1. Field of the Invention
The present invention relates generally to devices, systems, and methods for containing biological samples, and more specifically to devices, systems, and methods for containing biological samples in a plurality of reaction sites for assessment.
2. Description of the Related Art
The use of microtiter plates have been used for monitoring, measuring, and/or analyzing multiple biological and biochemical reactions during a single experiment or assay. Such plates are commonly used in sequencing, genotyping, polymerase chain reactions (PCR), and other biochemical reactions to monitor progress and provide quantitative data. For example, an optical excitation beam may be used during real-time PCR (qPCR) processes to illuminate fluorescent DNA-binding dyes or fluorescent probes to produce fluorescent signals indicative of the amount of a target gene or other nucleotide sequence. Increasing demands to provide greater numbers of reactions per experiment or assay have resulted in instruments that are able to conduct much large numbers of reactions simultaneously.
Newer approaches such as digital PCR (dPCR) have increased the demand for devices, systems, and methods involving ever greater numbers of reaction sites that are much smaller than those used in more traditional quantitative PCR (qPCR). There is a need for systems and sample formats that will provide reliable, high quality, data in high-density sample formats with sample sites having volumes on the order of nanoliters or picoliters or even smaller.
Embodiments of the present invention may be better understood from the following detailed description when read in conjunction with the accompanying drawings. Such embodiments, which are for illustrative purposes only, depict novel and non-obvious aspects of the invention. The drawings include the following figures:
Embodiments of the present invention are generally directed devices, instruments, systems, and methods for monitoring or measuring a biological reactions for a large number of samples or solutions located at a plurality of reaction regions or reaction sites. Embodiments include the use of polymerase chain reaction (PCR) processes, assays, and protocols. While generally applicable to dPCR (digital PCR) or qPCR (real-time or quantitative PCR) where a large number of samples are being processed, it should be recognized that any suitable PCR method may be used in accordance with various embodiments described herein. Suitable PCR methods include, but are not limited to allele-specific PCR, asymmetric PCR, ligation-mediated PCR, multiplex PCR, nested PCR, quantitative or real-time PCR (qPCR), cast PCR, genome walking, bridge PCR, digital PCR (dPCR), or the like.
While embodiments of the present invention are generally directed to dPCR and qPCR, the present invention may be applicable to any PCR processes, experiment, assays, or protocols where a large number of samples or test volumes are processed, observed, and/or measured. In a dPCR assay or experiment according to embodiments of the present invention, a dilute solution containing a relatively small number of at least one target polynucleotide or nucleotide sequence is subdivided into a large number of small test samples or volumes, such that at least some of these samples or volumes contains none of the target nucleotide sequence. When the samples are subsequently thermally cycled in a PCR assay, process, or experiment, individual samples containing one or more molecules of the target are amplified and produce a positive, detectable signal, while those containing none of the target(s) do not produce a signal, or a produce a signal that is below a predetermined threshold or noise level. Using Poisson statistics, the number of target nucleotide sequences in the original solution may be correlated to the number of samples producing a positive detection signal. In some embodiments, the detected signal may be used to determine a number, or number range, of target molecules contained in an individual sample or volume. For example, a detection system may be configured to distinguish between samples containing one target molecule and samples containing two or at least two target molecules. Additionally or alternatively, the detection system may be configured to distinguish between samples containing a number of target molecules that is at or below a predetermined amount and samples containing more than the predetermined amount. In certain embodiments, both qPCR and dPCR processes, assays, or protocols are conducted using a single device, instrument, or system.
In various embodiments, the devices, instruments, systems, and methods described herein may be used to detect one or more types of biological components or targets of interest that are contained in an initial sample or solution. These biological components or targets may be any suitable biological target including, but are not limited to, DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, cells (e.g., circulating tumor cells), or any other suitable target biomolecule. In various embodiments, such biological components may be used in conjunction with one or more PCR methods and systems in applications such as fetal diagnostics, multiplex dPCR, viral detection, quantification standards, genotyping, sequencing assays, experiments, or protocols, sequencing validation, mutation detection, detection of genetically modified organisms, rare allele detection, and/or copy number variation.
