Background Art The polymerase chain reaction (PCR) method has been developed to amplify nucleic acid sequences by being subject to a periodical hot-cold temperature cycle. In PCR, 1 cycle of DNA amplification requires a biochemical sample to be exposed to varying temperatures, such as T1 (for denaturing)- T2 (for annealing) < T3 (for extension).

As shown in FIG. 1, a conventional PCR system has a structure where polymerase chain reaction is made by controlling the temperatures (T1 for denaturing: 94 °C, T2 for annealing: 55 °C, T3 for extension: 72 °C) of a chamber retaining a biochemical fluid, such as a PCR fluid. In this system, the repetition of heating and cooling the chamber causes a time delay for heating and cooling, thus complicated circuits are needed for an accurate control of the temperatures.

U. S. Pat. No. 5,270, 183 discloses an apparatus and method for the amplification of nucleic acids in a sample using polymerase chain reaction, as shown in FIG. 2, where a polymerase chain reaction is made by continuously flowing a biochemical fluid, such as a PCR fluid, in zigzags along different temperature zones. Therefore, this system may require an extraordinarily long channel for a biochemical fluid to follow an accurate temperature profile, because the movement from T3 section to T1 section should be passed through T2 section.

Further, as shown in FIG. 3, is disclosed a PCR system where polymerase chain reaction is made by continuously flowing a biochemical fluid, such as a PCR fluid, in concentric circles along different temperature zones (Proc. Miniaturized Total Analysis Systems (uTAS 2001), Luisiana State University, Steven A. Soper et al., pp. 459-461). In this system, a flow path becomes shorten as one complete cycling repeats. Thus, the flow rate of the biochemical fluid should be accurately controlled in order to follow a temperature profile.

Disclosure of the Invention The present invention provides an apparatus for circulating a carrier fluid having two or more chambers or sections maintained at different temperatures and a method for operating the same. Further, the present invention provides an apparatus for amplifying a nucleic acid using the same and a chip containing the same.

In one aspect of the present invention, there is provided an apparatus for circulating a carrier fluid comprising two or more chambers maintained at different temperatures, each chamber comprising an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber (inlet pneumatic air pressure port); and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber (outlet pneumatic air pressure port); wherein the chambers are sequentially connected such that the outlet valve of one chamber is connected to the inlet valve of an adjacent chamber in a direction the fluid flows.

In another aspect of the present invention, there is provided a method for operating the above apparatus for circulating a carrier fluid, which comprises applying a pressure to the inlet pneumatic air pressure port of a chamber and venting an outlet pneumatic air pressure port of an adjacent chamber in a fluid flow direction at the same time to allow the carrier fluid to move from the chamber to the adjacent chamber; controlling a pressure applied to the outlet pneumatic air pressure port of the adjacent chamber to retain the

carrier fluid in the adjacent chamber for a predetermined time; and repeating the applying and controlling steps in turn to circulate the carrier fluid.

In still another aspect of the present invention, there is provided an apparatus, for use in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction, comprising three chambers, each chamber comprising an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber; and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber; wherein the chambers are sequentially connected such that the outlet valve of one chamber is connected to the inlet valve of an adjacent chamber in a direction the fluid flows ; and wherein the three chambers include a first chamber maintained at a temperature for denaturing, a second chamber maintained at a temperature for annealing, and a third chamber maintained at a temperature for extension.

In still another aspect of the present invention, there is provided an apparatus, for use in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction, comprising two chambers, each chamber comprising an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber; and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber; wherein the outlet valve of one chamber is connected to the inlet valve of the other chamber; and wherein one chamber is maintained at a temperature for denaturing and the other chamber is maintained at a temperature for both annealing and extension.

In still another aspect of the present invention, there is provided an apparatus for circulating a carrier fluid, comprising a micro-channel having two or more sections maintained at different temperatures, one section retaining a sample fluid and the remaining one or more sections retaining a magnetic fluid ; an inlet/outlet valve connected to the micro-channel ; and a magnet disposed outside the micro-channel, forming a magnetic field to effect on the magnetic fluid.

In still another aspect of the present invention, there is provided a method for operating the above apparatus for circulating a carrier fluid, which comprises applying a power to the magnet to allow the magnetic fluid to move, thereby moving the carrier fluid toward an adjacent section.

