Filter and SPE plate clogged well detection

The invention provides a method, a system, and a computer readable medium for determining the fill-ready status of one or more wells of a plurality of wells formed in a filter or solid-phase extraction (SPE) plate. In the method, a signal is directed toward a well of the plurality of wells. A reflected signal is received from the well responsive to the directed signal. The fill-ready status of the well is determined based on the received reflected signal. The steps may be repeated until the fill-ready status of each of the plurality of wells has been determined. In the method and the apparatus, the directed signal may be generated by a piezoelectric crystal. Some of the reflected vibrations impact the piezoelectric crystal, which oscillates in response. The piezoelectric crystal transmits a signal to a processor, the signal corresponding to the height of the liquid or other material in the well.

FIELD OF THE INVENTION

This invention relates generally to preparing a sample for analysis using a filter or solid phase extraction (SPE) plate having a plurality of wells. More specifically, the invention relates to methods, systems, and computer-readable media for determining the fill-ready status of wells in a filter or SPE plate, thereby detecting clogged wells.

BACKGROUND OF THE INVENTION

Array trays and assemblies are used in analyzing liquids and solids to determine, for example, their chemical, biochemical, or biological nature (including, for example, DNA/RNA cleanups, PCR setup, protein precipitation, solid phase extraction, protein purification, solubility assays, kinase assays, solid-liquid extraction, protein separation, and cell-based assays). Such arrays include filter plates and solid phase extraction (SPE) plates, which typically contain a plurality of wells in which liquids are forced through a membrane or sorbent located at the bottom of each well using differential pressure across the well.

Conducting assays using these multiwell plates generally requires multiple additions of liquids into the wells in the plates alternating with removal of the liquids. During an assay, non-soluble material in the liquid may cause a blockage (also referred to as a “clog”) that prevents the liquid from migrating through the well at the preferred rate over the desired period of time, resulting in a clogged well (also referred to as a “blocked” well). Clogged wells can overflow with the subsequent addition of liquid, causing loss of the samples in the clogged wells as well as contamination of the surrounding wells.

It would, therefore, be desirable to have methods and systems for determining the fill-ready status of each well in a filter or SPE plate in order to detect any clogged well in a filter or SPE plate and protect against sample loss and contamination.

SUMMARY OF THE INVENTION

One aspect of the invention provides a method of determining the fill-ready status of one or more wells of a plurality of wells formed in a filter or solid-phase extraction (SPE) plate. A signal is directed toward a well of the plurality of wells formed in the plate. A reflected signal is received from the well responsive to the directed signal. The fill-ready status of the well is determined based on the received reflected signal. The steps of the method may be repeated to determine the fill-ready status of all of the plurality of wells formed in the filter or SPE plate.

Another aspect of the invention provides a system for determining the fill-ready status of one or more wells of a plurality of wells formed in a filter or solid-phase extraction (SPE) plate. The system comprises means for directing a signal toward a well of the plurality of wells formed in the filter or SPE plate, means for receiving a reflected signal from the well responsive to the directed signal, means for determining whether the well is fill-ready based on the reflected signal, and means for filling the well based on a determination that the well is fill-ready. The system may further comprise means for clearing a well or for notifying a system operator that a well is clogged based on a determination that the well is not fill-ready.

Yet another aspect of the invention is a computer-readable medium containing instructions for controlling a processor performing a method for determining the fill-ready status of one or more wells of a plurality of wells formed in a filter or solid-phase extraction (SPE) plate. The computer-readable medium includes instructions for sequentially directing a signal toward each of the plurality of wells formed in the filter or SPE plate, for receiving a reflected signal from each of the plurality of wells responsive to the directed signal, and for determining whether each of the plurality of wells is fill-ready based on the received reflected signal.

The foregoing and other features and advantages of the invention will become further apparent from the following detailed description of the presently preferred embodiments, read in conjunction with the accompanying drawings. The drawings are not to scale. The detailed description and drawings are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof.

Throughout the various figures, like reference numbers refer to like elements.

