample by using microchip and method thereof - Google Patents

Abstract

PURPOSE: An apparatus and method for isolating sample using a microchip are provided to detect sample of low concentration in an excellent separation performance and sensitivity. CONSTITUTION: An apparatus for isolating sample using a microchip comprises: a microchip(100) having a channel(102); a buffer solution(110) storage unit placed at the upper end of channel of the microchip; and a capillary tip(104) which is processed by tapering in an infection needle. A hydrophilic cation polymer is coated with other surface charge on the inner wall of channel and capillary. The channel is coated with a weak hydrophilic cation polymer solution having low charge density. The weak hydrophilic cation polymer solution is polydiallylammonium chloride or N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride.

Description

Sample separation device using microchip and its method {APPARATUS FOR SEPARATING SAMPLE BY USING MICROCHIP AND METHOD THEREOF}

The present invention relates to a technique for separating and analyzing a sample, and in particular, an ionic liquid that is not mixed with water for the analysis of a trace amount sample using a lab-on-a-chip made of various materials such as plastic or glass. The present invention relates to a sample separation device and method using a microchip suitable for carrying out the separation of the microscopically sampled sample without diffusion using.

Advances in science and technology allow for the analysis of trace amounts, increasing the research on techniques for handling very small volumes of samples or reagents. Recent advances in synthetic chemistry and life sciences have led to an increase in target materials that need to be analyzed in areas such as new drug development and diagnostics.As a result, a large amount of expensive reagents or samples are required. The need for it is increasing. In particular, in the analysis of biosamples such as single cells and single molecules, it is assumed that the handling of samples with very small volume requires sampling and separation methods corresponding thereto.

Accordingly, Lab-on-a-Chip technology among various trace sample analysis technologies has excellent characteristics in dealing with trace samples or reagents in bio analysis. Lab-on-a-chip is a chemical microprocessor that integrates devices on a few cm ² chip using photolithography or micromachining technology, which is widely used in the semiconductor field.

These lab-on-a-chips are mainly implemented on glass, silicon, and plastic substrates. Initially, many chips using glass were made. Recently, however, the transition to plastic chips suitable for mass production of reproducible, standardized chips is fast progressing. Polymer materials such as poly (dimethylsiloxane), polymethyl methacrylate (PMMA), and polycarbonate (PC) are used as materials used in plastic chips. Among them, PDMS is the most commonly used plastic material because of its excellent optical transmittance and easy molding.

Lab-on-a-Chip also enables automated, high-speed, high-efficiency, low-cost automated experiments, including organic synthesis, drug and new catalyst screening, high-speed separation and analysis of chemicals, environmental monitoring, analysis of pharmaceuticals, and military applications. Its application range is expanding. Among the most widely used applications are high speed / high performance separation and analysis of compounds / medical substances. An important point in the separation experiment is to increase the separation efficiency, which is strongly influenced by the shape of the injected sample plug. Therefore, sample injection is one of the most important processes in separation / analysis. In addition, in recent years, many studies on living single cell analysis have been conducted in the life sciences, so it is necessary to analyze the volume of sub-pL / fL.

Conventional methods of injecting a very small amount of sample into a lab-on-a-chip channel include a method using an electric field and a method of injecting a very small amount of sample into a separation channel using pressure. The method using an electric field is controlled by applying a voltage across a fine channel filled with a solution to control the flow of the solution, and injecting a sample using the microchannel geometry to separate the capillary electrophoresis on a chip. Can perform analysis, and these methods are most commonly used today.

However, the method using the electric field is a method of flowing only a certain amount to the separation channel while flowing the solution, so it is very expensive to waste the sample, and the device for manipulating the fluid is complicated. Not suitable for The method using pressure can be applied to the component analysis in single-cell cells, but it requires enormous pressure because of the micro-sized channel size. In addition, the construction of such a pressure system is limited to the application to microchip capillary electrophoresis.

