Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUSRE43365 E1
Publication typeGrant
Application numberUS 12/891,733
Publication dateMay 8, 2012
Filing dateSep 27, 2010
Priority dateMar 14, 2003
Fee statusPaid
Publication number12891733, 891733, US RE43365 E1, US RE43365E1, US-E1-RE43365, USRE43365 E1, USRE43365E1
InventorsBrian L. Anderson, Bill W. Colston, Christopher J. Elkin
Original AssigneeLawrence Livermore National Security, Llc
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Apparatus for chemical amplification based on fluid partitioning in an immiscible liquid
US RE43365 E1
Abstract
A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.
Images(4)
Previous page
Next page
Claims(58)
1. An apparatus for nucleic acid amplification of a sample, comprising:
means for partitioning said sample into partitioned sections, wherein said means for partitioning said sample into partitioned sections comprises an injection orifice, and
means for performing PCR on said partitioned sections of said sample.
2. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said injection orifice is an injection orifice that produces microdroplets.
3. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said injection orifice is an injection orifice that injects said sample and a PCR reagent.
4. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for performing PCR on said partitioned sections of said sample comprises a continuous tube for circulating said partitioned sections of said sample through a heater to perform PCR.
5. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for performing PCR on said partitioned sections of said sample comprises a continuous tube for circulating said partitioned sections of said sample through a heater and cooler to perform PCR.
6. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for performing PCR on said partitioned sections of said sample comprises a pump, a continuous tube, and a heater.
7. The apparatus for nucleic acid amplification of a sample of claim 1 including means for detection and analysis of said partitioned sections of said sample comprising a laser and a detector.
8. The apparatus for nucleic acid amplification of a sample of claim 1 including means for detection and analysis of said partitioned sections of said sample comprising a blue laser and a detector.
9. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for partitioning said sample into partitioned sections comprises means for separating said sample into immiscible slugs.
10. A method of nucleic acid amplification of a sample, comprising the steps of:
partitioning said sample into partitioned sections, wherein said step of partitioning said sample into partitioned sections comprises flowing said sample through an injection orifice, and
subjecting said partitioned sections of said sample to PCR.
11. The method of claim 10 wherein the nucleic acid amplification of a sample comprises PCR amplification of a DNA target.
12. The method of claim 11 wherein said partitioned sections contain, on average, a single template of a DNA target, and wherein said single template is amplified within said partitioned sections.
13. The method of claim 12 wherein said sample comprises multiple DNA targets, and wherein multiple partitioned sections have a single template of a different DNA target such that said single template is amplified within said multiple partitioned sections.
14. The method of claim 10, wherein the partitioned sections are passed by a detector to detect the amount of amplification.
15. The method of claim 14 wherein the detector is a light detector.
16. The method of claim 15 wherein an amount of amplification is indicated by fluorescence.
17. The method of claim 16 where a fluorophore dye is used.
18. The method of claim 15 wherein a laser is projected upon the partitioned sections as they pass between the laser and detector.
19. The method of claim 15 wherein the detector comprises a confocal imaging system.
20. The method of claim 15 wherein scattering profiles from the partitioned sections are used to eliminate background signals.
21. The method of claim 16 wherein the partitioned sections are probed for fluorescent signal at a rate of several thousand per second.
22. A nucleic acid amplification apparatus comprising a microdroplet generator comprising an orifice, wherein said orifice connects a sample flow pathway to a channel or tube comprising an immiscible fluid, and wherein said channel or tube passes through a heating element.
23. The apparatus of claim 22 further comprising a cooler.
24. The apparatus of claim 22 wherein said microdroplet generator is capable of producing microdroplets with volumes in the picoliter range.
25. The apparatus of claim 22 wherein said microdroplet generator is capabe of producing microdroplets having volumes of about 5×10−9 liters to 1×10−12 liters.
26. The apparatus of claim 22 wherein the immiscible fluid is mineral oil.
27. The apparatus of claim 22, further comprising a a pump for moving generated microdroplets in said immiscible fluid through the clannel or tube.
28. The apparatus of claim 27 further comprising a pump for moving the microdroplets through the channel or tube.
29. The apparatus of claim 27 wherein the tube is a continuous tube.
30. The apparatus of claim 27 wherein the channel is a micromachined channel.
31. The apparatus of claim 28 wherein the pump for moving the microdroplets comprises a magnetohydrodynamic (MHD) element.
32. The apparatus of claim 27 wherein the channel or tube is heated and cooled.
33. The apparatus of claim 27 wherein the channel or tube extends through a heater and a cooler.
34. A nucleic acid amplification apparatus comprising:
a microdroplet generator comprising an orifice wherein said orifice connects a sample flow pathway to a channel or tube comprising an immiscible fluid, wherein said channel or tube passes through a heating element; and wherein said apparatus further comprises a detector capable of detecting microdroplets in said immiscible fluid.
35. The apparatus of claim 34 wherein the detector is positioned such that generated microdroplets suspended in said immiscible fluid pass by the detector as they are moved through the channel or tube.
36. A method for nucleic acid amplification comprising:
producing microdroplets within an immiscible fluid in a channel or tube: wherein the microdroplets comprise nucleic acids and components for performing nucleic acid amplification;
moving the microdroplets through the channel or tube; and
thermal cycling the microdroplets in the channel or tube to amplify the nucleic acids.
37. The method of claim 36 wherein the nucleic acid amplification comprises PCR.
38. The method of claim 36 wherein the thermal cycling of the microdroplets comprises passing the microdroplets through a heater and a cooler.
39. The method of claim 36 wherein the thermal cycling of the microdroplets comprises heating and cooling the channel or tube comprising the microdroplets.
40. The method of claim 36 further comprising passing the microdroplets by a detector to detect an amount of amplification.
41. The method of claim 40 wherein the detector is a light detector.
42. The method of claim 41 wherein the amount of amplification is indicated by fluorescence.
43. The method of claim 42 where a fluorophore dye is used.
44. The method of claim 41 wherein a laser is projected upon the microdroplets as they pass between the laser and detector.
45. The method of claim 41 wherein the detector comprises a confocal imaging system.
46. The method of claim 41 wherein scattering profiles from the microdroplets are used to eliminate background signals.
47. A method comprising:
diluting a sample comprising a plurality of DNA targets and PCR reagents:
partitioning the sample into microdroplets in an immiscible fluid in a tube or channel of a microfluidic device, wherein a plurality of microdroplets containing a single template of the target DNA are formed; and amplifying the target DNA in the microdroplets by heating and cooling such that a plurality of single templates within the microdroplets are amplified.
48. A method comprising:
a. performing PCR on a microdroplet suspended in an immiscible fluid in a microchannel, wherein said PCR comprises a plurality of cycles;
b. passing said microdroplet through said microchannel past a detector; and
c. detecting a PCR amplification product in said microdroplet.
49. The method of claim 48, wherein said microdroplet is isolated from a bulk solution, and whereby the number of PCR cycles needed to detect said amplication product in said microdroplet is less than the number of PCR cycles needed to detect amplication product in said bulk solution.
50. The method of claim 48, wherein said microdroplet is isolated from a bulk solution, and whereby the time needed for each cycle of PCR on said microdroplet is less than the time needed for each cycle of PCR in said bulk solution.
51. The method of claim 48 wherein the volume of said microdroplet is about 5×10−9 liters to 1×10−12 liters.
52. A nucleic acid amplification apparatus comprising: a microdroplet generator comprising an orifice wherein said orifice connects a sample flow pathway to a channel or tube comprising an immiscible fluid, wherein said channel or tube passes through a heating element; and wherein said apparatus further comprises a detector capable of detecting microdroplets in said immiscible fluid and a pump for moving said microdroplets through the channel or tube.
53. The apparatus of claim 52 wherein the detector is positioned such that generated microdroplets suspended in said immiscible fluid pass by the detector as they are moved through the channel or tube.
54. The apparatus of claim 52 wherein the immiscible fluid is mineral oil.
55. The apparatus of claim 52 wherein the tube is a continuous tube.
56. The apparatus of claim 52 wherein the channel is a micromachined channel.
57. The apparatus of claim 52 wherein the pump for moving the microdroplets comprises a magnetohydrodynamic (MHD) element.
58. The apparatus of claim 52 wherein the channel or tube extends through a heater and a cooler.
Description

The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.

