Cassettes for use in automated parallel electrophoretic assays and methods for manufacturing and using same

A cassette for use in parallel electrophoretic assays and methods of manufacture and use of same are provided. The cassette comprises assay channels suitable for carrying out electrophoresis and sample ports for introducing samples into the assay channels. Each sample port comprises one or more tip registration features for guiding a pipette tip to a desired location, which corresponds to the position of a well for receiving the sample. The cassette is particularly suited for use with automated liquid handling systems.

FIELD OF THE DISCLOSURE

The present disclosure relates to an electrophoresis gel cassette for use in electrophoresis and to the use and manufacture thereof. More particularly, the present disclosure relates to an electrophoresis gel cassette suitable for use with an automated liquid handling device.

BACKGROUND OF THE DISCLOSURE

Automated liquid handling workstations may be used, for example, in applications involving repetitive, predictable pipetting operations. Automation of repetitive pipetting operations may facilitate higher throughput, lower operating costs, and/or, improved consistency in pipetting. However, laboratory protocols involving, for example, non-standardized labware, gelatinous media, and/or other non-standard pipetting target areas may not be well suited to automation. In such cases, an liquid handling by an individual practitioner may be advantageous relative to an automated machine, at least because an individual could see the target and make real time positional corrections to the position and directional movement of a pipette tip to ensure the pipette tip orifice is located in a desired position prior to aspirate and dispense operations. For example, known liquid handling devices are not desirable for use in loading samples into one or more wells of an agarose gel, at least due to the small size of the wells and the low rigidity of the walls that define the agarose wells.

When pipette tips are loaded onto a manual pipette or onto mandrels of a liquid handling device, there is typically variation in ‘straightness’ of tips relative to their respective shaft or mandrel (i.e., the longitudinal axis of the tip may not be in line with the intended longitudinal axis of the tip). Such variation in straightness is known in the art as “tip splay”. Without positional feedback provided by a manual practitioner, the presence of tip splay means that the size of a well that can be accurately targeted using an automated system is larger relative to the size of a well that can be accurately targeting using manual pipetting. Further, off-target insertion of a pipette tip into a wall defining an agarose well or into the agarose surrounding a well orifice may damage the well and/or plug the pipette tip with agarose, thereby interfering with subsequent aspiration or dispensing.

One mechanism to account for tip splay on a single channel pipetting head is to utilize a sensor that can provide feedback regarding the position of the tip. However, such a sensor would not be useful with a multichannel pipetting head comprising mandrels at fixed spacing, at least because the direction of tip splay may differ between tips, meaning that a single positional adjustment could not account for the splay in each tip.

There are examples of products and devices designed to address one or more of the above challenges associated with automated pipetting of samples into agarose gels. For example, the EGel™ 96 agarose gel (Invitrogen) comprises 96 preformed wells in a staggered pattern and is designed for use with an automatic 96 channel liquid handling manifold. Rather than inserting sample-bearing pipette tips into the wells of the agarose gel, pipette tips are positioned above the wells of the EGel and dispensed. The dispensed sample is then drawn into a section of the well that is adjacent to the position of the tip by capillary action. Thus, the EGel would seem to overcome the issue of inserting the pipette tip into the gel, as the pipette tip remains above the upper surface of the gel. Further, each well of an EGel is relatively large, consisting of a shoulder zone and an adjacent compartment that draws dispensed fluid into the wells. The relatively large wells may address tip splay, by providing a larger target for each pipette tip. However, the larger size of the wells in the EGel prevents maximization of the number of wells that can be positioned adjacent to one another in a gel, thereby limiting sample throughput.

It is desirable to mitigate and/or obviate one or more of the above deficiencies.

SUMMARY OF THE INVENTION

In a first aspect, a cassette for use in parallel electrophoretic assays is provided. In one embodiment, the cassette comprises a tray, the tray having a floor and two pairs of opposing side walls extending upwards from the floor, the tray at least partially defining a plurality of assay channels. Each of the assay channels comprises a first buffer reservoir; a second buffer reservoir; and a media channel, the media channel extending between, and in fluid communication with, the first and second buffer reservoirs. In this embodiment, the cassette further comprises a lid, the lid adapted to engage the side walls of the tray, thereby creating a space between the tray floor and the lid, the lid comprising a plurality of ports, each of the ports extending from an outer surface of the lid to an inner surface of the lid. The plurality of ports comprise a plurality of buffer reservoir ports for introducing and/or removing buffer; and a plurality of sample ports for introducing a plurality of samples into the media channels, each of the plurality of sample ports comprising one or more tip registration features for guiding a pipette tip to a desired location in the sample port. In this embodiment, when the lid is engaged with the tray, the buffer reservoir port is in fluid communication with one or more of the first and second buffer reservoirs, the plurality of sample ports are aligned with the plurality of media channels.