In the method of injecting a very small amount of sample into the lab-on-a-chip channel according to the prior art operating as described above, it is not suitable for the analysis of the very small amount of sample. The problem covered by noise could not be solved.

Accordingly, the present invention provides a sample separation device using a microchip capable of performing separation / detection on a microchip by electrophoresis so as to show a sample separated into an ionic liquid which is not mixed with water to have excellent separation performance and high theoretical singular value. And a method thereof.

In addition, the present invention, for electrophoretic analysis using a microchip with a sample sampled using a hydrophobic ionic liquid, by coating the channel of the microchip and the inner wall of the sample injection capillary tip with a different material to the segmentation state at the injection portion The present invention provides a sample separation device and method using a microchip that can maintain the sample and the sample is separated from the ionic liquid at the beginning of the separation channel to complete the sample injection.

In addition, the present invention, in order to separate the trace amount sample injected into the hydrophobic ionic liquid, the two different surface charge and hydrophilic cationic polymer (Cationic polymer) is coated on the sampling capillary tip and the separation channel, respectively, and then interfaced The present invention provides a sample separation device and method using a microchip that can be designed to be effectively separated immediately after sample injection is performed on the same chip after sampling.

In one embodiment of the present invention, a device includes a microchip including a buffer reservoir at one end of the channel, and a capillary tip tapered in the shape of an injection needle at one end of the capillary-shaped rod. And a cationic polymer having different surface charges and hydrophilicity is coated on the channel and the inner wall of the capillary, respectively, and the microchip and the capillary tip are interfaced with each other, and the hydrophobic liquid is not mixed with water by an electric field. Separation of the segment sample is performed.

In one embodiment of the present invention, a sample is formed using a microchip including a buffer reservoir at one end of the channel and a capillary tip tapered at one end of a rod formed with a capillary tube in the shape of a needle. In the separation method, the process of coating the cationic polymer having different surface charge and hydrophilicity on the inner wall of the channel and the capillary, respectively, after the completion of the coating, interfacing the microchip and the capillary tip with each other, Using an interfaced microchip and capillary tip to perform hydrophobic liquids that are not mixed with water by an electric field, and to perform phase separation of the sample and separation of the segmented sample.

In the present invention, the effects obtained by the representative ones of the disclosed inventions will be briefly described as follows.

The present invention can be used in a capillary electrophoresis system for lab-on-a-chip as a sample separation device that performs segment collection using an ionic liquid, and can be used for various detection methods of various samples without limitations on the characteristics of the sample. Can be. This can be used as it is without changing the conventional detection method, and provides the advantage that the device can be performed simply, with high efficiency and high sensitivity.

Since the present invention can detect very low concentrations of samples with good separation performance and sensitivity, it can be applied to the sampling of microvolume cells or samples in any organ in the cell, especially in life science research. . In addition, the on-chip derivatization reaction can be carried out without time limitation or at various temperatures, and it can also function as a sample storage function after taking a sample, thereby allowing the analyst to have time to spare. This is useful for mass detection, multi-sampling, and so on.

Hereinafter, a microchip separation analysis method of phase separation and segment samples of the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, if it is determined that a detailed description of a known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. The following terms are defined in consideration of the functions of the present invention, and may be changed according to the intentions or customs of the user, the operator, and the like. Therefore, the definition should be made based on the contents throughout the specification.

The present invention utilizes a method of collecting a sample having a very low concentration without loss and diffusion as a method of segmentation using an ionic liquid which is not mixed with water for the analysis of a trace amount sample. As such, the method of collecting sample using ionic liquid that is not mixed with water has excellent advantages in introducing low concentration of trace sample (including multiple sampling), storage and injection, but is separated by hydrophobic ionic liquid surrounding the sample. Difficulties can be followed, in order to analyze the microchip electrophoresis of a sample sampled using a hydrophobic ionic liquid, coating the channel of the microchip and the inner wall of the sample injection capillary tip with different materials to This allows the sample to be retained and the sample is separated from the ionic liquid at the beginning of the separation channel to complete the sample injection.

