Systems and Methods for Multiple Analyte Detection
Systems and methods for multiple analyte detection include a system for distribution of a biological sample that includes a substrate, wherein the substrate includes a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber. The system may further include a preloaded reagent contained in each sample chamber and configured for nucleic acid analysis of a biological sample that enters the substrate and a sealing instrument configured to be placed in contact with the substrate to seal each sample chamber so as to substantially prevent sample contained in each sample chamber from flowing out of each sample chamber. The substrate can be constructed of detection-compatible and assay-compatible materials.
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This application claims the benefits of priority to U.S. Provisional Application No. 60/674,750, filed on Apr. 26, 2005, which is incorporated by reference in its entirety herein.
This application makes cross-reference to U.S. Provisional Application No. 60/674,876, entitled “System for Population Security and Epidemiological Analysis,” concurrently filed with U.S. Provisional Application No. 60/674,750, and later filed 60/696,157 of the same title, both of which are also incorporated by reference herein in their entirety.
FIELDThe present teachings relate to systems and methods for multiple analyte detection.
BACKGROUNDBiochemical testing for research and diagnostic applications can require simultaneous assays including a large number of analytes in conjunction with one or a few samples and can include extended sample manipulation, multiple test substrates, multiple analytical instruments, and other steps. It is desirable to provide a method for analyzing one or a few biological samples using a single test device with a large number of analytes while requiring a small amount of sample. It is desirable to provide a device that is small in size while providing high-sensitivity detection for the analytes of interest with minimal sample manipulation. It is desirable to provide a method of loading the sample(s) into chambers on the substrate and individually sealing each chamber. It is further desirable to provide a mechanism for venting of the substrate during filling, while also avoiding and/or minimizing leakage of fluid (e.g., biological sample and/or reagents) from the test device.
SUMMARYIn various embodiments, the present teachings can provide a system for distribution of a biological sample including a substrate with a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber, wherein the substrate is constructed of detection-compatible and assay-compatible materials, and a sealing plate with sealing protrusions for sealing the sample introduction channels and the venting channels for each sample chamber.
In various embodiments, the present teachings can provide a method for distribution of a biological sample including providing an injection molded base, wherein the base includes a plurality of sample cavities, a sample introduction trench for each sample cavity, and a venting trench for each sample cavity, and wherein the base is constructed of detection-compatible and assay-compatible materials, providing a film to adhere to the base forming a substrate, wherein film includes a plurality of vents, and wherein the film forms a plurality of sample chambers from each sample cavity, a sample introduction channel for each sample introduction trench, and a venting channel for sample venting trench, providing the biological sample to the substrate, forcing the biological sample to the sample chambers through the sample introduction channels, providing a sealing plate comprising sealing protrusions for each sample chamber, heating the sealing plate, sealing the sample chambers by contacting the sealing protrusions with the sample introductory channels and the venting channels of each sample chamber.
In various embodiments, the present teachings may provide a system for distribution of a biological sample that includes a substrate, wherein the substrate includes a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber. The system may further include a preloaded reagent contained in each sample chamber and configured for nucleic acid analysis of a biological sample that enters the substrate, and a sealing instrument configured to be placed in contact with the substrate to seal each sample chamber so as to substantially prevent sample contained in each sample chamber from flowing out of each sample chamber. The substrate can be constructed of detection-compatible and assay-compatible materials.
In still further various embodiments, a method for distribution of a biological sample may include providing a base, wherein the base includes a plurality of sample cavities containing a preloaded reagent therein, a sample introduction trench for each sample cavity, and a venting trench for each sample cavity, and wherein the base is constructed of detection-compatible and assay-compatible materials. The method also may include providing a film to adhere to the base to form a substrate, wherein the film and base form a plurality of sample chambers from each sample cavity, a sample introduction channel from each sample introduction trench, and a venting channel from each sample venting trench. The method may further include supplying the biological sample to the substrate, filling the sample chambers with the biological sample via the sample introduction channels, passing gas out of the substrate via at least one venting mechanism in the substrate, and sealing the sample chambers to substantially prevent sample in the sample chambers from flowing out of the sample chambers.
According to yet other embodiments, the present teachings may provide a system for distribution of a biological sample that includes means for distributing the sample to a plurality of sample chambers containing a preloaded reagent, means for venting each of the sample chambers, and means for sealing each of the sample chambers.
In various embodiments, the present teachings also can provide a device for testing a biological sample that includes a substrate defining a plurality of distribution portions configured to distribute biological sample throughout the substrate, the plurality of distribution portions comprising a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber. The device also may include a substance disposed in at least one of the distribution portions, the substance being configured to seal each sample chamber so as to substantially prevent sample disposed in each sample chamber from flowing out of each sample chamber during testing of the biological sample.
Additional embodiments are set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the various embodiments described herein.
BRIEF DESCRIPTION OF THE DRAWINGSVarious embodiments of the present teachings are exemplified in the accompanying drawings. The teachings are not limited to the embodiments depicted, and include equivalent structures and methods as set forth in the following description and known to those of ordinary skill in the art. In the drawings:
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide a further explanation of the various embodiments of the present teachings.
DESCRIPTION OF VARIOUS EMBODIMENTSIn this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.
The section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter described. All documents cited in this application, including, but not limited to patents, patent applications, articles, books, and treatises, are expressly incorporated by reference in their entirety for any purpose.
The term “sample chamber” as used herein refers to any structure that provides containment to a sample. The chamber can have any shape including circular, rectangular, cylindrical, etc. Multi-chamber arrays can include 12, 16, 24, 36, 48, 96, 192, 384, 3072, 6144, or more sample chambers. The term “channel” as used herein refers to any structure that is smaller than a chamber. A channel can have any shape. It can be straight or curved, as necessary, with cross-sections that are shallow, deep, square, rectangular, concave, or V-shaped, or any other appropriate configuration. A “distribution portion” of a substrate may refer to any portion of the substrate configured to contain, flow, receive, or otherwise hold sample and/or gas in the substrate. Examples of distribution portions include main fluid supply channels, venting channels, venting chambers, sample chambers, sample introduction channels, overfill chambers, and virtually any other channels and/or chambers of the substrate.
The term “biological sample” as used herein refers to any biological or chemical substance, typically in an aqueous solution with luminescent dye that can produce emission light in relation to nucleic acid present in the solution. The biological sample can include one or more nucleic acid sequence(s) to be incorporated as a reactant in a polymerase chain reaction (PCR) and other reactions such as ligase chain reactions, antibody binding reactions, oligonucleotide ligations assay, hybridization assays, and invader assays (e.g., for an isotherm reaction). The biological sample can include one or more nucleic acid sequences to be identified for DNA sequencing.
The term “luminescent dye” as used herein may refer to fluorescent or phosphorescent dyes that can be excited by excitation light or chemiluminscent dyes that can be excited chemically. As used herein, “luminescent dye” may also include the use of energy transfer pairs (intramolecular or intermolecular). For example, excitation of one energy transfer pair member and emission of the other member may expand the range of emission wavelengths for multiplexing. Quenchers selected should be suitable for both members of the energy transfer pair. Luminescent dyes can be used to provide different colors depending on the dyes used. Several dyes will be apparent to one skilled in the art of dye chemistry, including, for example, intercalating dyes. One or more colors can be collected for each dye to provide identification of the dye or dyes detected. The dye can be a dye-labeled fragment of nucleotides. The dye can be a marker triggered by a fragment of nucleotides. The dye can provide identification of a nucleic acid sequence in the biological sample by association, for example, bonding to or reacting with a detectable marker, for example, a respective dye and quencher pair. The respective identifiable component can be positively identified by the luminescence of the dye. The dye can be normally quenched, and then can become unquenched in the presence of a particular nucleic acid sequence in the biological sample. The fluorescent dyes can be selected to exhibit respective and, for example, different, excitation and emission wavelength ranges. The luminescent dye can be measured to quantitate the amount of nucleic acid sequences in the biological sample. The luminescent dye can be detected in real-time to provide information about the identifiable nucleic acid sequences throughout the reaction. Examples of fluorescent dyes with desirable excitation and emission wavelengths can include 5-FAM™, TET™, and VIC™. The term “luminescence” as used herein refers to low-temperature emission of light including fluorescence, phosphorescence, electroluminescence, and chemiluminescence.
In various embodiments, sample chambers can be dimensioned to hold from 0.0001 μL to 10 μL of sample per chamber, or between 0.001 μL and 2 μL. Conveniently, the volume of each detection chamber is between 0.001 μL and 1 μL. For example, a chamber having a volume of 0.2 μL may have dimensions of 1 mm×1 mm×0.2 mm, where the last dimension is the chamber's depth. As a further example, a chamber may have a substantially cylindrical shape having a diameter of about 1.96 mm and a depth of about 0.5 mm and a volume of about 1.35 μL.
In various embodiments, the sample introduction channels can be dimensioned to facilitate rapid delivery of sample to the sample chambers, while occupying as little volume as possible. For example, cross-sectional dimensions for the channels can range from 0.5 μm to 250 μm for both the width and depth. In some embodiments, the channel path lengths to the sample chambers can be minimized to reduce the total channel volume. For example, the network can be substantially planar, i.e., the sample introduction channels and sample chambers in the substrate intersect a common plane.
“Venting mechanisms” as used herein may refer to any mechanism configured and arranged to permit gas to escape therethrough and leave the substrate. Venting mechanisms may be the last structure the gas passes through prior to leaving the substrate or be a structure that passes the escaping gas therethrough to another structure that is the last structure before the gas escapes the substrate. According to various embodiments, venting mechanisms may permit the passage of gas while substantially preventing the passage of liquid. Also, venting mechanisms may be used in combination with one another to permit gas to escape the substrate. Examples of venting mechanisms include, but are not limited to, gas-permeable or porous membranes, vent through holes either in a film layer, a base or both, porous fibers, and hydrophobic liquid stops.
In various embodiments, there are a variety of means for distributing the biological sample to the plurality of sample chambers. All of these include applying a force. The force can be a pulling force or a pushing force, depending on whether it provides a negative (pulling) or positive (pushing) force relative to the direction of fluid flow. Examples of forces and how the force is enacted upon the biological sample include spinning the substrate to provide centrifugal force to push the liquid, sizing the sample introductory channels to provide capillary force to pull the liquid, aspirating the sample through the vents to pull the liquid, evacuating the sample chamber to pull the liquid, and/or providing pressure, such as by pumping, compressing, plunging, etc. to push the liquid. In each of these configurations, the venting channels and vents can be used to accommodate the displaced venting gas, whether air or other gas such as nitrogen, that is pushed out by the sample or the venting channels and vents can be used to evacuate the gas in the sample chambers to create a vacuum for the sample or aspirate sample itself.
