Silicon-based sleeve devices for chemical reactions

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BACKGROUND OF THE INVENTION

The present invention relates to instruments for chemical reaction control and detection of participating reactants and resultant products, particularly to integrated microfabricated instruments for performing microscale chemical reactions involving precise control of parameters of the reactions, and more particularly to silicon-based sleeve devices as reaction chambers for chemical reactions and which can be utilized inlarge arrays of individual chambers for a high-throughput microreaction unit.

Current instruments for performing chemical synthesis through thermal control and cycling are generally very large (table-top) and inefficient, and often they work by heating and cooling of a large thermal mass (e.g., an aluminum block). In recent years efforts have been directed to miniaturization of these instruments by designing and constructing reaction chambers out of silicon and silicon-based materials (e.g., silicon, nitride, polycrystalline silicon) that have integrated heaters and cooling via convection through the silicon.

Microfabrication technologies are now well known and include sputtering, electrodeposition, low-pressure vapor deposition, photolithography, and etching. Microfabricated devices are usually formed on crystalline substrates, such as silicon and gallium arsenide, but may be formed on non-crystalline materials, such as glass or certain polymers. The shapes of crystalline devices can be precisely controlled since etched surfaces are generally crystal planes, and crystalline materials may be bonded by processes such as fusion at elevated temperatures, anodic bonding, or field-assisted methods.

Monolithic microfabrication technology now enables the production of electrical, mechanical, electromechanical, optical, chemical and thermal devices, including pumps, valves, heaters, mixers, and detectors for microliter to nanoliter quantities of gases, liquids, and solids. Also, optical waveguide probes and ultrasonic flexural-wave sensors can now be produced on a microscale. The integration of these microfabricated devices into a single systems allows for the batch production of microscale reactor-based analytical instruments. Such integrated microinstruments may be applied to biochemical, inorganic, or organic chemical reactions to perform biomedical and environmental diagnostics, as well as biotechnological processing and detection.

The operation of such integrated microinstruments is easily automated, and since the analysis can be performed in situ, contamination is very low. Because of the inherently small sizes of such devices, the heating and cooling can be extremely rapid. These devices have very low power requirement and can be powered by batteries or by electromagnetic, capacitive, inductive or optical coupling.

The small volumes and high surface-area to volume ratios of microfabricated reaction instruments provide a high level of control of the parameters of a reaction. Heaters may produce temperature cycling or ramping; while sonochemical and sonophysical changes in conformational structures may be produced by ultrasound transducers; and polymerizations may be generated by incident optical radiation.

Synthesis reactions, and especially synthesis chain reactions such as the polymerase chain reaction (PCR), are particularly well-suited for microfabrication reaction instruments. PCR can selectively amplify a single molecule of DNA (or RNA) of an organism by a factor of 10.sup.6 to 10.sup.9. This well-established procedure requires the repetition of heating (denaturing) and cooling (annealing) cycles in the presence of an original DNA target molecule, specific DNA primers, deoxynucleotide triphosphates, and DNA polymerase enzymes and cofactors. Each cycle produces a doubling of the target DNA sequence, leading to an exponential accumulation of the target sequence.

The PCR procedure involves: 1) processing of the sample to release target DNA molecules into a crude extract; 2) addition of an aqueous solution containing enzymes, buffers deoxyribonucleotide triphosphates (dNTPS), and aligonucleotide primers; 3) thermal cycling of the reaction mixture between two or three temperatures (e.g., 90.degree.-96.degree., 72.degree., and 37.degree.-55.degree. C.); and 4) detection of amplified DNA. Intermediate steps, such as purification of the reaction products and the incorporation of surface-bending primers, for example, may be incorporated in the PCR procedure.

