KR20050065895A - Biomolecular filter and method thereof - Google Patents

Abstract

The present invention includes a first flow path structure having a flow path therein, and a dry film resist for forming a plurality of holes to allow fluid to pass through the holes, and using the plurality of holes to size the biomolecule. It provides a biomolecular filter separating each star.

By preparing a biomolecular filter on the lab-on-a-chip that can concentrate the analyte molecule in a sample mixed with several components, it is possible to reduce analysis time and cost compared to conventionally preparing an analytical sample by performing pretreatment outside the chip. The microfluidic device can be manufactured in a portable and portable form.

Description

Biomolecular Filter and Method for Manufacturing the Same {Biomolecular Filter And Method Thereof}

The present invention relates to a biomolecular filter used for the analysis of biomolecules, and includes a plurality of holes formed on a flow path structure having a flow path therein, so as to block a filter so that fluid can pass through the holes. By attaching a DFR filter having a plurality of holes formed in the same but actually formed in such a manner that the fluid passes through the plurality of holes, a biomolecular filter for separating the biomolecules by size is provided.

Currently, samples used in DNA chips are mainly purified DNA samples or white blood cells. However, because the DNA chip is used as a point-of-care diagnostic chip, it must be a system that can directly analyze blood to obtain rapid information in places such as homes or emergency rooms (Lab-on -a-chip)).

Summary of the prior art is as follows. First, attempts have been made to produce microfilters using silicon-based microfabrication techniques and then use these filters to separate white blood or red blood cells from the blood. However, the technology for manufacturing such a microfilter requires sophisticated techniques such as a high aspect ratio deep reactive active etching process, which requires much further research. A similar system is to separate samples according to the size of the cells by making certain weirs on the flow path.

After filling microbeads into microfluidic channels, there are also attempts to use principles similar to chromatography of these bead packed columns. These microbeads are in fact used in many commercial kits that extract genomic DNA from blood off-chip.

However, since the process of filling the microbeads into the microchannels has to be added to the existing microfabrication process, an easier method is required.

In addition, attempts have been made to produce porous polymer structures in channels and use them as filters. However, in order to separate the desired white blood cells in the blood it is necessary to control the size and distribution of the pore reproducibly, a method of controlling the structure more precisely is required.

Enrichment of biomolecules containing cells in samples is a major obstacle to the miniaturization of lab-on-a-chips, including biochips currently under research and development. This causes additional cost and time consumption.

Samples of lab-on-a-chip (hereinafter referred to as biochips) that target biomolecules can be purified from blood, tissue cell extracts, serum, DNA, proteins, etc., to purified samples. Most of the samples are concentrated and purified through the first pretreatment process outside the biochip. Preprocessing is the biggest obstacle to miniaturization and portability of biochips, and it is a major cause of cost and analysis time consumption.

For example, in the case of amplifying biochips for DNA amplification, pretreatment processes for extracting genomic DNA from blood are mostly performed outside the chip, and some of these pretreatments are manufactured as part of the chip parts (for DNA amplification using Nanogen's SDA method). In the case of Lab-on-a-Chip, the volume becomes very large, making it impossible to carry.

In order to solve the above problems, an object of the present invention is to use only a desired material from an unrefined mixture using a method of preparing a fine filter in the sample input of the biochip using a photo-resist (PR) and a semiconductor process It is possible to perform partial purification of the biochip by performing the pretreatment and analysis of the sample in the biochip, and improve the miniaturization and portability of the chip.

Another object of the present invention is to enable a 'concentration' process, which is a pretreatment process of a sample, which is an obstacle to analysis time and miniaturization problem in the development of a lab-on-a-chip or micro-analysis module for the analysis of biomolecules, in a chip or a module. It is to provide a technique for manufacturing a filter in the flow path structure.

Biochips can be of various types and forms as desired. In the present invention, a case in which a specific biomolecule is required to be purified and concentrated in a lab-on-a-chip for analyzing biomolecules will be described as an example. However, it will be apparent to those skilled in the art that the technique of the present invention can be applied when a similar purpose is required among the lab-on-a-chips.

The DFR filter manufactured according to the present invention is essential for implementing a sample pretreatment (including Lysis) process, a PCR reaction, and a sensing function for a purified DNA sample or a biological material such as white blood cells in a miniaturized micro diagnostic chip. Element.