DETAILED DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS

One aspect of the present invention is a system for determining the fill-ready status of one or more wells of a plurality of wells formed in a filter or solid phase extraction (SPE) plate.FIGS. 1 and 2are top and side views, respectively, of a filter or SPE plate designated10. Plate10comprises a unitary tray12having a number of spaced apart, discrete filter or SPE wells14defined therein.

FIG. 3is a detailed cross-section of a filter or SPE well14such as those illustrated inFIGS. 1 and 2. Well14has opposing ends: first end or top16and second end or bottom18. Side walls20are shown extending between first and second ends16and18and defining well cavity19. A porous layer22is typically formed at the bottom end18of each well14, as shown inFIG. 3; however, other arrangements are possible. For example, the side walls of the well may be porous as shown inFIG. 2. In a filter plate, porous layer22acts as a filter. In a SPE plate, porous layer22includes a material bound to the porous layer for solid phase extraction.

In practice, a liquid is loaded, entered, or injected into well14through or at first end16. In filtration applications, the liquid migrates through porous layer22as it moves from first end16through second end18, leaving unwanted particulates in or on the filter. In SPE extractions, the liquid may interact with the stationary phase bound to porous layer22to extract a desired analyte from the liquid. Any bound analyte is retained by the stationary phase until it is eluted from porous layer22using the appropriate reagent.

FIG. 4depicts one embodiment of an automated liquid handling workstation or fill apparatus50that may be used to deliver one or more liquids into the wells of a filter or SPE plate. In the present embodiment, fill apparatus50comprises a control unit, head or gripper52, a deck54, and a support member56adapted to move one or both of head52and deck54with respect to each other. In addition, fill apparatus50includes a plurality of tips or nozzles58fluidly communicating with head52and adapted to load, enter, or inject liquid into one or more of the filter or SPE wells14of plate10.

In at least one embodiment, fill apparatus50includes a vacuum system (not shown). The vacuum system is adapted to create a pressure differential between the first and second ends16and18of each filter or SPE well14. The pressure differential causes a liquid to migrate through each well14, from first end16to second end18. The pressure differential may be produced by applying a positive pressure to first end16or a negative pressure to second end18.

FIG. 5depicts a side elevational view of fill apparatus50engaging and filling plate10. In the depicted embodiment, head52is movable with respect to deck54and plate10, enabling fill apparatus50to inject liquids into the wells14of plate10using one or more nozzles58.

Conducting assays or other procedures using filter or SPE plates typically requires multiple additions of liquids to wells with intervening removal of the liquids from the wells, e.g., by the liquids passing through the porous layer22. It is not uncommon that non-soluble material in the liquid may prevent a well from being evacuated at the preferred rate over a specific period of time. This results in a clogged well that can overflow with the subsequent addition of liquid, causing cross-contamination with the surrounding wells and a subsequent loss of not only the sample in the clogged well, but also contaminated samples in the surrounding wells.

FIG. 6is a block diagram of a sensing apparatus60adapted to determine whether the wells in a filter or SPE plate are not clogged and, therefore, fill-ready. Sensing apparatus60includes a signal processing module62(e.g., a processor or computer) coupled to and communicating with at least one transducer64via coupling or transmitting wire66. In the illustrated embodiment, transducer64includes at least one piezoelectric crystal68adapted to generate vibrations that serve as signals for determining the fill-ready status of the wells (i.e., for indicating whether the well is or is not clogged).

In general, sensing apparatus60is used to scan the nearest surface, where the nearest surface may be the second end or bottom18of an empty well14, the top surface of a liquid or other material in a well14, or the top surface13of tray12. Sensing apparatus60may scan in either a continuous or intermittent motion.

Signal processing module62includes a computer-readable medium containing instructions for directing one or more signals (sonic or ultrasonic) at filter or SPE wells14formed in plate10.

In at least one embodiment, signal processing module62produces or generates a brief series or plurality of high-voltage pulses. The pulses are communicated or transmitted to the piezoelectric crystal68via transmitting wire66. These pulses cause the piezoelectric crystal68to oscillate at its fundamental frequency. Such piezoelectric crystal oscillation causes or creates vibrations that travel through the surrounding media (air or cover gas, for example) at a constant velocity for a given set of environmental conditions. The physical shape of piezoelectric crystal68, its orientation, its fundamental frequency, and the overall physical properties of transducer64are optimized to focus the vibrational energy of piezoelectric crystal68toward the desired areas of plate10.

