Systems and Methods for Assessing of Biological Samples

A system for determining the number of target nucleotide molecules in a sample includes a sample holder, an excitation optical system, an optical sensor, and an emission optical system. The sample holder is configured to receive an article comprising at least 20,000 separate reaction sites. The excitation optical system comprises a light source configured to simultaneously illuminate the at least 20,000 separate reaction sites. The optical sensor comprises a predetermined number of pixels, the predetermined number of pixels being at least 20 times the number of separate reaction sites. The emission optical system comprises a system working distance from the sample holder, wherein the working distance is less than or equal to 60 millimeters.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to devices, systems, and methods for assessing biological samples, and more specifically to devices, systems, and methods for simultaneously assessing a plurality of biological samples.

2. Description of the Related Art

Optical systems for biological and biochemical reactions have been used to monitor, measure, and/or analyze such reactions. Such systems 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 test or experiment have resulted in instruments that are able to conduct large numbers of reactions simultaneously.

The increase in the number sample sites in a single test or experiment has led to microtiter plates and other sample formats that provide ever smaller sample volumes. In addition, techniques such as digital PCR (dPCR) have increased the demand for smaller sample volumes that contain either zero or one target nucleotide sequence in a majority of a large number of test samples. There is a need for systems and sample format that will provide reliable data in even high-density sample format with sample sites having volumes on the order of nanoliters or picoliters.

DETAILED DESCRIPTION OF THE DRAWINGS

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 small 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, which may include, without limitation, 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 devices, instruments, systems, and methods according to 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 solutions test volumes are processed, observed, and/or measured, embodiments of the present invention are particularly well suited for dPCR. 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 very small test samples or volumes, such that the vast majority at least some of these samples or volumes contains either one molecule of the target nucleotide sequence or none of the target nucleotide sequence. When the samples are subsequently thermally cycled in a PCR protocol, procedure, assay, process, or experiment, the individual samples containing the one or more molecules of the target nucleotide sequence are greatly amplified and produce a positive, detectable detection signal, while the samples those containing none of the target(s) nucleotide sequence are not amplified and do not produce a no detection 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 the same devices, instruments, or systems, and methods.

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 containing the biological components of interest. These biological components or targets of interest 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 various one or more PCR methods and systems in applications such as fetal diagnostics, multiplex dPCR, viral detection, and 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 targets of interest may be contained in distributed or divided between a plurality of a small sample volumes or reaction volume sites. The samples or solutions for embodiments of the present invention sample volumes or reaction sites disclosed herein are generally illustrated as being contained in through-holes located in a substrate material; however, where applicable, other forms of sample volumes or reaction sites according to embodiments of the present invention may include be used, including reaction volumes located within wells or indentations formed in a substrate, spots of solution distributed on the surface a substrate, or other types of reaction chambers or formats, such as samples or solutions located within test sites or volumes of a microfluidic system, or within or on small beads or spheres.

In certain embodiments, order to conduct a dPCR protocol, assay, procedure, process, or experiment included distributing or dividing according to embodiments of the present invention, an initial sample or solution may be divided 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 tens of thousands, hundreds of thousands, or even millions of reaction sites, each having 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 10's or 100's of picoliters or less), in a way that is simple and cost effective. When Because the number of target nucleotide sequences contained in the initial sample or solution may be 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 in such circumstances that the entire content, or nearly the entire content, of the initial solution be accounted for and contained in or received by one of the sample volumes or reaction sites being processed. For example, where there are only a few target nucleotides present in the initial solution, many some or all of these target nucleotide could potentially be contained in a small residual fluid volume that are that is not successfully loaded into one not located in any of the reaction sites and, therefore, would not be detected, measured, or counted. Thus, efficient transfer of the initial solution helps reduces 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 missing the 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 efficiently 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. and load an initial sample solution into a large number of reaction sites or through-holes in a way that results in all, or essentially all, of the sample or solution being contained in one of a predetermined reaction sites.

Referring toFIGS. 1-3, in certain embodiments of the present invention, an article, device, substrate, slide, or plate100comprises a substrate102containing a plurality of partitions, through-holes, reaction regions, or reaction sites104located in substrate102. In certain embodiments, article100may comprise a chip. Additionally or alternatively, article100may comprise a microfluidic device which, for example, may further include a plurality of channels or paths for transferring reagents and/or test solutions to reaction sites104. In other embodiments, reaction sites104comprise a plurality of droplets or beads and article100may comprise one or more chambers and/or channels containing some or all of the droplets or beads104. In such embodiments, droplets or beads104may form an emulsion, where some or all of droplets or beads104contain one or more target of at least one polynucleotide or nucleotide sequence. Where reaction sites104are beads, the beams may optionally include an attached optical signature or label. Droplets or beams104may be inspected, monitored, or measured either one at time or in groups containing one or more droplets or beads104, for example using an imaging system according to embodiments of the present invention.

In the illustrated embodiment, article100comprises a first surface110and an opposing second surface112. In the illustrated embodiment, each reaction site104extends from an opening114in first surface110to an opening116in second surface112. While the illustrated embodiment shown inFIG. 3shows a substrate containing through-holes104, substrate102may additionally or alternatively comprise other types of reaction sites. For example, reaction sites104may include reaction volumes located within wells or indentations formed in substrate102, spots of solution distributed on the surfaces110or112, or other types of reaction chambers or formats, such as samples or solutions located within test sites or volumes of a microfluidic system, or within or on small beads or spheres.

Reaction sites104may 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. Article100may 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.

