FIELD OF THE INVENTION
The present invention relates to an improvement in the field conventional PCR system. More particularly, the present invention provides a Micro Thermal Cycler chip for DNA amplification, a method for fabrication of such chip, a device comprising said chip and a method for amplification of DNA followed by detection and quantification of the same.
BACKGROUND AND PRIOR ART OF THE INVENTION
Miniaturization and automation have revolutionized the world of microelectronics. Whereas computers once were room-sized, steady technological advance has led to laptops, palmtops, and even game consoles that are much more powerful than were those behemoths of the 1950s and 1 960s. But until recently few of these cutting-edge engineering technologies have been applied to the needs of the medical diagnostics laboratory. Consequently, much laboratory work long remained-and some still is inefficient, laborious, and time-consuming.
Over the past five years, however, research and development for clinical diagnostic systems based on lab-on-a-chip technologies have increased tremendously. To date, more than 2000 research papers on labs-on-a-chip, sometimes also called micro total-analysis systems (fiT AS), have been published. Such systems hold great promise for clinical diagnostics. They consume sample material and reagents only in extremely low volumes. Individual small chips can be inexpensive and disposable. Time from sampling to result tends to be very short. The most advanced chip designs can perform all analytical functions-sampling, sample pretreatment; separation, dilution, and mixing steps; chemical reactions; and detection-in a single integrated micro fluidic circuit. Lab- on-a chip systems allow designers to create small, portable, rugged, low-cost, and easy-to-use diagnostic instruments that offer high levels of capability and versatility. Microfluidics-fluids flowing in micro channel makes possible the design of analytical devices and assay formats that would not function on a larger scale.
Lab-on-a-chip technologies attempt to emulate the laboratory procedures that would be performed on a sample within a Microfabricated structure. The most successful devices have been those that operate on fluid samples. A large number of chemical processing, purification, and reaction procedures have been demonstrated on these devices. Some degree of monolithic integration of chemical processes has been demonstrated to produce devices that perform a complete chemical measurement procedure. These devices are based upon accepted laboratory procedures of analysis and thus are able to accommodate more complex sample matrices than conventional chemical sensing.
Recent advances in molecular and cell biology have been produced in great part as a result of the development of rapid and efficient analytical techniques. Due to miniaturization and multiplexing, techniques like gene chip or biochip enable the characterization of complete genomes in a single experimental setup. PCR (Polymerase chain reaction) is a molecular biology method for the in-vivo amplification of nuclear acid molecules. The PCR technique is rapidly replacing other time consuming and less sensitive techniques for identification of biological species and pathogens in forensic, environmental, clinical and industrial samples. Among the biotechniques, PCR has become the most important analytical step in life sciences laboratories for a large number of molecular and clinical diagnostics. Important developments made in PCR technology like real-time PCR, have led to rapid reaction processes compared to conventional methods. During the past several years, micro fabrication technology has been expanded to the miniaturization of the reaction and analysis system such as PCR analysis with the intention of further reducing analysis time and consumption of reagents. Several research groups have been working on the 'lab-on-a-chip' devices and have led to number of advances in the fields of miniaturized separation and reaction systems.
Microchip Fabrication and Deployment
Lab-on-a-chip microchips consist of a Microfabricated channel network that includes fluidic elements to allow execution of the basic chemical analysis process necessary to an analytic procedure. The microchips are fabricated using methods similar to those used to make printed circuit boards. Currently, as many as 40 chips are being fabricated on a single wafer of glass at a cost of a few dollars each. Once commercial applications start, chips will likely be made from plastics and cost pennies each to fabricate. The cost of a ready-to-use commercial microchip will run from $1, to under $100. Different chips, designed to accomplish different measurement goals, will all fit into a common controller device. This approach allows new and improved analysis procedures on advanced microchip designs to be deployed on existing hardware. Moreover, new microchips designed to detect new targets or suites of targets can also be utilized in existing hardware deployments.
Lab-on-a-chip technologies can be utilized in two distinct detection and monitoring scenarios: in-situ remote monitoring and field applications. In-situ measurements are accomplished by placing the microchip in contact with the fluid stream to be sampled. Liquid is drawn onto the analysis chip by electrokinetic forces and analyzed according to the fluidic circuit design included on the chip. Field monitoring is an application where a sample is collected manually and placed onto a chip for analysis in the field environment. Results are generally available in a I-minute time scale.