The signal transmitted from piezoelectric crystal68reduces in amplitude as it travels to and from the reflecting surface. It is therefore advantageous to increase the gain of piezoelectric crystal68over time in order to easily measure such lower amplitude signals.

Piezoelectric crystal68stops oscillating shortly after the high-voltage pulses end or are terminated. The vibrations, however, continue to travel outward from piezoelectric crystal68and are reflected back from the nearest surface, forming reflected signals. Some of the reflected vibrations impact piezoelectric crystal68, causing the piezoelectric crystal to oscillate. The oscillating piezoelectric crystal transmits a corresponding signal via transmitting wire66to module62, where the signal is processed.

The vibrations are directed at, and reflected back from, the second end or bottom18of an empty filter or SPE well14, the top surface of a liquid or other material in a filled well14, and/or the top surface13of tray12of filter or SPE plate10. Signal processing module62is adapted to determine the distance from the piezoelectric crystal to the encountered surface by measuring the signal transmission and return time and computing the distance based on the known velocity of the signal vibrations through the media.

The fill-ready status of a well may be determined solely on the basis of the computed distance from the piezoelectric crystal to the top surface of the liquid or other material in such well. Alternatively, because changes in humidity and temperature can cause slight variations in the velocity of the outgoing and reflected vibrations, accuracy of the determination may be improved by also computing the distance from the piezoelectric crystal to the top surface of the tray12and/or the bottom18of an empty well14. Signal processing module62may store this information as a standard measure or, alternatively, determine this distance at the beginning of each application. Signal processing module62may then determine the position of the surface of the liquid or other material in a well relative to the top surface of the plate or tray and/or the bottom of an empty well, thereby determining if one or more wells are clogged.

Signals transmitted from the piezoelectric crystal generally diverge as they travel away from the system. Reflected signals from objects located directly in front of the sensing apparatus are generally of higher amplitude than signals reflected from objects not located directly in front of the sensing apparatus. This may make it difficult to determine the distance to the bottom of a well because a weak signal may reflect peripherally from the top of the tray and be falsely interpreted as the level of liquid or other material in the well. One approach to eliminating this effect is to reduce the gain of the sensing apparatus until the signal has traveled past the top of the tray and to also establish an amplitude threshold below which signals will be ignored. This approach allows the weaker peripheral reflections from the top of the tray to be ignored when the sensing apparatus is located approximately above the center of a well while still allowing the distance to the surface of liquids or other materials in a well to be accurately determined.

Another approach is to ignore all reflected signals from objects that are as close as or closer than the top of the tray. The disadvantage of this second approach is that signals reflected from a liquid or other material in a well that is even with or above the top of the tray will be ignored.

Plate10is scanned in a continuous or intermittent motion. During such scanning operations, the plate may be moved with respect to one or more fixed sensing apparatuses, or the sensing apparatus(es) may be moved with respect to a fixed plate, or both the sensing apparatus(es) and the plate may be simultaneously moved so that multiple wells within the plate are ultimately scanned. In one example in which the wells of the plate are arranged in rows, the signal is directed toward each of the wells in a row in sequence, thereby scanning the row of wells. The row of wells may be scanned across and back in a continuous motion. Scanning steps may be repeated until the fill-ready status of each of the wells in a plate has been determined.

It is contemplated that signal processing module62is further adapted to map and store the positions of plate10and the filter or SPE wells14therein, thereby providing a mapped grid. The processing module generates and receives signals as provided previously. The signal processing module62, using the received reflected signals as provided previously, stores the positions of the plate10and each well14in the plate.

