Patent Description:
Testing within diagnostic laboratories, for example, may involve extracting and quantifying one or more constituents in a biological sample obtained from a patient, such as from blood serum or blood plasma.

PCR testing, in particular, is a technique used to amplify a targeted DNA or RNA sequence from a few extracted DNA or RNA fragments (hereinafter the "DNA/RNA template") that have been extracted from the biological sample to billions of copies within a short period of time. For example, PCR testing can be used for identification of DNA/RNA sequences involved in cancer or genetic disorders, such as cystic fibrosis, or for the identification and diagnosis of diseases caused by fungi, bacteria, and viruses.

In such PCR processing, cycles of heating and cooling are repeated many times on a PCR solution containing the extracted DNA/RNA templates, a master mix, possibly and reagent, and possibly water, leading to a large number of (e.g., more than one billion in some cases) exact copies of the originally-extracted DNA/RNA templates. Once the replication has occurred, an optical technique such as fluorescence staining may be used to determine the amount of replicated DNA/RNA that is present and/or analyze sequences thereof.

<CIT> discloses a cuvette comprising a liquid-confining chamber. The chamber is defined by two opposing walls and spaced apart a distance t<NUM>. The shape of side walls is a gradual concavity, so that they diverge at end <NUM> at an angle alpha of about <NUM>°, and at a point halfway between ends <NUM> and <NUM>, start to reconverge again at an angle of about <NUM>°. The distance t<NUM> is selected to minimize the quantity of liquid that is retained in the cuvette upon removal of liquid.

<CIT> discloses a disposable sample holding and processing device dimensioned for being operated in a nucleic acid amplification apparatus. The device comprises a rigid body and at least one channel, the binding chamber and the amplification chamber. The binding chamber and the amplification chamber are situated on side-surfaces of the body. The body has typically an outer volume between <NUM> and <NUM>. The volume of the sample preparation chamber is much larger than the volume of the amplification chamber.

According to the present invention, a sample holder is provided. The sample holder includes a body having a top surface and a bottom surface, the body further comprising: an inlet groove formed into the bottom surface; an outlet groove formed into the bottom surface alongside the inlet groove; a detection recess formed into the bottom surface and connected to the inlet groove and the outlet groove; a fill port interconnected to both the inlet groove and the outlet groove; and a cover connected to the bottom surface wherein the cover interfaces with the body to form an inlet channel interconnected to the fill port, a detection region interconnected to the inlet channel, and an outlet channel interconnected to the detection region and the fill port.

Still other aspects, features, and advantages of the present disclosure may be readily apparent from the following detailed description by illustrating a number of example embodiments and implementations. The present invention may also be capable of other and different embodiments, and its several details may be modified in various respects. The disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the claims.

The disclosure will be better understood by referring to the detailed description taken in conjunction with the following drawings. The drawings and descriptions are to be regarded as illustrative in nature, and not as restrictive. The drawings are not necessarily drawn to scale. Like numerals are used throughout to denote like elements.

The present disclosure is directed at sample holders for use in, for example, PCR testing. In particular, the sample holders can provide a low-cost design that can be used with automated PCR processing for both the amplification phase and detection phases of PCR processing. The sample holder can be intended for a single use, and disposed thereafter. Optionally, the sample holder may be washed and reused.

In another aspect, the sample holder is miniaturized in that the sample holder can perform detection (e.g., fluorescence or other detection) on very small volumes of the PCR solution including the extracted DNA/RNA templates, master mix, and possibly a reagent, such as less than or equal to 20µL, or even from 5µL to 20µL in the detection region.

The PCR station assembly is provided that is configured to hold the sample holder in a defined position for the amplification and detection phases of PCR. During the amplification phase, the PCR solution is subjected to multiple heating and cooling cycles to replicate the DNA/RNA templates. Thereafter, the PCR station assembly holds the sample holder containing the replicated DNA/RNA templates for interrogation by an optical interrogation apparatus, such as a fluorescence detection apparatus.