Polymerase Chain Reaction Instruments
The original PCR process is based on heat stable DNA polymerase enzyme from Thermus aquaticus (Taq), which can synthesize a complimentary strand to a given DNA strand in a mixture containing four DNA bases and two primer DNA fragments flanking the target sequence. The mixture is heated to separate the strands of double-helix DNA containing the target sequence and then cooled to allow the primers to find and bind to their complimentary sequences on the separate strands and the Taq polymerase to extend the primers into new complimentary strands. Repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two templates for further synthesis.
A typical temperature profile for the polymerase chain reaction is as follows:
1. Denature at 93°C for 15 to 30 seconds
2. Anneal Primer at 55°C for 15 to 30 seconds
3. Extend primers at 72°C for 30 to 60 seconds
The primer extension step has to be increased by roughly 60sec/kbase to generate products longer than a few hundred bases. The above are typical instrument times; in fact, the denaturing and annealing steps occur almost instantly, but the temperature rates in commercial instruments usually are less than 1°C /sec when metal blocks or water are used for thermal equilibration and samples are contained in plastic micro centrifuge tubes. Instantaneous temperature changes are not possible because of sample, container, and cycler heat capacities, and extended amplification times of 2 to 6 hours result. During the periods when sample temperature is making a transition from one temperature to another, extraneous, undesirable reactions occur that consume important reagents and create unwanted interfering compounds.
By micromachining thermally isolated, low mass PCR chambers; it is possible to mass produce a much faster, more energy efficient and a more specific PCR instrument. Moreover, rapid transitions from one temperature to another ensure that the sample spends a minimum amount of time at undesirable intermediate temperatures do that the amplified DNA has optimum fidelity and purity.
OBJECTIVES OF THE PRESENT INVENTION
The principle object of the present invention is to improve the conventional PCR systems in time taken, portability, sample volume and the ability to perform through put analysis and quantitation.
Another object of the present invention is to provide a micro thermal cycler chip for DNA amplification.
Yet another object of the present invention is to provide a method for the fabrication of micro thermal cycler chip for DNA amplification.
Still another object of the present invention is to provide a device having micro thermal cycler chip for DNA amplification, detection and quantification.
Still another object of the present invention is to provide a method for DNA amplification, detection and quantification using the device and micro thermal cycler chip developed in the instant invention.
STATEMENT OF THE INVENTION
Accordingly, the present invention provides a micro thermal cycler chip for DNA amplification, said chip comprising: micro channels (7a, 7b) for flow of sample from input port (4a) to output port ( 4b) via reaction chamber (6); check valves (3a, 3b) to control flow of the sample through the micro channels (7a, 7b) into the reaction chamber (6); the reaction chamber (6) for DNA amplification is lined with heater elements (5) for heating the sample in the reaction chamber (6); a thermal pathway (13) for heat dissipation from the reaction chamber (6) to heat sink (14); and voltage sources (19a, 19b) to control the check valves (3a, 3b) wherein said sources are maintained by control units (20a, 20b); a method for fabrication of micro thermal cycler chip for DNA amplification said method comprising steps of anchoring fixed comb elements (15) of check valve (3a, 3b) in a frame; providing reaction chamber (6) with micro channels (7a, 7b) and connecting it to input and output ports (4a. 4b); positioning micro heaters (5) along periphery of the reaction chamber (6); placing thermal pathway (13) and heat sink (14) in serial to the reaction chamber (6); providing DC voltage sources (19a, 19b) and control units (20a, 20b) to actuate and deactuate check valves (3a, 3b); and covering the chip with a transparent cover (11) to obtain the fabricated chip; a device for DNA amplification, detection and quantification, said device comprising micro thermal cycler chip; Electrophoresis system; data acquisition processing circuit and output on portable system for detection and quantification of amplified DNA; and a method for DNA amplification, detection and quantification using a device comprising micro thermal cycler chip; Capillary Electrophoresis system; required electronics, data acquisition processing and output on portable system, said method comprising steps of: loading DNA sample mixture via inlet port (4a); actuating check valve (3a) to allow the loaded DNA sample mixture to flow into reaction chamber (6) through micro channel (7a); thermal cycling of the loaded DNA sample mixture in the reaction chamber (6) to denature DNA sample mixture; annealing and extension of the denatured DNA sample mixture; and actuating check valve (3b) to allow unloading of processed DNA sample from the reaction chamber (6) through outlet port (4b) followed by Electrophoresis for detection and quantification.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1: shows the exploded view of the micro thermal cycler according to this
invention.