In still another aspect of the present invention, there is provided an apparatus for use in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction, the apparatus comprising a micro-channel having three sections, one section retaining a sample fluid and the remaining sections retaining a magnetic fluid ; an inlet/outlet valve connected to the micro-channel ; and a magnet disposed outside the micro-channel, forming a magnetic field to effect on the magnetic fluid, wherein the three sections include a first section maintained at a temperature for denaturing, a second section maintained at a temperature for annealing, and a third section maintained at a temperature for extension.

In still another aspect of the present invention, there is provided an apparatus for use in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction, the apparatus comprising a micro-channel having two sections, one section retaining a sample fluid and the other section retaining a magnetic fluid ; an inlet/outlet valve connected to the micro-channel ; and a magnet disposed outside the micro-channel, forming a magnetic field to effect on the magnetic fluid, wherein one section is maintained at a temperature for denaturing and the other section is maintained at a temperature for both annealing and extension.

In still another aspect of the present invention, there is provided a chip comprising a substrate, one of the above apparatus for amplifying a nucleic acid disposed on the substrate and an electrophoresis means operatively-interconnected with the apparatus.

Brief Description of the Drawings FIG. 1 illustrates a conventional PCR system; FIG. 2 illustrates another form of a conventional PCR system;

FIG. 3 illustrates still another form of a conventional PCR system; FiGs. 4 and 5 illustrate a schematic view where a biochemical fluid, such as a PCR fluid, is circulated through two or more sections maintained at different temperatures for PCR; FIGs. 6 and 7 illustrate basic components of each chamber unit in a pneumatic air pressure type of PCR system; FIG. 8 schematically illustrates a principle of operation in an apparatus having one chamber; FiGs. 9 and 10 schematically illustrate a principle of operation in an apparatus having two or three chamber units interconnected, respectively; FIG. 11 illustrates a schematic view of an apparatus for circulating a carrier fluid having three chambers interconnected; FIG. 12 schematically illustrates a principle of operation in an apparatus for circular PCR; and FIG. 13 schematically illustrates a principle of operation for circulating a biochemical fluid, such as a PCR fluid, using a magnetic fluid in a magnetic fluid type of PCR system.

Best mode for carrying out the Invention The apparatus of the present invention includes two or more chambers maintained at different temperatures, through which a carrier fluid circulates.

That is, the apparatus for circulating a carrier fluid includes two or more chambers maintained at different temperatures, each chamber comprising an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber (inlet pneumatic air pressure port); and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber (outlet pneumatic air pressure port); wherein the chambers are sequentially connected such that the outlet valve of one chamber is connected to the inlet valve of an adjacent chamber in a direction the fluid flows.

A carrier fluid includes any fluid to be retained in a temperature-maintained zone for reaction for a predetermined time. The carrier fluid may include a biochemical fluid, such as a fluid for polymerase chain reaction comprising a template DNA, an oligonucleotide primer, dNTP [deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanidine triphosphate (dGTP), deoxythymidine triphosphate (dTTP)], and a thermostable DNA polymerase.

In an apparatus for circulating a carrier fluid of the present invention, the outlet valve of each chamber may be integrated with the inlet valve of a subsequent chamber.

Both the inlet valve and the outlet valve may be a passively operative valve. Further, the passively operative valve may be a valve where a channel of an outlet valve is formed to be narrower than that of an inlet valve or a valve where an inner surface of an outlet valve is treated with a hydrophobic material to control flow of a carrier fluid.

In an apparatus of the present invention, the carrier fluid is circulated by controlling a pressure applied to each chamber. The method for operating an apparatus for circulating a carrier fluid comprises applying a pressure to the inlet pneumatic air pressure port of a chamber and venting an outlet pneumatic air pressure port of an adjacent chamber in a fluid flow direction at the same time to allow the carrier fluid to move from the chamber to the adjacent chamber; controlling a pressure applied to the outlet pneumatic air pressure port of the adjacent chamber to retain the carrier fluid in the adjacent chamber for a predetermined time; and repeating the applying and controlling steps in turn to circulate the carrier fluid.

The carrier fluid may be introduced and discharged through the inlet and outlet pneumatic air pressure port of a chamber, respectively.

The present invention also includes, within its scope, an apparatus for amplifying a nucleic acid using a carrier fluid circulating apparatus. The amplifying apparatus is used in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction and may comprise three

chambers. Each chamber comprises an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber. The chambers are sequentially connected such that the outlet valve of one chamber is connected to the inlet valve of an adjacent chamber in a direction the fluid flows. The three chambers include a first chamber maintained at a temperature for denaturing, a second chamber maintained at a temperature for annealing, and a third chamber maintained at a temperature for extension.