As a technical means for solving the above problems, one aspect of the present invention is a biomolecular filter, comprising: a first flow path structure having a flow path therein; And a DFR formed to block the flow path such that a plurality of holes are formed to allow fluid to pass through the holes, and the biomolecular filter separating the biomolecules by size using the plurality of holes. To provide.

The biomolecular filter may be formed with a structure in which at least two are connected, and the holes formed therein may have the same size or different sizes. The diameter of the holes can be formed from tens of nm to thousands of nm.

Meanwhile, the biomolecular filter of the present invention is applicable to a gene delivery and drug delivery system in combination with a micro device.

According to another aspect of the present invention, there is provided a method of manufacturing a first flow path structure having a flow path therein; And forming a DFR on the first flow path structure to cover the flow path. And it provides a method for producing a biomolecular filter comprising forming a plurality of holes to allow fluid to pass through the DFR formed on the flow path.

Hereinafter, a method of manufacturing a biomolecular filter according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings. One of the key processes in this embodiment is laminating a film-type resist DRY (Dry Film Resist), which is a tenting to form a lid shaped like a tent on top of a flow channel of liquid. Using the equipment, it is attached to the top of the flow path to form micro holes.

1 is a perspective view of a flow channel to be used in a biomolecular filter according to an embodiment of the present invention.

Referring to FIG. 1, a flow path structure 10 having a two-stage trench having a stepped flow path 20 is formed. The shape of the cross section of the flow path 10 is not particularly limited and can induce the flow of the fluid. The material of the flow path structure 10 may be a silicon or plastic material, and the method of forming the flow path 20 may be etched using a deep RIE or KOH solution when the material is silicon, and in the case of plastic, drilling or Hot pressing can be used.

Referring to FIG. 2, a DFR (dry film resist : 30) having a thickness of about 5 to 200 μm is adhered to a top of the flow path structure 10 by a laminating method under a pressure of about 5 to 10 kg / cm ² at a bonding temperature of about 70 to 100 ° C. do. Laminating methods are often used in the printed circuit board (PCB) industry. 3 shows a detailed structure of the DFR film used in the embodiment of the present invention. DFR (dry film resist) is a form of a photosensitive material (photopolymer) applied to the peel (peel sheet) to a suitable thickness and a cover sheet (cover sheet) attached thereto. The fill sheet is a polyolefin release liner, the photosensitive material that performs the two elements of photosensitive and adhesive function is a photodevelopable dry film, and the cover sheet is protective and oxygen resistant. Have

The DFR attaching process will be described in detail. First, the DFR is attached using a laminating apparatus at a temperature of 90 ° C. and a pressure of 10 kg / cm ² , facing the substrate and the photosensitive material layer of the DFR having the peeled sheet on the substrate to be attached. do. Laminating equipment is used in the yellow room for general equipment used in PCB substrate processing. The DFR at the top of the trench after lamination does not sag in the trench top plane despite its thickness of several micrometers.

Referring to FIG. 4, micro holes are formed on the DFR by lithography. Since DFR is a UV-gloss material, it is lithography using g-line or i-line steppers that can precisely control the pore diameter from tens of nm to thousands of nm. After exposing the UV, the hole is melted in the developer to form a plurality of holes on the DFR.

In the present specification, after the DFR is attached to the trench structure by laminating, the diameter of the DFR is formed according to the size of the biomaterial by lithography.

Meanwhile, as described above, another flow path structure may be additionally formed in the flow path structure having the DFR formed thereon such that the flow path may be continuously formed on the upper part of the DFR.

The following lists examples of applying the manufacturing method of the present invention.

5A is a structural diagram of a single DFR filter according to an embodiment of the present invention, and 5b and 5c are schematic diagrams of a multiple DFR filter according to an embodiment of the present invention.

The multi-DFR filter is a micropore or nanopore membrane (nanopore membrane) is made of a laminated structure by using an example as a multi-DFR filter is shown. In this case, by adjusting the size or shape of the pore, various compounds can be separated according to the size .

Referring to FIG. 5A, the single DFR filter has a structure including a laminated DFR 110 having micropores 115 formed in a middle portion of the flow path structures 100 and 120 in which the flow paths are formed.