This application is a Reissue of application Ser. No. 10/389,130, filed Mar. 14, 2003, issued as U.S. Pat. No. 7,041,481 on May 9, 2006.

Notice: More than one reissue application has been filed for the reissue of U.S. Pat. No. 7,041,481, which claims the benefit of U.S. patent application Ser. No. 10/389,130, filed Mar. 14, 2003. The reissue applications are application Ser. Nos. 12/891,733 (the present application), and 12/118,418, filed May 9, 2008 and issued as U.S. Pat. No. Re. 41,780. The present application is a continuation reissue application of U.S. Pat. No. Re. 41,780, and adds new claims relative to U.S. Pat. No. 7,041,481.

BACKGROUND

1. Field of Endeavor

The present invention relates to chemical amplification and more particularly to chemical amplification based on fluid partitioning.

2. State of Technology

U.S. Pat. No. 4,683,202 issued Jul. 28, 1987; U.S. Pat. No. 4,683,195 issued Jul. 28, 1987; and U.S. Pat. No. 4,800,159 issued Jan. 24, 1989 to Kary B. Mullis et al provide background information. The patents describe processes for producing any particular nucleic acid sequence from a given sequence of DNA or RNA in amounts which are large compared to the amount initially present. The DNA or RNA may be single-or-double-stranded, and may be a relatively pure species or a component of a mixture of nucleic acids. The process utilizes a repetitive reaction to accomplish the amplification of the desired nucleic acid sequence. The extension product of one primer when hybridized to the other becomes a template for the production of the desired specific nucleic acid sequence, and vice versa, and the process is repeated as often as is necessary to produce the desired amount of the sequence.

U.S. Pat. No. 6,503,715 for a nucleic acid ligand diagnostic biochip issued Jan. 7, 2003 provides the following background information, “Methods are provided in the instant invention for obtaining diagnostic and prognostic Nucleic acid ligands, attaching said ligands to a Biochip, and detecting binding of target molecules in a Bodily to said Biochip-bound Nucleic acid ligands.” In one embodiment of the instant invention, one or more Nucleic acid ligands are chosen that bind to molecules known to be diagnostic or prognostic of a disease; these ligands are then attached to the Biochip. Particular methods for attaching the Nucleic acid ligands to the Biochip are described below in the section entitled “Fabrication of the Nucleic Acid Biochip.” The Biochip may comprise either (i) Nucleic acid ligands selected against a single target molecule; or more preferably, (ii) Nucleic acid ligands selected against multiple target molecules.

U.S. Patent Application No. 2002/0197623 for nucleic acid detection assays published Dec. 26, 2002 provides the following background information, “means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences . . . methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.”

SUMMARY

Features and advantages of the present invention will become apparent from the following description. Applicants are providing this description, which includes drawings and examples of specific embodiments, to give a broad representation of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description and by practice of the invention. The scope of the invention is not intended to be limited to the particular forms disclosed and the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.

The present invention provides an apparatus for nucleic acid amplification of a sample comprising means for partitioning the sample into partitioned sections and means for performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides an apparatus for nucleic acid amplification and detection of a sample comprising means for partitioning the sample into partitioned sections, means for performing PCR on the partitioned sections of the sample, and means for detection and analysis of the partitioned sections of the sample. The present invention also provides a method of nucleic acid amplification of a sample comprising the steps of partitioning the sample into partitioned sections and subjecting the partitioned sections of the sample to PCR. Another embodiment of a method of the present invention provides a method of nucleic acid amplification and detection of a sample comprising the steps of partitioning the sample into partitioned sections, subjecting the partitioned sections of the sample to PCR, and detecting and analyzing the partitioned sections of the sample.

The invention is susceptible to modifications and alternative forms. Specific embodiments are shown by way of example. It is to be understood that the invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate specific embodiments of the invention and, together with the general description of the invention given above, and the detailed description of the specific embodiments, serve to explain the principles of the invention.

FIG. 1 is a flow diagram illustrating one embodiment of a system constructed in accordance with the present invention.

FIG. 2 is a flow diagram illustrating another embodiment of a system constructed in accordance with the present invention.

FIG. 3 is a diagram of another embodiment of a system constructed in accordance with the present invention.

FIG. 4 is a diagram of another embodiment of a system constructed in accordance with the present invention.

FIG. 5 is a diagram of another embodiment of a system constructed in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, to the following detailed description, and to incorporated materials; detailed information about the invention is provided including the description of specific embodiments. The detailed description serves to explain the principles of the invention. The invention is susceptible to modifications and alternative forms. The invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.

Referring now to the drawings, and in particular to FIG. 1, a flow diagram of one embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral 100. The system 100 provides a method and apparatus for performing extremely rapid nucleic acid amplification. The flow diagram illustrating system 100 shows block 101 “partitioning” the sample and block 102 performing “CR” on the sample. The system 100 provides an apparatus for nucleic acid amplification of a sample comprising means for partitioning the sample and means for performing PCR on the sample. The system 100 also provides a method of nucleic acid amplification of a sample comprising the steps of partitioning the sample and subjecting the sample to PCR. The system 100 has application wherever current PCR-type systems exist.

In block 101 a chemical reagent and an input sample are “partitioned” into a large number of microdroplets or other forms of fluid partitions prior to amplification in block 102. The partitioning 101 involves dispersing the DNA-containing solution. For example the partitioning 101 can be accomplished by dispersing the DNA-containing solution in an immiscible carrier liquid. The DNA-containing solution is dispersed in the immiscible carrier fluid as microdroplets. The DNA-containing solution can be partitioned in other ways, for example, by being dispersed as liquid slugs separated by the carrier fluid, as an emulsion with the carrier fluid, or by using a gelling agent that prevents transfer of DNA between partitioned regions. The DNA-containing solution can also be partitioned mechanically by partitioning the fluid into micro-tubes or capillaries, or into micro-wells.

With the system 100, each partitioned DNA-containing fluid volume contains the necessary biochemical constituents for selectively amplifying a specified portion of a sample DNA via polymerase chain reaction (PCR). The target DNA can be detected by monitoring for the colorimetric indicator (e.g., flourescence or optical absorption) generated with each DNA template duplicaton sequence.

In block 102 selected portions of each nucleic acid sample are amplified using polymerase chain reaction (PCR), with the product contained in each partitioned fluid volume. This results in much more concentrated amplification product, since the volume containing the reaction is so small.

The polymerase chain reaction (PCR), is a cyclic process whereby a large quantity of identical DNA strands can be produced from one original template. The procedure was developed in 1985 by Kerry Mullis, who was awarded the 1993 Nobel prize in chemistry for his work. In PCR, DNA is immersed in a solution containing the enzyme DNA polymerase, unattached nucleotide bases, and primers, which are short sequences of nucleotides designed to bind with an end of the desired DNA segment. Two primers are used in the process: one primer binds at one end of the desired segment on one of the two paired DNA strands, and the other primer binds at the opposite end on the other strand. The solution is heated to break the bonds between the strands of the DNA, then when the solution cools, the primers bind to the separated strands, and DNA polymerase quickly builds a new strand by joining the free nucleotide bases to the primers in the 5′-3′ direction. When this process is repeated, a strand that was formed with one primer binds to the other primer, resulting in a new strand that is restricted solely to the desired segment. Thus the region of DNA between the primers is selectively replicated. Further repetitions of the process can produce a geometric increase in the number of copies, (theoretically 2n if 100% efficient whereby n equals the number of cycles), in effect billions of copies of a small piece of DNA can be replicated in several hours.