Substrate102may be a flat plate or comprise any form suitable for a particular application, assay, or experiment. Substrate102may 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, substrate102may comprise a polymer material such as an acrylic, styrene, polyethylene, polycarbonate, and polypropylene material. Substrate102and reaction sites104may be formed by one or more of machining, injection molding, hot embossing, laser drilling, photolithography, or the like.

In certain embodiments, surfaces110,112may 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 sites104may 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 substrate102using 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 surface110,112during 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 sites104. 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 sites104.

Referring toFIG. 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. 5demonstrates 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 of reaction sites104of a given cross-sectional dimension provides both an increase in the number of test samples for a given size substrate102and decreases or eliminates residual fluid left on surfaces110,112(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 sites104are 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 sites104in a substrate102, 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 sites104, leading to a reduction in the amount of residual liquid or solution left behind on surfaces110,112after loading of a test solution or sample. Referring toFIGS. 6 and 7, an array of hexagonal reaction sites104having 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 the reaction sites104is a function of a minimum edge distance S between adjacent reaction sites. Thus, the geometry shown inFIG. 7represents 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. 7inside 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. 7shows 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 5show 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 inFIG. 7is 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 site104(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 inFIG. 1, article100has a square shape and an overall dimension of 15 millimeter by 15 millimeter. Article100also has an active area, region, or zone120with 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 the article100, over which reaction sites or solution volumes are contained or distributed. In certain embodiments, the active area of article100may 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 on substrate102. Article100may have other shapes and dimensions. For example, surfaces110,112may be rectangular, triangular, circular, or some other geometric shape. The overall dimensions of article100and active area120may 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 ofFIG. 1, reaction sites104may have a characteristic diameter of 75 micrometer and be distributed over active area120with a pitch of 125 micrometers between adjacent reaction sites. In other embodiments, reaction sites104have 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 sites104have 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, substrate102has a thickness between surface110and surface112that is at or about 300 micrometer, so that each reaction site104has a volume of about 1.3 nanoliters. Alternatively, the volume of each reaction site104may be less than 1.3 nanoliters, for example, by decreasing the diameter of reaction sites104and/or the thickness of substrate102. For example, each reaction site104may 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 site104is in a range from 1 nanoliter to 20 nanoliters.

In certain embodiments, the density of reaction sites104over surfaces110,112is at least 100 reaction sites per square millimeter. Higher densities are also anticipated. For example, a density of reaction sites104over surfaces110,112may 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, or 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 sites104in active area120may be simultaneously imaged and analyzed by an optical system. In certain embodiments, active area120imaged and analyzed by the optical system comprises at least 12,000 reaction sites104. In other embodiments, active area120imaged 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 sites104comprise 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 site104size on a single substrate102may be used to increase the dynamic range of a dPCR process, assay, or experiment. For example, article100may comprise two or more subarrays of reaction sites104, where each group is characterized by a diameter or thickness that is different a diameter or thickness of the reaction sites104of 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 substrate102or may be interspersed so that two or more subarrays extend over the entire active area of article100or over a common portion of active area of article100.

In certain embodiments, at least some of the reaction sites104are tapered over all or a portion of their walls. For example, referring toFIG. 8, at least some of reaction sites104may comprise a chamfer130at surface110. Additionally or alternatively, at least some of reaction sites104may comprise a chamfer130at surface112(not shown). The use of chamfered and/or tapered reaction sites have been found to reduce the average distance or total area between adjacent reaction sites104, 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 between adjacent reaction sites104may result in a reduction in the amount liquid solution that is left behind on surfaces110,112during a loading process. Thus, a higher sample loading efficiency may be obtained, while still maintaining a larger effective spacing between adjacent solution sites or test samples for the optical system.

In the embodiment shown inFIG. 9, an article, device, array, slide, or plate100aincludes an inactive area, region, or zone132athat does not contain any reaction sites104a. The inactive area may be a peripheral zone that surrounds the active zone containing reaction sites104a. 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, article100ahas 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 sites104ahave 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 to article100may be incorporated into article100a, or vice versa.

Referring toFIG. 10, in certain embodiments, an article, device, array, slide, or plate100bincludes an active area, region, or zone120bcomprising a plurality of reaction sites and an inactive area132b, wherein inactive area132bcomprises a partition, divider, or separator,134bthat is located between adjacent active areas120b. As illustrated inFIG. 10, inactive zone132bmay also include a peripheral zone that surrounds active zone120b. The dimensions shown inFIG. 10for the various features of article100bare an example of a particular embodiment and may be different, depending on the requirements of a particular design. For example, partition134bmay have a thickness between active areas120bthat 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.

Partition134bmay 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 partition134b. In certain embodiments, the surface of active areas120band partition134bare flush with one another on one or both faces of article100b. Additionally or alternatively, at least a portion of partition134bmay be raised or offset from active area120bon one or both faces of article100b. In other embodiments, at least a portion of partition134bforms a trough relative to active areas120bfor one or both faces of article100b. Where appropriate, features and/or dimensions discussed above in relation to articles100,100amay be incorporated into article100b, or vice versa.

In certain embodiments, substrate102comprises a photostructurable material, such as certain glass or ceramic materials. In such embodiments, a method140shown inFIG. 11may be used to fabricate substrate102. Advantageously, last optional element of method140shown inFIG. 11may be used to provide a substrate102that is opaque or nearly opaque, so that light emitted from one reaction site104does not enter an adjacent reaction site104.

Method140may be used to provide a substrate102having an opacity sufficient prevent any, or nearly any, light emitted in one reaction site104from being transmitted into an adjacent reaction site104. Method140may further comprise removing material from substrate102by an amount sufficient to reduce thickness between surfaces110,112, for example, removing material from substrate104by an amount sufficient to reduce the thickness between surfaces110,112by at least 20 percent over an initial thickness or by at least 30 percent or 40 percent over an initial thickness. Method140may also include heating substrate102to a temperature of at least 500 degrees Celsius during fabrication. In certain embodiments, the patterned mask used in method140comprises 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 method140may be hydrofluoric acid.

