KR20030075359A - Multiplex nucleic acid amplification device using micro electro mechanical system component and method for constructing same - Google Patents

Abstract

PURPOSE: A multiplex nucleic acid amplification device using a micro electro mechanical system(MEMS) component and a method for constructing the same device are provided, thereby selectively amplifying various nucleic acids using a small amount of sample. CONSTITUTION: A multiplex nucleic acid amplification device using a micro electro mechanical system(MEMS) component comprises a thin layer-formed board; a micro heater and RTD sensor portion for generating and detecting heat, which is positioned on the board; an electrode portion for forming the electric field, positioned on the heater and sensor portion; a mixed reaction vessel comprising a reaction vessel for covering the electrode portion, and mixing and storing PCR solution, and inlet and outlet ends; and a peltier component positioned under the board.

Description

Selective multinucleic acid amplification apparatus using MEMS device and manufacturing method thereof {MULTIPLEX NUCLEIC ACID AMPLIFICATION DEVICE USING MICRO ELECTRO MECHANICAL SYSTEM COMPONENT AND METHOD FOR CONSTRUCTING SAME}

The present invention relates to a selective multinucleic acid amplification apparatus using a MEMS device and a method for manufacturing the same. Specifically, a micro heater and an RTD sensor are used to control a temperature cycle used in a polymerase chain reaction (PCR). The present invention relates to a selective multinucleic acid amplification apparatus using a MEMS device, including an electrode integrated into a MEMS device, and including an electrode for forming a virtual multiple reaction vessel, and a method of manufacturing the same.

In general, nucleic acid amplification is divided into mononucleic acid amplification and polynucleic acid amplification. Mononucleic acid amplification is performed by dissolving or immobilizing a sample nucleic acid and a primer to be amplified in a liquid or solid matrix, respectively, in a reaction chamber. Only a portion of the nucleic acid of the sample having a base sequence complementary to the base sequence of the immobilized primer is combined with the primer to form a double stranded-helix nucleic acid (ds-DNA: double strand-deoxyribo nucleic acid). Therefore, since the type of nucleic acid to be amplified is determined according to the primer, only one nucleic acid is amplified for one primer. On the other hand, amplification of polynucleic acid can amplify various kinds of polynucleic acids of interest by adding a set of various primers in a reaction vessel.

Induced complementary binding of nucleic acids by electric fields in micro-chips to amplify mononuclear and polynucleic acids (Carl F. Edman, et al., Nucleic Acids Research 25:24, 1997), by heat Lysis and sizing through electrophoresis on a chip of multiple PCR amplification products (Larry C. Waters, et al, Anal Chem . 70: 158-162, 1998), Simultaneous PCR on microchips for four DNA samples (Larry C. Waters, et al., Anal Chem . 70: 5172-5176, 1998), single nucleic acid polymorphism detection (Patrick N. Gilles, et al., Nature Biotechnology 17, 1999), PCR in multiple reaction vessels (Hidenori Nagain et al., Anal. Chem. 73 (5): 1043-1047, 2001), PCR on microchips (Mann A. Shoffner, et al. , Nucleic Acid Research 24: 2, 1996; Jing Cheng, et al., Nucleic Acid Research 24: 2, 1996), Rapid DNA Amplification on Microchips (Thomas M. H. Lee, et al., Anal. Chem . 72 : 4242-4247, 2000), multiple amplification using strand displacement amplification (SDA) reaction of DNA immobilized on solid matrix (Lorelei Westin, et al., Nature Biotechnology 18: 2, 2000), complementary binding of DNA by electric field (Carl F. Edman, et al., Nucleic Acids Research 25:24, 1997), DNA multiplexing of whole cells (Larry C. Waters, et al., Anal. Chem . 70: 158-162, 1998).

The amplification and testing of the sample nucleic acid by the conventional PCR method requires a lab-scale macro-scale equipment by requiring a temperature cycle between 40 and 95 ° C, and thus, the sample nucleic acid required for the PCR reaction. And reagents are consumed in large quantities. In addition, PCR in a single reaction vessel for amplification of the polynucleic acid is deteriorated in PCR efficiency due to hybridization between multiple primers. To compensate for this, a method has been developed that uses electric fields to isolate DNA that does not exactly complementarily bind or induces complementary binding by electric fields (Lorelei Westin, et al., Nature Biotechnology 18: 2, 2000). However, this method also performs PCR in a single reaction vessel using one primer immobilized in a solid phase or liquid phase, and the limitation of amplification of polynucleic acid has been pointed out.

Accordingly, the present inventors have diligently researched to develop a device capable of selectively amplifying several types of target nucleic acids. As a result, a micro heater and a resistance thermometer device sensor (RTD sensor) are used to control a temperature cycle used for PCR. ), A multi-nucleic acid amplification apparatus that can efficiently perform independent PCR for multi-nucleic acid in each reaction vessel formed from this, including the electrode for the formation of a virtual multiple reaction vessel in a single MEMS device By this, the present invention was completed.