A PCR reaction is comprised of (a) a double-stranded DNA molecule, which is the “template” that contains the sequence to be amplified, (b) primer(s), which is a single-stranded DNA molecule that can anneal (bind) to a complimentary DNA sequence in the template DNA; (c) dNTPs, which is a mixture of dATP, dTTP, dGTP, and dCTP which are the nucleotide subunits that will be put together to form new DNA molecules in the PCR amplification procedure; and (d) Taq DNA polymerase, the enzyme which synthesizes the new DNA molecules using dNTPs.

Current amplification systems are limited in practice to half hour type amplification and detection windows (−30 cycles, 1 minute/cycle). The system 100 provides faster amplification. This has many applications, for example, in Homeland Defense applications, faster detection methods (a few minutes) can push the deployment of these sensors from “detect to treat” to “detect to protect,” having a serious impact on the number of casualties from a massive bioagent release.

The system 100 has significant advantages over typical bulk DNA detection techniques (even microscale bulk solution approaches), including (1) much faster detection time through a reduction in the total number of temperature cycles required, (2) a reduction in the time for each cycle, and (3) removing interference from competing DNA templates. The system 100 achieves a reduction in the total number of cycles by limiting the dilution of the optically generated signal (e.g., fluorescence or absorption). The formation of partitioned fluid volumes of the DNA-containing solution effectively isolates the fluid volumes which contain the target DNA from the fluid volumes that do not contain the target DNA. Therefore, the dilution of the optical signal is largely eliminated, allowing much earlier detection. This effect is directly related to the number of fluid partitions formed from the initial sample/reagent pool.

The system 100 achieves a reduction in the total number of cycles that are needed by limiting the dilution of the optically generated signal (e.g., fluorescence or absorption). The formation of partitioned fluid volumes of the DNA-containing solution effectively isolates the fluid volumes which contain the target DNA from the fluid volumes that do not contain the target DNA. Therefore, the dilution of the optical signal is largely eliminated, allowing much earlier detection. This effect is directly related to the number of fluid partitions formed from the initial sample/reagent pool. The effect of the number of fluid partitions on the number of cycles required for detection can be described by the following Equation E1:

N = 1 n [ D L A N ( V X ) ] 1 n ( 2 )
where: N=number of cycles; DL,=detection limit for optical signal [moles/liter]; X=initial number of DNA molecules; V=volume containing DNA molecules [liters]; AN=Avagadro's number [6.023×1023 molecules/mole]. From Equation E1 it is clear that N, the number of cycles until detection, decreases as V, the partitioned fluid volume, decreases.

The system 100 reduces the duration of each temperature cycle by effectively increasing the concentration of reactants by enclosing them in picoliter type volumes. Since reaction rates depend on the concentration of the reactants, the efficiency of a partitioned fluid volume or droplet should be higher than in an ordinary vessel (such as a test tube) where the reactant quantity (DNA quantity) is extremely low. It is estimated that through the reduction in the number of cycles and the reduction in the time required for each cycles that the FPDD technique can reduce the detection time by an order of magnitude as compared to bulk solution DNA detection techniques.

The system 100 facilitates removal of interference from competing DNA templates. Given the extremely small volumes involved with Fluid-Partitioned DNA Detection (FPDD), it is possible to isolate a single template of the target DNA in a given partitioned volume or microdroplet. For example, the formation of 2000 partitioned fluid volumes or microdroplets (each with a volume of 5×10′9 9 liters) made by dividing a bulk solution of 10 microliters containing 2002000 DNA molecules, would result in one DNA molecule per microdroplet on average. This makes it possible to amplify only one template in mixtures containing many kinds of templates without interference. This is extremely important in processing of real world aerosol samples containing complex mixtures of DNA from many sources, and has direct application in screening of cDNA libraries.

Referring now to FIG. 2, a flow diagram of another embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral 200. The flow diagram illustrating system 200 shows block 201 “partitioning” the sample, block 202 performing “PCR” on the sample, and block 203 “detection and analysis.” The system 200 provides a method and apparatus for performing extremely rapid nucleic acid amplification and detection. The system 200 provides an apparatus for nucleic acid amplification of a sample comprising means for partitioning the sample into partitioned sections, means for performing PCR on the partitioned sections, and means for detection and analysis of the partitioned sections. The system 200 also provides a method of nucleic acid amplification of a sample comprising the steps of partitioning the sample into partitioned sections, subjecting the partitioned sections to PCR, and detecting and analyzing the partitioned sections of the sample.

In block 201 a chemical reagent and an input sample are “partitioned” into a large number of microdroplets or other forms of fluid partitions prior to amplification. The system 200 achieves a reduction in the total number of cycles by limiting the dilution of the optically generated signal (e.g., fluorescence or absorption). The formation of partitioned fluid volumes of the DNA-containing solution effectively isolates the fluid volumes which contain the target DNA from the fluid volumes that do not contain the target DNA. Therefore, the dilution of the optical signal is largely eliminated, allowing much earlier detection. This effect is directly related to the number of fluid partitions formed from the initial sample/reagent pool.

In block 202 selected portions of each nucleic acid sample are then amplified using polymerase chain reaction (PCR), with the product contained in each partitioned fluid volume. This results in much more concentrated amplification product, since the volume containing the reaction is so small. If a Taqman type detection approach is used, fluorescent dye molecules unquenched by the PCF amplification are also more concentrated, making possible earlier optical based detection. Since it is possible to contain very amounts of the starting target DNA in each partition fluid volume, inhibitory competition from near-neighbor DNA templates is less allowing screening of very dilute samples.

In block 203 partitioned portions of the sample are detected by monitoring for the calorimetric indicator (e.g., fluorescence or optical absorption) generated with each DNA template duplication sequence. The partitioned portions of the sample are optically probed to detect the colorimetric indicator which signals the presence of the target DNA. The partitioned portions of the sample can also be scanned optically to detect the colorimetric indicator signaling the presence of the target DNA. In one embodiment, fluorescence, generated by degradation of the dye/quencher pair on the primer, is detected using a confocal imaging system such as that employed in conventional flow cytometers. Scattering profiles from individual microdroplets, as in conventional flow cytometers, can be used to eliminate background signal from other particles.

The system 200 has application wherever current PCR-type systems exist, including medical, drug-discovery, biowarfare detection, and other related fields. Biowarfare detection applications include identifying, detecting, and monitoring bio-threat agents that contain nucleic acid signatures, such as spores, bacteria, etc. Biomedical applications include tracking, identifying, and monitoring outbreaks of infectious disease. The system 200 provides rapid, high throughput detection of biological pathogens (viruses, bacteria, DNA in biological fluids, blood, saliva, etc.) for medical applications. Forensic applications include rapid, high throughput detection of DNA in biological fluids for forensic purposes. Food and beverage safety applications include automated food testing for bacterial contamination.

Referring now to FIG. 3, a diagram of another embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral 300. The system 300 provides an instrument for performing Fluid-Partitioned DNA Detection (FPDD) with PCR based detection and amplification. The system 300 includes a partitioning section 301, a PCR section 302, and a detection and analysis section 303.

