Patent Description:
In some protocols for cell lysis and nucleic acid isolation using magnetic beads, a sample is moved by a pipette system to a well within a multi-well plate, or sample holder, along with a cell lysis buffer and a quantity of magnetic beads. The beads are functionalized, for example with silica surfaces, to allow selective binding of nucleic acid molecules such as DNA. A succession of mixing by external vibration, magnetic bead separation, supernatant aspiration, and dilution/washing steps are repeated. These may be performed in the same well, or the magnetic beads may be moved from well to well for various steps. Heating of one or more of the wells of the sample holder may also be employed to facilitate lysis and/or binding. The sample transfer, washing, and elution steps require separate aspiration and dispensing tips to avoid cross-contamination.

Using magnetic beads in a sample fluid contained within a well to capture and extract nucleic acids from the specimens requires magnetic devices as the tools for accomplishment of workflow. Magnets may be used inside a probe that is inserted into the well to collect the magnetic beads prior to transfer to another well. Magnets may also be used outside the well to manipulate magnetic beads. Manipulating magnets in a fluid by magnetic movement and shaking the well itself, as well as by vortex mixing, are also known.

Effective mixing is critical in cell lysis and washing steps for sample preparation to ensure that an adequate amount and quality of nucleic acids are extracted from the sample. Vortex mixing is one of the most effective mixing techniques, but to generate a vortex in a small volume is difficult without a bulky setup.

It is known in the art cell culture vessel for the automated processing of cell cultures (<CIT>). The cell culture vessel assembly aids aeration and allows for reading of the optical density of the culture without removing the culture from the vessel. It is suitable for use in the production and purification of cell culture products and in particular to the automated production and purification of protein. A culture aeration unit for aerating liquid media, comprises a culture vessel and a mixing rod. The culture vessel has an open end for receiving liquid media and a closed end. The mixing rod has a first and second end. The open end of the culture vessel is being adapted to receive a first end of the mixing rod so that the mixing rod extends into the culture vessel. The mixing rod is being adapted for movement such that movement of the first end of the mixing rod is greater than movement of the mixing rod adjacent the open end of the culture vessel.

An internal medicine test device for convenient replacement of stirring structure is known in <CIT>. The medical testing and agitating device comprise bottom plate, switch, first rotating shaft, a sleeve, a connecting rod, a horizontal plate, support rod, the second rotating shaft, a bearing bottom, straight rod, motor and a stirring rod. The bearing bottom is connected with the support rod. The support rod is fixedly connected with the horizontal plate. The device is suitable for mixing of a sample with different lengths of test tubes.

In the field of molecular diagnostics, there is a need for an efficient and costeffective system and method for lysing cells and purifying samples for amplicon detection. There is further a need for mixing magnetic beads in multiple processing steps that minimizes liquid handling, contamination and reagent carryover.

A system and method for extracting nucleic acids from specimens using both magnetic and mechanical oscillation to enhance the speed and efficiency of mixing is disclosed. Vortex generation in a multi-well sample holder is performed by vibration rods. Vibration rods attached to cantilevers and inserted into wells may generate a high-speed vortex in a very confined space with small volume of fluid. The driving unit or oscillation source is a rotating cam (or non-round profile action such as rotating spur gear) that interacts with tabs on the cantilevers. In embodiments, a vibration rod method does not vibrate the containers or tubes, but simply transfers the oscillation energy directly to the fluid with the vibration of rods so that a vortex is generated in a very small confined space at high speed. Also, the magnetic beads for target nucleic acid extraction may be agitated physically with the rod vibration, leading to less aggregation in real biological samples, i.e. whole blood. Magnetic beads and associated nucleic acid molecules may be moved between wells using a magnetic that is external to the sample holder.

A sample holder and cover may be assembled easily and flexibly. The sample holder is an array of wells that hold fluid and magnetic beads during processing and moving from well to well. The sample holder cover is an array of vibration rods corresponding to some or all of the wells.

Illustrative embodiments of the disclosed technology are described in detail below with reference to the attached drawing figures:.

