MXPA96002775A

MXPA96002775A - Amplification devices, medium scale, polinucleoti

Info

Publication number: MXPA96002775A
Application number: MXPA/A/1996/002775A
Authority: MX
Inventors: Wilding Peter; J Kricka Larry
Original assignee: Trustees Of The University Of Pennsylvania
Priority date: 1994-11-14
Filing date: 1995-11-13
Publication date: 1997-12-01

Abstract

The present invention provides devices for amplifying a preselected polynucleotide in a sample by carrying out an amplification reaction of the polynucleotide. The devices are provided with a microfabricated substrate, which includes a polynucleotide amplification reaction chamber, having at least one cross-sectional dimension, of about 0.1 to 1,000æm. The device also includes at least one door, in fluid communication with the reaction chamber, to introduce a sample to the chamber, to ventilate the chamber, when necessary, and, optionally, to remove waste products or materials from the chamber. device. This reaction chamber may be provided with the reagents required for the amplification of a preselected polynucleotide. The device may also include elements for thermally regulating the contents of the reaction chamber, and amplifying a preselected polynucleotide. Preferably, the reaction chamber is manufactured with a high ratio of surface to volume, to facilitate thermal regulation. The amplification reaction chamber may also be provided with a composition, which decreases the inhibition of the amplification reaction by the material comprising the wall of the reaction chamber, when such treatment is required.

Description

AMPLIFICATION DEVICES, MEDIUM SCALE, OF POLYUCLEOTIDES Reference to Related Requests This request is a partial continuation of E. ü's request. A., Serial No. 08 / 308,199, filed September 19, 1994, which is a continuation of the application of E. U. A., Serial No. 07 / 877,662, filed May 1, 1992, the descriptions of which are incorporated herein by reference. here for reference. This application is being filed contemporaneously with the US application, Serial No. [Attorney File No. H-1203/1222], which is a partial continuation of applications Nos. Series 07 / 877,702 (filed on May 1992), 08 / 196,021 (deposited on February 14, 1994) and 08 / 250,100 (filed on May 26, 1994), whose descriptions are - * "". incorporate here as a reference.

Background of the Invention This invention relates, generally, to methods and apparatuses for carrying out amplifications and various analyzes of polynucleotides. More particularly, the invention relates to the design and construction of small modules, typically of simple use, for use in analyzes 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 equipment and cartridges to carry out the analysis of biological samples for purposes of various diagnostics and inspections. Immunoassays, immunometric assays, agglutination assays and analysis based on amplification assays. of polynucleotides (such as the polymer chain reaction), or in 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 types of cells.

Recently, small disposable devices have been developed to handle biological samples and carry out certain clinical tests. Shoji and colleagues reported the use of a miniature gas analyzer for blood, made on a silicon wafer. Shoji and collaborators, 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 laser photometer, controlled by a microprocessor, to detect blood clots.

Micromachinery technology, which uses, for example, silicon substrates, has made it possible to manufacture icroengineering devices, which have structural elements with minimum dimensions ranging from tens of microns (the dimensions of biological cells) to nanometers (the dimensions of some biological macromolecules). Angelí and collaborators, 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 refer to micromechanisms, ie the properties of mechanical movement and flow. The potential capacity of these structures has not been fully exploited 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, polymers coated with titanium, and the like. collaborators (Cancer Res. 41: 3046-3051 (1981)) examined the behavior of tumor cells in grooved plastic substrates LaCelle (Blood Cells 12: 179-189 (1986)) studied the flow of leukocytes and erythrocytes in micro- capillaries to gain penetration in the microcirculation.

Hung and eissman presented a study of fluid dynamics in micromachined channels, but did not produce the data associated with the analytical device. Hung et al., Med. And Biol. Ensineerins. 9_: 237-245 (1971); and Weissman et al. Am. Inst. Chem. Enq. J .. 17: 25-30 (1971). Columbus and colleagues used a sandwich composite of two orthogonally oriented, v-grooved, v-grooved sheets in the control of capillary flow from biological fluids to discrete ion-selective electrodes in a multi-channel experimental test device. Columbus et al., Clin. Chem .. 33: 1531-1537. Masuda et al. And Washizu et al. Have presented the use of a fluid flow chamber for the manipulation of cells (for example cell fusion). Masuda et al., Proceedings IEEE / IAS Meeting, pages 1549-1533 (1987); and Washizu et al., Proceedinss IEEE / IAS Meeting. pages 1735-1740 (1988). Silicon substrates have been used to develop microdevices to measure pH and biological sensors. McConnell et al., Science, 257-1906-12 (1992); and Eric are and collaborators, Clin. Chem .. 39: 283-7 (1993). however, the potential to use these devices for the analysis of biological fluids has, until now, remained largely unexplored.

The methodologies for using the polymerase chain reaction (PCR) to amplify a DNA segment are well established. (See, for example, Maniatis et al., Molecular Clonins: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pages 14.1-14.35.) A PCR amplification reaction can be performed in a DNA model, with the use of a thermostable DNA polymerase, for example the Taq polymerase of DNA (Chien et al. J. Bacteriol. 127: 1550 (1976)), nucleoside triphosphates, and two oligonucleotides with different sequences, complementary to the sequences that are placed in opposite strands of the model DNA and that flanks the segment of the DNA that is going to be amplified ("aprestos"). The components of the reaction are cycled between a higher temperature (for example of 94dC) to dehybridize ("melt") the double strand model DNA, followed by lower temperatures (eg 40-600C, to strengthen the sizing and, for example of 70-750C for polymerization). A repeated reaction cycle between the dehybridization, strengthening and polymerization temperatures provides an approximately exponential amplification of the model DNA. For example, up to 1 μg of the target DNA up to 2 kb in length, 30-35 amplification cycles can be obtained with only 10 ~ 6 μg of the starting DNA. Machines for performing automatic PCR chain reactions, using a thermal cycle device, are available (Perkin Elmer Corp.). The amplification of polynucleotides has been applied to the diagnosis of genetic disorders (Engelke et al., Proc. Nati, Acad. Sci.f 85: 544 (1988)), 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, for example sperm (Li et al.

* U) Nature. 335: 414 (1988)) the analysis of mutations in activated oncogenes (Farr et al., Proc. Nati, Acad. Sci. 85: 1629 (1988) and in many aspects of molecular cloning (Oste, BioTechniques. 1988).) Polynucleotide amplification assays can be used in a wide range of range of applications, such as the generation of specific sequences of cloned double-stranded DNA, for use as probes, the generation of specific probes for • - * - * genes not cloned by the selective amplification of particular segments of the cDNA, the generation of sets of cDNA of small amounts of mRNA, the generation of large amounts of DNA for the sequence, and the analysis of mutations. A wide variety of devices and systems have been described in the art for conducting polynucleotide amplification reactions with the use of thermal cycling procedures. Templeton, Diaq. Mol. Path. 1: 58-72 (1993); Lizardi et al., Biotechnolocry. 6: 1197-1202 (1988), Backman et al., European 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 drying blocks, such as aluminum. Haff et al., BioTechniques 10: 102-12 (1991); Findlay et al., Clin. Chem..391927-33 (1993); Wittwer et al., Nucí. Acids Res .. 17: 4353-7 (1989); The reactions of PCT have been described in small volumes. Wittwer et al., Anal. Biochem. 186: 328-31 (1990), and Wittwer et al. Clin. Chem. 39: 804-9 (1993). The poly-nucleotide amplification microdevices, made of silicon, have also been described. Northrup and collaborators, in Diaest of Technical Papers: Transducers 1993 (Proc. 7th International Conference on Solid State Sensors and Actuators) [Procedures of the 7th International Conference on Sensors and Solid State Actuators], Institute of Electrical and Electronic Engineers, New York , NY, USA, pages 924-6; and Northrup et al., PCT patent WO 94/05414 (1994). It has been shown that silicon particles bind to nucleic acids and have been used to isolate nucleic acids prior to PCR analysis. Zeillinger and collaborators, BioTechniques. 14: 202-3 (1993). While the use of silicon and other substrates made with microchannels and chambers for use in a variety of analyzes has been described in the art, little attention has been given to methods for the modification of icromachined silicon or other surfaces to decrease the binding or other properties of the surfaces, which could inhibit reactions such as polynucleotide amplification reactions, conducted in the devices. Northrup et al. Describe the chemical silanization of the PCR reaction chamber on a silicon substrate having a depth of 0.5 mm. Northrup and collaborators in: Diqest of Technical Papers: Transducers 1993 (Proc. 7th International Conference on Solid State Sensors and Actuators) [Proceedings of the 7th International Conference on Sensors and Solid State Actuators], Institute of Electrical and Electronic Engineers, New York , NY, USA, pages 924-6, and Northrup and co-workers in PCT patent WO 94/05414 (1994). The reference of Northrup and collaborators (in Diqest of Technical Papers: Transducers 1993). however, it reveals that, in the absence of silanization, the untreated silicon surfaces of the reaction chambers do not have an inhibitory effect on the reaction of a PCR: There is a need for rapid, convenient systems for polynucleotide amplification assays, which can be used clinically in a wide range of potential applications in clinical trials, such as paternity tests, and genetic and infectious diseases, and a wide variety of other tests in the 5 environmental and life sciences. There is a need for the development of microdevices made of substrates, such as silicon, which allow the polynucleotide amplification reactions to be conducted with high yields, without effects of interference in the reaction, caused by the substrate surfaces.

An object of the invention is to provide microscale analytical devices, with optimal reaction environments, to drive the polynucleotide amplification reactions, which can be used to detect very low concentrations of a polynucleotide and quickly produce analytical results. Another object is to supply small, disposable devices (for example minors - ~ about 1 cc in volume), easily produced in bulk, capable of rapid and automatic polynucleotide amplification analyzes of a previously selected sample of cells or cell-free, in a range of applications. It is a further object of the invention to provide agents for use in micro-scale reaction chambers, made of solid substrates, such as silicon, to decrease the potential inhibitory effects of substrate surfaces in an amplified reaction. - Polynucleotide sorting It is a further object of the invention to provide apparatus for delivering sample reagents and fluids to and from microscale polynucleotide amplification chambers, manufactured in solid substrates, such as silicon, and to provide an apparatus for sealing the reaction chamber during a Amplification reaction. It is still another object of the invention to provide an apparatus that can be used to carry out a range of rapid clinical tests, for example tests for viral or bacterial infection, tests for cell culture contaminants, or tests for the presence of a DNA re-combining or a gene in a cell, and the like. These and other objects and features of the invention will be apparent from the following description, drawings and claims. Brief Summary of the Invention The invention provides a family of typically single-use, small, mass-produced devices (sometimes referred to herein as "chips") to drive a reaction and enable rapid amplification of a polynucleotide in a sample. In one embodiment, the device comprises a solid substrate, which is fabricated by including a medium-scale amplification reaction chamber of a polynucleotide. The device may also include a cover, e.g., a transparent cover, disposed on the substrate, to seal at least a portion of the reaction chamber during an amplification reaction. The device further includes at least one door in fluid communication with the reaction chamber, for introducing a sample into the chamber (sometimes referred to herein as a "sample entry door" or "entry door"). This device may include one or more flow channels, which extend from the doors to the reaction chamber and / or connect two or more reaction chambers. The device can also include one or more additional doors, in fluid combination with the reaction chamber, to serve as access doors, entry / exit doors and / or ventilation doors. One or more doors and / or flow channels of the device can be manufactured on the cover or on the substrate. In the device, the reaction chamber may be provided with a composition that decreases the inhibition of an amplification reaction of polynucleotides by the wall (s) that define the reaction chamber. The device may also include elements for thermal cycling of the contents of the chamber, to allow amplification of a sample polynucleotide.