In view of the foregoing, there is an unmet for simple and cost-effective sample holders for PCR testing, and that can process even very small volumes of the PCR solution containing thin a detection region thereof (e.g., ≤ <NUM>µL).

These and other aspects and features of embodiments of the disclosure will be described with reference to <FIG> herein.

Referring now to <FIG>, an example embodiment of a sample holder <NUM> will now be described. The sample holder <NUM> comprises a body <NUM> having a top surface <NUM> and a bottom surface <NUM>. The bottom surface <NUM> may be a planar surface. The body <NUM> may be manufactured from an optically transparent or translucent material such as glass or plastic. Molded plastics may include transparent (e.g., thermoplastic materials) or translucent materials (e.g., white or frosted) plastics that are compatible with the particular PCR master mix and possibly a reagent being used. Example materials may include elastomers and olefins, polyvinyl chloride (PVC), polycarbonate, polyethylene terephthalate glycol (PETG), styrene, polyethylene, polystyrene, acrylonitrile butadiene styrene (ABS), and polypropylene or combinations. Polycarbonate and polypropylene are excellent choices for inert and transparent/translucent properties for PCR. White and frosted may be used for qPCR. The body <NUM> can be injection molded, compression molded, or the like.

The body <NUM> further comprises an inlet groove <NUM> formed into the bottom surface <NUM> and an outlet groove <NUM> also formed into the bottom surface <NUM>. The outlet groove <NUM> is positioned alongside the inlet groove <NUM>. The groves <NUM>, <NUM> may run in parallel and may be formed by molding. The inlet and outlet grooves <NUM>, <NUM> can have groove depths of from <NUM> to <NUM> in depth. Grooves <NUM>, <NUM> can have a variable groove width such as from <NUM> to <NUM>, for example.

The body <NUM> further comprises a detection recess <NUM> formed into the bottom surface <NUM>. The detection recess <NUM> is sufficiently large for the particular optical interrogation system (e.g., optical interrogation system <NUM>) to be able to measure light emission readings (e.g., fluorescence emission readings) therefrom, for example. The detection recess <NUM> may have a semi-circle of diameter between about <NUM> to <NUM>, for example. The detection area may be greater than <NUM><NUM>. Structurally, the inlet groove <NUM> leads to and is connected to the detection recess <NUM> and the outlet groove <NUM> is connected to and leads away from the detection recess <NUM>. The groves <NUM>, <NUM>, and the detection recess <NUM> may be of the same depth from the bottom surface <NUM>.

A fill port <NUM> can be interconnected to both the inlet groove <NUM> and the outlet groove <NUM> and provides both a fill and overflow function. The top surface <NUM> of the fill port <NUM> may be a planar surface and can be located above the top surface <NUM> and thus can be sealed with any suitable sealing membrane once the PCR solution <NUM> is received therein. Sealing may occur prior to insertion of the sample holder <NUM> in the PCR station assembly <NUM> (<FIG>) or after insertion therein.

The sample holder <NUM> can include a cover <NUM> connected to and sealed to the bottom surface <NUM> of the body <NUM> by any suitable means. The cover <NUM> can be a thin film cover in some embodiments, such as a generally planar sheet of constant thickness plastic or metal film. The thickness may be between <NUM> and <NUM>, for example. Other suitable thicknesses and other non-planar configurations of the cover <NUM> may be used. Depending on the configuration of the optical interrogation apparatus <NUM> (<FIG>) used, the cover <NUM> may be translucent or transparent if the light detector is positioned below the body <NUM> and cover <NUM>, or opaque if the light detector is positioned above the body <NUM>. In the depicted embodiment of <FIG>, the cover <NUM> can be opaque, such as a black plastic. The cover <NUM> interfaces with the body <NUM> to close the bottoms of the inlet groove <NUM>, detection recess <NUM>, and outlet groove <NUM> and thus form interconnected inlet channel <NUM>, reservoir <NUM>, and outlet channel <NUM>.