In various embodiments, the substrate that defines the sample-distribution network can be constructed from any solid material that is suitable for conducting analyte detection. Materials that can be used will include various plastic polymers and copolymers, such as polypropylenes, polystyrenes, polyimides, COP, COC, and polycarbonates. Inorganic materials such as glass and silicon are also useful. Silicon is especially advantageous in view of its high thermal conductivity, which facilitates rapid heating and cooling of the substrate if necessary. The substrate can be formed from a single material or from a plurality of materials. Examples of this are described at U.S. Pat. No. 6,126,899.
In various embodiments, the sample-distribution network including cavities and trenches, for example, formed in a substrate base portion, can be formed by any suitable method known in the art. For plastic materials, injection molding can be suitable to form sample cavities and connecting channels having a desired pattern. For silicon, standard etching, RIE, DRIE, and wet-etching techniques from the semiconductor industry can be used as known in the art of photo-lithography.
In various embodiments, the substrate can be prepared from two or more laminated layers. The term “detection-compatible material” as used herein refers to the optical detection with a substrate that includes one or more layers which provide an optical transparency for each sample chamber. By way of example, the optical transparency may permit detection of a luminescent dye. Silica-based glasses, quartz, polycarbonate, or an optically transparent plastic layer may be used, for example. Selection of the particular detection-compatible material depends in part on the optical properties of the material and the detection mechanism. For example, in luminescent dye-based assays, the material should have low fluorescence emission at the wavelength(s) being measured. The detection-compatible material should also exhibit minimal light absorption for the signal wavelengths of interest. However, in some cases, for example, to minimize cross-talk, it may be desirable to provide a substrate material that has relatively high absorption. Examples of such materials are described at U.S. Pub. No. 2005/0226779A1.
In various embodiments, other layers in the substrate can be formed using the same or different materials. The term “assay-compatible material” as used herein refers to the interaction of assay reagents and assay conditions (heat, pressure, pH, etc.) with the substrate material (hydrophobic, hydrophilic, inert, etc.). For example, the layer or layers, such as a film defining the sample chambers can be formed predominantly from a material that has high heat conductivity, such as silicon, a heat-conducting metal, or carbon fill in plastic. The silicon surfaces that contact the sample can be coated with an oxidation layer or other suitable coating, to render the surface more inert and make it an assay-compatible material. Similarly, where a heat-conducting metal is used in the substrate, the metal can be coated with an assay-compatible material, such as a plastic polymer, to prevent corrosion of the metal and to separate the metal surface from contact with the sample. The suitability of a particular surface should be verified for the selected assay as known by the conditions and reagents used in the assay.
In various embodiments, for optical detection, the opacity or transparency of the substrate material defining the sample chambers, for example, the base, can have an effect on the permissible detector geometries used for signal detection. For the following discussion, references to the “upper wall” of a detection chamber may refer to the chamber surface or wall through which the optical signal is detected, and references to the “lower wall” of a chamber may refer to the chamber surface or wall that is opposite the upper wall. For example, the upper wall can be formed by the base or the film, and the lower wall by the other, respectively.
In various embodiments, in fluorescence detection the substrate material defining the lower wall of the sample chambers can be optically opaque, and the sample chambers can be illuminated and optically scanned through the same surface (i.e., the top surfaces of the chambers which are optically transparent). Thus, for fluorescence detection, the opaque lower wall material can exhibit low reflectance properties so that reflection of the illuminating light back toward the detector can be minimized. Opacity also may prevent collection of background signals from a thermal cycler block or other instrumentation used to test the substrates. In other cases, the substrate lower wall of the sample chambers may be reflective so that more fluorescent signal is collected.
In various embodiments, in fluorescence detection the substrate material defining the upper wall of the sample chambers can be optically clear, the chambers can be illuminated with excitation light through the sides of the chambers (in the plane defined collectively by the sample chambers in the substrate), or more typically, diagonally from above (e.g., at a 45 degree angle), and emitted light is collected from above the chambers (i.e., through the upper walls, in a direction perpendicular to the plane defined by the detection chambers). The upper wall material can exhibit low dispersion of the illuminating light in order to limit Rayleigh scattering.
In various embodiments, in fluorescence detection the substrate material defining the entirety of the substrate can be optically clear, or at least the upper and lower walls of the chambers can be optically clear, the chambers can be illuminated through either wall (upper or lower), and the emitted or transmitted light is measured through either wall as appropriate. Illumination of the chambers from other directions can also be possible as already discussed above.
In various embodiments, in chemiluminescence detection, where light of a distinctive wavelength is typically generated without illumination of the sample by an outside light source, the absorptive and reflective properties of the substrate can be less important, provided that the substrate provides at least one optically transparent window for detecting the signal.
In various embodiments, the substrate can be designed to provide a vacuum-tight environment within the sample-distribution network for sample loading, and also to provide sample chambers having carefully defined reaction volumes. It is desirable to ensure that the network and associate sample chambers do not leak. Accordingly, lamination of substrate layers to one another can be accomplished so as to ensure that all chambers and channels are well sealed.
In various embodiments, the substrate layers can be sealably bonded in a number of ways. A suitable bonding substance, such as a glue or epoxy-type resin, can be applied to one or both opposing surfaces that will be bonded together. The bonding substance may be applied to the entirety of either surface, so that the bonding substance (after curing) can come into contact with the sample chambers and the distribution network. In this case, the bonding substance is selected to be compatible with the sample and detection reagents used in the assay. Alternatively, the bonding substance can be applied around the distribution network and detection chambers so that contact with the sample can be minimal or avoided entirely. The bonding substance may also be provided as part of an adhesive-backed tape or membrane, which is then brought into contact with the opposing surface. In yet another approach, the sealable bonding is accomplished using an adhesive gasket layer, which is placed between the two substrate layers. In any of these approaches, bonding may be accomplished by any suitable method, including pressure-sealing, ultrasonic welding, and heat curing, for example.
In various embodiments, a pressure-sensitive adhesive (PSA) can be used in constructing the substrate. PSA films which can be applied to a surface and adhered to that surface are obtained by applying pressure to the film. Normally, pressure is applied throughout the whole film, so that the whole film can adhere to the surface. PSA films can have threshold pressure in order to activate the adhesion. The threshold pressure can be very low. By applying pressure to some selected regions, the bonding can be limited to those regions only, thus allowing obtaining a bonding pattern. This way channels and chambers can be defined. The elastic properties of the film can then be used to pressure-drive a fluid through the unbonded regions, since the film would deform under the liquid pressure, thus opening up a channel. Eventually, the channel could be sealed by applying pressure on the portion of the film defining the channel. PSA films can be either hydrophobic or hydrophilic. PSA films can have hydrophobic and hydrophilic areas on the same film to provide areas of different wetting characteristics, properly patterned, to provide, for example fluid flow in sample introduction channels and gas venting in venting channels. Thus, by providing differing regions of hydrophobicity and hydrophilicity of the films, control over fluid flow through a device may be achieved. In various embodiments, PSA films that are hydrophilic can have the hydrophilic properties deteriorate in a matter of days. The lack of stability (hydrophilic film turning into hydrophobic) can provide controllable, irreversible or reversible, changes (upon temperature change, heat addition, UV exposure, or just time delay after curing) in the wetting nature of the film. In various embodiments, PSA films can have different porosities and permeabilities to a gas. A highly permeable PSA film can be more advantageous than a low-permeability one for instance to vent the sample chambers. Further, a PSA film whose permeability/porosity can be modified in a reversible fashion with temperature change, and/or in an irreversible fashion by heat addition or UV exposure can be used for sample distribution and then sealed for sample processing. In various embodiments, PSA films can be hydrophilic, provide solvent resistance, maintain the adhesion characteristics at a high temperature (95-100 degree Celsius), and can be optically clear with low auto-fluorescence. In various embodiments, PSA films can be thermally expandable to swell at desired locations and close off channels.
In various embodiments, the substrate of the present teaching can be adapted to allow rapid heating and cooling of the sample chambers to facilitate reaction of the sample with the analyte-detection reagents, including luminescent dyes. In one embodiment, the substrate can be heated or cooled using an external temperature-controller. The temperature-controller is adapted to heat/cool one or more surfaces of the substrate, or can be adapted to selectively heat the sample chambers themselves. To facilitate heating or cooling with this embodiment, the substrate can be formed of a material that has high thermal conductivity, such as copper, aluminum, or silicon. Alternatively, bases can be formed from a material having moderate or low thermal conductivity, while the film can be formed form a conductive material such that the temperature of the sample chambers can be conveniently controlled by heating or cooling the substrate through the film, regardless of the thermal conductivity of the base. For example, the film can be formed of an adhesive copper-backed tape.
In various embodiments, the sample chambers of the substrate can be pre-loaded with detection reagents that are specific for the selected analytes of interest. By way of example, such reagents may be deposited in the sample chambers in liquid form and dried (e.g., lyophilized). For example, reagents for nucleic acid analysis of a biological sample may be preloaded in the substrate, for example, in the sample chambers. In such embodiments, the substrate may then be loaded with sample when biological testing is desired to be performed by supplying sample to the substrate containing the pre-loaded reagent(s). The detection reagents can be designed to produce an optically detectable signal via any of the optical methods known in the field of detection. It will be appreciated that although the reagents in each detection chamber can contain substances specific for the analyte(s) to be detected in the particular chamber, other reagents for production of the optical signal for detection can be added to the sample prior to loading, or may be placed at locations elsewhere in the network for mixing with the sample. Examples of such reactions are described in U.S. Pub. No. 2005/0260640A1. Whether particular assay components are included in the detection chambers or elsewhere will depend on the nature of the particular assay, and on whether a given component is stable to drying. Pre-loaded reagents added in the detection chambers during manufacture of the substrate can enhance assay uniformity and minimize the assay steps conducted by the end-user.
In various embodiments, the analyte to be detected may be any substance whose presence, absence, or amount is desirable to be determined. The detection means can include any reagent or combination of reagents suitable to detect or measure the analyte(s) of interest. It will be appreciated that more than one analyte can be tested for in a single detection chamber, if desired.