A problem with standard PCR laboratory techniques is that the PCR reactions may be contaminated or inhibited by the introduction of a single contaminant molecule of extraneous DNA, such as those from previous experiments, or other contaminants, during transfers of reagents from one vessel to another. Also, PCR reaction volumes used in standard laboratory techniques are typically on the order of 50 microliters. A thermal cycle typically consists of four stages: heating a sample to a first temperature, maintaining the sample at the first temperature, cooling the sample to a second lower temperature, and maintaining the temperature at that lower temperature. Typically, each of these four stages of a thermal cycle requires about one minute, and thus to complete forty cycles, for example, is about three hours. Thus, due to the large volume typically used in standard laboratory procedures, the time involved, as well as the contamination possibilities during transfers of reagents from one vessel to another, there is clearly a need for microinstruments capable of carrying out the PCR procedure.

Recently, the cycling time for performing the PCR reaction has been reduced by performing the PCR reaction in capillary tubes and using a forced air heater to heat the tubes. Also, an integratged microfabricated reactor has been recently developed for in situ chemical reactions, which is especially advantageous for biochemical reactions which require high-precision thermal cycling, particularly DNA-based manipulations such as PCR, since the small dimensions of microinstrumentation promote rapid cycling times. This microfabricated reactor is described and claimed in copending U.S. application Ser. No. 07/938,106, filed Aug. 31, 1992, entitled "Microfabricated Reactor", assigned to the same assignee. Also, an optically heated and optically interrograted micro-reaction chamber, which can be utilized, for example, in the integrated microfabricated reactor of the above-referenced copending application Ser. No. 07/938,106, has been developed for use in chemical reactors, and is described and claimed in copending U.S. application Ser. No. 08/489,918, filed Jun. 13, 1995, entitled Diode Laser Heated Micro-Reaction Chamber With Sample Detection Means", assigned to the same assignee.

The present invention is directed to a particular geometry of silicon-based micro-reactors that have shown to be very efficient in terms of power and temperature uniformity. The micro-reactor of this invention, which is broadly considered as a silicon-based sleeve device for chemical reactions, can be effectively utilized in either of the reactor systems of the above-referenced copending applications. The present invention utilizes doped polysilicon for heating and bulk silicon for convective cooling. The present invention allows the multi-parameter, simultaneous changing of detection window size, in situ detection, reaction volumes, thermal uniformity, and heating and cooling rates. In addition, it enables the use of large arrays of the individual reaction chambers for a high-throughput microreaction unit.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an improved chemical reaction chamber.

A further object of the invention is to provide a silicon-based sleeve device for chemical reactors.

A further object of the invention is to provide a chemical reaction chamber that combines to use of doped polysilicon and bulk silicon.

A further object of the invention is to provide chemical reaction chambers that combines the use of doped polysilicon and bulk silicon to provide flexibility in thermal and optical properties allowing the implementation into small and large instruments.

Another object of the invention is to provide a silicon-based reaction sleeve that combines a criticial ratio of silicon and silicon nitride to the volume of material to be heated (e.g., liquid) in order to provide uniform heating, yet low power requirement.

Another object of the invention is to provide a silicon-based reaction sleeve that will allow the introduction of a secondary tube (e.g., plastic) into the reaction sleeve that contains the reaction mixture, thereby eleviating any potential materials incompatiblity issues.

Another object of the invention is to provide an array of individual reaction chambers for a high-throughput microreaction unit.

Another object of the invention is to provide a hand-held instrument that uses silicon-based sleeve-type reaction chambers with integrated heaters.

Another object of the invention is to provide a reaction chamber with automated detection and feedback control.

Another object of the invention is to provide for artificial intelligence control of reactions in a reaction chamber.

Another object of the invention is to provide pulse-width modulation as a feedback control for reaction chamber.

