KR20100020394A - Integrated micro bio chip - Google Patents

Abstract

PURPOSE: An integrated micro biochip is provided to perform sample pre-treatment and PCR on one chip and reduce labor, cost, time and sample amount. CONSTITUTION: An integrated micro biochip(100) comprises: a sample pre-treatment reaction bath(120) for removing residues from injected sample cells; a PCR container(140) which is formed at the exit of the sample pre-treatment reaction bath and is able to perform PCR; a check valve(160) which is formed between the reaction bath and PCR container and prevents reverse flow of DNA or PCR mixture; a heating unit(180) which regulates temperature of the reaction bath and PCR container; and thin glass chip(102).

Description

Integrated Micro Biochips {INTEGRATED MICRO BIO CHIP}

The present invention relates to an integrated micro biochip capable of performing a PCR process from a pretreatment process by integrating a sample pretreatment process such as cell lysis and DNA extraction and a PCR process into one chip.

In general, polymerase chain reaction (PCR) is a very well known deoxyribonucleic acid (DNA) cloning method that allows for the rapid and mass replication of any DNA using this technique. It is essentially used in various genetic fields such as therapeutic or forensic medicine. This is to replicate the DNA to be replicated by using a DNA polymerase and having a reaction temperature for each stage of replication.

This replication process uses a cyclic cycle of thermally controlled reactions, and the initial starting molecules repeat the temperature cycle, eventually increasing by about 1 billion.

DNA replication process through PCR is carried out through a step-by-step replication process. In other words, PCR begins with double-stranded DNA, and the first reaction of each cycle is the separation of two strands through heat treatment. This process is called denaturing and is usually performed at 94 ° C. Next is the cooling process, which heterologizes the complementary sequence of two DNA strands with primers separated, which is called annealing and is run at 55 ° C. The final step is the polymerization process, where the DNA polymerase in the mixture begins synthesis of DNA from two primers using four deoxyribonucleotides, which is called an extension and is 72 ° to 74 ° C. Will run on.

This in vitro DNA amplification process, the PCR process, is considered to be the most suitable biochemical analysis process for miniaturizing the entire experimental system because of the simplicity of the experimental step and the simple configuration of additional devices for the experiment. Unlike conventional, the consumption of the sample used in the experiment can be reduced, and the faster heating rate and cooling rate due to the much smaller heat capacity than the conventional can shorten the entire experiment time, as well as a portable device It can also have advantages as.

However, the chip applied to the conventionally-conjugated silicon and glass bonded PCR has a large error caused by impurities because the cell lysis process and the PCR process are performed in one chamber without DNA extraction process, and the material of silicon and glass There was a problem that the biochemical stability is poor by the characteristics.

In addition, when the cell lysis and DNA extraction process is carried out in a separate reactor, the structure of the micro biochip is complicated and inconvenient to use because a valve that requires an external device is used for this purpose.

In addition, in order to perform a PCR process on a conventional micro biochip, cell lysis and DNA extraction process must be preceded, and cell membrane rupture and thermal residue are induced by using a cell lysis buffer such as a surfactant or thermally or electrically. It should go through the process or washing process, this process was also a problem that takes as much time and manpower as the PCR process.

In order to solve this problem, micro biochips that can perform sample pretreatment have been studied. However, these biochips are made of silicon materials with low biochemical stability, complicated manufacturing process, or for driving valves for chip operation. An external device such as a pneumatic device is required, and several procedures such as cleaning during the operation of the chip are required.

The present invention is to solve the problems of the prior art as described above, and provides an integrated micro biochip that can perform a sample pretreatment process and a PCR process including a cell lysis and DNA extraction in one chip.

The present invention also provides an integrated micro biochip with a manual check valve that does not require an external device for chip integration.

In addition, the present invention provides an integrated micro-biochip that can be easily used by the user because there is no need for an external device, and can be usefully applied to integrate various types of single micro-biochips.

The present invention is a sample pretreatment reactor for removing the lysis and residue of the injected sample cells in order to achieve the object as described above; A PCR reaction tank formed at an exit side of the sample pretreatment reactor and receiving DNA from the sample pretreatment reactor to perform a polymerase chain reaction; A check valve formed between the sample pretreatment tank and the PCR reactor to prevent backflow of the DNA or the PCR mixture by a PCR mixture supplied with the DNA to the PCR reactor; It provides an integrated micro-biochip comprising a; and a heating unit for controlling the temperature of the sample pretreatment reaction tank and the PCR reaction tank.

By configuring as described above, it is possible to reduce the time, manpower, cost or amount of sample cells required to perform the PCR process.