The outlet channel <NUM> is thus interconnected to the reservoir <NUM> and the included detection region <NUM> therein and may also be connected to the fill port <NUM>. The cover <NUM> may be bonded to the body <NUM> by any suitable means. For example, the cover <NUM> may be bonded by thermal bonding, ultrasonic welding, adhesive bonding, solvent bonding, or by including a pressure sensitive adhesive layer on the body <NUM>. If a metal layer is used, and additional bonding layer of polymer can be used for thermal bonding.

In some embodiments, sealing of a PCR solution <NUM> in the reservoir <NUM> can involve at least one sealing member comprising heat sealing or deformation sealing along the lengths of one or more of the inlet channel <NUM> and outlet channel <NUM>, or providing a sealing member such as a sealing film <NUM> sealing the top of the fill port <NUM>, for example. Other sealing means may be used prior to PCR processing, such as deformation of the ports <NUM>, <NUM>, sealing of the ports <NUM>, <NUM> by adhesive, or sealing with heavy oil (e.g., a mineral oil) in ports <NUM>, <NUM> or channels <NUM>, <NUM>. If heat sealed, the thermal formed seals can be at two discreet locations along the lengths of the channels <NUM>, <NUM> at locations that can minimize displacement of the plastic volume and maintain acceptable flatness.

The grooves <NUM>, <NUM> may be locally modified to allow improved sealing. In some embodiments, more than one sealed area may be provided along each of the channels <NUM>, <NUM> to provide for a primary and secondary seal for backup. The seals may avoid trapped air in the channels <NUM>, <NUM>. The second seal can be used to contain any displaced solution <NUM> that has been displaced by the first seal. In some embodiments, the fill port <NUM> can include one or more funnels connected one or more of the inlet and outlet ports <NUM>, <NUM>. The included cone angle can be less than <NUM> degrees, for example. The funnels aid in ensuring proper fill with PCR solution <NUM>. The inlet and outlet ports <NUM>, <NUM> may be located approximately equidistant from the detection area <NUM> along the length of the body <NUM> so that they can be easily sealed and both are uninterrupted and uncovered by any part of the PCR station <NUM> (<FIG>).

In operation, as shown in <FIG>, an inlet port <NUM> located in the fill port <NUM> receives the PCR solution <NUM> via a pipette or other liquid dispensing mechanism into the inlet channel <NUM>. The PCR solution <NUM> is a suitable solution allowing amplification of the DNA/RNA (DNA/RNA means DNA, RNA or both as the case may be) templates extracted via conventional PCR sample processing operations. The PCR solution <NUM> includes PCR master mix, DNA/RNA templates, possibly a suitable reagent and possibly water. A PCR master mix is a premixed concentrated solution that has all of the components for a real-time PCR reaction that are not sample-specific. A master mix is a commercially available solution that contains a thermostable DNA/RNA polymerase, dNTPs, MgCl<NUM>, and/or other additives in a buffer optimized for efficient PCR amplification of the DNA/RNA templates.

<FIG> shows the flow path for the PCR solution <NUM> in isolation for illustration purposes. The PCR solution <NUM> flows from the inlet port <NUM> through the inlet channel <NUM> and then into and fills the reservoir <NUM> and the detection region <NUM> therein. The PCR solution <NUM> then exits through the outlet channel <NUM>, which connects to the outlet port <NUM>, which can be co-located in the fill port <NUM> with the inlet port <NUM>. Outlet port <NUM> acts as a vent. When the flow reaches the outlet port <NUM> of the outlet channel <NUM>, the sample holder <NUM> is adequately full. In some embodiments, the inlet port <NUM> may be approximately the size of the pipette tip and air <NUM> may be inserted to move the PCR solution <NUM> further into the inlet channel <NUM>, so as to further minimize the amount of the PCR solution <NUM> needed for replication and detection. The volume of the inlet channel <NUM>, reservoir <NUM>, and outlet channel <NUM> together can be less than <NUM>µL, and from <NUM>µL to <NUM>µL in some embodiments. The volume of the PCR liquid in the reservoir during detection phase can be ≤ <NUM>µL, or even from <NUM>µL to <NUM>µL in other embodiments. The fill port <NUM> can include a volume sufficient to reduce any splashing and ensure proper fill. If the inlet channel <NUM> and outlet channel <NUM> are sealed by heat sealing, the heat sealing should be close to the liquid-air interface, such as <NUM> to <NUM> therefrom to minimize any trapped air.