The term "medium scale" 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 and 1,000 μm. The flow channels leading to the reaction chamber have preferred widths and depths of the order of about 2.0 to 5 500 μm. The chambers in the substrate, where the amplification takes place, may have one or more larger dimensions, for example, widths and / or lengths of around 1 to 20 mm. Preferred widths and lengths of the reaction chamber are of the order of about 5 to 15 mm. The reaction chambers are manufactured with depths in the order of about 0.1 up to at most about 1,000 μm. Typically, the reaction chambers are manufactured with depths less than 500 μm, for example less than about 300 μm and optionally less than about 80 μm. The manufacture of the camera of reaction, with a shallow depth, for example less than 300 μm, advantageously facilitates the transfer of heat to the contents of the reaction chamber, for example through the substrate, and allows efficient thermal cycling during an amplification reaction requiring he thermal cycling. However, in some embodiments, the reaction chambers can be manufactured with depths between about 500 and 1,000 μm. The overall size of the device varies from microns to a few millimeters in thickness, depending on the material from which it is constructed and approximately with a length or width of 0.2 to 5.0 centimeters.

The devices can be used to amplify and / or analyze micro volumes of a sample, introduced into the flow system through an entry port, defined, for example, by a hole communicating through the substrate or cover. The volume of the medium-scale flow system will typically be less than 50 μl and the volume of the reaction chambers is often less than 20 μl, for example 10 μl or less. The volume of the channels and individual cameras, in another modality, can be smaller of 1 μl, for example in the range of nanoliters or pico-liters. The polynucleotides, present in very low concentrations (for example in amounts of nanograms) can be amplified rapidly (for example in less than ten minutes) and detected. After completing a trial of the amplification of a polynucleotide, the devices can be discarded or they can be cleaned and reused. "- In one embodiment, the reaction chambers can be manufactured with a ratio of the surface area of the walls, which define the reaction chamber, to the volume of the reaction chamber, is greater than about 3 mm2 / μl. The chambers can also be manufactured with even higher ratios of surface area to volume, such as 5 mm2 / μl or, optionally, greater than 10 mm2 / μl. According to the The ratio of the surface area to the volume increases, the transfer of heat through the substrate to and from the contents of the reaction chamber is facilitated and thus the thermal cycling of the reaction becomes more efficient and the productivity of the reaction becomes more efficient. reaction is increased. However, additionally, as the ratio of the surface area to the volume increases, the potential inhibitory effects of the substrate walls in the amplification reaction of the polynucleotides also increase. Depending on the material from which the device is made, the wall surfaces of medium-scale channels and chambers can interfere with the amplification of polynucleotides, for example by means of binding interactions between the material and the sample polynucleotides or the reagents of the amplification. The invention provides a range of compositions which can be provided in the reaction chamber, to diminish the potentially inhibitory effects of the surfaces of the reaction chamber wall, such as the silicon surfaces, in the reaction. Compositions are particularly useful in reaction chambers that have a surface area to volume ratio greater than about 3 mm2 / μl or 5 mm2 / μl or, in another embodiment, in chambers where the ratio exceeds about 10. mm2 / μl. The device may also include a cover, disposed on the reaction chamber, to seal this reaction chamber during the amplification reaction. The cover may comprise a material, such as glass or silicon, or a plastic material. The use of a cover, disposed on the reaction chamber, increases the total amount of the surface area in contact with the fluid in the reaction chamber. The surfaces of the cover exposed to the reaction chamber can also be treated with compositions, as disclosed herein, to reduce the potentially inhibitory effects of the material of the cover surface in the amplification reaction. A composition provided in the reaction chamber for decreasing the inhibition of an amplification reaction by a wall of the reaction chamber can be covalently or non-covalently adhered to the surface of the wall of the reaction chamber, or it can be provided in solution in the reaction chamber, during an amplification reaction. In one embodiment, the wall surfaces of one or more chambers and / or reaction channels in the device can be coated with a silane, using a silanization reagent, such as dimethylchlorosilane, dimethyl dichlorosilane, hexamethyldisilazane or trimethylchlorosilane (available , for example, of Pierce, of Rockford, IL., USA.). Alternatively, the surface of the walls of the chambers and / or reaction flow channels, for example, fabricated within a silicon substrate, may be provided with a relatively inert coating, for example, using a siliconization reagent, such as Aquasil ™ or Surfasil ™ (Pierce, Rockford, IL., USA) or Sigmacote ™ (Sigma Chemical Co., of St. Louis, MO., USA). Siliconizing reagents, available from commercial manufacturers, such as Pierce (Rockford, IL., USA) or Sigma Chemical Co. (St. Louis, MO, USA) are organosilanes containing a hydrolysable group, which can be hydrolysed in solution for forming a silanol, which can polymerize and form a film on the surface of the chamber and can react with the hydroxyl groups on this surface of the chamber, so that the film adheres firmly over the entire surface. The coating may further include a macromolecule (sometimes referred to herein as a "blocking agent"), associated non-covalently or covalently with the silicone coating, to further reduce the inhibitory effects of the reaction chamber wall in the amplification reaction. . Useful macromolecules include an amino acid polymer or polymers such as polyvinyl pyrrolidone, polyadenylic acid or polymaleimide. A silicon oxide film can be supplied on the surface of the reaction chamber and / or channel walls, on a silicon substrate, to reduce the inhibition of the amplification reaction by the surfaces of the wall. The silicon oxide film can be formed by a thermal process, in which the silicon substrate is heated in the presence of oxygen. Alternatively, an intensified plasma oxidation or intensified plasma chemical vapor deposition process may be used. Additionally, the reaction chamber and / or channel walls can be coated with a relatively inert polymer, such as a polyvinyl chloride.

Prior to the addition of the sample polynucleotide and the amplification reagents to the reaction chamber, another polynucleotide (sometimes referred to herein as a "blocker" polynucleotide) can be added to the chamber, such as a genomic DNA or a polyadenylic acid, preferably at a higher concentration than the concentration of the sample polynucleotide. This allows the blocking polynucleotide to occupy any site on the wall surfaces that could potentially bind to the sample polynucleotide and reduce the yield of the reaction or the accuracy of the assay. Thus, in one embodiment, a blocking polynucleotide can be supplied within a reaction chamber, made within a silicon substrate, so that the blocking polynucleotide can occupy any binding site of the polynucleotide, such as the free hydroxyl groups, in the wall surfaces of the reaction chamber. To avoid interference with the amplification reaction, the blocking polynucleotide must comprise sequences unrelated to those of the sample polynucleotide. Other compositions that bind to the chamber wall surfaces, such as poly-guanilic acid or various polypeptides, such as casein or serum albumin, can also be used as a blocking agent. The devices can be used to carry out the amplification reaction of the polynucleotides, such as a polymerase chain reaction (PCR), within the reaction chamber. The reaction chamber may be provided with reagents for PCR, including a sample polynucleotide, the polymerase, nucleoside triphosphates, a first sizing hybridizable with the sample polynucleotide, and a second hybridizable sizing with a sequence that is complementary to the sample polynucleotide, in which the first and second sizes define the terms of the amplified polynucleotide product. The device can also include resources for the thermal cycling of the contents of the amplification reaction chamber, so that, in each cycle, for example, the temperature is controlled to 1) dehybridize ("melt") the polynucleotide of double cord, 2) strengthen the sizes to a single-stranded polynucleotide and 3) synthesize the amplified poly-nucleotide between the sizes. Other amplification methods available in the art may also be used, including, but not limited to: 1) target polynucleotide amplification methods, such as auto sequence replication, sustained (3SR) and cord displacement amplification (SDA); (2) methods based on the amplification of a signal attached to the target DNA, such as the amplification of "branched-chain" DNA (Chiron Corp.); (3) methods based on the amplification of the probe DNA, such as the ligase chain reaction (LCR), and the amplification of QB replicase (QBR); and (4) various other methods, such as ligation-activated transcription (LAT), amplification based on the nucleic acid sequence (NASBA), repair chain reaction (RCR) and cycling probe reaction (CPR). ) (for a review of these methods, see pages 2 to 7 of The Genesis Reportf DX, Vol. 3, No. 4, Feb. 1994, Genesis Group, Montclair, NJ, USA.).

The reaction chamber can be manufactured with a section, which is thermally cycled in sequence between the temperatures required for the polynucleotide amplification reactions, which require thermal cycling, such as conventional PCR. Alternatively, the reaction chamber may comprise two or more sections, placed at the different temperatures required for the dehybridization, strengthening and polymerization, in this case, the device also comprises resources for the transfer of the contents of the chamber, between the sections, to carry out the reaction, for example a pump controlled by a computer. The reaction chamber can be attached, in at least a portion of the chamber, by a cover arranged on the substrate. The device may also include resources for detecting the amplified polynucleotide, as described herein. The device can be used to carry out a variety of automatic, sensitive and rapid polynucleotide analyzes, which include analyzes for the presence of polynucleotides in cells or in solution, or for the analysis of a virus or cell types that use the presence of a particular polynucleotide as a marker.

Flow channels and reaction chambers, of medium scale, can be designed and manufactured from solid substrates, using established micromachining methods, such as photolithography techniques, chemical etching and arrangement, laser machining, LIGA process (Becker and collaborators, Microelec, in., 4; 35-36, 1986) and plastic molding. Medium-scale flow systems in the devices can be constructed by fabricating flow channels and one or more reaction chambers on the surface of the substrate, and then adhering or holding a cover on the surface. The solid substrate and / or the cover may comprise a material, such as silicon, polysilicon, glass, gallium arsenide, polyimide, silicon nitride, and silicon dioxide. The cover and / or the substrate can alternatively comprise a plastic material, such as an acrylic material, polycarbonate, polystyrene or polyethylene. Optionally, the cover and / or the substrate may comprise a transparent material. An instrument may also be supplied for use with the device, which contains a nesting site for retaining the substrate of the device and which optionally corresponds to one or more entry ports on the substrate with one or more Flow lines in the instrument After applying to the entrance door a sample of biological fluid that is suspected to contain a particular polynucleotide, the substrate is placed in the instrument and the pumps, for example, arranged in the instrument, are operated to force the sample through the flow system. Alternatively, a sample can be injected into the substrate by the instrument (for example by a syringe equipped with the instrument). Reagents required for the assay, such as a polymerase enzyme, can be added (in liquid or dried form) to the polynucleotide sample, prior to injection into the substrate. Alternatively, the reagents necessary to complete the test can be injected into the reaction chamber from a separate entrance door, for example by the instrument. Fluid samples and reagents can also enter the medium-scale flow system by capillary action or by gravity. The invention also provides an element for sealing one or more of the fluid inlet / outlet ports in the device, during an amplification reaction. This advantageously prevents evaporation of the liquids during thermal cycling and thus maintains the preferred concentrations of the reaction during the amplification reaction. In one embodiment, an apparatus is provided that includes resources to deliver the fluid to and from the reaction chamber through a door in the device and adapted for internal adjustment and / or locking with the door, which can reversibly seal the door after delivering the fluid to the reaction chamber. For example, the apparatus that delivers the fluid may comprise a syringe or pipette. In one embodiment, the fluid delivery apparatus may comprise a pipette including a tip provided with an opening, for transferring the fluid between the pipette and the door. This pipette tip may be optionally releasable from the pipette and may be disposable to prevent contamination between samples.

The device may include a substrate comprising a heat conducting material, such as silicon, as well as a cover disposed on the substrate, which may comprise a transparent material, such as glass or a plastic. The device also includes a polynucleotide amplification chamber, of medium scale, fabricated within the substrate or the cover. This cover can include a cavity to receive and adjust internally with the pipette used to deliver the -4.0 sample and Reagent solutions to and from the reaction chamber. The device may further include a flow channel communicating through the substrate and / or the cover, between the opening of the pipette tip and the reaction chamber, when the pipette is accommodated within the cavity.