Other objects and advantages of the present invention will become apparent from the following description and the accompanying drawings. Basically, the invention is a silicon-based sleeve for chemical reactions. The invention encompasses a chemical reaction chamber that combines the use of polysilicon for heating and bulk silicon for convective cooling. The reaction sleeve combines a critical ratio of silicon and silicon nitride to the volume of material to be heated in order to provide uniform heating, yet low power requirements. The reaction sleeve also allows for the introduction therein of a secondary tube that contains the reaction mixture thereby eleviating any potential materially incompatibility issues. The present invention is an extension of the above-referenced integrated micofabricated reactor of above-referenced copending application Ser. No. 07/938,106 and the above-references optically integrated micro-reaction chamber of above-referenced copending application Ser. No. 08/489,819. The silicon-based sleeve reaction chamber can be utilized in chemical reaction systems for synthesis or processing of organic, inorganic, or biochemical reactions, such as the polymerase chain reaction (PCR) and/or other DNA reactions (such as the ligose chain reaction), or other synthetic, thermal-cycling-based reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a partial cut-away perspective view of a microfabricated chemical reaction instrument mounted in a power source / control apparatus.

FIG. 2 is a schematic of the reaction instrument of FIG. 1.

FIG. 3 schematically illustrates a heating and detection arrangement for a microfabricated reaction chamber.

FIG. 4 illustrates an embodiment of a microfabricated silicon-based sleeve reaction chamber made in accordance with the present invention.

FIG. 5 is an array of the sleeve reaction chambers of FIG. 4 operatively connected to a microelectrophoresis array.

FIG. 6 is an enlarged end view of another embodiment of a sleeve microreaction chamber similar to FIG. 4.

FIG. 7 illustrates in cross-section embodiment of an enlarged section of FIG. 6 using an isolated heater version, fixed window.

FIG. 8 illustrates in cross-section another embodiment of the same enlarged section of FIG. 6 using a non-isolated heater version variable window.

FIG. 9 is a view of a hand-held instrument (PCR man) which utilizes the reaction chambers of FIG. 6 as inserts to change reactions.

FIGS. 10A and 10B illustrate a thermal cycling instrument utilizing several hundreds of individually-controlled silicon-based microreaction chambers.

FIG. 11 illustrates a schematic representation of high-throughput DNA amplification, sample-handling, and electrophoreseis system.

FIG. 12 is an embodiment of an insert/lining for a reaction chamber with optical window, with the top/cover open.

FIG. 13 illustrates external filling of a reaction chamber insert/liner.

FIG. 14 illustrates immobilized reagents/probes for detection of specific products directly on windows or within reaction fluid a s "test strip" detected optically in the hand held instrument (PCR man) of FIG. 9.

FIGS. 15 and 16 schematically illustrate optical detection systems for use with the microreaction chambers of FIG. 6.

FIG. 17 schematically illustrates the use of integrated detection for an artificial intelligent feedback system.

FIG. 18 is a diagram showing the electrochemical oxidation and chemical reduction reactions for tris (2,2'bipyridyl) ruthenium (II) (TBR) and tripropylamine (TPA).

FIG. 19 illustrates a method for tagging and separating DNA for detection and quantification by electrochemiluminescence (ECL).

FIG. 20 illustrates cell voltage and ECL intensity versus time, with the voltage being increased, then decreased.

FIG. 21 illustrates an embodiment of a micromachined ECL cell with a thin film anode, and an associated photodiode detector.

FIG. 22 is an enlarged cross-sectional view of the ECL cell of FIG. 21 with the electrical leads.

FIGS. 23-30 illustrate the fabrication process for producing an ECL cell, as illustrated in FIG. 21.

FIG. 31 illustrates an embodiment using Al on ITO on glass which reduces resistance of the ITO electrode.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a micro-fabricated silicon-based sleeve chemical reaction chamber that combines heaters, such as doped polysilicon for heating and bulk silicon for conventive cooling. The microreaction chambers can be used in an array for a high-throughput microreaction unit, or in a hand-held unit. It combines a critical ratio of silicon and silicon nitride to the volume of material to be heated (e.g., liquid) in order to provide uniform heating, yet low power requirements. It also will allow the introduction of a secondary tube (e.g., plastic) into the reaction sleeve that contains the reaction mixture thereby alleviating any potential materials incompatibility issues. The present invention utilizes a particular geometry of silicon-based micro-reactors that have been shown to be very efficient in terms of power and temperature uniformity. The particular embodiment of the microfabricated reactor described has been experimentally used as a thermal cycling instrument for use in the polymerase chain reaction (PCR) and other chemical reactions, and has shown to be superior to present commercial instruments on thermally-driven chemical reactors. The silicon-based sleeve reaction chamber of this invention can be utilized in place of the reaction chamber of the microfabricated system of above-referenced copending application Ser. No. 07/938,106; and can be utilized with the integrated heater and detection arrangement of above-referenced copending application Ser. No. 08/489,819; and thus constitutes an extension of the microfabricated chemical reaction systems in these copending applications.

