Patent Description:
The disclosure herein relates generally to the field of molecular detection. More particularly, the present disclosure relates to an amplification and detection module and automated molecular testing system for quick and low-cost equipment.

Polymerase Chain Reaction (PCR) is a gene amplification technique used in molecular testing of biological samples to identify a vector of interest. Specialized equipment for performing molecular testing with PCR is often expensive and only operable by trained clinicians. Also, it is typically not available "on demand" but runs in batches. The molecular PCR IVD (in vitro diagnostic) industry started with batch processes of <NUM> well formats. While this can deliver mass parallelism and high throughput, especially in cases with higher replicates/batch, the large number of wells affords little sample-to-sample variation and can lead to processing delays for some sample sizes smaller than <NUM> wells. Some companies have developed systems with smaller "batches" such as <NUM> replicates/module in the sample prep section and <NUM> reactions in the amplification detection system. The cost of the "amplification-detection" function may be distributed by requiring that reactions within a module process on synchronized PCR steps.

Another solution in the industry is parallel multiple POC (Point of Care) modules via a robotic feeder. These are based on integrated sample-prep-assay cartridges which combine sample preparation with assays and can be very expensive because they are prepackaged with bulk liquids and reagents. Manufacturing a cartridge with several types of materials to meet the different processing needs is also difficult. While each POC module could technically run independently, a large variety of cartridge types would be needed resulting in an assay library storage facility that was very bulky, large and expensive. Also, the reaction volumes in these POC systems were typically <NUM>µl or more and had rather slow ramp times associated with air cooling (approx. <NUM> degree/s). Therefore, such systems could never be fast and compact. For example, quantitative assays on these types of systems required about an hour of turnaround time (TAT) per sample.

It is known in the prior art a quantitative DNA fragment detection system which includes a DNA amplification module for carrying out a DNA amplification system (<CIT>). The DNA amplification module comprises a chip base and an upper cover. The chip base of the DNA amplification module is placed in the sample well. The upper cover faces the side of a transparent window. A heating ceramic is mounted on the left and right sides of the transparent window. The base of a sample cell is provided with a sample tank. The sample cell is used to place a DNA amplification module.

<CIT>describes a PCR chip assembly that has a thermal gradient that is generated across the surface of an electrode array. The PCR chip assembly may be fabricated by patterning chrome electrodes on glass slides using standard photolithographic techniques. The chip connector/heater assembly can include an aluminum plate that contains two separate heater blocks each contacting a different area of PCR chip assembly and each connected to independently controlled Peltier thermoelectric devices. During the fluorescence detection operation, the chip connector/heater assembly may be mounted on the microscope stage and the fluorescence inside the droplet may be quantified using a sensitive photomultiplier tube (PMT),.

<CIT> provides a microfluidic PCR instrument including a control unit, a PCR chip and temperature control. The PCR chip can be inserted into a card slot formed on the side of the micro heater. The PCR chip is clamped in a clamping mechanism. The PCR chip clamping mechanism composed of a fixing screw is connected between two bracket plates which are sandwiched between two bracket plates.

Most PCR systems provide, at best, "mini-batching" capability where assays are run together with similar or identical protocols. This forces customers to accumulate tests which require identical protocols thus creating a queue of samples. In the case of urgent tests, such as STAT samples, speed is accomplished by occupying a fraction of the available batch process. Most commercial systems have a turnaround time of not less than <NUM> minutes. This total time includes the time to prepare the sample or extract the nucleic acid (sample prep) and the time to perform PCR. Most commercial systems have a batch-processing capability of at least <NUM> samples in the amplification and detection section and four samples in the sample preparation region. It should be emphasized that some systems claim a "flex-batch" process - but typically this involves using a subset of available reaction sites and reduces system throughput per space and increases cost. Most commercial assays were developed to perform tests on <NUM>µl PCR reactions, with some having as low as <NUM> to <NUM>µl reactions. This has negative impacts on theoretical limitations on cost effective speed for PCR. Up until now, there are no high throughput commercial systems capable of truly running random access PCR tests in reaction volumes less than <NUM>µl in a cost-effective manner with assay delivery flexibility into low-cost consumables.