In one embodiment, the analytes are selected-sequence polynucleotides, such as DNA or cDNA, RNA, and the analyte-specific reagents include sequence-selective reagents for detecting the polynucleotides. The sequence-selective reagents include at least one binding polymer that is effective to selectively bind to a target polynucleotide having a defined sequence. The binding polymer can be a conventional polynucleotide, such as DNA or RNA, or any suitable analog thereof, which has the requisite sequence selectivity. Other examples of binding polymers known generally as peptide nucleic acids may also be used. The binding polymers can be designed for sequence specific binding to a single-stranded target molecule through Watson-Crick base pairing, or sequence-specific binding to a double-stranded target polynucleotide through Hoogstein binding sites in the major groove of duplex nucleic acid. A variety of other suitable polynucleotide analogs are also known in the art of nucleic acid amplification. The binding polymers for detecting polynucleotides are typically 10-30 nucleotides in length, with the exact length depending on the requirements of the assay, although longer or shorter lengths are also contemplated.
The present teachings can find utility in a wide variety of amplification methods, such as PCR, Reverse Transcription PCR (RT-PCR), Ligation Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), self-sustained sequence replication (3SR), strand displacement activation (SDA), Q (3replicase) system, isothermal amplification methods, and other known amplification method or combinations thereof. Additionally, the present teachings can find utility for use in a wide variety of analytical techniques, such as ELISA; DNA and RNA hybridizations; antibody titer determinations; gene expression; recombinant DNA techniques; hormone and receptor binding analysis; and other known analytical techniques.
In one embodiment, the analyte-specific reagents include an oligonucleotide primer pair suitable for amplifying, by polymerase chain reaction, a target polynucleotide region of the selected analyte that is flanked by 3′-sequences complementary to the primer pair. In practicing this embodiment, the primer pair is reacted with the target polynucleotide under hybridization conditions which favor annealing of the primers to complementary regions of opposite strands in the target. The reaction mixture is then thermal cycled through several, and typically about 20-40, rounds of primer extension, denaturation, and primer/target sequence annealing, according to well-known polymerase chain reaction (PCR) methods. Typically, both primers for each primer pair are pre-loaded in each of the respective sample chambers. The primer also may be loaded along with the standard nucleotide triphosphates, or analogs thereof, for primer extension (e.g., ATP, CTP, GTP, and TTP), and any other appropriate reagents, such as MgCl2 or MnCl2. A thermally stable DNA polymerase, such as Taq, Vent, or the like, may also be pre-loaded in the chambers, or may be mixed with the sample prior to sample loading. Other reagents may be included in the detection chambers or elsewhere as appropriate. Alternatively, the detection chambers may be loaded with one primer from each primer pair, and the other primer (e.g., a primer common to all of sample chambers) can be provided in the sample or elsewhere. If the target polynucleotides are single-stranded, such as single-stranded DNA, cDNA, or RNA, the sample is preferably pre-treated with a DNA- or RNA-polymerase prior to sample loading, to form double-stranded polynucleotides for subsequent amplification. Also, a reverse transcription enzyme may be used to pretreat RNA to cDNA. This pre-treatment can be provided in the cartridge.
In various embodiments, the presence and/or amount of target polynucleotide in a sample chamber, as indicated by successful amplification, is detected by any suitable means. For example, amplified sequences can be detected in double-stranded form by including an intercalating or crosslinking dye, such as ethidium bromide, acridine orange, or an oxazole derivative, such as, Cyber Green, for example, which exhibits a fluorescence increase or decrease upon binding to double-stranded nucleic acids. The level of amplification can also be measured by fluorescence detection using a fluorescently labeled oligonucleotide. In this embodiment, the detection reagents include a sequence-selective primer pair as in the more general PCR method above, and in addition, a sequence-selective oligonucleotide (FQ-oligo) containing a fluorescer-quencher pair. The primers in the primer pair are complementary to 3′ regions in opposing strands of the target analyte segment which flank the region which is to be amplified. The FQ-oligo is selected to be capable of hybridizing selectively to the analyte segment in a region downstream of one of the primers and is located within the region to be amplified. The fluorescer-quencher pair can include a fluorescent dye and a quencher which are spaced from each other on the oligonucleotide so that the quencher is able to significantly quench light emitted by the fluorescer S at a selected wavelength, while the quencher and fluorescer are both bound to the oligonucleotide. The FQ-oligo can include a 3′-phosphate or other blocking group to prevent terminal extension of the 3′ end of the oligo. The fluorescer and quencher dyes may be selected from any dye combination having the proper overlap of emission (for the fluorescer) and absorptive (for the quencher) wavelengths while also permitting enzymatic cleavage of the FQ-oligo by the polymerase when the oligo is hybridized to the target. Suitable dyes, such as rhodamine and fluorscein derivatives, and methods of attaching them, are well known in the art of nucleic acid amplification.
In another embodiment, the detection reagents include first and second oligonucleotides effective to bind selectively to adjacent, contiguous regions of a target sequence in the selected analyte, and which can be ligated covalently by a ligase enzyme or by chemical means as known in the art of oligonucleotide ligation assay, (OLA). In this approach, the two oligonucleotides (oligos) can be reacted with the target polynucleotide under conditions effective to ensure specific hybridization of the oligonucleotides to their target sequences. When the oligonucleotides have base-paired with their target sequences, such that confronting end subunits in the oligos are base-paired with immediately contiguous bases in the target, the two oligos can be joined by ligation, e.g., by treatment with ligase. After the ligation step, the detection wells are heated to dissociate unligated probes, and the presence of a ligated, target-bound probe is detected by reaction with an intercalating dye or by other means. The oligos for OLA may also be designed so as to bring together a fluorescer-quencher pair, as discussed above, leading to a decrease in a fluorescence signal when the analyte sequence is present. In the above OLA ligation method, the concentration of a target region from an analyte polynucleotide can be increased, if necessary, by amplification with repeated hybridization and ligation steps. Simple additive amplification can be achieved using the analyte polynucleotide as a target and repeating denaturation, annealing, and ligation steps until a desired concentration of the ligated product is achieved.
In another embodiment, the ligated product formed by hybridization and ligation can be amplified by ligase chain reaction (LCR). In this approach, two sets of sequence-specific oligos are employed for each target region of a double-stranded nucleic acid. One probe set includes first and second oligonucleotides designed for sequence-specific binding to adjacent, contiguous regions of a target sequence in a first strand in the target. The second pair of oligonucleotides is effective to bind (hybridize) to adjacent, contiguous regions of the target sequence on the opposite strand in the target. With continued cycles of denaturation, reannealing and ligation in the presence of the two complementary oligo sets, the target sequence is amplified exponentially, allowing small amounts of target to be detected and/or amplified.
In various embodiments, it will be appreciated that since the selected analytes in the sample can be tested for under substantially uniform temperature and pressure conditions within the substrate, the detection reagents in the various sample chambers should have substantially the same reaction kinetics. This can be accomplished using oligonucleotides and primers having similar or identical melting curves, which can be determined by empirical or experimental methods as are known in the art. In another embodiment, the analyte is an antigen, and the analyte-specific reagents in each detection chamber include an antibody specific for a selected analyte-antigen. Detection may be by fluorescence detection, agglutination, or other homogeneous assay format. As used herein, “antibody” is intended to refer to a monoclonal or polyclonal antibody, an Fc portion of an antibody, or any other kind of binding partner having an equivalent function. For fluorescence detection, the antibody may be labeled with a fluorescent compound such that specific binding of the antibody to the analyte is effective to produce a detectable increase or decrease in the compound's fluorescence, to produce a detectable signal (non-competitive format). In an alternative embodiment (competitive format), the detection means includes (i) an unlabeled, analyte-specific antibody, and (ii) a fluorescer-labeled ligand which is effective to compete with the analyte for specifically binding to the antibody. Binding of the ligand to the antibody is effective to increase or decrease the fluorescence signal of the attached fluorephore. Accordingly, the measured signal can depend on the amount of ligand that is displaced by analyte from the sample. In a related embodiment, when the analyte is an antibody, the analyte-specific detection reagents include an antigen for reacting with a selected analyte antibody which may be present in the sample. The reagents can be adapted for a competitive or non-competitive type format, analogous to the formats discussed above. Alternatively, the analyte-specific reagents can include a mono- or polyvalent antigen having one or more copies of an epitope which is specifically bound by the antibody-analyte, to promote an agglutination reaction which provides the detection signal.
In various embodiments, the selected analytes can be enzymes, and the detection reagents include enzyme substrate molecules which are designed to react with specific analyte enzymes in the sample, based on the substrate specificities of the enzymes. Accordingly, detection chambers in the device may each contain a different substrate or substrate combination, for which the analyte enzyme(s) may be specific. This embodiment is useful for detecting or measuring one or more enzymes which may be present in the sample, or for probing the substrate specificity of a selected enzyme. Examples of detection reagents include chromogenic substrates such as NAD/NADH, FAD/FADH, and various other reducing dyes, for example, useful for assaying hydrogenases, oxidases, and enzymes that generate products which can be assayed by hydrogenases and oxidases. For esterase or hydrolase (e.g., glycosidase) detection, chromogenic moieties such as nitrophenol may be used, for example.
In various embodiments, the analytes are drug candidates, and the detection reagents include a suitable drug target or an equivalent thereof, to test for binding of the drug candidate to the target. It will be appreciated that this concept can be generalized to encompass screening for substances that interact with or bind to one or more selected target substances. For example, the assay device can be used to test for agonists or antagonists of a selected receptor protein, such as the acetylcholine receptor. In a further embodiment, the assay device can be used to screen for substrates, activators, or inhibitors of one or more selected enzymes. The assay may also be adapted to measure dose-response curves for analytes binding to selected targets. The assays also may be immunoassays.
Reference will now be made to various exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.
In various embodiments, as illustrated in
Examples of suitable membranes include gas-permeable membranes and/or porous membranes. For example, suitable porous membranes may include Gortex® and other similar materials known in the art of micro-porous membranes. Suitable gas permeable membranes may include, for example, PDMS membranes. The membrane materials also can be liquid impermeable to prevent a sponging effect of the liquid that can reduce the volume in the sample chamber. Some examples of suitable porous membrane materials are described in U.S. Pat. No. 5,589,350 and some examples of suitable gas-permeable membrane materials are described in U.S. Pat. Pub. No. 2005/0164373 Titled “Diffusion-Aided Loading System for Microfluidic Devices,” the entire disclosures of both of which are incorporated by reference herein.
