US20150044686A1 - Systems and Methods for Containing Biological Samples - Google Patents
Systems and Methods for Containing Biological Samples Download PDFInfo
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- US20150044686A1 US20150044686A1 US14/385,765 US201314385765A US2015044686A1 US 20150044686 A1 US20150044686 A1 US 20150044686A1 US 201314385765 A US201314385765 A US 201314385765A US 2015044686 A1 US2015044686 A1 US 2015044686A1
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
- B01L3/50857—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using arrays or bundles of open capillaries for holding samples
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
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- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
- G03F7/20—Exposure; Apparatus therefor
- G03F7/2002—Exposure; Apparatus therefor with visible light or UV light, through an original having an opaque pattern on a transparent support, e.g. film printing, projection printing; by reflection of visible or UV light from an original such as a printed image
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- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0642—Filling fluids into wells by specific techniques
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- B01L2200/14—Process control and prevention of errors
- B01L2200/142—Preventing evaporation
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- B01L2300/00—Additional constructional details
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- B01L2300/046—Function or devices integrated in the closure
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- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0654—Lenses; Optical fibres
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
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- B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
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- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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Abstract
An article for holding a plurality of biological samples includes a substrate a substrate comprising a first surface and an opposing second surface and a plurality of reaction sites in the substrate. Each of the reaction sites extends from an opening in the first surface to an opening in the second surface. The reaction sites comprise a hexagonal shape and are configured to provide sufficient surface tension by capillary action to hold respective biological samples. The reaction sites have a density over at least a portion of the surfaces that is at least 170 holes per square millimeter. At least one of the surfaces may have a surface roughness characterized by an arithmetic average roughness (Ra) that is less than or equal to 5 nanometers.
Description
- 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:
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FIG. 1 is a top view of an article of manufacture according to an embodiment of the present invention. -
FIG. 2 is a side view of the article shown inFIG. 1 . -
FIG. 3 is a cross-sectional view of a portion of the article shown inFIG. 1 . -
FIG. 4 is a schematic representation of model of an embodiment of the present invention -
FIG. 5 is a schematic representation result using the model shown inFIG. 4 -
FIG. 6 is a representation of a pattern for distribution of reaction sites according to an embodiment of the present invention -
FIG. 7 is a representation of the geometric layout showing a comparison between circular reaction sites and hexagonal reaction sites. -
FIG. 8 is a cross-sectional view of a substrate according to an embodiment of the present invention. -
FIG. 9 is a perspective view of a portion of a substrate according to an embodiment of the present invention. -
FIG. 10 is a top view of an article of manufacture according to an embodiment of the present invention. -
FIG. 11 is a flow chart of a method according to an embodiment of the present invention. -
FIG. 12 is a cross-sectional view of a carrier and associated substrate according to an embodiment of the present invention. -
FIG. 13 is a top view of the carrier and substrate shown inFIG. 12 . -
FIG. 14 is a perspective view of a carrier according to an embodiment of the present invention. -
FIG. 15 is a perspective view of a carrier according to an embodiment of the present invention. -
FIG. 16 is a flow chart of a method according to an embodiment of the present invention. -
FIG. 17 is a schematic representation of a system according to an embodiment of the present invention. -
FIG. 18 is a front view of an article of manufacture according to an embodiment of the present invention. -
FIGS. 19-20 are magnified front views of the article of manufacture shown inFIG. 18 . -
FIG. 21 is a front view of the article of manufacture shown inFIG. 18 showing various dimensions of the article. -
FIG. 22 is a front view of an article of manufacture according to an embodiment of the present invention. -
FIG. 23 is a front views of the article of manufacture shown inFIG. 22 showing various dimensions of the article. -
FIG. 24 is a flow chart illustrating a method of making an article of manufacture according to an embodiment of the present invention. -
FIGS. 25A-C are a cross-sectional views of an embodiment of the article shown inFIGS. 18-21 illustrating an embodiment of the method shown inFIG. 24 . -
FIG. 26 is an article of manufacture and associated case or holder according to an embodiment of the present invention. - 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