The opening can be placed on a wall of the tip of the pipette, to allow it to move between a first position, which allows the transfer of fluid from the tip through the opening and the channel to the reaction chamber , and a second position, to allow that the opening faces a wall of the cavity, whereby it seals the flow channel and the reaction chamber during a reaction. Additionally, an oppressible element can be provided, which extends from the substrate and can seal the door by pressing the counter element. the door.

The temperature of one or more sections in the reaction chamber can be regulated, for example, by supplying one or more electric resistance heaters in the substrate, near the reaction chamber, or by using a pulsed laser or other source of directed electromagnetic energy to the reaction chamber. The instrument may include electrical contacts in the nesting region, which correspond to the contacts, integrated into the substrate structure, for example, for the supply of energy for heating by electrical resistance of the reaction chamber. A cooling element can also be supplied in the instrument, to help the thermal regulation of the reaction chamber. This instrument can be provided with a conventional circuit system in communication with the sensors in the device, to thermally regulate the temperature cycles required for the dehybridization and polymerization reactions.

The amplified polynucleotide, produced by the amplification reaction of the polynucleotide in the medium-scale reaction chamber, can be picked up through a gate in the substrate and detected. Alternatively, specific reagents and methods known in the art can be used to directly detect the amplification products in the reaction chamber ("Taq Man" ™ PCR reagents and kits, available from Perkin Elmer Corp., for example). As another alternative, a medium-scale detection region can be microfabricated in the substrate, in fluid communication with the reaction chamber in the device, as a part of the medium-scale flow system. The detection region may include a labeled binding portion, such as a labeled polynucleotide or antibody probe, capable of binding, in detectable form, to the amplified polynucleotide. The presence of the polymerized polynucleotide product in the detection region can be detected, for example by the optical detection of the agglutination the polymerized polynucleotide and the binding portion through the glass cover over the detection region or through a section translucent or transparent of the substrate itself. Alternatively, the detection region may comprise a series of microlithographic channels or arrays to electrophoretically separate and detect an amplified polynucleotide.

A positive test can also be indicated by detectable changes in the fluid flow properties of the sample, such as changes in pressure or electrical conductivity at different points in the flow system in the production of an amplified polynucleotide in the chamber of reaction. In one embodiment, the device comprises a medium-scale flow system, which includes a polynucleotide amplification reaction chamber (e.g. a chamber or a portion of a flow channel), used in combination with an instrument, the which includes a sensor equipment, such as a spectrophotometer, capable of reading a positive result through an optical window, for example, arranged over the detection region. The instrument may also be designed to receive electrical signals indicative of a pressure reading, conductivity, or the like, detected in the reaction chamber, the detection region or some other region of the flow system.

The substrate may comprise a plurality of reaction and / or detection chambers, to enable rapid, parallel amplification and / or detection of several polynucleotides in a mixture. The medium-scale flow system may include prominences, or a section of reduced area in cross-section, to cause lysis or destruction of the cells in the microsample, before delivery to the reaction chamber. Pieces with sharp edges of silicon, trapped in the flow path, can be used as a lysis resource. The medium-scale flow system may also include a cell capture region, comprising a binding portion, for example immobilized on a wall of a flow channel, which binds to a particular type of cell in a heterogeneous population of cells. cells at a relatively slow fluid flow rate, and at a higher flow rate or changing the nature of the solvent, for example, that releases the cell type before delivering the cells to a lysis region of the cells, then to the reaction chamber. In this embodiment, the intracellular DNA or RNA is isolated from the selected subpopulation of cells and delivered to the medium-scale reaction chamber for the analysis of polynucleotides in a device. In an alternative embodiment, the binding reagent can be immobilized on a solid particle, such as a latex or a magnetic bead, as described below.

The complexing agents, such as magnetic beads coated with a polynucleotide probe may be provided within the flow system medium scale, which can be moved along the flow system by an external magnetic field, for example in the instrument. The polynucleotide probe, immobilized on the magnetic beads, makes it possible for these beads to bind to an amplified polynucleotide in the reaction chamber or in a separate detection chamber. Magnetic beads containing immobilized polynucleotide probe may be, for example, carried through flow system or otherwise introduced to the reaction chamber at the end of an assay to bind to amplified polynucleotide product. This bound polynucleotide can then be transported on the magnetic beads to a detection or purification chamber in the flow system, or to a collection door. Alternatively, the magnetic beads can be retained in place, at a predetermined location on the device, then transported to a detection or purification chamber, after binding to the polynucleotide product.

Some of the features and benefits of the devices are illustrated in Table 1. The devices can provide a rapid test for the detection of pathogenic bacteria and viruses, or for the presence of certain cell types, or the presence of a gene or a virus. sequence of the recombinant DNA in a cell. The devices disclosed herein are all characterized by a system of flow medium scale, including a reaction chamber polynucleotide amplification, preferably having at least one dimension of medium scale, which is used to amplify a polynucleotide in a sample, and that is provided with required amplification reagents. The device can be used to amplify a polynucleotide in a wide range of applications. At the conclusion of the test, the device can be discarded or cleaned and reused.

TABLE 1 Feature Benefit Flexibility Does not limit the available number of designs or applications of the device.

Reproducible Enables reliable, standardized mass production of devices. Bass Production Supplies a competitive price with the existing systems cost. Disposable in nature for single use processes Small Size No bulky instrumentation required. By themselves, they lead to portable units and systems, designed for use in unconventional laboratory media. Minimum storage and transport costs. Scale Sample volumes and minimum reagents Micrometer required, Reduce reagent costs, especially for specialized, more expensive test procedures. Allows simplified instrumentation schemes. Sterility The devices can be sterilized for use in microbiological tests and other procedures that require clean environments. Sealed System Minimizes biological hazards, Ensures the integrity of the process.

Capacities can perform multiple processes or analysis circuits in a single device. It allows Multiple panel tests. Capacities Increase capabilities for multiple inspection of test processes for virtual detection of any system. It allows a wide range of applications. Devices Reduces the cost per user process Reusable in certain applications.

Brief Description of the Drawings Figure IA is a schematic view in longitudinal cross section 5 of a device 10, according to the invention, which includes a solid substrate 14, on which a medium-sized flow channel 20 is machined, connected to the doors of inlet 16 and the polynucleotide amplification reaction chamber 22, with a cover 12 adhered to the surface of the substrate.

Figure IB is a schematic view in longitudinal cross-section of an alternative embodiment of the device 10, according to the invention, which includes a solid substrate 14, on which the polynucleotide amplification reaction chamber 22 is machined, medium scale, and the entrance doors 16, with the cover 12 adhered to the surface of the substrate.

Figure IC is a schematic longitudinal cross-sectional view of another embodiment of the device 10, which includes a solid substrate 14, made with a median-scale polynucleotide amplification reaction chamber 22, and the cover 12 made with doors 16 and channels 20, in fluid communication with the chamber 22.

Figure 2A is a perspective view of the device of Figure IA.

Figure 2B is a perspective view of the device of Figure IB.

Figure 3A is a schematic illustration of an analytical device 10, nested within an instrument 50, schematically illustrated, which can be used to support the device 10 and includes a heating element 57, to regulate the temperature of the chamber reaction 22 in the device 10.

Figure 3B is a schematic illustration of an analytical device 10, nested within the instrument 50, which can be used to support the device 10 and which includes the heating element 53 for regulating the temperature of the reaction chamber 22 in the device 10 Figure 4 is a schematic longitudinal cross-sectional view, according to the invention, including a solid substrate 14, on which a medium-scale flow channel 20 is machined, connected to the inlet ports 16 and the sections of the reaction chamber 22, with the cover 12 adhered to the surface of the substrate. Figure 5 is a perspective view of the device of Figure 4.

Figure 6A is a schematic illustration of the analytical device 10, nested within the instrument 50, which can be used to support the device 10 and which includes heating elements 57 for regulating the temperature of the sections of the reaction chamber 22 in the device 10.

Figure 6B is a schematic illustration of the analytical device 10, nested within the instrument 50, which can be used to support the device 10 and which includes the heating element 57, to regulate the temperature of the section of the reaction chamber 22A on device 10.

Figure 7 is a schematic plan view of a microfabricated substrate 14 with reaction chamber sections, 22A and 22B, of medium scale, in fluid communication with a detection chamber, comprised of a divergent flow channel system. of dimensions in transverse sections, decreasing progressively, arranged on the substrate.

Figure 8 is a perspective view, in cross section, of a flow channel 20 in the substrate 14, with the protrusions 80 filtering the cells or debris, which extend from a wall of the channel.

Figure 9 is a perspective view, in cross section, of a flow channel 20 in the substrate 14, with protrusions 90 passing through the cells, which extend from a wall of the channel.

Figure 10A is a schematic plan view of a medium-scale analytical device, including the sections 22a and 22b of the reaction chamber, and the detection chamber 22C, microfabricated in the substrate 14.

Figure 10B is a schematic plan view of another medium-scale analytical device, including sections 22A and 22B of the reaction chamber, and the detection region 26. Figure 11 is a schematic plan view of another analytical device of medium scale, which includes a reaction chamber 2A, microfabricated in the substrate 14.

Figure 12 is a schematic plan view of an analytical device, fabricated with a series of medium-scale cameras, suitable for carrying out a variety of functions, including cell sorting, cell lysis or destruction and polynucleotide analysis .

Figure 13 is a schematic plan view of an analytical device, fabricated with two separate flow channel systems 40.

Figures 14, 15 and 16 illustrate plan views of different embodiments of a mid-scale filter 24, microfabricated in the flow channel 20 in an analytical device 10. Figure 17 is a schematic perspective view of an apparatus 60, used in combination with device 10 (not shown) to view the contents of this device 10.

Figure 18 is a schematic cross-sectional view of the apparatus 60 of Figure 17.

Figure 19 is a schematic cross-sectional view of a device including a substrate 14 and a transparent cover 12, which includes the cavity 87 that receives the pipette 86.

Figure 20 is a schematic cross-sectional view of a pipette tip 84, including the opening 88.

Figure 21 is a schematic cross-sectional view of a substrate 14, provided with the element 85, which can be compressed to seal the door 16 and the channel 20. Figure 22 is a schematic perspective view of an apparatus including a transparent cover 12, provided with the cavity 87 and the flow passage 20A, which leads to the flow channel 20B and the reaction chamber 22 in the substrate 14.

Figure 23 is a drawing illustrating an agarose gel electrophoresis model of amplified polynucleotide samples in a medium scale amplification chamber.

Similar reference characters in the respective figures of the drawings indicate corresponding parts. The drawings are not necessarily to scale.

Detailed Description A, General Data The invention provides a family of small, typically single-use, mass-produced devices for carrying out polynucleotide amplification reactions, and enabling rapid amplification of the polynucleotides in fluid samples. The device comprises a solid substrate, fabricated including at least one polynucleotide amplification reaction chamber and typically having a length and / or width ranging from about 0.1 to 5.0 centimeters. The channels and chambers in cross section, through the thickness of the device, may be triangular, truncated, square, rectangular, circular or any other configuration. The device also includes a sample inlet port, in fluid communication with the reaction chamber. This device may also include additional doors (which may function as access or entry / exit doors, or as vents), arranged at any location on the flow system, and one or more flow channels of the samples, in fluid communication with the reaction chamber. One or more of the doors may be open to the atmosphere or attached to appropriate pressure or suction devices, for example, to fill or evacuate the device, or they may be sealed, for example during an amplification reaction. The doors and channels can be manufactured within the substrate or, alternatively, in a cover arranged on the substrate, or both. The device may further include a system for thermal cycling of the contents of the reaction chamber, to allow amplification of a sample polynucleotide.