Figure 2: shows the, system schematic of the micro thermal cycler Chip according to the invention
Figure 3: shows the assembled view of the micro thermal cycler according to this invention.
Figure 4: shows the top view of micro thermal cycler according to this invention. Figure 5: shows the check valve with comb elements and spring elements Figure 6: shows the Operation Schematic of Micro Thermal Cycler Chip
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The present invention is in relation to a micro thermal cycler chip for DNA amplification, said chip comprising:
a) micro channels (7a, 7b) for flow of sample from input port (4a) to output port ( 4b) via reaction chamber (6);
b) check valves (3a, 3b) to control flow of the sample through the micro channels (7a, 7b) into the reaction chamber (6);
c) the reaction chamber (6) for DNA amplification is lined with heater elements (5) for heating the sample in the reaction chamber (6);
d) a thermal pathway (13) for heat dissipation from the reaction chamber (6) to heat sink (14); and
e) voltage sources (19a, 19b) to control the check valves (3a, 3b) wherein said sources are maintained by control units (20a, 20b).
In another embodiment of the present invention, said sample is a DNA sample mixture which is pumped using an external system or a pump which is integrated within the system.
In yet another embodiment of the present invention, the check valves (3a, 3b) consists of a set of fixed comb elements (15) anchored to the substrate copper and a set of movable comb elements (16) coupled with spring element (17) for guiding the valve arm (18) in and out of the micro channels (7a, 7b).
In still another embodiment of the present invention, said heater element (5) shape is selected from a group comprising zigzag, pi and circular and is made up of polysilicon.
In still another embodiment of the present invention, said thermal pathway (13) is made up of a metal copper and the heat sink (14) is made up of ceramic material.
In still another embodiment of the present invention, said chip is covered with a transparent cover (11) made up of poly dimethylsiloxane or polymethylmethacrylate.
The present invention is in relation to a method for fabrication of micro thermal cycler chip for DNA amplification said method comprising steps of anchoring fixed comb elements (15) of check valve (3a, 3b) in a frame; providing reaction chamber (6) with micro channels (7a, 7b) and connecting it to input and output ports (4a, 4b); positioning micro heaters (5) along periphery of the reaction chamber (6); placing thermal pathway (13) and heat sink (14) in serial to the reaction chamber (6); providing DC voltage sources (19a, 19b) and control units (20a, 20b) to actuate and deactuate check valves (3a, 3b); and covering the chip with a transparent cover (11) to obtain the fabricated chip.
In yet another embodiment of the present invention, the check vaiyes_ (3a, 3b) consists of a set of movable comb elements (16) coupled with spring element (17) for guiding the valve arm (18) in and out of the micro channel (7a, 7b). In still another embodiment of the present invention, the heater element (5) shape is selected from a group comprising zigzag, pi and circular and is made up of polysilicon.
In still another embodiment of the present invention, said transparent cover (1 1) is made up of poly dimethylsiloxane or polymethylmethacrylate.
In still another embodiment of the present invention, said thermal pathway (13) is made up of a metal copper and the heat sink (14) is made up of ceramic material.
In still another embodiment of the present invention, said frame is a copper frame having good thermal conductivity.
In still another embodiment of the present invention, said micro heaters (5) are two symmetric micro heaters (5).
The present invention is in relation to a device for DNA amplification, detection and quantification, said device comprising micro thermal cycler chip; Electrophoresis system; data acquisition processing circuit and output on portable system for detection and quantification of amplified DNA.