In various embodiments, as illustrated in
The number and positioning (spacing) of the disks 2930 may be selected such that the roller 2920 may pass over the substrate 2910 once to isolate all of the sample chambers 2980, sealing both inlet and outlet channels 2975 and 2976 simultaneously. The number and positioning of the disks 2930 may thus depend on a variety of factors, including but not limited to, for example, the number of sample chambers, the arrangement of the sample chambers, and the arrangement of the inlet channels and outlet channels. According to various exemplary embodiments, the number of disks may be reduced by staking the shared main fluid channels 2970 and 2971 between two lines of chambers 2980 in the embodiment of
With reference now to
It should be understood that the protrusions provided on the rollers in the embodiments of
In the exemplary embodiments of
In addition, it should be understood that in the embodiments of
It also is envisioned that, in the case of an integrated instrument where the substrate passes through various stations, the sealing rollers or plates of the may be placed in the transfer path of the substrate. For example, the sealing rollers or plates may be placed at a location after the substrate has been filled (e.g., at a filling station) and before the next station, such as, for example, a thermocycling block or instrument delivery port.
The exemplary sealing roller embodiments of
In various embodiments, sample preparation instrument 150 can take raw biological sample from syringe 170 and prepare the sample for introduction into substrate 10. Preparation of sample can include extraction of nucleic acids and pre-treatment for detection as described above. The instrument 150 docks with several cartridges 160 that provide preparation.
Various other exemplary embodiments may provide mechanisms for sealing, venting, controlling pressure, sample preparation, mixing, and/or other features useful in multiple analyte detection in a substrate in accordance with teachings of the disclosure and are described in further detail below.
As discussed above, once the sample chambers of a substrate have been filled, it may be desirable to seal filled chambers from flow communication with each other and the various distribution channels. Such sealing may be desirable, for example, before various operations, such as, for example, PCR, may be performed, and to prevent cross-contamination between wells. It also may be desirable to provide a mechanism for sealing that is relatively easily performed by a user of the biological testing device. Further, it may be desirable to provide a mechanism for sealing the substrate that does not require the use of sensors, heaters, and/or other components that may be relatively difficult and costly to implement.
Referring to
The substrate 810 may include a base 830, which may have a configuration like the bases described above. The base 830 may be covered with an adhesive-backed film 820. In various embodiments, the adhesive-backed film 820 may be, for example, a PSA film. With reference to the cross-section of the substrate 810 shown in
By way of example, in the case of a consumable product, the adhesive-backed film 820 may be loosely applied during manufacturing and a user of the substrate 810 may apply the pressure required for sealing after loading the substrate with sample. Various mechanisms may be used to apply pressure, for example, a substantially uniform pressure, over substantially the entire adhesive-backed film. For example, the pressure could be applied by the user's hand pressing on the film 820. Other techniques for applying the pressure include using a motorized stepping plate 825 (as schematically depicted in
According to various exemplary embodiments, in addition or as an alternative to providing pressure to force the adhesive 822 to fill the channels 875, 876, and 870, heat also may be used to facilitate the filling of the channels with the adhesive 822. However, it is envisioned that the use of heat is not necessary. The appropriate thickness of the adhesive layer 822 may be selected so as to perform adequate channel closing without injecting too much adhesive into the chambers 880. By way of example, the thickness of the adhesive layer 822 may be such that the entire depths of the channels are filled with adhesive, leaving no air pockets within the channels. Also, if sample is displaced during sealing, it may be desirable to provide an adhesive thickness that results in a substantially consistent amount of fluid being displaced, while also minimizing the amount of wasted sample due to displacement.
According to various exemplary embodiments, the protruding portions 850 may be made of the same material as the base 830 and may be formed via injection molding of the base 830. Other materials and techniques for forming the protruding portions 850 also may be used and would be understood to those having ordinary skill in the art. By way of example, and not limitation, the protruding portions 850 may have a height equal to about one-half the depth of the channels 875 and 876, and may span across the width of the channels (e.g., in the left to right direction shown in
In various exemplary embodiments, for example, when using a motorized roller 826 such as that depicted in
In various embodiments, a stationary rotating cam may be used, for example, instead of the motorized plate or roller of
The biological testing device may further include a sealing carrier 1120, having a plate-like structure, that includes a plurality of staking blades 1122 on a side of the carrier 1120 facing the substrate 1110. Prior to filling the substrate 1110, the carrier 1120 may be separated from the substrate 1110 via a temporary mechanical mechanism, such as, for example, a film hinge, or may be separate form the substrate 1110 with no connection. The carrier 1120 may be brought into contact with the substrate 1110 when sealing is desired. In various embodiments, the carrier 1120 and substrate 1110 may be provided with mating pins and holes or other fastening mechanisms that are configured for insertion in one direction but prevent separation in the opposite direction. The carrier 1120 may be separated such that the blades 1122 are above and at a distance from the upper surface (as shown in
Once the sample chambers 1180 of the substrate 1110 have been filled, as shown in
The blades 1122 may pierce, deform, or otherwise alter the structure of the substrate 1110 at the locations so as to prevent flow communication between the chambers 1180 and between the channels 1190 and the chambers 1180. By way of example, the blades 1122 may pierce through film layer 1135 and enter the channels 1190 so as to block flow between the channels 1190 and corresponding chambers 1180. In addition to blocking flow communication between the channels 1190 and the chambers 1180, the carrier 1120 may be configured to provide a seal (e.g., prevent flow communication) between the substrate 1110 and the exterior, for example, through the fill port in the substrate 1110.
The staking blades 1122 may be positioned relative to the carrier 1120 such that they are properly aligned with the channels 1190 as desired to prevent flow communication between the channels 1190 and the sample chambers 1180 when the carrier 1120 is placed into the contacting position with the substrate 1110 via the applied clamping force. Providing the blades 1122 as part of the carrier 1120 (e.g., an integral part of the carrier 1120) may facilitate manufacturing and alignment of the blades 1122, as the appropriate alignment can be assured prior to sealing the substrate 1110 with the carrier 1120. The appropriate alignment of the blades 1122 with the channels 1190 may ensure reliable sealing of the substrate 1110 and may permit the use of relatively small staking blades 1122. Relatively small staking blades in turn may require less force to drive the blades into the substrate 1110, for example, as compared to larger staking blades. The shape, size, and material of the blades may be selected based on the thickness, material, and other properties of the cover 1135.
The carrier 1120 also may include optical apertures 1123, for example, windows, that are in substantial alignment with the sample chambers 1180 when the carrier 1120 is in the sealing position, as depicted in
In accordance with various exemplary embodiments, when using the device of
In various embodiments, for example, in the case of PCR, the sealing of the substrate sample chambers may be implemented using the thermal block that is placed in contact with the substrate to perform thermocycling. Using the thermal block to perform the sealing function reduces a step in the processing of the substrate, allowing the step of thermocycling and sealing to be performed at the same time. Further, as will be explained, using the thermal block to perform the sealing function may enhance thermal contact and heat transfer between the thermal block and the sample chambers, which may thereby reduce thermocycling times.
To perform the sealing function, the thermal block 3250 is provided with a plurality of sealing protrusions (e.g., bumps) 3260 (which may be in the form of an array), only one of which is depicted in
The sealing protrusions 3260 of the thermal block 3250 may have various configurations. In an exemplary embodiment, the dimensions of the protrusions 3260 should be such that sufficient contact is made to seal the chambers 3280 from the channels 3275 and 3276. By way of example, the sealing protrusions 3260 may have a substantially circular configuration with a radius that is slightly larger than the radius of the chambers 3280. The top of the sealing protrusions 3260 may be substantially flat, as shown by protrusions 3260a and 3260b in
According to exemplary embodiments, the sealing protrusions 3260 may be machined directly on the thermal block 3250 or may be formed on a metal insert secured to the thermal block 3250.
Due to the sealing protrusions of a thermal block entering the sample chambers to perform sealing, as discussed above, it may be desirable to permit a relatively small amount of displaced sample contained in the chambers to escape.
Referring first to
Although the embodiments of
Once the substrate 3910 has been filled with sample S, as depicted in
In an exemplary aspect, the pumping of the sample into the substrate 3910 may be at a substantially constant pressure so that the pressure is not excessive so as to burst the seals during the time between filling the last well and breaking through the plug 3900. To allow the sample S to reach the plug 3900, a vent hole (not shown) that permits gas (e.g., air) to escape the substrate 3910 may be provided. According to various embodiments, a mechanical sealing mechanism may be desired at the inlet and outlet of channels 3970 and 3972 to prevent oil from being pumped out due to potentially expanding sample S, for example, during PCR and/or thermocycling. In various embodiments, it also may be desirable to exert pressure on the oil to pressurize the fluids to reduce bubble formation. In an exemplary aspect, such force may be placed on the film layer covering the base.
According to various embodiments, the oil sealing approach described above may include variations. By way of example, and not limitation, instead of the dissolvable plug 3900, a burst valve or a Timavo valve can be used. In this case, rather than waiting for the plug to dissolve, the sample slug can be immediately followed with an oil slug. Also, rather than utilizing the vents described above, membranes (porous or gas permeable) can be used between the venting channel and the main fluid outlet channel. In yet another exemplary embodiment, in place of the vents described with reference to
The exemplary embodiment of
Prior to filling the substrate 4010 with sample, the bead 4000 may be positioned within the venting chamber 4090 such that it does not occupy the entire volume of the venting chamber 4090, as depicted in
The beads 4000 may be configured so as not to block the connection channels 4073 when they have absorbed the sample S, thereby permitting escape of gas (e.g., air) into the main outlet (vent) channel 4072. In various embodiments, it may not be necessary to permit gas to escape the venting chambers and out of the substrate, however, since even at elevated temperatures, the super-absorbent beads 4000 may retain water without the tendency for the water to evaporate. This may be especially true when the beads are used for a relatively small fraction of their absorptive capacity.
In various embodiments, the beads 4000 may be substantially spherical and have a diameter of about 1 mm prior to absorbing sample. It is envisioned, however, that other shapes and sizes of the beads 4000 may be used. In particular, the shape and size of the beads 4000, as well as the configuration of the venting chamber 4090, may be selected such that the beads may swell and deform to substantially match the surface of the opening of the venting channel 4076 to the venting chamber 4090. Further, venting chamber 4090 may be small enough so that the bead 4000 may only expand to a limited extent to prevent the bead 4000 from absorbing more than a predetermined amount of sample. In the exemplary embodiment of
According to various other embodiments, the venting channel 4076 may be provided in the surface of the base 4030, as previously described herein, rather than having the cylindrical configuration shown in
In yet further exemplary embodiments, the bead 4000 may be in the form of a superporous hydrogel bead that acts to absorb sample and permit passage of gas.
