Patent Application
20080311585
The present invention relates to methods, devices, systems for fluid handling, specifically for multiplex liquid handling in microfluidic lab cards. Genetic information is critical in continuation of life processes. Life is substantially informationally based and its genetic content which controls the growth and reproduction of the organism. The amino acid sequences of polypeptides, which are critical features of all living systems, are encoded by the genetic material of the cell. Further, the properties of these polypeptides, e.g., as enzymes, functional proteins, and structural proteins, are determined by the sequence of amino acids which make them up. As structure and function are integrally related, many biological functions may be explained by elucidating the underlying structural features which provide those functions, and these structures are determined by the underlying genetic information in the form of polynucleotide sequences. In addition to encoding polypeptides, polynucleotide sequences can also be specifically involved in, for example, the control and regulation of gene expression.
The study of this genetic information has proved to be of great value in providing a better understanding of life processes, as well as diagnosing and treating a large number of disorders. In particular, disorders which are caused by mutations, deletions or repeats in specific portions of the genome, may be readily diagnosed and/or treated using genetic techniques. Similarly, disorders caused by external agents may be diagnosed by detecting the presence of genetic material which is unique to the external agent, e.g., bacterial or viral DNA.
While current genetic methods are generally capable of identifying these genetic sequences, such methods generally rely on a multiplicity of distinct processes to elucidate the nucleic acid sequences, with each process introducing a potential for error into the overall process. These processes also draw from a large number of distinct disciplines, including chemistry, molecular biology, medicine and others. It would therefore be desirable to integrate the various process used in genetic diagnosis, in a single process, at a minimum cost, and with a maximum ease of operation.
Interest has been growing in the fabrication of microfluidic devices. Typically, advances in the semiconductor manufacturing arts have been translated to the fabrication of micromechanical structures, e.g., micropumps, microvalves, and the like, and microfluidic devices including miniature chambers and flow passages.
A number of researchers have attempted to employ these microfabrication techniques in the miniaturization of some of the processes involved in genetic analysis in particular. For example, published PCT Application No. WO 94/05414, to Northrup and White, incorporated herein by reference in its entirety for all purposes, reports an integrated micro-PCR apparatus for collection and amplification of nucleic acids from a specimen. Conventional approaches often involve extremely complicated fluidic networks as more reagents are introduced into these systems and more samples are processed. By going to a smaller platform, such fluidic complexity brings many concerns such as difficulty in fabrication, higher manufacture cost, lower system reliability, etc. Thus, there's a need to have a simpler way to process many samples and perform the reactions in a controlled fashion. However, there remains a need for an apparatus which simplifies and automates the processing of multiple samples through numerous reactions, steps and operations involved in preparing samples for nucleic acid analysis. Various embodiments of the present invention meet one or more of these and other needs.
An embodiment of the present invention provides systems, method, and devices related to lab cards. In one aspect of the present invention, systems, methods, devices and computer software products are provided related to multiplex liquid handling systems utilizing lab cards related to biological assays. Merely by way of example, the invention is described as it applies to utilizing lab cards for preparing nucleic acid samples for hybridization with microarrays, but it should be recognized that the invention has a broader range of applicability.
According to an embodiment of the present invention, a microfabricated device and a method for simultaneously transporting a plurality of aliquots of at least one fluid a known distance in a lab card are provided. The microfabricated device includes a first and a second structure separated from a common chamber by a flexible diaphragm. The flexible diaphragm covers the first and second structures, where each chamber has a known volume. The first and second structures are each in connection with a different reaction port. A valve is actuated to allow a pressurized gas to fill the common chamber to deform the flexible diaphragm into the first and second structures so that the aliquots of the fluid are simultaneously transported the known distance in the lab card.
According to another embodiment of the present invention, a microfabricated device and method for simultaneously mixing a plurality of aliquots of a mixture of fluids in a lab card are provided. The microfabricated device includes a plurality of pairs of a first structure and a second structure. The first structures are separated from the second structures by at least one flexible diaphragm. The first and second structures are each a chamber having a known volume. The first structures are each in connection with a reaction port and the second structures are connected by a common chamber. A first valve is actuated to allow a pressurized gas to fill the common chamber with a gas to deform the flexible diaphragm into the first structures. The displaced gas causes the aliquots of the mixture of fluids to be transported a known distance from point A to point B in the lab card. A second valve is then actuated to create a vacuum in the common chamber to deform the flexible diaphragm into the second structures. The deformation of the flexible diaphragm causes the aliquots of the mixture of fluids to be transported back a known distance from point B to point A. The movement of the aliquots of fluids from point A to point B, back to point A results in mixing of the mixture of fluids in the lab card.
According to an alternate embodiment of the present invention, a microfabricated device and method for simultaneously transporting a plurality of aliquots of at least one fluid a known distance in a lab card that can be repeated are provided. The device includes a plurality of pairs of a first and a second structure connected by one common channel. The first structures are separated from a first common chamber by a first flexible diaphragm. The first structures are each in connection with a reaction port. The first structures are chambers having a known volume. The second structures are separated from a second common chamber by a second flexible diaphragm and are connected to a vent.
The aliquots of a fluid are simultaneously transported a known distance in the lab card by performing the following steps: Step 1) actuate a second valve while a first valve is deactuated to allow a pressurized gas to fill the second common chamber. The pressurized gas causes the flexible diaphragm to deform into the second structure, closing the vent and the openings between the common channels and the second structures, Step 2) actuate the first valve while maintaining the actuation of the second valve to allow the pressurized gas to fill the first common chamber. The pressurized gas causes the flexible diaphragm to deform into the first structure, initiating the transportation of the aliquots of the fluid a know distance in the lab card, Step 3) deactuate the first valve while maintaining the actuation of the second valve to finish transporting the aliquots of the fluid a known distance in the lab card and Step 4) deactuate the second valve while maintaining the deactuation of the first valve to equilibrate the pressure in the lab card. Steps 1-4 are repeated to transport the plurality of aliquots of the fluid in the lab card according to a preferred embodiment of the present invention.
The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
a-1b illustrate a system of channels and valves for introducing multiple samples and performing a number of reactions and steps according to an embodiment of the present invention.
a-4d illustrate a method for making and using a valve mechanism with an air driven flexible membrane valve according to an embodiment of the present invention.
a-6d illustrate a method for making and using a 3-layer flexible membrane valve mechanism according to an embodiment of the present invention.
a-8d illustrate a method for making and using a gas permeable fluid barrier mechanism according to an embodiment of the present invention.
a-9b illustrate the steps to operate a valve utilizing a gas permeable fluid barrier mechanism according to an embodiment of the present invention.
a-d illustrate steps of a prior art method of creating a through hole in a substrate.
a-c illustrate a method of creating a through hole in a substrate according to an embodiment of the present invention.
a-b illustrate an embodiment of the present invention of a method of creating a plurality of stabilized pins.
a-e illustrate an embodiment of the present invention of a method of a one-step nano structure embossing method.
a-19c illustrate images of chip-to-chip interface structures according to some embodiments of the present invention.
a-21e illustrate an overall system which performs a plurality of processes within a closed system according to an embodiment of the present invention.
a and b illustrate an embodiment of the present invention of a microfabricated device for simultaneously transporting a plurality of aliquots of at least one fluid to a known distance in a lab card.
a-d illustrate an embodiment of the present invention of a method of simultaneously mixing a plurality of aliquots of a mixture of fluids in a lab card.
a-d illustrate another embodiment of the present invention of another method of a microfabricated device for simultaneously transporting a plurality of aliquots of a fluid a known distance in a lab card.
The figures are merely examples, which should not unduly limit the scope of the claims. One of ordinary skill in the art would recognize many variations, alternatives, and modifications.
The present invention cites certain patents, applications and other references. When a patent, application, or other reference is cited or repeated below, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited.
As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.
An individual is not limited to a human being but may also be other organisms including but not limited to mammals, plants, bacteria, or cells derived from any of the above.
Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.
The present invention can employ solid substrates, including arrays in some preferred embodiments. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication Number WO 99/36760) and PCT/US01/04285 (International Publication Number WO 01/58593), which are all incorporated herein by reference in their entirety for all purposes.
Patents that describe synthesis techniques in specific embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098.
Nucleic acid arrays are described in many of the above patents, but the same techniques are applied to polypeptide arrays.
Nucleic acid arrays that are useful in the present invention include those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip®. Example arrays are shown on the website at affymetrix.com.
The present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Gene expression monitoring and profiling methods can be shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos. 15 10/442,021, 10/013,598 (U.S. Patent Application Publication 20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.
The present invention also contemplates sample preparation methods in certain preferred embodiments. Prior to or concurrent with genotyping, the genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. The sample may be amplified on the array. See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300, which are incorporated herein by reference.
Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporated herein by reference.
Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al., Genome Research 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent Application Publication 20030096235), Ser. No. 09/910,292 (U.S. Patent Application Publication 20030082543), and 10/013,598.
Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor, N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S. 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference.
The present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.
Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194, 60/493,495 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.
The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g. Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001). See U.S. Pat. No. 6,420,108.
The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.
Additionally, the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (United States Publication No. 20020183936), 10/065,856, 10/065,868, 10/328,818, 10/328,872, 10/423,403, and 60/482,389.
An “array” is an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, e.g., libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.
Nucleic acid library or array is an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically and screened for biological activity in a variety of different formats (e.g., libraries of soluble molecules; and libraries of oligos tethered to resin beads, silica chips, or other solid supports). Additionally, the term “array” is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (e.g., from 1 to about 1000 nucleotide monomers in length) onto a substrate. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Thus the terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein. These analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleoside sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.