In other words, in order to separate the trace amount sample injected into the hydrophobic ionic liquid, two different surface charges and a hydrophilic cationic polymer are coated on the sampling capillary tip and the separation channel, and then interfaced. As soon as the sample has been injected, it is designed to perform effective separation.

On the other hand, the ionic liquid is a hydrophobic liquid which is not mixed with water, and cationic ionic liquids such as alkylimidazolium or N-alkylpyridinium, hexafluorophosphate, bis (trifluoromethylsulfonyl) imide, And anionic liquids such as trifluoromethylsulfonate or tetrafluoroborate. In addition, the samples that can be separated through the present invention, as a variety of biologically active substances in cells, tissues, organs, neurotransmitters, DNA, RNA, proteins, enzymes, metabolites, sugars, inorganic ions, etc. Trace amounts and expensive natural materials or environmental analytical samples are also included, but are not limited to these.

1 is a view showing a segment sample injection and separation device for microchip analysis according to an embodiment of the present invention, Figure 2 is a photograph of the actual sample injection and separation device produced.

Referring to FIG. 1, a segment sample injection and separation device for microchip separation analysis of a sample injected by using an ionic liquid that is not mixed with water, and the segment sample injection and separation device has a channel 102 formed therein. Microchip 100, a buffer reservoir 102 located at the end of the channel 102 on the top of the microchip 100, and a micropipette in the shape of a needle, positioned at the other end of the channel, to allow the biological sample to be pierced And a capillary tip 104 made of a micropipette tip into which a sample is introduced. At this time, reference numeral 106 of the capillary tip 104 is a segment injected sample, 108 is an ionic liquid, 110 is a buffer solution contained in the channel.

The segment sample injection and separation device may be formed in one piece or separate type of the microchip 100 and the capillary tip 104, the microchip 100 may be formed in a single or array type.

In the embodiment of the present invention, the microchip 100 may be manufactured by selecting any one or more of materials such as poly (dimethylsiloxane), rubber, silicone rubber, plastic, glass, silica, and the like. Etching, molding, pressing, machining, laser processing, etc. may be used to form the channel.

The sample separation device manufactured in actual separation type is as shown in FIG. 2. A method of manufacturing the sample separation device will be described in detail with reference to the following drawings.

3A to 3I are process flowcharts illustrating a manufacturing process of a sample separation device including a microchip and a sampling capillary tip according to an embodiment of the present invention.

First, referring to FIG. 3A, a microchip manufacturing process is performed. In order to manufacture a microchip substrate for a lab-on-a-chip, first, a negative photoregist layer 302 is coated on the silicon wafer 300. At this time, any material of the silicon wafer 300 may be used, such as rubber, silicon-based rubber, plastic, glass, silica. The negative photosensitive layer 302 may be, for example, SU-8 (Microchem), AL-217 (Aldrich) or KTFR (Kodak). Coating methods include spin coating or dip coating, preferably spin coating. In addition, the number of coating is not limited, but is preferably 1 to 2 times.

After the negative photosensitive layer 302 is coated on the silicon wafer 300, as shown in FIG. 3B, the photomask 304 is covered thereon and exposed to ultraviolet rays to perform exposure. When the exposed silicon wafer is developed, an embossed mold having a desired pattern is formed as shown in Fig. 3C.

Thereafter, as shown in FIG. 3D, a prepolymer such as PDMS is applied to the silicon wafer 300 having the embossed frame formed thereon, the top plate is cast and cured, and then peeled off from the silicon wafer.

Then, as shown in FIG. 3e, the replicated top plate is oxidized by surface treatment with a corona discharge formed using tesla coils, and then, as shown in FIG. By fixing with a flat bottom plate and an adhesive, a microchip substrate for lab-on-a-chip with a sample injection tip holder can be completed.