FIGS. 1-3 , in certain embodiments of the present invention, an article, device, substrate, slide, orplate 100 comprises asubstrate 102 containing a plurality of partitions, through-holes, reaction regions, orreaction sites 104 located insubstrate 102. In certain embodiments,article 100 may comprise a chip. Additionally or alternatively,article 100 may comprise a microfluidic device which, for example, may further include a plurality of channels or paths for transferring reagents and/or test solutions toreaction sites 104. In other embodiments,reaction sites 104 comprise a plurality of droplets or beads andarticle 100 may comprise one or more chambers and/or channels containing some or all of the droplets orbeads 104. In such embodiments, droplets orbeads 104 may form an emulsion, where some or all of droplets orbeads 104 contain one or more target of at least one polynucleotide or nucleotide sequence. Wherereaction sites 104 are beads, the beams may optionally include an attached optical signature or label. Droplets or beams 104 may be inspected, monitored, or measured either one at time or in groups containing one or more droplets orbeads 104, for example using an imaging system according to embodiments of the present invention. - In the illustrated embodiment,
article 100 comprises afirst surface 110 and an opposingsecond surface 112. In the illustrated embodiment, eachreaction site 104 extends from an opening 114 infirst surface 110 to an opening 116 insecond surface 112. While the illustrated embodiment shown inFIG. 3 shows a substrate containing through-holes 104,substrate 102 may additionally or alternatively comprise other types of reaction sites. For example,reaction sites 104 may include reaction volumes located within wells or indentations formed insubstrate 102, spots of solution distributed on thesurfaces -
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 andreaction 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 withinsubstrate 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 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 designatedreaction sites 104. - Referring to
FIG. 4 , this increase in loading efficiency was demonstrated with a computer model of a hydrophobic surface containing a plurality of hydrophilic reaction sites. The model was used to analyze the distribution of a sample into the plurality of reaction sites as a function of the reaction site pitch (or density) for through-holes having a diameter of 75 micrometers.FIG. 5 demonstrates that as the spacing between reaction sites is decreased (increased density), a greater percentage of an initial liquid sample is captured by the reaction sites, and a lesser amount of residual liquid is left behind on the hydrophobic surface after the loading process. Thus, a higher density ofreaction sites 104 of a given cross-sectional dimension provides both an increase in the number of test samples for a givensize substrate 102 and decreases or eliminates residual fluid left onsurfaces 110, 112 (which may contain a rare allele or other target molecule of interest). - 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 ofreaction sites 104 in asubstrate 102, a close-packed hexagonal matrix pattern may be used, for example, as illustrated inFIGS. 6 and 7 . - 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 onsurfaces FIGS. 6 and 7 , an array ofhexagonal reaction sites 104 having a vertex-to-vertex diameter D are arranged in a hexagonal pattern in which the spacing or pitch between adjacent reaction sites is P. In certain embodiments, cross-talk between adjacent reaction sites in an optical system used to measure a fluorescence signal from thereaction sites 104 is a function of a minimum edge distance S between adjacent reaction sites. Thus, the geometry shown inFIG. 7 represents a minimum pitch P between reaction sites that can be used and still maintain the cross-talk between adjacent reaction sites at or below a predetermined value. A dash-lined circle is also shown inFIG. 7 inside each hexagon. This represents a circular reaction site of diameter D′ having the same values of pitch P and the same edge spacing S as that of the hexagonal reaction site. The grayed portion inFIG. 7 shows the area between adjacent reaction sites over some width W for both the circular and hexagonal reaction sites. As is clearly seen inFIG. 7 , the area between adjacent reaction sites over width W is greater for the circular reaction sites than between the hexagonal reaction sites, when the pitch P and the edge spacing S are the same. The modeling results discussed in regards toFIGS. 4 and 5 show that a smaller area between adjacent reaction sites lead to higher loading efficiency. Thus, based on the results illustrated inFIG. 7 , a higher loading efficiency is provided, under the same spacing conditions (P and S), for a hexagonal shaped reaction site than for a circular reaction site. - This result also provides an unexpected advantage for an optical system configured to inspect the reaction sites. Since the minimum edge spacing S in
FIG. 7 is the same for both the circular and hexagonal reaction sites, the cross-talk between adjacent reaction sites would be the same or similar for either type of reaction site. However, the cross-sectional area of the hexagonal reaction sites is greater than that of the circular reaction sites, for the same pitch P and edge spacing S. Thus, the image produced by an optical system would have a greater area for hexagonal reaction sites than for circular reaction sites. Accordingly, the larger image produced by the hexagonal reaction site may potentially span a greater number of pixels. A greater number of pixels per reaction site aids in making a more accurate calculation of the signal produced a reaction site. Thus, in addition to providing a higher loading efficiency, the use of hexagonal reaction sites, as shown inFIGS. 6 and 7 , may also produce more accurate measurement or calculation of an optical signal or output produce by each reaction site 104 (e.g., measurement or calculation of a fluorescence signal produced in proportion to an amount of a target or dye molecule). - In the illustrated embodiment shown in