Biopolymer or biological polymer: is intended to mean repeating units of biological or chemical moieties. Representative biopolymers include, but are not limited to, nucleic acids, oligonucleotides, amino acids, proteins, peptides, hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides, phospholipids, synthetic analogues of the foregoing, including, but not limited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, and combinations of the above. “Biopolymer synthesis” is intended to encompass the synthetic production, both organic and inorganic, of a biopolymer.
Related to a bioploymer is a “biomonomer” which is intended to mean a single unit of biopolymer, or a single unit which is not part of a biopolymer. Thus, for example, a nucleotide is a biomonomer within an oligonucleotide biopolymer, and an amino acid is a biomonomer within a protein or peptide biopolymer; avidin, biotin, antibodies, antibody fragments, etc., for example, are also biomonomers. initiation Biomonomer: or “initiator biomonomer” is meant to indicate the first biomonomer which is covalently attached via reactive nucleophiles to the surface of the polymer, or the first biomonomer which is attached to a linker or spacer arm attached to the polymer, the linker or spacer arm being attached to the polymer via reactive nucleophiles.
Complementary: Refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.
Combinatorial Synthesis Strategy: A combinatorial synthesis strategy is an ordered strategy for parallel synthesis of diverse polymer sequences by sequential addition of reagents which may be represented by a reactant matrix and a switch matrix, the product of which is a product matrix. A reactant matrix is a 1 column by m row matrix of the building blocks to be added. The switch matrix is all or a subset of the binary numbers, preferably ordered, between 1 and m arranged in columns. A “binary strategy” is one in which at least two successive steps illuminate a portion, often half, of a region of interest on the substrate. In a binary synthesis strategy, all possible compounds which can be formed from an ordered set of reactants are formed. In most preferred embodiments, binary synthesis refers to a synthesis strategy which also factors a previous addition step. For example, a strategy in which a switch matrix for a masking strategy halves regions that were previously illuminated, illuminating about half of the previously illuminated region and protecting the remaining half (while also protecting about half of previously protected regions and illuminating about half of previously protected regions). It will be recognized that binary rounds may be interspersed with non-binary rounds and that only a portion of a substrate may be subjected to a binary scheme. A combinatorial “masking” strategy is a synthesis which uses light or other spatially selective deprotecting or activating agents to remove protecting groups from materials for addition of other materials such as amino acids.
Effective amount refers to an amount sufficient to induce a desired result.
Genome is all the genetic material in the chromosomes of an organism. DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA.
A genomic library is a collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism. Hybridization conditions will typically include salt concentrations of less than about 1M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures can be as low as 5.degree. C., but are typically greater than 22.degree. C., more typically greater than about 30.degree. C., and preferably in excess of about 37.degree. C. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
Hybridizations, e.g., allele-specific probe hybridizations, are generally performed under stringent conditions. For example, conditions where the salt concentration is no more than about 1 Molar (M) and a temperature of at least 25 degrees Celsius (° C.), e.g., 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4 (5×SSPE) and a temperature of from about 25 to about 30° C.
Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25° C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridizations. For stringent conditions, see, for example, Sambrook, Fritsche and Maniatis. “Molecular Cloning A laboratory Manual” 2nd Ed. Cold Spring Harbor Press (1989) which is hereby incorporated by reference in its entirety for all purposes above.
The term “hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide; triple-stranded hybridization is also theoretically possible. The resulting (usually) double-stranded polynucleotide is a “hybrid.” The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the “degree of hybridization.”
Hybridization probes are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991), and other nucleic acid analogs and nucleic acid mimetics.
Hybridizing specifically to: refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
Isolated nucleic acid is an object species invention that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).
Mixed population or complex population: refers to any sample containing both desired and undesired nucleic acids. As a non-limiting example, a complex population of nucleic acids may be total genomic DNA, total genomic RNA or a combination thereof. Moreover, a complex population of nucleic acids may have been enriched for a given population but include other undesirable populations. For example, a complex population of nucleic acids may be a sample which has been enriched for desired messenger RNA (mRNA) sequences but still includes some undesired ribosomal RNA sequences (rRNA).
Monomer: refers to any member of the set of molecules that can be joined together to form an oligomer or polymer. The set of monomers useful in the present invention includes, but is not restricted to, for the example of (poly) peptide synthesis, the set of L-amino acids, D-amino acids, or synthetic amino acids. As used herein, “monomer” refers to any member of a basis set for synthesis of an oligomer. For example, dimers of L-amino acids form a basis set of 400 “monomers” for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer.
The term “monomer” also refers to a chemical subunit that can be combined with a different chemical subunit to form a compound larger than either subunit alone. mRNA or mRNA transcripts: as used herein, include, but not limited to pre-mRNA transcript(s), transcript processing intermediates, mature mRNA(s) ready for translation and transcripts of the gene or genes, or nucleic acids derived from the mRNA transcript(s). Transcript processing may include splicing, editing and degradation. As used herein, a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template. Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample. Thus, mRNA derived samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
Nucleic acid library or array is an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically and screened for biological activity in a variety of different formats (e.g., libraries of soluble molecules; and libraries of oligos tethered to resin beads, silica chips, or other solid supports). Additionally, the term “array” is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (e.g., from 1 to about 1000 nucleotide monomers in length) onto a substrate. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Thus the terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein. These analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleoside sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.
Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally-occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging from at least 2, preferable at least 8, and more preferably at least 20 nucleotides in length or a compound that specifically hybridizes to a polynucleotide. Polynucleotides of the present invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, recombinantly produced or artificially synthesized and mimetics thereof. A further example of a polynucleotide of the present invention may be peptide nucleic acid (PNA). The invention also encompasses situations in which there is a nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix. “Polynucleotide” and “oligonucleotide” are used interchangeably in this application.
Probe: A probe is a surface-immobilized molecule that can be recognized by a particular target. See U.S. Pat. No. 6,582,908 for an example of arrays having all possible combinations of probes with 10, 12, and more bases. Examples of probes that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
Primer is a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions e.g., buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template. The primer site is the area of the template to which a primer hybridizes. The primer pair is a set of primers including a 5′ upstream primer that hybridizes with the 5′ end of the sequence to be amplified and a 3′ downstream primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.
“Solid support”, “support”, and “substrate” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See U.S. Pat. No. 5,744,305 for exemplary substrates.
Target: A molecule that has an affinity for a given probe. Targets may be naturally occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended. A “Probe Target Pair” is formed when two macromolecules have combined through molecular recognition to form a complex.
WGSA (Whole Genome Sampling Assay) Genotyping Technology: A technology that allows the genotyping of hundreds of thousands of SNPS simultaneously in complex DNA without the use of locus-specific primers. In this technique, genomic DNA, for example, is digested with a restriction enzyme of interest and adaptors are ligated to the digested fragments. A single primer corresponding to the adaptor sequence is used to amplify fragments of a desired size, for example, 500-2000 bp. The processed target is then hybridized to nucleic acid arrays comprising SNP-containing fragments/probes. WGSA is disclosed in, for example, U.S. Provisional Application Ser. Nos. 60/319,685; 60/453,930, 60/454,090 and 60/456,206, 60/470,475, U.S. patent application Ser. Nos. 09/766,212, 10/316,517, 10/316,629, 10/463,991, 10/321,741, 10/442,021 and 10/264,945, each of which is hereby incorporated by reference in its entirety for all purposes.
Whole Transcript Assay (WTA): is used herein, a WTA is an assay protocol that can representatively sample entire transcripts (i.e., all exons in a transcript). WTA is disclosed in, for example, U.S. Provisional Application Ser. Nos. 60/683,127 and U.S. patent application Ser. Nos. 11/419,459, each of which is hereby incorporated by reference in its entirety for all purposes.
Reference will now be made in detail to exemplary embodiments of the invention. While the invention will be described in conjunction with the exemplary embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modification and equivalents, which may be included within the spirit and scope of the invention.
Some embodiments of the present invention are systems and methods for using multiplex liquid handling to make lab cards, such as microfluidic lab cards. In one aspect of the present invention, systems, methods, and computer software products are provided for using the multiplex liquid handling for making lab cards related to biological assays. Merely by way of example, the invention is described as it applies to making lab cards for preparing nucleic acid samples for hybridization with microarrays, but it should be recognized that the invention has a broader range of applicability. In another example, the lab cards are suitable for performing complex chemical and/or biochemical reactions.
Some conventional techniques for handling multiple liquids simultaneously often require several number of valves and sensors which can grow linearly to the number of reactions. In one aspect of the present invention, systems, methods, and computer software products are provided for a driving mechanism by using one valve/sensor set to control multiple fluidics flow at the same time. In another aspect of the present invention, the lab cards using the multiplex liquid handling system are suitable for performing complex chemical and/or biochemical reactions.
In other aspect of the invention, methods, devices, systems and computer software products for automated biological assay and/or reduce reagent volume are provided. For example, the biological assay is related to sample preparation which is provided with respect to illustrative, non-limiting, implementations. Various alternatives, modifications and equivalents are possible. Some embodiments of the present invention are systems and methods for controlling lab cards, such as microfluidic lab cards. In another aspect of the present invention, the lab cards are suitable for performing complex chemical and/or biochemical reactions. They are particularly suitable for performing the WGSA assay as an example, however, they are not limited to such uses.