The substrate 4110 may have a configuration similar to that described in the embodiment of
After filling the substrate 4110 with sample S, the pellet 4100 may prevent the sample from flowing past it, as shown in
In various embodiments, the sample introduction channels 4175 leading to the sample chambers 4180 may be sealed via a fluid that is immiscible with the sample, such as, for example, oil, in a manner similar to that described with reference to
According to still further embodiments, a material capable of breaking down to a gas, for example, with elevated temperatures may be used to seal sample chambers of a substrate. With reference to
The material 4200 may break down into a gas, like a blowing agent, at elevated temperatures, for example, at temperatures associated with PCR and/or thermocycling. By way of example, the material 4200 may turn to gas at temperatures of about 90° C. Thus, as shown in
Upon further processing of the sample in the sample chambers, for example, during thermocycling and/or PCR, elevating the temperature of the substrate 4310 causes the trapped air in the chamber 4382 to expand, introducing an air pocket P in the portion of the introduction channel 4375 slightly upstream and downstream of the chamber 4382, as shown in
Although
The embodiment of
With reference to
The base 1330 and film layer 1320 may be made from any of the materials described herein for the bases and film layers, respectively. By way of example, the film layer 1320 may be a COP film or a PSA film and may be thermally bonded to the base 1330, which may be manufactured from a plastic material, for example, such as COP. As described above with reference to the embodiment of
As shown in
The membrane strips 1350 may be gas-permeable or porous and also liquid impermeable so as to prevent leakages of the sample fluid from the substrate 1310. In various exemplary embodiments, the membrane strips may be made of materials such as those described above for the membranes 40 of the embodiment of
In various exemplary embodiments (not shown in the figures), instead of providing adhesive strips 1325 to bond the membrane strips 1350 to the film layer 1320, the film layer 1320 may be a double-sided adhesive PSA layer such that adhesive on one side of the layer 1320 is used to bond the film 1320 to the base 1330 and adhesive on the opposite side is used to bond the membrane strips 1350 to the film layer 1320. In such an embodiment, the vent holes 1340 would be formed through the entire film layer 1320 including both adhesive sides of the layer 1320.
According to various exemplary embodiments, when using multi-chamber devices for parallel processing of plural fluid samples, it may be desirable to cycle the device through various temperatures. For example, it may be desirable to perform PCR, which requires thermal cycling of the device over a range of temperatures, for example from about 60° C. to about 95° C. In such cases, relatively precise temperature control in the individual sample chambers of a substrate, as well as temperature uniformity over the entire substrate area may be desired. Further, as discussed above, the ability to isolate the individual chambers after filling the chambers (e.g., to prevent flow communication between the chambers and between the chambers and channels that lead to and from each chamber, such as fluid introduction and venting channels), may be desired in order to prevent cross-contamination during a biological testing process such as PCR.
In order to perform thermal cycling, in accordance with various exemplary embodiments, a thermal block may be placed in contact with the multi-chambered substrate. Typically, the thermal block is placed in contact with the film layer of the substrate that, together with the cavities formed in the base of the substrate form the fluid distribution network made up of, for example, main fluid channels, a plurality of sample chambers (e.g., in an array) in flow communication with the main fluid channel by a plurality of sample introduction channels, and a plurality of venting chambers in flow communication with the plurality of sample chambers via a plurality of venting channels. In other words, the thermal block may be positioned in contact with the substrate on the side of the base of the substrate that defines the various channel and chamber openings. For example, in the exemplary embodiments of
Placing the thermal block on the same side of the base of the substrate that the membranes are located, however, may impair the ability to achieve effective and uniform thermal conductivity between the thermal block and the sample chambers. In particular, the presence of membranes, whether disposed between the film layer and the base or on the side of the film layer facing away from the base, may cause an irregular surface (e.g., a “bumpy” surface). Such an irregular surface may prevent uniform contact of the thermal block with the substrate, and in some cases, it may be desirable to remove the membranes and add a metal lamination layer instead of the film layer to perform PCR after the substrate has been loaded with sample.
Further membranes in the form of strips of material may have a potential to leak around the borders of the strips.
With reference to
In the embodiment of
With reference again to
As shown in
Gas-permeable or porous membrane strips 1450 may be placed in contact with the film layer 1425 and in alignment with the vent holes 1426. Each membrane strip 1450 may be arranged and configured so as to cover a row of vent holes 1426. Examples of porous membranes include Gortex® and other similar materials known in the art and examples of selectively permeable membrane materials include, for example, PDMS. The membrane strips 1450 can be liquid impermeable so as to prevent leakage of sample from the substrate and to prevent a sponging effect of the liquid that can reduce the volume in the sample chamber. Other suitable porous membrane materials are described in U.S. Pat. No. 5,589,350 and other suitable gas-permeable membrane materials are described in U.S. Pat. Pub. No. 2005/0164373 entitled “Diffusion-Aided Loading System for Microfluidic Devices,” both of which are incorporated herein.
According to various embodiments, the film layer 1425 may be a double-sided adhesive film layer, for example, a double-sided PSA polymer film, and the membrane strips 1450 may be adhered to the film layer 1425 via the adhesive provided on the side of the film layer 1425 facing the strips 1450. In alternative exemplary embodiments (not shown) the film layer 1425 may be a PSA film having only one adhesive layer facing the base 1430. The membrane strips 1450 may be adhered to the opposite side of the film layer 1425 via adhesive strips (e.g., PSA strips) having substantially the same length and width as the membrane strips 1450. Thus, for example, the membrane strips 1450 may be adhered to the film layer 1425 via adhesive strips similar to adhesive strips 1325 shown and described with reference to the embodiment of
With reference now to
As described above with reference to the exemplary embodiment of
Yet another exemplary embodiment of a substrate for parallel processing of biological samples that utilizes a venting membrane on the backside of the substrate is shown in
Providing a single membrane 1650 may facilitate positioning and attaching of the membrane 1650 to the substrate 1410, may reduce the number of components, and thus also may facilitate manufacturing. The membrane 1650 also may include a plurality of optical apertures 1655 configured and arranged to be substantially aligned such that the sample chambers 1480 (shown in
According to various embodiments, in a manner similar to that described with reference to
By providing the venting membranes 1450, 1550, and 1650 on the side of the substrate 1410 opposite to the side that is placed in contact with the thermal block during thermal cycling, as shown in the exemplary embodiments of
According to various embodiments, a heated cover used for processing (e.g., a thermal block in a thermocycler), if placed in contact with the venting side of the substrate in
In some circumstances, it may be desirable to eliminate venting membranes at each of the venting chambers. For example, by eliminating the need for such membranes, manufacturing may be facilitated and less costly since handling and assembly of the membranes is not needed. Further, precise alignment of the membranes will not be required and the chances of misalignment of a membrane and potential consequent leakage of sample may be avoided. Also, isolation (e.g., sealing) of the sample chambers of the substrate may be facilitated and improved due to a reduction in force needed to deform and/or penetrate substrate layers to achieve isolation, as removal of the membranes may provide less layers to deform and/or penetrate. Finally, removal of such venting membranes may improve thermal conductivity and thermal uniformity, for example, during PCR thermal cycling, due to the provision of a substantially flat surface with which a thermal block may be placed in contact and/or a decrease in thickness of the layers of the substrate that a thermal block must act on.
According to various embodiments, a multi-chambered substrate may include a plurality of micro-sized vent holes in the film layer that, together with the base, forms the fluid distribution network (e.g., sample chambers, main fluid channel, sample introduction channels, venting channels, and venting chambers) in the substrate. The micro-sized vent holes may function both as capillary stops to prevent leakage of sample from a filled substrate and as vents to release gas from the substrate.
A partial, isometric, perspective view of a multi-chambered substrate 1710 that eliminates the need to provide a membrane over each venting chamber is depicted in
The film layer 1720 is provided with a plurality of micro-sized vent holes 1742 configured and arranged to be aligned with the venting chambers 1790 when the film layer 1720 is in position on the base 1730. By way of example only, the film layer 1720 may be a PSA film layer with adhesive on one side used to attach the film layer 1720 to the base 1730. The film layer 1720 may be, for example, a PSA polymer film or a PSA metal film. The vent holes 1742 may be sized so as to allow gas to escape from the substrate 1710 while creating a fluidic stop that prevents the sample within the substrate from leaking through the holes 1742. For example, capillary forces may prevent the sample from passing through the holes 1742 and out of the substrate 1710. In various exemplary embodiments, the vent holes 1742 may have a dimension (e.g., a diameter) ranging from about 1 μm to about 10 μm, for example, about 5 μm. In some embodiments, areas surrounding the vent holes 1742 may be substantially free of adhesive to prevent adhesive from flowing (e.g., cold-flowing) into and reducing the diameter of the vent holes.
In some cases, it may also be desirable to provide venting at the end of the fill channel 1770. Thus, the exemplary embodiment of
Various techniques may be used to provide the micro-sized vent holes 1742 in the film layer 1720. According to various embodiments, laser micro-machining (e.g., drilling) may be used to form the holes 1742 in the film layer 1740. For example, a laser micro-machining process may be used to drill holes through the film layer 1740 after it has been attached to the base 1730, without penetrating the base 1730. One exemplary laser micro-machining process developed by Oxford Lasers, Inc. (Oxon, United Kingdom) uses an ultraviolet cold laser process capable of drilling holes having a dimension (e.g., diameter) ranging from about 5 μm to about 10 μm in the film layer 1720. This process may form about 10 holes to several hundred holes per second, for example about 16 holes per second, in the film layer 1720 after the film layer 1720 has been bonded to the base 1730, without damage to the base 1730.
The laser drilling process can be applied to a variety of materials, including, but not limited to, for example, silicon, glass, metal, and/or polyimide. Examples of holes laser-drilled in various materials using Oxford Lasers, Inc. instruments are shown in
A technique that permits vent holes 1742 to be formed in the film layer 1720 after the film layer 1720 has been bonded to the base 1730 eliminates the need to precisely align the film layer with the base, which may thereby facilitate manufacturing. In other words, in a film layer that has pre-formed holes, precise alignment of the film layer with the base during bonding is needed to ensure alignment of the pre-formed holes with the venting chambers. Moreover, a micro-machining technique for forming the holes, such as that described above, for example, permits the size (e.g., diameter) of the vent holes to be altered as desired and progressively. This may permit control over the pressure gradient along the fill path during filling of the substrate.