Looking at the manufacturing process of the capillary tip with reference to Figure 3g, in order to taper the capillary tip, using a micro CO ₂ laser pipette puller (puller) as shown in FIG. The capillary is pulled, ie tapered. Thereafter, the pooled capillary is cut using a cutter as shown in FIG. 3i, and polished with a 0.3 μm alumina plate for 3 hours using a micro pipette beveler. The resulting capillary tip is cut to length and the angled part is ground with a soft sandpaper (0.3 micron).

Thereafter, as shown in FIG. 3J, after the inner coating is performed with a cationic polymer on the microchip generated through FIG. 2F and the capillary tip generated as shown in FIG. 3I, the microchip holder is separated from the capillary tip by interfacing phase separation, A segment sample injection lab-on-a-chip, that is, a sample separation device, is formed.

4 is an enlarged view illustrating a connection portion between a separation chip substrate and a capillary tip according to an exemplary embodiment of the present invention.

Referring to FIG. 4, the connecting portion between the separation chip substrate and the capillary tip, that is, the interfaced portion, expands the channel 102 by placing an organic solvent in a chip having a chip width of 80% of the capillary diameter, and then expands the capillary tip ( By positioning and securing 106).

FIG. 5 is a diagram illustrating a state in which a silica capillary tube is tapered according to an embodiment of the present invention.

Referring to FIG. 5, the capillary tip 104 may be made of any one of rubber, silicone rubber, plastic, metal, glass, or silica. In the case of using glass or silica, the capillary tip 104 may be formed using a carbon dioxide laser. Can be produced by tapering into a needle shape.

6 is a diagram illustrating an inner wall structure after coating the inner wall of the sampling tip and the separation channel with different ionic polymers according to an embodiment of the present invention.

Referring to FIG. 6, a cross-sectional view of a polymer coating of a capillary inner wall for sample sampling and a microchip (eg PDMS) channel for separation, the surface of the capillary 606 and the channel of the microchip 604 with different surface charges It is for coating each of the cationic polymer having a hydrophilic property.

To this end, the inner wall of the capillary 606 for sample sampling was coated 602 with a strong hydrophilic cationic polymer with high charge density, and the inner wall of the microchip 604 channel for separation was a weak hydrophilic cation with low charge density. It was coated 600 with a polymeric polymer.

At this time, the coating process is as follows. The separated chip substrate and the capillary were washed with 0.1 M NaOH solution for 20 minutes. After deprotonation is completed, the solution is washed with clean tertiary distilled water and coated with 0.5% of each cationic polymer solution.

That is, the inner wall of the capillary 606 flows a strong hydrophilic cationic polymer solution having a high charge density, and the channel inner wall of the microchip 604 flows a weak hydrophilic cationic polymer having a low charge density to perform coating. Accordingly, the capillary tube 606 and the microchip 604 encoded with the cationic polymer solution are left to stand for 1 hour, and then washed thoroughly with tertiary distilled water and a buffer solution, and then used.

Here, examples of strong hydrophilic cationic polymers include polyethyleneimine (33% cationic charge density / nitrogen vs. other atoms), polyallyamine (31% cationic charge density / nitrogen), and relatively weak hydrophilic cations. The polymer may be polydiallylammonium chloride (11% cation charge density / nitrogen atom), aminosilane having a relatively long carbon chain.

In addition, if the (CH2) n lengths of amino sealants are selected, various sealants with large hydrophilic differences can be used as coating materials.These cationic polymers are used as coating materials for capillary sampling tips and PDMS separation channels, respectively. Can be replaced. Specifically, for example, 3-aminopropyltriethoxysilane can be used as a coating material for sampling capillary tips, and N-octadecyldimethyl [3- (trimethoxysilyl) propyl] ammonium chloride has a long carbon chain and is hydrophobic so that it can be used as a coating material for separation channels. Can be used as

FIG. 7 is a view illustrating a state in which an ionic liquid and a sample are transferred using an electric field according to an embodiment of the present invention, in which a phase separated and segmented sample maintains phase separation in a capillary tube using an electric field. It is showing movement.

FIG. 8 is a view showing a state in which a sample is separated from an ionic liquid according to an embodiment of the present invention. FIG. Electrophoretic separation will begin.