For example, certain systems, methods, and computer software products are described herein using exemplary implementations for analyzing data from arrays of biological materials such as, for instance, Affymetrix® GeneChip® probe arrays. However, these systems, methods, and products may be applied with respect to many other types of probe arrays and, more generally, with respect to numerous parallel biological assays produced in accordance with other conventional technologies and/or produced in accordance with techniques that may be developed in the future. For example, the systems, methods, and products described herein may be applied to parallel assays of nucleic acids, PCR products generated from cDNA clones, proteins, antibodies, or many other biological materials. These materials may be disposed on slides (as typically used for spotted arrays), on substrates employed for GeneChip® arrays, or on beads, bead arrays, optical fibers, or other substrates or media, which may include polymeric coatings or other layers on top of slides or other substrates. Moreover, the probes need not be immobilized in or on a substrate, and, if immobilized, need not be disposed in regular patterns or arrays. For convenience, the term “probe array” will generally be used broadly hereafter to refer to all of these types of arrays and parallel biological assays. Certain embodiments of the present invention are described in the simplified figures of this application.
The device of the present invention is generally capable of carrying out a number of preparative and analytical reactions on a number of samples. In a preferred embodiment, to achieve this end, the device generally comprises a number of inlet channels, a common channel and a set of control valves within a single unit, body or system.
According to one aspect of the present invention, a system for introducing multiple samples and performing multiple reactions and steps is provided as shown in
Typically, the body of the device defines the various inlet channels, common channel(s) and measuremetric channels in which the above described operations are carried out according to certain embodiments of the present invention. Fabrication of the body and thus the various channels and chambers disposed within the body may generally be carried out using one or a combination of a variety of well known manufacturing techniques and materials as described in U.S. Pat. Nos. 6,197,595 and 6,830,936. These references are incorporated herein by reference in its entirety. The body of the device is generally fabricated using one or more of a variety of methods and materials suitable for micro fabrication techniques such as embossing, injection molding, thermal bonding thermal forming, etc. Typical plastic materials used for microfluidics are thermal-plastics: polycarbonate, polymethyl methacrylate (PMMA), COC, etc. and elastomers: polydimethylsiloxane (PDMS). For example, in a preferred embodiment, the body of the device may be injected molded parts from Polycarbonate.
As shown in
In another preferred embodiment, a measuremetric channel and a valve mechanism may be used to precisely measure fluid volumes for introduction into a subsequent sample chamber. In such cases, the location of the valve mechanism of the channel will be dictated by measuremetric needs of a given reaction. Furthermore, the measuremetric channel(s) may be fabricated to include a number of valve mechanisms to provide a number of volumes. In a preferred embodiment, the controllable valves will stop the liquid at a desired location to provide the desired volume.
According to an embodiment of the present invention, an apparatus for providing a plurality of predetermined volumes of liquids is illustrated in
According to one aspect of the present invention,
In a preferred embodiment, a liquid is provided to all the samples in the various chambers in the same or different volumes. The smallest volume required of any of the liquids may be indicated by the first valve. When the pressure is applied, the pressure can be applied equally such that volume of liquid is equally split between the channels. This process may be accomplished based on the design of the valve mechanism, the operation of the microfluidics and the characteristics of the liquid.
According to yet another embodiment, a method for providing a plurality of predetermined volumes of a liquid includes providing a volume of a liquid to a channel. The channel is directly or indirectly connected to a plurality of channels. The plurality of channels is coupled to a plurality of valve and each of the plurality of channels includes a plurality of channel segments. A first segment of the plurality of channel segments is capable of being connected to or disconnected from a second segment of the plurality of channel segments in response to at least one of the plurality of valves. Additionally, the method includes receiving information associated with a plurality of predetermined volumes for a liquid corresponding to the plurality of channels respectively, and processing information associated with the plurality of predetermined volumes for the liquid. Moreover, the method includes selecting one valve from a plurality of valves based on at least one information associated with the plurality of predetermined volumes, opening the selected valve, and transporting the liquid through the plurality of channels up to the opened valve. In another example, the method also includes closing the selected valve after transporting the liquid through the plurality of channels up to the opened valve. The process for closing the selected valve is performed so that the liquid flows in the plurality of channels and the plurality of channels holds the plurality of predetermined volumes of the liquid respectively. In yet another example, the process for transporting the liquid through the plurality of channels up to the opened valve includes applying a pressure to the liquid in the channel.
a-4d illustrate a method for making and using a valve mechanism with an air driven flexible membrane valve according to an embodiment of the present invention. These diagrams are merely examples, which should not unduly limit the scope of the claims. One of ordinary skill in the art would recognize many variations, alternatives, and modifications. As shown in
According to another embodiment of the present invention, a valve mechanism design utilizing a gas permeable fluid barrier is provided as shown in
In a preferred embodiment,
An illustration of another method, according to an embodiment of the present invention, of a valve mechanism utilizing a gas permeable fluid barrier is shown in
In another preferred embodiment, a schematic of another system for introducing multiple samples and performing a number of reactions and steps is shown in
In one aspect of the invention, a system for splitting a plurality of liquids comprising a housing which comprises a liquid cavity is provided. The liquid cavity comprises a plurality of inlet channels, at least one common channel, a plurality of measuremetric channels, a computer and a controlling device. The inlet channels comprise a plurality of inlets which are fluidly connected by at least one common channel. The common channel is fluidly linked to a number of measuremetric channels which comprise a set of valve mechanisms and gates. The pressure of a gas introduces the liquid into a measuremetric channel. A computer is used with the controlling device to control the valve mechanisms and gates such that the liquid is split into the plurality of measuremetric channels and the desired volume of liquid is produced. In a preferred embodiment, the housing is made of plastic. In another preferred embodiment, the liquid contains at least one target molecule.
In another preferred embodiment of the present invention, a system as described above is provided wherein the valve mechanism comprises a gas permeable fluid barrier, a valve and housing. The gas permeable fluid barrier comprises a first surface and a second surface, wherein the first surface is exposed to an air cavity. The valve permits air to flow out of the air cavity. The housing comprises a mounting surface, an air cavity, and a liquid cavity. The liquid cavity comprises an inlet port constructed to permit air flow into the air cavity through said inlet port. The second surface of the gas permeable fluid barrier is sealably mounted with respect to the mounting surface of the housing. The valve is used as the control unit either sealing the cavity or allowing the air to flow. In a preferred embodiment, the gas permeable fluid barrier is a hydrophobic membrane.
In one aspect of the present invention, an apparatus for controlling liquids is provided which comprises a gas permeable fluid barrier and a valve. The gas permeable fluid barrier comprises a first surface and a second surface, such that the first surface is exposed to an air cavity. The valve permits air to flow out of the air cavity. The housing comprises a mounting surface, the air cavity, and a liquid cavity. The liquid cavity comprises an inlet port constructed to permit air flow into said air cavity through said inlet port. The second surface of the gas permeable fluid barrier is sealably mounted with respect to the mounting surface of the housing whereby the valve is located inside the air cavity. In a preferred embodiment, the apparatus as described above is provided wherein the housing is made of plastic. In another preferred embodiment, the apparatus as described above is provided wherein the gas permeable fluid barrier is a hydrophobic membrane.
In another aspect of the present invention, a method for controlling liquids is provided which comprises providing a gas permeable fluid barrier which comprises a first surface and a second surface, the first surface exposed to an air cavity. The method then involves providing a valve, wherein the valve permits air to flow out of the air cavity, sealably mounting the second surface of the gas permeable fluid barrier to a housing which comprises a mounting surface, the air cavity, and a liquid cavity. The liquid cavity comprises an inlet port constructed to permit air flow into said air cavity through said inlet port, wherein the sealably mounting step assist in preventing a liquid to pass through the gas permeable fluid barrier. The method continues by controlling the valves to introduce the liquid inside the liquid cavity and stopping the liquid at a desired location. In a preferred embodiment, the method described above is provided wherein the housing is made of plastic. In another preferred embodiment, the method described is provided wherein the gas permeable fluid barrier is a hydrophobic membrane.
The inclusion of gas permeable fluid barriers, e.g., poorly wetting filter plugs or hydrophobic membranes, in these devices also permits a sensorless fluid direction and control system for moving fluids within the device. For example, such filter plugs, incorporated at the end of a reaction chamber opposite a fluid inlet will allow air or other gas present in the reaction chamber to be expelled during introduction of the fluid component into the chamber. Upon filling the chamber, the fluid sample will contact the hydrophobic plug thus stopping net fluid flow. Fluidic resistances, may also be employed as gas permeable fluid barriers, to accomplish this same result, e.g., using fluid passages that are sufficiently narrow as to provide an excessive fluid resistance, thereby effectively stopping or retarding fluid flow while permitting air or gas flow. Expelling the fluid from the chamber then involves applying a positive pressure at the plugged vent. This permits chambers which may be filled with no valves at the inlet, i.e., to control fluid flow into the chamber. In most aspects however, a single valve will be employed at the chamber inlet in order to ensure retention of the fluid sample within the chamber, or to provide a mechanism for directing a fluid sample to one chamber of a number of chambers connected to a common channel.
In a preferred embodiment of the present invention, the apparatus, method and system of the present invention is directed towards a hand held disposable device for performing a plurality of processes wherein the reagents are stored within the device according to an embodiment of the present invention. After performing the plurality of processes, the reacted solution is collected in a collection chamber and the generated waste is stored in a waste chamber within the hand held disposable device. According to a preferred embodiment of the present invention, the processing of reagents is directed by a gas and vacuum.