Here, the sample pretreatment reactor, the PCR reactor and the check valve may be formed by one PDMS chip and one thin glass chip bonded to one surface of the PDMS chip. As a result, sample pretreatment and PCR can be integrated in one chip.

The check valve may include a PDMS thin film formed inside the PDMS chip, and a hole may be formed in the PDMS thin film, and the hole may be opened and closed by a stopper formed between the PDMS chip and the PDMS thin film. At this time, the PDMS thin film may move in the opposite direction to the stopper and open the hole. By such a configuration, it is possible to prevent the sample introduced into the check valve from flowing in the reverse direction.

Meanwhile, the sample pretreatment reactor and the PCR reactor include a diagonal channel formed in the PDMS chip, and a micro filter for extracting pure DNA from the dissolved sample cells is formed in the channel of the sample pretreatment reactor. Can be. Here, the micro filter includes micro-pillars formed at regular intervals along the width direction of the channel and micro-beads provided between the micro-pillars.

As described above, pure DNA and impurities can be easily separated by using a micro filter including micro pillars and micro beads of a minute size.

In addition, the sample pretreatment reaction tank, the check valve and the PCR reaction tank may be formed to be separated from the heating unit. For this reason, the reactor according to an embodiment of the present invention is used for one-time, the heating unit can be used by recycling. That is, by separating the reactor portion to be suitable for single use can be used by the user without subsequent procedures, such as washing the reactor.

In this case, the heating unit may include a glass chip placed under the thin film glass chip and a micro heater and a temperature sensor formed by depositing chromium and gold on one surface of the glass chip. As such, by adjusting the calorific value of the micro heater using the temperature sensor, cell lysis of the sample cells injected into the reactor can be efficiently performed, and there is no need to provide a separate external device to heat the sample cells.

In addition, the present invention is a sample pretreatment reaction tank for performing a sample pretreatment process of the injected sample cells to achieve the object as described above; A PCR reactor formed of the same chip as the sample pretreatment tank and performing a polymerase chain reaction using DNA extracted from the sample pretreatment reactor; And a check valve formed of the same chip as the sample pretreatment reactor and the PCR reactor and provided between the sample pretreatment reactor and the PCR reactor.

Here, the check valve is a manual valve for preventing the DNA or the PCR mixture from flowing back by the PCR mixture supplied to the PCR reactor. By using the manual valve in this way it is not necessary to provide a separate external device such as a pneumatic device to drive the valve.

To this end, the check valve is a chamber into which the DNA or the PCR mixture is introduced, a flap member for flowing the DNA or the PCR introduced into the chamber in only one direction, and formed inside the chamber to open and close the flap member. It may include a stopper.

In addition, a micro filter for extracting pure DNA from the sample cells is further provided between the sample pretreatment reactor and the check valve, and the micro filter may be formed of the same chip as the sample pretreatment reactor and the PCR reactor.

As described above, the integrated micro-biochip according to the present invention can reduce the time, manpower, cost and the amount of samples required by performing sample pretreatment and PCR in one chip.

In addition, the integrated micro biochip according to the present invention can be used by the user easily and conveniently.

In addition, the integrated micro-biochip according to the present invention can easily integrate several types of single micro-biochip by using a valve that does not require an external device.

In addition, since the micro biochip according to the present invention is manufactured by separately separating the reactor portion and the heating portion which can be used permanently to be suitable for single use, the micro biochip can be conveniently used by the user without subsequent procedures such as washing the reactor.

Hereinafter, with reference to the accompanying drawings will be described in detail with respect to the configuration and operation according to an embodiment of the present invention. The following description is one of several aspects of the patentable invention and the following description forms part of the detailed description of the invention.

However, in describing the present invention, a detailed description of known functions or configurations will be omitted for clarity of the gist of the present invention.

1 is a perspective view illustrating an integrated micro biochip according to an embodiment of the present invention, and FIG. 2 is a plan view schematically illustrating the integrated micro biochip according to FIG. 1.

1 and 2, the integrated micro-biochip 100 according to an embodiment of the present invention is a sample pretreatment tank 120 for performing a sample pretreatment process, including the injection of the sample cells, cell lysis or DNA extraction, etc. ), A PCR reactor 140 for performing PCR (polymerase chain reaction) using DNA extracted from the sample pretreatment reactor 120 and a sample pretreatment reactor 120 and a PCR reactor 140 for efficient experimentation. It includes a heating unit 180 to adjust the temperature of).

The sample pretreatment reactor 120 includes a PDMS (Polydimethylsiloxane) chip 101 and a PDMS chip in which a serpentine-shaped channel 122 having a micro filter 150 for extracting pure DNA from sample cells is formed. It may include a thin glass chip 102 bonded to one surface of the (101). In the channel 122, an inlet 121 for injecting sample cells into the sample pretreatment reactor 120 is formed.