In a first aspect, a random access automated molecular testing system is used with a planar polymerase chain reaction (PCR) chip to provide molecular detection covering a wide variety of assays/tests in a small footprint. A plurality of amplification and detection (AD) modules comprises: a heating block operatively coupled to a controller for controlling the heating block to cycle through a plurality of temperatures; a clip for retaining the planar PCR chip adjacent to the heating block, said clip further comprising a viewing window, wherein the clip comprises a passive spring; and a detection platform adjacent to the viewing window and operatively coupled to the controller for identifying a characteristic of interest of the aliquot of fluid. An automated transport mechanism moves the PCR chip between a pipette loading station, a sealing station and an amplification and detection module to provide batchless and random access amplification and detection of a biological sample fluid.

In another aspect, an amplification and detection module includes a heating block operatively coupled to a controller for causing controlling the heating block to cycle through a plurality of temperatures; a clip for retaining a planar polymerase chain reaction (PCR) chip holding an aliquot of fluid to be tested in a sealed channel adjacent to the heating block, said clip including a viewing window, wherein the clip comprises a passive spring; and a detection platform adjacent to the viewing window and operatively coupled to the controller for identifying a content characteristic of interest of the aliquot of fluid.

In general, PCR (polymerase chain reaction) testing has two main processes: sample preparation (SP) and amplification and detection (AD). Testing and/or identifying nucleic acids, for example, in a biological fluid sample requires sample preparation to isolate nucleic acids for further processing. In general, sample preparation involves lysing or liberating nucleic acids (NA) from sample material in a liquid state then separating the NAs in an eluate in a process that may involve several steps.

A lower standard cost of PCR assays and faster TAT is provided by random access testing and low reagent usage that concentrate the nucleic acids from biological sample into a smaller elution volume. In embodiments, the nucleic acids from standard working volumes, for example, <NUM>µl, are concentrated into a smaller liquid volume, such as approximately <NUM> to <NUM>µl. In other words, if a larger volume of eluate contained <NUM> target nucleic acid molecules and was processed in <NUM>µl PCR reactions, the random access automated molecular testing system disclosed herein operates on the same <NUM> molecules in a smaller <NUM> or <NUM>µl reaction volume. Therefore, there is no loss in sensitivity as all the nucleic acids available in the sample are captured as efficiently as in a larger eluate.

For purposes of illustration, a representative example of sample preparation will now be described below. Each patient sample is processed within a consumable. The sample prep consumable typically would contain separate chambers (process element volumes) to process each of the steps. Once a patient sample has been aspirated into the sample prep consumable, a sample preparation process may be broadly described as including the following steps:.

The steps described above are representative. Variations may be made to prepare samples for different assays.

The eluate, or concentrated nucleic acids, is then combined with other reagents for amplification and detection (AD). In embodiments, a random access automated molecular testing system includes modules, systems and methods for performing PCR AD without batch processing. Batchless processing provides complete flexibility in running AD protocols. This includes being able to run a melt assay on one AD module while performing an entirely different protocol on another module without any requirement that the protocols be synchronized.

<FIG> depict a top and bottom perspective views of a PCR chip <NUM> for random access automated molecular testing. PCR chip <NUM> includes a planar body <NUM> that is generally rectangular and a gripping feature <NUM> laterally extended from an upper surface of one end of planar body <NUM>. Planar body <NUM> includes internal features generally indicated at <NUM>, including ports <NUM> and <NUM>, that are used for filling and retaining an eluate for PCR amplification and detection. PCR chip <NUM> is approximately <NUM> long by <NUM> wide. Planar body <NUM> and gripping feature <NUM> are molded from a plastic such as polypropylene but any plastic that can withstand temperatures of PCR thermal cycling and that is not autofluorescent may be used. Dimensions used herein are for purposes of illustration.