Typically, a device for performing a plurality of process can be referred to a microfluidic device, for example, as described in U.S. Pat. No. 6,168,948 which is incorporated herein in its entirety. In general, a lab card is a disposable part, for example, where reagents are stored, controlled and processed. A microfluidic device generally incorporates a lab card with the necessary instruments (for example, reusable components) required to control the fluidics and reaction conditions (for example, pressure regulator, valves, computers, mechanical hardware, heaters, coolers, etc).
The body of the lab card, in general, defines the various storage, reaction chambers and fluid passages or channels. According to an embodiment of the present invention, a lab card is fabricated with microfluidic features to incorporate a plurality of processes, for example, reagent delivery, storage, reaction, mixing, bubble removing, purification, drying, waste storage and the like. Fabrication of the body, and thus the various chambers and channels may generally be carried out using one or a combination of a variety of well known manufacturing techniques and materials. Generally, the material from which the body is fabricated will be selected so as to provide maximum resistance to the full range of conditions to which the device will be exposed, e.g., extremes of temperature, salt, pH, application of electric fields and the like, and will also be selected for compatibility with other materials used in the device. Additional components may be later introduced, as necessary, into the body. Alternatively, the device may be formed from a plurality of distinct parts that are later assembled or mated.
The body of the lab card is generally fabricated using one or more of a variety of methods and materials suitable for microfabrication techniques. For example, in preferred aspects, the body of the device may comprise a number of planar members that may individually be injection molded parts fabricated from a variety of polymeric materials, or may be silicon, glass, or the like. In the case of substrates like silica, glass or silicon, methods for etching, milling, drilling, etc., may be used to produce wells and depressions which make up the various reaction chambers and fluid channels within the device. Microfabrication techniques, such as those regularly used in the semiconductor and microelectronics industries are particularly suited to these materials and methods. These techniques include, e.g., electrodeposition, low-pressure vapor deposition, photolithography, wet chemical etching, reactive ion etching (RIE), laser drilling, and the like. Where these methods are used, it will generally be desirable to fabricate the planar members of the device from materials similar to those used in the semiconductor industry, i.e., silica, silicon, gallium arsenide, polyimide substrates. U.S. Pat. No. 5,252,294, to Kroy, et al., incorporated herein by reference in its entirety for all purposes, reports the fabrication of a silicon based multiwell apparatus for sample handling in biotechnology applications.
Some conventional techniques for plastic embossing often can only construct micro features that have certain aspect ratios, such as below 1. Usually, microfluidic structures each use a structure that has many through holes for inlets, outlets, and/or via connections. But certain conventional embossing techniques cannot provide through holes with high aspect ratios in embossed plastic slides. Subsequently, such holes often have to be drilled after embossing and hence cause several difficulties and/or disadvantages. For example, the multiple process steps requires extra time and costs. In anther example, it is often difficult to align the mechanical-drilled holes to the embossed micro features, thus causing chip-to-chip variations. In yet another example, contaminations during the drilling process, such as one performed in a machine shop, is often undesirable. In yet another example, the drilling process may generate debris, which can block or even damage the micro features. In yet another example, one or more extra cleaning processes may be needed. Thus, a one-step through hole embossing technique is highly desirable.
In a preferred embodiment of the present invention, the apparatus, method and system of the present invention is directed towards creating a hole with a high aspect ratio in a substrate which is used for example, as a channel. Typically, through holes in substrates is utilized in microfluidic systems such as, for example, lab cards or the microfluidic devices described in U.S. Pat. No. 6,168,948 which is incorporated herein in its entirety. In general, a lab card is a disposable part, for example, where reagents are stored, controlled and processed. A microfluidic device generally incorporates a lab card with all the necessary instruments (for example, reusable components) required to control the fluidics and reaction conditions (for example, pressure regulator, valves, computers, mechanical hardware, heaters, coolers, etc). The body of the lab card, in general, defines the various storage, reaction chambers and fluid passages or channels. Fabrication of the body, and thus the various chambers and channels may generally be carried out using one or a combination of a variety of well known manufacturing techniques and materials. Generally, the material from which the body is fabricated will be selected so as to provide maximum resistance to the full range of conditions to which the device will be exposed, e.g., extremes of temperature, salt, pH, application of electric fields and the like, and will also be selected for compatibility with other materials used in the device. Additional components may be later introduced, as necessary, into the body. Alternatively, the device may be formed from a plurality of distinct parts that are later assembled or mated.
The number, shape and size of the channels included within the device will also vary depending upon the specific application for which the device is to be used. The apparatus, method and system according to an embodiment of the present invention refers to a hole with a high aspect ratio wherein the aspect ratio is in the range of 1 to 20, preferably in the range of 1 to 50 and most preferably in the range of in the range of 1 to 100. In general, the holes corresponding to these high aspect ratios, in general, for example, typically range from about 10 to 5000 μm in depth, and from about 10 to 5000 μm in diameter, preferably about 50 to 1000 μm in depth and 100 to 1000 μm in diameter and most preferably about 100 to 1000 μm in depth and 100 to 500 μm in diameter. In this example, the hole is describe as a cylinder, defining the aspect ratio as the material thickness to the hole diameter. It is to be understood that the above description is intended to be illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description and figures. Such variation may include the shape which include those well known in the art, e.g., (i.e. the shape of the end of the hole, for example, being in the shape of a square, rectangle, circle, oval, star, free shape, pentagon, hexagon and the like). Thus, the corresponding aspect ratio is the depth to the smallest width of the hole. In addition, although described in terms of holes, it will be appreciated that these holes may perform a number of varied functions, e.g., as storage chambers/channels, incubation chambers/channels, mixing chambers/channels, and the like. According to preferred embodiment of the invention, the shape of the hole will correspond to the shape of the pin that penetrates through the substrate. As described above, in general, the hole is part of the lab card, therefore, it is usually fabricated directly onto the body of the microfluidic device. Although primarily described in terms of producing a fully integrated body of the device, the above described methods can also be used to fabricate individual discrete components of the device which are later assembled into the body of the device.
Photolithographic methods of etching substrates are particularly well suited for the microfabrication of these substrates and are well known in the art. For example, the first sheet of a substrate may be overlaid with a photoresist. An electromagnetic radiation source may then be shone through a photolithographic mask to expose the photoresist in a pattern which reflects the pattern of chambers and/or channels on the surface of the sheet. After removing the exposed photoresist, the exposed substrate may be etched to produce the desired wells and channels. Generally preferred photoresists include those used extensively in the semiconductor industry. Such materials include polymethyl methacrylate (PMMA) and its derivatives, and electron beam resists such as poly(olefin sulfones) and the like (more fully discussed in, e.g., Ghandi, “VLSI Fabrication Principles,” Wiley (1983) Chapter 10, incorporated herein by reference in its entirety for all purposes).
As an example, the wells manufactured into the surface of one planar member make up the various reaction chambers of the device. Channels manufactured into the surface of this or another planar member make up fluid channels which are used to fluidly connect the various reaction chambers. Another planar member is then placed over and bonded to the first, whereby the wells in the first planar member define cavities within the body of the device which cavities are the various reaction chambers of the device. Similarly, fluid channels manufactured in the surface of one planar member, when covered with a second planar member define fluid passages through the body of the device. These planar members are bonded together or laminated to produce a fluid tight body of the device. Bonding of the planar members of the device may generally be carried out using a variety of methods known in the art and which may vary depending upon the materials used. For example, adhesives may generally be used to bond the planar members together. Where the planar members are, e.g., glass, silicon or combinations thereof, thermal bonding, anodic/electrostatic or silicon fusion bonding methods may be applied. For polymeric parts, a similar variety of methods may be employed in coupling substrate parts together, e.g., heat with pressure, solvent based bonding. Generally, acoustic welding techniques are generally preferred. In a related aspect, adhesive tapes may be employed as one portion of the device forming a thin wall of the reaction chamber/channel structures.
In additional embodiments, the body may comprise a combination of materials and manufacturing techniques described above. In some cases, the body may include some parts of injection molded plastics, and the like, while other portions of the body may comprise etched silica or silicon planar members, and the like. For example, injection molding techniques may be used to form a number of discrete cavities in a planar surface which define the various reaction chambers, whereas additional components, e.g., fluid channels, arrays, etc, may be fabricated on a planar glass, silica or silicon chip or substrate. Lamination of one set of parts to the other will then result in the formation of the various reaction chambers, interconnected by the appropriate fluid channels.
In one embodiment of the present invention, the body of the device in which the hole is made is from a material that has a glass transition temperature (Tg), for example, glasses and plastics. According to a preferred embodiment of the present invention, the material of the device is a thermoplastic. Examples of suitable polymers for embossing include, e.g., acrylic, polymethylmethacrylate (PMMA), thermoplastic polyimide, polyamide, cyclic olefin copolymer (COC), polyester, polycarbonate, polyetherimide, polyethylene (LDPE, HDPE, LLDPE), polypropylene, polysulfone, polyvinylchloride (PVC), polyrurethane, polystyrene, acrylonitrile-butadiene-styrene copolymer (ABS) plastic, and commercial polymers such as AUREM.™, NYLON.™, PEBAX.™, LEXAN.™, MAKROFOL.™, CALIBRE.™, HYTREL.™, VALOX.™, TEFLON.™, DELRIN.™, KALREZ.™, VALOX.™ and the like.