FIG. 1 ,article 100 has a square shape and an overall dimension of 15 millimeter by 15 millimeter.Article 100 also has an active area, region, orzone 120 with a dimension of 13 millimeter by 13 millimeter. As used herein, the term “active area”, “active region”, or “active zone” means a surface area, region, or zone of an article, such as thearticle 100, over which reaction sites or solution volumes are contained or distributed. In certain embodiments, the active area ofarticle 100 may be increased to 14 millimeter by 14 millimeter or larger, for example on a 15 millimeter by 15 millimeter substrate dimension, in order to increase the total number of reaction sites contained onsubstrate 102.Article 100 may have other shapes and dimensions. For example, surfaces 110, 112 may be rectangular, triangular, circular, or some other geometric shape. The overall dimensions ofarticle 100 andactive area 120 may be smaller or larger than that for the illustrated embodiment inFIG. 1 , depending on the particular design parameters for a given system, assay, or experiment. - In the illustrated embodiment of
FIG. 1 ,reaction sites 104 may have a characteristic diameter of 75 micrometer and be distributed overactive area 120 with a pitch of 125 micrometers between adjacent reaction sites. In other embodiments,reaction sites 104 have a characteristic diameter of that is less than or equal 75 micrometers, for example, a characteristic diameter that is less than or equal to 60 micrometers or less than or equal to 50 micrometers. In other embodiments,reaction sites 104 have a characteristic diameter that is less than or equal to 20 micrometers, less than or equal to 10 micrometers, less than or equal to 1 micrometer, or less than or equal to 100 nanometers. The pitch between reaction sites may be less than 125 micrometers, for example, less than or equal to 100 micrometers, less than or equal to 30 micrometers, less than or equal to 10 micrometers, or less than or equal to 1 micrometer. - In certain embodiments,
substrate 102 has a thickness betweensurface 110 andsurface 112 that is equal to or about 300 micrometer, so that eachreaction site 104 has a volume of about 1.3 nanoliters. Alternatively, the volume of eachreaction site 104 may be less than 1.3 nanoliters, for example, by decreasing the diameter ofreaction sites 104 and/or the thickness ofsubstrate 102. For example, eachreaction 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 thereaction site 104 is in a range from 1 nanoliter to 20 nanoliters. - In certain embodiments, the density of
reaction sites 104 oversurfaces reaction sites 104 oversurfaces - Advantageously, all the
reaction sites 104 inactive 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 12,000reaction 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 inreaction site 104 size on asingle 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 ofreaction sites 104, where each group is characterized by a diameter or thickness that is different a diameter or thickness of thereaction 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 ofsubstrate 102 or may be interspersed so that two or more subarrays extend over the entire active area ofarticle 100 or over a common portion of active area ofarticle 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 toFIG. 8 , at least some ofreaction sites 104 may comprise achamfer 130 atsurface 110. Additionally or alternatively, at least some ofreaction sites 104 may comprise achamfer 130 at surface 112 (not shown). The use of chamfered and/or tapered reaction sites have been found to reduce the average distance or total area betweenadjacent reaction sites 104, yet without exceeding optical limitations for minimum spacing between solution sites or test samples. As discussed above in relation toFIG. 5 , a decrease in the area betweenadjacent reaction sites 104 may result in a reduction in the amount liquid solution that is left behind onsurfaces - In the embodiment shown in
FIG. 9 , an article, device, array, slide, or plate 100 a includes an inactive area, region, orzone 132 a that does not contain anyreaction sites 104 a. The inactive area may be a peripheral zone that surrounds the active zone containingreaction sites 104 a. Alternatively, the inactive area may comprise an area that boarders the active zone on one, two, or more sides or zones. In the illustrated embodiment shown inFIG. 9 ,article 100 a has a thickness equal to, or about equal to, 0.3 millimeter and the distance from the edge of the inactive area to the active area is equal to, or about equal to, 1 millimeter; however, other dimensions may be used. In the illustrated embodiment shown inFIG. 9 ,reaction sites 104 a have a diameter that is equal to, or about equal to, 0.075 millimeter and a pitch spacing that is equal to, or about equal to, 0.100 millimeter; however, other dimensions may be used. Where appropriate, features and/or dimensions discussed above in relation toarticle 100 may be incorporated intoarticle 100 a, or vice versa. - Referring to
FIG. 10 , in certain embodiments, an article, device, array, slide, orplate 100 b includes an active area, region, orzone 120 b comprising a plurality of reaction sites and aninactive area 132 b, whereininactive area 132 b comprises a partition, divider, or separator, 134 b that is located between adjacentactive areas 120 b. As illustrated inFIG. 10 ,inactive zone 132 b may also include a peripheral zone that surroundsactive zone 120 b. The dimensions shown inFIG. 10 for the various features ofarticle 100 b are an example of a particular embodiment and may be different, depending on the requirements of a particular design. For example,partition 134 b may have a thickness betweenactive areas 120 b that less than or equal to 500 micrometers, less than or equal to 1 millimeter, or less than or equal to 2 millimeters or 3 millimeters. -