At least one of the reaction chambers and the flow channels of the device, and preferably both, have a medium scale dimension, ie at least one cross-sectional dimension of the order of 0.1 to 1,000 μm. The preferred depth of the reaction chamber is less than about 500 μm. more preferably less than 300 μm and especially preferred less than 80 μm. The reaction chambers may have larger widths and lengths, for example of the order of about 1 to 20 mm, preferably about 5 to 15 mm.

The shallowness of the reaction chamber advantageously facilitates the transfer of heat to the contents of the reaction chamber, for example from a heater, placed close to the substrate, and allows efficient thermal cycling during an amplification reaction. In one embodiment, the reaction chamber can be manufactured so that the ratio of the surface area of the walls of the reaction chamber to the volume of this reaction chamber varies from about 3 mm2 / μl to more than about 10 mm2 / μl . As the ratio of surface area to volume increases, the heat transfer through the substrate and the efficiency of the thermal cycling of the reaction are improved. However, the potential inhibitory effects of the substrate walls in the amplification reaction may also increase, depending on the material from which the substrate is constructed. Therefore, compositions are provided which are useful in decreasing the inhibitory effects of wall surfaces, such as silicon surfaces, in reaction chambers in which such treatment is guaranteed.

Compositions provided to decrease the inhibition of an amplification reaction by a wall of the reaction chamber, can be adhered covalently or noncovalently, on the surface of the chamber. Alternatively, a composition in solution can be supplied in the reaction chamber during an amplification reaction. In one embodiment, flow channels and medium-scale reaction chambers can be fabricated on a silicon substrate. The walls of the reaction chambers and / or the channels can then be coated with a composition which reduces the inhibition of the reaction by the silicon surfaces in the device. For example, the silicon surfaces of the device can be coated with any of a range of silanization reagents, available in the art, as described herein.

In one embodiment, the devices of the invention can be used to carry out a polymerase chain reaction (PCR) within the reaction chamber. The chamber is provided with reagents required for this polymerase chain reaction, which includes the sample polynucleotide, a polymerase, such as Taq polymerase, nucleoside triphosphates, a first sizing hybridizable with the sample polynucleotide and a second hybridizable sizing. with a sequence complementary to the polynucleotide, in which the first and second sizes define the terms of the polynucleotide of the polymerized product. The reagents can be added to a sample and then delivered through a gateway to a medium-scale reaction chamber, or the reagents can be delivered to the reaction chamber regardless of the sample, through a gateway separated.

The polymerase chain reaction can be carried out according to the methods established in the art (Maniatis et al., Molecular Cloninq: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). Any thermostable polynucleotide polymerase, available in the art, can be used. The device may include elements for thermal cycling of the contents of the chamber, so that, in each cycle, the temperature is controlled to dehybridize the double-stranded polynucleotide and produce a single-stranded polynucleotide, and then strengthen the sizes and make possible the polymerization of the polynucleotide.

Although amplification of the polynucleotide by the polymerase chain reaction has been described and exemplified here, those skilled in the art will appreciate that the devices and methods of the present invention can equally be used effectively for a variety of other amplification reactions. polynucleotides. Such additional reactions may be thermally dependent, such as the polymerase chain reaction, or they may be carried out at a single temperature (eg, nucleic acid sequence base amplification (NASBA)). Also, such reactions can employ a wide variety of amplification reagents and enzymes, including DNA ligase, T7 RNA polymerase and / or reverse transcriptase, among others. Additionally, the denaturing of the polynucleotides can be achieved by known chemical and physical methods, alone or in combination with temperature change. The polynucleotide amplification reactions that can be practiced in the device of the invention include, but are not limited to: (1) methods of amplifying target polynucleotides, such as self-sustained sequence replication (3SR) and amplification of cord displacement (SDA)); (2) methods based on the amplification of a signal attached to the target polynucleotide, such as the amplification of "branched-chain" DNA (Chiron Corp. Emeryville, CA., USA); (3) methods based on the amplification of the probe DNA, such as the ligase chain reaction (LCR) and the amplification of the QB replicase (QBR); (4) methods based on transcription, such as ligation-activated transcription (LAT) and nucleic acid sequence base amplification (NASBA); and (5) various other amplification methods, such as repair chain reaction (RCR) and cycling probe reaction (CPR) (for a summary of these methods and their commercial sources, see pages 2 to 7 of The Genesis Report, DX, Vol.3 No. 4, Feb. 1994, Genesis Group, Montclair, NJ, USA.).

The capacity of the devices of the invention is small, making it possible to perform tests in very small quantities of a liquid sample (for example less than 50 μl and preferably less than 10 μl). The medium scale flow systems of the devices can be microfabricated with microliter volumes or alternatively volumes of nanoliters or less, which advantageously limit the amount of the sample and / or the reagent fluids required for an assay. The devices can be used to carry out a variety of polynucleotide analyzes in an automatic, sensitive and rapid manner, including analysis of the polynucleotides in cells or in solution. At the end of the test, the devices can be cleaned and reused, or discarded. The use of disposable devices eliminates contamination and reduces biological hazards. The sample and the reaction mixture, at all times, can remain buried and the low volumes simplify the disposition of the waste. B. Substrate Manufacturing Analytical devices comprising a solid substrate and, optionally, a cover on the substrate, can be designed and manufactured with flow channels and reaction chambers of medium scale, of a wide range of materials. The devices can, optionally, be made of a material which can be easily sterilized. Silicon supplies a useful material, due to the well-developed technology, which allows its precise and efficient manufacture, but a wide range of other materials can be used within the scope of the invention. Other materials that may be used include, for example, gallium arsenide, indium phosphide, aluminum, polysilicon, silicon nitride, silicon dioxide, polyimide, and thermal pairs such as chromium / aluminum, as well as than quartz, glass, diamond, polycarbonate, polystyrene and other polymers, such as polytetrafluoroethylenes. Other possible materials include superalloys, zircaloy, steel, gold, silver, copper, tungsten, molybdenum, 5 tantalum, KOVAR, ceramics, KEVLAR, KAPTON, MYLAR, brass, sapphire, or any of a range of plastics and organic polymeric materials, available in art.

The doors, the medium-scale flow system, which include the flow channels of the sample and the reaction chambers, and other functional elements, can be manufactured cheaply in large quantities from, for example, a silicon substrate. , by any of a variety of micromachining methods, known to those skilled in the art. Micromachining methods available include film deposition processes, such as chemical vapor deposition, laser-based fabrication or photolithographic techniques, such as the processes "" UV, X-ray, LEAGUE and plastic molding, or chemical etching methods, which can be performed by either wet chemical processes or plasma processes. (See, for example, Manz et al., Trends in Analvtical Chemistry, 10; 144-149 (1991)). The arrangement of the channels, chambers and multiple doors facilitate the sequence, properly synchronized, and the correct addition of the sample and reagents within the device volumetrically. In one embodiment, channels or flow chambers of various widths and depths can be manufactured, with at least one having a medium scale dimension, on a substrate, such as silicon. This substrate, which contains a medium-sized flow channel and reaction chamber, may be covered and sealed with a glass cover attached, anodally bonded or otherwise adhered to "? Or substrate.Other clear or opaque roofing materials can be used.Alternatively, two substrates can be walled, or a substrate can be walled between two glass covers.The use of a transparent cover results in a window that facilitates the dynamic vision of the contents in the medium-scale flow system.

Other manufacturing methods can be used.

C. Methods of Passivation A composition can be supplied within the amplification reaction chamber or a flow channel, of medium scale, to passivate the surfaces of the wall, that is, to decrease the inhibition of the amplification reaction by the wall surfaces, if the nature of the wall material needs this treatment. The composition can be adhered to the surface of the reaction chamber or the walls of the channel, either covalently or non-covalently. For example, the wall surfaces can be coated with any of a range of silanization agents, known in the art. Alternatively, the composition may be provided in the chamber in solution, together with the sample polynucleotide and the amplification reagents, during an analysis. Medium-scale reaction chambers can be manufactured when the ratio of the surface area of the wall defining the reaction chamber to the chamber volume varies from around 3 mm2 / μl to more than 10 mm2 / μl or, optionally, higher of 20 mm2 / μl. As the ratio of the surface area to the volume increases, the heat transfer to the reaction chamber through the substrate is improved, and an amplification reaction, thermally dependent, can be cycled more rapidly. However, concurrently, the inhibitory effect of the wall surfaces can also be intensified as the ratio of surface area to volume increases. Compositions for reducing the inhibition of the expansion reaction by a wall of the reaction chamber are particularly useful in chambers with a high ratio of surface area to volume, for example greater than about 3 mm2 / μl. Those skilled in the art will appreciate that the compositions and passivation methods described herein are applicable to only certain materials, where it has been observed that amplification reactions can be improved by the passivation reaction of the chamber surfaces. Some materials considered for use in the devices of the invention are naturally inert and thus will not benefit from the passivation treatments described herein.

The substrate may comprise a heat conductive material, such as silicon or glass. The reaction chamber and / or channel walls can be passivated by coating the surface with a silane, using a silanization agent available in the art. Useful silanization agents include dimethylchlorosilane (DMCS), dimethyldichlorosilane (DMDCS), hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). These chlorosilanes can react covalently with the surface hydroxyl groups on the walls comprising the silicon or other material that could potentially interfere with the reaction, for example, by binding to the sample polynucleotide or the amplification reagents.

Additionally, the walls of the reaction chambers and / or the channels may be provided with a silicone coating, using a commercially available siliconizing-cyclin reagent, such as Aquasil ™ or Surfasil ™ (Pierce, of Rockford, IL. , USA :), or Sigmacote ™ (Sigma Chemical Co., of St. Louis, MO., USA.). Siliconization reagents available from commercial manufacturers, such as Pierce (Rockford, IL., USA) or Sigma Chemical Co. (St. Louis, MO., USA) are organosilanes containing a hydrolyzable group, which can be hydrolysed in solution for forming a silanol, which can polymerize and form a film on the surface of the chamber, and can react with hydroxyl groups on the surface of the chamber, so that the film adheres strongly over the entire surface of the chamber.

The coating may also include a macromolecule, associated in a non-covalent or covalent manner with the coating, to further reduce the inhibitory effects of the reaction chamber wall in the amplification reaction. amino acid, or polymers such as polyvinyl pyrrolidone, polyadenylic acid or polymaleimide, or compositions such as maleimide, etc. Other useful macromolecules include, for example, poly-L-alanine, poly-L-aspartic acid, polyglycine , poly-L-phenylalanine or poly-L-tryptophan. supplying a silicon oxide film in the reaction chamber and / or channel walls, to reduce the inhibition of the amplification reaction by the silicon wall surfaces. The silicon oxide film can be formed by a thermal process, in which the substrate is heats in the presence of oxygen. Alternatively, an intensified plasma oxidation or chemical vapor deposition process may be used. Additionally, the reaction chamber and / or the walls of the channel can be coated with a polymer, such as polyvinyl chloride. For example, a solution of polyvinyl chloride in chloroform can be added to the medium-scale flow system, and then the coating can be formed by evaporating the solvent.

In another embodiment, a blocking agent, such as a polynucleotide or polypeptide, can be added to the chamber. For example, genomic DNA or polyadenylalic acid can be added to the solution within the reaction chamber, at a concentration preferably higher than the concentration of the sample polynucleotide. This allows the polynucleotide to occupy any site on the wall surfaces that could potentially bind to the sample polynucleotide or test reagents and reduce the reaction yield. If the DNA or RNA is used as the blocking polynucleotide, it must be effectively deprived of sequences that could interfere with the amplification reaction (i.e., it must substantially contain only sequences unrelated to those of the sample polynucleotide). Other compositions that can be used as blocking agents include bovine serum albumin or an amino acid polymer or polymers such as polyvinylpyrrolidone or poly-aleimide, or compositions such as maleimide.