Referring to FIG. 5B, the multi-DFR filter includes two laminating DFRs 210 and 230 having fine holes 215 and 225 formed in two middle portions of the flow path structures 200, 220, and 240, respectively. Have In this case, two DFR filters are connected to each other, and the micropores 215 may be configured to have the same size or may have different hole sizes.

Referring to FIG. 5C, the multi-DFR filter includes two laminating DFRs 310 and 330 in which each of the flow path structures 300, 320, and 340 has fine holes 315 and 325 formed in two middle portions thereof. It has a structure. As a result, the multiple DFR filter of FIG. 5C has a structure in which a plurality of multiple DFR filters of FIG. 5B are connected. In this case, the micro holes 315 and 325 of each of the flow path structures 300, 320 and 340 may be configured by a combination of various modifications in the number and size of holes, such as the same size and different hole sizes. That is, each DFR filter may be selectively separated from a specific component by modifying the surface, for example, hydrophilic or hydrophobic, by modifying the surface according to the characteristics of the material to be separated.

FIG. 6 is a schematic conceptual view illustrating a situation of using the multiple DFR filter of FIG. 5C. The sample shows a structure that can be filtered using each filter (filters 1, 2, 3) to integrate information obtained on one chip through preprocessing, reaction, and sensing. For example, in biological materials such as DNA, a variety of useful genetic information can be obtained through amplification reactions and appropriate sensing methods.

7 is a conceptual diagram illustrating the principle of separating the actual white blood cells, red blood cells, circulating DNA in the DFR filter according to an embodiment of the present invention.

Blood composed of various components such as white blood cells, red blood cells, and circulating DNA is separated according to the size of the components. As shown in Figure 7, the size of the white blood cells, red blood cells, circulating DNA can be listed as white blood cells> red blood cells> Circulating DNA. Therefore, the DFR filter 1 (Filter1) forms a micropore 500 having a size between the white blood cells and the red blood cells, and the white blood cells are filtered and sent to OUT1, and the remaining red blood cells and the circulating DNA are passed to the next DFR filter. In the same principle, DFR filter 2 (Filter2) forms a microhole 510 having a size between red blood cells and circulating DNA to send red blood cells to OUT2 and circulating DNA to OUT3. Through this process, white blood cells, red blood cells, and circulating DNA are separated.

FIG. 8 is a conceptual diagram illustrating an example of using the DFR filter mentioned in FIG. 7.

Referring to FIG. 8, by using a genomic DNA, the cells are lysed and detected through a PCR process to obtain various useful genetic information. In the blood separated from leukocytes and red blood cells, By using circulating DNA, it can be usefully used for diagnosis of diseases such as cancer. This process may use a PCR amplification method or a general Immunoassay method for the purpose of detection.

9 shows a microfabricated microfabrication device 600 using a DFR filter. The microfabrication device 600 includes a signaling part 610, a drug control part 640 having a membrane structure having a micropore or a nanopore, and an actuation part 630. ), And may include a signal analyzer 620. The device can be applied to gene delivery, drug delivery systems (DDS).

In the current drug delivery system, it is possible to inject the drug mainly into the drug carrier and then injected into the body. However, as in diabetes, when the relative amount of glucose in the body changes from time to time, there may be the hassle of having to inject the drug every time. From this point of view, by detecting and analyzing the amount of glucose occurring in the body automatically, there is an urgent need for the development of a micro device that can deliver the required amount of drug in case of abnormalities.

By introducing such micropore or nanopore structures into such micro drug delivery devices, the amount of drug delivered can be controlled. The detector 610 detects a change in a signal (eg, a change in the amount of glucose) occurring in a living body, and the detected signal is quantitatively or qualitatively detected by the signal analyzer 620. By analyzing, the control unit 630 for controlling the movement of the drug of the micro-pores or nano-pores can be configured to be integrated into one micro-device so that only a corresponding amount of drug in the device to the body. In particular, membranes having nanopore sizes have recently been of great interest in the field of DNA structure, characterization, and molecular sensing.

DFR filter produced by the method of the present invention is expected to be applicable to the diagnostic field, because the pore size can be adjusted in various ways, it can be used in the manufacture of a chip for diagnosing the infection of the disease virus in the blood samples. Currently, virus infection is a test using an antigen-antibody reaction. By applying this, a fixed antibody is attached to the chip, and when the blood is injected into the chip, the permeation of the filter removes various blood cells that may slow down the antigen-antibody reaction. And more purified serum (serum) samples will be able to enter the antigen antibody reaction site. If several antigen-antibody reactions are made and various diagnostic antibodies are attached to it, it is expected that the collected blood can be easily used to diagnose several viral or bacterial diseases simultaneously.

