Mesoscale polynucleotide amplification devices

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BACKGROUND OF THE INVENTION

This invention relates generally to methods and apparatus for conducting amplification and various analyses of polynucleotides. More particularly, the invention relates to the design and construction of small, typically single-use, modules for use in analyses involving polynucleotide amplification reactions such as the polymerase chain reaction (PCR).

In recent decades, the art has developed a very large number of protocols, test kits, and cartridges for conducting analyses of biological samples for various diagnostic and monitoring purposes. Immunoassays, immunometric assays, agglutination assays and analyses based on polynucleotide amplification assays (such as polymerase chain reaction), or on various ligand-receptor interactions and/or differential migration of species in a complex sample, all have been used to determine the presence or concentration of various biological compounds or contaminants, or the presence of particular cell types.

Recently, small, disposable devices have been developed for handling biological samples and for conducting certain clinical tests. Shoji et al. reported the use of a miniature blood gas analyzer fabricated on a silicon wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988). Sato et al. reported a cell fusion technique using micromechanical silicon devices. Sato et al., Sensors and Actuators, A21-A23:948-953 (1990). Ciba Corning Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser photometer for detecting blood clotting.

Micromachining technology, using, e.g., silicon substrates, has enabled the manufacture of microengineered devices having structural elements with minimal dimensions ranging from tens of microns (the dimensions of biological cells) to nanometers (the dimensions of some biological macromolecules). Angell et al., Scientific American, 248:44-55 (1983). Wise et al., Science, 254:1335-42 (1991); and Kricka et al., J.Int. Fed. Clin. Chem., 6:54-59 (1994). Most experiments involving structures of this size relate to micromechanics, i.e., mechanical motion and flow properties. The potential capability of these structures has not been exploited fully in the life sciences.

Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986)) studied the behavior of fibroblasts and epithelial cells in grooves in silicon, titanium--coated polymers and the like. McCartney et al. (Cancer Res., 41:3046-3051 (1981)) examined the behavior of tumor cells in grooved plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986)) studied leukocyte and erythrocyte flow in microcapillaries to gain insight into microcirculation. Hung and Weissman reported a study of fluid dynamics in micromachined channels, but did not produce data associated with an analytic device. Hung et al., Med. and Biol. Engineering, 9:237-245 (1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17:25-30 (1971). Columbus et al. utilized a sandwich composed of two orthogonally orientated v-grooved embossed sheets in the control of capillary flow of biological fluids to discrete ion-selective electrodes in an experimental multi-channel test device. Columbus et al., Clin. Chem., 33:1531-1537 (1987). Masuda et al. and Washizu et al. have reported the use of a fluid flow chamber for the manipulation of cells (e.g., cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting pp. 1735-1740 (1988). Silicon substrates have been used to develop microdevices for pH measurement and biosensors. McConnell et al., Science, 257:1906-12 (1992); and Erickson et al., Clin. Chem., 39:283-7 (1993). However, the potential of using such devices for the analysis of biological fluids heretofore has remained largely unexplored.

Methodologies for using polymerase chain reaction (PCR) to amplify a segment of DNA are well established. (See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp. 14.1-14.354.) A PCR amplification reaction can be performed on a DNA template using a thermostable DNA polymerase, e.g., Taq DNA polymerase (Chien et al. J. Bacteriol., 127:1550 (1976)), nucleoside triphosphates, and two oligonucleotides with different sequences, complementary to sequences that lie on opposite strands of the template DNA and which flank the segment of DNA that is to be amplified ("primers"). The reaction components are cycled between a higher temperature (e.g., 94.degree. C.) for dehybridizing ("melting") double stranded template DNA, followed by lower temperatures (e.g., 40.degree.-60.degree. C. for annealing of primers and, e.g., 70.degree.-75.degree. C. for polymerization). A repeated reaction cycle between dehybridization, annealing and polymerization temperatures provides approximately exponential amplification of the template DNA. For example, up to 1 .mu.g of target DNA up to 2 kb in length can be obtained from 30-35 cycles of amplification with only 10.sup.-6 .mu.g of starting DNA. Machines for performing automated PCR chain reactions using a thermal cycler are available (Perkin Elmer Corp.)