To provide an understanding of a microfabricated chemical reaction instrument and the integrated heating/detection arrangement, prior to the description of the embodiment of the sleeve reaction chamber of the present invention, a description is set forth of a microfabricated chemical reactor and an integrated heating/detection arrangement of the two-referenced copending applications.

FIG. 1 illustrates an embodiment of a microfabricated chemical reaction instrument generally indicated at 10, shown above a recessed section thereof, indicated generally at 11, in a power source/control system of the microfabricated reaction instrument, generally indicated at 12. A hypodermic needle 13 is shown inserting a sample through a silicone rubber window 14 into the reaction instrument 10. The reaction is controlled and powered by: induction coupling, such as that between coil L.sub.CL in the instrument 10 and a magnetic coil 15; by capacitive coupling, such as that between the plates of capacitor C.sub.3 and plates 16 and 17; and by electromagnetic coupling between a resonant circuit, see FIG. 2, in instrument 10 and a radio frequency antenna 18.

A schematic of the instrument 10 of FIG. 1 is illustrated in FIG. 2, and comprises three reagent chambers 19, 20 and 21, which, for example, may contain the DNA primers, the polymerase, and the nucleotides and any detection-tag molecules, such as magnetic beads. The target DNA molecule is placed in reagent chamber 19 by insertion of a hypodermic needle 13 (FIG. 1) or the like through a silicone rubber or other type material window 14. The reactants chambers 19, 20 and 21 are respectively connected by channels 22, 23, and 24, having narrow midsections, not shown, to a reaction chamber 25. Typically the chambers 19-21 and 25 have a volume ranging from microliter to nanoliters. The channels 22-24 are equipped with Lamb-wave pumps LW.sub.1, LW.sub.2 and LW.sub.3, respectively, for pumping reactants in chambers 19-21 through channels 22-24 in the direction of the arrows into reaction chamber 25. The Lamb-wave pumps may be located on any wall, or on multiple walls, of the channels 22-24. The Lamb-wave pumps LW.sub.1, LW.sub.2, and LW.sub.3 are connected respectively to capacitors C.sub.1, C.sub.2, and C.sub.3. The surface tension across the narrow midsections of the channels 22-24 prevents the reactants in chambers 19-21 from flowing into reaction chamber 25 until pumping is initiated. The inner surfaces of the channels 22-24 may be treated to raise the surface tension thereby further inhibiting flow of the reagents when the Lamb-wave pumps are not activated.

The reaction chamber 25 may be equipped with a Lamb-wave transducer LW.sub.C and a heater H.sub.C. The Lamb-wave transducer LW.sub.C is connected to inductor L.sub.CL (also shown in FIG. 1). The heater H.sub.C is connected to a resonant circuit consisting of an inductor L.sub.CH and a capacitor C.sub.CH. The Lamb-wave transducer LW.sub.C acts as an agitator, mixer, or sonochemical inducer, as indicated by the connected arrows 26 in chamber 25.

A channel 27 connects the reaction chamber 25 to a detection chamber 28. The channel 27 is equipped with a Lamb-wave pump LW.sub.DP, which is connected to a resonant circuit consisting of an inductor L.sub.DP and a capacitor C.sub.DP. The detection chamber 28 is equipped with a Lamb-wave sensor LW.sub.D, which is connected to a capacitor C.sub.D.