Referring toFIGS. 12 and 13, in certain embodiments, article100is housed within a carrier150comprising a first cover152having a bottom surface154and a second cover156having a top surface158. Carrier150may further include one or more side walls159configured to maintain a predetermined spacing between the covers152,154. The covers152,154and the walls159together form a cavity160sized to contain article100. During use, article100is disposed within the cavity160formed between surfaces154,158. The thickness of cavity160may be greater than the thickness of article100such that there is a gap between article100and bottom surface154and/or between article100and top surface158. As shown in the illustrated embodiment ofFIG. 12, there may also be a gap between one or more of side walls159. Additionally or alternatively, at a portion of article100may be attached to one or more of covers152,156and one or more of side walls159.

Carrier150may 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 carrier150may 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 covers152,156comprises a suitably transparent material for providing a window configured to allow optical access to and/or from reaction sites104. Additionally or alternatively, the entire carrier150may be made of one or more transparent or nearly transparent materials.

Referring toFIG. 14, in certain embodiments, a carrier150acomprises an aperture, port, or opening162that may be disposed generally perpendicular to a cover or optical access window152aand sized to allow passage of article100into carrier150a. Carrier150amay further comprise a wiper or blade164disposed along at least one long edge of opening162. Blade164may be configured to contact or engage at least one of surfaces110,112of article100when article100is loaded into carrier150a. Carrier150amay further comprise a film or membrane (not shown) disposed over all or a portion of opening162that helps to seal cavity160aand is pierced when article100is loaded into carrier150a. In certain embodiments, the membrane and blade164form a single piece.

In certain embodiments, blade164is configured to aid in distributing sample fluid into some or all of reaction sites104as article100is inserted into carrier150through opening162. For example, blade164may be configured to contact one or both surfaces110,112during loading of article100, so that liquid does not pass blade164, but is instead pushed, and/or pulled by capillary forces, into reaction sites104as surface110,112moves past blade164. Additionally or alternatively, blade164may be configured to cover one or both surfaces110,112of article100with a liquid, gel, or the like, for example to reduce or eliminate contamination and/or evaporation of sample fluid contained inside reaction sites104.

Where appropriate, carrier150amay incorporate any of the structures or features discussed above in relation to carrier150, or vice versa.

Referring toFIG. 15, in certain embodiments, a carrier150bcomprises a body170, which may include some or all of the structures and features of carrier150and/or carrier150a. Carrier150bfurther comprises a loader or insertion tool172for holding article100, for aiding in loading article100into a body170, and/or for loading a test solution into reaction sites104. Tool172may have a U-shaped body, wherein article100is held inside the “U” prior to loading into body170. Tool172may include tabs174on opposite arms175that are configured to engage or press into corresponding tabs or similar structure176of body170.

Portions of cavity160between article100and surfaces154,158may be filled with an immiscible fluid170(e.g., a liquid or a gel material) that does not mix with test solution contained in reaction sites104and configured to prevent or reduce evaporation of the test solution contained from reaction sites104. One suitable fluid170for 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 cavity160if 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 plate100that 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 toFIG. 16, a method200of preparing a plurality of biological samples comprises providing a substrate of an article such as article100,100a, or100b. Method200further comprises providing a carrier such as carrier150,150a, or150b, and providing an insertion tool such as insertion tool172, the insertion tool comprising a U-shaped body that includes a pair of arms configured to slideably engage the carrier. Method200also 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, method200includes 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 method200, 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 sites104is selected to be greater than the volume of the liquid sample to be loaded into reaction sites104. 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 sites104is less than or equal to 95 percent. In other embodiments, the ratio of the liquid volume sample to the total volume of all reaction sites104is 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 site104contains liquid sample after loading, then the ratio of the liquid volume sample to the total volume of all reaction sites104may 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.

Referring toFIGS. 17-20, in certain embodiments an article, device, array, slide, or plate200comprises a substrate202containing a plurality of through-holes or reaction sites204located in substrate102. Substrate202comprises a first surface and an opposing second surface. In the illustrated embodiment, each reaction site204extends from an opening in the first surface to an opening in the second surface. As illustrated inFIGS. 18 and 19, reaction sites204may have a hexagonal shape and/or be arranged in a close-packed hexagonal matrix pattern. Alternatively, some or all of reaction sites204may have a shape, diameter, density, thickness, pitch spacing, or the like discussed above in relation to reaction sites104. Article200further comprises one or more tabbed, cutout, or blank regions206in which no reaction sites204are present. As discussed below, blank regions206may be located in support regions for article200. In the illustrated embodiment, blank regions define four semi-circular shape; however, other shapes and sizes are anticipated. In addition, article200may include a blank perimeter208in which no reaction sites are located.

In certain embodiments, substrate202comprise silicon, which may be configured to provide an even temperature distribution across article200during use. Alternatively, substrate202comprises a glass material, such as a photo-structured glass ceramic, or a metal, such as aluminum, copper, or stainless steel.

Referring toFIG. 18, reaction sites204may be arranged so as to define one or more dropout regions209located within the array of reaction sites204. In some embodiments, drop regions209have 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, article200comprises one dropout region204located in a first quadrant of article200; however, multiple dropout regions on a single article200may be incorporated. One or more dropout regions209may define an overall shape that is longer along one axis than along an orthogonal axis, as illustrated inFIG. 19. Thus, the use of the single elongated dropout region209shown ifFIG. 18that is located away from a center of article200allows a uses to determine the orientation of article200(e.g., to determine which side is the front and back, and determine the proper orientation about an axis perpendicular to the page ofFIGS. 17-19). Dropout regions209may also be configured to provide a reference signal, for example, a reference optical signal used during optical inspection of reaction sites204.