The partitioning section 301 includes a sample introduction unit 304 and a unit 305 where the sample and a PCR reagent are combined. The sample and a PCR reagent are injected through a small orifice 306. The injection of the sample through the small orifice 306 produces microdroplets 308.

The PCR section 302 includes a continuous tube 309 for circulating the microdroplets 308 and suspended in an immiscible carrier fluid 314. The microdroplets 308 suspended in an immiscible carrier fluid 314 are pumped through the continuous tube 309 by pump 311. The microdroplets 308 suspended in an immiscible carrier fluid 314 are cycled through heater 310 and cooler 315 to perform PCR.

The detection and analysis section 303 includes a blue laser 312 and a detector 313. The laser 312 is projected upon the droplets 308 as they pass through tube 308 between the laser 312 and the detector 313.

In the system 300, the DNA-containing solution is partitioned into many microdroplets 308 and suspended in an immiscible carrier fluid 314. The microdroplets 308 are formed by forcing the PCR mix (sample and reagent) through the small orifice or microjet 306. These microdroplets 308 are then captured in the immiscible fluid 314, such as mineral oil, and flowed past the heating element 310 and cooler 315. An optical signal (e.g., fluorescence or optical absorption), generated by degradation of the dye/quencher pair on the primer, is detected using a confocal imaging system such as that employed in conventional flow cytometers. Scattering profiles from individual microdroplets, as in conventional flow cytometers, can be used to eliminate background signal from other particles. Once exposed to multiple heating cycles, the microdroplets can be identified and probed for an optical signal at rates of several thousand per second.

The FPDD system achieves a reduction in the total number of cycles by limiting the dilution of the optically generated signal (e.g., fluorescence or absorption). The formation of partitioned fluid volumes of the DNA-containing solution effectively isolates the fluid volumes which contain the target DNA from the fluid volumes that do not contain the target DNA. Therefore, the dilution of the optical signal is largely eliminated, allowing much earlier detection. This effect is directly related to the number of fluid partitions formed from the initial sample/reagent pool. The effect of the number of fluid partitions on the number of cycles required for detection is described by the Equation E1 set out earlier.

The FPDD technique reduces the duration of each temperature cycle by effectively increasing the concentration of reactants by enclosing them in picoliter type volumes. Since reaction rates depend on the concentration of the reactants, the efficiency of a partitioned fluid volume or droplet should be higher than in an ordinary vessel (such as a test tube) where the reactant quantity (DNA quantity) is extremely low. It is estimated that through the reduction in the number of cycles and the reduction in the time required for each cycles that the FPDD technique can reduce the detection time by an order of magnitude as compared to bulk solution DNA detection techniques

The FPDD technique facilitates removal of interference from competing DNA templates. Given the extremely small volumes involved with FPDD, it is possible to isolate a single template of the target DNA in a given partitioned volume or microdroplet. For example, the formation of 2000 partitioned fluid volumes or microdroplets (each with a volume of 5×10−9 9 liters) made by dividing a bulk solution of 10 microliters containing 2002000 DNA molecules, would result in one DNA molecule per microdroplet on average. This makes it possible to amplify only one template in mixtures containing many kinds of templates without interference. This is extremely important in processing of real world aerosol samples containing complex mixtures of DNA from many sources, and has direct application in screening of cDNA libraries.

With this new bioassay technique, each partitioned DNA-containing fluid volume contains the necessary biochemical constituents for selectively amplifying a specified portion of a sample DNA via polymerase chain reaction (PCR). The target DNA is detected by monitoring for the colorimetric indicator (e.g., fluorescence or optical absorption) generated with each DNA template duplication sequence.

The system 300 provides a fast, flexible and inexpensive high throughput, bioassay technology based on creation and suspension of microdroplets in an immiscible carrier stream. Each microdroplet contains the necessary biochemical constituents for selectively amplifying and fluorescently detecting a specified portion of a sample DNA via polymerase chain reaction (PCR). Once exposed to multiple heating cooling cycles, the microdroplets can be identified and probed for fluorescent signal at rates of several thousand per second.

Isolating the PCR reaction in such small (picoliter) volumes provides an order of magnitude reduction in overall detection time by:

    • (1) reducing the duration of each temperature cycle—the concentration of reactants increases by enclosing them in picoliter type volumes. Since reaction kinetics depend on the concentration of the reactant, the efficiency of a microdroplet should be higher than in an ordinary vessel (such a test tube) where the reactant quantity is infinitesimal
    • (2) reducing the total number of cycles—dilution of the fluorescently generated signal is largely eliminated in such a small volume, allowing much earlier detection. This effect is directly related to the number of microdroplets formed from the initial sample/reagent pool. Since PCR is an exponential process, for example, 1000 microdroplets would produce a signal 10 cycles faster than typical processing with bulk solutions.
    • (3) removing interference from competing DNA templates—given the extremely small volumes involved, it is possible to isolate a single template of the target DNA in a given microdroplet. A pL microdoplet filled with a 1 pM solution, for example, will be occupied by only one molecule on average. This makes it possible to amplify only one template in mixtures containing many kinds of templates without interference. This is extremely important in processing of real world aerosol samples containing complex mixtures of DNA from many sources, and has direct application in screening of precious cDNA libraries.

Referring now to FIG. 4, an illustration of another embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral 400. The system 300 provides system for nucleic acid amplification of a sample. The system 400 includes means for partitioning the sample into partitioned sections and means for performing PCR on the partitioned sections of the sample.

The sample is separated into immiscible slugs 406, 407, and 408. The immiscible slugs 406, 407, and 408 are formed through a system of microfluidics. Background information on microfluidics is contained in U.S. Pat. No. 5,876,187 for micropumps with fixed valves to Fred K. Forster et al., patented Mar. 2, 1999. As stated in U.S. Pat. No. 5,876,187,“ Miniature pumps, hereafter referred to as micropumps, can be constructed using fabrication techniques adapted from those applied to integrated circuits. Such fabrication techniques are often referred to as micromachining. Micropumps are in great demand for environmental, biomedical, medical, biotechnical, printing, analytical instrumentation, and miniature cooling applications.” Microchannels 403, 404, and 405 are formed in substrates 401 and 402. The disclosures of U.S. Pat. Nos. 5,876,187 and 5,876,187 are incorporated herein by reference.

The immiscible slugs 406, 407, and 408 can be moved through the microchannels using magnetohydrodynamics. Background information on magnetohydrodynamics is contained in U.S. Pat. No. 6,146,103 for micromachined magnetohydrodynamic actuators and sensors to Abraham P. Lee and Asuncion V. Lemoff, patented Nov. 14, 2000. As stated in U.S. Pat. No. 6,146,103, “Microfluidics is the field for manipulating fluid samples and reagents in minute quantities, such as in micromachined channels, to enable hand-held bioinstrumentation and diagnostic tools with quicker process speeds. The ultimate goal is to integrate pumping, valving, mixing, reaction, and detection on a chip for biotechnological, chemical, environmental, and health care applications. Most micropumps developed thus far have been complicated, both in fabrication and design, and often are difficult to reduce in size, negating many integrated fluidic applications. Most pumps have a moving component to indirectly pump the fluid, generating pulsatile flow instead of continuous flow. With moving parts involved, dead volume is often a serious problem, causing cross-contamination in biological sensitive processes. The present invention utilizes MHDs for microfluid propulsion and fluid sensing, the microfabrication methods for such a pump, and the integration of multiple pumps for a microfluidic system. MHDs is the application of Lorentz force law on fluids to propel or pump fluids. Under the Lorentz force law, charged particles moving in a uniform magnetic field feel a force perpendicular to both the motion and the magnetic field. It has thus been recognized that in the microscale, the MHD forces are substantial for propulsion of fluids through microchannels as actuators, such as a micropump, micromixer, or microvalve, or as sensors, such as a microflow meter, or viscosity meter. This advantageous scaling phenomenon also lends itself to micromachining by integrating microchannels with micro-electrodes.” The disclosure of U.S. Pat. No. 6,146,103 is incorporated herein by reference.