Partition 134 b 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 bypartition 134 b. In certain embodiments, the surface ofactive areas 120 b andpartition 134 b are flush with one another on one or both faces ofarticle 100 b. Additionally or alternatively, at least a portion ofpartition 134 b may be raised or offset fromactive areas 120 b on one or both faces ofarticle 100 b. In other embodiments, at least a portion ofpartition 134 b forms a trough relative toactive areas 120 b for one or both faces ofarticle 100 b. Where appropriate, features and/or dimensions discussed above in relation toarticles article 100 b, or vice versa. - In certain embodiments,
substrate 102 comprises a photostructurable material, such as certain glass or ceramic materials. In such embodiments, amethod 140 shown inFIG. 11 may be used to fabricatesubstrate 102. Advantageously, last optional element ofmethod 140 shown inFIG. 11 may be used to provide asubstrate 102 that is opaque or nearly opaque, so that light emitted from onereaction site 104 does not enter anadjacent reaction site 104. -
Method 140 may be used to provide asubstrate 102 having an opacity sufficient prevent any, or nearly any, light emitted in onereaction site 104 from being transmitted into anadjacent reaction site 104.Method 140 may further comprise removing material fromsubstrate 102 by an amount sufficient to reduce thickness betweensurfaces substrate 104 by an amount sufficient to reduce the thickness betweensurfaces Method 140 may also includeheating substrate 102 to a temperature of at least 500 degrees Celsius during fabrication. In certain embodiments, the patterned mask used inmethod 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 inmethod 140 may be hydrofluoric acid. - Referring to
FIGS. 12 and 13 , in certain embodiments,article 100 is housed within acarrier 150 comprising afirst cover 152 having abottom surface 154 and asecond cover 156 having atop surface 158.Carrier 150 may further include one ormore side walls 159 configured to maintain a predetermined spacing between thecovers covers walls 159 together form acavity 160 sized to containarticle 100. During use,article 100 is disposed within thecavity 160 formed betweensurfaces cavity 160 may be greater than the thickness ofarticle 100 such that there is a gap betweenarticle 100 andbottom surface 154 and/or betweenarticle 100 andtop surface 158. As shown in the illustrated embodiment ofFIG. 12 , there may also be a gap between one or more ofside walls 159. Additionally or alternatively, at a portion ofarticle 100 may be attached to one or more ofcovers side walls 159. -
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 ofcarrier 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 thecovers reaction sites 104. Additionally or alternatively, theentire carrier 150 may be made of one or more transparent or nearly transparent materials. - Referring to
FIG. 14 , in certain embodiments, acarrier 150 a comprises an aperture, port, or opening 162 that may be disposed generally perpendicular to a cover oroptical access window 152 a and sized to allow passage ofarticle 100 intocarrier 150 a.Carrier 150 a may further comprise a wiper orblade 164 disposed along at least one long edge ofopening 162.Blade 164 may be configured to contact or engage at least one ofsurfaces article 100 whenarticle 100 is loaded intocarrier 150 a.Carrier 150 a may further comprise a film or membrane (not shown) disposed over all or a portion ofopening 162 that helps to sealcavity 160 a and is pierced whenarticle 100 is loaded intocarrier 150 a. In certain embodiments, the membrane andblade 164 form a single piece. - In certain embodiments,
blade 164 is configured to aid in distributing sample fluid into some or all ofreaction sites 104 asarticle 100 is inserted intocarrier 150 throughopening 162. For example,blade 164 may be configured to contact one or bothsurfaces article 100, so that liquid does not passblade 164, but is instead pushed, and/or pulled by capillary forces, intoreaction sites 104 assurface past blade 164. Additionally or alternatively,blade 164 may be configured to cover one or bothsurfaces article 100 with a liquid, gel, or the like, for example to reduce or eliminate contamination and/or evaporation of sample fluid contained insidereaction sites 104. - Where appropriate,
carrier 150 a may incorporate any of the structures or features discussed above in relation tocarrier 150, or vice versa. - Referring to
FIG. 15 , in certain embodiments, acarrier 150 b comprises abody 170, which may include some or all of the structures and features ofcarrier 150 and/orcarrier 150 a.Carrier 150 b further comprises a loader orinsertion tool 172 for holdingarticle 100, for aiding inloading article 100 into abody 170, and/or for loading a test solution intoreaction sites 104.Tool 172 may have a U-shaped body, whereinarticle 100 is held inside the “U” prior to loading intobody 170.Tool 172 may includetabs 174 onopposite arms 175 that are configured to engage or press into corresponding tabs orsimilar structure 176 ofbody 170. - Portions of
cavity 160 betweenarticle 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 inreaction sites 104 and configured to prevent or reduce evaporation of the test solution contained fromreaction sites 104. Onesuitable 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
FIG. 16 , amethod 200 of preparing a plurality of biological samples comprises providing a substrate of an article such asarticle Method 200 further comprises providing a carrier such ascarrier insertion tool 172, the insertion tool comprising a U-shaped body that includes a pair of arms configured to slideably engage the carrier.Method 200 also includes mounting or attaching the substrate to the insertion tool and the insertion tool to the carrier. In certain embodiments, the substrate is mounted or attached to the insertion tool, then the insertion tool and substrate together are mounted to the carrier. In other embodiments, the insertion tool is mounted to the carrier without the substrate, then the substrate is later mounted to the insertion tool and/or the carrier. - 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 themethod 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 intoreaction 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 allreaction 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 allreaction 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 eachreaction site 104 contains liquid sample after loading, then the ratio of the liquid volume sample to the total volume of allreaction 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