Although the exemplary embodiment of
Filling tests were performed on substrates defining a 24 sample-chamber array having a configuration substantially as shown in the partial schematic representation of the substrate 1910 depicted in
In a first test configuration, a single vent hole of approximately 10 μm in diameter was laser-drilled in the film layer and aligned with each venting chamber. In a second test configuration, three vent holes of approximately 10 μm in diameter were laser-drilled in the film layer and aligned with each venting chamber. In a third test configuration, six vent holes of approximately 10 μm in diameter were laser-drilled in the film layer and aligned with each venting chamber. A syringe pump was used to supply a red dye fluid to each substrate at a pump speed of 40 μl/minute. Red dye was used to assist in observing the flow and filling in the substrate.
For the first test configuration using a single hole for each venting chamber 1990, no leakage was observed during filling. For the second test configuration using three holes for each venting chamber 1990, single droplet leakage was observed for three vent locations. For the third test configuration using six holes for each venting chamber 1990, single droplet leakage was observed in two vent locations. Those skilled in the art would understand that the number and/or size of vent holes provided for each venting chamber may vary based on a variety of factors, including, the sample being introduced, the pressure in the substrate, and other factors. Overall, the size and number of vent holes may be chosen so as to substantially prevent leakage of sample through the one or more vent holes and out of the device, while permitting gas (e.g., air) to escape through the one or more vent holes.
Various exemplary embodiments may utilize a hydrophobic, porous filter, substantially in the form of a fiber-like configuration, in lieu of a venting membranes described earlier, to permit gas (e.g, air) to escape the substrate while preventing sample leakage therethrough. Although the embodiments described below use a hydrophobic, porous fiber member, it may also be possible to utilize a porous or gas-permeable membrane material formed into a fiber-like structure. With reference to
The hydrophobic porous fibers 4400 may have a configuration similar to such fibers used in the filtration industry to filter impurities from water pumped into the fiber at pressures higher than the outside of the fiber. Such fibers permit impurities to flow through the pores of the fiber wall while water is retained. In various other exemplary embodiments, the fibers 4400 may be in the form of a resilient fiber cord that has a porous hydrophobic coating. Similarly, in the case of use with a substrate for biological testing, the filter 4400 can permit gas (e.g., air) to pass therethrough while retaining sample. Thus, with the fibers 4400 in place in the substrate 4110, gas may be permitted to pass through the main venting channels 4172 and fibers 4400 and out of the substrate 4410 through vent through holes.
As shown in
In yet further various embodiments, the instead of the weld spots 4650, a two layer laminated material may be used.
To further improve sealing of the vent through holes 4640 with the fiber 4600, a circular seal ring 4642 may be provided around the vent through hole opening in the channel 4672, as shown in
Although
With reference to
A hydrophobic, porous fiber 4800 may be provided in the main venting channel 4872 in a manner similar to that described above with reference to the exemplary embodiments of
Vented air may pass into the porous fiber 4800, which may be in the form of a hollow tube as shown or may have other configurations, as described above. The air may pass down the fiber 4800 and/or exit the fiber 4800 into the channel 4872 that the fiber 4800 lies in. According to various embodiments, a single vent hole 4825, shown in
Providing the sample chambers 4880 and 5180 in two rows, as shown in the exemplary embodiments of
Although the exemplary substrates 4810 and 5110 include an array of two rows of chambers 4880 and 5180, the substrates may be formed with any number of chamber rows. By way of example only, the substrates may be formed with four rows of chambers, in which case a main fluid supply channel may be positioned between a first pair of chamber rows and a second pair of chamber rows. Two venting channels may then be provided at the two opposite edges of the substrate in conjunction with each of the pair of rows of chambers. Any number of rows may be used, with the porous, hydrophobic filters disposed inward from the edges of the substrate being secured in position by a separate film or series of strips of film on the underside of the substrate. The substrates also may include negative template control sections, described in more detail with reference to the embodiments of
The substrates 4810 and 5110 may be assembled in a manner similar to that described above with reference to
In yet other embodiments, a thermocycler may be configured to accommodate two sample substrates, for example, substrates 4810 or 5110. A perspective view of such a thermocycler 5300 is depicted in
As discussed above, control over the pressure gradient along the fill path during filling of the substrate may be provided, for example, by controlling the size of vent holes, such as vent holes 1742 provided in the substrate 1710, as was described in relation to the embodiment of
By way of example, the hydrophobicity of the venting channels of the substrate may be modified, for example increased, to control the pressure gradient while filling the substrate with sample. The hydrophobicity may be modified, for example, by adding texture and/or increasing roughness (e.g., on a nano-scale level) to the surface defining the venting channels. Such texturing and/or increasing roughness may be introduced during the injection molding process, for example, by texturing the mold as desired in the area that forms the venting channels. Other techniques for modifying the hydrophobicity of a surface defining the venting channels may include providing a coating, or chemically treating the surface. By way of example only, Kim et al., “Nanostructured Surfaces For Dramatic Reduction Of Flow Resistance In Droplet-Based Microfluidics,” IEEE 2002, hereby incorporated by reference in its entirety herein, teaches one technique for providing nanostructures on a surface to alter hydrophobicity. It is envisioned that the hydrophobicity of all or a portion of the venting channels may be altered.
According to various embodiments, the venting channel configuration (e.g., geometry) also may be modified in order to control the pressure gradient during filling of the substrate. For example, the venting channels may be provided with a region of reduced cross-section so as to increase the pressure within the venting channel and reduce the potential for leakage. With reference to the exemplary embodiments of
According to various embodiments, for example, when filling a multi-chambered substrate via positive pressure (e.g., via pumping, syringe, etc.), it is desirable to know when to stop the filling once the various chambers and channels have been filled in order to control over-pressurization and/or sample leakage. An exemplary mechanism for determining when to stop filling the substrate includes providing optical sensors in association with the sample chambers. The sensors, which in an exemplary embodiment may be an optical sensor including a photodiode and LED, can detect the presence of the sample by a difference in the index of refraction and send a signal to stop the filling process (which may occur either manually or automatically). Due to potential increased costs and manufacturing complexity associated with such a sensor/feedback mechanism, it may be desirable to provide a relatively simple substrate design configured to passively and automatically stop sample delivery so as to avoid over-pressurization and/or leakage of the substrate.
Referring to
Eventually, due to the continued positive pressure applied at the end of the channel 2190 proximate the dashed arrow, as shown in
In
Leakage of sample out of the substrate may depend upon various factors, including, for example, the configuration of the substrate, the applied pressure, the method of pressure generation (e.g., via syringe, pump, constant pressure source, etc.), properties of the membrane material and/or configuration of vent holes, and other factors that may influence the extent to which the substrate becomes over-pressurized during filling with sample.
According to various embodiments, providing an additional vent upstream of the vent 2150 of
In
In
Eventually, due to the continued positive pressure applied at the end of the channel 2190 proximate the dashed arrow, as shown in
In order to provide the proper functioning of the upstream bypass venting mechanism, as described with reference to
Referring to
On the other hand, as depicted in
Referring now to
Upstream of the chambers 2480, the substrate 2410 is provided with a venting mechanism 2455, which may be, for example, in the form of a vent hole in a film layer of the substrate 2410 covered with a venting membrane. This upstream venting mechanism 2455 may allow gas (e.g., air) to escape from the substrate 2410 after the various channels and chambers have been filled such that over-pressurization and sample leakage out of the venting mechanisms associated with the venting chambers do not occur.
In accordance with the exemplary embodiment of
The overfill channel 2475 may have a smaller cross-sectional area than the main fluid channel 2470. The smaller cross-section will increase the fluidic resistance (e.g., pressure) encountered by the sample S as it fills the substrate 2410. As such, the overfill chamber 2495 will fill only after the remaining chambers 2480 and 2490 and channels 2470, 2420, and 2400. In various embodiments, rather than a straight overfill channel of reduced cross-section, an overfill channel having a serpentine configuration may be used as depicted in
According to various embodiments, the inlet sample volume requirement may be calculated as follows to ensure that all of the sample chambers 2480 are filled and the substrate is not overpressurized. Assuming that the substrate 2480 has 24 sample chambers, as shown in
According to various embodiments, aspiration of sample into the chambers can be assisted by moving the overfill chamber to the inlet to provide protection against over-aspirating the sample.
As discussed above, in various embodiments, it may be desirable to integrate sample preparation with a multi-chamber array substrate. For example, it may be desirable to elute nucleic acid from a membrane and supply the eluted sample volume directly to a substrate. Before filling the sample chambers with the eluted sample, however, it is desirable to ensure that the eluted sample has been sufficiently mixed to substantially homogenize the concentration of the sample prior to filling the sample chambers. If the eluted sample is not sufficiently homogenized, a concentration gradient may result in the substrate chambers. For example, the concentration of nucleic acid may be higher in upstream chambers than in downstream chambers of the substrate. Such a concentration gradient in the substrate may impair detection, quantization (e.g., for gene expression) and/or analysis of the biological sample being tested.
To mix the eluted sample so as to obtain a substantially homogenized concentration, an external mixing force, for example, via a vortex or the like, may be applied to the collected sample and the mixed sample may then be introduced into the substrate. In an alternative embodiment, however, it may be desirable to provide a mechanism for mixing eluted sample as part of the substrate itself.
In the multi-chamber array substrate 2510 shown in
In addition to the various features listed above, the substrate 2510 also defines a serpentine mixing channel 2565 that connects the inlet port 2560 and the main fluid channel 2570 in flow communication with each other. The serpentine channel 2565 serves to lengthen the distance the sample travels between being supplied to the inlet port 2560 and filling the sample chambers 2580. By increasing the distance, and thus time, the sample travels prior to filling the chambers 2580, diffusion may be increased in the sample thereby mixing the sample and promoting homogenization of the sample concentration. In other words, moving a plug of liquid, such as a volume of eluted sample, through a channel of sufficient length prior to introducing the sample into the sample chambers may take advantage of the recirculation patterns that occur along the axis of the microfluidic channel that results from the plug of liquid having a parabolic velocity profile with substantially flat menisci at both ends of the plug. With a long enough mixing channel, and thus time, such recirculation patterns may act to mix the sample plug and provide a substantially uniform concentration prior to the sample being introduced into the sample chambers. Enhanced mixing may also occur by providing the mixing channel 2565 with sharp corners rather than rounded corners and/or by applying various surface finishes configured to enhance mixing.