It is an object of the present invention to provide an ultra-small multinucleic acid amplification apparatus incorporating a MEMS device for amplifying selective multinucleic acid by multiple primers.

1 shows a temperature profile of a commonly used PCR,

Figure 2 shows a summary of the amplification process of the sample nucleic acid using PCR,

Figure 3 shows a plan view of the electrode position in a single reaction vessel according to the selective multinucleic acid amplification apparatus using the MEMS device of the present invention,

Figure 4 shows the process of forming a single reaction vessel by the electrophoresis method and electrode polarization electrophoresis method in the selective multinucleic acid amplification apparatus using the MEMS device of the present invention,

5 illustrates a process of forming a multi-reaction vessel by changing the outer electrode into a circle in the selective multinucleic acid amplification apparatus using the MEMS device of the present invention.

6 shows the basic principle of performing PCR using the selective multinucleic acid amplification apparatus using the MEMS device of the present invention,

7 is a cross-sectional view of a selective multinucleic acid amplification apparatus using the MEMS device of the present invention.

8 shows the shape and size of the electrode for efficient control of the electric field in the selective multinucleic acid amplification apparatus using the MEMS device of the present invention,

Figure 9 shows the size of a single reaction vessel formed in the selective multinucleic acid amplification apparatus using the MEMS device of the present invention,

10 shows the structure of a micro heater and a resistance thermometer device (RTD) sensor used for PCR in a selective multinucleic acid amplification apparatus using the MEMS device of the present invention.

11A to 11C illustrate a process of manufacturing a selective multinucleic acid amplification apparatus using the MEMS device of the present invention.

In order to achieve the above object, the present invention uses a MEMS device, including an electrode for integrating a micro heater and RTD sensor in one MEMS device and forming a virtual multiple reaction vessel to control the temperature cycle used for PCR Provided is a selective multinucleic acid amplification device.

In addition, the present invention provides a method of manufacturing a selective multinucleic acid amplification apparatus using the MEMS device.

Hereinafter, the present invention will be described in detail.

The present invention provides a selective multinucleic acid amplification apparatus using a MEMS device, including an electrode for integrating a micro heater and an RTD sensor into a single MEMS device and forming a virtual multiple reaction vessel for controlling a temperature cycle used for PCR. do.

Specifically, the multinucleic acid amplifying apparatus of the present invention comprises a substrate on which a thin film is formed; A micro heater and an RTD sensor unit positioned on the substrate and used for heat generation and heat detection; An electrode unit positioned on the heater and sensor unit to form an electric field; A mixed reaction container surrounding the electrode part and including a reaction container and an outlet of a sample for mixing and storing a PCR solution; And a peltier element located under the substrate.

The heater and the sensor unit are fixed with a thin film or a substrate to form a top electrode and an insulating layer to detect heat absorption and heat generation, and the electrode unit forms an electric field for mixing PCR solution molecules and sealing the target DNA. This results in the formation of a virtual multi-amplification reaction vessel controlled by an electric field inside the mixing reaction vessel.

In the multinucleic acid heavy-weight apparatus of the present invention, an electrically negative nucleic acid is led to a positive electrode in a direct current electric field, and in the alternating electric field, polarization electrophoresis occurs due to polarization, and thus it is led to a constant space according to the intensity of the electric field. Attention is drawn to the method of confining the desired nucleic acid in the desired space.

The multinucleic acid heavy-duty device of the present invention manufactured on the same principle includes an RTD sensor, which is a temperature sensing element located on the same plane, including a microheater for a thermal cycle for basic PCR amplification as a heat source on a substrate. Also included under the device is a Peltier device as a heat sink. First, the microheater is supplied with a power source of 0 to 10V from the outside as a heat source required for the temperature cycle to emit heat of 25 to 300 ℃ by the coil-type resistance wire. The heat generated therefrom is transferred to the PCR solution and used as an energy source for performing PCR. At this time, the heat generated by the resistance wire is absorbed by the Peltier heat absorption source installed in the lower part of the element or is dissipated into the air by natural convection to be consumed.

In addition, the RTD sensor measures the temperature in the current reaction vessel and detects the change in resistance of the lead due to heat to form an accurate temperature cycle. In this way, the required energy is output in the form of voltage by the control of an externally attached controller according to the measured temperature, and amplified to apply a voltage to a coil-type resistance wire, thereby providing PID (proportion-integration-derivation). , Proportional-differential-integral) control. Here, PID control means a value that is proportional to an error, that is, a proportional control, a derivative value, that is, a derivative control, an integral value, that is, integral control, simultaneously outputting a correction value. The temperature value is read from to control the output value of the corresponding heater. When heat absorption by natural convection and heat generation by coils do not reach the heat capacity for PCR, heat absorption and heat generation are performed in parallel by the Peltier element disposed below as an auxiliary means.