In certain embodiments, a plurality of dropout regions209may be configured provide information about the article200based on, for example, the dropout shape(s), number of dropout regions209, and/or the relative position of one dropout region209to another dropout region209. For example, the number of dropout regions on a particular article200may be used to determined the diameter of reaction sites204and/or the distance between dropout regions209, or the geometry of the dropout regions209to one another, may be used to determine the number of reaction sites204or the pitch between reaction sites204. Many other combinations of dropout regions size, shape, and distribution are anticipated.

Referring toFIG. 20, article200may have an overall dimension of or about 10 mm by 10 mm.FIG. 20also shows the value of other dimensions relevant to the particular embodiment shown inFIG. 20.

Referring toFIGS. 21 and 22, an article200′ may be configured similar to article200inFIG. 17, expect that article200′ also includes one or more landing regions230that may be size to provide more favorable loading properties. Thus, one or more edges of article200′ have wider zones without reaction sites204than other edges of article200′.

Article200may incorporate, where appropriate, various of the elements and/or features discussed in relation to article100, or vice versa. In addition, article200may be used in carrier150or other carriers according to embodiments of the current invention. Article200may be used in conjunction with system400or method140in ways similar to those in which article100has been disclose herein, as well as in or with other systems and methods disclosed herein in relation to article100.

Various methods and devices may be used to provide detection of one or more biological components of interest that are contained in reaction sites104. For example, various fluorescent dyes or markers 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 toFIG. 23, a system300may be used to optically view, inspect, or measure one or more samples or solutions containing biological components of interest that are located in reaction sites104of article100. System300comprises an optical head or system400configured to read or monitor some or all of the reaction sites104of article100, article200, or the like. In certain embodiments, system100may further include one or more of a thermal control system500, an integrated controller, computer, computational system, or processor530located on or within the optical head or system400and/or thermal control system500, and/or an external controller, computer, or processor560located external to optical head or system400and thermal control system500.

Either or both computers530,560may include electronic memory storage containing instructions, routines, algorithms, test and/or configuration parameter, test or experimental data, or the like. Either or both computers530,560may be configured, for example, to operate various components of optical system400or to obtain and/or process data provided by system300. For example, either or both computers530,560may be used to obtain and/or process optical data provided by one or more photodetectors of optical system400. In certain embodiments, integrated computer530may communicate with external computer560and/or transmit data to external computer560for further processing, for example, using a hardwire connection, a local area network, an internet connection, cloud computing system, or the like. External computer560may be physical computer, such as a desktop computer, laptop computer, notepad computer, tablet computer, or the like. Additionally or alternatively, either or both computers530,560may comprise a virtual device or system such as a cloud computing or storage system. Data may be transferred or shared between computers530,560via a wireless connection within a local area network, a cloud storage or computing system, or the like. Additionally or alternatively, data from system300(e.g., from optical system400and/or thermal controller500) may be transferred to an external memory storage device, for example, an external hard drive, a USB memory module, a cloud storage system, or the like. System300may include both computers530,560. Alternatively, system300may include only one of either computer530or computer560. In such embodiments, data from computer530or computer560may be stored, transferred, or processed via a hardwire connection, a local area network, an internet connection, cloud computing system, or the like.

In certain embodiments, system300does not include thermal control system500within a common housing or instrument. For example, optical system400may be configured to receive a chip or plate such as article100or200that was processed in a separate thermal control system, such as a PCR thermal cycler. Optical system400may then be used to perform an endpoint PCR or dPCR experiment or assay. In certain embodiments, the external thermal controller is configured to process a plurality of chips or plates, for example, in the form of article100,200, or the like. In such embodiments, optical system400is configured to receive one or more of the plurality of chips or plates in order to perform a dPCR experiment or assay. A computer system such as computer530and/or computer560may be used to perform a dPCR analysis based on data from the plurality of chips or plates. In such embodiments, the computer system may be configured to pool data from the chips or plate, for example, to increase the effective sample size of a dPCR experiment or assay to create a “virtual chip”. For example, a plurality of articles100,200comprising at least 15,000 or at least 20,000 through-holes or partitions for containing samples may be processed in a separate PCR thermal cycler (either together, in groups, or separately), then examined using optical system400, either one chip or plate at a time or in groups thereof, to provide pooled dPCR data or a virtual chip comprising at least 100,000 samples, at least 1,000,000 samples, or at least 10,000,000 samples.

In certain embodiments, optical system400comprises a light source410and an associated excitation optic system412configured to illuminate at least some of samples contained in the reaction sites of article100or200. Excitation optical system412may include one or more lenses414and/or one or more filters416for conditioning light directed to the samples. Optical system400may further comprise a photodetector or optical sensor420and an associated emission optical system or imaging system422configured to receive radiation emitted by at least some of the reaction sites104,204and to direct this radiation onto optical sensor420, for example, by forming an image of article100or200or reaction sites104or204at or near optical sensor420. Radiation received by the emission optical system may comprise fluorescent emissions produced within reaction sites104,204in response to one or more excitation beams produced by light source410. Additionally or alternatively, radiation received by the emission optical system may be other types of luminescence produce within reaction sites104,204, for example, bioluminescence, chemiluminescence, phosphorescence, or the like. For example, when system300is configured to perform a qPCR and/or a dPCR procedure, assay, or process, reaction sites104,204may contain one or more fluorescent dyes or markers that provide a fluorescent signal that varies according to an amount of a target nucleotide sequence or molecule contained in various or all of the reaction sites of article100,200.