In the event that signal processing module62determines that a well is not fill-ready (i.e., clogged) based on the distance from the piezoelectric crystal to the top surface of the liquid or other material in the well, the signal processing module may facilitate readying the clogged well by clearing the blockage using the vacuum, for example. In another embodiment, the signal processing module may facilitate filling only those wells determined to be fill-ready (i.e., not clogged), using the stored positions of the wells, for example. In still another embodiment, the signal processing module may notify the operator that one or more of the wells are not fill-ready by generating a signal or alarm, allowing the operator to clear the blockage(s). If signal processing module62has mapped the wells, it may indicate the wells that need to be cleared by showing the location of any clogged well(s) on the mapped grid using lights or a display (not shown). In still another embodiment, the processing module may facilitate another mechanical operation that will clear the blockage, for example piercing the clogged well(s) with a sharpened object.

Referring now toFIGS. 7 and 8, top and side views of filter or SPE plate110are depicted having a plurality of filter or SPE wells114similar to those provided previously with respect toFIGS. 1 and 2. As illustrated, one of the wells, shown at130inFIGS. 7 and 8, is clogged. The addition of liquid to clogged well130, without some intervention, will result in an overflow, causing contamination of the surrounding wells and loss of the affected samples.

FIGS. 9,10, and11depict side views of a fill apparatus150that is similar to the fill apparatus shown inFIGS. 4 and 5at50. Fill apparatus150includes a sensing apparatus60such as that shown inFIG. 6. Fill apparatus150further comprises head152having a plurality of nozzles158, deck154, and support member156adapted to move head152and deck154with respect to each other.

Fill apparatus150includes a sensing apparatus60adapted to determine whether the wells in a filter or SPE plate are fill-ready in accordance with one embodiment of the present invention as provided previously. In the illustrated embodiment, sensing apparatus60of fill apparatus150comprises a signal processing module62(as seen inFIG. 6) coupled to and communicating with at least one transducer64via coupling or transmitting wire66(as seen inFIG. 6). Transducer64of fill apparatus150includes at least one piezoelectric crystal68adapted to generate vibrations. In one embodiment, signal processing module62is incorporated into head152. Signal processor62may, alternatively, be a separate unit.

Sensing apparatus60is used to scan the nearest surface, where the nearest surface is the second end or bottom118of an empty well114, the top surface of a liquid or other material in a properly filled or clogged well, or the top surface113of tray112(as seen inFIGS. 7 and 8). Signal processing module62produces or generates a brief series or plurality of high-voltage pulses. These pulses cause piezoelectric crystal68to oscillate at its fundamental frequency.

As previously described, piezoelectric crystal68stops oscillating shortly after the high-voltage pulses end or are terminated. The vibrations continue to travel through the media. As seen inFIG. 10, vibrations170travel outward from piezoelectric crystal68and are reflected back by the encountered object(s), forming reflected vibrations172. The reflected vibration signals cause piezoelectric crystal68to oscillate. The oscillating piezoelectric crystal68transmits a corresponding signal to processing module62, where the signal is processed.

Signal processing module62is adapted to receive the signals reflected from wells114and determine whether one or more of the plurality of wells114are fill-ready based on such reflected signals. In at least one embodiment, plate110is placed or positioned on deck154. One or more signals are generated, and the reflected signals are received. The processing module is adapted to determine or measure the distance from the piezoelectric crystal to the top surface113of tray112, to the bottoms of wells114, and/or to the nearest surface of the liquid or other material within a well by measuring the signal transmission and return time and computing the distance based on the known velocity of the signal vibrations through the air or other gaseous medium.

Signal processing module62is adapted to determine if one or more of the wells are clogged (e.g., clogged well130) based upon such measurements. For example, signal processing module62may determine the position of the surface of the liquid or other material in a well relative to the top surface of the plate or tray and/or the bottom of the well, thereby determining if one or more wells are clogged.

In the event that signal processing module62determines one or more of the wells are clogged (i.e., not fill-ready), signal processing module62may provide instructions to ready the clogged well(s) by clearing the blockage by, for example, using the vacuum. In another embodiment, signal processing module62may provide instructions to fill only those wells114determined to be not clogged (i.e., fill-ready), using the stored positions of the wells, for example. In still another embodiment, signal processing module62may notify the operator that one or more of the wells are not fill-ready by generating a signal or alarm, allowing the operator to clear the blockage. If signal processing module62has mapped the wells, it may indicate the wells that need to be cleared by showing the position of each clogged well using lights or a display (not shown). In still another embodiment, the processing module may facilitate another mechanical operation that will clear the blockage, for example by piercing the clogged well or wells with a sharpened object.