Further, the amplifying apparatus is used in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction and may comprise two chambers. Each chamber comprises an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber. The outlet valve of one chamber is connected to the inlet valve of the other chamber. One chamber is maintained at a temperature for denaturing and the other chamber is maintained at a temperature for both annealing and extension.

An apparatus for amplifying a nucleic acid of the present invention may be a miniaturized circular PCR cycler, in which a biochemical fluid, such as a PCR fluid, circulates along two or three chambers maintained at different temperatures. For example, 1 cycle of DNA amplification may be completed by circulating a sample fluid along a first chamber (maintained at a temperature for denaturing, T1) a second chamber (maintained at a temperature for annealing, T2)-"a third chamber (maintained at a temperature for extension, T3) or by circulating a sample fluid along a first chamber (maintained at a temperature for denaturing, Tu a second chamber (maintained at a temperature for both annealing and extension, T2'). By running a plurality of cycles in the apparatus for PCR, the DNA amount in a sample is exponential amplified.

Alternatively, two or more sections maintained at different temperatures may be implemented in a micro-channel. That is, an apparatus for circulating a carrier fluid comprises a micro-channel having two or more sections maintained at different temperatures. One section retains a sample fluid and the remaining one or more sections retain a magnetic fluid. An inlet/outlet valve is connected to the micro-channel and a magnet is disposed outside the micro-channel, forming a magnetic field to effect on the magnetic fluid.

The magnet may be a magnet located in a center of the micro-channel or an electromagnet located along the micro-channel.

The magnetic fluid includes any fluid to be moved by a magnetic force of a simple magnet or an electromagnet. For example, the magnetic fluid may be a mixture of a ferromagnetic particle in aqueous medium (an aqueous-based ferrofluid), in oil (an oil-based ferrofluid), or in polymeric gel (a polymeric gel-based ferrofluid). Among them, an oil-based ferrofluid is preferred.

A power either magnetic or electric is applied to the magnet to cause a movement thereof. As the magnet moves, the magnetic fluid moves, which allows the carrier fluid to move toward an adjacent section.

Where the micro-channel includes three sections, there is provided an apparatus for amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction. The three sections include a first section maintained at a temperature for denaturing, a second section maintained at a temperature for annealing, and a third section maintained at a temperature for extension.

Where the micro-channel includes two sections, there is also provided an apparatus for amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction. One section is maintained at a temperature for denaturing and the other section is maintained at a temperature for both annealing and extension.

An apparatus for amplifying a nucleic acid of the present invention may be a miniaturized circular PCR cycler, in which a biochemical fluid, such as a PCR fluid, circulates along two or three sections maintained at different

temperatures of micro-channel. For example, 1 cycle of DNA amplification may be completed by circulating a carrier fluid along a first section (maintained at a temperature for denaturing, Tu a second section (maintained at a temperature for annealing, T2)-"a third section (maintained at a temperature for extension, T3) or by circulating a carrier fluid along a first section (maintained at a temperature for denaturing, T1) a second section (maintained at a temperature for both annealing and extension, T2'). By running a plurality of cycles in the apparatus for PCR, the DNA amount in a sample is exponential amplified.

The amplifying apparatus can be implemented in a chip. The chip comprises a substrate, an apparatus for amplifying a nucleic acid disposed on the substrate and an electrophoresis means operatively interconnected with the apparatus. And, the substrate may comprise a heating means deposited thereon. The heating means includes a thermoelectric device, an infrared light, or a pre-heated metal block.

For example, the amount of DNA in the sample introduced to the chip of the present invention is amplified. And then, the amplified DNA is supplied to an electrophoresis means to be isolated according to a molecular weight or a charge thereof and finally identified as a specific DNA.

The substrate of the chip may be selected from the group consisting of glass, quartz, silicon, plastic, ceramic, and metal. The electrophoresis means may be a multi-channel form for capillary electrophoresis. The apparatus for PCR amplification and the electrophoresis means may be embodied on a substrate using a photolithography technique.

The present invention is described in more detail referring to the attached drawings hereinafter.