In more detail, the inlet channel <NUM> can comprise an inlet first channel portion 118A and an inlet second channel portion 118B that is wider than the inlet first channel portion 118A, and thus has a larger cross-sectional area. The inlet channel <NUM> can further comprise an inlet transition portion 118T that allows the inlet first channel portion 118A to generally smoothly transition to the larger inlet second channel portion 118B. The transition portion 118T can allow the PCR solution <NUM> to expand to the larger area of the inlet second channel portion 118B with less turbulence that might undesirably introduce bubbles in the PCR solution <NUM>.

Likewise, the outlet channel <NUM> comprises an outlet first channel portion 122A and an outlet second channel portion 122B that is wider than the outlet first channel portion 122A, and thus of a larger cross-sectional area. The outlet channel <NUM> can further comprise an outlet transition portion 122T that allows the transition from the larger outlet second channel portion 122B to the smaller outlet first channel portion 122A. The transition portion 122T allows the PCR solution <NUM> to contract from the larger area outlet second channel portion 122B to minimize the amount of PCR solution <NUM> in the sample holder <NUM>.

The detection region <NUM> is a region that contains a volume of the PCR solution within the reservoir <NUM> that is adapted to contain the PCR solution <NUM> and replicated DNA/RNA templates after the amplification phase and that viewable by the optical interrogation apparatus <NUM>, as shown in <FIG>.

<FIG> illustrates a PCR station assembly <NUM> of a PCR station <NUM> and sample holder <NUM> mounted therein. The PCR station <NUM> is a fixture configured to hold the sample holder <NUM> during portions of the PCR processing including the amplification phase and detection phase. Amplification phase involves a large number of heating and cooling steps wherein DNA/RNA templates are replicated. Detection involves interrogation with an optical interrogation apparatus, such as optical interrogation apparatus <NUM> as shown in <FIG>. A fluorescence detection interrogation apparatus is shown in <FIG>; however, other suitable configurations and types of optical interrogation apparatus may be used.

In the depicted embodiment, the PCR station <NUM> can include a base <NUM> and a clamp member <NUM>. The base <NUM> and clamp member <NUM> cooperate to form a recess that is appropriately sized to receive and retain, via clamping, the sample holder <NUM> therein. Any suitable clamp initiator <NUM>, such as a screw or electro-, hydraulic- or pneumatic-actuator may be used to initiate the clamping. The clamping ensures intimate thermal contact of the bottom of the sample holder <NUM> with the base <NUM>. The base <NUM> and the clamp member <NUM> may be made out of a highly thermally-conductive material, such as aluminum, copper, or the like.

The PCR station <NUM> can include a temperature-controlling element <NUM>. Temperature-controlling element <NUM> can be a thermoelectric element such as a Peltier device that can rapidly heat and cool the base <NUM> that is in intimate thermal contact with the sample holder <NUM>. Thus, rapid temperature cycling between heating and cooling can be provided as controlled by drive signals from one or more drivers of a controller <NUM> (<FIG>). For example, temperature cycles between a lower nominal temperature of about <NUM> and an upper nominal temperature of about <NUM> can be implemented. Other suitable upper and lower nominal temperatures can be used. About as used herein means +/- <NUM>%.