In still another embodiment of the present invention, said micro thermal cycler chip comprising:
a) micro channels (7a, 7b) for flow of sample from input port (4a) to output port ( 4b) via reaction chamber (6);
b) check valves (3a, 3b) to control flow of the sample through the micro channels (7a, 7b) into the reaction chamber (6);
c) the reaction chamber (6) for DNA amplification is lined with heater elements (5) for heating the sample in the reaction chamber (6);
d) a thermal pathway (13) for heat dissipation from the reaction chamber (6) to heat sink (14); and
e) voltage sources (19a, 19b) to control the check valves (3a, 3b) wherein said sources are maintained by control units (20a, 20b).
In still another embodiment of the present invention, the check valves (3a, 3b) consists of a set of fixed comb elements (15) anchored to the substrate copper and a set of movable comb elements (16) coupled with spring element (17) for guiding the valve arm (18) in and out of the micro channels (7a, 7b).
In still another embodiment of the present invention, said Electrophoresis system is ^capillary electrophoresis system.
The present invention is in relation to a method for DNA amplification, detection and quantification using a device comprising micro thermal cycler chip; Capillary
Electrophoresis system; required electronics, data acquisition processing and output on portable system, said method comprising steps of:
a) loading DNA sample mixture via inlet port (4a);
b) actuating check valve (3a) to allow the loaded DNA sample mixture to flow into reaction chamber (6) through micro channel (7a);
c) thermal cycling of the loaded DNA sample mixture in the reaction chamber (6) to denature DNA sample mixture;
d) annealing and extension of the denatured DNA sample mixture; and
e) actuating check valve (3b) to allow unloading of processed DNA sample from the reaction chamber (6) through outlet port (4b) followed by Electrophoresis for detection and quantification.
In still another embodiment of the present invention, said denaturation is carried out at a temperature ranging from 94° - 95° C.
In still another embodiment of the present invention, said extension is carried out at a temperature of about 72° C.
To achieve the said objective this invention provides micro-thermal cycler for DNA amplifications, which comprises DNA sample loading and unloading ports, a reaction chamber containing the appropriate primers and dNTP connected to said ports using micro-fluidic channels, said reaction chamber is provided with heaters and temperature sensors for DNA sample heating and accurate thermal cycling for DNA amplification, check valves provided in said micro-fluidic channels for controlling flow of said sample into said chamber and reaction products out of said chamber, a thermal pathway placed below and around the said reaction chamber for conducting heat from said reaction chamber, a heat sink placed below said thermal pathway covering the entire thermal pathway to facilitate fast cooling of said sample
The said check valves are active valves comprising of moveable and fixed comb elements coupled with spring elements which open or close the valve depending on the applied actuation voltage to the comb elements and the spring elements to facilitate fast opening and closing of the valve and prevent leakage of DNA sample and reaction product from said valve.
The heaters are pi-shaped, zig-zag shape or circular heating elements made of polysilicon. The temperature sensors are thermistors. The thermal pathway is made of metals, which have high thermal conductivity. The heat sink is made of ceramic material. The polymer cover is placed over said micro-thermal cycler. The polymer cover is made of polydimethyl siloxane (PDMS) or polymethylmethacrylate (PMMA).
Array of micro-thermal cycler for DNA amplification each running on its own thermal protocol independently comprising of a series of micro-thermal cycler for DNA amplification.
Referring to Figure 1 which shows the exploded view of the micro thermal cycler wherein A shows the complete thermo cycler, B shows the BDMS cover, C shows silicon reaction chamber assembly, D shows poly silicon micro heater, E shows copper thermal pathway and F shows ceramic heat sink.
Referring to figure 2 which shows the stem schematic of the micro thermal cycler chip wherein 3a & 3b shows the two active check valves provided for the control of sample flow in and out of the reaction chamber( 6) through the input and output micro channels (7a & 7b) respectively. The two check valves (3a & 3b) are actively controlled by the actuation voltage applied through the DC voltage sources (19a & 19b) maintained by control units (20a & 20b). The reaction chamber (6) is lined with micro heater element (5) which is used for the heating of the sample in the said chamber. The reaction chamber (6) is fixed atop a thermal pathway (13) for effective heat dissipation into the heat sink (14). These parts are assembled and the assembly of the micro thermal cycler is shown in Figure 3.
In figure 3, item 9 shows the inlet pipe and item 10 shows the outlet pipe. Item 11 shows the PDMS transparent covcr for PCR functional components. Item 12 shows the PCR functional components and item 13 copper conductors between the PCR functional components (12) and heat sink (14).