D. Thermal Cycling A polynucleotide amplification reaction, such as a PCR reaction, can be carried out within the reaction chamber of device 10, shown in Figures IA and 2A. An alternative embodiment of the device 10 is illustrated in Figures IB and 2B. As illustrated schematically in Figures IA, Ib, 2A, and 2B, the arrangement? The substrate 10 may include a silicon substrate 14, microfabricated with inlet ports 16, a medium-scale flow channel 20 and the reaction chamber 22. The sample of the polynucleotide and the reagents required for the polymerization reaction are aggregated and the products are removed (if is necessary) through the flow channel 20 from the reaction chamber 22 through the inlet ports 16, which are manufactured at one end of the flow channel 20. The substrate 14 is covered, for example, with a glass cover or plastic 12. The device 10 can be used in combination with an instrument, such as the instrument 50, shown schematically in Figure 3A. This instrument 50 includes a nesting site 58 for retaining the device 10 and for registering the doors, for example the doors 16, in the device 10, with a flow line 56 in the instrument. A pump 52 in the instrument 50 is used t, to deliver a sample and / or reagents from the flow line 56 in the instrument to the reaction chamber 22, by means of the entry doors 16.

The instrument 50 may include a heating / cooling element 57, for controlling the temperature inside the PCR chamber, for example, an electric heating element and / or a cooling coil. This electrical heating element can alternatively be integrated in the substrate 10, with contacts for "- * 0" the energy that are coupled with the corresponding electrical contacts in the instrument, below the reaction chamber 22. Alternatively, as shown in FIG. Figure 3B, the instrument may include a heating element 53, such as a laser heater, Peltier, or a source of electromagnetic energy, arranged on or adjacent to the reaction chamber in the device 10. The heater can also be arranged inside the instrument, below the reaction chamber. A microprocessor inside the instrument, can be used to regulate the heating element, for the purpose of providing a temperature cycle in the amplification chamber, between a temperature suitable for dehybridization, for example 94oc, and a temperature suitable for annealing or strengthening and polymerization, for example 40 to 60ac, for the strengthening and 70 to 752C for the polymerization. A thermal pair, thermistor or resistance thermometer can also be provided on the substrate, in electrical contact with the instrument, to allow the microprocessor to detect and maintain the temperature cycles in the reaction chamber. The heating and detection can advantageously be combined using a single element, for example a resistance thermometer, for both purposes, combining heating and detection simultaneously or in a multiple base.

A cooling element, such as a miniature thermoelectric heat pump (Materials Electronic Products Corporation, of Trenton, New Jersey, USA), a Peltier thermal pair or a Joule Thompson cooling device, may also be included in the instrument, to adjust the temperature of the reaction chamber. In another embodiment, in the instrument shown in Figure 3B, the temperature of the reaction chamber can be regulated by a laser pulse in synchronism, directed to the reaction chamber through the glass cover 12, to thereby allow the heating and sequential cooling of the sample at the temperatures required for the amplification cycle of the polynucleotide. Additionally, heating and cooling can be advantageously combined by the use of Peltier thermal pairs, to supply both of these functions. The thermal properties of silicon make possible a rapid heating and cooling cycle. It is advantageous to use the reaction chambers manufactured with a high ratio of surface area to volume, for example greater than 3 mm2 / μl, since the transfer of heat to and from the contents of the reaction chamber is facilitated. This increases the efficiency of thermal cycling and the productivity of the amplification reaction within the chamber. As illustrated schematically in Figures 4, 5 and 6A, a medium-sized polynucleotide amplification reaction chamber can be microfabricated with multiple sections, eg, two sections 22A and 22B, connected by the flow channel 20B. In this embodiment, the section 22A is heated to, or maintained at, a temperature suitable for dehybridization and the sections 22B are heated to, or maintained at, a temperature suitable for annealing or strengthening and polymerization. During an analysis, the device 10 can be placed inside the instrument 50 (Figure 6A). This instrument 50 is provided with elements 57 for controlling the temperature of the sections of the reaction chamber. Alternatively, a laser can be used to heat the sections. A thermal pair or other device that senses the temperature can be included in the substrate to inspect the temperatures of the sections of the reaction chamber and its output can be used to control the thermal input, for example with the help of a microprocessor. During the operation, a pump 52 in the instrument is used to deliver the polynucleotide sample and the required reagents from the flow line 56 through the entry port 16A to the section 22A. The pump 52, which can also be controlled by a microprocessor in the instrument, is then used to transfer the sample periodically, between the sections 22A and 22B, through the channel 20B, to carry out a repetitive cycle of amplification reaction. polynucleotides, while door 16B serves as a ventilation. When the reaction is complete, the pump 52 in the instrument 50 can be used to deliver the sample through the door 16B and line 56 in the instrument to the door 59, to recover the product. Of course, three or more chambers can be used, each of which is maintained at a suitable temperature to carry out a particular reaction.

In the device 10, shown in Figures 4, 5 and 6B, a heating element can be used to heat the section 22A to a temperature suitable for dewatering the double-stranded DNA, for example from 942C, while the section 22B and the channel 20B, which connect sections 22A and 22B, are spaced apart from section 22A so that in transporting a sample heated from section 22A to section 22B, the heat is sufficiently dissipated to allow the temperature of the sample to fall at the temperature reqd for strengthening and polymerization, before the sample returns to section 22A for further cycling. This can be easily achieved, since silicon has a relatively high thermal conductivity and the area of the interface between the liquid sample and the substrate is quite high. In this embodiment, the microprocessors in the instrument 50 are used to control the pump 52, which regulates the flow cycle of the sample between the sections 22A and 22B. Thus, a dynamic thermal equilibrium creates a temperature gradient along the flow path between the chambers and the appropriate temperatures are achieved in both, using a single heating source. Other designs are possible. For example, the strengthening and polymerization reactions can be carried out in different sections of a single chamber, placed at different opti- mized temperatures.

E. Fluid Sealer Transfer Doors The devices include a solid substrate fabricated with a medium-scale polynucleotide amplification chamber. The devices further include at least one sample inlet port and a sample flow channel, which connects the entrance port to the reaction chamber. One or more flow gates and channels in the device may be manufactured within the substrate (Figure IA) or in a cover disposed on the substrate (Figure 1C9) This cover may comprise, for example, a transparent material, such as glass or any of a range of plastic materials available in the art The invention provides elements for sealing one or more of the doors during an amplification reaction to impede the evaporation of the liquids during thermal cycling. it supplies a fluid delivery apparatus for the delivery of fluid to and from the reaction chamber, through the door, which is adapted for internal adjustment with and / or internal locking with the door, and which can reversibly seal the door after delivery of fluid to the reaction chamber A syringe or pipette, capable of internal adjustment with and sealing the fluid inlet / outlet door in the substrate, can be used.

As illustrated in Figures 19 to 22, in one embodiment, the cover 12 can be manufactured with a cavity 87 for internal adjustment with and receiving a pipette 86. This pipette 86 can be provided with a tip 84, which includes an opening 88 for transferring fluid from the tip 84 of the pipette through the flow channel 20A in the cover, to the flow channel 20B and the amplification reaction chamber 22 in the substrate 14, when the pipette is internally adjusted in the cavity 87. The tip of the pipette can, optionally, be released from the pipette and can be disposable to prevent contamination between the samples. As illustrated in Figure 20, the opening 88 can be placed on a side wall of the tip 84 of the pipette, to allow this tip, on a pipette fitted internally in the cavity 87 in the device 10, shown in the Figure 22, moves between a first position, which allows the transfer of fluid from the tip through the opening 88 to the flow channel 20A and the reaction chamber 22, and a second position, to allow the opening to face the a wall of the cavity 87, in order to seal the channel 20A and the chamber 22 during a reaction. Additionally, a pressure element can be supplied, which extends from the substrate and is capable of sealing the door by pressing the element 85, as illustrated in Figure 21.

The devices comprising the fluid transfer ports, as described above, can be used for a variety of purposes, in addition to the amplification of polynucleotides. For example, such doors may be employed in a separate device for sample preparation, immunoassay, or both, as described in the also pending, commonly-owned patent application, Serial No. [ unassigned], the description of which has been incorporated herein by reference. F. Detection of Amplified Polynucleotide The amplified polynucleotide, present in the reaction chamber, can be detected by various methods known in the art to detect polynucleotides, such as electrophoresis on an agarose gel, in the presence of ethidium bromide. In one embodiment, the amplified polynucleotide product can be detected directly in the reaction chamber, using reagents, commercially available, developed for that purpose (for example, "Taq Man" ™ reagents, from Perkin Elmer Corporation). The devices may also be provided with an element for detecting the amplified polynucleotide, arranged on either the substrate or on an instrument used in combination with the substrate. The presence of the amplified polynucleotide product in the device can be detected by any of a number of methods, including, but not limited to: (1) inspecting the pressure or electrical conductivity of incoming sample fluids and / or leave the reaction chamber in the medium-scale flow system; (2) forming a detectable complex, for example, by attaching the polynucleotide product to a labeled probe, such as a labeled oligonucleotide or anti-body probe; and (3) electrophoretically separating the polynucleotide product from the reagents and other components of the sample.

Analytical devices can also be used in combination with an instrument to view the contents of medium-scale channels in the devices. The instrument, in this modality, can comprise a microscope to see the contents of medium-scale channels in the devices. In another embodiment, a camera may be included in the instrument, as illustrated in the instrument 60 shown in Figures 17 and 18 schematically. This instrument 60 is provided with a housing 62, a vision screen 64 and a slit 66, for inserting a device into the instrument. As shown in cross section in Figure 18, the instrument 60 also includes a video camera 68, an optical system 70 and a tumbling mechanism 72 for retaining the device 10 and allowing the positioning and angle of the device 10 to adjust manually The optical system 70 may include a lens system for amplifying the contents of the channel, as well as a light source. The video camera 68 and the screen 64 allow changes in the properties of the sample fluid, such as the flow properties or its color, induced by the presence of a polynucleotide amplification product, to be visually inspected and recorded optionally using the instrument. Also, the addition or removal of fluid samples to and from the reaction chambers can be inspected, for example, optically, using the instrument.

In one embodiment, the amplified polynucleotide product can be detected using a detection chamber made in the medium scale flow system in the substrate, in fluid communication with the reaction chamber. This detection chamber is provided with a complexing agent, for example a binder capable of binding to the amplified polynucleotide, to form a detectable complex. This binder part may comprise, for example, a polynucleotide or antibody probe. The detection chamber can be manufactured according to the methods described in the patent application of E. U. A., Serial No. 07 / 877,702, filed May 1, 1992, the description of which is incorporated herein by reference. In another embodiment, the agent forming a complex can be added to the reaction chamber, after completing this reaction, to form a detectable complex in that chamber. The device can be used in combination with a detector, such as an instrument containing a microprocessor, to detect and record the data obtained during a test.