The means for performing PCR on the partitioned sections of the sample can be a system for alternately heating and cooling the immiscible slugs 406, 407, and 408. Alternatively, the means for performing PCR on the partitioned sections of the sample can be a system for alternately heating and cooling the immiscible slugs 406, 407, and 408 can be a system for moving the immiscible slugs 406, 407, and 408 through zones for heating and cooling. An example of such a system is shown in U.S. patent application No. 2002/0127152 published Sep. 12, 2002 for a convectively driven PCR thermal-cycling system described as follows: “A polymerase chain reaction system provides an upper temperature zone and a lower temperature zone in a fluid sample. Channels set up convection cells in the fluid sample and move the fluid sample repeatedly through the upper and lower temperature zone creating thermal cycling.” The disclosure of U.S. Patent Application No. 2002/0127152 is incorporated herein by reference.

In another embodiment of the invention, the DNA-containing solution is partitioned by adding a gelling agent to the solution to form cells of partitioned volumes of fluid separated by the gelling agent. Using this approach for fluid partitioning, the DNA-containing solution is gelled in a tube or as a very thin layer. For example, it can be in a thin layer between flat plates and the surface of the thin film can be optically probed spatially in directions parallel to the film surface to detect micro-regions in the film where the colorimetric indicator suggests the presence of the target DNA.

Another embodiment of the invention is to partition the DNA-containing solution as microdroplets in an immiscible fluid where the droplets are arranged in a two-dimensional array such that the array of microdroplets can be optically probed to detect the colorimetric indicator which signals the presence of the target DNA. In this approach a solid hydrophobic substrate supports the microdroplets. For example, in small indentations, and the immiscible “partitioning” fluid is less dense than the aqueous DNA-containing solution.

In another embodiment of the invention the DNA-containing solution is partitioned using mechanical means. For example, the DNA-containing solution can be partitioned into an array of capillaries, microtubes, or wells. In this approach, the micro vessels holding each partitioned fluid volume can be scanned optically to detect the colorimetric indicator signaling the presence of the target DNA.

Referring now to FIGS. 5A, 5B, and 5C example representations of the mechanical partitioning approach for DNA detection using fluid partitioning are shown. In FIG. 5A a line of capillaries or micro-tubes 501 are used for partitioning and holding the DNA containing solution. In FIG. 5B an array 502 of capillaries or micro-tubes are used for partitioning the DNA-containing solution. In FIG. 5C a micro-wells or micro-vessels unit 503 is used for partitioning and holding the DNA-containing solution.