Disclosed herein is an apparatus for extracting nucleic acids such as DNA molecules from biological samples. Use of the presently disclosed and described apparatus enables simplified, easy, and reliable mixing and transfer of magnetic beads between wells in a fashion particularly suitable to automated sample preparation techniques. In embodiments, magnetic beads may be magnetic micron- or nano-particles with a surface modification which binds target nucleic acids released during a sample preparation process of biological specimens. In the systems and methods described herein, a magnetic field is the only driving force for sample and/or magnetic bead handling and transportation. Liquid handling is eliminated resulting in minimization of cross-contamination and liquid carryover. Magnetic beads are transferred from well to well within a consumable sample holder using a single module to drag beads along an internal plastic surface of the consumable, providing improved cleaning and drying. In further embodiments, the consumable sample holder is self-contained, cleaning of other components of the system is not required, thereby minimizing biohazard exposure.

<FIG> illustrates an exemplary embodiment of an apparatus <NUM> for extracting nucleic acids from specimens according to the present disclosure. The apparatus <NUM> is comprised of a base, indicated generally at <NUM>, <NUM>, <NUM> and <NUM>. Base <NUM> is used to retain vortex generator assembly <NUM> in position together with heating blocks <NUM> and <NUM>. In the embodiment of <FIG>, a second vortex generator assembly <NUM> is retained in position by heating block <NUM> and heating block <NUM>. Heating blocks <NUM>, <NUM> and <NUM> each include a cavity <NUM> for receiving a lysis well and a cavity <NUM> for receiving an elution well of vortex generator assemblies <NUM> or <NUM>, discussed in more detail with reference to <FIG>. Cavity <NUM> of heating block <NUM> is empty as shown in <FIG>, but may receive a lysis well of a third vortex generator assembly. Any number of vortex generator assemblies may be connected in sequence, within the range of motion of the stepper motors described below.

Apparatus <NUM> further includes a stepper motor <NUM>. Selective actuation of motor <NUM> causes rotating cam <NUM> to spin and translate rotary motion into linear motion of vibration rod <NUM>. In an exemplary embodiment, the rotating cam spins at approximately <NUM> to <NUM>. The contact angle between rotating cam <NUM> and a vibration rod <NUM> may also be finely adjusted to optimize the vortex within wells. A horizontal actuator (not shown) moves motor <NUM> along an x-axis as defined by axes <NUM>, repositioning motor <NUM> along vortex generator assemblies <NUM> and <NUM> during sample processing. Motor <NUM> may also include a vertical actuator for moving motor <NUM> along the z-axis.

In addition, the apparatus <NUM> may include an magnet <NUM> external to vortex generator assemblies <NUM> and <NUM> that may be selectively translated by stepper motor <NUM> in all three of x, y and z axes. Magnet <NUM> may be moved along vortex generator assemblies <NUM>, <NUM> along the x-axis, closer and farther away from a side wall of a vessel along the y-axis as defined by axes <NUM>, in order to attract and release, respectively, magnetic beads disposed within a vessel, and vertically along the z-axis, as will be discussed subsequently. Operation and oscillation of motors <NUM> and <NUM> may be synchronized or individual for optimization and different operating modes.

An embodiment of vortex generator assembly <NUM>, <NUM> is shown in <FIG>, Vortex generator assembly may also be referred to as a multi-well plate, or sample holder, for use in cell lysing and nucleotide purification together with a sample holder cover. <FIG> illustrates an embodiment of a sample holder <NUM> having a body member and a plurality of wells extending in a downwardly direction from the floor of the body member, according to the present disclosure. In this embodiment, the body member is a channel <NUM> and the process wells include a lysis well <NUM>, wash wells <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and elution well <NUM>. Channel <NUM> may help inhibit the unintended flow of working fluid off sample holder <NUM>.

Lysis well <NUM> is disposed at a first end <NUM> of the sample holder <NUM> while the wash wells are disposed intermediate the first end and an opposite second end <NUM> where elution well <NUM> is located. Each well extends in a substantially orthogonal direction from the floor of the channel <NUM> and has an interior volume communicating with the channel via an aperture in the channel floor. The illustrated apertures are circular and coplanar with the floor surface, although embodiments of differing shapes and orientations are also contemplated. The apertures are also substantially colinear along the floor surface and are centered about a longitudinal axis <NUM> of symmetry of the sample holder.