As shown in FIGs. 4 and 5, a biochemical fluid, such as a PCR fluid, is circulated along two or more sections maintained at different temperatures for PCR. In FIGs. 4 and 5, the circle shows a channel to circulate a carrier fluid and T1, T2, and T3 show different temperature zones, respectively. The arrow shows a direction to circulate or introduce/discharge a carrier fluid.

According to the present invention, there is no need for a long channel and/or a complicated circuit for the accurate control of temperatures as required in conventional systems.

FIGs. 6 and 7 illustrate basic components of each chamber unit in a pneumatic air pressure type of PCR system. In FIGs. 6 and 7, a temperature-maintained chamber (or micro-chamber) (11) retains a carrier fluid for polymerase chain reaction for a predetermined time. The basic components include a chamber (11), an inlet valve (12) comprising a pneumatic air pressure port (13), an outlet valve (12') comprising a pneumatic air pressure port (13'). The chamber units may be interconnected to form an apparatus where the outlet valve of each chamber may be integrated with the inlet valve of a subsequent chamber. A flow of the carrier fluid is controlled by a passively operative valve, such as a valve where a channel of the outlet valve is formed to be narrower than that of the inlet valve, thereby giving an abrupt pressure drop effect, or a valve where an inner surface of the outlet valve is treated with a hydrophobic material to control flow of the carrier fluid.

Where a higher pressure is applied to the inlet pneumatic air pressure port (13) in the inlet valve (12) than the outlet valve (12'), the carrier fluid in the chamber (11) moves toward the outlet valve (12'). At that time, by lowering the air pressure applied to the outlet pneumatic air pressure port (13'), the air may be discharged.

Those basic components of each chamber unit make the carrier fluid flow in one direction by a pneumatic air pressure. Two or more chamber units may be interconnected to form an apparatus for circulating the carrier fluid by a pneumatic air pressure.

FIG. 8 schematically illustrates a principle of operation in an apparatus having one chamber. A carrier fluid in a chamber (11) moves to an outlet by an air pressure applied to the inlet pneumatic air pressure port (13). Where the air pressure applied to the inlet pneumatic air pressure port (13) is higher than the air pressure applied to outlet valve, the carrier fluid moves toward

outlet valve (22). A hydrophobic treatment or an abrupt pressure drop due to a narrower channel structure may passively operate the outlet valve.

FIG. 9 schematically illustrates a principle of operation in an apparatus having two chamber units interconnected. Applying an air pressure to an inlet pneumatic air pressure port (13) and venting an outlet pneumatic air pressure port (33) cause a pressure difference (P1 i-P3o). Where the air pressure (P1 i) of the inlet pneumatic air pressure port (13) is higher than the air pressure (P2) of a valve (22), the carrier fluid in a chamber (11) moves toward the adjacent chamber (21). Further, where the air pressure (P3) of a valve (32) is higher than the air pressure (P1 i), the carrier fluid is retained in a chamber (21) while air is easily discharged.

FIG. 10 schematically illustrates a principle of operation in an apparatus having three chamber units interconnected. This is operated in accordance with the same process as described referring to FIG. 9. Applying an air pressure successively to pneumatic air pressure ports (13,23, and 33) makes a carrier fluid successively move through the chambers (11,21, and 31).

FIG. 11 illustrates a schematic view of an apparatus for circulating a carrier fluid having three chambers interconnected. The principle of operation is the same as described referring to FIG. 10. That is, applying an air pressure successively to pneumatic air pressure ports makes a carrier fluid successively moved through the chamber (11) (Temp Zone 1), the chamber (21) (Temp Zone 2), and the chamber (31) (Temp Zone 3) according to the arrow direction.

FIG. 12 schematically illustrates a principle of operation in an apparatus for circular PCR. A carrier fluid is introduced, via a plug, to a chamber (11).

During the first cycle, the introduced carrier fluid is circulated through the chambers (denaturing chamber (11) < annealing chamber (21)- extension chamber (31) ) to be subject to polymerase chain reaction. In the same way, the second PCR cycle is made. The repetition of the cycle causes sufficient polymerase chain reactions as desired. After a predetermined number of

cycles, the carrier fluid is discharged through the plug to move to a channel or a chamber for analysis, such as electrophoresis.

FIG. 13 schematically illustrates a principle of operation for circulating a biochemical fluid, including a PCR fluid, using a magnetic fluid in a magnetic fluid type of PCR system. This apparatus uses a magnetic fluid, in place of pneumatic air pressure, for circulating a biochemical fluid. A biochemical fluid (1) is circulated along the sections maintained at different temperatures (T1, T2, T3), by moving a magnetic fluid (2) along the micro-channel, which is successively operated by a magnet located in the center of the micro-channel or an electromagnet located along the micro-channel.