While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3575220Aug 12, 1968Apr 20, 1971Scientific IndustriesApparatus for dispensing liquid sample
US4283262Jul 1, 1980Aug 11, 1981Instrumentation Laboratory Inc.Analysis system
US4801529Jun 18, 1985Jan 31, 1989Brandeis UniversityIncubation, separation, detection; enzymes, antigens, antibodies
US4948961Apr 5, 1988Aug 14, 1990Biotrack, Inc.Capillary flow device
US5176203Jul 31, 1990Jan 5, 1993Societe De Conseils De Recherches Et D'applications ScientifiquesApparatus for repeated automatic execution of a thermal cycle for treatment of samples
US5376252Nov 10, 1992Dec 27, 1994Pharmacia Biosensor AbMicrofluidic structure and process for its manufacture
US5422277Mar 27, 1992Jun 6, 1995Ortho Diagnostic Systems Inc.Cell fixative composition and method of staining cells without destroying the cell surface
US5585069Nov 10, 1994Dec 17, 1996David Sarnoff Research Center, Inc.Substrates with wells connected by channels controlled by valves, for concurrent analysis of several liquid samples
US5587128Nov 14, 1994Dec 24, 1996The Trustees Of The University Of PennsylvaniaPolymerase chain reaction
US5602756Dec 8, 1995Feb 11, 1997The Perkin-Elmer CorporationThermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5736314Nov 16, 1995Apr 7, 1998Microfab Technologies, Inc.Inline thermo-cycler
US5827480Mar 25, 1997Oct 27, 1998The Perkin-Elmer CorporationNucleic acid amplification reaction apparatus
US5842787Oct 9, 1997Dec 1, 1998Caliper Technologies CorporationMicrofluidic systems incorporating varied channel dimensions
US5856174Jan 19, 1996Jan 5, 1999Affymetrix, Inc.Integrated nucleic acid diagnostic device
US5858187Sep 26, 1996Jan 12, 1999Lockheed Martin Energy Systems, Inc.Apparatus and method for performing electrodynamic focusing on a microchip
US5912945Jun 23, 1997Jun 15, 1999Regents Of The University Of CaliforniaX-ray compass for determining device orientation
US5928907Dec 2, 1996Jul 27, 1999The Perkin-Elmer Corporation., Applied Biosystems DivisionSystem for real time detection of nucleic acid amplification products
US5945334Jun 7, 1995Aug 31, 1999Affymetrix, Inc.For packaging a device with probe array surface; used in high throughput hybridization assay systems
US5972716Dec 5, 1995Oct 26, 1999The Perkin-Elmer CorporationWith sample tube having surface roughness which reduces background due to contamination of tube holder in apparatus; for polymerase chain reactions
US6057149Sep 15, 1995May 2, 2000The University Of MichiganMerging microdroplets by using housing of silicon with etched transport channels and delivering each liquid drop by separate channel to reaction region in third transport channel by differential heating
US6126899Apr 2, 1997Oct 3, 2000The Perkins-Elmer CorporationDevice for multiple analyte detection
US6130098Sep 26, 1997Oct 10, 2000The Regents Of The University Of MichiganMoving microdroplets
US6143496Apr 17, 1997Nov 7, 2000Cytonix CorporationMethod of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly
US6146103Oct 9, 1998Nov 14, 2000The Regents Of The University Of CaliforniaMicromachined magnetohydrodynamic actuators and sensors
US6156181Oct 26, 1998Dec 5, 2000Caliper Technologies, Corp.Controlled fluid transport microfabricated polymeric substrates
US6174673Jun 16, 1998Jan 16, 2001Diversa CorporationHigh throughput screening for novel enzymes
US6175669Mar 30, 1998Jan 16, 2001The Regents Of The Universtiy Of CaliforniaOptical coherence domain reflectometry guidewire
US6176609Oct 13, 1998Jan 23, 2001V & P Scientific, Inc.Magnetic tumble stirring method, devices and machines for mixing in vessels
US6177479Mar 1, 1999Jan 23, 2001Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And FisheriesContinuous manufacturing method for microspheres and apparatus
US6221654Sep 23, 1997Apr 24, 2001California Institute Of TechnologyChip of substrate and analysis unit; unit has two branch channels, detector, flow controller, and main channel with sample inlet, detection region for one polynucleotide at a time, and branch point discrimination region
US6281254Mar 1, 1999Aug 28, 2001Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And FisheriesMicrochannel apparatus and method of producing emulsions making use thereof
US6337740Aug 19, 1999Jan 8, 2002Caliper Technologies Corp.Microfluidic devices for electrophoretic analysis of materials
US6344325Feb 8, 2000Feb 5, 2002California Institute Of TechnologyMethods for analysis and sorting of polynucleotides
US6357907Jun 15, 1999Mar 19, 2002V & P Scientific, Inc.Magnetic levitation stirring devices and machines for mixing in vessels
US6384915Mar 30, 1998May 7, 2002The Regents Of The University Of CaliforniaCatheter guided by optical coherence domain reflectometry
US6391559May 2, 2000May 21, 2002Cytonix CorporationApparatus that can hold a small sample volume and allow amplification without evaporation
US6403338Jun 27, 2000Jun 11, 2002Mountain ViewFluid flowing sample containing nucleic acid into microscale chamber; amplification of plurality of sequences; detecting polymorphisms; dna sequencing
US6429025Jun 24, 1997Aug 6, 2002Caliper Technologies Corp.High-throughput screening assay systems in microscale fluidic devices
US6440706Jul 11, 2000Aug 27, 2002Johns Hopkins UniversityDigital amplification
US6466713Jun 18, 2001Oct 15, 2002The Regents Of The University Of CaliforniaOptical fiber head for providing lateral viewing
US6479299Aug 12, 1998Nov 12, 2002Caliper Technologies Corp.Pre-disposed assay components in microfluidic devices and methods
US6488895May 9, 2000Dec 3, 2002Caliper Technologies Corp.Multiplexed microfluidic devices, systems, and methods
US6494104Mar 21, 2001Dec 17, 2002Sumitomo Wiring Systems, Ltd.Bend test for a wire harness and device for such a test
US6509085Apr 10, 2000Jan 21, 2003Caliper Technologies Corp.Fabrication of microfluidic circuits by printing techniques
US6521427Sep 16, 1998Feb 18, 2003Egea Biosciences, Inc.Annealing oligonucleotides in assembling artificial organisms
US6522407Mar 18, 2002Feb 18, 2003The Regents Of The University Of CaliforniaOptical detection dental disease using polarized light
US6524456Sep 29, 1999Feb 25, 2003Ut-Battelle, LlcDrawing and transporting segmenting liquid through microchannels; combinatorial libraries, medical diagnosis, gene expression of proteins
US6540895May 21, 1999Apr 1, 2003California Institute Of TechnologyMicrofabricated cell sorter for chemical and biological materials
US6551841Jan 27, 1999Apr 22, 2003The Trustees Of The University Of PennsylvaniaSmall, typically single-use, modules capable of receiving and rapidly conducting a predetermined assay protocol on a fluid sample
US6558916Feb 7, 2001May 6, 2003Axiom Biotechnologies, Inc.Cell flow apparatus and method for real-time measurements of patient cellular responses
US6575188Sep 18, 2001Jun 10, 2003Handylab, Inc.Methods and systems for fluid control in microfluidic devices
US6602472May 15, 2000Aug 5, 2003Agilent Technologies, Inc.Microfluidic microchip for chemical, physical, and/or biological analysis or synthesis; deformable substrate carrying a channel structure
US6637463May 10, 2002Oct 28, 2003Biomicro Systems, Inc.Flow path configurations are designed to control pressure drops on selected flow path segments to balance fluid flow among multiple flow paths, leading to efficient operation of the wetted microfluidic circuit
US6660367Oct 24, 2002Dec 9, 2003Caliper Technologies Corp.Surface coating for microfluidic devices that incorporate a biopolymer resistant moiety
US6663619Jul 9, 2001Dec 16, 2003Visx IncorporatedMethod and systems for laser treatment of presbyopia using offset imaging
US6664044May 29, 1998Dec 16, 2003Toyota Jidosha Kabushiki KaishaAn extremely minute amount of a solution introduced by an ink-jet method is retained on a substrate for a long period of time without evaporation.
US6670153Sep 13, 2001Dec 30, 2003Caliper Technologies Corp.Improved channel and reservoir configurations; for performance of temperature mediated reactions
US6767706Jun 5, 2001Jul 27, 2004California Institute Of TechnologyIntegrated active flux microfluidic devices and methods
US6773566Aug 30, 2001Aug 10, 2004Nanolytics, Inc.Electrostatic actuators for microfluidics and methods for using same
US6833242Apr 4, 2001Dec 21, 2004California Institute Of TechnologyMethods for detecting and sorting polynucleotides based on size