In one embodiment of the first aspect, the lid further comprises a plurality of media ports for introducing media into the media channels.

In one embodiment of the first aspect, the one or more tip registration features comprise at least one surface extending in one or more of x- and y-axes towards the desired location in the sample port, the x- and y-axes being in line with the plane of the lid. In a preferred embodiment, the at least one surface of the one or more registration features comprises a sloped surface for receiving a pipette tip.

In one embodiment of the first aspect, each of the plurality of sample ports comprises a tip landing zone. In a preferred embodiment, the tip landing zone is adjacent to the desired location in the sample port. In a preferred embodiment, the at least one surface of the one or more registration features comprises a projection extending into the sample port from a wall in the sample port opposite the tip landing zone.

In one embodiment of the first aspect, the cassette further comprises a gel medium, the gel medium being disposed in the media channels of the tray, the gel medium in each media channel comprising a well for receiving a sample, wherein, when the lid is engaged with the tray, each well is aligned with the desired location in one of the plurality of sample ports. In a preferred embodiment, the cassette further comprises a buffer, the buffer being disposed in the first and second buffer reservoirs. In a preferred embodiment, the gel medium in each media channel further comprises a tip landing zone adjacent to the well for receiving a sample.

In one embodiment of the first aspect, the plurality of ports further comprises: a plurality of barrier ports for positioning at least a portion of first and second barriers into each of the assay channels, wherein, when the lid is engaged with the tray, the plurality of barrier ports facilitate positing of the first and second barriers between the first buffer reservoir and the media channel and the second buffer reservoir and the media channel, respectively. In a preferred embodiment, the plurality of ports further comprises: a plurality of sample extraction ports for extracting a plurality of samples from the media channel, each of the plurality of sample extraction ports comprising one or more tip registration features for guiding a pipette tip to a second desired location in the sample extraction port. In a preferred embodiment, each of the plurality of sample extraction ports comprises a tip landing zone. In a preferred embodiment, the tip landing zone is adjacent to the desired location in the sample port.

In one embodiment, the gel medium in each media channel further comprises a second well for extracting a sample. In a preferred embodiment, the gel medium in each media channel further comprises a tip landing zone adjacent to the well for extracting a sample.

In one embodiment, the plurality of assay channels comprises 6, 12, 24, 48 or 96 assay channels.

In a second aspect, a method for manufacturing various embodiments of the cassette provided herein is provided. In one embodiment, the method comprises: forming the tray and lid from an optically neutral material; positioning a first removable barrier between the first buffer reservoir and the media channel in each of the assay channels of the formed tray and a second removable barrier between the second buffer reservoir and the media channel in each of the assay channels of the formed tray; introducing media into each media channel in the plurality of assay channels of the formed tray; positioning a comb into the introduced media in each media channel, each tooth in the comb configured to form a well in each media channel upon gelification of the media; introducing buffer into each first and second buffer reservoir in each assay channel of the formed tray; removing the comb from the introduced media after gelification of the introduced media; removing the barrier after gelification of the media; engaging the formed lid on the side walls of the formed tray, thereby creating a space between the floor of the formed tray and the lid, the space being occupied at least partially by the introduced media and the introduced buffer.

In one embodiment of the first aspect, the engaging step is completed prior to the positioning of the first and second removable barriers, the introducing of media, the positioning of the comb and the introducing of the buffer.

In one embodiment of the first aspect, the first and second removable barriers do not sealably separate the first buffer reservoir from the media channel and the second buffer reservoir from the media channel, respectively, in each of the assay channels of the manufactured tray.

In one embodiment of the first aspect, the introduction of the media and the introduction of the buffer are carried out substantially simultaneously.

In a third aspect, a method of using various embodiments of the cassette provided herein is provided. In one embodiment, the method comprises: positioning the cassette in a liquid handling device; inserting a plurality of pipette tips in the tip landing zones of the plurality of sample ports, the plurality of pipette tips corresponding to the plurality of sample ports; moving the plurality of pipette tips inserted in the tip landing zones in one or more axes towards one or more of the tip registration features, the one or more axes being co-planar with the plane of the cassette lid; registering the plurality of pipette tips against the one or more tip registration tip features, at least one of the one or more tip registration features per sample port being adjacent to the desired location in each of the sample ports; and dispensing a plurality of samples from the plurality of pipette tips registered against the registration features adjacent to the desired location in each of the sample ports.

In one embodiment of the third aspect, the method further comprises lowering the plurality of registered pipette tips into the corresponding wells for receiving a sample prior to dispensing the plurality of samples.