Lamb-wave transducers have high mechanical Q values and can therefore be powered by only a narrow range of alternating voltage frequencies. The Lamb-wave pumps (LW.sub.1, LW.sub.2, LW.sub.3) and Lamb-wave sensor (LW.sub.D) are powered capacitively by generating an electric field between the plates (such as plates 16 and 17 of FIG. 1 for example) at the resonant frequencies of the Lamb-wave transducers (LW.sub.1, LW.sub.2, LW.sub.3, and LW.sub.D). But, because the transducers have high Q values, only when the frequency of the imposed field is near the resonant frequency of a transducer do the transducer vibrate with any substantial magnitude. Similarly, the Lamb-wave mixing chamber transducer LW.sub.C is provided by an alternating frequency magnetic field generated by the coil (15 in FIG. 1) at the mechanical resonant frequency of the transducer LW.sub.C. The heater H.sub.C and the Lamb-wave pump LW.sub.DP are activated by directing an electromagnetic wave from the antenna (18 in FIG. 1) to the resonant circuit C.sub.CH and L.sub.CH, and resonant circuit C.sub.DP and L.sub.DP, respectively. The frequency of the incident electromagnetic radiation must correspond to the mechanical resonant frequency of the transducer LW.sub.DP, to activate the pump LW.sub.DP. The frequency of the incident electromagnetic radiation must correspond to the resonant frequency of the electrical elements C.sub.H, L.sub.CH and H.sub.C to activate the heater H.sub.C.

A PCR reaction, for example, is initiated by pumping the reagents in the chamber 19, 20 and 21 along the directions of the arrows through respective channels 22, 23 and 24 to the reaction chamber 25 by activating pump LW.sub.1, LW.sub.2, and LW.sub.3. A series of about twenty to forty thermal cycles, for example, are then initiated, and during each cycle the temperature of the reactants in the reaction chamber 25 goes from 55.degree. C. to 96.degree. C., and back to 55.degree. C., for example. The temperature of the reaction chamber 25 is determined by the power of the incident electromagnetic signal at the frequency corresponding to the resonant frequency of the circuit composed of the capacitor CC.sub.H, and the inductor L.sub.CH, together with the heater H.sub.C. The Lamb-wave device LW.sub.C of the reaction chamber 25 acts as an agitator or mixer, as indicated by arrows 26, to mix the reagents and promote the reaction.

When the thermal cycling is complete, the contents of the reaction chamber 25 are pumped by the Lamb-wave perm LW.sub.DP through channel 27 in the direction of the arrow to the detection chamber 38, which utilizes a Lamb-wave sensor LW.sub.D. Alternatively, the detection chamber 28 may be provided with an optical window and testing may be performed by fluorescence-based or absorption-based optical spectroscopy.

FIG. 3 illustrates a heating/detection arrangement that can be incorporated into the microfabricated reactor of FIGS. 1 and 2. As shown in FIG. 3, a chemical reaction chamber, such as a PCR chamber, of a miniaturized, microfabricated instrument, generally indicated 30, is illustrated in cross-section, with chamber 31 being formed in a housing 32, constructed of Pyrex for example, and having silicon inserts 33 and 34 therein, with an inlet 35 and an outlet 36. Energy from two different energy (light) sources is directed onto the housing 32, one source 37 being infrared (IR) source, and the second source 38 being an ultra-violet (UV) source. The IR source 17 applies heat more uniformly through the bulk of the solution in chamber 31. The UV source 18 induces fluorescence of the reaction products in the visible (Vis) spectrum, which can be detected by a visible (Vis) detector 39 located external of the housing 32 defining reaction chamber 31. Housing 32 must be constructed of a material transparent to UV and/or the visible spectrum. By incorporating an integrated excitation (heating) and detection system in the reaction chamber itself, confirmation of the presence of a sample in the reaction chamber can be confirmed, and the dual reaction and detection chambers 25 and 28 of the microfabricated reactor of FIG. 2 can be consolidated, thus reducing fabrication costs by reducing components.