US6900021May 18, 1998May 31, 2005The University Of AlbertaMicrofluidic system and methods of use
US6905885Jun 12, 2001Jun 14, 2005The Regents Of The University Of CaliforniaPortable pathogen detection system
US6960437Apr 5, 2002Nov 1, 2005California Institute Of TechnologyApparatus for use in the amplification of preferential nucleotide sequences
US6964846Apr 7, 2000Nov 15, 2005Exact Sciences CorporationDiagnosis of colorectal cancer; amplification of nucleic acid
US7010391Mar 28, 2001Mar 7, 2006Handylab, Inc.Methods and systems for control of microfluidic devices
US7041481Mar 14, 2003May 9, 2006The Regents Of The University Of CaliforniaPartitioned DNA replication apparatus comprising blue laser for monitoring hybridization and polymerase chain reaction amplification products; biochip assays and genetic polymorphisms
US7052244Jun 10, 2003May 30, 2006Commissariat A L'energie AtomiqueDevice for displacement of small liquid volumes along a micro-catenary line by electrostatic forces
US7081336Jun 25, 2002Jul 25, 2006Georgia Tech Research CorporationDual resonance energy transfer nucleic acid probes
US7091048Oct 24, 2002Aug 15, 2006Parce J WallaceIncludes microfluidic channels and electroosmosis for fluorescent detection and monitoring receptor/ligand interactions on substrates; immunoassays; drug screening
US7094379Oct 23, 2002Aug 22, 2006Commissariat A L'energie AtomiqueDevice for parallel and synchronous injection for sequential injection of different reagents
US7118910Nov 27, 2002Oct 10, 2006Fluidigm CorporationMicrofluidic device and methods of using same
US7129091May 9, 2003Oct 31, 2006University Of ChicagoDevice and method for pressure-driven plug transport and reaction
US7188731Aug 25, 2003Mar 13, 2007The Regents Of The University Of CaliforniaVariable flexure-based fluid filter
US7192557Dec 14, 2001Mar 20, 2007Handylab, Inc.Methods and systems for releasing intracellular material from cells within microfluidic samples of fluids
US7198897Dec 17, 2002Apr 3, 2007Brandeis UniversityLate-PCR
US7238268Feb 24, 2003Jul 3, 2007Ut-Battelle, LlcMicrofluidic devices for the controlled manipulation of small volumes
US7244567Jan 28, 2004Jul 17, 2007454 Life Sciences CorporationAnnealing an unblocked primer to a first strand of nucleic acid; annealing a second blocked primer to a second strand of nucleic acid; elongating the nucleic acid along the first strand with a polymerase; terminating the first sequencing primer; deblocking the second primer; and elongating nucleic acid
US7252943Jun 3, 2004Aug 7, 2007Medical Research CouncilIn Vitro sorting method
US7268167Feb 13, 2002Sep 11, 2007Japan Science And Technology AgencyProcess for producing emulsion and microcapsules and apparatus therefor
US7268179Sep 30, 2003Sep 11, 2007Cytonix CorporationDurability, weatherproofing, wear resistance protective coatings; fluoropolymers
US7270786Dec 14, 2001Sep 18, 2007Handylab, Inc.Methods and systems for processing microfluidic samples of particle containing fluids
US7279146Apr 19, 2004Oct 9, 2007Fluidigm CorporationCrystal growth devices and systems, and methods for using same
US7294503Sep 14, 2001Nov 13, 2007California Institute Of TechnologyMicrofabricated crossflow devices and methods
US7312085Apr 1, 2003Dec 25, 2007Fluidigm CorporationApparatus for manipulation and/or detection of cells and/or beads
US7323305Sep 22, 2004Jan 29, 2008454 Life Sciences CorporationDetermining sequence of preferential nucleotide sequences comprising, fragmenting large template nucleic, deliver to microreation chamber filled with water-in-oil emulsion, amplify and bind copies to beads, incubate with array and sequence
US7368233Dec 7, 2000May 6, 2008Exact Sciences CorporationMethods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
US7459315Apr 25, 2002Dec 2, 2008Cytonix CorporationMiniaturized apparatus for use in analyzing sample associated with fertility, immunology, cytology, gas analysis and drug screening
US7595195Feb 11, 2004Sep 29, 2009The Regents Of The University Of CaliforniaApparatus for controlling the size and composition of emulsified droplets, multi-lamellar and asymmetric vesicles, encapsulation of reagents, membrane proteins and sorting of vesicles/droplets
US7622280Jun 11, 2004Nov 24, 2009454 Life Sciences CorporationSilicone based surfactant comprising hydrophobic and hydrophilic phases containing functional in vitro eukaryotic expression systems; compartmentalisation of transcription/translation reactions
US7682565Dec 22, 2003Mar 23, 2010Biotrove, Inc.Assay apparatus and method using microfluidic arrays
US7833708May 19, 2005Nov 16, 2010California Institute Of TechnologyNucleic acid amplification using microfluidic devices
US7842457Jan 28, 2004Nov 30, 2010454 Life Sciences CorporationBead emulsion nucleic acid amplification
US7927797Jan 28, 2005Apr 19, 2011454 Life Sciences CorporationNucleic acid amplification with continuous flow emulsion
US20010039014Jan 10, 2001Nov 8, 2001Maxygen, Inc.Integrated systems and methods for diversity generation and screening
US20010046701May 24, 2001Nov 29, 2001Schulte Thomas H.Nucleic acid amplification and detection using microfluidic diffusion based structures
US20020021866Jun 18, 2001Feb 21, 2002The Regents Of The University Of CaliforniaOptical fiber head for providing lateral viewing
US20020058332Sep 14, 2001May 16, 2002California Institute Of TechnologyMicrofabricated crossflow devices and methods
US20020093655Mar 18, 2002Jul 18, 2002The Regents Of The University Of CaliforniaOptical detection of dental disease using polarized light
US20020141903 *Dec 14, 2001Oct 3, 2002Gene ParunakMethods and systems for processing microfluidic samples of particle containing fluids
US20020164820Apr 25, 2002Nov 7, 2002Brown James F.Miniaturized apparatus for use in analyzing sample associated with fertility, immunology, cytology, gas analysis and drug screening
Non-Patent Citations
Reference
13M Fluorinert(TM) Electronic Liquid FC-3283, 3M product information, 2001.
23M Fluorinert™ Electronic Liquid FC-3283, 3M product information, 2001.
3Abdelgawad et al., All-terrain droplet actuation, Lab Chip (2008) vol. 8, pp. 672-677.
4Abdelgawad, M. et al., "All-terrain droplet actuation," Lab on a Chip, 2008, pp. 672-677, vol. 8.
5Abil® EM 90, Goldschmidt Personal Care product literature, 2003, seven pages.
6Abil® EM 90, Goldschmidt Personal Care product literature, 2003.
7Baroud et al., Thermocapillary valve for droplet production and sorting. Physical Review (2007) E 75, 046302, pp. 1-5.
8Baroud, C. et al., "Thermocapillary Valve for Droplet Production and Sorting," Physical Review E, 2007, pp. 046302-1 to 046302-5, vol. 75.
9Beer et al., On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets. Anal Chem, (2008) vol. 80 No. 6, pp. 1854-1858.
10Beer et al., On-Chip, Real-Time, Single-Copy Polymerase Chain Reaction in Picoliter Droplets, Anal. Chem, (2007) vol. 79 No. 22, pp. 8471-8475.
11Beer, N. et al., "On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets," Anal. Chem., 2008, pp. 1854-1858, vol. 80 No. 6.
12Beer, N. et al., On-Chip, Real-Time, Single-Copy Polymerase Chain Reaction in Picoliter Droplets. Anal. Chem., 2007, pp. 8471-8475, vol. 79, No. 22.
13Bransky et al., A microfluidic droplet generator based on a piezoelectric actuator, Lab Chip (2009) vol. 9, pp. 516-520.
14Bransky, A. et al., "A Microfluidic Droplet Generator Based on a Piezoelectric Actuator," Lab Chip, 2009, pp. 516-520, vol. 9.
15Carroll et al., Droplet-Based Microfluidics for Emulsion and Solvent Evaporation Synthesis of Monodisperse Mesoporous Silica Microspheres, Langmuir (2008) vol. 24, pp. 658-661.
16Carroll, N. et al., "Droplet-Based Microfluidics for Emulsion and Solvent Evaporation Synthesis of Monodisperse Mesoporous Silica Microspheres," Langmuir, 2008, pp. 658-661, vol. 24.
17Chabert et al., "Droplet fusion by alternating current (AC) field electrocoalescence in microchannels," Electrophoresis, 2005, vol. 26, pp. 3706-3715.
18Chabert et al., Droplet fusion by alternating current (AC) field electrocoalescence in microchannels, Electrophoresis (2005) vol. 26, pp. 3706-3715.
19Chen et al., "Using Three-Phase Flow of Immiscible Liquids To Prevent Coalescence of Droplets in Microfluidic Channels: Criteria To Identify the Third Liquid and Validation with Protein Crystallization," Langmuir, 2007, vol. 23, pp. 2255-2260.