In order to optimize vortexing, lysis well <NUM> may have a larger volume than the wash wells in order to provide sufficient space for the biological sample, lysis buffer, and magnetic beads. Conversely, elution well <NUM> may have a smaller volume than the wash wells in order to minimize dilution of the final nucleic acid product and may be characterized by a conical cross-section to facilitate removal of the product with a pipettor or other devices for transferring fluids.

Lysis well <NUM> of sample holder <NUM> may be subjected to heating, depending upon the characteristics of the lysis process implemented therewith. For example, the outer surface of the lower extent of the lysis well <NUM> may be configured to be received within a heater external to the unitary structure. Such a heater may be a heating block <NUM> placed beneath the holder, receiving the outer surface of the lower extent of lysis well <NUM> within cavity <NUM> and heating lysis well <NUM> for a required or desired time period. Similarly, the elution well <NUM> of the sample holder <NUM> may be heated with another heater external to the unitary structure, such as cavity <NUM> of heating block <NUM>, depending upon the elution process implemented therewith. Heating blocks may provide temperatures up to approximately <NUM>.

In one embodiment, the wells are pre-filled with appropriate buffers and other components and then sealed off, for example with a peel-away layer that is removed at the time of use. In another embodiment, the wells each have a tapered lower extent. This enables multiple sample holders to be vertically stacked, whereby the outer surface of a lysis well of a first holder is received within the lysis well of a lower, second holder. Similarly, the outer surfaces of the wash wells of the first holder are each received within a respective wash well of the lower, second holder.

Sample holder <NUM> may be provided with retention features, such as tab <NUM> projecting from the upper rim of channel <NUM> or other lateral projections extending from the sample holder on either side of sample holder <NUM>. During processes such as heating and vortexing, when external devices move relative to sample holder <NUM>, the retention features may be selectively engaged by external releasable gripping mechanisms, thereby maintaining the multi-well plate in a fixed position relative to the external devices. The retention features may also be of use during the introduction of samples, buffers, beads or other components in the wells or eluted product retrieval as a pipetting system presses down on the inner surface of the elution well <NUM>. Alternatively, sample holder <NUM> and associated heating blocks and support structures, i.e., base <NUM>, may be configured for lateral, horizontal translation relative to the motors <NUM> and <NUM>, thus obviating the need for enabling horizontal translation of the rotor mixer and associated components.

In embodiments, sample holder <NUM> may handle a wide range of fluid quantities, from <NUM> to 3µL in a single piece because the sample preparation procedure of biological specimens may vary widely among different matrices from whole blood, plasma, serum, stool, urine, sputum, swabs. Sample holder <NUM> provides flexibility to cover all those but not limited to the aforementioned specimen types. In embodiments, sample holder <NUM> may include seven compartments in one molded piece of polymer selected from, for example, polypropylene, polyethylene, polyethylene terephthalate (PET), cyclic olefin copolymer, polycarbonate or polyacrylates.

In embodiments, a volume size of wells in sample holder <NUM> ranges from <NUM> to <NUM> to <NUM> to <NUM>µL for different applications with different fill liquid. The compartments can be assigned to Lysis, Incubation, Washing, Drying and Extraction functions with different programming.

<FIG> illustrates a sample holder cover <NUM> for use with sample holder <NUM> of <FIG>, in an embodiment. Sample holder cover <NUM> includes a base member <NUM> sized and shaped for insertion into channel <NUM>. Base member <NUM> may be retained within channel <NUM> in several ways. It may be sized to fit snugly into channel <NUM> with a friction fit, it may snap into channel <NUM> or it may be retained with channel <NUM> using one or more clips. In embodiments, base member <NUM> includes a plurality of vibration rods <NUM>, <NUM> extending in a downwardly direction from base member <NUM>. As shown in <FIG>, vibration rods <NUM> correspond to wash wells <NUM>, <NUM>, <NUM>, <NUM> and <NUM> of sample holder <NUM>, however, any number of vibration rods may be provided. Vibration rod <NUM> is intended for insertion into lysis well <NUM> and thus, is proportionally bigger than vibration rods <NUM> in the same way that lysis well <NUM> is proportionally larger than wash wells <NUM> - <NUM>. Vibration rods <NUM>, <NUM> are positioned on base member <NUM> so as to be generally centered in the interior volume of a respective well when base member <NUM> is inserted into channel <NUM>.