According to embodiments of the present invention, one or more samples or solutions containing at least one biological target of interest may be distributed or divided between a plurality of small sample volumes or reaction sites. The sample volumes or reaction sites disclosed herein are generally illustrated as through-holes located in a substrate material; however, where applicable, sample volumes or reaction sites according to embodiments of the present invention may include wells or indentations formed in a substrate, spots of solution distributed on the surface a substrate, or samples or solutions located within test sites or volumes of a microfluidic system, or within or on small beads or spheres.
In certain embodiments, a dPCR protocol, assay, process, or experiment included distributing or dividing an initial sample or solution into at least ten thousand reaction sites, at least a hundred thousand reaction sites, at least one million reaction sites, or at least ten million of reaction sites. Each reaction site may have a volume of a few nanoliters, about one nanoliter, or that is less than or equal to one nanoliter (e.g., less than or equal to 100 picoliters, less than or equal to 10 picoliters, and/or less than or equal to one picoliter). When the number of target nucleotide sequences contained in the initial sample or solution is very small (e.g., less than 1000 target molecules, less than 100 target, less than 10 target molecules, or only one or two target molecules), it may also be important in certain cases that the entire content, or nearly the entire content, of the initial solution be contained in or received by the sample volumes or reaction sites being processed. For example, where there are only a few target nucleotides present in the initial solution, some or all of these target nucleotide could potentially be contained in a small residual fluid volume that are not located in any of the reaction sites and, therefore, would not be detected, measured, or counted. Thus, efficient transfer of the initial solution may aid in reducing the chances or possibility of a miscalculation in the number count of a rare allele or target nucleotide or of failing to detect the presences at all a rare allele or target nucleotide if none of the target molecules are successfully located into one of the designated reaction sites. Accordingly, embodiments of the present invention may be used to provide a high loading efficiency, where loading efficiency is defined as the volume or mass of an initial sample or solution received within the reaction sites divided by the total volume or mass of the initial sample or solution.
Referring to
In the illustrated embodiment, article 100 comprises a first surface 110 and an opposing second surface 112. In the illustrated embodiment, each reaction site 104 extends from an opening 114 in first surface 110 to an opening 116 in second surface 112. While the illustrated embodiment shown in
Reaction sites 104 may be configured to provide sufficient surface tension by capillary action to draw in respective amounts of liquid or sample containing a biological components of interest. Article 100 may have a general form or construction as disclosed in any of U.S. Pat. Nos. 6,306,578; 7,332,271; 7,604,983; 7,6825,65; 6,387,331; or 6,893,877, which are herein incorporated by reference in their entirety as if fully set forth herein. Substrate 102 may be a flat plate or comprise any form suitable for a particular application, assay, or experiment. Substrate 102 may comprise any of the various materials known in the fabrication arts including, but not limited to, a metal, glass, ceramic, silicon, or the like. Additionally or alternatively, substrate 102 may comprise a polymer material such as an acrylic, styrene, polyethylene, polycarbonate, and polypropylene material. Substrate 102 and reaction sites 104 may be formed by one or more of machining, injection molding, hot embossing, laser drilling, photolithography, or the like.
In certain embodiments, surfaces 110, 112 may comprise a hydrophobic material, for example, as described in US Patent Application Publication Numbers 2006/0057209 or 2006/0105453, which are herein incorporated by reference in their entirety as if fully set forth herein. In such embodiments, reaction sites 104 may comprise a hydrophilic material that attracts water or other liquid solutions. An array of such hydrophilic regions may comprise hydrophilic islands on a hydrophobic surface and may be formed on or within substrate 102 using any of various micro-fabrication techniques including, but are not limited to, depositions, plasmas, masking methods, transfer printing, screen printing, spotting, or the like.