In one embodiment, the medium-scale detection chamber may be provided with an inert substrate, for example, a bead or other particle, capable of binding to the polynucleotide product, to cause detectable agglomeration of the beads, in the presence of the product. of polymerized polynucleotide. The particles induced by the agglomeration can be increased by the binding of a binder part, such as an antibody, to the particle. Antibodies, or other binding portions, capable of binding to the polynucleotide product, can be introduced into the detection chamber or can be coated, either chemically or by adsorption, on the surface of the detection region, or alternatively on the surface of an inert particle in the detection region, to induce binding, giving a positive test for the polynucleotide. Techniques for the chemical activation of silicon surfaces are well developed, particularly in the context of chromatography. (See, for example, Haller in: Solid Phase Biochemistry, WH Scouten, Ed., John Wiley, New York, pp. 535-597 (1983); and Mandenius et al., Anal. Biochem. 170? 68-72 (1988 )). In one embodiment, the binding part may comprise an antibody and the immunoassay techniques, known in the art, in the detection region may be carried out. (See, for example, Bolton et al., Handbook of Experimental Immunoloqyf Weir D.M., Ed. Blackwell Scientific Publications, Oxford, 1986, Vol. 1, Chapter 26, for a general discussion of immunoassays). An optically detectable label, such as a fluorescent molecule or fluorescent bead, can be attached to the binder part, to increase the detection of the amplified polynucleotide product. Alternatively, a second labeling substance, such as a fluorescent labeling antibody, can be delivered through the flow system for binding to the polynucleotide / binder part complex, in the detection region, to produce a "sanh", which it includes an optically detectable part, indicative of the presence of the analyte. The binding of the amplified polynucleotide to the binder part, in the detection region, can be detected, for example optically, either visually or by a machine, through a transparent window, arranged over the detection region. In one embodiment, the production of the amplified polynucleotide can be detected by the addition of a dye, such as ethidium bromide, which exhibits increased fluorescence at binding to the double-stranded polynucleotide. Higuchi et al., Biotechnoloqy. 10: 413 (1992).

The detection chamber may also be provided with a labeled complementary polynucleotide capable of binding to one of the strands of the amplified polynucleotide, for example, a labeled polynucleotide, immobilized on a bead, to enable detection of the amplified polynucleotide product by means of the agglutination of pearls. Hybridization techniques of the polynucleotide, known in the art, can be used. Maniatis et al., Molecular Cloninq A Laboratorv Manual. 2nd Ed., Cold Spring Harbor Press, 1989); Vener et al., Anal. Chem .. 19.8: 308-311 (1991 = .The polynucleotide probes can be attached, for example, to a submicron size latex particle Wolf et al., Nucleic Acids Research 15: 2911-2926 (1987). .

In another embodiment, the polynucleotide amplification products can be separated from the reactants and other components of the original sample by electrophoretic methods, adaptable to the medium-scale devices of the invention. These techniques are well known in the art. For example, microlithographic arrays have been fabricated in SIO2 in order to electrophoretically separate the DNA molecules (Volkmuth and Austin, Nature 358: 600-602, 1992). Various combinations of channels for carrying out capillary electrophoresis to separate several biological molecules (Harrison et al., Science 261: 895-897, 1993).

In this embodiment, the devices of the invention can be fabricated with a detection region comprising a microlithographic array or a series of channels, and electrophoresis can be performed on the chip, providing an appropriate electric field across the region (eg. example, placing the microelectrodes at either end of the detection region). The region is provided «« any end with a loading area, to collect the contents of the reaction chamber before electrophoresis. The various components of the reaction mixture are then separated from each other by electrophoresis. The amplification product of the polynucleotide can be identified by comparing its size with molecules of known size. In one embodiment, size markers are entered into the detection region (by means of an access door), electrophoretically separated, and the results recorded and stored (for example, in the memory of a computer). The contents of the reaction chamber are then transferred to the detection region, separated electrophoretically, and the recorded results are compared with the results of the electrophoresis of the size markers. In this manner, the polynucleotide amplification product can be identified, as can be purified for later use, without the use of inert substances and binding portions to capture the polynucleotide product. The amplification of the polynucleotide can also be detected using a sensing region sensitive to flow restriction, caused by the presence of the polynucleotide produced in the reaction chamber, as described in the US patent application, Serial No. 08 / 250,100, the description of which has been incorporated herein by reference. The presence of the amplified polynucleotide can also be detected by means of the pressure or electrical conductivity of the fluid samples, which enter and leave the flow system. The conductivity can be measured, for example, by using electrical contacts, which extend through the substrate and which correspond to the electrical contacts in an instrument used in combination with the device. The electrical contacts can be manufactured by known techniques, such as by various methods of melting the thermal gradient zone. (See Zemel et al in: Fundamentals and Applications of Chemical Sensors, D. Schuetzle and R. Hammerle, Eds., ACS Symposium Series 309, Washington, DC, 1986, p.2) The polynucleotide amplified in the reaction chamber may be detected by inspecting the pressure of the sample fluids. For example, in a device 10, nested in the instrument 50, schematically illustrated in Figure 6A, the pressure detectors 54, connected to the sample fluid entering and leaving the medium-scale flow system, through the gates 16 , will allow the detection of pressure drops, caused by the presence of the polymerized product and the resulting obstruction or restriction of flow. A medium-scale pressure sensor can also be manufactured directly on the silicon substrae. Angelí et al., Scientifis American 248: 44-45 (1983).

The amplification of the polynucleotide can be detected by the use of a medium-scale flow system, sensitive to flow restriction, constructed with a "fractal" model, ie a model of divergent flow channels. The channels can be manufactured on a silicon substrate with dimensions that are progressively reduced, providing increasingly narrow flow channels. It will be appreciated by those skilled in the art that, although bifurcated channels are exemplified, the devices can be manufactured with different numbers of parallel flow channels or other symmetrical or asymmetric patterns of the flow channels, with reduced cross-sectional areas. Alternatively, a single channel comprising a narrow region may be used, as described in commonly owned U.S. Patent Application Serial No. 08 / 250,100 (incorporated herein by reference).

Figure 7 shows a schematic plan view of a substrate 14 made with a system of flow channels 40 connected via channel 20 to doors 16 and to a reaction chamber, comprising sections 22A and 22B. The presence of the amplified polynucleotide product in a sample will influence the flow characteristics within the flow channels. O Channels 40 in this mode are arranged symmetrically and have progressively narrower diameters towards the center of the pattern. The flow through this channel model is sensitive to changes in fluid viscosity, caused by the presence of the amplified polynucleotide product. Alternatively, a more complex channel flow system can be used, as illustrated in Figure 13. Figure 13 illustrates a pair of flow channel systems, 40A and 40B. The channel system 40A is constructed with progressively narrower flow channels towards the center of the model, resulting in increased sensitivity to flow restriction. The flow restriction can be detected, for example optically, through a transparent cover over the detection region. Alternatively, one or more pressure sensors can be used to detect changes in pressure, due to changes in fluid properties, caused by the accumulation of the amplified polynucleotide at or beyond the restricted flow paths. The changes in conductivity in the polynucleotide amplification can also be easily detected through the sensors of the electrical conductivity, in contact with the flow region. For example, the obstruction of the restricted region 40, which blocks the flow from the entry door 16A to the door? of output 16B, can be detected by a conventional conductivity probe 17, whose output is indicative of the presence or absence of the aqueous fluid in the external flow channel. Binders, such as labeled antibodies or polynucleotide probes, can be included in the restricted flow region, eg, immobilized, or in a solid phase reagent, such as a bead, to bind to a polynucleotide amplified to induce restriction of flow reduction in the restricted flow path.

In one embodiment, the medium-scale flow system includes a chamber for the destruction (lysis) of cells from a sample being prepared, for analysis of downstream polynucleotides. The device may also include a region adapted to separate a particular type of cell in a population of heterogeneous cells. The cell separation region includes immobilized binding moieties in structures within the substrate, which bind, selectively and reversibly, to a target cell, by means of a molecule characteristic of the cell surface, such as a protein. Other cells in the sample pass downstream and are channeled into a sump or through an exit port. The flow can be continued to wash the cells, for example with a regulation flow. At flow rates? and higher pressures, or changing the composition of the solvent, the washed cells are released from the structures on which they are immobilized and then move from the downstream cell separation region to a lysis element, which destroys the cells before the analysis of the PCR of the intracellular RNA or DNA.

The destruction (lysis) element of the cells is arranged in the flow path, between the separation region of the cells (if any) and the polynucleotide amplification reaction chamber, to allow These cells are destroyed before the analysis of an intracellular polynucleotide. As illustrated in Figure 9, the lysis element of the cells can comprise protrusions 90 that pierce the cell membrane, which extend from a surface of a flow channel 20. According to FIG. the flow of the fluid is forced through the prominence 90 ^ ^ of perforation, the cells are broken. In another embodiment, the lysis element of the cells can simply comprise a region with restricted dimension in cross section, which carries out the destruction of the cells by applying a sufficient flow pressure. This cell lysis element can also comprise pieces with sharp edges of silicon, trapped inside the chamber of lysis of the scaphoid. median. An instrument, which includes elements, such as a pump, to force the sample containing the cells within the cell lysis element, causes the destruction of the cells by applying a sufficient flow pressure and then delivers the sample to -through the flow system to the reaction chamber. In another embodiment, the lysis element of the cells may comprise a cell killing agent. Cell destruction agents, known in the art, can be used.

Reagents can be added to the reaction chamber from a separate inlet port on the substrate, in fluid communication with the reaction chamber. A filter, microfabricated in the deluxe channel on the substrate, can be used to filter the cellular waste, before the analysis of the polynucleotide. In one embodiment, shown in Figures 14, 15 and 16, the filter 24 in the device 10 may comprise a scale flow channel Medium, reduced diameter compared to channel 20. In operation, the sample flows from the flow channel 20A thereof through the filter 24. The filtrate of the sample then leaves the filter 24 and flows through the channel 20B. The filter 24 is microfabricated with straight or tortuous channels, having preferred depths and widths in the order of 0.1 to 50 μm, and the extension flow channels, 20A and 20B, which have maximum depths and widths in the order of approximately 500 μm. . As illustrated in Figure 8, the surface of a flow channel 20 may also include protrusions 80, which constitute a cell screen for separating cells by size, upstream of the PCR analysis chamber. As the cell samples flow through the flow channel, typically under reduced pressure, only cells suf fi ciently small to pass between the prominences 80, arrive at the functional elements, downstream. These cells can then be delivered through a lysis region of cells and then into a polynucleotide amplification reaction chamber, for analysis.

In another embodiment, paramagnetic or ferromagnetic beads can be supplied within the medium scale flow system, which can be moved along the flow system by an external magnetic field, for example inside the instrument. These beads can be used to transport the rectifiers between the functional elements in the device, or to displace a sample, a reagent or a reaction mixture. In one embodiment, a polynucleotide probe can be immobilized on said magnetic beads to enable them to bind to the amplified polynucleotide. These magnetic beads, comprising a coating of the polynucleotide probe, can be transported through the flow system to the reaction chamber at the end of an assay, for binding to the amplified polynucleotide product. This bound amplified polynucleotide can then be transported in the magnetic beads to a detection or purification chamber in the flow system, or to a collection door.

G. Exemplary Apparatus One embodiment of the invention, illustrated in Figure 10, comprises a device 10 that includes a microfabricated substrate 14, with a medium-scale polynucleotide amplification chamber, having sections 22A and 22B, which are connected by the path of flow 20B. The device 10 is used in combination with an instrument, such as instrument 50, shown in Figure 6A, which contains a nesting site for retaining the device. The instrument 50 is provided with flow paths 56 corresponding to the doors 16A, 16B, 16C and 16D in the device 10. This instrument also includes valves that allow the doors 16A, 16B, 16C and 16D to be mechanically opened and closed. Gate 16E is included to add reagents to detection chamber 22C. In one embodiment, the flow systems of the devices can be maintained at a full volume hydraulically, and the valves in the instrument, or alternatively in the devices, can be used to direct the flow of the fluid. Sections 22A and 22B of the PCR chamber are heated to, for example, 94fiC and 40 to 65QC, respectively, to supply a melting temperature and an annealing or strengthening temperature, as required for PCR and other reactions of amplification that are thermally dependent. As discussed above, the sections of the reaction chamber can be heated by means of an electrical element, integrated in the substrate, below the sections, which can correspond to the electrical elements in the instrument. Alternatively, an optical laser can be used to heat the sections of the reaction chamber through a glass cover, disposed on the substrate. A heat sensor can be supplied to the substrate, in electrical contact with the instrument. A microprocessor in the instrument can be used to control the temperature of the sections of the reaction chamber and the flow of fluid in the flow system.