As a method for performing multiple PCR by multiple reaction vessels, separation and formation of each reaction vessel by an electric field is performed by a center electrode and an outer electrode located in the uppermost layer, and the electrodes of each container are controlled by an electric field received from an external controller. The electric field is formed to form a virtual electric barrier to separate each reaction vessel. The center electrode is used to fix the primer for PCR and to mix the PCR solution by electric field formation and fluid rotation. The outer electrode is used as the counter electrode to form the fluid wall and the physical wall. The outer and outermost electrodes located around the PCR electrode prevent the incorporation of other nucleic acids that may occur during the PCR reaction and increase the efficiency of PCR by controlling the electric field of the central electrode to which the primer is fixed. The reaction vessel for PCR is coupled on top electrode protected by an insulating layer and contains the entrance and exit of the fluid required for the reaction.

Unlike the amplification by the conventional PCR method, the multinucleic acid heavy device of the present invention applies an electric field to an electrode to selectively fix primers, each of which is a part of a gene to be examined, in a desired space. The immobilized primer is hybridized with a gene having a sequence complementary to that of the primer among the genes present in the sample to selectively amplify only this gene.

Conventional PCR methods using MEMS devices (Northrup, MA, et al., Solid-State Sensors and Actuators 1995 and Eurosensors Ⅸ. Transducers '95. The 8th International Conference 1: 764-767, 1995) Each primer is immobilized on the electrode in order to detect the desired mononucleic acid. In this case, since primers having different base sequences are fixed to individual electrodes, a portion of an amplification sample corresponding to each primer causes interference between each electrode when performing nucleic acid amplification such as PCR.

In order to solve this problem, the MEMS device of the present invention forms an electric field by arranging another electrode around the electrode so that multiple PCR by multiple primers may occur only in a partial electrode, thereby changing the electrode to which the primer is fixed. Separation from the electrodes minimizes the interference between PCR and the mixing of different genes. Therefore, the multinucleic acid amplification apparatus employing the MEMS device of the present invention enables the electric barrier to selectively amplify the polynucleic acid by mixing the biological components and electric field formation required for the PCR while at the same time giving the effect of the physical wall.

In addition, the MEMS device of the present invention not only controls the exact complementary coupling between the primer and the sample by controlling the electric field of the center electrode, but also supplies energy used for PCR by arranging a heater at the bottom of the device and using it as a lower heat source. In addition, complementary binding can be controlled. Furthermore, unlike the conventional heat supply method, the MEMS device of the present invention has an advantage of using a thin film micro heater and a Peltier device at the same time to realize more accurate and faster temperature response.

The multinucleic acid amplification apparatus using the MEMS device of the present invention improves low PCR efficiency due to the use of multiple primers in a single reaction vessel, which has been pointed out as a problem in the prior art, and complements the formation of the multiple reaction vessel as a physical wall. Controlling each electrode enables PCR in highly integrated multiple reaction vessels without physical walls. In addition, the MEMS device is very economical because it can selectively amplify a variety of target nucleic acids only with a sample and a reagent of a micro unit.

In addition, the present invention generally uses a micro heater and a Peltier element as a heat source by supplementing the disadvantage that it is difficult to control the temperature of the reaction vessel due to the limitation of the heat capacity when only the Peltier element is used as the heat source necessary for the temperature cycle. Cooling is characterized by natural convection and Peltier elements.

The selective multinucleic acid amplification apparatus using the MEMS device according to the present invention will be described in detail with reference to the accompanying drawings.

1 shows a temperature profile of a PCR generally used in the related art. The Y axis represents the temperature required for each step of the PCR and the X axis represents the duration required for each step. The temperature profile in the PCR using the multinucleic acid amplification apparatus of the present invention was reduced to 1 to 3 seconds by substituting the microheater for 1 to 3 minutes, which is a necessary time for each step in the existing temperature profile.

Figure 2 shows the process of amplifying the sample nucleic acid using PCR. In each step, the sample DNA is denatured from a double stranded helix to a single stranded helix by high temperature, hybridized with complementary primers, and then amplified and re-separated through a series of cycles, such that only desired DNA can be amplified.

Figure 3 shows a plan view of the electrode position in a single reaction vessel formed according to the selective multinucleic acid amplification apparatus using the MEMS device of the present invention, the entire vessel consists of a single circle including the outlet of the sample and the electrode portion As shown in the enlarged view, it is composed of an outer electrode, a center electrode, and an outer electrode. The center electrode is where the primer is fixed, and the outer electrode is where the reaction solution is trapped during PCR to form an electric field for mixing. In addition, in order to prevent sample mixing between different single reaction vessels, an outer electrode or four outermost electrodes may be installed.