The PCR station <NUM> can also include one or more heat sinks <NUM> coupled thermally to the base <NUM> and/or possibly to the temperature-controlling element <NUM>. The one or more heat sinks <NUM> may be coupled to one or more sides or top of the base <NUM> and/or to the sides and/or bottom of the temperature-controlling element <NUM>. Any suitable construction of the one or more heat sinks <NUM> may be used. The one or more heat sink may be aluminum or other conductive metal and may include a plurality of fins.

<FIG> illustrates the clamp member <NUM>, base <NUM>, and temperature-controlling element <NUM> in more detail. The clamp member <NUM> includes a viewing aperture <NUM> formed there through, which defines a viewing window for the optical interrogation apparatus <NUM>. The aperture <NUM> may include angled side walls <NUM>, which may be angled at from <NUM> degrees to <NUM> degrees to a central axis of the aperture <NUM>, for example. The viewing aperture <NUM> may be slightly smaller than the dimensions of the reservoir <NUM> and is the window through which fluorescence readings can be taken. The clamp member <NUM> can further include a bore <NUM> formed therein and adapted to receive the clamp initiator <NUM> (e.g., screw, actuator or the like). Spring beams 232B can flex and allow the holding portion <NUM> of the clamp member <NUM> located outboard from the beams 232B to secure the sample holder <NUM> in the pocket 230P (<FIG>) of the base <NUM>. This the clamp member <NUM> acts as a spring clip to ensure good thermal contact between the sample holder <NUM> and the base <NUM>.

Pocket 230P may include a stop <NUM> configured to limit the extent of insertion of the sample holder <NUM> in the pocket 230P. The pocket 230P can include lateral sidewalls 230W that aid in positioning the reservoir <NUM> of the sample holder <NUM> relative to the viewing aperture <NUM>. The holding portion <NUM> can register against lateral sidewalls 230W. Base <NUM> further can include extenders 230E that extend to the width and length of the temperature-controlling element <NUM> to maximize thermal contact therewith.

In some embodiments, the base <NUM> may include a temperature sensor <NUM> in thermal contact with the base <NUM>, such as by being mounted therein or thereon, such as in a hole formed therein proximate the detection region <NUM>. The temperature sensor <NUM> may provide feedback information to estimate the temperature of the PCR solution <NUM> in the detection region <NUM>. The temperature sensor <NUM> may be a thermocouple or a thermistor, for example, and may be used by the controller <NUM> to maintain the upper and lower temperatures of the heating and cooling cycles.

<FIG> illustrates a temperature-controlling element <NUM>, such as a Peltier device that is configured to provide rapid heating and cooling cycles to the base <NUM> and thus to the PCR solution <NUM> contained in the reservoir <NUM> and at the detection region <NUM> of the sample bolder <NUM> mounted therein.

Referring now to <FIG>, a PCR testing system <NUM> is shown and described. The PCR testing system <NUM> includes the PCR assembly <NUM> of the PCR station <NUM> and sample holder <NUM> and the optical interrogation apparatus <NUM>. Optical interrogation apparatus <NUM> is configured to measure the optical emissions from tagged fluorescence (fluorophores) of the PCR solution <NUM> at one or more wavelengths after replication of the DNA/RNA templates. The optical interrogation apparatus <NUM> is an optical system including light source <NUM> such as a white light LED that projects light through collimating optics (e.g., one or more lenses), through a suitable color filter <NUM> that cuts out multiple spectra and allows one spectra to pass (e.g., blue light). This blue light spectrum is reflected off from a suitable dichroic mirror <NUM> and is focused by focusing optics (e.g., one or more appropriate lenses) onto the PCR solution <NUM> in the detection region <NUM> within the reservoir <NUM>. This causes the tagged fluorophores responsive to the blue light to fluoresce. This fluorescent emission is then collimated and passed through the dichroic mirror <NUM>, further filtered with filter <NUM> to remove any stray excitation light and then focused onto a light detector <NUM> with focusing optics (e.g., one or more suitable lenses). The relative intensity of the fluorescent emission measured by the detector <NUM> can render a relative amount of the tagged DNA sequence that is present. At least the filter <NUM> and dichroic mirror <NUM> can be changed out to enable discrimination at one or more other wavelengths, and to allow analysis of sequences.