Thus, the mixing channel 2565 provides a passive mixing feature that is integral with the substrate 2510. This permits direct loading of the substrate 2510 with eluted sample without prior mixing, while ensuring that the sample filling the chambers 2580 will have a substantially uniform concentration for all of the chambers 2580. The serpentine mixing channel 2565 may be relatively large in comparison to the main fluid channel 2570. By way of example only, the main fluid channel 2570 may be approximately 150 μm wide by approximately 50 μm deep, while the width of the mixing channel 2565 may range from approximately 1 mm to approximately 2 mm and have the same depth as the main fluid channel 2570.
A straight mixing channel may be used rather than the serpentine mixing channel shown in
Yet another exemplary embodiment of a multi-chamber array substrate 2610 is depicted in
The substrate 2610 of
As noted above, the exemplary embodiment of
This configuration of two inlet ports 2660 for supplying two differing sample chamber networks that are not in flow communication with each other permits two differing samples to be supplied to the substrate 2610 and/or differing biological testing (analysis) to be performed within the same substrate 2610. Moreover, according to various embodiments, the dual fluid distribution network provided in the substrate 2610 may provide a negative template control mechanism in order, for example, to test for false positives. By way of example, the inlet portion 2660 connected to the main fluid channels 2670a and 2670b may be supplied with a biological sample for which PCR analysis may be desired, while the inlet port 2660 in flow communication with the main fluid supply channel 2671 may be supplied with a blank sample (such as, for example, an elution buffer such as deionized water or Tris HCl). Analysis, such as via optical detection (for example, by detection of a fluorescent signal), of both groups of sample chambers may be performed and if a signal is detected in the sample chambers filled with the blank sample, this may indicate that the substrate is contaminated or otherwise susceptible to giving a false positive result.
Although the exemplary embodiment of
As mentioned above, for an inlet supply port supplying sample to more than one main fluid channel, those main fluid channels may be connected either serially or in parallel. The exemplary embodiment of
The exemplary embodiment of
An upstream venting mechanism in conjunction with the inlet supply ports 2660 and 2760 may be provided in order to protect against overfill and over-pressurization of the substrate, as discussed above with reference to
Using an upstream venting mechanism, the sample volume that may be used to fill the sample chambers 2680 and 2780 associated with the main fluid supply channels 2670a, 2670b and 2770a, 2770b may range from a minimum determined by adding the total volume of the sample chambers (the 16 chambers in the case of
According to other exemplary embodiments, instead of or in addition to providing an upstream venting mechanism to protect against over-pressurization of the substrate, optical detection of sample reaching the overfill chambers 2695 and 2795 may be implemented. In an exemplary aspect, a dried, colored, fluorescence dye (e.g., a red dye) may be deposited in the overfill chambers 2695 and 2705, for example, proximate an inlet of the chambers 2695 and 2795. Thus, when the sample begins filling the overfill chambers 2695 and 2795, an optical detection mechanism may detect a change in color in the overfill chamber 2695 and 2795 and a feedback control mechanism may send a signal indicating to stop the application of pressure used for filling the substrate 2610 and 2710. For example, a feedback signal may be sent to a pressure-providing device (e.g., a pump, syringe, etc.) to automatically stop the pressure being used to fill the substrate and/or to an individual to manually stop the pressure.
The optical detection system may, for example, include an optical cover over the substrates 2610 and 2710 such that only the sample chambers 2680 and 2780, and the outlet portion of the overfill chambers 2695 and 2795 are viewable by an optical reading mechanism. The optical detection system may use, for example, an LED beam to illuminate the overfill chambers 2695 and 2795 and the optical reading mechanism may monitor the overfill chambers 2695 and 2795 near their respective outlets 2696 and 2697 for a change in fluorescence. After all of the chambers 2680 and 2780 have been filled, the sample will move into the overfill chambers 2695 and 2795, dissolve the predeposited dye and carry it to the outlets 2696 and 2796. The optical reading mechanism may then detect a fluorescence signal change and send a feedback signal, for example, to an operator or a filling device, to stop the application of pressure for supplying sample to the substrate 2610 and 2710. In other embodiments, the detector could detect the presence of an internal standard in the mastermix mixed with sample (which may be ROX), rather than spotting additional dye in the overfill chambers.
It has been observed that flowing deionized water into an empty chamber also causes a signal increase, which may be contributed by air and water having differing optical background signals and/or by the meniscus of the traveling water causing a signal change through both reflection and diffraction effects. Thus, in various exemplary embodiments, rather than using a red dye, LED beam, and fluorescence detecting mechanism in the overfill chamber, an optical sensor configured to detect the presence of liquid may be used. By way of example, a refractive index sensor may be used to detect liquid filling the overfill chamber. Because the refractive index of water (e.g., sample) differs from that of air, the light is deflected in a way that differs when the sample enters an overfill chamber and can be recognized by the detector. When the detector senses the change, a signal can be sent to stop the application of pressure and supply of sample.
Yet further exemplary embodiments for detection of the presence of sample in an overfill chamber include the use of a capacitance sensor or the use of an infrared sensor. Regarding the former, a capacitance sensor may be used to measure the capacitance between the film layer (e.g., an aluminum film layer) covering an overfill chamber and the opposite side of the substrate at the location of the overfill chamber. Since the dielectric constant of water is much greater than air, when sample fills the overfill chamber, the sensed capacitance may change and the capacitance sensor may send a signal indicating to stop filling (e.g., pressure application to) the substrate. FIG. 37 depicts an exemplary embodiment of using a capacitance sensor to detect the presence of sample in an overfill chamber 3895 in a substrate 3810. In
In various other embodiments, an infrared sensor may be used to detect sample filling of an overfill chamber. Because water and air have differing absorbance in the infrared range, infrared absorbance and/or reflection may be measured at the overfill chamber to detect the presence of sample (which contains water) entering the chamber. For example, in the case of an aluminum film layer covering the base of a substrate, infrared reflection off the aluminum layer over an overfill chamber may be detected.
Those having skill in the art will recognize various other detection mechanisms that may be used to detect the presence of the sample filling the overfill chamber, and the exemplary embodiments above should not be construed as limiting. Also, it should be understood that the various optical detection mechanisms described with reference to the embodiments of
Using an optical detection mechanism, the volume of sample that may be supplied to the sample distribution networks associated with the first group of sample chambers 2680 and 2780 (the 16 chamber group) of the substrates 2610 and 2710 may range from a minimum determined by adding the total volume of the sample chambers (the 16 chambers in the case of
Whether implementing the upstream venting mechanism approach or the optical detection approach, using an overfill chamber and channel, as described in the embodiments of
Although various exemplary substrate embodiments described above and shown in the figures depicted partial views, schematic views, or substrates defining a 12-chamber, 16-chamber, or 24-chamber array, it should be understood that the various configurations and features of those embodiments can be applied to substrates of varying sizes and chamber arrays, including, for example, substrates defining multi-chamber arrays including various number of chambers, including, but not limited to, 12, 24, 36, 48, 96, 192, 384, 3072, 6144, or more sample chambers. In an exemplary configuration, the various substrates described above can have dimensions of, for example, about 127.0 millimeters by about 85.7 millimeters and define 384 sample chambers.
In some cases, for example when performing biological testing using a substrate defining a chamber array that differs from a conventional substrate, it may be desirable to use existing instrumentation for such conventional substrate configurations to perform biological analysis on substrates of other sizes. For example, at least some substrates in accordance with the present teachings may define 12-, 16-, or 24-chamber arrays, which have fewer chambers, and thus require fewer assays, than are available in a conventional low density array substrate (e.g., a substrate including a 96-chamber or 384-chamber array). When performing biological testing, such as, for example, real-time PCR, on the substrates with fewer chambers, a solution may be to perform testing on more than one substrate and thereby enable analysis of multiple samples during the same testing step. However, this may result in a mismatch between the number of samples presenting for analysis and the number of positions available on a substrate. Further, using a low density array substrate that does not use all of the chambers available may not be desirable since once subject to biological analysis, the substrates may not be suitable for further use. Also, in some cases, it may be desirable to subject one or more samples to differing assay panels (e.g., biological analysis) during a single processing routine (e.g., a single real time PCR assay step).
In order to achieve at least some of the desirable features above and in accordance with various embodiments of the present teachings, substrates that define a subset of chambers (e.g., “subcards”) of a standard low density array substrate (e.g., 96- or 384-chamber array) may be provided that are configured to be combined and positioned into a holding fixture compatible for use with standard low density array testing instrumentation.
The holding fixtures 5400 and 5600 of
The configurations of the subcards and holding fixtures of
In various embodiments, when performing biological testing, differing numbers of substrates maybe processed in each testing run. For example, one, two, three, four, or more cards may be processed (e.g., thermocycled) during a given biological testing run. This may be particularly true in benchtop testing of substrates.
In this situation, it is desirable to achieve substantial chamber-to-chamber thermal uniformity within and between substrates being processed. Moreover, it may be desirable to provide information to the testing instrumentation regarding the number of substrates being processed in a given run.
With reference to
Each of the differing carriers in
To use the carriers of
The present teachings provide a variety of structural arrangements, techniques, and/or methodology useful for performing biological analysis, including multiple analyte detection. It should be understood that although in some cases the embodiments described herein may focus on a particular aspect, various embodiments may be combined to form a system and/or substrate configuration useful for multiple analyte detection. By way of example only, various sealing approaches may be combined with various venting approaches. The various embodiments described herein are not intended to be mutually exclusive.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of “less than 10” includes any and all subranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all subranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5.
It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent. Thus, for example, reference to “a layer” includes two or more different layers. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
Various embodiments of the teachings are described herein. The teachings are not limited to the specific embodiments described, but encompass equivalent features and methods as known to one of ordinary skill in the art. Other embodiments will be apparent to those skilled in the art from consideration of the present specification and practice of the teachings disclosed herein. It is intended that the present specification and examples be considered as exemplary only.
Claims
1. A system for distribution of a biological sample, the system comprising:
- a substrate, wherein the substrate comprises a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber;
- a preloaded reagent contained in each sample chamber and configured for nucleic acid analysis of a biological sample that enters the substrate; and
- a sealing instrument configured to be placed in contact with the substrate to seal each sample chamber so as to substantially prevent sample contained in each sample chamber from flowing out of each sample chamber,
- wherein the substrate is constructed of detection-compatible and assay-compatible materials.