Internal features <NUM> provide PCR AD on an aliquot of eluate, such as <NUM> or <NUM>µl, by leveraging advances in component technology. For example, advances in electronics components in analog and digital processing, communications, LED, photodetectors and general-purpose processors, magnetics, sample preparation technology, and micromolding allowed for high-throughput platforms to be created from replicas of unit process module subsystems and small volume PCR chips. Internal features <NUM> are formed in a bottom surface of planar body <NUM>, then sealed with a film laminated to the bottom of planar body <NUM>. The film is an aluminum tape with silicone adhesive but any laminate with low autofluorescence and good adhesion to polypropylene may be used.

In the following description, internal features <NUM> of PCR chip <NUM> will be described in more detail followed by a description of the modular processing system for performing assays using PCR chips <NUM>.

<FIG> depicts a top view of the PCR chip of <FIG>. <FIG> depicts a cross-sectional view along line 2B-2B of <FIG> depicts a detail view from <FIG> do not depict the film laminated to the bottom of planar body <NUM>. <FIG> are best viewed together in the following description.

<FIG> shows gripping feature <NUM> includes two overlapping cylinders <NUM> and <NUM>. Ports <NUM> and <NUM> are centered with cylinders <NUM> and <NUM> respectively. Inlet port <NUM> receives a PCR eluate through a pipette or other filling device. Cylinder <NUM> ends in a curved or tapered surface <NUM> where it meets inlet port <NUM>. This surface provides a seal with a tip of a pipette and also helps compensate for chips that may be off axis during automated processing. <FIG> depicts a detail view of <FIG> showing curved surface <NUM>. Although a specific curvature is shown, a variety of profiles may be used to provide sealing and alignment of chip <NUM> during a filling process. Vent port <NUM> in cylinder <NUM> serves as a vent during a filling process. Cylinders <NUM> and <NUM> of gripping feature <NUM> provide a mechanism for gripping and moving chip <NUM> during automated processing, and also serve as a containment or overflow reservoir for fluid during a filling process.

<FIG> depicts a bottom view of the PCR chip <NUM> including internal features <NUM>. <FIG> depict cross-sectional views and <FIG> depicts a detailed view of the PCR chip of <FIG> do not depict the film laminated to the bottom of planar body <NUM>.

An eluate is introduced into PCR chip <NUM> through inlet port <NUM> while vent port <NUM> provides a vent as described above. From inlet port <NUM>, eluate travels through passage <NUM>, reservoir <NUM> and passage <NUM> to one end of U-shaped channel <NUM>. The other end of U-shaped channel <NUM> is connected to vent port <NUM> through passage <NUM>, reservoir <NUM> and passage <NUM>. U-shaped channel <NUM> is sized to hold approximately <NUM>µl of eluate. The fluid volume within the PCR chip <NUM> is less than <NUM>µl and typically less than <NUM>µl and more than <NUM>µl. Furthermore, PCR chip <NUM> is thermally sealed with a largely planar construction providing a fluid thickness in U-shaped channel <NUM> not exceeding approximately <NUM>.

<FIG> depicts a cross-sectional view of passages <NUM> and <NUM> along line 3B-3B. <FIG> depicts a cross-sectional view of U-shaped channel <NUM> along line 3C-3C. Passages <NUM> and <NUM> have a width of approximately <NUM>. Each arm of U-shaped channel <NUM> in <FIG> has a width of approximately <NUM>. The heights of passages <NUM> and <NUM> as measured relative to the overall height of planar body <NUM> are flexible as long as they provide unimpeded flow for eluate. Passages <NUM> and <NUM> are similar to passages <NUM> and <NUM>. The height and width of U-shaped channel <NUM> are flexible as long as a volume of approximately <NUM>µl is provided.