In one embodiment of the third aspect, the method further comprises engaging the cassette comprising the plurality of dispensed samples with electrodes and a power supply, thereby creating one or more electric fields in the cassette, the one or more electric fields being sufficient to case migration of the plurality of samples through the gelified media in the plurality of assay channels.

In one embodiment of the third aspect, the method further comprises extracting a plurality of migrated samples from the cassette following exposure to the electric field.

In one embodiment, the extracting comprises: inserting a second plurality of pipette tips in the second tip landing zones of the plurality of sample extraction ports, the second plurality of pipette tips corresponding to the plurality of sample extraction ports; moving the second plurality of pipette tips inserted in the second tip landing zones in one or more axes towards one or more of the second tip registration features, the one or more axes being co-planar with the plane of the cassette lid; registering the second plurality of pipette tips against the one or more second tip registration tip features, at least one of the one or more second tip registration features per sample extraction port being adjacent to the second desired location in each of the sample extraction ports; moving the registered second plurality of pipette tips downward into the gelified media below the second desired location in each of the sample ports; aspirating a the plurality of migrated samples from the gelified media below the second desired location in each of the sample ports; and retracting the second plurality of pipette tips from the gelified media and the sample ports of the cassette.

DETAILED DESCRIPTION OF THE INVENTION

Cassettes

The present disclosure is generally directed to a cassette for use in parallel electrophoretic assays, and more particularly to a cassette for use in automated parallel electrophoretic assays.

Liquid handling systems are configured to manipulate a plurality of fluid samples, automatically and concurrently, enabling high-throughput operations. Liquid handling devices are marketed as being highly reliable and accurate. However, sometimes a device may malfunction during the liquid handling process or not perform to a user's expectations. For example, a sample may not be properly loaded into a well in an electrophoresis gel because one or more pipette tips may not be aligned with a well. Such a malfunction may lead to inaccurate or misleading results.

In one aspect, the cassette provided herein may be used with an automated liquid handling device to guide a plurality of pipette tips to a desired location relative to the cassette, such as, for example, above a plurality of wells for receiving samples in a gel disposed in the cassette.

In general, various embodiments of the cassette provided herein comprise a tray portion, which functions as a base for the cassette and at least partially defines a plurality of assay channels, and a lid portion, which functions as a cover for the tray. The lid and tray portions may be discrete components or may be integral with each other.

In general, each assay channel in the cassette comprises an elongate media channel separating, but in fluid communication with (during functional operation of the cassette), a pair of spaced apart buffer reservoirs (i.e., first and second buffer reservoirs), which are functionally identical unless otherwise indicated. While the tray may define substantially all aspects of the assay channels, such arrangement is not considered to be necessary to the use or functionality of the various invention embodiments. In some embodiments, various features of the lid and tray together may together define the assay channels of the cassette.

Referring toFIG. 1, which illustrates one embodiment of a cassette provided herein, the cassette100comprises a tray120and a lid110. The tray120has a floor122and two pairs of opposing side walls extending upwards from the floor122. The lid110is adapted to engage the side walls of the tray120, thereby creating a space between the tray floor122and the lid110. For example, a lid may be adapted to engage a tray via mating parts (e.g., tongue and groove features), but such mating parts are not required. An electrophoresis gel and buffer may be disposed in the space between the tray floor122and the lid110, as described further below. The cassette100may be configured for communication with electrodes for use in one or more electrophoretic assays.

Referring toFIG. 2, which illustrates the tray of the cassette provided inFIG. 1, the tray220tray at least partially defines a plurality of assay channels202. Twelve assay channels202are at least partially defined by the tray220ofFIG. 2. However, in other embodiments of the cassette provided herein, various numbers of assay channels202may be at least partially defined by a tray of the cassette (e.g., 6, 12, 24, 48, 96 or more assay channels). Each of the assay channels202comprises a first buffer reservoir230and a second buffer reservoir232for receiving and holding buffer (e.g., buffer suitable for use in an electrophoretic assay). Each of the assay channels202further comprises a media channel240extending between the first and second buffer reservoirs230,232. Each media channel240is in fluid communication with the first and second buffer reservoirs230,232, such that in use, an electric field may be applied to the cassette, such that the field is present throughout each assay channel202in the cassette.

Referring further toFIG. 2, the tray220comprises a plurality of walls224extending upwards from the floor of the first and second buffer230,232portions of the tray and the media channel240portions of the tray. These walls224separate one assay channel202from another, thereby inhibiting fluid and optionally electric communication between the assay channels202.

In one preferred embodiment, various portions of surfaces in the media channels240may be textured242, as discussed further below.