Emission optical system422may include a lens or lens system424and/or one or more filters426for conditioning light directed to the samples. In the illustrated embodiment shown inFIG. 23, excitation/emission optical systems412,422both comprise one or more common optical elements. For example, excitation/emission optical systems412,422may both comprise a beamsplitter430that reflects excitation light and transmits emission light from the samples to optical sensor420. In certain embodiments, excitation/emission optical systems412,422both comprise a field lens (not shown) disposed between beamsplitter430and article100,200, which may be used improve optical performance, for example, by providing even illumination and/or emission of light to and from the reaction sites104,204. In certain embodiments, for example where even illumination or emission (for example as provided by a telecentric optical system) is less critical (e.g., some dPCR applications), the common field lens may be omitted, as shown in the illustrated embodiment ofFIG. 23. Omission of the field lens may help to reduce the size and complexity of optical system400. Exemplary embodiments of excitation and emission optical systems are discussed in U.S. Pat. Nos. 6,818,437; 7,498,164; 7,387,891; 7,635,588; or 7,410,793, which publications are herein incorporated by reference in their entirety.

Lens424may comprise a single lens or simple lens, for example, a plano-convex lens, a plano-concave lens, a bi-convex lens, a bi-concave lens, a meniscus lens, or the like. Additionally or alternatively, lens424may comprise a compound lens and/or lens system, for example, an achromatic lens, a multi-element lens, a camera lens, a lenslet array, or the like. In certain embodiments, lens424may comprise one or more reflective optical elements or diffractive optical elements, such as a diffractive lens, a diffractive grating, a holographic optical element, or the like.

Optical sensor420may comprise one or more photodiodes, photomultiplier tubes (PMTs), or the like. Such photodetectors may be used, for example, where optical system400is configured to scan individual reaction sites104,204or subsets of reaction sites104,204. In other embodiments, optical sensor420may 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 sites104,204are simultaneously imaged or inspected. In order to provide a plurality of pixels per each reaction site, optical sensor420may comprise at least 4,000,000 pixel or more than 10,000,000 pixels. In embodiments where optical sensor420comprises a segmented detector and/or comprises one or more common optical elements, optical system400may be configured so that a plurality of the reaction sites (e.g., reaction sites104,204, or the like) are each imaged onto a single pixel or photo-element, or a group of pixels or photo-elements, of optical sensor420. For embodiments where optical sensor420comprises a segmented detector, imaging optical system422may be configured to form an image of individual reaction regions of article100,200, or the like. Additionally or alternatively, emission optical system422may be configured to from an image comprising a plurality of the reaction regions of article100,200, or the like

Any or all of the components or elements of optical system400may be mounted to one or more common frames (not shown). In certain embodiments, any or all of the components or elements of optical system400may be enclosed within a housing or case450, for example, to prevent or reduce the introduction of external or stray light, to protect optical system400from external dust and debris, and/or to shield from electrical or magnetic noise. In the illustrated embodiment ofFIG. 23, light source410and optical sensor420are contained within housing450. Alternatively, light source410and/or optical sensor420may be mounted on an external surface of housing450and/or may be partially located within housing450.

In certain embodiments, optical system400is configured to provide simultaneous imaging of a large number reaction sites104,204of article100,200, for example, a sufficiently large number of reaction sites104,204to provide a dPCR analysis of one or more target molecules, sequence, genes, biological micro-organisms, or the like. For example, system400may be configured to simultaneously image at least 20,000 reaction sites104,204, wherein a density of reaction sites104,204may be at least 100 reaction sites per square millimeter and/or each reaction site104,204may have a characteristic diameter that is less than or equal to 100 micrometers. For example, an example embodiment of the instant invention (herein referred to as Example A), a two-dimensional array of 29,760 reaction sites104,204, each having a characteristic diameter of less than 70 micrometers, were simultaneously imaged and processed to provide a dPCR analysis of a target nucleotide sequence or molecule. In the Example A embodiment, reaction sites104,204were arranged in a 160×186, hexagonally arrange pattern (like that shown inFIG. 6or13) over active area of an article100,200that was less than 14 millimeters by about 14 millimeters (about 13 millimeters by about 13 millimeters). The reaction volume of each reaction site may be less than one nanoliter. In other embodiments, the active area of article100,200comprises at least 15,000, at least 20,000 reaction sites104,204, at least 30,000 reaction sites104,204, at least 100,000 reaction sites104,204, or at least 1,000,000 reaction sites104,204.

Referring toFIG. 24, an external photodetector, camera, or portable electronic device600may be mounted onto optical system400for obtaining optical images of article100,200and/or associated optical signals or data from reaction sites104,204of article100,200. Device600may be used in place of (e.g.,FIG. 24), or in conjunction with (not shown), optical sensor420. Device600may be removably mounted to optical system400. For example, device600may be temporarily attached to optical system400to obtain images of article100,200and/or reaction sites104,204before, during, or after processing. Subsequent to obtaining one or more such images, device600may be detached for removed from optical system400, for example, to view the images, transfer the images, and/or process the images (e.g., to enhance the images and/or obtain or calculate information related to biochemical reactions in one or more of the reaction sites104,204).