Meanwhile, the heating unit 180 is a micro heater 181 formed by depositing chromium (Cr) and gold (Au) on one surface of the glass chip 190 and the glass chip 190 positioned below the thin film glass chip 102. ) And a temperature sensor 186.

By constructing as described above, it is possible to perform a sample pretreatment process such as cell lysis and DNA extraction without a separate external device, it is possible to perform a PCR experiment quickly and simply.

Here, the micro filter 150 may be formed in the channel 122 of the sample pretreatment reactor 120 and may be integrally formed with the sample pretreatment reactor 120, and may be provided at the front end of the outlet of the sample pretreatment reactor 120. It is also possible to be formed as a separate part from the sample pretreatment reactor 120. Hereinafter, the micro filter 150 will be described as an example in which the micro filter 150 is integrally formed in the channel 122 of the sample pretreatment reactor 120.

Meanwhile, a check valve 160 is provided at an outlet of the sample pretreatment reactor 120 or an outlet of the micro filter 150, and the check valve 160 is a sample pretreatment reactor 120 or the micro filter 150 and the PCR reactor ( 140 is connected to allow the DNA extracted from the sample pretreatment reactor 120 to be introduced into the PCR reactor 140.

Similar to the sample pretreatment tank 120, the PCR reactor 140 also includes a channel 142 having a serpentine shape, and an outlet portion for extracting DNA from which the PCR reaction is completed is formed at the end of the channel 142. 141a is formed.

The check valve 160 is provided with two, the check valve for DNA (160a) is a countercurrent to the flow of the pure DNA extracted from the sample pretreatment reactor 120 or the micro filter 150 flows into the PCR reactor (140) In order to prevent the PCR mixture check valve 160b is to prevent the backflow of the PCR mixture (mixture) that is injected together when the extracted DNA is transferred to the PCR reactor (140). At this time, the PCR mixture injector 170 for injecting the PCR mixture into the PCR mix check valve 160b is connected to the PCR mix check valve 160b.

The DNA check valve 160a connects the sample pretreatment reactor 120 or the micro-filter 150 to the check valve 160a to connect the first flow passage 168 and the PCR reactor 140 to the check valve 160a. The second flow path 169 is connected.

Reference numeral “175” denotes a mixer for mixing the DNA and the PCR mixture.

As shown in FIG. 1 and FIG. 2, briefly looking at the mutual positions of the chips constituting the integrated micro biochip 100 according to the present invention, the heating unit 180 of the glass chip 190 is located at the bottom thereof. The thin glass chip 102 of thin glass is positioned thereon, and the PDMS chip 101 is attached to the upper surface of the thin glass chip 102.

The PDMS chips 101 constituting the reactors 120 and 140 are formed with channels 122 and 142 into which sample cells are injected, and the channels 122 and 142 are serpentine in consideration of the hydrophobicity of the PDMS chips. It is formed in the form. That is, it is formed in the S-shape bent several times. Here, the channels 122 and 142 are provided with an inlet 121 for injecting sample cells and an outlet 141a for discharging the amplified DNA.

The channel 122 of the sample pretreatment reactor 120 is required to have a sufficient capacity to obtain accurate experimental results, the height and width of the channel 122 of the sample pretreatment reactor 120 according to the present invention is 150㎛ and It is preferably 600 µm and a total capacity of 20 µl. On the other hand, the overall size of the reaction tank 120 is preferably 25mm wide, 18mm long.

The inlet portion 121 of the channel 122 is provided with a circular shape having a diameter of about 1 mm to easily inject sample cells by a syringe.

Hereinafter, the micro filter 150 provided in the integrated micro biochip 100 according to an embodiment of the present invention will be described with reference to the drawings. 3 is an exploded perspective view illustrating a sample pretreatment reaction tank part of the micro biochip according to FIG. 1, and FIG. 4 is an enlarged view of the micro filter of the micro biochip according to FIG. 3.

3 and 4, the channel 122 of the sample pretreatment reactor 120 is provided with a micro filter 150 for extracting only DNA from the sample cells, the micro filter 150 is a channel 122 Micro-beads 155 provided between the micro pillars to micro-pillar 151 and the micro pillars 151 spaced at regular intervals along the width (see W of FIG. 4) direction It may be configured to include. Since the DNA is extracted using the micro filter 150 configured as described above, an external device for extracting DNA is not required, and since a chip having a simple structure is used, it is easy to manufacture and has the advantage of extracting DNA only by sample injection. .