Figure 4 shows the formation process and control flow of the vessel by the electrophoresis method and electrode polarization electrophoresis method for a single reaction vessel in the selective multinucleic acid amplification apparatus using the MEMS device of the present invention, the electrode control method is electrophoresis Control the movement and mixing of DNA and PCR samples. The negatively charged (-) biomolecule is moved by the electric field between the center electrode and the outer electrode, and as the externally controlled voltage is sequentially applied to the outer electrode, a circular electric field is formed with a constant frequency. As a result, the biomolecule inside the central electrode is trapped in the virtual reaction vessel formed by the electric field, and the PCR solution around the outer electrode is mixed. At this time, the sample mixing with the other reaction vessel is prevented by the outer electrode or the outermost electrode.

In order to form a multiple reaction vessel, the negative electrode is repelled toward the center electrode by repelling negatively charged DNA using the outer electrode or the outermost electrode as a cathode. At this time, the PCR product generated in the central electrode is not moved to another reaction vessel by the outer electrode or the outermost electrode. If the positive and negative electrodes are applied to the outer electrode in sequence while changing the positive and negative poles, electropolarization electrophoresis occurs, where the electric field of the negative or positive electrode is distributed at the maximum or minimum value according to the polarization amount of the biomolecule and the nature of the solution. When the electric field is distributed at the maximum or minimum value, the biomolecules are collected in a virtual reaction vessel formed at the central electrode.

FIG. 5 illustrates a flow in which a reaction vessel is formed according to an application sequence of an electric field and a negatively charged biomolecule is moved when the outer electrode is changed into a circle to form a reaction vessel in the multinucleic acid amplification apparatus of the present invention.

Figure 6 shows a PCR reaction made in a virtual reaction vessel formed according to the multinucleic acid amplification apparatus of the present invention.

Figure 7 shows a cross-sectional view of a selective multinucleic acid amplification device using a MEMS device made in accordance with the present invention, Figure 8 is different for efficient control of the electric field to form a single reaction vessel in the multinucleic acid heavyweight apparatus of the present invention It shows the shape and size of the electrode, Figure 9 shows the size of a single reaction vessel formed in accordance with the present invention. A single reaction vessel of FIG. 9 may be integrated after excluding a contact site for applying a voltage in the single reaction vessel to form a multiple reaction vessel. The shape of the electrode used in the multiple reaction vessel can be designed differently than a single reaction vessel, but basically each reaction vessel is designed to prevent mixing with biological molecules in the other reaction vessel.

Figure 10 shows the structure of the micro-heater and RTD (resistance thermometer device) sensor used in the PCR in the multinucleic acid amplification device of the present invention, Figure 11a to 11c shows the manufacturing process of the multinucleic acid amplification device of the present invention.

In addition, the present invention provides a method of manufacturing a selective multinucleic acid amplification apparatus using the MEMS device.

The manufacturing method of the multinucleic acid amplification apparatus according to the present invention

1) forming a low stress nitride thin film on the substrate surface;

2) depositing a metal layer on the nitride thin film to form a heat source and a sensor shape;

3) forming a first layer by rotationally coating the photosensitive polymer on the metal layer and then etching the shape of the heat source and the sensor with an ultraviolet mask;

4) forming a first insulating layer over the heat source and sensor structure of the first layer;

5) depositing a metal layer to form a top electrode on the first insulating layer;

6) forming a second layer by rotating the photosensitive polymer on the metal layer and etching the shape of the top electrode with an ultraviolet mask;

7) spin-coating a photosensitive polymer on the back surface of the substrate obtained in step 6) and etching the shape of the etching mask with an ultraviolet mask;

8) etching the silicon and forming a low stress nitride thin film to remove the insulating silicon oxide film;

9) forming a second insulating layer between the exposed portion and the insulating portion of the top electrode of the second layer;

10) removing the insulating layer other than the electrode pad of the heat source and the sensor after exposing the exposed portion of the top electrode and the electrode pad;

11) preparing a structure having a sample outlet and a reaction vessel shape by applying SU-8;

12) silanizing the surface of the SU-8 structure, and then pouring polymethylsiloxane (PDMS) to obtain a reaction vessel structure to construct a MEMS device; And

13) connecting the external controller to the MEMS device fabricated as described above, and combining the Peltier device at the bottom to fabricate the entire multinucleic acid amplification apparatus (see FIGS. 11A to 11C ).

When the manufacturing method is described step by step, a substrate made of silicon, glass, or polycarbonate (PC, Polycarbonate) may be used as the substrate in step 1), and a silicon substrate having a thickness of 500 to 550 μm is preferable. First, impurities on the surface of the substrate were removed and an oxide film was applied, and then a low stress nitride film was additionally coated to improve insulation and form a thin film (see A of FIG. 11A ). Low stress nitride film is applied to a thickness of 1 to 1.5 ㎛ using a low pressure chemical vapor deposition (LPCVD) deposition apparatus.