Polynucleotide amplification has been applied to the diagnosis of genetic disorders (Engelke et al., Proc. Natl. Acad. Sci., 85:544 (19884), the detection of nucleic acid sequences of pathogenic organisms in clinical samples (Ou et al., Science, 239:295 (1988)), the genetic identification of forensic samples, e.g., sperm (Li et al., Nature, 335:414 (1988)), the analysis of mutations in activated oncogenes (Farr et al., Proc. Natl. Acad. Sci., 85:1629 (1988)) and in many aspects of molecular cloning (Oste, BioTechniques, 6:162 (19884)). Polynucleotide amplification assays can be used in a wide range of applications such as the generation of specific sequences of cloned double-stranded DNA for use as probes, the generation of probes specific for uncloned genes by selective amplification of particular segments of cDNA, the generation of libraries of cDNA from small amounts of mRNA, the generation of large amounts of DNA for sequencing, and the analysis of mutations.

A wide variety of devices and systems has been described in the art for conducting polynucleotide amplification reactions using thermal cycling procedures. Templeton, Diag. Mol. Path., 1:58-72 (1993); Lizardi et. al., Biotechnology, 6:1197-1202 (1988); Backman et al., Eur. Patent No. 320308 (1989); and Panaccio et al., BioTechniques, 14:238-43 (1993). The devices use a wide variety of design principles for transfer, such as water baths, air baths and dry blocks such as aluminum. Haff et al., BioTechniques, 10:102-12 (1991); Findlay et al., Clin. Chem., 39:1927-33 (1993); Wittwer et al., Nucl. Acids Res., 17:4353-7 (1989). PCR reactions in small reaction volumes have been described. Wittwer et al., Anal. Biochem., 186:328-31 (1990); and Wittwer et al., Clin. Chem., 39:804-9 (1993). Polynucleotide amplification micro-devices fabricated from silicon also have been described. Northrup et al., in: Digest of Technical Papers: Transducers 1993 (Proc. 7th International Conference on Solid State Sensors and Actuators) Institute of Electrical and Electronic Engineers, New York, N.Y., pp. 924-6; and Northrup et al., PCT WO 94/05414 (1994).

Silica particles have been shown to bind to nucleic acids, and have been used to isolate nucleic acids prior to PCR analysis. Zeillinger et al., BioTechniques, 14:202-3 (1993). While the art has described the use of silicon and other substrates fabricated with microchannels and chambers for use in a variety of analyses, little attention has been focused on methods for the modification of micromachined silicon or other surfaces, to diminish binding or other properties of the surfaces, which can inhibit reactions, such as polynucleotide amplification reactions, conducted in the devices. Northrup et al. describe the chemical silanization of a PCR reaction chamber in a silicon substrate having a depth of 0.5 mm. Northrup et al., in: Digest of Technical Papers: Transducers 1993 (Proc. 7th International Conference on Solid State Sensors and Actuators) Institute of Electrical and Electronic Engineers, New York, N.Y., pp. 924-6; and Northrup et al., PCT WO 94/05414 (1994). The reference of Northrup et al., (in: Digest of Technical Papers:Transducers 1993), however, discloses that, in the absence of silanization, untreated silicon surfaces of the reaction chambers had no inhibitory effect on the PCR reaction.

There is a need for convenient, rapid systems for polynucleotide amplification analyses, which could be used clinically in a wide range of potential applications in clinical tests such as tests for paternity, and genetic and infectious diseases and a wide variety of other tests in the environmental and life sciences. There is a need for the development of micro-devices fabricated in substrates such as silicon which permit polynucleotide amplification reactions to be conducted in high yields without interfering effects on the reaction caused by the surfaces of the substrate.