Another embodiment of the present invention utilizes equipment and processes that may heat, emboss and cool the substrate while in a planar condition to provide a large volume of constructed parts. One advantage of embossing the features into the substrate is that the stress relaxation problems associated with injection molded substrates are avoided. There is substantially better alignment of the polymer strands from the polymer material because the embossed substrates are not flowed or injected into a mold. Accordingly, there is substantially less relaxation of the overall substrate when the pins penetrate into the substrate during the embossing process. Therefore, there is substantially better alignment as a result of the significant reduction of channel deformation. The published article, J. Narasimhan et al., “Polymer Embossing Tools for Rapid Prototyping of Plastic Microfluidic Devices”, Journal of Micromechanisc and Microengineering, 14 (2004) 96-103), which is incorporated herein by reference for all purposes), describes an example of an embossing tool (MTP-10, Tetrahedron Associates Inc., San Diego, Calif.) and method used to created microchannels in a thermoplastic device.
An embodiment of the present invention provides devices, systems and methods for creating holes in lab cards, such as microfluidic lab cards for the example, sample preparation of biological assays. Merely by way of example, the invention is described as it applies to preparing nucleic acid samples for hybridization with microarrays, but it should be recognized that the invention has a broader range of applicability.
Certain embodiments of the present invention are described in simplified figures of the application.
Constructing the through hole using the method according to the present invention simplifies construction for the fabrication of internal channels and the like, and can also be made at a relatively low cost. In particularly preferred embodiments, at least one planar member or substrate of the body of the device is made from at least one embossed molded part that has one or more depressions manufactured into its surface to define a wall of a well, chamber, channel or through holes constructed by the method according to an embodiment of the present invention. The through holes can act as channels, or chambers and the like.
In a preferred embodiment of the present invention, an apparatus, method and system for constructing at least one hole in a substrate for a microfluidic device is provided.
It is to be understood that the above description is intended to be illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description and figures. Such variation may include the method of providing the pin(s) in to the plate which include those well known in the art, e.g., (i.e. press fitting the pin into the plate and the like).
According to an embodiment of the present invention, a delay mechanism is provided to allow some time to pass in a controlled fashion. The delay mechanism may be driven by time, pressure, heat and the like according to another embodiment of the present invention. An example of a delay mechanism using time, is using a computer program to control the time. A preferred embodiment of the present invention is a delay mechanism using a component that is pressure sensitive is, for example, using a spring. In a most preferred embodiment, the delay mechanism comprises a heating component wherein the heating component is at least two spacers. As illustrated in
In a preferred embodiment, the middle plate (1202) is stationary while the top plate (1201) and the back plate (1203) are movable. The substrate raw material is fed between the top plate (1201) and the middle plate (1202) as shown in
According to an embodiment of the present invention, a back plane (1203) with metal pins (1207) is used during an embossing process. The metal pins (1207) are held underneath the micro features (1209) by the spacers (1206) made from the same plastic material used in embossing. During the embossing process, the entire structure which includes the mold, top plate (1201), middle plate (1202) and back plate (1203) with at least one pin (1204) are heated above Tg (e.g., the glass transient temperature) of the plastic. The plastic then becomes soft and starts to fill the entire cavity. Also, the spacer becomes soft and presses into thin films. Such changes allow the backplane and the mold to get come together as the pins penetrates through the entire plastic layer (1103). According to an embodiment of the present invention, a device, a method and a system for making through holes from an embossing process is provided by phasing the embossing process with a delayed hole penetration step. As a result, an embossed plastic substrate with self-aligned holes is created. Hence, certain embodiments of the present invention provide a quick, accurate, and clean process.
c illustrates the final molded substrate (1103) with the constructed through holes. The applied temperature and pressure will depend on the type of thermoplastic. In addition, the material densities and thickness of the substrate may also affect the apparatus and process. In a preferred embodiment of the present invention, the design of the molded piece is such that the required pins provide balanced pressure distribution across the substrate when penetrating through the substrate. In one example, four pins penetrate the substrate to provide two sets of through holes with different diameters in the molded substrate (1103). The number of pins that penetrate the substrate will depend on, for example, the application requirements of the device. The surfaces of the plates used for molding may be coated with any number of materials to assist in separating the pieces apart, for example, a releasing agent according to another embodiment of the present invention. Such releasing agent may include those well known in the art, e.g., teflon or the like.
According to another embodiment of the present invention, besides constructing through holes for the fabrication of internal channels and the like, devices, methods and systems are provided for embossing nanoscale fluid structures or other delicate structures at a relatively low cost.
As a miniaturized device, the body of the device, for example a lab card, will typically be in the range of 1 to 20 cm in length by in the range of 1 to 15 cm in width by in the range 0.1 to 2.5 cm thick, preferably in the range of 2 to 15 cm in length by in the range of 2 to 10 cm in width by in the range 0.1 to 1.5 cm thick, most preferably about 7.6 cm in length by about 5.1 cm in width by about 0.64 cm. Although indicative of a rectangular shape, it will be readily appreciated that the devices of the present invention may be embodied in any number of shapes depending upon the particular need. Additionally, these dimensions will typically vary depending upon the number of operations to be performed by the device, the complexity of these operations and the like. As a result, these dimensions are provided as a general indication of the size of the device. The number, shape and size of the channels included within the device will also vary depending upon the specific application for which the device is to be used.
The transportation of fluid within the device of the invention may be carried out by a number of various methods. Internal pump elements which are incorporated into the device may be used to transport fluid samples through the device. Alternately, fluid transport may be affected by the application of pressure differentials provided by either external or internal sources as described in U.S. Pat. No. 6,168,948, Miniaturized Genetic Analysis Systems and Methods, which is hereby incorporated by reference in its entirety for all purposes.
According to an embodiment of the present invention, an air-driven microfluidics mechanism is provided as shown in
In one embodiment of the present invention, the device will include a pressure/vacuum manifold for directing an external vacuum source to the various reaction/storage/analytical chambers/channels to direct and process the reagents within the hand held disposable device. According to another embodiment of the present invention, all fluid transport or processing is performed within lab card(s) by utilizing pressurized gas, vacuum and vents with a pneumatic manifold. Performing an assay can require several types of processes. Examples of processes that are directed by a gas driven source include reagent delivery, storage, reaction, mixing, bubble removing, purification, drying, waste storage and the like according to some embodiments of the present invention. According to a preferred embodiment, the gas is air. Introducing a reagent and moving a reagent forward in a channel are examples of processes where a pressurized air is applied and a valve is opened for a specific desired time. Mixing is an example of a process where a combination of a vacuum and air pressure is utilized. Reversing the flow of reagent is an example of a process when a vacuum is utilized. Another example, is applying a vacuum into the system, for example, to remove alcohol after purification. One other example is carrying out a number of preparative and analytical reactions for introducing multiple samples and performing multiple reactions and steps as described in U.S. patent application Ser. No. 11/553,944, which is hereby incorporated by reference in its entirety for all purposes. It is to be understood that the above examples are intended to be illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description and figures. Such variation may include other ways of utilizing pressurized air, vacuum and vents to process the fluids.
The configuration and air requirements of a lab card will depend on several factors, such as, for example, number of reagents, type of assay, number of assay, number of reactions, etc. according to an embodiment of the present invention. An example of a lab card configuration according to an embodiment of the present invention is illustrated in
Most assays require multiple reagents to be added to multiple reactions. In many embodiments, the liquid is a reagent for a biochemical reaction. In this example, the lab card is being used to perform a WTA assay according to an embodiment of the present invention. This diagram is merely an example, which should not unduly limit the scope of the claims. One of ordinary skill in the art would recognize many variations, alternatives, and modifications. As shown in
According to a preferred embodiment of the present invention, an example of utilizing the above specified sections of the lab card is illustrated in
In another aspect of the present invention, a microfluidic system may comprise of a number of different types of devices or lab cards as described below according to some embodiments of the present invention.
According to an embodiment of the present invention, a reagent card may include both the samples and the reagents to perform an assay as discussed previously in this application. All the reagents and sample are transferred to the reaction card such that all the reagents including the sample required to perform an assay are provided within the lab card according to an embodiment of the present.
According to another embodiment of the present invention, a sample card is provided wherein at least one sample of at least one patient is included in a lab card which is separate from the reagent card. The sample from the sample card may be transferred into the reagent storage card or directly to the reaction card.
The reagent card can be a universal reagent card wherein the reagents to perform the desire assays are standard according to another embodiment of the present invention. These universal reagent cards may be used with different sample cards, reagent cards and array processing cards.
The lab cards that are used to store sample and reagents may include an apparatus that provides a cold storage mechanism designed to keep the store sample(s) and reagent(s) within the lab card(s) at the desired temperature storage conditions according to an embodiment of the present invention.
According to an embodiment of the present invention, a reaction card is provided wherein processes or reactions are directed by a gas or vacuum and processed with the contained reagent within the lab card. This provides a device, method and system where the contamination and error from handling reagents is significantly reduced or eliminated. A waste and collection chambers are provided within the lab card to assure that all the reagents are self contained in the lab card. Examples of reactions or processes that are performed by a reaction card are sample prep, target prep, and other various assays.
A universal card is provided by having components that are common across a number of assays in a lab card and the other components that are specific to an assay on a separate card according to an embodiment of the present invention. For example, typically for assay development, the same reagents are used with different reactions. Thus, a universal reagent card may be utilized with various reaction cards. On the other hand, for performing different assays, in general, different reagents are required while performing the same reactions. Users can use different reactions cards for different assays with a universal reaction card.