It has been discovered that a high reaction site density may be configured to reduce the amount of a solution that is left on surface 110, 112 during a loading process, thus leading to higher loading efficiency or transfer of the initial solution. For example, by reducing ratio of the value of the spacing between adjacent well to the value of the well diameter, the amount of solution left on the surface of a plate may be significantly reduced so that, all, or nearly all, of an initial solution or sample containing biological components of interest is located inside reaction sites 104. In this way the possibility is reduced of missing a rare allele or other target molecule, since it would be less likely that one or more target molecule would remain on the substrate surface instead of being received in one of the designated reaction sites 104.
Referring to
In certain embodiments, a lower bound in the spacing between adjacent reaction sites may exist, for example, due to optical limitations when reaction sites 104 are being imaged by an optical system. For example, the lower bound in spacing between adjacent reaction sites may exist because of limitations in the ability of the optical system to distinctly image adjacent reaction sites. To increase the density of reaction sites 104 in a substrate 102, a close-packed hexagonal matrix pattern may be used, for example, as illustrated in
It has been discovered that reaction sites having a non-circular cross-section may advantageously reduce an average distance or spacing between adjacent reaction sites 104, leading to a reduction in the amount of residual liquid or solution left behind on surfaces 110, 112 after loading of a test solution or sample. Referring to
This result also provides an unexpected advantage for an optical system configured to inspect the reaction sites. Since the minimum edge spacing S in
In the illustrated embodiment shown in
In the illustrated embodiment of
In certain embodiments, substrate 102 has a thickness between surface 110 and surface 112 that is equal to or about 300 micrometer, so that each reaction site 104 has a volume of about 1.3 nanoliters. Alternatively, the volume of each reaction site 104 may be less than 1.3 nanoliters, for example, by decreasing the diameter of reaction sites 104 and/or the thickness of substrate 102. For example, each reaction site 104 may have a volume that is less than or equal to 1 nanoliter, less than or equal to 100 picoliters, less than or equal to 30 picoliters, or less than or equal to 10 picoliters. In other embodiments, the volume some or all of the reaction site 104 is in a range from 1 nanoliter to 20 nanoliters.
In certain embodiments, the density of reaction sites 104 over surfaces 110, 112 is at least 100 reaction sites per square millimeter. Higher densities are also anticipated. For example, a density of reaction sites 104 over surfaces 110, 112 may be greater than or equal to 150 reaction sites per square millimeter, greater than or equal to 200 reaction sites per square millimeter, greater than or equal to 500 reaction sites per square millimeter, greater than or equal to 1,000 reaction sites per square millimeter, greater than or equal to 10,000 reaction sites per square millimeter, or greater than or equal to 1,000,000 reaction sites per square millimeter.
Advantageously, all the reaction sites 104 in active area 120 may be simultaneously imaged and analyzed by an optical system. In certain embodiments, active area 120 imaged and analyzed by the optical system comprises at least 12,000 reaction sites 104. In other embodiments, active area 120 imaged and analyzed by the optical system comprises at least 15,000, at least 20,000, at least 30,000, at least 100,000, at least 1,000,000 reaction sites, or at least 10,000,000 reaction sites.
In certain embodiments, reaction sites 104 comprise a first plurality of the reaction sites characterized by a first characteristic diameter, thickness, and/or volume, and a second plurality of the reaction sites characterized by a second characteristic diameter, thickness, and/or volume that is different than that of the corresponding the first characteristic diameter, thickness, or volume. Such variation in reaction site size or dimension may be used, for example, to simultaneously analyze two or more different nucleotide sequences that may have different concentrations. Additionally or alternatively, a variation in reaction site 104 size on a single substrate 102 may be used to increase the dynamic range of a dPCR process, assay, or experiment. For example, article 100 may comprise two or more subarrays of reaction sites 104, where each group is characterized by a diameter or thickness that is different a diameter or thickness of the reaction sites 104 of the other or remaining group(s). Each group may be sized to provide a different dynamic range of number count of a target polynucleotide. The subarrays may be located on different parts of substrate 102 or may be interspersed so that two or more subarrays extend over the entire active area of article 100 or over a common portion of active area of article 100.