A tab, indicated at <NUM>, corresponds to each vibration rod <NUM>, <NUM> and is used to cause vibration rods <NUM>, <NUM> to vibrate in conjunction with rotating cam <NUM> of <FIG>. Tabs <NUM> extend from base member <NUM> in an upwardly direction relative to vibration rods <NUM>, <NUM>. Tabs <NUM> also extend above rim <NUM> of base member <NUM> for engagement with rotating cam <NUM>. In embodiments, tabs <NUM> may be recessed below rim <NUM> and rotating cam <NUM> may be positioned to extend down into cover <NUM> slightly to engage with tabs <NUM>.

<FIG> shows a bottom perspective view of sample holder cover <NUM>. Each vibration rod <NUM> is attached to base member <NUM> at one end of a cantilever <NUM>. A tab <NUM> is located on the opposite side of each cantilever <NUM> as shown in <FIG>. Cantilever <NUM> allows vibration rod <NUM> to be vibrated at a selectable speed and duration through control of rotating cam <NUM>.

<FIG> shows a bottom perspective view of a portion of sample holder <NUM> and sample holder cover <NUM> combined to form a vortex generator assembly <NUM>, <NUM>. A more detailed view <NUM> is shown in <FIG>.

<FIG> shows a close-up bottom view of a vortex generator assembly <NUM>. Sample holder <NUM> is shown transparently so details of sample holder cover <NUM> may be illustrated. Sample holder cover <NUM> has been inserted into sample holder and held in place using snapping mechanism <NUM>. Mechanism <NUM> is depicted as a tab on sample holder cover <NUM> that is inserted and retained in a slot on sample holder <NUM>, but other mechanisms and designs may be used. Other mechanisms for retaining the sample holder cover in the sample holder are also contemplated.

Vibration rod <NUM> is attached to cantilever <NUM>, which has been formed from a floor of base member <NUM> by cutout areas <NUM> and <NUM>. In embodiments, cutout area <NUM> allows the oscillation/vibration from rotating cam <NUM> through tab <NUM> to vibration rod <NUM> to be performed with less energy loss. It may also provide access for liquid dispensing and aspiration. Cutout areas <NUM>, <NUM> and <NUM> create cantilever <NUM> which is able to oscillate up and down in response to engagement of rotating cam <NUM> with tab <NUM>. As explained above with reference to <FIG> and <FIG>, the oscillation driving force from spinning cam <NUM> motion engages with tab <NUM> on the opposite side of cantilever <NUM> (not visible in this view) which leads to vibration at the rod end <NUM>, as shown by motion arrows <NUM>. The design of vibration rod <NUM> causes vibration energy to be directly transferred to fluid in sample holder <NUM>, creating a vortex <NUM> in fluid in sample holder <NUM>. Further, the dimension of the rod allows generation of vortex in very confined and small volume.

Rod <NUM> has an asymmetric cross section along its length extending from the base member to its tip, as shown at rod end <NUM>. Flanges <NUM> and <NUM> on either side of rod end <NUM> enhance the creation of vortex <NUM>. Although a representative cross-section and flange arrangement has been shown, this is for purposes of illustration and other designs for vibration rod <NUM> are contemplated. The shape and dimension of vibration rods may be customized for different fluids and methods of mixing without requiring changes to the overall system.