Device600may comprise an photodetector or optical sensor (e.g., a CCD or CMOS sensor) configured to receive one or more images of article100,200and/or reaction sites104,204when mounted to optical system400. Device600may also include one or more lenses, filters, or other optical elements for providing images of article100,200and/or reaction sites104,204. Len424may be configured to provide images on the photodetector of device600. In certain embodiments, device600may include a lens or lens system (e.g., a camera lens including a system of lens elements). In such embodiments, the lens or lens system of device600may be configured to work with lens424of optical system400to provide images of a predetermined quality or characteristic. Alternatively, device600includes one or more lenses, or the like, for forming an image of article100,200and/or reaction sites104,204, while optical system400contains no lenses such as lens424in the optical path between article100,200and the photodetector of device600.

In certain embodiments, device600is designed or configured specifically for use with optical system400for obtaining images of article100,200and/or reaction sites104,204. Alternatively, device600may be configured for other uses besides obtaining images of article100,200and/or reaction sites104,204. For example, device600may include a camera that may be configured for personal, commercial, and/or scientific photography. In certain embodiments, device600is a commercial or consumer device that may include uses apart from photographic applications. For example, device600may be a consumer or professional camera or video recorder, camera phone, smartphone (e.g., a mobile phone based on a mobile operating system including, but not limited to, Google's Android OS (operating system), Apple's iOS, Nokia's Symbian, RIM's BlackBerry OS, Samsung's Bada, Microsoft's Windows Phone, Hewlett-Packard's webOS, or an embedded Linux distributions such as Maemo and MeeGo, or other mobile phone built on a mobile computing platform), Apple iPod Touch or similar device, personal digital assistant (PDA), portable media player, personal computer or electronic table with a photo input, or the like.

In certain embodiments, optical system400(e.g., the illustrated embodiments ofFIGS. 23 and 24) is configured to be relatively small, while still providing an imaging quality of all of the reaction sites104,204of article100,200or nearly all of the reaction sites104,204of article100,200(e.g., at 90 percent, at least 95 percent, or at least 99 percent of the reaction sites104,204of article100,200). For example, the size of optical system400may be configured to be sufficiently small so as to provide a compact system that is more readily portable that existing systems for conducting qPCR and/or dPCR tests, experiments, or analyses. Additionally or alternatively, the size of optical system400may be configured to be sufficiently small so as to maintain the cost of optical elements within a predetermined budget or target costs. For example, the focal length or effective focal length an imaging or camera lens, such as lens424shown inFIGS. 23 and 24by be selected to provide a relatively short working distance. In such embodiments, a relatively sort focal length or effective focal length may also provide the ability to use relatively small lenses, beamsplitter(s), excitation filter(s), emission filters, and/or other systems components, thereby reducing overall material and/or system cost of goods.

Importantly, it has been discovered that decreasing the size of optical system400(e.g., the illustrated embodiments ofFIGS. 23 and 24) and/or a decreasing a characteristic imaging system focal length or effective focal length, for example as compared to prior art PCR systems used for similar purposes, may be restricted by design or system constraints to maintain reaction site104,204image quality at or above a predetermined amount. For example, optical aberrations such as astigmatism, distortion and field curvature have been found to decrease image quality, particularly of images of peripheral reaction sites in a two-dimensional array of reaction sites104,204, by an amount that may preclude obtaining a desired accuracy in dPCR calculations used to determine the number count of one or more target nucleotide sequences or molecules. Surprisingly, it has been discovered that the use of certain design methodologies and/or optical figures of merit may result in systems and instruments that produce sufficient image quality of an array of reaction sites104,204to allow detection and/or quantification of target nucleotide sequences or molecules in a sample, for example, through the use of amplification processes such as qPCR and/or dPCR.

In certain embodiments, emission optical system or imaging system422is configured according to a design method to provide a miniaturized optical system for detecting and/or quantifying target nucleotide sequences or molecules contained in at least some of reaction sites104,204. The design of emission optical system422may be complicated by the existence of various components that interact in a non-linear way. The design of emission optical system422may also be complicated when limited to only a finite number of commercially available components, for example, where cost constraints preclude or reduce the use of custom optics. Miniaturized optical elements available from certain consumer market driven applications, such cell phone and smartphone cameras, may be utilized in the current design method to provide very low component prices. For example, the specification and characteristics of commercially available hardware and optic elements produced for these markets is finite and may not match desired or preferred values for use in the development of a miniaturized instruments to identify and/or quantify target nucleotide sequences or molecules.

In certain embodiments, the current design method may use a scanning microscope approach to provide systems and instruments according to embodiments of the present invention. Alternatively, design method may use a camera design approach to provide systems and instruments according to embodiments of the present invention. The camera design method may consist of an object, a photographic lens or objective, and a pixilated sensor such as a CCD or CMOS sensor. In the case of camera design methods, an entire field of regard (FOR) may be sensed at once over a time interval herein referred to as an “exposure time”.

The design method may include incorporation of an optical “object”. Without limiting the scope of embodiments of the present invention, the object may comprise a well array plate, which may consist of as many as 96 or 384 individual wells in an 80×120 mm gridded format. The wells may be spaced at a 9 millimeter or 4.5 millimeter pitch. Alternatively, smaller numbers of wells spaced at these pitches may also be used. In certain embodiments, the object may be a substrate with an array of through-holes that are 350 microns in diameter and spaced 500 microns apart. The through-holes may be gridded into 8×8 sub grids arranged in a 4×12 pattern. Additionally or alternatively, the object used in the current design method may comprise one or more of configurations of article100,200discussed above herein.