Step 2) is thermally depositing a metal layer to form a single heat source and a temperature sensing sensor structure on the oxide thin film silicon insulating layer fabricated in step 1). In the present invention, the chromium / gold (Cr / Au) metal layers are sequentially It is deposited to a thickness of 2000 GPa to 5000 GPa. Chromium improves adhesion between the substrate and gold, and gold used as an electrode prevents corrosion by electrolysis or redox of a solution and facilitates modification of the primer when the primer is fixed to the electrode ( B of FIG. 11A ) . Reference). Titanium (Ti) may be used instead of chromium and platinum (Pt) may be used instead of gold.

In step 3), HMDS (hexamethyldisilazane) is rotatably coated on the upper surface of the insulating layer on which the metal layer is deposited in step 2) to a thickness of 0.2 to 0.3 μm. The photoresist is spin-coated to a thickness of 1 to 1.5 μm, and then exposed to an ultraviolet (UV) mask to pattern the heat source and the sensor (see C of FIG. 11A ). In this case, AZ5214, AZ94562 or AZ9260 (all manufactured by AZ) may be used as the photosensitive polymer.

In step 4) spin-on-glass (SOG) is applied to a thickness of 0.3 to 0.8 [mu] m to form an insulating layer on the single heat source and the temperature sensor of the first layer formed by the above steps ( FIG. 11A). See D ).

Step 5) is to deposit a chrome / gold metal layer, as shown in a step of depositing a metal layer using an evaporator column to form a top electrode on the insulating layer, step 2) in this order (see E of Fig. 11).

In step 6), HMDS is coated on the upper surface of the substrate on which the metal layer is deposited in a thickness of 0.2 to 0.3 μm, and the photosensitive polymer is rotated to a thickness of 1 to 1.5 μm, and the top electrode shape is etched by exposing with a UV mask ( See FIG. 11A F ).

In step 7), HMDS is 0.2-0.3 μm thick and photosensitive polymer is rotated 1-1.5 μm thick on the back side of the substrate in step 6), and then the etching mask shape is etched on the back side by exposing with an ultraviolet mask ( FIG. 11A). G )).

In step 8), the silicon is etched to form a low stress nitride thin film, and then the insulating silicon oxide film is removed (see H of FIG. 11A ).

In step 9), SOG is applied to a thickness of 0.3 to 0.8 mu m to form an insulating layer between the exposed portion and the insulating portion of the top electrode of the second layer (see I in FIG. 11B ).

In step 10), HMDS is 0.2 to 0.3 μm thick and the photosensitive polymer is rotated to 1 to 1.5 μm thick on the upper surface of the substrate, and then exposed by an ultraviolet mask to expose the exposed portion of the top electrode and the electrode pad ( J in FIG. 11B ) . Reference).

In step 11) SU-8 50 (Nano ™ SU-8, Micro Chem) is spun onto 90-110 μm thick. Here, the SU-8 structure is fabricated by etching the UV mask to form a sample outlet of the channel and a reaction vessel in a circular shape (see K and L of FIG. 11B ). The SU-8 structure manufactured as described above has the opposite shape of the PDMS mold as a template for the PDMS shape.

In step 12), the surface of the SU-8 structure is silanized so that the structure is easily separated from the mold. At this time, trichloro (3,3,3 trifluoropropyl) silane] is preferable as the compound used for silanization. PDMS mold is prepared by pouring PDMS made by mixing dimethylsiloxane monomer (monomer) and curing agent (1:10) onto SU-8 structure mold. The PDMS reaction vessel structure obtained from this can be combined with the SU-8 structure to form a complete MEMS device. At this time, since the PDMS structure is removed from the mold by the silanization treatment, it is easy to obtain a large amount of channel structures by repeating the process of pouring and heat-processing the PDMS mixed with the curing agent again without a separate process. The produced PDMS structure and SU-8 structure can be easily bonded by a reversible reaction as well as oxidize the surface and can be bonded by irreversible bonding (see M of FIG. 11B ).

In step 13), an external controller and a commercial Peltier device are combined with the MEMS device fabricated as described above to manufacture an overall multinucleic acid amplification apparatus (see N to P of FIG. 11C ).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Fabrication of multinucleic acid medium width device using MEMS device

(Step 1)

A silicon substrate (500 μm thick, <100> direction, N-type, LG Siltron) was prepared at 120 ° C. in a 1: 2 (v / v) dilution of sulfuric acid (H ₂ SO ₄ ) and hydrogen peroxide (H ₂ O ₂ ). After cleaning for 10 minutes to remove contaminants such as metal residue and metal / organic, put into an oxidation furnace and oxidized with deionized water (DI water) at 1,000 ℃ for 6 hours at about 1.2 ㎛ Oxide film (silicon oxide, SiO ₂ -Silicon Oxide) was formed. In order to improve insulation and to form a thin film after oxide coating, a low stress nitride film was additionally added to 4,000 으로 by a low pressure chemical vapor deposition (LPCVD) process (Lee Seung-Seop, Ph.D. Thesis , UC Berkeley, 1995,). coated (a in Fig. 11a).