Referring to figure 4, which shows the top view of the micro thermal cycler, item 1 shows the thermal frame and item 2 shows the thermal isolation between the thermal frame and micro heater (5). Check valves (3a, 3b) is provided between the micro heater (5) and the thermal frame (1). The reaction chamber (6) is provided inside the micro heater (5). The fluid port input (4a) and output port (4b) are provided in the thermal frame (1).
Micro thermal cycler for DNA amplifications consists of DNA fluid sample input and output ports (4), a reaction chamber (6) containing the appropriate primers and dNTP connected to said ports using micro fluidic channels (7a, 7b). Said reaction chamber (6) is provided with heaters (5) for DNA sample heating and accurate thermal cycling for DNA amplifications.
Check valves (3a, 3b) provided on said micro fluidic channel (7a, 7b) for controlling flow of said sample into said chamber (6) and reaction products out of said chamber (6). A thermal pathway (13) placed below and around said reaction chamber (6) for conducting heat from the reaction chamber (6). A heat sink (14) is placed below said thermal pathway covering the entire thermal pathway to facilitate fast cooling of said sample.
The said check valves (3a, 3b) are active valves consisting of movable comb elements (16) and fixed comb elements (15), (16) coupled with spring element (17) connected to the valve arm (18) which open or close depending on the actuation voltage to the comb elements (15& 16) and the spring elements (17) to facilitate fast opening and closing of the valve and prevent leakage DNA samples and reaction product from said check valve (3a, 3b) see figure 5.
The said heaters (5) are pi-shaped, zigzag shaped or circular elements made of poly silicon. The thermal pathway (13) is made of metals such as copper, which has high thermal conductivity. The heat sink (14) is made of ceramic material. The PDMS transparent cover (11) is made of poly dimethylsiloxale PDMS or polymethylmethacrylate (PMMA) and is placed over said micro thermal cycler.
WORKING
The operation schematic of the Micro thermal cycler is shown in a self- explanatory illustration below in four steps I, II, III, IV. Sample loading followed by thermal cycling and sample unloading are the basic steps performed by the chip with an external or onchip control. The fluid is assumed to be under pumping pressure provided by an external system level pump, which also can be integrated within the system.
The flow of the sample into the reaction chamber where the thermal cycling takes place is controlled by the said active check valves (3a & 3b). The check valve consists of a set of fixed combs anchored to the substrate below and a set of movable combs for guiding the valve arm in and out of the micro channel to check or release flow. An electrostatic mechanism is used to actuate these valves. A difference in potential applied to the two sets of movable and the fixed combs causes a lateral force on the movable assembly towards the fixed assembly causing back and forth motion depending on whether the actuation voltage is applied or not.
In Step I, both the check valves are shown to be in the closed mode wherein there is no actuation voltage applied to both the valves. In Step II the check valve (3a) is shown in the actuated mode allowing the sample to flow into the reaction chamber (6).The reactioxLchamber is designed as a thermal island to achieve good thermal isolation. Two symmetric micro heaters are placed along the periphery of the chamber for efficient heating with high temperature uniformity within the chamber. The heat transfer paths are kept to a minimum but retained just enough for fast cooling and uniform heat retention in the entire chamber. The chamber is provided with inlet and outlet channels for sample loading and unloading and the entire assembly is placed on a frame of copper that acts as a thermal conduction path to the heat sink frame.
Step III shows the two check valves again in the closed or unactuated mode. This is when the thermal cycling of the sample takes place in the reaction chamber lined with Polysilicon micro heaters which heat up using the principle ofjoule_ heating for DNA sample heating and accurate thermal cycling for DNA amplifications.
The sample undergoes repetitive thermal cycling in the said reaction chamber. Each cycle has three steps:
1. Melting: The PCR mixture is heated to a high temperature (typically 94-95°) to denature DNA-DNA, DNA-primer, and primer-primer complexes.
2. Annealing: The mixture is cooled to a temperature that allows for the annealing of primers to target DNA sequences. If the temperature is too high, no annealing occurs and no product is formed. Low annealing temperatures promote non-specific priming and result in a "smear" of DNA on agarose gels.