An object of the invention is to provide microscale analytical devices with optimal reaction environments for conducting polynucleotide amplification reactions which can be used to detect very low concentrations of a polynucleotide and to produce analytical results rapidly. Another object is to provide easily mass produced, disposable, small (e.g., less than about 1 cc in volume) devices having functional elements capable of rapid, automated polynucleotide amplification analyses of a preselected cell or cell-free sample, in a range of applications. It is a further object of the invention to provide agents for use in microscale reaction chambers fabricated in solid substrates such as silicon, to diminish potential inhibitory effects of the substrate surfaces on a polynucleotide amplification reaction. It is a further object of the invention to provide apparatus for delivering reagents and sample fluids to and from microscale polynucleotide amplification chambers fabricated in solid substrates such as silicon, and to provide apparatus for sealing the reaction chamber during an amplification reaction. It is yet another object of the invention to provide apparatus that can be used to implement a range of rapid clinical tests, e.g., tests for viral or bacterial infection, tests for cell culture contaminants, or tests for the presence of a recombinant DNA or a gene in a cell, and the like.

These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.

SUMMARY OF THE INVENTION

The invention provides a family of small, mass produced, typically one-use devices (sometimes referred to herein as "chips") for conducting a reaction to enable the rapid amplification of a polynucleotide in a sample. In one embodiment, the device comprises a solid substrate that is fabricated to comprise a mesoscale polynucleotide amplification reaction chamber. The device also may include a cover, e.g., a transparent cover, disposed over the substrate, to seal at least a portion of the reaction chamber during an amplification reaction. The device further includes at least one port in fluid communication with the reaction chamber, for introducing a sample into the chamber (sometimes referred to herein as a "sample inlet port" or "inlet port"). The device may include one or more flow channels extending from the ports to the reaction chamber, and/or connecting two or more reaction chambers. The device also may include one or more additional ports in fluid communication with the reaction chamber, to serve as access ports, inlet/outlet ports and/or vents. One or more ports and/or flow channels of the device may be fabricated in the cover or in the substrate. In the device, the reaction chamber may be provided with a composition which diminishes inhibition of a polynucleotide amplification reaction by the wall(s) defining the reaction chamber. The device may also include means for thermally cycling the contents of the chamber to permit amplification of a sample polynucleotide.

The term "mesoscale" is used herein with reference to reaction chambers or flow channels, at least one of which has at least one cross-sectional dimension between about 0.1 .mu.m and 1,000 .mu.m. The flow channels leading to the reaction chamber have preferred widths and depths on the order of about 2.0 to 500 .mu.m. Chambers in the substrate wherein amplification takes place may have one or more larger dimensions, e.g., widths and/or lengths of about 1 to 20 mm. Preferred reaction chamber widths and lengths are on the order of about 5 to 15 mm. The reaction chambers are fabricated with depths on the order of about 0.1 to at most about 1000 .mu.m. Typically, the reaction chambers are fabricated with depths less than 500 .mu.m, e.g., less than about 300 .mu.m, and optionally less than about 80 .mu.m. Fabrication of the reaction chamber, with shallow depths, e.g., less than 300 .mu.m, advantageously facilitates heat transfer to the reaction chamber contents, e.g., through the substrate, and permits efficient thermal cycling during an amplification reaction requiring thermal cycling. However, in some embodiments, the reaction chambers may be fabricated with depths between about 500 .mu.m and 1000 .mu.m. The overall size of the device ranges from microns to a few millimeters in thickness, depending on the material from which it is constructed, and approximately 0.2 to 5.0 centimeters in length or width.

The devices may be used to amplify and/or analyze microvolumes of a sample, introduced into the flow system through an inlet port defined, e.g., by a hole communicating through the substrate or the cover. The volume of the mesoscale flow system typically will be less than 50 .mu.l, and the volume of the reaction chambers is often less than 20 .mu.l, e.g., 10 .mu.l or less. The volume of the individual channels and chambers in another embodiment may be less than 1 .mu.l, e.g., in the nanoliter or picoliter range. Polynucleotides present in very low concentrations, (e.g., nanogram quantities) can be rapidly amplified (e.g., in less than ten minutes) and detected. After a polynucleotide amplification assay is complete, the devices may be discarded or they may be cleaned and re-used.

In one embodiment, reaction chambers may be fabricated wherein the ratio of the surface area of the walls defining the reaction chamber to the volume of the reaction chamber is greater than about 3 mm.sup.2 /.mu.l. Chambers also may be fabricated with even higher surface area to volume ratios, such as 5 mm.sup.2 /.mu.l or, optionally, greater than 10 mm.sup.2 /.mu.l. As the ratio of the surface area to volume increases, heat transfer through the substrate to and from the rea