According to a preferred embodiment, an array processing card including a lab card comprising at least one probe array (1906) is provided as illustrated in
Alternately, the array processing card can perform a plurality of these steps within the array processing card according to another embodiment of the present invention. Integrating bioarrays into a microfluidic card makes the entire assay fully automated and/or minimize the difficulties in transferring small amount liquid from microfluidic card to a, for example, hybridization card.
The device, systems and methods of the present invention has a wide variety of uses in the manipulation, identification and/or sequencing of nucleic acid samples according to certain embodiments of the present invention. These samples may be derived from plant, animal, viral or bacterial sources. For example, the device, method and system of the invention may be used in diagnostic applications, such as in diagnosing genetic disorders, as well as diagnosing the presence of infectious agents, e.g., bacterial or viral infections. Additionally, the device, method and system be used in a variety of characterization applications, such as forensic analysis, e.g., genetic fingerprinting, bacterial, plant or viral identification or characterization, e.g. epidemiological or taxonomic analysis, and the like.
According to another embodiment of the present invention, high-throughput lab card (e.g., microcard) (e.g., 10× or 100×) for various applications, such as 96 wells are provided. A lab card, for example, a reaction card can perform, for example, 1, 2, 3, 4 . . . 96 reactions on an individual card. For example, a reaction card may be used for a plurality of assays, for example, WTA and WGSA. Typically, with more reactions, the number of ports and the size of the lab card will increase with the increased number of reactions.
According to another embodiment, at least one individual component insert (for example, reagent, array, etc.) is incorporated into a lab card.
An array component, for example, can be placed and positioned in various ways within a lab card. For example, the array(s) can be located within a chamber as illustrated in
In another embodiment of the present invention, a universal array processing card is provided where the array processing card includes card features where various number and types of array pegs can be inserted. Different arrays can be assembled into this universal array processing card depending on the application. According to another embodiment of the present invention, a plurality of samples can be processed simultaneously using a lab card.
Although primarily described in terms of producing a fully integrated body of the device for performing a particular assay wherein all the reagents including the sample is provided in the device along with all the microfluidic features required to perform an assay, the above described methods can also be used to fabricate additional lab cards which are used to perform separate process steps according to a preferred embodiment of the present invention. The lab card is design such that the reagents can be transferred from one to the other according to an embodiment of the present invention.
A system for controlling liquids is provided which comprises a plurality of lab cards according to an embodiment of the present invention. According to an embodiment of the present invention, a two stage platform is provided where fluid reagents are transferred from a reaction/storage/analytical chamber from a first lab card to a another chamber in a second lab card. The first lab card comprises at least one outlet port and the second lab card comprises at least one inlet port. A positive pressure source may be applied to the originating chamber in the first lab card to push the reagent into the chamber in the second lab card.
A gasket comprises a first surface and a second surface, wherein the first surface is flat around the inlet port to assure a liquid tight seal when mated to the second device. The gasket (1902) can be press fit or attached, for example, by adhesive, into the port before or after the mating of the lab cards. A housing may also be provided which includes an alignment and a clamping mechanism to align and clamp the two lab cards together according to another embodiment of the present invention. The compression of the gasket permits liquid to flow from the first device to the second device.
According to another embodiment, as illustrated in
According to another embodiment of the present invention, more than two cards can to assembled simultaneously to transfer the reagents. For example, a first lab card that is used to store a set of reagents, a second lab card that is used to perform a first assay, and a third lab card that is used to perform the next assay, can be assembled together such that both assays are performed without any manual intervention. It is to be understood that the above description is intended to be illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description and figures. Such variation may include a sample card, a reagent storage card, a reaction card and an array processing card to perform a complete assay, and the like according to embodiments of the present invention. According to another embodiment, the apparatus to perform the two-stage process is incorporated in the base plate assembly.
A system to operate a microfluidic card which may also be called a microfluidic or lab card system is shown in
a-e illustrate an overall enclosed system (2000) including a pneumatic manifold assembly (2001) placed on top of a lab card (1500) mounted onto a base plate assembly (2003) controlled by a computer (not shown) according to an embodiment of the present invention. In a preferred embodiment, all fluid transport or processing is performed within lab card(s) by utilizing pressurized air, vacuum and vents with a pneumatic manifold (2001). All the reagents that are required to perform an assay is stored in a lab card(s) so that there is no handling of any liquids while performing an assay according to an embodiment of the present invention. The reagents can be transferred from a lab card that stores the reagents into storage chambers of a lab card that performs a reaction. The processed reagents from a lab card can also be transferred into another lab card that is configured to perform the next process step. Waste chambers are provided in the lab card to collect the generated waste. Thus, the reagents are contained within the lab card throughout the entire process. Application of a positive pressure to the fluid inlet, combined with the selective opening of the other valves may introduce the sample into the channels/chambers. The combination of providing a vacuum, air pressure or vent provides the various processes described above that is required to perform an assay. According to an embodiment of the present invention, all the external processing components are mounted onto a base plate.
The overall system excluding the computer and manifold will typically be approximately 11 inches in length×8 inches in width×6 inches in height or smaller according to a preferred embodiment of the present invention. Although indicative of a rectangular shape, it will be readily appreciated that the devices of the invention may be embodied in any number of shapes depending upon the particular need. Additionally, these dimensions will typically vary depending upon the number of operations to be performed by the device, the complexity of these operations and the like. As a result, these dimensions are provided as a general indication of the size of the device. The number, shape and size of the channels included within the device will also vary depending upon the specific application for which the device is to be used. According to an embodiment of the present invention, an imaging/scanning instrument is integrated into the fluidic control instrumentation.
According to another embodiment of the present invention, the system, in general is portable. The base plate assembly, manifold and the lab cards that do not require extra storage conditions are placed into a transporting apparatus such that a user can carry the system from one location to the next according to an embodiment of the present invention. The system can be shipped back for trouble shooting or maintenance as required according to an embodiment of the present invention. The automated system requires minimal training and can be operating with minimal operator intervention.
Computer software products are provided to control various active components (i.e. the valves, or liquids, microfluidic system, etc.), temperature and measurement devices. The system may conveniently be controlled by any programmable device, preferably a digital computer such as a Dell personal computer. The computers typically have one or more central processing unit coupled with a memory. A display device such as a monitor is attached for displaying data and programming. A printer may also be attached. A computer readable medium such as a hard drive or a CD ROM can be attached. Program instructions for controlling the liquid handling can be stored on these devices.
According to an another embodiment of the present invention, a barcode reader, Radio Frequency Identification (RFID), magnetic strip, or other means of electronic identification is integrated into the microfluidic or lab card system. Use of the electronic identification mechanism may assure that the proper components (for example, lab cards, reagents, array pegs, individual reagent components, manifold, base plate assembly) are used and the components are placed properly. The identification mechanism may also provide data related to the design or conduct of experiments. A lab card (1500) may include, for example, a barcode label which identifies the particular lab card and the components within the lab card. Further, a barcode reader (not shown) may be disposed within base plate assembly (2100) to read the barcode label as the lab card is being removed from/or placed into the base plate assembly (2100). In this manner, the lab cards may include a barcode label that is scanned with a fixed barcode reader. The use of barcodes is also described in U.S. Pat. No. 6,399,365 and U.S. Pat. No. 7,108,472 which are hereby incorporated by reference herein in their entirety for all purposes.
The PC board may be configured to control all of the operations so that scanning takes place in a fully automated manner. Conveniently, a barcode scanner may be employed to identify the lab card contents to the host computer. Conveniently, a computer having a display screen may be coupled to the PC board and may include a networking interface to permit convenient interaction with the scanner and the other apparatuses. Further, the host computer may include appropriate display screens to permit manual operation of any of the above steps and to permit tracking of a specific components (for example, lab card) based on the barcode information.
a-e illustrate an example of a preferred pneumatic manifold system according to an embodiment of the present invention.
According to an embodiment of the present invention, the weight of the manifold is utilized to assist in creating an air tight connection between the manifold and the lab card by placing the manifold on top of the lab card as shown in
A universal manifold assembly is provided by separating all the gas or air requirements from the rest of the processing components that do not require a gas drive source according to an embodiment of the present invention. A set of air requirements (2200) is illustrated in
The manifold is configured such to provide the necessary combination of air requirements to provide flexibility among the ports according to an embodiment of the present invention. For example, the same pneumatic manifold is used during the processing of a sample card, a reagent card, a reaction card, and an array processing card (hybridization, wash, and stain) on a first base plate assembly and then used to process an array processing card for scanning on the second base plate assembly wherein the external component assembly is a scanning mechanism according to an embodiment of the present invention. The same pneumatic manifold can deliver different air requirements by operating a different computer program according to another embodiment of the present invention.
According to an embodiment of the present invention, a base plate assembly is provided for system integration. The base plate assembly is used to apply an external processing component to a lab card with a plurality of reagents is provided. In general, performing an assay requires exposure to external processing components such as, for example, cooling, heating, a magnetic field, exposure to a cold area for storage, and scanning. According to another embodiment of the present invention, an imaging instrument is integrated into the base plate assembly.
a-e illustrates different views of the base plate assembly showing other components. For example,
For PCR amplification methods, denaturation and hybridization cycling will preferably be carried out by repeated heating and cooling of the sample. Accordingly, PCR based amplification chambers will typically include a temperature controller for heating the reaction to carry out the thermal cycling. For example, a heating element or temperature control block may be disposed adjacent the external surface of the amplification chamber thereby transferring heat to the amplification chamber. In this case, preferred devices will include a thin external wall for chambers in which thermal control is desired. This thin wall may be a thin cover element, e.g., polycarbonate sheet, or high temperature tape, i.e. silicone adhesive on Kapton tape (commercially available from, e.g., 3M Corp.). Micro-scale PCR devices have been previously reported. For example, published PCT Application No. WO 94/05414, to Northrup and White, which is hereby incorporated by reference herein in its entirety for all purposes, reports a miniaturized reaction chamber for use as a PCR chamber, incorporating microheaters, e.g., resistive heaters. The high surface area to volume ratio of the chamber allows for very rapid heating and cooling of the reagents disposed therein. Similarly, U.S. Pat. No. 5,304,487 to Wilding et al., which is hereby incorporated by reference herein in its entirety for all purposes, also discusses the use of a microfabricated PCR device.