3. Extension: The mixture is warmed to 72° to permit optimal dNTP incorporation by the polymerase. Longer annealing times are required for less processive polymerases
Step IV shows the check valve (3b) in the actuated mode and valve (3a) in the closed mode allowing the processed sample out of the reaction chamber (6) into the outlet port.
ADVANTAGES Time:
By reducing thermal delay, and eliminating the wait until reaching the balanced uniform temperature in the sample, we would reduce the time taken from 2 to 3 hours to finish a 30 to 40-cycle reaction, even for a moderate sample volume of 5-25 JLI 1, to a few seconds.
By micro machining low thermal mass PCR chambers we will be able to mass- produce a faster, more energy efficient, and most importantly a more specific PCR instrument. Moreover rapid transitions from one temperature to another ensure that the sample spends a minimum of time at undesirable intermediate temperatures so that the amplified DNA has optimum fidelity and purity.
Portability and sample size
Advances in micro fabrication have allowed the miniaturization and integration of a myriad of systems making them more powerful and less expensive, as well as smaller. Historically, electronics were miniaturized first, producing complicated integrated circuits such as modem microprocessors. More recently the benefits of miniaturization have been extended to mechanical systems. Tiny,
inexpensive, resonating devices have been produced and integrated with electronics for a variety of purposes such as inertial and chemical sensors; silicon membranes have been used for pressure sensors; and micro mirrors have been used for a number of optical applications such as beam manipulation and optical switching.
The benefits of miniaturization and integration extend to the world of fluidics as well. For most applications, the sample size required for a conventional chemical sensing system is much larger than is required to obtain statistically significant results. Miniaturization would allow accurate readings with smaller sample sizes and consumption of smaller volumes of costly reagents. The small thermal masses of Microsystems and the small sample sizes allows rapid low -power thermal cycling increasing the speed of many processes such as DNA replication through micro PCR. In addition, chemical processes that depend on surface chemistry are greatly enhanced by the increased surface to volume ratios available on the micro-scale. The advantages of micro fluidics have prompted calls for the development of integrated micro systems for chemical analysis.
The MEMS advantage would translate into a device that would be handheld, thereby removing the PCR machine from a sophisticated laboratory, thus increasing the reach of this extremely powerful technique, be it for clinical diagnostics, food testing, blood screening at blood banks or a host of other application areas.
High throughput:
. C
Existing micro thermal cyclers with multiple reaction chambers provide multiple ^ \ c '
- - - vAts'
DNA experiment sites all running the same thermal protocol and hence are not
time efficient. The need arises, to minimize reaction time and intake sample
volume.
Our device would be so designed that in future, we could have an array of devices with very quick thermal response and highly isolated from the adjacent thermal cyclers to be able to effectively and independently run multiple reactions with different thermal protocols with minimum cross talk.
Quantification
The analysis or quantification of the PCR products can be realized by practical integration of a micro fluidic capillary electrophoresis chip. This system could also be integrated with quantification and sensing systems to detect diseases like AIDS, tuberculosis, etc. Our device would consist of two parts: the thermal cycler and the other a Capillary Electrophoresis system, with complete micro fluidics, pumps and valves to enable detection and quantification

We claitn:
1) A micro thermal cycler chip for DNA amplification, said chip comprising:
a} micro channels (7a, 7b) for flow of sample from input port (4a) to output port ( 4b) via reaction chamber (6);
b) ' check valves (3a, 3b) to control flow of the sample through the micro channels (7a, 7b) into the reaction chamber (6);
c) the reaction chamber (6) for DNA amplification is lined with heater elements (5) for heating the sample in the reaction chamber (6);
d) a thermal pathway (13) for heat dissipation from the reaction chamber (6) to heat sink (14); and
e) voltage sources (19a, 19b) to control the check valves (3a, 3b) wherein said sources are maintained by control units (20a, 20b).
2) The chip as claimed in claim 1, wherein said sample is a DNA sample mixture which is pumped using &n external system or a pump which is integrated within the system. 1
3) The chip as claimed in claim 1, wherein the check valves (3a, 3b) consists of a set of fixed comb elements (15) anchored to the substrate copper and a set of movable comb elements (16) coupled with spring element (17) for guiding the valve arm (18) in and out of the micro channels (7a, 7b).