FIG. 17 , asystem 400 may be used to optically view, inspect, or measure one or more samples or solutions containing biological components of interest contained inreaction sites 104 ofarticle 100.Article 100 may be contained in a carrier such ascarrier System 400 comprises an optical head orsystem 402.System 400 further comprises a controller, computer, orprocessor 404 configured, for example, to operate various components ofoptical system 402 or to obtain and/or process data provided bysystem 400. For example,computer 404 may be used to obtain and/or process optical data provided by one or more photodetectors ofoptical system 402. In certain embodiments,processor 404 may transmit data to one or more computing systems for further processing. Data may be transmitted fromprocessor 404 to the computing systems via an internet connection or some other network system. - In certain embodiments,
system 400 further comprises athermal 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 inarticle 100.Systems article 100. In such embodiments,computer 404 may be used to controlsystems systems thermal control system 406 may be completely separate fromoptical system 402 and/or fromcomputer 404. In such embodiments,optical system 402 may be used to perform a dPCR or end-point PCR procedure on the samples contained inreaction sites 104 after thermal cycle has been performed on the samples usingthermal 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 ofthermal control system 406 may be integrated with or intoarticle 100. For example,article 100 may include one or more heating elements distributed along one or bothsurfaces 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 tosubstrate 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 alight source 410 and an associatedexcitation optic system 412 configured to illuminate at least some of samples contained in the reaction sites ofarticle 100. Excitationoptical system 412 may include one ormore lenses 414 and/or one ormore filters 416 for conditioning light directed to the samples.Optical system 402 may further comprise aphotodetector 420 and an associatedemission optic system 422 configured to receive optical data emitted by at least some of samples contained in the reaction sites ofarticle 100. For example, whensystem 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 ofarticle 100. Emissionoptical system 422 may include one ormore lenses 424 and/or one ormore filters 426 for conditioning light directed to the samples. - In the illustrated embodiment of
FIG. 17 , excitation/emissionoptical systems optical systems beamsplitter 430 that reflects excitation light and transmits emission light from the samples tophotodetector 420. In certain embodiments, excitation/emissionoptical systems beamsplitter 430 andarticle 100, which may be used improve optical performance, for example, to provide more even illumination and reading of light to and from the samples contained inarticle 100. In certain embodiments, for example where even illumination is less critical (e.g., some dPCR applications), the common field lens may be omitted, as shown in the illustrated embodiment ofFIG. 17 . Omission of the field lens may help to reduce the size and complexity ofoptical system 402. -
Photodetector 420 may comprise one or more photodiodes, photomultiplier tubes (PMTs), or the like. Such photodetectors may be used, for example, whereoptical system 402 is configured to scanindividual reaction sites 104 or subsets ofreaction 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 ofreaction 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
FIGS. 18-21 , in certain embodiments an article, device, array, slide, orplate 500 comprises asubstrate 502 containing a plurality of through-holes orreaction sites 504 located insubstrate 102.Substrate 502 comprises a first surface and an opposing second surface. In the illustrated embodiment, eachreaction site 504 extends from an opening in the first surface to an opening in the second surface. As illustrated inFIGS. 19 and 20 ,reaction sites 504 may have a hexagonal shape and/or be arranged in a close-packed hexagonal matrix pattern. Alternatively, some or all ofreaction sites 504 may have a shape, diameter, density, thickness, pitch spacing, or the like discussed above in relation toreaction sites 104.Article 500 further comprises one or more tabbed, cutout, orblank regions 506 in which noreaction sites 504 are present. As discussed below,blank regions 506 may be located in support regions forarticle 500. In the illustrated embodiment, blank regions define four semi-circular shape; however, other shapes and sizes are anticipated. In addition,article 500 may include ablank perimeter 508 in which no reaction sites are located. - In certain embodiments,
substrate 502 comprise silicon, which may be configured to provide an even temperature distribution acrossarticle 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