In certain embodiments, at least some of the reaction sites 104 are tapered over all or a portion of their walls. For example, referring to
In the embodiment shown in
Referring to
Partition 134b may be configured to aid in isolating the reaction sites in one active area, region, or zone from those in a separate active area, region, or zone. Such configurations may be used, for example, to facilitate the loading of a first sample in a first active area and a different second sample in a second active area, where the two areas are separated by partition 134b. In certain embodiments, the surface of active areas 120b and partition 134b are flush with one another on one or both faces of article 100b. Additionally or alternatively, at least a portion of partition 134b may be raised or offset from active areas 120b on one or both faces of article 100b. In other embodiments, at least a portion of partition 134b forms a trough relative to active areas 120b for one or both faces of article 100b. Where appropriate, features and/or dimensions discussed above in relation to articles 100, 100a may be incorporated into article 100b, or vice versa.
In certain embodiments, substrate 102 comprises a photostructurable material, such as certain glass or ceramic materials. In such embodiments, a method 140 shown in
Method 140 may be used to provide a substrate 102 having an opacity sufficient prevent any, or nearly any, light emitted in one reaction site 104 from being transmitted into an adjacent reaction site 104. Method 140 may further comprise removing material from substrate 102 by an amount sufficient to reduce thickness between surfaces 110, 112, for example, removing material from substrate 104 by an amount sufficient to reduce the thickness between surfaces 110, 112 by at least 20 percent over an initial thickness or by at least 30 percent or 40 percent over an initial thickness. Method 140 may also include heating substrate 102 to a temperature of at least 500 degrees Celsius during fabrication. In certain embodiments, the patterned mask used in method 140 comprises a quartz plate with chrome pattern. The mask may be removed prior to exposing the at least portion of the substrate to the corrosive agent. The corrosive material used in method 140 may be hydrofluoric acid.
Referring to
Carrier 150 may be made or formed from a metallic material, such as stainless steel, aluminum, copper, silver, or gold, or a semimetal such as graphite. Additionally or alternatively, all or portions of carrier 150 may be made of a non-metallic material including, but are not limited to, glass, acrylics, styrenes, polyethylenes, polycarbonates, and polypropylenes. In certain embodiments, at least one of the covers 152, 156 comprises a suitably transparent material for providing a window configured to allow optical access to and/or from reaction sites 104. Additionally or alternatively, the entire carrier 150 may be made of one or more transparent or nearly transparent materials.
Referring to
In certain embodiments, blade 164 is configured to aid in distributing sample fluid into some or all of reaction sites 104 as article 100 is inserted into carrier 150 through opening 162. For example, blade 164 may be configured to contact one or both surfaces 110, 112 during loading of article 100, so that liquid does not pass blade 164, but is instead pushed, and/or pulled by capillary forces, into reaction sites 104 as surface 110, 112 moves past blade 164. Additionally or alternatively, blade 164 may be configured to cover one or both surfaces 110, 112 of article 100 with a liquid, gel, or the like, for example to reduce or eliminate contamination and/or evaporation of sample fluid contained inside reaction sites 104.
Where appropriate, carrier 150a may incorporate any of the structures or features discussed above in relation to carrier 150, or vice versa.
Referring to
Portions of cavity 160 between article 100 and surfaces 154, 158 may be filled with an immiscible fluid 170 (e.g., a liquid or a gel material) that does not mix with test solution contained in reaction sites 104 and configured to prevent or reduce evaporation of the test solution contained from reaction sites 104. One suitable fluid 170 for some applications is Fluorinert, sold commercially by 3M Company. However, in certain embodiments, Fluorinert may be problematic for certain PCR applications due to its propensity to readily take up air that may be later released during PCR cycling, resulting in the formation of unwanted air bubbles.