The flow channels of the device 10 are equipped with filters 24A, 24B and 24C. The 24A filter is designed to prevent cellular debris and other unwanted particulate matter in the sample from entering the reaction chambers. The filters 24B and 24C are included for the purpose of restricting the complexing agent (i.e., the beads 92) within the detection chamber 22C. Therefore, filters 24A, 24B and? C do not need to be identical. During the operation, for a thermally dependent amplification reaction, such as PCR, initially, with the channels and chambers filled with the regulator, the door 16A and 16C open, while the doors 16B and 16D are closed. A pump 52 in the instrument delivers the sample fluid and / or reagents required for amplification, such as Taq polymerase, sizes and nucleoside triphosphates, through the gate 16A through the filter 24A, to the section 22A of the reaction chamber. The door 16A is then closed and the 16B is opened, and the pump 52 in the instrument is used for the reciprocal movement of the fluid flow in cycles, through the flow channel 20B, between the section 22A, where the deshibridi occurs. -zation of the polynucleotide, and section 22B, where the strengthening and polymerization occurs. Gate 16C can be used to ventilate the system and also, optionally, to deliver Taq polymerase, nucleoside triphosphates, sizing and other reagents. When the cyclic amplification reaction is finished, for example within 30 to 35 cycles, the door 16C is closed, the door 16D is opened and the pump in the instrument is operated to deliver the reaction products from the sections, 22A and 22B , from the reaction chamber to the detection chamber 22C, which contains, for example, a polynucleotide complementary to the amplified sensitivity and / or antisensitivity strand, immobilized in the beads 92. The amplification product is detected by observing the agglutination of the beads 92, for example visually through a transparent cover, disposed over the detection region.

Another embodiment is illustrated in Figure 10B. The function, structure and operation of this device is similar to those shown in Figure 10A, except that it comprises a detection region 26, where the channels or arrays (not shown) can be manufactured to perform the electrophoretic separation of the amplification product of the polynucleotide. The device includes a gate 16E for adding or removing materials from the detection region. The device is used in combination with an instrument similar to instrument 50, shown in Figure 6A, which comprises an element for applying an electric field through the detection region 26.

Another embodiment is illustrated in Figure 11. The function, structure and operation of this device are identical to those shown in Figure 10, except that it comprises a single reaction chamber 22A. The device is used in combination with an instrument, such as instrument 50 shown in Figure 3A. The device includes elements for heating and cooling the reaction chamber 22A alternatively, at a temperature required for melting and at a temperature required for strengthening and polymerization.

During the operation, the instrument is used to deliver a sample containing the polymerase and other reagents required for the reactions, such as PCR, through the gate 16A to the reaction chamber 22A. The doors 16A and 16D are then closed using a valve connected to the instrument. The heating element in the instrument is then used for the thermal cycle of the reaction chamber between a temperature suitable for dehybridization and temperatures suitable for annealing or strengthening and polymerization. When the amplification cycles are completed, the doors 16B and 16D are opened and the sample is delivered to the detection chamber 22B, which contains a polynucleotide probe, for example immobilized on the beads 92 or other solid substrate. A positive assay for the polynucleotide is indicated by the agglutination of the solid substrate (for example the beads) in the detection chamber. In the embodiment shown in Figure 10B, the contents of the sections, 22A and 22B, of the reaction chamber are delivered to the detection region 26, where the polynucleotide product is separated and identified electrophoretically.

The invention will be better understood from the following non-limiting examples.

Example 1 A polymerase chain reaction was carried out in the device illustrated schematically in Figure 11, provided with a medium-sized reaction chamber 22A. To perform a PCR analysis and detect a polynucleotide in a cell, a sample of cell lysate was added to the buffered solution of Taq polymerase, nucleoside triphosphates, polynucleotide sizes and other reagents, required for PCR. The lysate of the cell sample was delivered via the instrument through the entry port 16A to the reaction chamber 22A of the PCR. The doors 16A and 16D were closed by means of the valves included in the instrument. A microprocessor and a temperature control element were used in the instrument to carry out a temperature cycle in the reaction chamber 22A, between 942C for the dehybridization of the polynucleotide, 40 to 602C for annealing or strengthening and to 75se for the extension of the sizing.

After completion of the polymerase chain reaction, gates 16B and 16D were opened, and the pump in the instrument was connected to gate 16B, used to deliver the sample from the PCR reaction chamber 22A, through the channel of flow 20B to the detection chamber 22B. This detection chamber 22B contains the beads 92, which include a complementary polynucleotide immobilized on the surface, capable of binding to the amplified polynucleotide. The agglutination of the beads, caused by the hybridization reaction between the amplified polynucleotide and the complementary polynucleotide, was observed through a window arranged on the detection region 22B, and provides a test of the presence of the amplified polynucleotide product. .

Example 2 Figure 12 schematically illustrates a device 10 that includes a substrate 14, used to separate a nucleic acid from a subpopulation of cells in a mixture in a biological fluid sample, and then carry out an assay for a particular nucleotide sequence. Microfabricated on the device 10 is the medium-scale flow path 20, which includes a cell separation chamber 22A, a cell destruction chamber 22B (lysis), a filter region 24, a PCR reaction chamber , comprising sections 22C and 22D, and a region 40 for detection of restricted flow. The medium-scale flow system 20 is also provided with fluid inlet / outlet ports, 16A, 16B, 16C and 16D. The device is used in combination with an instrument, such as instrument 50, shown in Figure 6A.

Initially, the valves in the instrument are used to close the doors 16C and 16D, while the doors 16A and 16B are opened. A sample, which contains a mixture of cells, is directed to the entry port 16A of the sample by a pump 52 in the instrument, and flows through the medium-scale flow path 20, into the separation chamber 22A. This chamber 22A contains binding portions immobilized on the wall of the chamber, which selectively binds to the surface molecule in a desired type of cell in the sample. The remaining cellular components leave the substrate through gate 16B. After binding to the desired cell population in chamber 22A, the flow with the buffer is continued, to wash and ensure isolation of the cell population. Then door 16B is closed and 16C is opened. The flow then increases sufficiently to dislodge the immobilized cells. The flow is continued, the cells are forced through the prominences 90 that perforate the membranes, in the chamber 22B, which open by tearing the cells, releasing the intracellular material. The flow continues to pass filter 24, which filters out large components of the cell membrane and other debris, to the section 22C of the medium-sized PCR chamber, which is connected to the section 22D of the PCR chamber via the flow channel 20B. The Taq polymerase, the sizing and other reagents required for the PCR assay, are then added to section 22D through gate 16B, from a corresponding gate, and the flow path in the instrument, which allows mixing of the soluble intracellular components from the separate subpopulation of cells and the PCR reagents. With the door 16A closed, a pump is used, in the instrument, connected by the gate 16B, for the cycle of the PCR sample and the reagents through the flow channel 20B, between the sections 22C and 22D, set to , for example, 94 and 65se, respectively, to carry out several cycles of polynucleotide fusion, annealing and polymerization, which make possible the amplification of the product polynucleotide. Alternatively, all the doors can be closed during the amplification reaction and the thermal cycle can be performed as described in Example 1 above. The valves in the instrument are used immediately to open the door 16D. The pump in the instrument, connected to gate 16B, is then used to direct the amplified polynucleotide, isolated from the cell population, to a detection region, comprising bifurcated series of flow paths 40. The flow restriction in the detection region 40 serves as a positive indicator of the presence of the amplified polynucleotide product and is detected optically through a glass cover, disposed over the detection region.

Example 3 The amplification of a sample polynucleotide (bacteriophage lambda DNA) in a medium-sized reaction chamber having dimensions of 80 μm in depth, 8 mm in width and 14 mm in length, made on a silicon substrate was examined and passivated using different passivation methods.

To perform the reaction, PCR reagents (eg, nucleotides, AmpliTaq DNA polymerase, size and sample of bacterio-phage lambda DNA) were mixed in tubes and transferred to the medium-sized reaction chamber. , on the silicon substrate. The final concentrations of the reagents were: nucleotides, 200 mM each, Taq polymerase, 0.25 U / 10 ml; sizing, 1.0 mM each; DNA model, 0.1 ng per 10 ml. The thermal cycling (normally 35 cycles) was carried out automatically, using a Peltier heater-cooler, controlled by computer.

The results of this PCR reaction, using different methods of passivation of the walls of the reaction chamber, of medium scale, made in the silicon substrate, are illustrated in Figure 23. This Figure 23 is a drawing of a gel of agarose containing ethidium bromide, after electrophoresis of the reaction products in the gel. The bands in the gel are as follows: (l and 7) molecular weight markers (1000, 750, 500, 300, 150 and 50 bp); (2) products of a control amplification reaction conducted in a Perkin-Elmer thermal cycle device, Model 9600; (3) products of an amplification reaction in an untreated reaction chamber; (4) products of an amplification reaction in the reaction chamber having a thermal film of silicon oxide on the surfaces of the wall, (5) products of an amplification reaction in the reaction chamber, having a coating of silicon nitride on the surface formed by a process of chemical vapor deposition, intensified with plasma (PECVD), which uses a mixture of silane and ammonia; and (6) products of an amplification reaction in a reaction chamber, which has a surface coating of a silicon oxide film, formed by the PECVD process. Methods for thermal oxidation of silicon are described, for example, by Runyan and Bean, in "Semiconductor Interated Circuit Processing Technology", Addison-Wesley Publishing Co., 1990, Chapter 3 The methods for depositing films on the surfaces by the process of chemical vapor deposition, assisted by plasma, are described, for example, in Sze, "VLSI Technology" (VLSI Technology) McGraw-Hill Book Co., 1983, Chapter 3 .

As illustrated in Figure 23, the reaction product increased substantially in the silicon reaction chambers, provided with a thermal oxide coating (belt 4) or a PECVD oxide coating (belt 6), as compared to the carbon dioxide chamber. untreated silicon reaction (strip 3). In contrast, the silicon nitride coating (strip 4) did not have a positive passivation effect on the amplification reaction.

Example 4 A medium-sized polynucleotide amplification reaction chamber fabricated on a silicon substrate was provided with a coating to passivate the wall surfaces of the chamber. A silicon substrate was provided, which was manufactured with fluid inlet and outlet ports and a medium-scale flow system, which includes a flow channel, in fluid communication with the doors, and a reaction chamber of amplification of polynucleotides. This amplification reaction chamber, of medium scale, has dimensions of 80 μm depth, 8 mm width and 14 mm length, and was treated with a siliconization reagent and, optionally, a macromolecule, to form a coating , which passivates the surface of silicon. The amplification chamber was filled with a silico-nization reagent, such as AquaSil ™ or Surfasil ™ (Pierce, of Rockford, IL., USA, or Sigmacote ™ (Sigma Chemical Co., of St. Louis, MO., USA.), using a 100 μl pipette and applying a Negative pressure to the exit hole of the chip The siliconization reagent was allowed to remain on the chip for at least 30 minutes, at room temperature A constant negative pressure was applied to the exit door to remove the siliconization reagent, by At least about four hours, about 100 μl of distilled water or a 0.1 M TE regulator, were delivered through the flow system through the entrance door to the amplification chamber, using a 100 μl pipette, and a pressure was applied negative to the exit door The washing was repeated for about 6 times After the last washing, a negative pressure was applied to the exit door for around 10 to 15 minutes, to empty the channels.