FIG. 19 ,reaction sites 504 may be arranged so as to define one ormore dropout regions 509 located within the array ofreaction sites 504. In some embodiments, dropregions 509 have a dimension suitable of viewing with an unaided eye (e.g., visible to the unaided eye without the use of a magnifying device). In the illustrated embodiment,article 500 comprises onedropout region 504 located in a first quadrant ofarticle 500; however, multiple dropout regions on asingle article 500 may be incorporated. One ormore dropout regions 509 may define an overall shape that is longer along one axis than along an orthogonal axis, as illustrated inFIG. 19 . Thus, the use of the singleelongated dropout region 509 shown ifFIG. 19 that is located away from a center ofarticle 500 allows a uses to determine the orientation of article 500 (e.g., to determine which side is the front and back, and determine the proper orientation about an axis perpendicular to the page ofFIGS. 18-20 ).Dropout regions 509 may also be configured to provide a reference signal, for example, a reference optical signal used during optical inspection ofreaction sites 504. - In certain embodiments, a plurality of
dropout regions 509 may be configured provide information about thearticle 500 based on, for example, the dropout shape(s), number ofdropout regions 509, and/or the relative position of onedropout region 509 to anotherdropout region 509. For example, the number of dropout regions on aparticular article 500 may be used to determined the diameter ofreaction sites 504 and/or the distance betweendropout regions 509, or the geometry of thedropout regions 509 to one another, may be used to determine the number ofreaction sites 504 or the pitch betweenreaction sites 504. Many other combinations of dropout regions size, shape, and distribution are anticipated. - Referring to
FIG. 21 ,article 500 may have an overall dimension of or about 10 mm by 10 mm.FIG. 21 also shows the value of other dimensions relevant to the particular embodiment shown inFIG. 21 . - Referring to
FIGS. 22 and 23 , anarticle 500 b may be configured similar toarticle 500 inFIG. 18 , expect thatarticle 500 b also includes one ormore landing regions 530 that may be size to provide more favorable loading properties. Thus, one or more edges ofarticle 500 b have wider zones withoutreaction sites 504 than other edges ofarticle 500 b. -
Article 500 may incorporate, where appropriate, various of the elements and/or features discussed in relation toarticle 100, or vice versa. In addition,article 500 may be used incarrier 150 or other carriers according to embodiments of the current invention.Article 500 may be used in conjunction withsystem 400 ormethod 140 in ways similar to those in whicharticle 100 has been disclose herein, as well as in or with other systems and methods disclosed herein in relation toarticle 100. - In certain embodiments,
substrate 502 comprises silicon material andreaction sites 504 may comprise through-holes that are formed using a hexamethyldisilazane (HMDS) vapor coating process. Referring toFIG. 24 , amethod 600 may be used to form through-hole reaction sites 504 in thesilicon substrate 502.Method 600 includes aprocess 605 comprising applying or forming a pattern on a first surface of a substrate material.Method 600 further includes aprocess 610 comprising using the pattern to etch a plurality of wells in the first surface of the substrate.Method 600 also includes aprocess 615 comprising removing material from a second surface that is opposite the first surface.Method 600 additionally includes aprocess 620 comprising etching and/or polishing the second surface.Method 600 further includes aprocess 625 comprising coating at least one of the surfaces, for example, to form a hydrophobic surface. - Referring to
FIGS. 25A-C ,method 600 may comprise a deep reactive-ion etching (DRIE) process to formreaction sites 504 inarticle 500. As seen inFIG. 25A ,article 500 comprises afirst surface 510 and an opposingsecond surface 512.FIG. 25A shows amask 550 applied tosecond surface 512 in accordance withmethod 600. Using at etching process such as DRIE,mask 550 may be configured to form a plurality ofwells 504′ insecond surface 512. For illustrative purposes,article 500 is shown in a horizontal orientation, withsecond surface 512 being located belowfirst surface 510; however, it will be appreciated that during fabrication and/or use,second surface 512 may be located abovefirst surface 510 and/orarticle 500 may have a different orientation, such as a vertical orientation. As seen inFIG. 25B ,wells 504′ do not penetrate through tofirst surface 510, but have a depth that is less than the thickness ofarticle 500. Alternatively,etching process 610 may produce through-holes that completely penetrate the thickness ofarticle 500. In such embodiments, further processing offirst surface 510 may or may not be conducted in accordance withmethod 500. - Referring to
FIG. 25C ,first surface 510 may be further processed in accordance withprocess 615 ofmethod 600 so as to reduce the thickness ofarticle 500 by an amount sufficient to form through-holes 504 fromwells 504′.First surface 512 may be further processed in accordance withprocesses 620 and/or 625 ofmethod 600 so as be preparefirst surface 510 so that a sample or solution applied tofirst surface 510 is efficiently received by through-holes 504. Additionally or alternatively, processes 620 and/or 625 may be performed onsecond surface 512. In such embodiments,etching process 615 may be excluded altogether or may performed so that the final form ofarticle 500 is a substrate with the plurality ofwells 504′ instead of the through-holes 504 shown inFIG. 25C . - In certain embodiments, processes 620 and/or 625 are performed so that at least one of the
surfaces 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) onfirst surface 510. This residual film may provide a “bridge” between adjacent or neighboringreaction sites 504. The bridging layer can result in contamination of onereaction site 504 by one or more adjacent or neighboringreaction sites 504. To solve this problem, it has been discovered whenfirst 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, whenarticle 500 comprises a silicon material and a reaction site geometry as shown inFIG. 20 , it has been determined that the bridging problem is eliminated if the roughness offirst surface 510 meets any of the following roughness criteria: -
- Ra (arithmetic average): less than or equal to 5 nanometers.