The present invention is a technique for manufacturing a cut-off filter using a PR and a semiconductor process so that only the analyte can be concentrated from the unpurified sample at the sample inlet side of the biochip. One microfilter may be manufactured in one chip, and filters having different cut-off sizes may be manufactured in one chip as desired. For example, if there are two or more kinds of substances to be concentrated and purified in a mixture, filters having different cut-off sizes may be manufactured in each structure, and the two structures may be bonded to form a single chip. In addition, the surface properties of the final filter may vary according to the PR used, and this characteristic is expected to bring about an increase in the concentration and purification effect depending on the usage.

The DFR filter prepared as described above can be manufactured in a fine size, and when applied to a PCR chamber using a property of easily attaching DFR, DNA can be separated from blood on a microchip capable of performing a PCR reaction and a detection reaction at the same time. The system can be integrated into one device.

In addition, the DFR filter manufactured according to the present invention is formed integrally with a gene chip such as a PCR chip, DNA chip, protein chip, etc. to form a generic name rep on a chip (Lab. On a chip).

On the other hand, in addition to biomolecules (i.e., biomaterials or drugs), they may be used for separation of general industrial materials such as fine powders.

In addition to saving time and cost when performing purification and enrichment during the pre-processing of samples to be analyzed in Lab-on-A-Chip outside the chip, Lab-A-Chip itself can perform all processes. It is expected to increase portability.

1, 2 and 4 are perspective views schematically showing the manufacturing process of the biomolecular filter according to an embodiment of the present invention.

Figure 3 is an example of the DFR film structure used in the embodiment of the present invention

5A is a structural diagram of a single DFR filter according to an embodiment of the present invention, and 5b and 5c are schematic diagrams of a multiple DFR filter according to an embodiment of the present invention.

FIG. 6 is a schematic conceptual view illustrating a situation of using the multiple DFR filter of FIG. 5C.

7 is a conceptual diagram illustrating the principle of separating the actual white blood cells, red blood cells, circulating DNA in the DFR filter according to an embodiment of the present invention.

FIG. 8 is a conceptual diagram illustrating an example of using the DFR filter of FIG. 7.

9 is a schematic diagram of a microfabricated microfabrication device using a DFR filter according to an embodiment of the present invention.

Claims (12)

In the biomolecular filter, A first flow path structure having a flow path therein; And A plurality of holes are formed, including a dry film resist that allows fluid to pass through these holes, Biomolecular filter, characterized in that for separating the biomolecule by size using the plurality of holes. The method of claim 1, The DFR is a biomolecular filter, characterized in that having a thickness of 5um to 1000um. The method of claim 1, And the first flow path structure is made of a silicon or plastic material. The method of claim 1, Biomolecular filter, characterized in that the second flow path structure is further manufactured on the DFR. The method of claim 1, The biomolecular filter has a structure in which at least two or more are connected, and the holes formed in the biomolecular filter have the same size or different sizes. The method of claim 1, The DFR is a biomolecular filter, characterized in that it comprises a fill sheet, a photosensitive material and a cover sheet. The biomolecule filter according to claim 1, wherein the holes have a diameter of several tens of nm to several thousand nm. The method of claim 1, Biomolecular filter, characterized in that applied to the gene delivery, drug delivery system coupled to the micro device. Preparing a first flow path structure having a flow path therein; And Forming a DFR on the first flow path structure to cover the flow path; And And forming a plurality of holes to allow fluid to pass through the DFR formed on the flow path. The method of claim 9, Formation of the DFR is a method for producing a biomolecular filter, characterized in that the adhesion temperature by laminating method under a pressure of about 70 ~ 100 ℃ 5 ~ 10kg / cm ² pressure. The method of claim 9, The forming of the plurality of holes in the DFR is a method of manufacturing a biomolecular filter, characterized in that formed by exposure, development, and etching by UV lithography method used in the semiconductor process. The method of claim 9, The biomolecule filter is a method for producing a biomolecule filter, characterized in that used for the separation of biomaterials, drugs or fine powder.