By forming micro pillars 151 in the form of pillars in the order of tens of micrometers in the channel 122, and filling the micro beads 155 having a diameter of 50 μm between the micro pillars 151 to entangle each other, The micro filter 150 may filter the DNA.

Referring to FIG. 4A, it can be seen that a columnar micropillar 151 is formed in one of the serpentine-shaped channels 122 in the width direction of the channel 122. have. Here, the micro pillars 151 positioned at both ends may be formed by protruding the wall of the channel 122. As shown in FIG. 4B, the micro pillars 151 may be formed to have a predetermined gap G. Here, the gap G and the diameter of the micro pillars 151 may be determined according to the diameter or the size of the micro beads 155.

As shown in FIG. 4C, the microbeads 155 preferably have a diameter such that they cannot pass through the gap G between the micropillars 151. That is, when the diameter of the microbead 155 is 50 μm, the gap G of the micropillars 151 should be at most 50 μm, and the diameter of the micro pillars 151 may be about 100 μm.

As such, the diameters of the microbeads 155 are larger than or equal to the gap G between neighboring micropillars 151 and the diameters of the micropillars 151 are larger than the diameters of the microbeads 155. As a result, the microbead 155 is caught by the micropillar 151 as the sample flows through the channel 122, so that the micropillar 151 and the microbead 155 may function together as the microfilter 150. The DNA may pass between the entangled micropillars 151 and the microbeads 155 but only DNA may be extracted because other cell residues may not pass.

FIG. 3 is an enlarged schematic view of the extraction of DNA generated from the micro filter 150 of the micro biochip 100 according to FIG. 1. Referring to this, the operation of the micro pillars 151 and the micro beads 155 is further described. I can see clearly.

As shown in FIG. 3, when the microbead 155 is caught by the micropillar 151, the DNA may pass between the micropillar 151 and the microbead 155 that is entangled, but relatively relative to the DNA. Remaining residues after large cell lysis do not pass between the microbeads 155 and are caught by the microbeads 155. Through this process, only DNA is extracted.

On the other hand, the PDMS chip 101 is bonded to the thin film glass chip 102 in order to increase the biochemical stability of the sample. As such, the reaction tanks 120 and 140 may be integrally formed using the PDMS chip 101 and the thin film glass chip 102 to increase the biochemical stability of the micro biochip.

The manufacturing process of the PDMS chip 101 and the thin film glass chip 102 forming the reactors 120 and 140, the micro filter 150, and the check valve 160 on one chip will be described later.

In addition, the micro filter 150 is preferably formed near the outlet 121 of the channel 122 of the sample pretreatment reactor 120 as shown in FIG. By forming a micro filter 150 for extracting DNA near the outlet 121 of the channel 122, the sample cells can be sufficiently lysed, and by reducing the length of the channel through which the extracted DNA must pass, DNA can be extracted.

In order for DNA to be extracted by the micro filter 150, the cell wall of the sample cell must first be ruptured. That is, cell lysis should be made before DNA extraction, and a heating unit 180 is required for this purpose.

The heating unit 180 is 200Å and 1000Å thick chromium and gold, respectively, by thermal evaporation on the glass chip 190 and the glass chip 190 where the thin glass chips 102 of the reactors 120 and 140 are placed. After the deposition may include a micro heater 181 and the temperature sensor 186 formed using a photolithography technique.

Here, the micro heater 181 performs a cell lysis process of rupturing the cell wall by heating the sample cells injected into the channel 122 of the sample pretreatment reactor 120, the temperature sensor 186 is a sample pretreatment reactor 120 Alternatively, the power supplied to the micro heater 181 may be adjusted by sensing the temperature of the PCR reactor 140.

1 and 3, the micro heater 181 is formed of a thin film of metal such as chromium and gold on one of the upper and lower surfaces of the glass chip 190, and the heat in the center of the micro heater 181. There is provided a temperature sensor 186 operating in a resistive manner.

The micro heater 181 is supplied with power to the micro heater 181 through an electrode or a power applying unit 181a, and a current unit 181b for applying a 1 mA current to measure the resistance by the temperature sensor 186. Is provided, and the voltage detector 181c detects the voltage value generated by the relationship between the resistance and the current according to Ohm's law.

The micro heater 181 is positioned at a portion corresponding to a portion where the channel 122 of the sample pretreatment reactor 120 is formed, thereby heating the sample injected into the channel 122. On the other hand, although not shown, the heating unit provided in the lower portion of the PCR reaction tank 140 is also formed in the same manner as the heating unit 180 provided in the lower portion of the sample pretreatment reaction tank 120. Therefore, hereinafter, the heating unit 180 positioned below the sample pretreatment reactor 120 will be described.