Another aspect of the present invention is a method of detecting a clogged well in a filter or SPE plate.FIG. 12depicts a flow diagram, generally designated200, illustrating one method of using a filter or SPE plate in accordance with one embodiment. Method200comprises positioning a filter or SPE plate on a fill apparatus (Block210) and mapping a grid of the plate (Block212) by recording the position of each well with respect to the other wells formed in the filter or SPE plate. One or more signals (sonic or ultrasonic) are directed at one or more filter or SPE wells formed in the plate (Block214).

The signals are reflected from at least one of the wells and received (Block316). The fill-ready status of the well is determined based on the received reflected signals (Block218). Method200further comprises filling at least one of the wells in the plate based on the determination (Block220).

In one embodiment, the processing module fills only those wells that are not clogged. In another embodiment, the fill apparatus unclogs any clogged well using a vacuum (not shown) or other device. In yet another embodiment, the fill apparatus operator is notified of the clogged well(s).

FIG. 13depicts a scanned profile of a single row of wells generated in accordance with one embodiment of the present invention.FIG. 13uses shading to indicate levels of liquid (or other material) present in the wells. In the illustrated example, every other well contains some finite volume of liquid, while the remaining wells are empty. The face of the sensing apparatus is positioned at position 0 mm in the illustration. The top surface of the plate or tray measures about 20 mm from the face of the sensing apparatus, as may be seen on the left portion of the illustration. In this illustration, the well separators appear at approximately 20 mm, and these separators clearly differentiate each well. Each data point shown in this illustration represents the processed data for one transmitted and received signal as the sensing apparatus was moved with respect to the fixed location of the plate. Alternatively, the plate could be moved with respect to the fixed position of the sensing apparatus or both the sensing apparatus and plate could be simultaneously moved with respect to each other.

Still another aspect of the present invention is a computer readable medium containing instructions for controlling a processor performing a method of forming a sample for analysis. As previously noted, signal processing module62may include such a computer-readable medium. The computer-readable medium contains instructions for carrying out some or all of the steps outlined above and in flow diagram200ofFIG. 12.

EXAMPLE

The following example serves to illustrate, but not to limit, the present invention. In the present example, a sensing apparatus is mounted into or otherwise attached to the head or gripper of a liquid handling workstation, e.g., a Zephyr® or Sciclone Liquid Handling Workstation, available from Caliper Life Sciences, Inc. The sensing apparatus includes a signal processing module coupled to and communicating with a transducer via a transmitting wire. The signal processing module generates a series of high-voltage pulses that are transmitted to the piezoelectric crystal via the transmitting wire. In this example, a series of pulses is generated at intervals of about 2 milliseconds. The pulses cause the piezoelectric crystal to oscillate at its fundamental frequency, in this example about 800 kHz.

The sensing apparatus scans a filter or SPE plate placed onto the deck of the liquid handling workstation, sending out vibrations from the piezoelectric crystal that are reflected back from encountered surfaces of the filter or SPE plate, e.g., from the tray of the plate as well as from the wells of the plate. In the present example, the plate is a 96-well plate having a standard 8×12 configuration.

The sensing apparatus continuously scans a row of the plate, scanning across the row more than once if necessary, until vibrations reflected back from the tray and wells of that row indicate all of the wells have emptied properly (i.e., are not clogged) or, where wells are clogged (also referred to as “blocked”), for a specified period of time, e.g., 30 seconds, after which the scan of that row times out. The sensing apparatus then goes on to scan all of the rows in the plate until either all of the wells in each row have emptied (are fill-ready) or the time-out period has expired for that row.

The sensing apparatus maps and stores the positions of the filter or SPE plate and the wells therein. Using a grid obtained from mapping the plate, the sensing apparatus indicates any wells that need to be cleared by showing the location of the clogged well(s) on the mapped grid using a display of the grid. The liquid handling workstation is configured either to continue processing of only those wells that have drained properly or to pause so that the workstation operator can clear the clogged wells and signal the workstation to continue processing of all of the wells.