The present invention, an embodiment of which is illustrated generally in FIGS. 4 and 5 involves a microfabricated reactor generally indicated at 40 which includes a silicon-based sleeve as a chemical reaction chamber, generally indicated at 41, constructed of two bonded silicon parts, and which utilizes doped polysilicon for heating and bulk silicon for convective cooling, as described in greater detail hereinafter. The sleeve 41 includes a slot or opening 42 into which reaction fluid, indicated at 43, from a hypodermic needle 44 is inserted into the reaction chamber, or into which a secondary tube 45 containing a reaction mixture 46 may be inserted. The tube 45 is constructed of plastic, for example, or other material which is inert with respect to the reaction mixture, thereby alleviating any potential material incompatibility issues. The sleeve is also provided with an opening 47 in which is located an optical window 48, made, for example, of silicon nitride, silicon dioxide, or polymers. The silicon sleeve reaction chamber 41 includes doped polysilicon for heating and bulk silicon for convective cooling, and combines a critical ratio of silicon and silicon nitride to the volume of material to be heated (e.g., liquid) in order to provide uniform heating, yet low power requirements.

FIG. 6 is an enlarged view of microreaction chamber, similar to the FIG. 4 embodiment, but utilizing two windows. The reaction chamber of FIG. 6, generally indicated at 50, is composed of two silicon wafers or substrates 51 and 52 bonded together as indicated at 53, and configured to define a slot or opening 54 therein. Each of wafers 51 and 52 include a layer of silicon nitride 51' and 52' which define a window, indicated generally at 55 and 56, respectively. Window 55 in wafer 51, constructed of silicon nitride, is provided with a heater 57 having electrical leads 58 and contacts 59 which extend along the edges of heater 57 to provide uniform heating. Window 56 in wafer 52 has a heater not shown in FIG. 6 but which is secured by metal contacts 60 and 61 as illustrated in either of FIGS. 7 and 8. The silicon nitride layers 51' and 52' are very thin (about 1 .mu.m) and vapor-deposited onto the bulk silicon wafers 51 and 52. The silicon nitride only becomes a window, as indicated at 55 and 56, when the bulk silicon wafers 51 and 52 are etched away to form the opening or slot 54. Heater 57 is transparent to energy passing through window 55, for example.

FIG. 7 is a greatly enlarged view of an embodiment of a section of silicon wafer 52 and window 56, as indicated by the circle 62 in FIG. 6. As seen in FIG. 7, the section of the silicon wafer 52, indicated at 63, is composed of bulk or single crystal silicon and is in contact with a low (100 to 500 MPa) stress silicon nitride membrane or window 64 (52' in FIG. 6) which in turn is in contact with a doped polysilicon heater 65 and metal contact 60 and 61. The FIG. 7 embodiment comprises an isolated heater version fixed window.

FIG. 8 is a greatly enlarged view of another embodiment of a section of silicon wafer 52 and window 56, as indicated by the circle 62. As seen in FIG. 8, the sections of the silicon substate 52, indicated at 66 are composed of bulk or single crystal silicon. As in the FIG. 7 embodiment, a low (100 to 500 MPa) stress silicon nitride member or window 69 (52' in FIG. 6) is in contact with silicon section 66, a doped polysilicon heater 70 is in contact with window membrane 69 and metal contacts 71 are mounted to heater 70. The FIG. 8 embodiment comprises a non-isolated heater version. The window size relative to the chamber can be varied to ensure thermal uniformity and optical access to the reaction chamber.

By way of example, the silicon wafers or substrates 51 and 52 may have a length of 5 to 50 mm, width of 2 to 10 mm, thickness of 0.1 to 1.0 mm, with the slot 54 having a cross-sectional area of 5 to 500 mm.sup.2. Slot 54, which shown to be of a six-sided configuration, may be a round, oblong, square, rectangular, or other configuration. Windows 55 and 56 may have a length of 0.1 to 1 mm, width of 0.1 to 50 mm, thickness of 0.1 to 10 .mu.m, and in addition to silicon nitride, may be composed of silicon dioxide, silicon, or polymers. The doped polysilicon heater 65 of FIG. 7 may have a thickness of 0.05 to 5 .mu.m, with the heater 70 of FIG. 8 having a thickness of 0.05 to 5 .mu.m. The metal contacts 60-61 and 61' of FIGS. 6 and 7 may be composed of gold or aluminum, with a thickness of 0.01 to 5 .mu.m, with the metal contact 71 of FIG. having a thickness of 0.01 to 5 .mu.m and composed of gold or aluminum. The heater 57 in silicon wafer or substrate 51 is composed of doped polysilicon having a thickness of 0.05 to 5 .mu.m, with the electrical leads and contacts 58 and 59 being composed of gold or aluminum.