2. The system of claim 1, wherein the sealing instrument is configured to be placed in contact with an exterior portion of the substrate.
3. The system of claim 1, wherein the sealing instrument is configured to be placed in at least one of pressure contact and thermal contact with the substrate.
4. The system of claim 1, wherein the sealing instrument is configured to seal the sample introduction channels and venting channels for each sample chamber.
5. The system of claim 1, wherein the sealing instrument is configured to be placed in contact with the substrate at least one of before a reaction process that occurs in the sample chambers and during a reaction process that occurs in the sample chambers.
6. The system of claim 5, wherein the sealing plate is configured to be placed in contact with the substrate before a reaction process that occurs in the sample chambers to seal each sample chamber.
7. The system of claim 5, wherein the sealing plate is configured to be placed in contact with the substrate during a reaction process that occurs in the sample chambers to seal each sample chamber.
8. The system of claim 7, further comprising a thermal block, wherein the thermal block comprises the sealing plate.
9. The system of claim 1, wherein the sealing instrument comprises one of a plate and a roller.
10. The system of claim 1, wherein the substrate comprises a film layer and wherein a portion of the film layer fills the sample introduction channels and the venting channels upon contact of the sealing instrument with the substrate.
11. The system of claim 1, wherein the sealing instrument comprises a plurality of sealing protrusions.
12. The system of claim 11, wherein the sealing protrusions are configured to seal the sample introduction channels and venting channels for each sample chamber.
13. The system of claim 11, further comprising an additional sealing instrument, wherein the plurality of sealing protrusions comprises a first plurality of sealing protrusions associated with the sealing instrument and a second plurality of the sealing protrusions associated with the additional sealing instrument, the first plurality and second plurality of sealing protrusions being configured to seal differing groups of sample chambers when the sealing instrument and the additional sealing instrument are respectively placed in contact with the substrate.
14. The system of claim 13, wherein the first plurality of sealing protrusions is configured to seal the sample introduction channels for each sample chamber and the second plurality of sealing protrusions are configured to seal the venting channels for each sample chamber.
15. The system of claim 11, wherein each sealing protrusion is configured to contact a respective sample introduction channel and venting channel for each sample chamber when the sealing instrument is placed in contact with the substrate.
16. The system of claim 11, wherein the plurality of sealing protrusions are configured to respectively align with the plurality of sample chambers.
17. The system of claim 16, wherein each sample chamber is configured to receive a respective sealing protrusion such that the sealing protrusions seal the sealing chambers substantially at an inner peripheral surface defining each sample chamber.
18. The system of claim 11, wherein the sealing instrument further comprises a plurality of optical apertures configured to provide optical access to the sample chambers.
19. The system of claim 18, wherein the sealing protrusions are configured to pierce the substrate and enter the introduction and venting channels for each sample chamber.
20. The system of claim 11, wherein the sealing protrusions comprise one of pins, disks, blades, and bumps.
21. The system of claim 1, wherein the substrate further comprises a film layer and a base, and at least one venting mechanism configured to permit gas to escape the substrate.
22. The system of claim 21, wherein the substrate further comprises a sample supply channel in fluid communication with a plurality of the sample introduction channels.
23. The system of claim 22, wherein the sample supply channel comprises a serpentine portion.
24. The system of claim 22, wherein the sample supply channel comprises a bifurcation.
25. The system of claim 22, wherein the sample supply channel comprises a U-shape portion.
26. The system of claim 21, wherein the substrate further comprises a plurality of sample supply channels, each sample supply channel being in flow communication with a differing group of sample introduction channels and corresponding sample chambers.
27. The system of claim 26, wherein the substrate further comprises a plurality of sample inlet ports, each sample inlet port being in flow communication with a differing sample supply channel.
28. The system of claim 21, wherein the at least one venting mechanism comprises a plurality of venting mechanisms respectively corresponding to the plurality of sample chambers.
29. The system of claim 21, wherein the substrate further comprises a venting chamber in flow communication with each venting channel.
30. The system of claim 21, wherein the substrate further comprises at least one overfill chamber corresponding to the at least one venting mechanism.
31. The system of claim 30, further comprising a detector configured to detect sample in the overfill chamber.
32. The system of claim 31, wherein the detector comprises one of an optical sensor, a refractive sensor, and a capacitance sensor.
33. The system of claim 30, further comprising an additional venting mechanism disposed upstream of the at least one vent mechanism corresponding to the overfill chamber.
34. The system of claim 21, wherein the substrate further comprises a venting chamber for each sample chamber, the venting chambers being in flow communication with the sample chambers via the venting channels, wherein the at least one venting mechanism comprises a plurality of venting mechanisms each corresponding to a respective venting chamber.
35. The system of claim 21, wherein the substrate further comprises a main venting channel in flow communication with the venting channels.
36. The system of claim 35, wherein the at least one venting mechanism comprises a porous fiber disposed in the main venting channel.
37. The system of claim 36, wherein the fiber is a hollow fiber or a solid fiber.
38. The system of claim 21, wherein the at least one venting mechanism comprises a gas-permeable or porous membrane.
39. The system of claim 38, wherein the substrate further comprises a venting chamber for each sample chamber, the venting chambers being in flow communication with the sample chambers via the venting channels, each venting chamber being configured to receive a gas-permeable or porous membrane.
40. The system of claim 38, wherein the gas-permeable or porous membrane is in contact with the film layer at a surface of the film facing away from the base.
41. The system of claim 40, wherein the gas-permeable or porous membrane has a sheet-like or strip-like configuration.
42. The system of claim 41, wherein the substrate further comprises a venting chamber for each sample chamber, the venting chambers being in flow communication with the sample chambers via the venting channels, and wherein the at least one gas-permeable or porous membrane corresponds to a plurality of venting chambers.
43. The system of claim 42, wherein the at least one gas-permeable or porous membrane comprises a plurality of membranes, each membrane corresponding to differing groups of venting chambers.
44. The system of claim 40, wherein the substrate further comprises an additional film layer, the film layers being disposed on opposing surfaces of the base.
45. The system of claim 44, wherein the additional film layer and the base together define the sample chambers, sample introduction channels, and venting channels.
46. The system of claim 21, wherein the at least one venting mechanism comprises at least one through hole in the substrate.
47. The system of claim 46, wherein the at least one through hole is provided in one of the base and the film layer.
48. The system of claim 47, wherein the at least one through hole is provided in the film layer and is configured to prevent sample from passing therethrough via capillarity.
49. The system of claim 21, wherein the at least one venting mechanism is configured to substantially prevent sample from passing through the at least one venting mechanism and out of the substrate.
50. A method for distribution of a biological sample, the method comprising:
- providing a base, wherein the base comprises a plurality of sample cavities containing a preloaded reagent therein, a sample introduction trench for each sample cavity, and a venting trench for each sample cavity, and wherein the base is constructed of detection-compatible and assay-compatible materials;
- providing a film to adhere to the base to form a substrate, wherein the film and base form a plurality of sample chambers from each sample cavity, a sample introduction channel from each sample introduction trench, and a venting channel from each sample venting trench;
- supplying the biological sample to the substrate;
- filling the sample chambers with the biological sample via the sample introduction channels;
- passing gas out of the substrate via at least one venting mechanism in the substrate; and
- sealing the sample chambers to substantially prevent sample in the sample chambers from flowing out of the sample chambers.
51. The method of claim 50, wherein the sealing the sample chambers comprises placing a sealing instrument in contact with the substrate.
52. The method of claim 50, wherein filling the sample chambers comprises at least one of spinning the substrate to provide centrifugal force, sizing the sample introductory channels to provide capillary force, and aspirating the sample through the vent channels.
53. The method of claim 50, wherein filling the sample chambers comprises providing positive pressure to the sample.
54. The method of claim 53, further comprising venting gas in the sample chamber through the venting channels and the at least one venting mechanism.
55. The method of claim 54, wherein the venting mechanism comprises at least one gas-permeable or porous membrane.
56. The method of claim 50, further comprising substantially preventing the sample from passing through the at least one venting mechanism and out of the substrate.
57. The method of claim 56, wherein preventing the sample from passing through the at least one venting mechanism comprises holding the sample in the venting mechanism via capillarity.
58. A system for distribution of a biological sample, the system comprising:
- means for distributing the sample to a plurality of sample chambers containing a preloaded reagent;
- means for venting each of the sample chambers; and
- means for sealing each of the sample chambers.
59. A device for testing a biological sample, the device comprising:
- a substrate defining a plurality of distribution portions configured to distribute biological sample throughout the substrate, the plurality of distribution portions comprising a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber; and
- a substance disposed in at least one of the distribution portions, the substance being configured to seal each sample chamber so as to substantially prevent sample disposed in each sample chamber from flowing out of each sample chamber during testing of the biological sample.
60. The device of claim 59, wherein the substance comprises one of an adhesive, a gas, a substance configured to dissolve into a gas upon contact with the biological sample, a fluid immiscible with the biological sample, an absorbent material, and porous hydrophobic material.
61. The device of claim 59, wherein the substance is disposed in the sample introduction and venting channels for each sample chamber.
62. The device of claim 59, wherein the plurality of distribution portions further comprises a vent chamber in flow communication with each venting channel and wherein the substance is disposed in each vent chamber.
63. The device of claim 59, wherein the plurality of distribution portions further comprises a main fluid supply channel in flow communication with the sample introduction channels, and wherein the substance is disposed in the main fluid supply channel.
64. The device of claim 59, wherein the substance is configured to seal each sample chamber upon sample contacting the substance.
65. The device of claim 59, wherein the substance is configured to seal each sample chamber in response to elevating a temperature of the substrate during testing of the biological sample.
Type: Application
Filed: Apr 26, 2006
Publication Date: Jan 18, 2007
Applicant: Applera Corporation (Foster City, CA)
Inventors: Min Yue (Belmont, CA), David Liu (Los Altos, CA), Yuh-Min Chiang (Foster City, CA), Joon Mo Yang (Redwood City, CA), Dennis Lehto (Santa Clara, CA), Charles Vann (El Granada, CA), Nigel Beard (Redwood City, CA), Ian Harding (San Mateo, CA), John Van Camp (San Ramon, CA), Alexander Dromaretsky (Davis, CA), Sergey Ermakov (Hayward, CA), Mark Oldham (Los Gatos, CA), Joy Roy (San Jose, CA), Maryam Shariati (Sunnyvale, CA), Umberto Ulmanella (Foster City, CA)
Application Number: 11/380,327
International Classification: B01L 3/00 (20070101);