(Step 2)

In order to form a single heat source and a temperature sensing sensor structure on the oxide thin film silicon insulating layer fabricated in step 1), a chromium / gold (Cr / Au) metal layer is in turn thermal evaporator (ULVAC: high vacuum coater MH89-4504) Was deposited using. In order to improve the adhesion between the substrate and the gold, chromium was deposited in total 100 mA at a deposition rate of 1 mA / sec for about 2 minutes between 55 and 60 A of current, and gold was approximately 10 to 15 between 50 and 55 A of current. A total of 1,000 mW was deposited at a deposition rate of 1-1.5 mW / sec for minutes ( B in FIG. 11A ).

(Step 3)

In step 2), HMDS (hexamethyldisilazane, giraffe reagent) was coated on the upper surface of the insulating layer on which the metal layer was deposited (0.3 seconds at 4 rpm at 250 rpm, 35 seconds at 5000 rpm), and then softbaked at 90 ° C. for 100 seconds. soft baking). The AZ 5214 photosensitive polymer (photoresist, Giraffe reagent) was spun onto a 1.3 μm thickness (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and then softbaked at 90 ° C. for 50 seconds. In order to pattern the heat source and the sensor shape, an ultraviolet (UV: Ultra Violet) mask was exposed to ultraviolet light for 15 seconds at 16 mA / cm 2 intensity. After developing for 1 minute in an AZ 300 MIF developer (giraffe reagent), it was hard baked at 120 ° C. for 100 seconds in an oven or hot plate. A gold etchant prepared by mixing KI (potassium iodide): I2 (iodine): DI (deionized water) in a ratio of 4 g: 1 g: 40 ml to remove the chromium / gold metal layer outside the shape of the heat source and sensor. Was treated at a rate of 1 μm / min for etching for about 10 seconds, and the gold shape was used as a mask and was used in a commercially available chromium-etched solution (Cr-etchant, Cr-7k, Zeus Co., Ltd.). The shape was prepared by etching for 5 seconds. The etched substrate was cleaned for 3 minutes using TCE (trichloroetylene) / aceton / methyl alcohol solution or a dedicated stripper (Zeus Co., Ltd.) for 3 minutes, and the organic photosensitive The polymer was removed and then dried over nitrogen (N ₂ ) ( C in FIG. 11A ).

(Step 4)

Spin-on-glass (T-12NEB, Honeywell, Inc.) was applied to form an insulating layer between the single heat source and temperature sensor structure of the first layer and the top electrode fabricated later. SOG may be applied under rotational conditions of 3,000 rpm / 11 seconds, 1,500 rpm / 3 seconds, and 3,000 rpm / 9 seconds to form a thickness of about 4,000 mm 3, or may form a thick insulating layer up to about 1.2 μm by double coating. The applied SOG was softbaked for 2 minutes for curing and then cured for about 60 minutes with a temperature change of 250-425 ° C. ( D in FIG. 11A ).

(Step 5)

In order to form the top electrode on the insulating layer, chromium / gold metal layers were sequentially deposited using a thermal evaporator. In order to improve the adhesion between the substrate and the gold, chromium was deposited in total 100 mA at a deposition rate of 1 mA / sec for about 2 minutes between 55 and 60 A current, and gold was approximately 10 to 15 between 50 and 55 A current. A total of 1,000 mW was deposited at a deposition rate of 1-1.5 mW / sec for minutes ( E in FIG. 11A ).

(Step 6)

In step 5), HMDS was coated on a top surface of the substrate on which the metal layer was deposited (0.3 seconds at 250 rpm for 4 seconds at 5000 rpm) and then softbaked at 90 ° C. for 100 seconds. Here AZ 5214 photosensitive polymer was spun onto a 1.3 μm thickness (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and then softbaked at 90 ° C. for 50 seconds. In order to etch the shape of the top electrode composed of the center electrode and the outer electrodes arranged in a circular shape, it was exposed to ultraviolet light for 15 seconds using an ultraviolet mask at 16 ㎽ / cm 2 intensity. After developing for 1 minute in AZ 300 MIF developer (giraffe reagent), it was hardbaked at 120 ° C. for 100 seconds in an oven or hotplate. In order to remove the chromium / gold metal layer other than the top electrode, the gold etchant prepared by mixing KI: I2: DI at a ratio of 4 g: 1 g: 40 ml was etched for about 10 seconds by treating at a rate of 1 μm / min. , The gold shape was used as a mask and etched in chromium-etched solution (Cr-7k, Zeus Co., Ltd.) for about 5 seconds to form the shape. The etched substrate was washed for 3 minutes using a TCE / acetone / methyl alcohol solution or a dedicated remover to remove the organic photosensitive polymer and then dried with nitrogen ( F of FIG. 11A ).