On the other hand, the present invention will be described in more detail by the test examples. However, the following test examples are provided only to more easily understand the embodiments of the present invention, and the present invention is not limited to the following test examples.

<Test Example 1>

The first experiment was a variety of application methods using lab-on-a-chip. The performance of the sample was separated and injected into the microchip from the fabricated sample separation device. For this purpose, a separation experiment of flain adenine nucleotide (FAD) and riboflavin (RF) was conducted using boric acid-sodium hydroxide buffer as a electrolyte.

After filling the microchip with buffer, the tip of the capillary tip is immersed in the buffer. For segmental sampling of samples with ionic liquids, the capillary tip ends are introduced in order of ionic liquid, buffer solution, and ionic liquid, respectively. At the time of sampling, we put a platinum electrode in the reservoir and applied a 0.7 kV voltage.

The ionic liquid or buffer solution containing the capillary tip was connected to the ground by connecting a platinum electrode. To monitor the sampling process of clear liquid samples, the Sudan III reagent was dissolved in an ionic liquid and designed to be red. The ionic liquid used here is 1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide, which is insoluble in water, has an inner diameter of 10 μm at the tip of the capillary tip, a sampling voltage of 700 V, and a sampling and time of 1 s. The separation voltage is 250 V / cm and the distance to the detection point is 2 cm.

9 is a diagram illustrating an electropherogram in which two phosphors are separated according to an embodiment of the present invention. FIG. 9 illustrates riboflavin and FAD as a sample in the sample separation device, and the sample is separated from the separation chip. have.

<Test Example 2>

In the second experiment, in order to proceed with the derivatization reaction in capillary tube using Lab-on-a-chip, 0.1mM protein and 1mV derivatization reagent (1 mM naphthalenedialdehyde, 1mM 2-mecaptoethanol) were injected, respectively. Injection for 1 second under The separation voltage is 250 V / cm and the distance to the detection point is 2 cm.

FIG. 10 is a diagram showing electropherograms after fluorescence of three proteins according to an embodiment of the present invention, on-chip fluorescence derivatization of myoglobin, cytochrome c, and lysozyme on capillary tips. FIG. After the reaction, separated and confirmed. Segmentally introduced samples (or reactants, products) will react with each other due to their fast diffusion in small volumes. This is very advantageous for small volume derivatization reactions.

As described above, the present invention provides a microchip electrophoretic analysis of a sample sampled using an ionic liquid which is not mixed with water. The segmentation state is maintained and the sample is separated from the ionic liquid at the beginning of the separation channel to complete the sample injection.

While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments, but is capable of various modifications within the scope of the invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined not only by the scope of the following claims, but also by those equivalent to the scope of the claims.

1 is a view showing a separation device of a sample sampled with an ionic liquid according to an embodiment of the present invention,

Figure 2 is a photograph showing a sample injection device manufactured according to an embodiment of the present invention,

3A to 3I are process flowcharts illustrating a manufacturing process of a separation device including a microchip and a sampling capillary tip according to an embodiment of the present invention;

4 is an enlarged view of a connection portion between a separation chip substrate and a capillary tip according to an embodiment of the present invention;

5 is a view showing a state taping the silica capillary (tapering) according to an embodiment of the present invention,

6 is a view showing an inner wall structure after coating the inner wall of the sampling tip and the separation channel with different ionic polymers according to an embodiment of the present invention;

7 is a view illustrating a state in which an ionic liquid and a sample are transferred using an electric field according to an embodiment of the present invention;

8 is a view showing a state in which a sample is separated from an ionic liquid according to an embodiment of the present invention;

9 is a diagram showing an electropherogram separating two phosphors according to an embodiment of the present invention;

10 is a diagram showing an electropherogram after fluorescence labeling of three proteins according to an embodiment of the present invention.