<FIG> depicts a detailed view of the connection between passage <NUM> and U-shaped channel <NUM>. The specific shape is illustrative and any transition between the smaller width of the passage and the larger width of the U-shaped channel may be used. The connection between U-shaped channel <NUM> and passage <NUM> is similar.

<FIG> depicts a bottom view of the PCR chip <NUM> including internal features <NUM>. <FIG> depicts a detailed view of reservoir <NUM> and <FIG> depicts a cross-sectional view of reservoir <NUM> along line 4C-4C. <FIG> depicts a detailed view of reservoir <NUM> and <FIG> depicts a cross-sectional view of reservoir <NUM> along line 4E-4E.

Reservoirs <NUM> and <NUM> serve as volume reservoirs for fluid overflow when eluate is sealed in U-shaped channel <NUM>, as described in more detail below. As eluate enters inlet port <NUM>, it flows through passage <NUM> to reservoir <NUM>. Although reservoir <NUM> is depicted as a square with rounded corners, this specific shape is not required so long as a sharp edge is provided at <NUM>. This sharp edge acts as a pinning region to prevent capillary flow and retains fluid in U-shaped channel <NUM>. As shown in <FIG>, reservoir <NUM> has a greater height than passages <NUM> and <NUM>. Sharp edge <NUM> forms an approximately <NUM>-degree angle with passage <NUM> in both the horizontal direction along the width of PCR chip <NUM> as shown in <FIG> and in the vertical direction along the height of PCR chip <NUM> as shown in <FIG>. Edge <NUM> between reservoir <NUM> and passage <NUM> is angled. The reservoir <NUM> of <FIG> has a circular shape and gradual transition between reservoir <NUM> and passages <NUM> and <NUM>.

PCR chip <NUM> may be used with a random access automated molecular testing system <NUM> as shown in <FIG>, in embodiments. <FIG> depicts a top view of system <NUM> and <FIG> depicts a side view. <FIG>, <FIG>, <FIG>, and <FIG> depict detailed views of various aspects of system <NUM>. <FIG> are best viewed together in the following description.

System <NUM> provides an automated transport mechanism for performing a PCR assay by moving PCR chips <NUM> between several processing stations. Elements of system <NUM> may be controlled with a controller including hardware and software for storing and executing computer-implemented instructions. Gripper <NUM> may be controlled to move in X and Y directions using chip transport system including X-axis drive <NUM> and Y-axis drive <NUM>. Other chip transport mechanisms are contemplated. Gripper <NUM> retrieves a chip from one of three chip feeders <NUM>. As shown in <FIG>, jaws <NUM> of gripper <NUM> are adapted for reversible lateral movement to selectively grasp gripping feature <NUM> of PCR chip <NUM>. Although three chip feeders with a capacity of approximately <NUM> chips each are shown, any number and capacity of chip feeders <NUM> may be provided. A cross-sectional side view of a chip feeder <NUM> is shown in <FIG>, in embodiments. As shown, each chip feeder is adapted to retain a substantially vertical plurality of PCR chips <NUM>, with the gripping feature <NUM> of each disposed towards an open end of the respective chip feeder, such that the gripping feature <NUM> of the lowest PCR chip <NUM> may be engaged by a gripper <NUM>. Other arrangements that allow an automated gripper to select a single chip are contemplated.

Gripper <NUM> moves a selected PCR chip <NUM> to pipette loading station <NUM> at which one or more pipettes (not shown) are used to introduce an eluate and assay reagents to inlet port <NUM> as described above. Next, PCR chip <NUM> is moved to sealing station <NUM>. Referring to <FIG>, passages <NUM> and <NUM> are heat sealed to prevent evaporation and leaks during thermal cycling. Heat sealing may also or alternatively be applied to passages <NUM> and <NUM>. A goal of heat sealing is also to minimize the air volume in U-shaped channel <NUM> because this creates internal pressure during cycling and causes chip <NUM> in the region of U-shaped channel <NUM> to flex. Reservoirs <NUM> and <NUM> provide for volume overflow when passages <NUM> and <NUM> are sealed.