Alternatively, in certain embodiments, it has been discovered that polydimethylsiloxane (PDMS) may be used in cavity 160 if the PDMS is not fully cross-linked. In such embodiment, PDMS has been found to have several characteristics that make it suitable for use with PCR, including low auto-fluorescing, thermal stability at PCR temperatures, and being non-inhibiting to polymerization processes. In addition, PDMS may contain an aqueous sample but be gas permeable to water vapor. A typical siloxane to cross linking agent used for general applications outside embodiments of the present invention is at a ratio of 10:1 (10 percent cross-linker) by weight.
It has been discovered that by under cross-linking a PDMS material, the resulting material can function as a suitable encapsulant for reducing evaporation, while also retaining the favorable attributes discussed above and associated with the fully cross linked material. More specifically, an under cross-linked PDMS material may be formed by using less than 10 percent of the cross-linker by weight. For example, a cross link level of less than or equal to 1% by weight has been shown to meet design requirements for certain PCR applications, such as for certain dPCR applications. Multiple dPCR responses have been demonstrated using a flat plate 100 that is encapsulated with an amount of cross-linker that is less than or equal to 0.8 percent by weight. Further, due to the higher viscosity of the under cross-linked PDMS material, as compared to Fluorinert, a PDMS encapsulant may also lend itself packaging requirements and customer workflow solutions.
Referring to
Once the substrate is mounted or attached, method 200 includes sliding the insertion tool along the carrier by an amount sufficient to locate the substrate inside the carrier, for example, by inserting the substrate through an opening and/or membrane of the carrier. Using the method 200, a solution or sample may be applied to a face of the substrate in such a way that the solution is deposited or drawn into reaction sites or through-holes in the substrate as the substrate is inserted into the carrier. In addition, one of both surfaces of the substrate may be covered with a liquid or gel, for example, in order to protect the solution from contaminants and/or evaporation.
In certain embodiments, at least 99 percent of the liquid sample is received by at least some of reaction sites. In other embodiments, at least 99.5 percent or 99.9 percent of the liquid sample is received by at least some of reaction sites. In certain embodiments, the total volume of reaction sites 104 is selected to be greater than the volume of the liquid sample to be loaded into reaction sites 104. This has been found to increase the loading efficiency, which can be critical in certain circumstances, as discussed above. In certain embodiments, the ratio of the liquid volume sample to the total volume of all reaction sites 104 is less than or equal to 95 percent. In other embodiments, the ratio of the liquid volume sample to the total volume of all reaction sites 104 is less than or equal to 90 percent, less than or equal to 80 percent, or less than or equal to 70 percent. In certain embodiments, the value of this ratio depends on the percent of the total volume of each reaction site that is filled with liquid after loading. For example, if only 90 percent of each reaction site 104 contains liquid sample after loading, then the ratio of the liquid volume sample to the total volume of all reaction sites 104 may be less than or equal to 90 percent, less than or equal to 80 percent, less than or equal to 70 percent, or less than or equal to 60 percent.
Various methods and devices may be used to provide detection of one or more biological components of interest that are contained in reaction sites 104. For example, various fluorescent dyes may be incorporated into solutions containing one or more biological components of interest, which may then be detected using an optical system to determine the presence or amount of the one or more biological components. In other embodiments, the presence of ions (positive or negative) may be detected and/or changes in pH, voltage, or current may be used to determine the presence or amount of one or more biological components of interest.
Referring to
In certain embodiments, system 400 further comprises a thermal control system 406 comprising, for example, a thermal cycler configured to perform a PCR procedure or protocol on at least some of the samples contained in article 100. Systems 402, 406 may combined or coupled together into a single unit, for example, in order to perform a qPCR and/or a dPCR procedure, assay, experiment, or protocol on at least some of the samples contained in article 100. In such embodiments, computer 404 may be used to control systems 402, 406 and/or to collect or process data provided or obtained by either or both systems 402, 406. Alternatively, thermal control system 406 may be completely separate from optical system 402 and/or from computer 404. In such embodiments, optical system 402 may be used to perform a dPCR or end-point PCR procedure on the samples contained in reaction sites 104 after thermal cycle has been performed on the samples using thermal control system 406 or some other thermal controller or thermal cycler. In certain embodiments, thermal control system 406 comprises a thermal cycler in which PCR is done using a traditional thermal cycler, isothermal amplification, thermal convention, infrared mediated thermal cycling, or helicase dependent amplification. In certain embodiments, at least a portion of thermal control system 406 may be integrated with or into article 100. For example, article 100 may include one or more heating elements distributed along one or both surfaces 110, 112. Additionally or alternatively, at least portions of substrate 102 may be a heating element, for example, by being made of a material with an electrical resistance configured to provide resistive heating upon application of a voltage potential to substrate 102.