Alternatively, the surface of the amplification chamber is passive with a silanization reagent, such as dimethyldichlorosilane (DMDCS) or dimethylchlorosilane (DMCS). Methods that can be used to treat surfaces with siliconization or silanization agents are described, for example, in Pierce, "Instructions: Siliconizing Agents". of Rockford, IL. , USA, 1993, whose description is incorporated herein by reference. The amplification reaction chamber was then optionally filled with a solution of a blocking agent, comprising macromolecules (about 10 mg / ml macromolecules in 0.1 M Tris buffer, pH 8.6), for example an amino acid polymer ( see Table 2), through the entrance door, using a 100 μl pipette and applying a negative pressure to the exit door. The solution of macromolecules was allowed to remain inside the amplification chamber for at least about one hour at 4 ° C. A negative pressure was then applied to the exit door of the device for about 10 to 15 minutes. This provides a coating of the macromolecule, associated non-covalently with the surface treated with silicone.

Example S The effectiveness of different coatings in reducing the inhibitory effect of silicon in the polynucleotide amplification reaction was tested.

A sample of silicon powder was coated with Surfasil ™ (Pierce, Rockford, IL., USA) or Sigmacote ™ (Sigma Chemical Co., St. Louis, MO., USA) and allowed to dry. The silicon particles were then coated with a variety of different macromolecules (obtained from Sigma Chemical Co., of St. Louis, MO., USA) listed in Table 2, as described in Example 4. About 4 mg. Each coated silicon preparation was then placed in separate reaction tubes, containing 45 μl of a PCR reaction mixture (see Example 3) and operated on a thermal cycle device, Perkin Elmer, Model 9600.

Additionally, it was supplied to a reaction chamber, medium scale, having dimensions of 80 μm deep, 8 mm wide and 14 mm long, with a coating of a silanization reagent or a siliconization reagent, associated with different molecules (Table 2), according to the procedure described in Example 4. A PCR reaction was carried out in the reassumed storage chambers, using the reastives dessritos in Example 3. The results, using different re-exposures, are shown in Table 2, which employs a classification table from 0 to 4, where the positive control (operated on the GeneAmp 9600 device) has a rating of 3. As illustrated in Table 2, the most effective coating was the Surfasil ™ (Pierce, Rockford, IL., USA) in combination are polyvinylpyrrolidone or polyadenylic acid. TABLE 2 No. Silicone Agent / acromolecule Effectiveness in Silicon Powder Efficiency PCR Chip 1 Sigmacote ™ Pol¡-L-alanine 2 - 2 Sigmacote ™ / Poly-L-aspartic acid or - 3 Sigmacote ™ / Polyglycine 3 > 1 4 Sigmacote ™ / Poly-L-leucine 3 or Sigmacote ™ / Poly-L-phenylalanine 2 - 6 Sigmacote ™ / Poly-L-tryptophan 2 - 7 Sigmacote ™ / Poly-L-lysine 0 - I 8 Sigmacote ™ / Polyvinylpyrrolidone > 1 - 9 Sigmacote ™ / Polyadenyl Acid 4 or Sigmacote ™ / Polymaleimide or - 11 Sgmacote ™ / Maleimide 1 - 12 Surfasil ™ / Poly-a-alanine 3 2 13 Surfasil ™ / Poly-L-aspartic acid or - 14 Surfasil ™ / Polyglycine 1 - Surfasil ™ / Poly-L-leucine 2 - 16 Surfasil ™ / Poly-L-phenylalanine 2 - TABLE 2 (Continued) No. Silicone Agent / Macromolecule Effectiveness in Silicon Powder Efficiency PCR Chip 17 Surfasil ™ / Poly-L-t? Iptophan 1 - 18 Surfasil ™ / Poli-L-lysine 0 - 19 Surfasil ™ / Polyvinylprolodone 4 2 to 3 Surfasil ™ / Polyadenylic Acid 4 3 to 4 21 Surfas¡l ™ / Polimaleimide 0 - 22 Surfasil ™ / Maleamide 1 - 23 Uncoated Silicon 0 or 2 24 Surfasil ™ 3 0 to 1 Sigmacote ™ 2 - 26 DMDCS - 0 27 DMDCS / polyadenylic acid - 1 28 AquaSil ™ in H2O, 1:99 - 1 It will be understood that the foregoing descriptions were made in illustrative form and that the invention may take other forms within the spirit of the structures and methods disclosed herein. Those skilled in the art may have variants and modifications, and all these variants and modifications are considered as part of the invention, as defined in the claims.

Claims

NOVELTY OF THE INVENTION Having discussed the present invention, it is considered a novelty and, therefore, it is claimed that this is maintained in the following: CLAIMS 1. A device for amplifying a polynucleotide in a sample, leading to You know a reassessment of amplifi- < "Polynucleotide ion", this device comprises: a solid substrate, manufactured including at least one polynucleotide amplification reaction chamber, this chamber has at least one dimension in transverse sessión, of median gap, between about 0.1 and 1,000 μm, The chamber has at least one reastive for the in vitro amplification of the polynucleotide, and at least one door, in somunisation of fluid, is the reassumment chamber, to introduce the sample into the re-entry chamber.
2. The device, according to claim 1, the sual also somprende a flow sanal, which are this door are the reassumment chamber.
3. The device, according to claim 1, in which the solid substrate comprises a material of the group consisting of glass, silisium, silica, polysilicon, silicon nitride, silicon dioxide, plastic and polymeric material.
4. The device, according to claim 1, wherein the reassignment chamber has a depth of less than about 500 μm.
5. The device, according to claim 1, wherein the reassignment chamber has a depth of less than about 300 μm.
6. The device, according to claim 1, wherein the reassignment chamber has a depth of less than about 80 μm.
7. The device, according to claim 1, wherein the reassumment chamber has a recession of the super-fisial area at a volume greater than about 3 mm2 / μl.
8. The device, according to claim 7, wherein said ratio is greater than about 5 mm2 / μl.
9. The device, according to claim 7, in which dishalation is greater than about 10 mm2 / μl.
10. The device, according to claim 1, in which the reassumment chamber includes at least one wall, the sual is associated with a somposission that decreases the inhibition, by disha wall, of an amplification reaction of polynucleotides, in the samara .
11. The device, according to claim 10, in which the somposission adheres to the surface of the wall.
12. The device, according to claim 11, wherein the somposission is covalently attached to the surface of the wall.
13. The device according to claim 11, wherein the composition comprises a polymer.
14. The device according to claim 13, wherein the polymer somersates the poly (vinyl slurry).
15. The device, according to claim 10, wherein the somposisance comprises a blocking agent, in a solution disposed within the reassuring chamber.
16. The device, according to claim 15, wherein the blocking agent is separated from the group consisting of polynucleotides and polypeptides.
17. The device, according to claim 16, wherein the blocking agent is a blocking polynucleotide, separated from the group of DNA, RNA, poly-guanilic acid and polyadenyly acidic.
18. The device, according to claim 17, in which the solution, which comprises the blocking polynucleotide, also contains a sample polynucleotide, which is to be amplified in the reassumment chamber.
19. The device, according to claim 10, in which the substrate suffers silisium.
20. The device, according to claim 19, in which the somposisance results in a silane re-covering on the surface of the wall.
21. The device, according to claim 20, in which the re-surfacing is formed by a reassessment of the wall super-fisie are a silane selessionate of the group consisting of dimethylslorosilane, dimethyldislorosilane, hexamethyldisilazane and trimethylslorosilane.
22. The device, according to claim 19, in which the somposisance assumes a silisone overlay on the surface of the wall.
23. The device, according to claim 22, in which the back-coating is formed by the interassion of the wall surface is a re-sewage of silison-sizing.
24. The device, according to claim 22, which also assumes a Masromolésula, asosiada is the silicone re-cover.
25. The device, according to claim 24, in which the masromolésula is an aminoaside polymer.
26. The device, according to claim 24, in which the masromolésula is separated from the group that is the polystyrene pyrrolidone, polyadenyly acid and polymaleate.
27. The device, according to claim 19, in which the somposission comprises a silicon oxide film, arranged on the surface of the wall.
28. The device, according to claim 1, which also includes a thermal regulator for regulating the temperature inside the reassumment chamber.
29. The device, according to claim 1, which also includes a retention system, to detest a produst of the amplification reaction of the polynucleotide.
30. The device, according to claim 29, in which the detest system appears: an agent that forms a somplex, the sual, to sontaste are the prodrug of the amplification reaction of the polynucleotide, forms a detectable complex with the product; a chamber, in which the reactive product of the amplification of the polynucleotide is put into sontaste, is the agent that forms the somplex, thus obtaining a detestable spleen disho; and a detestor, to determine the presensia of a sanctity of the detestable splendor in the samara.
31. The device, according to claim 30, in which the samara in the sual is formed the detestable spleen is the reassuring chamber of the amplification of the polynucleotide.
32. The device, according to claim 30, in which the samara in the sual is formed the detestable somplejo is a sámara of detessión, in somunisasión of fluid, are the chamber of reacsión of the amplifisasión of polinusleótido.
33. The device, according to claim 1, in which the reassumment chamber is at least partially subverted, disposed on the substrate.
34. The device, according to claim 2, in which the door and the flow sanal are manufactured within the substrate.
35. The device, according to claim 2, which also includes a sub-piece arranged on the substrate, where this door and the flow sanal are manufactured inside the sub-floor.
36. The device, according to claim 33, in which the sub-surface comprises a selessionate material from the group of glasses and organic polymeric materials.
37. The device, according to claim 34, which also includes an element extending from the substrate, capable of sealing the door when pressing this element on the door.
38. A device to carry a amplifisation reassión of polynucleotides, this device appears: a system of introduction of samples, to introduce a sample inside the device, the sual is a door of entrance and a ventilation; at least one sanal of sample flow, which extends from the entrance door; a solid substrate, fabricated including at least one amplification chamber of polynucleotide amplification, in somunisation of fluid are the flow sanal and the ventilation, this replacement chamber has at least one dimension in transverse section, of median gap, between approximately 0.1 and 1,000 μm; and a fluid delivery apparatus, for delivering the fluid to, and receiving the fluid from, the entry port, in which the fluid delivery apparatus is internally adjusted with the entry port and reversibly seals this entry port.
39. The device, according to claim 38, in which the delivery apparatus assumes a syringe.
40. The device, according to claim 38, in which the delivery apparatus aspirates a pipette, this pipette includes a tip, provided with an opening, for transferring the fluid between the tip of the pipette and the entrance port.
41. A device for carrying out an amplification reassumption of polynucleotides, this device is: a solid substrate; a cover, arranged on the substrate; and at least one polynucleotide amplification reaction chamber, fabricated in at least one of the substratum or the cover, this reaction chamber has at least one dimension in transverse cross-section, of median gap, between about 0.1 and 1,000 μm; where the sub-surface dips: a savity, to hold and adjust internally a pipette, the sual is included a tip provided are an opening; and a flow sanal, that somunisa between the opening of the tip of the pipette and the reassuring chamber, suando the pipette fits inside the cavity.
42. The device, according to claim 41, in which the sub-surface comprises a transparent material.
43. The device, according to claim 42, in which the transparent material comprises a semi-ionized material from the group of glass and polymeric organelle materials.
44. The device, according to claim 41, in which the opening is soldered on a wall of the tip of the pipette, to allow this pipette tip to move by sucking the pipette soldered into the pocket, between: a first position, the sual allows the transfer of the fluid from the tip through the opening and the sanal to the reassumment chamber; and a second position, the sual allows the opening to face a wall of the savity, thus sealing the sanal and the samara.