In addition, the total size of the heating unit 180 located below the sample pretreatment reactor 120 is preferably larger than the sample pretreatment reactor 120 having a width of 28 mm, a length of 20 mm, and a width of 25 mm and a length of 18 mm. As such, by forming the heating unit 180 larger than the sample pretreatment reactor 120, it is not only easy to position the sample pretreatment reactor 120 on the heating unit 180 in an experiment preparation process, but also the heating unit 180. The electrode to power supply unit 181a for supplying power to the micro heater 181 can be prevented from being covered by the reactor 120.

The temperature sensor 186 senses the temperature of the reaction tank 120 to adjust the power supplied to the micro heater 181, and the micro heater 181 performs cell lysis by inducing cell wall rupture of the sample cells. That is, by adjusting the amount of heat generated from the micro heater 181 to the amount of power supplied using the temperature sensor 186, cell lysis of the sample cells injected into the reactor 120 can be efficiently performed and the sample cells are heated. There is no need to provide a separate external device.

The manufacturing method of the heating unit 180 including the micro heater 181 and the temperature sensor 186 will be described later.

On the other hand, the reaction tank (120, 140) is preferably provided to be separated from the heating unit 180. Reactors 120 and 140 of the direct micro biochip 100 according to the present invention may be used for single use, but the heating unit 180 may be recycled. That is, by separating the parts of the reaction tank (120,140) to be suitable for use in a single use can be used conveniently by the user without subsequent procedures, such as washing the reaction tank (120,140). In this case, the reaction vessels 120 and 140 and the thin film glass chip 102 may be bonded by micro molding.

Hereinafter, with reference to the drawings, the configuration and operation of the check valve 160 provided in the integrated micro biochip 100 according to an embodiment of the present invention will be described.

FIG. 5 is a perspective perspective view schematically illustrating a check valve of the micro biochip according to FIG. 1, and FIG. 6 is a cross-sectional view of the check valve according to FIG. 5.

As mentioned above, the check valve 160 includes a DNA check valve 160a and a PCR mix check valve 160b. Since the two valves have the same configuration, the check valve 160a for DNA is described below. An example will be described.

As shown in FIG. 5, the check valve 160 has a substantially cylindrical shape and two flow paths 168 and 169 are connected to each other. Inside the cylindrical portion are first and second chambers 165 and 166 in communication with the two flow paths 168 and 169, respectively, and these two chambers 165 and 166 are defined by the flap member 163. Here, since the check valve 160 is formed on the PDMS chip 101, the check valve 160 may be formed on the same chip as the sample pretreatment reactor 120, the micro filter 150, and the PCR reactor 140.

The flap member 163 is formed of a PDMS thin film having a thickness thinner than that of the PDMS chip 101, and a hole 163a is formed in the flap member 163. The hole 163a may be opened and closed by a stopper 161 formed in one of the two chambers 165 and 166. The flap member 163 can be easily deformed because it is formed of a thin PDMS thin film.

Referring to the drawings will be described the operation of the check valve as follows.

FIG. 6 (a) shows a state in which DNA flows in the forward direction to the check valve 160, FIG. 6 (b) shows an open state of the check valve 160 and FIG. 6 (c) shows a closed state. .

6 (a) and 6 (b), the DNA extracted from the sample pretreatment reactor 120 or the micro filter 150 passes through the first channel 168 to the first chamber 165 of the check valve 160. Flows into). DNA introduced into the first chamber 165 may be introduced until the first chamber 165 fills up. When the DNA continues to be introduced while the first chamber 165 is full, the flap member 163 formed of the PDMS thin film is moved in the opposite direction of the stopper 161 so that the flap member (163) is blocked by the stopper 161. The hole 163a of 163 is opened. The introduced DNA is introduced into the PCR reactor 140 through the open hole 163a through the second chamber 166 and the second channel 169. DNA flow like this is normal flow.

DNA cannot be reversed by the check valve. That is, DNA cannot flow through the second channel 169 to the first channel 168. Referring to FIG. 6C, when the DNA flows back, DNA passing through the second channel 169 flows into the second chamber 166. Even when the second chamber 166 is filled with DNA, the hole 163a of the flap member 163 is not opened. This is because the hole 163a is opened only when the flap member 163 moves in the inflow direction of the DNA introduced into the second chamber 166, because the stopper 161 prevents the opening of the hole 163a. By such a structure, the check valve 160 can prevent the fluid from passing back.