<FIG> shows a vortex generator assembly with sample holder cover <NUM> is inserted in sample holder <NUM>. Lysis well <NUM> is shown in cross-section to illustrate the positions of vibration rod <NUM> and tab <NUM>. Rotating cam <NUM> engages with tab <NUM> to impart a vibration to vibration rod <NUM>. As stepper motor <NUM> moves between adjacent tabs, rotating cam <NUM> engages with adjacent vibration rods. Because of the cantilever attachment of vibration rod <NUM>, the vibration movement of vibration rod <NUM> will be largest at rod end <NUM>. This may provide improved fluid handling and less aggregation in some types of sample fluid. Further customization of vortex mixing may be provided by controlling the spin rate and length of rotation cam <NUM>.

Referring to <FIG>, operation of apparatus <NUM> will now be described. Stepper motor <NUM> causes rotating cam <NUM> to spin and translate rotary motion into linear motion of vibration rod <NUM>. A horizontal actuator (not shown), repositions motor <NUM> along vortex generator assemblies <NUM> and <NUM> during sample processing. Stepper motor <NUM> includes actuators for moving magnet <NUM> in three directions: horizontally along the x-axis to reposition magnet <NUM> along vortex generator assembly <NUM>, horizontally along the y-axis from positions closer to and farther away from assembly <NUM>, and vertically along the z-axis. Motors <NUM> and <NUM> may be independently controlled so that both magnetic and mechanical oscillation may be used to mix and manipulate magnetic beads and fluid in a sample holder.

<FIG> is a flowchart of a method of operating the components of apparatus <NUM>. Not all steps need be practiced in the order described below, nor be utilized at all, depending upon the embodiment.

Step <NUM> includes placing a sample, magnetic beads and other fluids or buffers in sample holder <NUM>. In an example of step <NUM>, one or more wash buffers are loaded into the wash wells <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, an elution buffer is loaded into elution well <NUM>, and lysis buffer is loaded into the lysis well <NUM>. Magnetic beads are also introduced in the lysis well <NUM>. In one example, the material of the beads may be optimized for genomic DNA extraction from blood samples, but its composition may vary to suit other types of bodily fluids or tissues or for extracting other types of nucleic acids such as RNA. A biological sample is then loaded into the lysis well, yielding a lysis mixture ready for vortexing. Typical samples include blood, sputum, hair, and other bodily fluids and tissues, optionally pretreated for example by freezing, homogenizing, or grinding. Those of skill in the art will recognize that the choice of buffers and other reactants may vary according to the type of sample and beads to provide optimal conditions for nucleic acid extraction. While this illustrated process depicts a certain order of loading the lysis well to form the lysis mixture, other orders may be employed, such as disposing the sample into the lysis well prior to adding the magnetic beads. In embodiments, sample holder <NUM> may be preloaded with fluids or they may be added with a pipette system, for example.

Step <NUM> includes installing a sample holder cover <NUM> on sample holder <NUM>. In an example of step <NUM>, sample holder cover <NUM> may be snapped or otherwise securely retained in sample holder <NUM>.

Step <NUM> includes moving rotating cam <NUM> into position over a lysis well <NUM> of sample holder <NUM>. In an example of step <NUM>, rotating cam <NUM> is positioned so that it will make contact with a tab <NUM> of a vibration rod <NUM> inserted into lysis well <NUM>.

Step <NUM> includes operating rotation cam <NUM> for a selected period of time to cause a vortex in lysis well <NUM>. In an example of step <NUM>, both a rotation speed and length of time for rotating are selectable depending on the sample being mixed and mixing may be performed either continuously or intermittently. For at least a portion of the vortexing step, the rotating cam <NUM> is spun at a rate sufficient to overcome attraction forces between magnetic beads, thereby freeing the beads to swirl about the lysis mixture and bind to nucleic acid molecules dispersed therein following cell lysis. In an exemplary embodiment, the rotating cam spins at approximately <NUM> to <NUM>. In embodiments, magnet <NUM> may also be moved along any of the x, y or z axes in coordination with rotating cam <NUM> to facilitate vortex generation and mixing.