In preferred embodiments, a chamber or channel used to contain a reagent to be heated will incorporate a thin bottom layer (e.g., thickness of 200 μm) for fast and accurate heat conduction from heaters/coolers. In another preferred embodiments, the chamber or channel will incorporate a controllable heater disposed adjacent to the external thin surface, for example, for thermal cycling of the sample. Thermal cycling is carried out by varying the current supplied to the heater to achieve the desired temperature for the particular stage of the reaction. Alternatively, thermal cycling for the PCR reaction may be achieved by transferring the fluid sample among a number of different reaction chambers or regions of the same reaction chamber, having different, although constant temperatures, or by flowing the sample through a serpentine channel which travels through a number of varied temperature ‘zones’. Heating may alternatively be supplied by exposing the amplification chamber to a laser or other light or electromagnetic radiation source.
A computer and a computer software product (for example, LabView) and at least one PCB are provided to control the various active components (i.e., valves, temperature control, etc.). For example, the programmable temperature control is realized by using, for example, a LabView program to send out an analog signal into the system as a temperature set point. According to an embodiment of the present invention, the lab card has three separate regions including a cold reagent storage region (1804) (e.g., temperature at 4° C.) and two heating (1505)/cooling (1504) regions (e.g., temperature varying from 16 to 70° C. for cooling or heating). The temperature of the three regions are controlled by three PCBs (421) as illustrated in
a. Transporting Mechanism
According to an embodiment of the present invention, microfluidic devices and methods are provided herein to perform multiple reactions simultaneously for conducting a variety of high throughput analyses. Typically, performing multiple assays simultaneously would require an increased number of valves by a factor of the number of assays to be performed. For example, if an individual assay that provides several reactions uses 36 valves, 432 valves would be required (36×12) to perform 12 assays simultaneously and 3,456 valves (36×96) would be required to perform 96 assays simultaneously. As the number of assays increase, the number of valves plus other requirements (i.e. operating mechanism, foot print etc.) significantly increases. In a preferred embodiment, multiple assays are performed simultaneously with a significantly reduced overall system footprint, minimal number of valves, and a significantly reduced control mechanism.
According to one aspect of the present invention, the apparatus, method and system of the present invention are directed toward a microfabricated device (2400) for simultaneously transporting a plurality of aliquots of at least one fluid a known distance in a lab card. The device comprises a first and a second structure (2410) separated from a common chamber (2411) by a flexible diaphragm (2412). The flexible diaphragm (2412) covers the first and second structures (2410). The first and second structures (2410), each chamber having a known volume, are each in connection with a different reaction port (2413). A valve (2414) is actuated to allow a pressurized gas to fill the common chamber (2411) to deform the flexible diaphragm (2412) into the first and second structures (2410) so that the aliquots of the fluid are transported the known distance in the lab card. According to another aspect of the present invention, the lab card is designed to perform the corresponding number of assays simultaneously.
a and b illustrate one embodiment of the present invention that of a microfabricated device (2400) for simultaneously transporting a plurality of aliquots of at least one fluid a known distance in a lab card (1500
In a preferred embodiment, the structures have a known volume allowing for precise transporting of a fluid in a lab card or other device. In this case, the volume of the structures will be dictated by the transportation needs of a given reaction. Further, the microfabricated device may be fabricated to include a range of volumetric chambers having varied, but known volumes according to the requirements of a given reaction, or a set of volumetric chambers to accommodate various different lab cards. The number, shape and size of the structures included within the microfabricated device (2400) will vary depending upon the specific application for which the device is to be used. In the Figures the structures are depicted as semi-circles but one would appreciate that many different shapes may be used. For example, the structure may be a circle, oval, star, free shape and the like. The microfabricated device (2400) may comprise a number of structures (2410), which will depend on the number of reactions or assays to be performed simultaneously. For example, a microfabricated device may have 1 to 96 structures. In the example, where 96 assays are performed simultaneously, there may be one flexible diaphragm that covers all the structures such that the aliquots of liquids for the 96 assays are all controlled by one valve according to an embodiment of the present invention. The flexible diaphragm may made of any material that will function as described in this application. In a preferred embodiment of the present invention, the flexible diaphragm is comprised of an elastomeric material, preferably silicone rubber. The flexible diaphragm is bonded to the corresponding substrate of the microfabricated device by methods known in the art, for example, thermal bonding, mechanical snapping features, ultra sonic welding and the like. In a preferred embodiment of the present invention, the flexible diaphragm is bonded to the substrate with an adhesive. Preferred adhesives are resistant to degradation under conditions to which the substrates will be subjected to. In preferred aspects, an ultraviolet cured adhesive bonds the flexible diaphragm to the substrate.
According to another aspect of the present invention, the method provides a microfabricated device (2400) having at least a first and a second structure (2410) separated from a common chamber (2411) by a flexible diaphragm (2412). The flexible diaphragm (2412) covers the first and second structures. Each of the structures (2410) has a known volume and is in connection with a different reaction port (2413). The microfabricated device (2400) is preferably in contact with a lab card where the reaction ports (2413) on the microfabricated device (2400) are connected to the reaction ports (1501) on the lab card which contains a plurality of aliquots of a liquid. According to another aspect of the present invention, the lab card is designed to perform the corresponding number of reactions simultaneously. The aliquots of liquid are transported a known distance in the lab card by actuating a valve (2414) to introduce a pressurized gas into the microfabricated device (2400). The pressurized gas fills the common chamber (2411) which then causes the flexible diaphragm (2412) to deform into the first and second structures (2410). The displacement of the gas in the structures (2410) causes the pressure in the channels to increase and the aliquots of fluid to be transported a known distance in the lab card. The gas is Nitrogen, Argon, CDA, etc. according to an embodiment of the present invention, In a preferred embodiment, the gas is air.
In a preferred embodiment of the present invention, the microfabricated device including the structures, flexible diaphragm and valve(s) are included in a subassembly in the base plate assembly (2100). According to an embodiment of the present invention, the microfabricated device is constructed for various different lab cards. In another aspect of the present invention, the microfabricated device can be interchanged with other microfabricated devices to be used for different lab cards.
b. Mixing Mechanism
According to an embodiment of the present invention, the apparatus, method and system of the present invention are directed toward a microfabricated device (2500) for simultaneously mixing a plurality of aliquots of a mixture of fluids in a lab card. The device comprises a plurality of pairs of a first structure (2410) and a second structure (2510). The first structures (2410) are separated from the second structures (2510) by at least one flexible diaphragm (2412). The first and second structures are each a chamber having a known volume. The first structures (2410) are each in connection with a reaction port (2413) and the second structures (2510) are connected by a common chamber. The microfabricated device (2500) is preferably in contact with a lab card. According to another aspect of the present invention, the lab card is designed to perform the corresponding number of reactions simultaneously. The reaction ports (2413) on the microfabricated device (2500) are connected to the reaction ports (1501) on the lab card which contains a plurality of aliquots of a mixture of fluids. The aliquots of a mixture of fluids are mixed in the lab card by alternately actuating a valve (2414) that introduces a pressurized gas and a valve (2520) that creates a vacuum in the microfabricated device (2500). The common chamber (2411) is pressurized with a gas (2512) which deforms the flexible diaphragm (2412) into the first structures (2410). The gas is displaced in the structures causing pressure in the channels to increase and the aliquots of fluid to transport a known distance, for example, point A to point B, in the lab card. The pressure is turned off and a vacuum valve (2520) is actuated to create a vacuum (2513) in the common chamber (2411) causing the flexible diaphragm (2412) to deform into the second structures (2510) such that the aliquots of the mixture of fluids are transported back a known distance from point B to point A. The transporting from point A to point B, back to point A results in mixing of the mixture of fluids in the lab card.
a-d illustrate an embodiment of the present invention of a method of simultaneously mixing a plurality of aliquots of a mixture of fluids in a lab card.
b-d illustrate the steps involved in the mixing process according to an embodiment of the present invention.
c. Rechargeable Transporting Mechanism
a-d illustrate another embodiment of the present invention of another microfabricated device for simultaneously transporting a plurality of aliquots of a fluid a known distance in a lab card that can be repeatedly used.
Step 4 (2653) includes deactuating the second valve (2414b) while maintaining the deactuating step of the first valve (2414a) to equilibrate the pressure in the lab card. Steps 1-4 are repeated to transport the plurality of aliquots of the fluid in the lab card.
According to another embodiment of the present invention, the apparatus, method and system of the present invention are directed toward a microfabricated device for simultaneously transporting a plurality of aliquots of a fluid a known distance in a lab card. The device comprises a plurality of pairs of a first and a second structure connected by one common channel. The first structures are separated from a first common chamber by a first flexible diaphragm. The first structures are each in connection with a reaction port. The first structures are chambers having a known volume. The second structures are separated from a second common chamber by a second flexible diaphragm and the second structures are connected to a vent. The second structures can be connected to a common vent or each second structure can be connected to a different vent or a combination thereof. A first valve and a second valve are provided to introduce pressurized gas. The second valve is connected to the second common chamber and the first valve is connected to the first common chamber.