The use of bulk silicon, polysilicon, silicon nitride enables flexibility in design for thermal and optical properties of each chamber. This enables individually controlled, thermally isolated reaction chambers in a small instrument (FIG. 9) or in large instrument (FIG. 10).

FIG. 9 is an embodiment of a miniature thermal cycling, battery operated, hand-held low-power, feedback-controlled instrument for PCR that uses microfabricated, silicon-based reaction chambers, such as those of FIGS. 4 and 6, the development of which addressed thermal uniformity and temperature precision of the reaction chambers, temperature ramp rates of the chambers, and biocompatibility of the materials in contact with the reagents.

As shown in FIG. 9, the hand-held, battery-operated instrument, coined "PCR man", generally indicated at 75, comprises a pressure-fit electrical contact controller holder, or housing 76, which for example may be 3.times.5 inches having a control-face-plate 77 with various indicators thereon, including a "status" window 78. The holder 76 is provided with a thermocouple-based temperature feedback control circuitry, heater electronics, computer interface, and power source connector, as described in greater detail hereinafter. The holder 76 is provided with batteries, indicated at 79, such as four nine-volt batteries, and at the upper end is provided with slots 80 for insertion of reaction chambers inside the holder (three slots shown), and into which one or more silicon-based reaction chambers 81, 82, 83 and 84, with integrated heaters (as shown in FIG. 6) are inserted as indicated by the arrow 85. The reaction chambers 81-84 may when constructed contain different reagents or chemicals, and can be selectively inserted into the handheld instrument 75 via slots 80 in holder or controller 76.

This instrument can be used to rapidly and repetitively provide controlled thermal cycles to the reaction mixture. The thermal conductivity properties of the silicon or similar semiconducting substrate, help speed up the thermal rise and fall times, and allow low power operation. While silicon is unique in its thermal properties, i.e., high thermal conductivity, a combination of silicon, silicon nitride, silicon dioxide, polymers and other materials would provide a combination of thermal conductivity and insulation that would allow thermal uniformity and low power operation.

The particular embodiment, such as FIG. 6, of a microfabricated reactor described can be used as a thermal cycling instrumentation for use inthe PCR and other chemical reactions, biochemical processes, microbiological processes, and incubators. As shown hereinafter the reaction chamber of this invention is superior to present commercial instruments used in thermally-driven chemical reactions.

During the experimental verification of the instrument of FIG. 9 and the microreaction chambers for use therein, such as illustrated in FIGS. 4 and 6, several different sizes of PCR reaction chamber designs were fabricated using integrated circuit (IC)-type silicon processing steps. The generalized fabrication process was as follows: Three-inch round, 0.5 mm thick single crystal silicon (SCS) wafers were processed inthe following way: low stress (200-300 MPa) silicon nitride (Si.sub.x N.sub.y) was low-pressure chemical vapor (LPCVD) deposited onto entire wafer (1.0-2.0 .mu.m thick). Photolithographic patterns for reaction chamber and subsequent processing steps were taken in the following order: 1) the silicon nitride was reactive ion etched (RIE) over the reaction chamber area, 2) the SCS was etched to the silicon nitride backside defining the chamber volume, 3) the wafer was patterned and the silicon nitride is chemically etched away everywhere except over the nitride membrane or left over the entire surface, depending upon the reaction chamber design, 4) the remaining silicon nitride membrane (side opposite the chamber) was LPCVD deposited with polycrystalline silicon (polysilicon) to a thickness of 3000.ANG., 5) the polysilicon was then high temperature doped with boron to a resistivity of 50-200 ohms per square, and 6) either aluminum or gold thin-film metal contacts were deposited defining the heater geometry.