In certain embodiments, article 100 comprises an electronic chip comprising integrated circuits and semiconductor. In such embodiment, a detection system may also be integrated into the chip to determine the presence and/or quantity of a biological components of interest.
In certain embodiments, optical system 402 comprises a light source 410 and an associated excitation optic system 412 configured to illuminate at least some of samples contained in the reaction sites of article 100. Excitation optical system 412 may include one or more lenses 414 and/or one or more filters 416 for conditioning light directed to the samples. Optical system 402 may further comprise a photodetector 420 and an associated emission optic system 422 configured to receive optical data emitted by at least some of samples contained in the reaction sites of article 100. For example, when system 400 is configured to perform a qPCR and/or a dPCR assay or experiment, the sample may contain fluorescent dyes that provide a fluorescent signal that varies according to an amount of target nucleotide sequence contained in various of the reaction sites of article 100. Emission optical system 422 may include one or more lenses 424 and/or one or more filters 426 for conditioning light directed to the samples.
In the illustrated embodiment of
Photodetector 420 may comprise one or more photodiodes, photomultiplier tubes (PMTs), or the like. Such photodetectors may be used, for example, where optical system 402 is configured to scan individual reaction sites 104 or subsets of reaction sites 104. In other embodiments, photodetector 420 may comprise one or segmented detector arrays, for example, one or more CCD (charge coupled device) or CMOS (complementary metal-oxide semiconductor) arrays. Segmented detector arrays may be advantageously used where all or large groups of reaction sites 104 are simultaneously imaged or inspected. In order to provide a plurality of pixels per each reaction site, photodetector 420 may comprise at least 4,000,000 pixel or more than 10,000,000 pixels.
In certain embodiments, article 100 comprises an electronic chip comprising integrated circuits and semiconductor. In such embodiment, a detection system may also be integrated into the chip to determine the presence and/or quantity of a biological component of interest.
Referring to
In certain embodiments, substrate 502 comprise silicon, which may be configured to provide an even temperature distribution across article 500 during use. Alternatively, substrate 502 comprises a glass material, such as a photo-structured glass ceramic, or a metal, such as aluminum, copper, or stainless steel.
Referring to
In certain embodiments, a plurality of dropout regions 509 may be configured provide information about the article 500 based on, for example, the dropout shape(s), number of dropout regions 509, and/or the relative position of one dropout region 509 to another dropout region 509. For example, the number of dropout regions on a particular article 500 may be used to determined the diameter of reaction sites 504 and/or the distance between dropout regions 509, or the geometry of the dropout regions 509 to one another, may be used to determine the number of reaction sites 504 or the pitch between reaction sites 504. Many other combinations of dropout regions size, shape, and distribution are anticipated.
Referring to
Referring to
Article 500 may incorporate, where appropriate, various of the elements and/or features discussed in relation to article 100, or vice versa. In addition, article 500 may be used in carrier 150 or other carriers according to embodiments of the current invention. Article 500 may be used in conjunction with system 400 or method 140 in ways similar to those in which article 100 has been disclose herein, as well as in or with other systems and methods disclosed herein in relation to article 100.