20Chen et al., Using Three-Phase Flow of Immiscible Liquids To Prevent Coalescence of Droplets in Microfluidic Channels; Criteria To Identify the Third Liquid and Validation with Protein Crystallization, Langmuir (2007) vol. 23, pp. 2255-2260.
21Clausell-Tormos et al., "Droplet-Based Microfluidic Platforms for the Encapsulation and Screening and Mammalian Cells and Multicellular Organisms," Chemistry and Biology, 2008, pp. 427-437, vol. 15.
22Clausell-Tormos et al., Droplet-Based Microfluidic Platforms for the Encapsulation and Screening and Mammalian Cells and Multicellular Organisms, Chemistry & Biology, (2008), vol. 15, pp. 427-437.
23Diehl et al., "Digital quantification of mutant DNA in cancer patients," Current Opinion in Oncology, 2007, pp. 36-42, vol. 19.
24Diekema et al., "Look before You Leap: Active Surveillance for Multidrug-Resistant Organisms," Healthcare Epidemiology, 2007, pp. 1101-1107, vol. 44.
25Dressman et al., "Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations," PNAS, 2003, pp. 8817-8822, vol. 100 No. 15.
26Fan et al., "Highly parallel genomic assays," Nature Reviews, Genetics, 2006, pp. 632-644, vol. 7.
27Fidalgo et al., "Coupling Microdroplet Microreactors with Mass Spectrometry: Reading the Contents of Single Droplets Online," Angew. Chem. Int. Ed., 2009, pp. 3665-3668, vol. 48.
28Halloran, P.J., Letter to John H. Lee, Assistant Laboratory Counsel, Lawrence Livermore National Laboratory, re U.S. Appl. No. 12/118,418, filed Jun. 4, 2010, 5 pages.
29Higuchi et al., "Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions," Bio/Technology, 1993, pp. 1026-1030, vol. 11.
30Jarvius et al., "Digital quantification using amplified single-molecule detection," Nature Methods, 2006, pp. 725-727, vol. 3, No. 9; includes supplementary information from www.nature.com website.
31Kalinina et al., "Nanoliter scale PCR with TaqMan detection," Nucleic Acids Res., 1997, pp. 1999-2004, vol. 25, No. 10.
32Katsura, S. et al., "Indirect Micromanipulation of Single Molecules in Water-In-Oil Emulsion," 2001, Electrophoresis, vol. 22, pp. 289-293.
33Kiss et al., "High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets," Anal. Chem., DOI: 10.1021/ac801276c, Nov. 17, 2008 .
34Kiss et al., "High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets," Anal. Chem., DOI: 10.1021/ac801276c, Nov. 17, 2008 <http://pubs.acs.org>.
35Kojima et al., "PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets," Nucleic Acids Res., 2005, p. e150, vol. 33 No. 17.
36Kopp, M., et al., "Chemical Amplification: Continuous-Flow PCR on a Chip," Science, vol. 280, May 15, 1998, pp. 1046-1048 [Online] [Retrieved on Sep. 22, 2009] Retrieved from the internet URL<http://www.sciencemag.org/cgi/content/full/280/5366/1046.
37Kumaresan et al., "High-Throughput Single Copy DNA Amplification and Cell Analysis in Engineered Nanoliter Droplets," Anal. Chem., DOI: 10.1021/ac800327d, Apr. 15, 2008 , plus supporting information.
38Kumaresan et al., "High-Throughput Single Copy DNA Amplification and Cell Analysis in Engineered Nanoliter Droplets," Anal. Chem., DOI: 10.1021/ac800327d, Apr. 15, 2008 <http://pubs.acs.org>, plus supporting information.
39Leamon et al., "Overview: methods and applications for droplet compartmentalization of biology,"Nature Methods, 2006, pp. 541-543, vol. 3, No. 7.
40Lin et al., "Droplet Formation Utilizing Controllable Moving-Wall Structures for Double-Emulsion Applications," Journal of Microelectromechanical Systems, 2008, pp. 573-581, vol. 17 No. 3.
41Link et al., "Electric Control of Droplets in Microfluidic Devices," Angew. Chem. Int. Ed., 2006, pp. 2556-2560, vol. 45.
42Liu et al., "Droplet-based synthetic method using microflow focusing and droplet fusion," Microfluid Nanofluid, 2007, pp. 239-243, vol. 3.
43Lo et al., "Digital PCR for the molecular detection of fetal chromosomal aneuploidy," PNAS, 2007, pp. 13116-13121, vol. 104 No. 32.
44Margulies et al., "Genome sequencing in microfabricated high-density picloitre reactors," Nature, 2005, pp. 376-380, vol. 437; includes supplementary information from www.nature.com website.
45Margulies et al., Supplementary figures from JM Rothberg, Nature, May 2005, twelve pages.
46Margulies et al., Supplementary methods from JM Rothbert, Nature, May 2005, thirty-four pages.
47Musyanovych et al., "Miniemulsion Droplets as Single Molecule Nanoreactors for Polymerase Chain Reaction," Biomacromolecules, 2005, pp. 1824-1828, vol. 6.
48Nagai et al., "Development of A Microchamber Array for Picoliter PCR," Anal. Chem., Mar. 1, 2001, pp. 1043-1047, vol. 73, No. 5.
49 *Nagai et al., Anal. Chem. 73, 1043-4047 (2001).
50Nagai et al., Development of A Microchamber Array for Picoliter PCR, Anal. Chem, Mar. 1, 2001, pp. 1043-1047, 7 pages, vol. 73, No. 5.
51Pamme, "Continuous flow separations in microfluidic devices," Lab Chip, 2007, pp. 1644-1659, vol. 7.
52Pohl et al., "Principle and applications of digital PCR," Expert Rev. Mol. Diagn., 2004, pp. 41-47, vol. 4, No. 1.
53Price, "Regular review: Point of care testing," BMJ, 2001, pp. 1285-1288, vol. 322.
54Roach et al., "Controlling Nonspecific Protein Adsorption in a Plug-Based Microfluidic System by Controlling Interfacial Chemistry Using Fluorous-Phase Surfactants," Anal. Chem., 2005, pp. 785-796, vol. 77 No. 3.
55Rutledge et al., "Mathematics of quantitative kinetic PCR and the application of standard curves," Nucleic Acids Res., 2003, p. e93, vol. 31 No. 16.
56Rutledge, "Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications," Nucleic Acids Res., 2004, p. e178, vol. 32 No. 22.
57Scheegabeta, I., "Miniaturized Flow-through PCR with Different Template Types in a Silicon Chip Thermocycler," Lab on a Chip, 2001, pp. 42-49, vol. 1.
58Scheegaβ, I., "Miniaturized Flow-through PCR with Different Template Types in a Silicon Chip Thermocycler," Lab on a Chip, 2001, pp. 42-49, vol. 1.
59U.S. Appl. No. 60/443,471, filed Jan. 29, 2003, 68 pages, 3M Fluorinert(TM) Electronic Liquid FC-3283, 3M product information, 2001.
60U.S. Appl. No. 60/443,471, filed Jan. 29, 2003, 68 pages, 3M Fluorinert™ Electronic Liquid FC-3283, 3M product information, 2001.
61U.S. Appl. No. 60/443,471, filed Jan. 29, 2003, sixty-eight pages.
62Vogelstein et al., "Digital PCR," PNAS, 1999, pp. 9236-9241, vol. 96.
63Williams et al., "Amplification of complex gene libraries by emulsion PCR," Nature Methods, 2006, pp. 545- 550 vol. 3, No. 7.
64Zhang et al., "Behavioral Modeling and Performance Evaluation of Microelectrofluidics-Based PCR Systems Using SystemC," IEEE Transactions on Computer-Aided Design of Integrated Circuits and Systems, 2004, pp. 843-858, vol. 23 No. 6.
65Zhang et al., "Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends," Nucleic Acids Res., 2007, pp. 4223-4237, vol. 35.
66Zhao et al., Microparticle Concentration and Separation by Traveling-Wave Dielectrophoresis (twDEP) for Digital Microfluidics, Journal of Microelectromechanical Systems, 2007, pp. 1472-1481, vol. 16, No. 6.
67Zhelev et al., "Heat Integration in Micro-Fluidic Devices," 16th European Symposium on Computer Aided Process Engineering and 9th International Symposium on Process Systems Engineering, 2006, pp. 1863-1868.
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US8367976 *Dec 10, 2008Feb 5, 2013Lawrence Livermore National Security, LlcLaser heating of aqueous samples on a micro-optical-electro-mechanical system
US20090261086 *Dec 10, 2008Oct 22, 2009Neil Reginald BeerLaser Heating of Aqueous Samples on a Micro-Optical-Electro-Mechanical System
Classifications
U.S. Classification435/6.12, 435/91.2, 435/287.2
International ClassificationC12M1/34, C12P19/34, C12Q1/68
Cooperative ClassificationC12Q1/6806
European ClassificationC12Q1/68A4
Legal Events
DateCodeEventDescription
Mar 6, 2014ASAssignment
Owner name: LAWRENCE LIVERMORE NATIONAL SECURITY LLC, CALIFORN
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THE REGENTS OF THE UNIVERSITY OF CALIFORNIA;REEL/FRAME:032371/0654
Effective date: 20080623
Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIF
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ANDERSON, BRIAN L.;COLSTON, BILLY W., JR.;ELKIN, CHRIS;SIGNING DATES FROM 20030206 TO 20030303;REEL/FRAME:032371/0625
Jan 7, 2014FPAYFee payment
Year of fee payment: 8
Jan 7, 2014SULPSurcharge for late payment
Year of fee payment: 7
Dec 20, 2013REMIMaintenance fee reminder mailed
Sep 17, 2013CCCertificate of correction