After sealing, gripper <NUM> moves the filled and sealed PCR chip <NUM> to one of amplification and detection (AD) modules <NUM>, shown in more detail in <FIG> and <FIG>. Each AD module <NUM> is comprised of a detection platform <NUM> oriented to receive a largely planar PCR chip <NUM>. In embodiments, detection platform <NUM> includes an LED and camera-based detection system such as CMOS cameras or photodetectors. Detection platform <NUM> interrogates a field of view through viewing window <NUM> corresponding to U-shaped channel <NUM> containing a volume of eluate and assay reagent to identify a characteristic of interest. PCR chip <NUM> is thermally sealed and the planar construction provides a small distance between the fluid volume and temperature controlled surface <NUM>, for example the thickness of fluid in U-shaped channel <NUM> does not exceed approximately <NUM>. In embodiments, temperature controlled surface <NUM> may be a Peltier heater. Further, PCR chip <NUM> allows for single - pipette based loading and with a thermal contact force generated without additional discrete bearings or linkages. Temperature sensing element <NUM> is used to provide feedback to a controller for control the thermal cycling of surface <NUM>.

<FIG> depicts an exploded view of the module <NUM> of <FIG>. The bottom of PCR chip <NUM> is laminated to aluminum foil <NUM> to retain fluid volume in U-shaped channel <NUM>. Aluminum clip <NUM> acts as a spring to retain PCR chip <NUM> against aluminum block <NUM>. The use of a passive spring force ensures thermal contact and sliding PCR chip <NUM> into clip <NUM> is automation friendly. In embodiments, heating block <NUM> is a thermoelectric cooler. Aluminum foil <NUM> acts as a thermal spreader to improve heat transfer from block <NUM> to U-shaped channel <NUM>. Aluminum foil <NUM> also acts as a reflective surface to enhance optical readings because it is opaque and thus blocks any debris or dust on block <NUM> that might impact analysis.

Automated molecular platform <NUM> includes a series of coplanar AD modules <NUM>. AD modules on platform <NUM> may be controlled individually or as a group.

After the AD process is completed, gripper <NUM> moves PCR chip <NUM> to waste chute <NUM> for disposal in a tube (not shown) retained in tube holder <NUM>.

In embodiments, several variations of the system described herein are contemplated. AD module <NUM> may be considered to be an assembly of submodules. These submodules facilitate design and manufacturing, service and calibration activities. AD module <NUM> may be:.

An amplification only module - in which case a chip is PCR amplified in one module but reading is accomplished at end-point (typical of a dPCR and certain qualitative assays) in another module.

A detection module - in which a pre-amplified chip is inserted into an "end-point" reading module. The detection module could be an image based detection of sub-reactions within the chip or integral detection, such as with a photodiode or small photomultiplier tube (sPMT).

A combined module - the AD thermal control and amplification is coupled to the detection module via a certain correspondence and appropriately calibrated. The trade-offs between a combined module and special purpose modules is that a combined module is not the most cost-effective approach if all the assays are an endpoint (for example a melt assay). However, this approach affords the greatest flexibility and one less transport step.

<FIG> depicts a flowchart illustrating a method <NUM> of random access automated molecular testing.

Step <NUM> includes retrieving a PCR chip from a chip feeder. In an example of step <NUM>, gripper <NUM> selects a PCR chip <NUM> from chip feeder <NUM>.

Step <NUM> includes filling the selected PCR chip with an eluate and assay reagent. In an example of step <NUM>, gripper <NUM> moves PCR chip <NUM> to pipette loading station <NUM> where it is filled from one or more pipettes.

Step <NUM> includes sealing the PCR chip. In an example of step <NUM>, gripper <NUM> retrieves PCR chip <NUM> from pipette loading station <NUM> and moves it to sealing station <NUM> where at least passages <NUM> and <NUM> are heat sealed.