(Step 7)

HMDS was coated on the back of the substrate in step 6) with a thickness of 0.3 μm (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and then softbaked at 90 ° C. for 100 seconds. The AZ 9260 photosensitive polymer was spun onto a thickness of 5.7 μm (45 seconds at 4000 rpm) and then softbaked at 120 ° C. for 100 seconds. It was left for about 10 minutes to solidify the photosensitive polymer. In order to etch the shape of the etching mask on the back surface of the low stress nitride thin film, UV exposure was performed for 100 seconds with 16 ㎽ / cm 2 intensity using an ultraviolet mask. After developing for 5 minutes in AZ 400K developer (giraffe reagent), it was hard-baked at 90 degreeC for 30 minutes in oven or a hotplate. The low stress nitride protective film other than the etching mask shape etched on the back side was removed by using reactive ion etching (RIE). Treatment conditions for RIE were etched RF (radio frequency) at 50 W for 50 minutes. After etching, the remaining oxide film was removed in a BHF (buffed HF) solution for 5 minutes. To remove AZ 9260 (giraffe reagent) used as a mask, each was washed for 5 minutes with acetone, methanol, and deionized water ( G of FIG. 11A ).

(Step 8)

In order to etch the silicon, the substrate of step 7) was placed in a tetramethyl ammonium hydroxide (TMAH) solution (giraffe reagent) for 10 hours at 90 ° C to etch silicon, and a low stress nitride nitride film was fabricated. To remove, washed with BHF solution for 5 minutes and dried with deionized water and nitrogen ( H in Fig. 11a ).

(Step 9)

SOG was applied to form an insulating layer between the exposed and insulating portions of the top electrode of the second layer. SOG may be applied under rotational conditions of 3,000 rpm / 11 seconds, 1,500 rpm / 3 seconds, and 3,000 rpm / 9 seconds to form a thickness of about 4,000 mm 3, or may form a thick insulating layer up to about 1.2 μm by double coating. The applied SOG was softbaked for 2 minutes for curing and then cured for about 60 minutes with varying temperature to 250-425 ° C. ( I in FIG. 11B ).

(Step 10)

HMDS was coated on the upper surface of the substrate in step 9) with a thickness of 0.3 μm (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and then softbaked at 90 ° C. for 100 seconds. Here AZ 5214 photosensitive polymer was spun onto a 1.3 μm thickness (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and then softbaked at 90 ° C. for 50 seconds. In order to expose the exposed portion of the top electrode and the electrode pad (pad), the ultraviolet light was exposed to ultraviolet light for 16 seconds with 16 ㎽ / ㎠ intensity with an ultraviolet mask. After developing for 1 minute in an AZ 300 MIF developer, it was hardbaked at 120 ° C. for 100 seconds in an oven or a hot plate. In order to remove SOG other than the single heat source and the electrode pad of the sensor, the SOG layer, which is an insulating layer, was removed by immersing in BHF (Buffered HF) solution for 15 minutes ( J in FIG. 11B ).

(Step 11)

To prepare a fluid container for a single reaction vessel, SU-8 50 (70 wt% EPON, 30 wt% GBL, Microchem Co., Ltd.) was coated with a rotary coating (5 seconds at 200 rpm, 35 seconds at 1000 rpm) to be about 85 μm thick. The container was formed. It was prebaked at 95 ° C. for 15 minutes ( K in FIG. 11B ).

(Step 12)

In order to make the sample outlet of the channel and the reaction vessel of a circular shape, it was exposed at around 365 nm with the intensity of 300-400 mJ / cm <2> with an ultraviolet mask. Postbaking at 95 ° C. for 15 minutes was developed and washed with PGMEA (propyleneglycol monomethylether acetate) solution (giraffe reagent) for 15 minutes. After hard-baking at 200 ℃ for 30 minutes, SU-8 is completely removed by boiling NMP (1-methyl-2-pyrrolidinone) or O ₂ ashing (ashing) to remove the inlet of the channel and the reaction vessel in the circular form. A shaped SU-8 structure was produced. The fabricated structure has a shape for PDMS (polydimethylsiloxane, SYLGUARD ™ 184, Dow Corning Co., Ltd.) shape and has the opposite shape of the PDMS mold ( L in FIG. 11B ).