- Rv (maximum valley depth): less than or equal to 15 nanometers.
- Rp (maximum peak height): less than or equal to 9 nanometers.
- Rt (maximum peak to trench): less than or equal to 24 nanometers.
- In certain embodiments,
articles FIG. 26 ,article 500 may be arranged in an enclosure, housing, orcase 700, according to an embodiment of the present invention and of provisional application No. 61/723,710.Case 700 may comprise abase 702 and a cover orlid 704 configured to sealably engagebase 702.Base 702 and cover 704 may be joined together to form a cavity orchamber 708, which may receive or contain aarticle 500.Article 500 may be part ofbase 702, or may be separate and/or distinct frombase 702 and be configured to be mounted or held bybase 702. In the illustrated embodiment, tabs -
Base 702 may comprise a plurality of bosses, tabs, staking sites, or support pads 720 (e.g.,tabs article 500 withinbase 702 andcavity 708. One or more tabs 182 may be staked so that material from the tab is deformed or moved to holdarticle 500 firmly withinbase 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 orsilicon 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 720 a correspond withblank regions 506 ofarticle 500. In certain embodiments, tabs 620 a andblank regions 506 are large enough to provide proper support ofarticle 500, but small enough so that the active area ofcorresponding article 500 provide a desired predetermined active area containing a predetermined number ofreaction 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.
Claims (34)
1. An article for holding a plurality of biological samples, the article comprising:
a substrate comprising a first surface and an opposing second surface; and
a plurality of reaction sites in the substrate, each of the reaction sites extending from an opening in the first surface to an opening in the second surface, the reaction sites being configured to provide sufficient surface tension by capillary action to hold respective biological samples;
wherein a density of reaction sites over at least a portion of one of surfaces is at least 170 holes per square millimeter.
2. The article of claim 1 , wherein at least one of the surfaces has a surface roughness characterized by one or more of an arithmetic average roughness (Ra) that is less than or equal to 5 nanometers, a maximum valley depth roughness (Rv) that is less than or equal to 15 nanometers, a maximum peak height roughness (Rp) that is less than or equal to 9 nanometers, or a maximum peak to trench roughness (Rt) that is less than or equal to 24 nanometers.
3. (canceled)
4. The article of claim 1 , wherein two or more of the reaction sites comprise a hexagonal shape at one of the surfaces or at each of the surfaces.
5-7. (canceled)
8. The article of claim 1 , wherein the openings of each surface are arranged in a close-packed hexagonal matrix.
9-11. (canceled)
12. The article of claim 1 , wherein each of the walls of the two or more of the reaction sites is tapered between the surfaces.
13. (canceled)
14. The article of claim 1 , wherein each of the walls of the two or more of the reaction sites includes a chamfer located at one of the surfaces or at each of the surfaces.
15. (canceled)
16. The article of claim 1 , wherein at least some of the reaction sites have a volume between the opposing surfaces that is less than or equal to 1 nanoliter.
17-18. (canceled)
19. The apparatus of claim 1 , wherein a reaction site volume of at a first plurality of the reaction sites is different than a reaction site volume of at a second plurality of the reaction sites.
20-27. (canceled)
28. The article of claim 1 , wherein the substrate comprises a photo-structured glass ceramic material.
29. The article of claim 1 , wherein the substrate comprises a silicon material.
30. The apparatus of claim 1 , further comprising a fiducial disposed on at least one of the surfaces.
31. An article for holding biological samples for analysis, the article comprising:
a substrate having a pair of opposing surfaces; and
a plurality of reaction sites in the substrate, each of the reaction sites extending from an opening in one of the opposing surfaces of the substrate to an opening in the other one of the opposing surfaces, the reaction sites being configured to provide sufficient surface tension by capillary action to hold respective biological samples;
wherein at least some of the reaction sites have a volume between the opposing surfaces that is less than or equal to 1 nanoliter.