Claims (18)

In the sample separation device using a microchip, A microchip in which a channel is formed, the microchip including a buffer reservoir at one end of the channel, One end of the capillary-shaped rod includes a capillary tip tapered in the shape of a needle, The channel and the inner wall of the capillary are coated with a cationic polymer having different surface charges and hydrophilicities, respectively, and the microchip and the capillary tip are interfacing with each other, and the hydrophobic liquid is not mixed with water by an electric field. A sample separation device using a microchip in which separation of a sample is performed. The method of claim 1, The channel is, A sample separation device using a microchip, characterized in that the coating is coated with a weakly hydrophilic cationic polymer solution having a low charge density. 3. The method of claim 2, The weak hydrophilic cationic polymer solution, A sample separation device using a microchip, characterized in that formed from polydiallylammonium chloride, N-octadecyldimethyl [3- (trimethoxysilyl) propyl] ammonium chloride. The method of claim 1, The capillary is, A sample separation device using a microchip, characterized in that the coating is coated with a strong hydrophilic cationic polymer solution having a high charge density. The method of claim 4, wherein The strong hydrophilic cationic polymer solution, Sample separation device using a microchip, characterized in that formed of polyethyleneimine, polyallyamine, 3-aminopropyltriethoxysilane. The method of claim 1, The microchip, Sample separation apparatus using a microchip, characterized in that formed by selecting from poly (dimethylsiloxane), rubber, silicone rubber, plastic, glass and silica. The method of claim 1, The capillary tip is, Sample separation device using a microchip, characterized in that formed of a material selected from rubber, silicon rubber, plastic, metal, glass and silica. The method of claim 1, The hydrophobic liquid is a sample separation device using a microchip, characterized in that the ionic liquid. The method of claim 1, The ionic liquid, Cationic ionic liquids of alkylimidazolium or N-alkylpyridinium and anionic liquids of hexafluorophosphate, bis (trifluoromethylsulfonyl) imide, trifluoromethylsulfonate, tetrafluoroborate Sample separation apparatus using a microchip, characterized in that the liquid selected from. In the sample separation method using a microchip including a buffer reservoir is formed at one end of the channel, and a capillary tip tapered in the shape of a needle in one end of the rod formed capillary, Coating cationic polymers having different surface charges and hydrophilicity on the inner wall of the channel and the capillary; After the coating is completed, interfacing the microchip and the capillary tip with each other, A process of performing phase separation of a sample and separation of a segmented sample from a hydrophobic liquid which is not mixed with water by an electric field using the interfaced microchip and capillary tip Sample separation method using a microchip comprising a. The method of claim 10, The channel is, A sample separation method using a microchip, characterized by coating with a weakly hydrophilic cationic polymer solution having a low charge density. The method of claim 11, The weak hydrophilic cationic polymer solution, A method for separating samples using a microchip, characterized in that formed from polydiallylammonium chloride, N-octadecyldimethyl [3- (trimethoxysilyl) propyl] ammonium chloride. The method of claim 10, The capillary is, A sample separation method using a microchip, characterized in that the coating with a strong hydrophilic cationic polymer solution having a high charge density. The method of claim 13, The strong hydrophilic cationic polymer solution, Sample separation method using a microchip, characterized in that formed of polyethyleneimine, polyallyamine, 3-aminopropyltriethoxysilane. The method of claim 10, The microchip, A sample separation method using a microchip, characterized in that formed of polydimethylsiloxane (poly (dimethylsiloxane)), rubber, silicone rubber, plastic, glass and silica. The method of claim 10, The capillary tip is, A sample separation method using a microchip, characterized in that formed of a material selected from rubber, silicone rubber, plastic, metal, glass and silica. The method of claim 10, The hydrophobic liquid is a sample separation method using a microchip, characterized in that the ionic liquid. The method of claim 10, The ionic liquid, Cationic ionic liquids of alkylimidazolium or N-alkylpyridinium and anionic liquids of hexafluorophosphate, bis (trifluoromethylsulfonyl) imide, trifluoromethylsulfonate, tetrafluoroborate Sample separation method using a microchip, characterized in that the liquid selected from.

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