Step <NUM> includes actuating magnet <NUM> to move magnetic beads and associated nucleic acids to an adjacent well. In an example of step <NUM>, magnet <NUM> is moved by motor <NUM> into a position adjacent to a side wall of a lysis well <NUM> in order to attract magnetic beads disposed within the well. Then motor <NUM> is actuated to move magnet <NUM> along motions arrows <NUM>, vertically to an aperture of lysis well <NUM>, horizontally across a floor of channel <NUM> to an adjacent wash well <NUM>, then vertically into down into wash well <NUM>. Magnet <NUM> may then be moved horizontally along the y-axis to release magnetic beads into the wash fluid in well <NUM>. In embodiments, the floor of channel <NUM> is lower than the floor of base member <NUM> of sample holder cover <NUM> so that magnetic beads may be moved from well to well in sample holder <NUM> without interference from the sample holder cover.

Step <NUM> includes moving rotating cam horizontally along the x-axis to tab <NUM> connected to vibration rod <NUM> inserted into wash well <NUM>. In step <NUM>, steps <NUM> - <NUM> are repeated until the last well of vortex generator assembly <NUM>, <NUM> is reached. In each well of sample holder <NUM>, a process similar to that executed within lysis well <NUM> may be carried out. After a desired number of washing steps have been completed, magnet <NUM> is actuated to move magnetic beads into elution well <NUM> of sample holder <NUM> where nucleic acids elute from the magnetic beads into the elution buffer.

As anticipated, the contents of the lysis well <NUM> may be heated prior to or during the illustrated step <NUM> of vortexing the contents of the lysis well. Following removal of the magnetic beads in step <NUM>, liquid residues in the lysis well and the wash wells may be aspirated by a pipetting system and dispensed to a waste receptacle. In embodiments, this may be done by removing sample holder cover <NUM> or through cutout area <NUM> of <FIG>. Similarly, elution well <NUM> may undergo heating at any point prior to removal of the final nucleotide product solution.

Embodiments described above have several advantages. The system provides improved vortex generation in a confined and small volume with easily fined tuned speed and low-cost assembly of driving source. As the effective mixing is critical for the Sample Preparation for qPCR, this mixing invention can be lead to significant improvement of time, efficiency, throughput and quality of PCR assays. The vibration source is a simple motor connected to a cam shaft. This allows the flexibility of speed selection and cost saving. One driving source is good for driving vortex mixing in different positions.

Further, a vortex generator assembly with multiple vibration rods in an array has low production cost and it is easy to modify and optimize the vortex generation by slightly changing the shape and dimension of the rod. In embodiments, apparatus and methods used above may be incorporated in a high throughput liquid handling robot such that every well on a plate having, for example, <NUM>, <NUM>, <NUM> wells may be agitated at the same time. This may reduce the cost of multiple batches using disposable pipette tips for liquid updown handling, with better and faster mixing. Only a single source of mechanical oscillation is needed to multiple throughputs. In another embodiment, multiple sources of mechanical oscillation may be used.

Claim 1:
A sample lysis and nucleic acid extraction apparatus (<NUM>), comprising:
(i) a base (<NUM>) for retaining a vortex generator assembly (<NUM>, <NUM>), the vortex generator assembly comprising:
a sample holder (<NUM>) comprising a body member forming a channel (<NUM>) extending from a first end (<NUM>) to an opposite second end (<NUM>) of the sample holder, the sample holder further comprising a first plurality of wells extending in a downwardly direction from a floor of the channel (<NUM>), each well having an interior volume; and
a sample holder cover (<NUM>) comprising a base member (<NUM>) comprising a floor extending from a first end to an opposite end of the sample holder cover and a second plurality of vibration rods (<NUM>, <NUM>) extending in a downwardly direction from the floor of the base member (<NUM>) into the interior volume of a corresponding one of the plurality of wells when the base member (<NUM>) is inserted into the channel (<NUM>);
(ii) a rotating cam (<NUM>) attached to a stepper motor (<NUM>) and configured to impart vibrational movement to one or more of the second plurality of vibration rods (<NUM>, <NUM>); and
(iii) a horizontal actuator attached to the motor (<NUM>) and configured to selectively impart horizontal movement of the motor (<NUM>) along an x-axis repositioning the motor (<NUM>) along vortex generator assemblies (<NUM>, <NUM>) during sample processing.