Step <NUM> includes moving the PCR chip to an AD module for analysis. In an example of step <NUM>, gripper <NUM> retrieves PCR chip <NUM> from sealing station <NUM> and automatically moves it to any AD module <NUM> in platform <NUM>. The selection and processing of an AD module <NUM> may be automatically controlled by a computer processor executing instructions stored in a non-transitory memory.

Step <NUM> includes performing an AD assay. In an example of step <NUM>, AD module <NUM> is thermally cycled while detection platform <NUM> takes an image through viewing window <NUM> every cycle.

Step <NUM> includes removing the PCR chip from the module for disposal. In an example of step <NUM>, gripper <NUM> removes PCR chip <NUM> from AD module <NUM> after thermal cycling is complete and places it in waste chute <NUM> for disposal in a tube (not shown) retained in tube holder <NUM>.

As disclosed herein, a PCR chip has a form factor such that many different future types of assays could be run by simply changing the assay supply with minimal modification to the balance of the system thereby supporting a product family. In embodiments, modified chips would be part of a library of chips and that could be processed using independently controlled modules or assay-specific modules that utilized similar technologies, power, communication architecture and size requirements. Other processing variations may be accommodated without having to fundamentally change the way assay reagents were loaded, and chips were used and transported. In addition, separating the sample preparation (SP) and amplification and detection (AD) processes into separate devices allows the most appropriate and minimal materials to be selected according to utilization (material specification, packaging, transport) for the AD consumable. It also provides maximum flexibility in materials selections for the PCR chip vs the SP consumable. For example, in the case where a random access automated molecular testing system requires a higher temperature grade and more stable plastic, or a plastic with certain wetting, autofluorescence or porosity requirements, this could be supported while maintaining minimum plastic costs on the SP consumable and other more commonly used PCR chips.

A random access automated molecular testing system <NUM> for amplification and detection may be used with a sample preparation process to provide a complete system. In embodiments, an assembly line sample preparation process, or extraction, may be delivered to individual detection channels. Sample extraction channels may have an approximately <NUM> minute total process time with a throughput of <NUM> extractions/hour/channel or <NUM> x <NUM> = <NUM> extractions/hour. AD channels receiving prepared samples may have an approximately <NUM> minute total process time with a throughput of <NUM> reactions/hour/channel or <NUM> x <NUM> = <NUM> reactions/hour.

Alternatively, individual sample extraction channels may be delivered to individual detection channels. Sample extraction channels may have an approximately <NUM> minute total process time with a throughput of <NUM> extractions/hour/channel or <NUM> x <NUM> = <NUM> extractions/hour. AD channels receiving prepared samples may have an approximately <NUM> minute total process time with a throughput of <NUM> reactions/hour/channel or <NUM> x <NUM> = <NUM> reactions/hour.

Changes may be made in the above methods and systems without departing from the scope of the claims. For example, PCR chips may be loaded into the system in a variety of ways, including bowl-fed, from a tape, a cartridge or a plate-based format. PCR chips may be provided with thermally conductive window for scanning, or may be scanned from both sides or either side. In addition, a PCR chip may be used with an amplification-only or a detection-only module as disclosed herein.

Claim 1:
An amplification and detection module, comprising:
a heating block (<NUM>) operatively coupled to a controller for controlling the heating block (<NUM>) to cycle through a plurality of temperatures;
a clip (<NUM>) for retaining a planar polymerase chain reaction (PCR) chip (<NUM>) holding an aliquot of fluid to be tested in a sealed channel adjacent to the heating block (<NUM>),
said clip (<NUM>) further comprising a viewing window (<NUM>), wherein the clip (<NUM>) comprises a passive spring; and
a detection platform (<NUM>) adjacent to the viewing window (<NUM>) and operatively coupled to the controller for identifying a characteristic of interest of the aliquot of fluid.