Since the check valve 160 is a passive or passive valve, that is, a valve that is opened and closed by the force of a flowing fluid, a separate structure such as a pneumatic device for opening and closing the valve is not necessary and the structure is simple. Easy to use

Since the PCR mixer channel is connected to the second channel 169 that introduces the DNA into the PCR reactor 140, when the pressure is changed in the second channel 169 due to the inflow of the PCR mixture, the DNA may flow back. Such a backflow can be prevented by the DNA check valve 160a. At this time, the PCR mixture can also flow back due to the influx of DNA, and the PCR mix check valve 160b can prevent such a backflow.

As described above, in the micro biochip 100 according to the embodiment of the present invention, the sample pretreatment reactor 120, the micro filter 150, the check valve 160, and the PCR reactor 140 are formed on one chip. All PCR processes can be performed on one chip without the need for external devices. That is, the integrated micro biochip 100 may be implemented.

The polymerase chain reaction step using the integrated micro biochip 100 according to the present invention is as follows.

Injecting the microbeads 155 into the diagonal channel 122 formed in the sample pretreatment reactor 120, causing the microbeads 155 to be caught by the micropillar 151 provided in the channel 122, the channel Injecting the sample cell to the 122, Cell lysis step of destroying the cell wall of the sample cell by heating the sample cell injected into the channel 122 with a micro heater 181 provided in the heating unit 180, Residues other than DNA contained in the ruptured sample cells are filtered out by the micropillars 151 and microbeads 155, and DNA is extracted through the micropillars 151 and the microbeads 155. Supplying a PCR mixture to the PCR reactor 140 through a check valve, supplying a PCR mixture to the PCR reactor 140 together with DNA, and performing a PCR in the PCR reactor 140.

By using the integrated micro-biochip 100 as described above, the polymerase chain reaction can be performed only by injection of sample cells.

The above-mentioned polymerase chain reaction step is just one example, it should be understood that it is not necessarily limited thereto.

Hereinafter, a method of manufacturing the reaction tanks 120 and 140, the micro filter 150, the check valve 160, and the heating unit 180 of the integrated micro biochip 100 according to the present invention will be briefly described.

FIG. 7 is a view illustrating a step of forming a reactor portion of the micro biochip according to FIG. 1, and FIG. 8 is a view illustrating a step of forming a heating unit of the micro biochip according to FIG. 1.

As shown in FIG. 7, the forming of the reaction tanks 120 and 140 may be performed by immersing the silicon wafer in a SPM (sulfuric acid and fruit tree) solution to remove foreign substances (a), followed by spin coating. On the upper surface of the silicon wafer, SU-8, which is a negative sensitizing film, is placed on the upper surface of the silicon wafer, followed by soft baking (b).

SU-8 is a negative photoresist. It is a photoresist based on chemically amplified epoxy for micromachining. It was originally developed by IBM and is now developed by Microchem and Sotec Microsystems. The company obtains sales rights from IBM and manufactures and sells them.

Accordingly, the SU-8 has good connection with glass, ceramic, etc., has excellent optical properties, and is capable of producing a solid-state ratio structure having a diameter of 1 to 1000 μm according to coating conditions, as well as a semiconductor manufacturing process. It also has excellent connectivity, so it can be easily exposed by using UV aligner, a semiconductor device, and is suitable for Plasma Ion Etching Process. In addition, it has the advantage of being able to withstand high temperatures above 200 ℃, and especially easy exposure with the existing UV aligner, so LIGA (Lithographie, Galvano-formung, Abformung) process that had to use expensive synchrotron X-Ray equipment It also leads to the development of a LIGA-like process that can be substituted for.

After the soft bake is completed, the shape is transferred to the SU-8 (c) and developed (d), and a SU-8 mold for making a chip is produced (e), and bubbles are removed in a vacuum. Done.

When the above operation is completed, the PDMS and the curing agent are mixed in a constant ratio (for example, 10: 1), poured into the upper part of the mold, cured at 65 ° C. for 3 hours and 30 minutes, and then the PDMS is removed from the mold. 10) is formed (f). After cutting and punching the removed PDMS small (g), and then bonded with a thin film glass chip (h), the final reactors 120 and 140 are obtained.

By the preparation method as described above, the sample pretreatment reactor 120, the micro filter 150, the check valve 160, and the PCR reactor 140 may be implemented in one chip, and may achieve integration of the micro biochip.

Next, as shown in FIG. 8, the forming of the heating unit 180 includes a cleaning step (a) in which a Pyrex glass wafer is immersed in a SPM (sulfuric acid and fruit water mixture) solution to remove foreign substances, and after the cleaning is performed, Chromium 200 Pa (b) and gold 1000 Pa (c) are deposited on the wafer by thermo-phase deposition.