4) The chip as claimed in claim 1, wherein said heater element (5) shape is selected from a group comprising zigzag, pi and circular and is made up of polysilicon.
5) The chip as claimed in claim 1, wherein said thermal pathway (13) is made up of a metal copper and the heat sink (14) is made up of ceramic material.
6) The chip as claimed in claim 1, wherein said chip is covered with a transparent covcr (11) made up of poly dimethylsiloxane or polymethylmethacrylate.
7) A method for fabrication of micro thermal cycler chip for DNA amplification said method comprising steps of anchoring fixed comb elements (15) of check valve (3a, 3b) in a frame; providing reaction chamber (6) with micro channels (7a, 7b) and connecting it to input and output ports (4a, 4b); positioning micro heaters (5) along periphery of the reaction chamber (6); placing thermal pathway (13) and heat sink (14) in serial to the reaction chamber (6); providing DC voltage sources (19a, 19b) and control units (20a, 20b) to
actuate and deactuate check valves (3a, 3b); and covering the chip with a transparent cover (11) to obtain the fabricated chip.
8) The method as claimed in claim 7, wherein the check valves (3a, 3b) consists of a set of movable comb elements (16) coupled with spring element (17) for guiding the valve arm (18) in and out of the micro channel (7a, 7b).
9) The method as claimed in claim 7, wherein the heater element (5) shape is selected from a group comprising zigzag, pi and circular and is made up of polysilicon.
10) The method as claimed in claim 7, wherein said transparent cover (11) is made up of poly dimethylsiloxane or polymethylmethacrylate.
1 l)The method as claimed in claim 7, wherein said thermal pathway (13) is made up of a metal copper and the heat sink (14) is made up of ceramic material.
12) The method as claimed in claim 7, wherein said frame is a copper frame having good thermal conductivity.
13) The method as claimed in claim 7, wherein said micro heaters (5) are two symmetric micro heaters"(^5). _
14) A device for DNW amplification, deletion and quantification, said device comprising micro thermal cycler chip; Electrophoresis system; data acquisition processing circuit and output on portable system for detection and quantification of amplified DNA./ f'"
15) The device as claimed kj eldim 14, wherein said micro thermal cycler chip comprising: - ^
a) micro channels (7a, 7b) for flow of sample from input port (4a) to output port ( 4b) via reaction chamber (6);
b) check valves (3a, 3b) to control flow of the sample through the micro channels (7a, 7b) into the reaction chamber (6);
c) the reaction chamber (6) for DNA amplification is lined with heater elements (5) for heating the sample in the reaction chamber (6);
d) a thermal pathway (13) for heat dissipation from the reaction chamber (6) to heat sink f 14); and
e) voltage sources (19a, 19b) to control the check valves (3a, 3b) wherein said sources are fnaintained by control units (20a, 20b).
16) The device as claimed in claim 15, wherein the check valves (3a, 3b) consists of a set of fixed comb elements (15) anchored to the substrate copper and a set of movable comb elements (16) coupled with spring element (17) for guiding
, the valve arm (18) in and out of the micro channels (7a, 7b).
17) The device as claimed in claim 14, wherein said Electrophoresis system is a capillary electrophoresis system.
18) A method for DNA amplification, detection and quantification using a device comprising micro thermal cycler chip; Capillary Electrophoresis system; required electronics, data acquisition processing and output on portable system, said method comprising steps of:
a) loading DNA sample mixture via inlet port (4a);
b) actuating check valve (3a) to allow the loaded DNA sample mixture to flow into reaction chamber (6) through micro channel (7a);
c) thermal cycling of the loaded DNA sample mixture in the reaction chamber (6) to denature DNA sample mixture;
d) annealing and extension of the denatured DNA sample mixture; and
e) actuating check valve (3b) to allow unloading of processed DNA sample from the reaction chamber (6) through outlet port (4b) followed by Electrophoresis for detection and quantification.
19) The method as claimed in claim 18, wherein said denaturation is carried out at a temperature ranging from 94° - 95° C.
20) The method as claimed in claim 18, wherein said extension is carried out at a temperature of about 72° C.
21) A micro thermal cycler chip, a method of fabricating a chip, a device and a method for DNA amplification, detection and quantification are substantially as herein described along with accompanying drawings and examples.