The design method may include the use of a commercially available photographic objective lens, since by design such lenses image a wide FOR with low aberrations. These lenses may be multi-element (at least 3 lens elements, up to as many as 11 or more lens elements). The limiting aperture (aperture stop) may be inside the lens assembly and controls a “speed” or f/# (light gathering power) of the lens, where f/# may be defined as the ratio of a lens or system focal length divided by the diameter of the limiting aperture. This lens design type is very mature and a large number of choices have been specifically designed various type of commercially available sensors (e.g., CCD and CMOS sensors). These sensors convert light energy into electrical energy and typically consist of millions of individual sensing elements, each element receiving energy imaged through the photographic objective lens from a small part of the FOR. The electrical signals from each of these sensing elements (called picture elements, or “pixels”) is assembled through software that allow a 2-dimensional representation of the object imaged by the objective.

Embodiments of the design method will now be describe in greater detail in which the optical object comprises article100,200described above as the Example A embodiment. A CMOS sensor may be used in embodiments of the current design method, due to the relatively low cost of these sensors compared to CCD sensors providing similar performance, for example, in terms of pixel resolution, frame rate, and/or dynamic range. It will be appreciated that in other embodiments of the design method, a CCD or other type of photodetector may be utilized.

In certain embodiments, hole or spot finding algorithms and quantifying algorithms may provide that an image of each reaction site104,204comprise at least 5 pixels across a hole diameter or about 20 pixels total. For the present embodiment, a value of about 8 pixels across a well or hole diameter is selected. For the Example A embodiment of article100,200, the holes are characterized by a diameter of 60 micrometers and are arranged in a hexagonal pattern in which adjacent reaction sites104,204are spaced at a pitch of 82 micrometers. Since 8 pixels span 60 microns and they are spaced at an 82 micron pitch, the hole spacing is 11 pixels, leaving approximately 3 dark pixels between adjacent reaction sites104,204. Along one axis of the CMOS detector, there are about 160 reaction site images on a side, which provide a CMOS pixel design parameter of about 1760 pixels on a side, or a total number of detector pixels of at least about 3.1 million for a square active area of article100,200. In certain embodiments, an even large number of sensor pixels may be selected to accommodate for positioning consideration and/or increased lens aberrations at the edges of the FOR.

The next consideration in the current design method may be pixel size. Currently available sensors have, for example, 1.1, 1.4, 2.2, 3, 5, 8, 11 micrometers and larger square pixel sizes. There are three factors which may influence choice of pixel size: dynamic range (DR), signal to noise ratio, and diffraction. DR may be determined by the maximum number of photoelectrons which can be stored by each pixel. The absolute number for 2.2 micrometer diameter pixels is about 3200 photoelectrons and about 20,000 photoelectrons for 5.5 micron pixels. As a general design parameter, larger pixels (greater number of photoelectrons) provide better DR, as well as better signal to noise ratio (SNR) characteristic, which is equal to the square root of the signal. A 20K DR has an SNR of 140; a 3.2K DR has an SNR of 57, which is 2.5× less.

Diffraction is a fundamental characteristic of electromagnetic waves in general and light waves in particular. It is caused by truncation of the wave's extent by a physical aperture such as a lens or system aperture stop. The effect in an optical system is that the image of a perfect point source infinitely far away (such as a star) is not itself infinitely small, but is a blob whose exact shape is determined by the size and shape of the aperture. The circular aperture of a most lenses create a bulls-eye type pattern with a bright, large central ring (the so called “Airy disk”) and infinite number of smaller and less bright surrounding circles. For the visible wavelengths, the size of the Airy disk is approximately equal to the f/# of the system objective lens in microns. Thus, diffraction considerations move an optical design toward larger lens diameters and system apertures. However, there is a practical limitation on the lens diameter or aperture, since lens aberrations (such as, but not limited to, astigmatism, distortion and field curvature) caused by the surface shapes of the lens elements increase as the f/# decreases or lens diameter increases. A practical value for f/# is about f/2.5. This means that the image of a point source will create a circular image of about 2.5 microns diameter. If a pixel is 2.5 microns square, the Airy Disk will just fit inside. If the pixel is 1.4 microns, then the Airy disk will occupy a 2×2 pixel grid. One evaluation criteria for calculating a lens or system resolution is called the Rayleigh criterion, which states that two point objects are just resolved by an optical system when the image the maximum of one Airy disk occurs at the minimum of the other. This would imply that two adjacent pixels in the current example should be separated by one Airy disk radius, or about 1.25 microns. Another resolution evaluation criteria is to separate adjacent signals by at least one Airy disk diameter. This latter criteria provides a limit of the size of the pixel to 2.5 microns square or larger. The overall size of a sensor with this pixel size and pixel count becomes 1760×2.5=4400 microns or 4.4 mm.

Two commercially available sensors meeting the above criteria for the current design method are the Aptina MT9P031 monochrome CMOS sensor (4.28 millimeters×5.7 millimeters and 1944×2592 pixels having a diameter of 2.2 micrometers by 2.2 micrometers) and the DX sized 16 Mp sensor of the Nikon D7000 camera (23.6×15.6 millimeters and 5 micrometer by 5 micrometer pixels). In the current example, the Aptina MT9P031 may be selected based on cost considerations.

Based on the smaller diameter of the Aptina CMOS sensor of 4.28 millimeters and an active area of 13 millimeters by 13 millimeters for article100,200in the Example A embodiment, the system magnification is 0.33 (4.28/13). This provides a reaction sites104,204image size of 60 micrometers×0.33=19.8 microns. Thus, each reaction site104,204image covers 19.8/2.5=7.92 pixels, which meets both criterion above of greater than 5 pixels and of about 8 pixels.