(Step 13)

In order to obtain a reaction vessel structure using the SU-8 structure as a template, the surface of the SU-8 structure was first silanized to harden the PDMS and then easily removed. The silanization was performed using trichloro (3,3,3 trifluoropropyl) silane (Trichloro (3,3,3 Trifluoro propyl) silane, Sigma) in about 10 μl into a vacuum vessel for 8 hours. Formation of PDMS template was made by mixing dimethylsiloxane monomer (monomer) and curing agent (SYLGOARD184) with 10: 1 to remove the bubble-free PDMS on a pre-made SU-8 structure template. After removing the bubbles generated during the pouring process and heat treatment at 100 ℃ for about 1 hour to remove the solidified PDMS to obtain a reaction vessel structure, combined with the structure made up to step 12) to form a complete MEMS device ( Fig. 11b M ).

(Step 14)

In order to connect the MEMS device manufactured as described above with an external controller, an outer PCB (printed circuit board (SME) Co., Ltd.) substrate is manufactured, and the connection part is a conductive polymer mixed with silver and epoxy resin. G) to supply a voltage to the top electrode ( N in FIG. 11C ).

(Step 15)

After connecting the prepared MEMS device with an external controller, PCR samples were injected and the reaction vessel was covered with a slide glass ( O of FIG. 11C ).

(Step 16)

In order to fabricate the overall multinucleic acid amplification apparatus, a commercial peltier element was bonded to the lower portion of the MEMS element fabricated according to the above step by using a thermal grease and a bond to prepare a heat absorption source ( P of FIG. 11C ).

As described above, the selective multinucleic acid amplification apparatus using the MEMS device of the present invention is capable of amplifying a plurality of DNAs in a micro-range sample capacity and is coupled to an external controller in a hand-held type. By using such a device, the size limitation of the physical reaction vessel can be eliminated, and the reaction vessel by the electrode can be miniaturized to improve the degree of integration and to generate multiple reactions. Therefore, the DNA amplified by the selective polynucleic acid amplification apparatus using the MEMS device of the present invention can be usefully used for analyzing the binding properties between DNA and proteins.

Claims (9)

A substrate on which a thin film is formed; A micro heater and an RTD sensor unit positioned on the substrate and used for heat generation and heat detection; An electrode unit positioned on the heater and sensor unit to form an electric field; A mixed reaction container surrounding the electrode part and including a reaction container and an outlet of a sample for mixing and storing a PCR solution; And a multi-nucleic acid amplification apparatus using a micro electro mechanical system (MEMS) device including a peltier device located under the substrate. The method of claim 1, Selective multinucleic acid amplification apparatus characterized in that the micro-heater and RTD sensor unit is fixed to a thin film or substrate to form the upper electrode and the insulating layer to detect heat absorption and heat generation. The method of claim 1, Selective polynucleic acid amplification apparatus, characterized in that the electrode portion is arranged to form an electric field capable of mixing the PCR solution molecules and seal the target DNA. The method of claim 1, Selective multinucleic acid amplification apparatus, characterized in that the mixed reaction vessel has a virtual multiple reaction vessel capable of amplifying the target DNA by the electric field controlled by the electrode portion located therein. 1) forming a low stress nitride thin film on the substrate surface; 2) depositing a metal layer on the nitride thin film to form a heat source and a sensor shape; 3) forming a first layer by rotationally coating the photosensitive polymer on the metal layer and then etching the shape of the heat source and the sensor with an ultraviolet mask; 4) forming a first insulating layer over the heat source and sensor structure of the first layer; 5) depositing a metal layer to form a top electrode on the first insulating layer; 6) forming a second layer by rotating the photosensitive polymer on the metal layer and etching the shape of the top electrode with an ultraviolet mask; 7) spin-coating a photosensitive polymer on the back surface of the substrate obtained in step 6) and etching the shape of the etching mask with an ultraviolet mask; 8) etching the silicon and forming a low stress nitride thin film to remove the insulating silicon oxide film; 9) forming a second insulating layer between the exposed portion and the insulating portion of the top electrode of the second layer; 10) removing the insulating layer other than the electrode pad of the heat source and the sensor after exposing the exposed portion of the top electrode and the electrode pad; 11) manufacturing a structure having a sample outlet and a reaction vessel shape by applying SU-8 50; 12) silanizing the surface of the SU-8 structure and then pouring polymethylsiloxane (PDMS) to obtain a reaction vessel structure to construct a MEMS device; And 13) fabricating the selective multinucleic acid amplification apparatus using the MEMS device of claim 1, comprising connecting the external controller to the MEMS device fabricated as described above and combining the Peltier element at the bottom to produce the entire multinucleic acid amplification apparatus. How to. The method of claim 5, In step 1) the substrate is selected from the group consisting of silicon, glass or polycarbonate. The method of claim 5, In steps 2) and 5), wherein the metal layer is formed by sequentially depositing chromium / gold (Cr / Au). The method of claim 5, In steps 4) and 9), wherein the insulating layer is formed by applying spin-on-glass (SOG) to a thickness of 0.3 to 0.8 μm. A method for selectively amplifying a polynucleic acid in a sample using the apparatus of any one of claims 1 to 4.