[not claimed] Referring now to <FIG>, a broad method of operating a PCR testing system is provided according to one or more embodiments of the disclosure. The method <NUM> includes, in <NUM>, providing a PCR station (e.g., PCR station <NUM>) comprising a base (e.g., base <NUM>), a clamp member (e.g., clamp member <NUM>), and a temperature-controlling element (e.g., temperature-controlling element <NUM>) thermally coupled to the base.

[not claimed] The method <NUM> includes, in <NUM>, securing a sample holder (e.g., <NUM>) between the base and clamp member, the sample holder including an inlet channel (e.g., inlet channel <NUM>) interconnected to a fill port (e.g., fill port <NUM>), a detection region (e.g., detection region <NUM>) interconnected to the inlet channel, and an outlet channel (e.g., outlet channel <NUM>) interconnected to the detection region and the fill port. Securing may be by way of a clamp initiator <NUM> or other suitable clamping or securement means.

[not claimed] The method <NUM> includes, in <NUM>, inserting a volume of PCR solution (e.g., PCR solution <NUM>) into the fill port thus filling the detection region. Insertion of the volume may be before or after the insertion of the sample holder <NUM> in the PCR station assembly <NUM>. Thereafter, the channels <NUM>, <NUM>, ports <NUM>, <NUM>, and/or fill port <NUM> may be sealed as described herein.

[not claimed] Next, in <NUM>, the PCR solution <NUM> is subjected to heating and cooling by subjecting the base <NUM> to cycles of heating and cooling with the temperature-controlling element <NUM> to replicate the tagged DNA/RNA templates and to produce tagged replicated DNA/RNA. The tagged replicated DNA/RNA are replicates of the tagged DNA/RNA templates extracted by the previously conducted PCR sample processing via known methods.

[not claimed] According to the method, in <NUM>, after a predetermined number of heating and cooling cycles, fluorescent emissions are detected and measured from the detecting region with an optical interrogation apparatus (e.g., interrogation apparatus <NUM>) by exciting the tagged replicated DNA/RNA with a particular wavelength of light. This can be used to monitor the progress of the PCR process. Other wavelengths of light may be used to excite other fluorescent dyes tagged to the tagged replicated DNA along with associated changes to the respective filter <NUM> (and possibly filter <NUM>) and dichroic mirror <NUM> in the optical interrogation apparatus <NUM> for analysis at other wavelengths as are known to those of ordinary skill in the art.

Claim 1:
A sample holder (<NUM>), comprising:
a body (<NUM>) having a top surface (<NUM>) and a bottom surface (<NUM>), the body (<NUM>) further comprising:
an inlet groove (<NUM>) formed into the bottom surface (<NUM>);
an outlet groove (<NUM>) formed into the bottom surface alongside the inlet groove (<NUM>);
a detection recess (<NUM>) formed into the bottom surface (<NUM>) and connected to the inlet groove (<NUM>) and the outlet groove (<NUM>);
a fill port (<NUM>) interconnected to both the inlet groove (<NUM>) and the outlet groove (<NUM>); and
a cover (<NUM>) connected to the bottom surface (<NUM>)
wherein the cover (<NUM>) interfaces with the body (<NUM>) to form an inlet channel (<NUM>) interconnected to the fill port (<NUM>), a detection region (<NUM>) interconnected to the inlet channel (<NUM>), and an outlet channel (<NUM>) connected to an outlet port (<NUM>) and interconnected to the detection region (<NUM>) and the fill port (<NUM>),
wherein the outlet channel (<NUM>) comprises an outlet first channel portion (122A) and an outlet second channel portion (122B) that is wider than the outlet first channel portion.