32. An apparatus, comprising:
the article of claim 1 ; and
a carrier comprising:
a first cover comprising a bottom surface; and
a second cover comprising a top surface;
wherein the article is disposed between the top surface and the bottom surface;
wherein there is a space between the first surface of the substrate and the top surface;
wherein there is a space between the second surface of the substrate and the bottom surface.
33. (canceled)
34. The apparatus of claim 32 , further comprising a PDMS material disposed within the spaces.
35. (canceled)
36. The article of claim 32 , wherein the carrier comprises an aperture disposed perpendicular to the surfaces and sized to allow passage of the substrate, wherein the aperture comprises a piercable membrane that is broken when the substrate is moved into the carrier.
37-39. (canceled)
40. A system comprising:
the apparatus of claim 32 ;
a light source configured to provide an excitation beam;
a photodetector configured to receive emission light from two or more of the reaction sites when the excitation beam interacts with a biological solution contained in the two or more of the reaction sites;
an optical system comprising an excitation optical system configured to deliver at least a portion of the excitation beam to the two or more of the reaction sites and emission optical system to deliver at least some of the emission light from the two or more of the reaction sites to the detector.
41. The system of claim 40 , further comprising a thermal control unit to control a temperature of the substrate or within the substrate.
42-45. (canceled)
46. A method of making an article for holding biological samples for analysis, the method comprising:
providing a substrate comprising glass and having a first surface and an opposing second surface;
covering the glass with a mask comprising a pattern;
passing an ultraviolet beam through the mask and onto at least portions of the substrate;
exposing the at least portion of the substrate to a corrosive agent;
removing material to provide a plurality of reaction sites extending from one of the surfaces of the substrate to the other one of the surfaces;
subsequently exposing the substrate to an ultraviolet beam so as to change a characteristic of the glass;
47-54. (canceled)
55. A method of making an article for holding biological samples for analysis, the method comprising:
providing a substrate comprising silicon and having a first surface and an opposing second surface;
applying a pattern to one of the surfaces;
etching a plurality of wells the first surface;
removing material from the second surface to form at least one through-hole from a corresponding well of the plurality of wells;
forming a surface roughness on at least one of the surfaces, the surface roughness characterized by one or more of an arithmetic average roughness (Ra) that is less than or equal to 5 nanometers, a maximum valley depth roughness (Rv) that is less than or equal to 15 nanometers, a maximum peak height roughness (Rp) that is less than or equal to 9 nanometers, or a maximum peak to trench roughness (Rt) that is less than or equal to 24 nanometers.
56. (canceled)
57. A method of preparing a plurality of biological samples, the method comprising:
providing the article of claim 1 ;
providing a carrier comprising:
a first cover comprising a bottom surface; and
a second cover comprising a top surface;
an aperture sized to receive the article;
providing an insertion tool comprising:
a U-shaped body;
a pair of arms configured to slideably engage the carrier;
attaching the article within the U-shaped body;
sliding the insertion tool by an amount sufficient to dispose the article between the covers;
58-63. (canceled)
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US14/385,765 US20150044686A1 (en) | 2012-03-16 | 2013-03-15 | Systems and Methods for Containing Biological Samples |
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CN (2) | CN104411408B (en) |
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Also Published As
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US11590506B2 (en) | 2023-02-28 |
SG11201406039WA (en) | 2014-11-27 |
IN2014DN08135A (en) | 2015-05-01 |
CN104411408B (en) | 2017-07-11 |
CN104411408A (en) | 2015-03-11 |
CN104302400B (en) | 2017-03-01 |
AU2013231910A1 (en) | 2014-10-30 |
US20150080247A1 (en) | 2015-03-19 |
RU2014141637A (en) | 2016-05-10 |
KR20140140080A (en) | 2014-12-08 |
CN104302400A (en) | 2015-01-21 |
WO2013138767A1 (en) | 2013-09-19 |
SG11201405785WA (en) | 2014-11-27 |
KR20150003738A (en) | 2015-01-09 |
US20230201839A1 (en) | 2023-06-29 |
WO2013138706A2 (en) | 2013-09-19 |
JP2015511015A (en) | 2015-04-13 |
RU2014140837A (en) | 2016-05-10 |
JP2015516802A (en) | 2015-06-18 |
WO2013138706A3 (en) | 2014-01-16 |
EP2825310A2 (en) | 2015-01-21 |
EP2825314A1 (en) | 2015-01-21 |
US20190381502A1 (en) | 2019-12-19 |
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