Further understanding of the nature and advantages of the present invention herein may be realized by reference to the following Examples. The following Examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention.

Example 1. Pneumatic air pressure type of PCR system having two chamber units The apparatus, for use in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction, had two chambers. Each chamber had an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber. The outlet valve of one chamber was integrated with the inlet valve of the other chamber. One chamber was maintained at about 94°C for denaturing, the other chamber was maintained at about 68 °C for both annealing and extension. The amount of a nucleic acid present in a sample was amplified by polymerase chain reaction.

Example 2. Pneumatic air pressure type of PCR system having three chamber units

The apparatus, for use in amplifying an amount of a nucleic acid present in a sample using a polymerase chain reaction, had three chambers. Each chamber had an inlet valve containing a pneumatic air pressure port for controlling inflow of the carrier fluid to the chamber and an outlet valve containing a pneumatic air pressure for controlling outflow of the carrier fluid from the chamber. The chambers were sequentially connected such that the outlet valve of one chamber was integrated with the inlet valve of an adjacent chamber in a direction the fluid flows. The three chambers included a first chamber maintained at 94°C for denaturing, a second chamber maintained at about 55°C for annealing, and a third chamber maintained at about 72°C for extension. The amount of a nucleic acid present in a sample was amplified by polymerase chain reaction.

Example 3. Magnetic fluid type of PCR system having a micro-channel with two sections The apparatus for use in amplifying an amount of a nucleic acid in a sample, using a polymerase chain reaction, had a micro-channel having two sections. One section retained a sample fluid and the other section retained a magnetic fluid. An inlet/outlet valve was connected to the micro-channel and a magnetic stirrer was located in the center of the micro-channel. One section was maintained at about 94°C for denaturing and the other section was maintained at about 68°C for both annealing and extension. The amount of a nucleic acid present in a sample was amplified by polymerase chain reaction.

Example 4. Magnetic fluid type of PCR system having a micro-channel with three sections The apparatus for use in amplifying an amount of a nucleic acid in a sample, using a polymerase chain reaction, had a micro-channel having three

sections. One section retained a sample fluid and the remaining two sections retained a magnetic fluid. An inlet/outlet valve was connected to the micro-channel and a magnetic stirrer was located in the center of the micro-channel. The three sections included a first section maintained at about 94°C for denaturing, a second section maintained at about 55°C for annealing, and a third section maintained at about 72°C for extension. The amount of a nucleic acid present in a sample was amplified by polymerase chain reaction.

The apparatus and method for circulating a carrier fluid according to the present invention have following advantages.

In a conventional PCR cycler, heating (usually 1-2 seconds) and cooling (usually 3-4 seconds) are required. In the present invention, temperature preset chambers are used and a sample fluid goes through a series of such chambers. Thus, a predetermined time is taken for the sample fluid to move from one chamber to another chamber. The moving time depends on a pneumatic air pressure or a magnetic force and is less than 1 second. Thus, run time of one cycle is greatly reduced compared with a conventional PCR cycler.

Further, a carrier fluid moves along temperature-maintained chambers or sections, which makes it possible to control PCR conditions according to characteristics of a biochemical fluid by varying a residence time of the carrier fluid in each of the chambers or sections.

And, there is no need for a complicated circuit. In a conventional PCR cycler, complicated circuits, such as PID (proportional/integral/differential), are needed for an accurate control of temperatures. Further, a high voltage for a rapid heating causes an overshoot effect increasing a temperature of a chamber, e. g. , by about 1-2°C.

There is no need for a cooling system. In a conventional PCR cycler, a cooling fan or a thermoelectric apparatus is required for rapid cooling.

However, in the present invention, there is no need for any circuits for cooling or cooling system.

There is no need for an extraordinarily long channel as in a continuous-flow PCR cycler. Therefore, it is possible to manufacture portable system as well as to reduce the size of the entire system of the present invention.

The present invention may be embodied on a microchip, such as lab-on-a-chip, which makes it possible to use a photolithography technique with silicon, glass, or plastic, etc.

The present invention may be embodied on a microchip, which makes it possible to use a small amount (mL-pL) of a biochemical fluid, such as a PCR fluid.