The current design method further comprises determining the accuracy of intensity calculations based on signals received from reaction sites104,204when emission optical system422is used to image article100,200onto photodetector420. In the current example, the intensity calculation is based on an image size of each reaction site that is about 5 sensor pixels in diameter. The effectiveness of an optical detection system such as emission optical system422is dependent on the signal to noise ratio (SNR). For fluorescence sensing instruments one rule of thumb is that the SNR be at least 3. The place where the SNR is measured is at the sensor transducer. This transducer transforms optical energy (photons) to electrical energy (electrons). These electrons are usually called “photoelectrons”—literally electrons produced by photons. SNR may comprise two parts: signal energy and noise energy. Signal energy (signal photoelectrons) results from photon flux (photon arrival rate, photons/second). Noise energy may be produced by several mechanisms, such as the signal itself, optical background energy (from sources other than the signal source), and transducer-related energy such as read noise or dark (thermal) noise. A more detailed discussion of read and dark noise is found in U.S. Pat. Nos. 7,233,393 and 7,045,756, which are herein incorporated by reference in their entirety.

When an accurate estimate of the noise energy is determined or provided, SNR calculations may be used to determine if a given signal energy is large enough to overcome the noise energy. Transducers (optical detectors) may be characterized for read noise and for thermal noise. The signal flux at the detector can be estimated from detector quantum efficiency (QE), optical throughput (the ratio of photons measured to the ratio of photons generated at the sample, for example), and the optical flux produced at the optical source (laser, LED, lamp, arc, etc. . . . ). Optical throughput may be composed of source spectral flux, optical filter spectral transmission characteristics, fluorescence conversion efficiency (proportion of incident flux at a wavelength 1 that gets converted to fluorescence emitted flux at a wavelength 2, angular extent of the emitted optical flux, optical gathering efficiency of camera lens, and other characteristics of windows, fluids and any other materials in the optical path. Optical background can be calculated by knowledge of the throughput characteristics of the optical components and the residual transmission of the optical filters at normally blocked wavelengths, (auto) fluorescence of materials in the optical path, and scattered light produced within the confines of the structure containing the optical system. Background may be difficult to model, but may be obtained from measurements in the system.

Based on the current design method, the imaging system comprises the Aptina CMOS sensor and a system magnification of 0.33. Using a general design criteria that the system be small, various commercially available objective lenses designed for surveillance systems and cell phones were evaluated in light of the design criteria that the imaging system provide a predetermined intensity calculation accuracy for all, or nearly all, reaction sites104,204. These lenses have the added advantage of being relatively inexpensive. It was determined from this evaluation that the Sunex lens model number DSL901 produced total aberrations that were low enough to meet the predetermined intensity calculation accuracy criteria. The DSL901 is an f/3 lens and has focal length of 15 millimeters, which results in a working distance of 60 millimeters, using the relationship,

As alternatives, other photographic objectives were identified that meet the predetermined intensity calculation accuracy criteria. These photographic objective have focal lengths that are less than or equal to the 15 millimeter focal length of the DSL901. In certain embodiments, the magnification may be slightly less than 0.33. This will result in a slightly larger system, due to an increase in working distance; however, the lower magnification advantageously reduces aberrations such as astigmatism, which are larger at peripherally located reaction sites104,204within the FOR.

In summary, it has been discovered that a system including an optical imaging system having a working distance that is less than or equal to 60 millimeters may be provided that is able to receive an article100,200comprising at least 20,000 separate reaction sites104,204, simultaneously illuminate the at least 20,000 separate reaction sites104,204, and calculate a number of target nucleotide particles contained in some or all of the at least 20,000 separate reaction sites. In certain embodiments, an accurate calculation of the number target nucleotide particles is obtained by providing a photodetector, such as photodetector420, comprising a predetermined number of pixels that is at least 20 times the number of separate reaction sites. In other embodiments, the predetermined number of pixels is at least 60 times the number of separate reaction sites or at least 100 times the number of separate reaction sites.

In certain embodiments, an imaging system according to embodiments of the design method discussed above may be characterized by one or more figures of merit (FOM). Some FOMs include:Sensor pixel square size>=camera lens f# (e.g., for systems with wavelengths in the visible part of the spectrum).Number of sensor pixels>=(n*n*l*w)/(p*p), where n=required pixels/feature in image, l=object field of regard length, w=object field of regard width, p=feature pitch.Choose magnification to be the smallest of the four ratios of sensor length/width to object length/width.Choose lens focal length and object to image distance pairs (determined by magnification) to provide best compromise between lens aberrations and overall optics package length.

In certain embodiments, system300further comprises a thermal control system500comprising, for example, a thermal cycler configured to perform a PCR procedure or protocol on some or all of the samples contained in article100,200. Systems400,500may 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 article100,200. In such embodiments, computer530,560may be used to control systems400,500and/or to collect or process data provided or obtained by either or both systems400,500. Alternatively, thermal control system500may be completely separate from optical system400and/or from computer530,560. In such embodiments, optical system400may be used to perform a dPCR or end-point PCR procedure on the samples contained in reaction sites104,204after thermal cycle has been performed on the samples using thermal control system500or some other thermal controller or thermal cycler. In certain embodiments, thermal control system500comprises 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 system500may be integrated with or into article100,200. For example, article100,200may include one or more heating elements distributed along one or both surfaces110,112. Additionally or alternatively, at least portions of substrate102may 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 substrate102.

According to various embodiments, emission optical system422and/or lens424may comprise a focal length that is less than or equal to 15 mm and/or a working distance that is less then or equal to 60 mm, where the working distance of the distance from articles100,200to lens424or a principal plane of lens424. Furthermore, in various embodiments, emission optical system422and/or lens424comprise an f-number that is less than or equal to 3. In some embodiments, emission optical system422and/or lens424may comprise both a focal length that is less than or equal to 15 mm and an f-number that is less than or equal to 3.

In certain embodiments, article100,200comprises 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.

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.

All of these applications are also incorporated herein in their entirety by reference.