According to an embodiment of the present invention, an apparatus, method, and system for constructing a microfabricated device for simultaneously transporting a plurality of aliquots of at least one fluid a known distance in a lab card are provided. The microfabricated device includes a first and a second structure separated from a common chamber by a flexible diaphragm. The flexible diaphragm covers the first and second structures, where each chamber has a known volume. The first and second structures are each in connection with a different reaction port. A valve is actuated to allow a pressurized gas to fill the common chamber to deform the flexible diaphragm into the first and second structures so that the aliquots of the fluid are transported the known distance in the lab card.
According to an embodiment of the present invention, an apparatus, method, and system for constructing a microfabricated device for simultaneously mixing a plurality of aliquots of a mixture of fluids in a lab card are provided. The microfabricated device includes a plurality of pairs of a first structure and a second structure. The first structures are separated from the second structures by at least one flexible diaphragm. The first and second structures are each a chamber having a known volume. The first structures are each in connection with a reaction port and the second structures are connected by a common chamber. A valve is actuated to allow a pressurized gas to fill the common chamber with a gas to deform the flexible diaphragm into the first structures. The displaced gas causes the aliquots of the mixture of fluids to be transported a known distance from point A to point B in the lab card. A vacuum valve is then actuated to create a vacuum in the common chamber to deform the flexible diaphragm into the second structures. The deformation of the flexible diaphragm causes the aliquots of the mixture of fluids to be transported back a known distance from point B to point A. The movement of the aliquots of fluids from point A to point B, back to point A results in mixing of the mixture of fluids in the lab card.
According to an another embodiment of the present invention, an apparatus, method, and system for constructing a microfabricated device for simultaneously transporting a plurality of aliquots of at least one fluid a known distance in a lab card that can be repeated are provided. The device includes a plurality of pairs of a first and a second structure connected by one common channel. The first structures are separated from a first common chamber by a first flexible diaphragm. The first structures are each in connection with a reaction port. The first structures are chambers having a known volume. The second structures are separated from a second common chamber by a second flexible diaphragm. The second structures are connected to a vent.
The aliquots of a fluid are simultaneously transported a known distance in the lab card by performing the following steps: Step 1) actuate a second valve while a first valve is deactuated to allow a pressurized gas to fill the second common chamber. The pressurized gas causes the flexible diaphragm to deform into the second structure, closing the vent and the openings between the common channels and the second structures, Step 2) actuate the first valve while maintaining the actuation of the second valve to allow the pressurized gas to fill the first common chamber. The pressurized gas caused the flexible diaphragm to deform into the first structure, initiating the transportation of the aliquots of the fluid a know distance in the lab card, Step 3) deactuate the first valve while maintaining the actuation of the second valve to finish transporting the aliquots of the fluid a known distance in the lab card and Step 4) deactuate the second valve while maintaining the deactuation of the first valve to equilibrate the pressure in the lab card. Steps 1-4 are repeated to transport the plurality of aliquots of the fluid in the lab card according to a preferred embodiment of the present invention.
Experiments were performed to perform the WTA assay protocol as illustrated in
The assay began with placing the reaction card (1100) into the pocket (1702) of the base plate assembly (1700). Lab View was used to operate the system and provide temperature control. A Total RNA (1410) sample was transferred from the storage chamber to the reaction chamber where the second reagent, 1st strand buffer (1411) was added to synthesize the 1st strand cDNA. After 2nd strand cDNA synthesis was initiated with the addition of the third reagent (1412), the solution was incubated with the addition of the fourth reagent (1413). EDTA (1414), the fifth reagent, was added to make a total volume of 16.2 μl. The bead purification step involved four reagents: magnetic beads, alcohol, alcohol, and water. Afterwards, cDNA fragmentation (1415) was completed with the addition of the tenth reagent. The eleventh reagent (1416) was added and the Terminal Labeling step was completed. Finally, the last reagent, EDTA (1417), was added and the resulting labeled and fragmented sample was then hybridized to an Affymetrix U133A chip. All 12 reagents were stored in the reaction card and all the reactions in this WTA assay protocol was processed within the reaction card.
Experiments were performed to perform the WTA assay protocol as illustrated in
The assay began with placing the reaction card (1100) into the pocket (1702) of the base plate assembly (1700). Lab View was used to operate the system and provide temperature control. The Total RNA (1410) sample was transferred from the storage chamber to the reaction chamber where the second reagent, 1st strand buffer (1411) was added to synthesize the 1st strand cDNA. After the 2nd strand cDNA was synthesized with the addition of the third reagent (1412), the solution was incubated with the addition of the fourth reagent (1413). EDTA (1414), the fifth reagent, was added to make a total volume of 16.2 μl. The beads purification step involved four reagents: magnetic beads, alcohol, alcohol, and water. Afterwards, cDNA fragmentation (1415) was completed with the addition of the tenth reagent. The eleventh reagent (1416) was added and the Terminal Labeling step was completed. Finally, the last reagent, EDTA (1417), was added and the resulting sample was then hybridized with an Affymetrix U133A chip. All 12 reagents were stored in the reaction card and all the reactions in this WTA assay protocol was processed within the reaction card.
A set of lab cards are utilized to perform an assay: a sample card, a reaction card, an array processing card for hybridizing, washing, and staining and an array processing card for scanning. All the lab cards are universal such that each lab card can be utilized in a plurality of various assays, applications, etc. For example, a universal reagent card provides a number of lab card features for a plurality of assays or reactions.
At least one sample from a patient is stored in a sample card. The sample is then transferred into a reagent card where all the reagents are stored to perform the assay, for example, the WTA assay as described in the first example. The resulting sample is then transferred to an array processing card that includes a plurality of array pegs for hybridizing, washing and staining. After processing the arrays, the array pegs are then transferred and scanned into an array processing card specifically for scanning.
A lab card was fabricated using convention embossing. The lab card was made from a thermoplastic and consisted of three pieces: top, middle and bottom. The middle piece required two through holes which were formed during the embossing process according to an embodiment of the present invention. The mold structure included a top plate (1201), a middle plate (1202), a back plate (1203) with two pins (1207) that will penetrate the substrate material of the middle piece during embossing as shown in
A microfabricated device and a lab card were constructed to facilitate the operation of nine sample preparations simultaneously. The microfabricated device included the mixing mechanism as shown in
It is to be understood that the above description is intended to be illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description and figures. All cited references, including patent and non-patent literature, are incorporated by reference herein in their entireties for all purposes. The figures are merely examples, which should not unduly limit the scope of the claims. One of ordinary skill in the art would recognize many variations, alternatives, and modifications.
The present application claims priority from U.S. Provisional Patent Application Ser. No. 60/942,792, filed Jun. 8, 2007, and is a continuation in part of U.S. patent application Ser. No. 11/761,062, filed Jun. 11, 2007, which is a continuation in part of U.S. patent application Ser. No. 11/761,007, filed Jun. 11, 2007, which is a continuation in part of U.S. patent application Ser. No. 11/760,948, filed Jun. 11, 2007 and a continuation in part of U.S. patent application Ser. No. 11/760,938, filed Jun. 11, 2007, which both claim priority from U.S. Provisional Patent Application Ser. No. 60/813,547, filed Jun. 13, 2006, and claim priority from U.S. Provisional Patent Application Ser. No. 60/814,014, filed Jun. 14, 2006, and claim priority from U.S. Provisional Patent Application Ser. No. 60/814,316, filed Jun. 15, 2006, and claim priority from U.S. Provisional Patent Application Ser. No. 60/814,474, filed Jun. 16, 2006, and claim priority from U.S. Provisional Patent Application Ser. No. 60/815,506, filed Jun. 20, 2006, and claim priority from U.S. Provisional Patent Application Ser. No. 60/816,099, filed Jun. 22, 2006, and claim priority from U.S. Provisional Patent Application Ser. No. 60/942,792, filed Jun. 11, 2007, and are continuation in part of U.S. patent application Ser. No. 11/553,944, filed Oct. 27, 2006. Each application is hereby incorporated by reference herein in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 60942792 | Jun 2007 | US | |
| 60813547 | Jun 2006 | US | |
| 60814014 | Jun 2006 | US | |
| 60814316 | Jun 2006 | US | |
| 60814474 | Jun 2006 | US | |
| 60815506 | Jun 2006 | US | |
| 60816099 | Jun 2006 | US | |
| 60942792 | Jun 2007 | US | |
| 60732538 | Nov 2005 | US | |
| 60813547 | Jun 2006 | US | |
| 60814014 | Jun 2006 | US | |
| 60814316 | Jun 2006 | US | |
| 60814474 | Jun 2006 | US | |
| 60815506 | Jun 2006 | US | |
| 60816099 | Jun 2006 | US | |
| 60942792 | Jun 2007 | US | |
| 60732538 | Nov 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11761062 | Jun 2007 | US |
| Child | 12134857 | US | |
| Parent | 11761007 | Jun 2007 | US |
| Child | 11761062 | US | |
| Parent | 11760948 | Jun 2007 | US |
| Child | 11761007 | US | |
| Parent | 11553944 | Oct 2006 | US |
| Child | 11760948 | US | |
| Parent | 11760938 | Jun 2007 | US |
| Child | 11761007 | US | |
| Parent | 11553944 | Oct 2006 | US |
| Child | 11760938 | US |