In certain embodiments, substrate 502 comprises silicon material and reaction sites 504 may comprise through-holes that are formed using a hexamethyldisilazane (HMDS) vapor coating process. Referring to
Referring to
Referring to
In certain embodiments, processes 620 and/or 625 are performed so that at least one of the surfaces 510, 512 has a roughness that is below a predetermined value. For example, it has been found that after a solution or sample has been introduced into reaction sites 504, a residual thin film of the solution or sample may be left behind or later formed (e.g., during a PCR thermal cycling process) on first surface 510. This residual film may provide a “bridge” between adjacent or neighboring reaction sites 504. The bridging layer can result in contamination of one reaction site 504 by one or more adjacent or neighboring reaction sites 504. To solve this problem, it has been discovered when first surface 510 is polished and/or coated to have roughness is less than or equal to a predetermined value, this bridging problem can be solved or eliminated. For example, when article 500 comprises a silicon material and a reaction site geometry as shown in
In certain embodiments, articles 100 or 500 may be configured for use in any of the enclosures, housings, or cases disclosed in copending U.S. provisional application No. 61/723,710, which is herein incorporated by reference in its entirety. For example, as shown in
Base 702 may comprise a plurality of bosses, tabs, staking sites, or support pads 720 (e.g., tabs 720a and 720b in the illustrated embodiment) that are configured to hold and/or locate article 500 within base 702 and cavity 708. One or more tabs 182 may be staked so that material from the tab is deformed or moved to hold article 500 firmly within base 702. Additionally or alternatively, article 500 may be glued to one or more tabs 182 using an adhesive, epoxy, or glue. In certain embodiments, gluing is used in conjunction with a glass or silicon article 500 in order to avoid possible cracking or damage to such holder materials, which might be induced by use of a crimping or holding force produced by tabs 720. In the illustrated embodiment, tabs 720a correspond with blank regions 506 of article 500. In certain embodiments, tabs 620a and blank regions 506 are large enough to provide proper support of article 500, but small enough so that the active area of corresponding article 500 provide a desired predetermined active area containing a predetermined number of reaction sites 504.
The above presents a description of the best mode contemplated of carrying out the present invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains to make and use this invention. This invention is, however, susceptible to modifications and alternate constructions from that discussed above which are fully equivalent. Consequently, it is not the intention to limit this invention to the particular embodiments disclosed. On the contrary, the intention is to cover modifications and alternate constructions coming within the spirit and scope of the invention as generally expressed by the following claims, which particularly point out and distinctly claim the subject matter of the invention.
Exemplary systems for methods related to the various embodiments described in this document include those described in following U.S. provisional patent applications:
U.S. provisional application No. 61/612,087, filed on Mar. 16, 2012; and
U.S. provisional application No. 61/723,759, filed on Nov. 7, 2012; and
U.S. provisional application No. 61/612,005, filed on Mar. 16, 2012; and
U.S. provisional application No. 61/612,008, filed on Mar. 16, 2012; and
U.S. provisional application No. 61/723,658, filed on Nov. 7, 2012; and
U.S. provisional application No. 61/723,738, filed on Nov. 7, 2012; and
U.S. provisional application No. 61/659,029, filed on Jun. 13, 2012; and
U.S. provisional application No. 61/723,710, filed on Nov. 7, 2012; and
U.S. provisional application No. 61/774,499, filed on Mar. 7, 2013; and
Life Technologies Docket Number LT00656 PCT, filed Mar. 15, 2013; and
Life Technologies Docket Number LT00657 PCT, filed Mar. 15, 2013; and
Life Technologies Docket Number LT00658 PCT, filed Mar. 15, 2013; and
Life Technologies Docket Number LT00668 PCT, filed Mar. 15, 2013; and
Life Technologies Docket Number LT00699 PCT, filed Mar. 15, 2013.
All of these applications are also incorporated herein in their entirety by reference.
Filing Document | Filing Date | Country | Kind |
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PCT/US2013/032002 | 3/15/2013 | WO | 00 |
Number | Date | Country | |
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61612087 | Mar 2012 | US | |
61612005 | Mar 2012 | US | |
61612008 | Mar 2012 | US | |
61659029 | Jun 2012 | US | |
61723759 | Nov 2012 | US | |
61723658 | Nov 2012 | US | |
61723738 | Nov 2012 | US | |
61723710 | Nov 2012 | US | |
61774499 | Mar 2013 | US |