Each wafer potentially contains many reaction chambers, depending upon geometry and volume desired. The etched depression in each wafer constitutes one-half of a dual-heater reaction chamber. Processed wafers are subsequently bound together forming an enclosed chamber with heaters on both sides.

The reaction chambers can be bonded together by depositing a thin film of low-temperature-curing polyimide between the two wafers directly or other bonding techniques such as eutectic metal bonding. A high precision computer-controlled silicon saw was used in each design to cut out each dual-heater chamber. The chambers were then rinsed repeatedly with de-ionized water and dried prior to treatment with silane.

The reaction chambers were inserted into a pressure-fit electrical contact holder that was part of the plexiglas backboard of the electronics components making up the controller. The controller electronics could be either/or anologue or digital and could use processes such as pulse-width modulation as a feedback control mechanism. The backboard was 3 inches by 5 inches and consisted of the thermocouple-based temperature feedback control circuitry, heater electronics, computer interface, and power source connector. The circuitry was designed to work from 8 to 32 volts. Thermal calibration was accomplished by correlating the temperature of the fluid with that of the silicon-measuring Type K thermocouple. Once calibrated, the instrument was capable of automated, feedback-controlled, thermal cycling operation without direct measurement of the reaction fluid. The thermal cycler output is to an Apple Centris 650 computer which displays the thermal cycle real-time along with storing the accumulated profiles. Four nine-volt batteries were abel to run the entire instrument continuously for over 2.5 hours.

Typical PCRs were set up as scaled-up master mixes, to assure uniformity between aliquotes thermocycled under different conditions. Reagent amounts were based on those ideal for 50 ul reactions. In general, master mixes contained: 50 Mm KCl, 10 mM Tris-HCl pH 8.3, 1.5-3.0 mM MgCl.sub.2, 200 uM each deoxynucleotide, or 800 uM dNTP total, 0.5 uM each of two oligonucleotide primers, 25 units/ml AmpliTaq.RTM. DNA polymerase, and target template at a specified copy number per 50 ul reaction. Template for some of the .beta.-globin PCRs was added as single strand DNA from a M13 bacteriophage clone of a portion of the human .beta.-globin gene. CF template was human genomic, double stranded, DNA derived from a cultured cell lines, HL60, GM07460, or GM08345. Each reaction mixture was aliquoted from the same master mix and thermocycled in the instrument of the present invention and a Perkin-Elmer GeneAmp.RTM. 9600 Thermal Cycler. Thermocycled reactions from both thermal cyclers were fractionated on 3% NuSeive, 1% Seakem agarose (FMC Corp.) using tris-borate buffer. The gels were stained with ethidium bromide and photographed under illumination with 302 nm UV light.

Although initially conceived as single use, disposable reaction chamber, the robust nature and stable properties allowed for repeated use of the reaction chambers.

The (MEMS) based thermal cycling instrument of this invention has been tested with a variety of PCR systems, including viral, bacterial, and human genomic templates. As well, various changes in both the reaction chamber design and controller instrumentation have been implemented and evaluated. A controller output real-time display of a thermal cycle from microfabricated thermal cycler has been prepared and it has been shown that with 15 volts input (average 1.2 Watts) that heating rates of over 5.degree. C./sec are attained. Cooling is slightly slower (2.5.degree. C./sec.) mostly due to the fact that the reaction chamber is held inside a plexiglass instrument board. Precision of .+-.0.5.degree. C. is maintained at the target temperatures. Higher heating and cooling rates have been achieved.

We have performed experiments that show the quantitative nature of the PCR process in both FIG. 9 and commercial instruments. These experiments consisted of removing 5 .mu.L aliquots out of a 105 starting copies, .beta.-globin PCR from both the instruments at 23, 25, 27, 29, and 31 cycles. These aliquots were subsequently run on an agarose electrophoresis gel. The results from both instruments are virtually identical. The same quantitative gel electrophoresis series results from the amplification of the 268-bp target