Subsequently, after spin coating a positive photoresist to photoresist (PR, photoresist) called AZ1512 on the thin film of chromium and gold (d), exposure (e) and development process (f) are performed.

The AZ1512 is manufactured by Clariant, U.S.A as a positive photoresist, and is widely used for patterning metal thin films.

After performing the above exposure and development process, the chromium and gold are sequentially wet-etched or etched, and then the dicing operation is performed to remove the photosensitive agent (g) and to cut the small (h) glass chip 190 of the heating unit 180. It is formed.

Hereinafter, an experiment for extracting and amplifying DNA of oral cells using the integrated micro biochip 100 according to an embodiment of the present invention will be described.

FIG. 9 is a diagram illustrating a heat transfer analysis result of the micro biochip according to FIG. 1, FIG. 10 is a graph illustrating a temperature distribution of the micro biochip along the AB direction of FIG. 9, and FIG. 11 is a diagram of the micro biochip using FIG. Gel electrophoresis pictures showing the results of PCR performed with the extracted DNA and the results of PCR according to the prior art, respectively.

First, in order to predict heat transfer efficiency of the micro biochip 100 according to the present invention, heat transfer analysis was performed using a commercial program (CFD-ACE +, ESI Group, France). 9 and 10 illustrate temperature flatness along the A-B direction of the micro biochip. When performing the experiment, it is possible to adjust the temperature inside the reactor with respect to the temperature of the heater surface by referring to the analysis result.

In FIG. 9, the portion marked in blue is relatively low in temperature, and the portion marked in red is relatively high in temperature. As shown in FIG. 9, it can be seen that the temperature of the micro heater 181 and the channel 122, which is a heating portion thereof, is relatively high. In addition, referring to FIG. 10, the surface temperature of the micro heater 181 is indicated by a dotted line, and the temperature inside the channel 122 is indicated by a solid line. From this graph, it can be seen that there is a temperature difference between the surface of the micro heater 181 and the channel 122.

On the other hand, the temperature control system for controlling the temperature of the micro heater 181 to the reactor (120, 140) is a power supply for supplying power to the biochip (E3631A, Agilent Technology, USA), the data acquisition board connected to the computer (PCI-6024E, National Instrument, USA), a signal conditioner (SC2345, National Instruments, USA). The thin film type temperature sensor 186 connected to the data collection board through the power supply unit supplies a certain amount of voltage to the micro biochip and collects the temperature inside the reactors 120 and 140. The temperature control system can use a commercial program (LabVIEW 7.0, National Instrument, U.S.A).

Oral cells that can be easily collected for sample pretreatment experiments were used. Cytolysis was performed on micro biochips to derive optimal temperature and time conditions for cytolysis. Evaluation of the optimum conditions was performed by measuring the concentration and purity of the DNA after the reaction using a DNA concentration measuring device NanoDrop ND-1000 (Nanodroptechnologies. Inc, U.S.A.), the result was determined to 80 ℃, 2 minutes as the optimum conditions. Table 1 below shows the results of improving the purity of DNA using the micro filter 150 of the present invention.

Before DNA Extraction After DNA Extraction  DNA concentration (ng / μl) 23.95 20.17  DNA Purity (260/280) 0.93 1.62

Meanwhile, PCR was performed on the SY158 gene present in the Y chromosome in the extracted DNA to confirm the performance of the sample pretreatment process in the micro biochip. The composition of the PCR sample is shown in [Table 2].

1 (machine) 2 (chip)  Nuclease free water 14.4 8.4  10X Buffer 2.0 2.0  dNTP 2.0 2.0  Primer (SY158 F) 0.2 0.2  Primer (SY158 R) 0.2 0.2  Additive [BSA] 0.0 3.2  Template 1.0 3.0  Taq polymerase (5unit / μl) 0.2 1.0  Total 20.0 20.0

Commercial PCR apparatus (Primus 96 Thermocycler, MWG, U.S.A) was used. PCR conditions were performed for pre-denature at 240 ° C. for 240 sec, denature at 95 ° C. for 15 sec, annealing at 60 ° C. for 15 sec, and extension ( extension) was performed at 65 ° C. for 30 sec to perform a total of 35 cycles and the results are shown in FIG. 11.

As shown in FIG. 11, a gel electrophoresis photograph showing the results of PCR performed with DNA extracted using the microbiochip of the present invention and the results of PCR using a conventional method, respectively, the center of which is the present invention microbiochip PCR results performed with the extracted DNA, and the left side shows the PCR results performed in a conventional manner. As can be seen in Figure 11 it can be seen that the result of using the micro biochip according to the present invention is superior to the conventional method.

The integrated micro biochip described so far should be understood as the technical spirit of the present invention or the minimum technology for the present invention, which can be grasped from various viewpoints, and should not be understood as the boundary limiting the present invention.

In addition, although the preferred embodiment of the present invention has been described above by way of example, the scope of the present invention is not limited only to such specific embodiments, and may be appropriately changed within the scope described in the claims.

1 is a perspective perspective view showing an integrated micro biochip according to an embodiment of the present invention;

2 is a plan view schematically illustrating the integrated micro biochip according to FIG. 1;

3 is an exploded perspective view illustrating a sample pretreatment reactor part of the micro biochip according to FIG. 1;

4 is an enlarged view of a micro filter of a micro biochip according to FIG. 3;

5 is a perspective perspective view schematically showing a check valve of the micro biochip according to FIG. 1;

6 is a cross-sectional view of the check valve according to FIG. 5, FIG.

7 is a view showing a step of forming a reaction tank of the micro biochip according to FIG.

8 is a view illustrating a step of forming a heating unit of the micro biochip according to FIG. 1;

9 is a view showing a heat transfer analysis result of the micro biochip according to FIG.

FIG. 10 is a graph showing a temperature distribution of a micro biochip along the A-B direction of FIG. 9;

FIG. 11 is a gel electrophoresis photograph showing the results of PCR performed with DNA extracted using the micro biochip according to FIG. 1 and the results of PCR according to the prior art, respectively.

<Explanation of symbols for the main parts of the drawings>

100 ： Integrated micro biochip

101: PDMS chip 102: thin film glass chip

120: sample pretreatment reactor 140: PCR reaction tank

150: micro filter 160: check valve

170: PCR mix injection unit 180: heating unit

Claims (12)

A sample pretreatment reactor for removing lysed and residues of the injected sample cells; A PCR reaction tank formed at an exit side of the sample pretreatment reactor and receiving DNA from the sample pretreatment reactor to perform a polymerase chain reaction; A check valve formed between the sample pretreatment tank and the PCR reactor to prevent backflow of the DNA or the PCR mixture by a PCR mixture supplied with the DNA to the PCR reactor; And Integrated micro biochip comprising a; heating unit for controlling the temperature of the sample pretreatment reaction tank and the PCR reaction tank. The method of claim 1, And said sample pretreatment reactor, said PCR reactor and said check valve are formed by one PDMS chip and one thin film glass chip bonded to one surface of said PDMS chip. The method of claim 2, The check valve includes a PDMS thin film formed inside the PDMS chip, wherein the hole is formed in the PDMS thin film, and the hole is opened and closed by a stopper formed between the PDMS chip and the PDMS thin film. . The method of claim 3, And the PDMS thin film moves in a direction opposite to the stopper and opens the hole. The method according to any one of claims 2 to 4, The sample pretreatment reaction vessel and the PCR reaction vessel include diagonal channels formed in the PDMS chip. Integrated micro-biochip, characterized in that a micro filter for extracting pure DNA from the dissolved sample cells in the channel of the sample pretreatment reactor. The method of claim 5, The micro-filter includes an integrated micro biochip, wherein the micro-pillar is formed at regular intervals along the width direction of the channel and micro-beads provided between the micro-pillars. . The method of claim 5, And the sample pretreatment reactor, the check valve and the PCR reactor are formed to be separated from the heating unit. The method of claim 5, The heating unit includes a microchip and a temperature sensor formed by depositing chromium and gold on one surface of the glass chip and the glass chip placed under the thin film glass chip. A sample pretreatment reactor for performing a sample pretreatment process of the injected sample cells; A PCR reactor formed of the same chip as the sample pretreatment tank and performing a polymerase chain reaction using DNA extracted from the sample pretreatment reactor; And And a check valve formed of the same chip as the sample pretreatment reactor and the PCR reactor and provided between the sample pretreatment reactor and the PCR reactor. The method of claim 9, The check valve is an integrated micro biochip, characterized in that the manual valve to prevent the DNA or the PCR mixture from flowing back by the PCR mixture supplied to the PCR reaction tank. The method of claim 10, The check valve may include a chamber into which the DNA or the PCR mixture flows, a flap member for flowing the DNA or the PCR introduced into the chamber in only one direction, and a stopper formed inside the chamber to open and close the flap member. Integrated micro-biochip comprising a. The method according to any one of claims 9 to 11, A micro filter is further provided between the sample pretreatment reactor and the check valve to extract pure DNA from the sample cells, wherein the micro filter is formed of the same chip as the sample pretreatment reactor and the PCR reactor. Biochip.

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