Referring toFIG. 3, which illustrates one embodiment of the tray of the cassette provided herein, some portions of the media channels340comprise a non-smooth, textured surface342, which facilitates improved adherence of a gel medium to the media channels340by providing binding sites for the media and/or areas of increased friction between the media and the media channel340. Other portions of the media channels340comprise a substantially smooth surface344, which facilitates optical transparency of that portion of the media channel340, thereby facilitating imaging of the media channel is desired by a user.FIG. 3Aillustrates a side elevation cross-sectional view taken along the line A-A of the tray ofFIG. 3, which depicts a single assay channel comprising first and second buffer reservoirs330,332and a media channel340extending between, and in fluid communication with, the first and second buffer reservoirs330,322.FIG. 3Billustrates a side elevation cross-sectional view taken along the line B-B of the tray ofFIG. 3, which depicts12media channels340, the media channels340being separated by walls324extending upwards from the floor322of the tray320.

Referring toFIG. 4, which illustrates the lid of the cassette provided inFIG. 1, the lid410comprises a plurality of ports, each of the ports extending from an outer surface of the lid410to an inner surface412of the lid410. The function of the plurality of ports is to facilitate the introduction and/or removal of fluids (e.g., media, buffer, samples) and/or electrical current.

In one embodiment, the plurality of ports comprises first and second buffer reservoir ports450,452for introducing and/or removing buffer. In the embodiment shown inFIG. 4, if the lid410were engaged with a tray, the first and second buffer reservoir ports450,452would substantially align with first and second buffer reservoirs of a common assay channel in the tray, thereby introduction or removal of buffer to the first and second buffer reservoirs of the assay channel via the first and second buffer reservoir ports450,452, respectively. In a preferred embodiment, the first and second buffer reservoir ports450,452may also accommodate removable electrodes for use in electrophoretic operations.

Referring further toFIG. 4, the plurality of ports comprises sample ports460for introducing a plurality of samples into the media channels. In the embodiment shown inFIG. 4, if the lid410were engaged with a tray, the sample ports460would substantially align with a proximal end of a media channel in the tray, and if the tray were to comprise an electrophoresis gel, the sample ports460would substantially align with wells for receiving a sample in the gel, thereby facilitating introduction of a sample into a well in a gel disposed in a media channel of the cassette. In one embodiment, the sample ports460may also be used for introduction of media into the media channel of each assay channel.

In the embodiment shown inFIG. 4, each of the plurality of sample ports460comprises a tip landing zone474and one or more tip registration features468for guiding a pipette tip to a desired location490in the sample port460.

In general, the tip landing zone is located in an enlarged portion of a sample port, which accommodates initial aberrant pipette tip alignments (i.e., tip splay) such that no pipette tips entering the sample ports are damaged by accidental impingement on the outer surface of the lid of the cassette due to, for example, misalignment. Once partially disposed within the tip landing zone of the enlarged opening, the pipette tips can be laterally moved to contact a tip registration feature, such as, for example, a portion of respective sample port orifice walls and be guided to a desired location in the sample port (e.g., over a sample well in a gel disposed therebelow). The tip landing zone and tip registration features will be discussed further below.

Referring further toFIG. 4, the plurality of ports comprises sample extraction ports462for extracting a plurality of samples from the media channels after an electrophoretic assay has been run. In the embodiment shown inFIG. 4, if the lid410were engaged with a tray, the sample extraction ports462would substantially align with a distal end of a media channel in the tray, thereby facilitating extraction of a sample that has been subjected to electrophoresis in a gel disposed in a media channel of the cassette. In general, sample extraction ports may be located at any location within a media channel that is a distance downstream (i.e., in a direction in which a sample would travel in the media channel when treated by electrophoresis) from a sample port. Sample extraction ports462are not required in the cassettes disclosed herein, but may be provided in one or more preferred embodiments of the cassette provided herein.

Referring further toFIG. 4, the plurality of ports further comprises ports referred to as barrier ports454. In the embodiment shown inFIG. 4, each assay channel comprises two barrier ports454,455, a first barrier port454being located between the first buffer reservoir port450and the sample port460, a second barrier port455being located between the second buffer reservoir port452and the extraction port462. Each barrier port454,455functions to receive a barrier that is used during manufacture of the cassette to separate the first buffer reservoir from the media channel and the second buffer reservoir from the media channel, respectively. In the embodiment shown inFIG. 4, in the absence of a barrier, if the lid410were engaged with a tray, the first and second barrier ports454,455would substantially align with portions of the assay channel bridging a media channel and first and second buffer reservoirs respectively, thereby facilitating introduction of media to the media channel and optionally facilitating introduction of buffer to one or more of the buffer reservoirs. Barrier ports454,455are not required in the cassettes disclosed herein (media may be introduced into the media channel by way of, for example, sample ports and/or sample extraction ports), but may be provided in one or more preferred embodiments of the cassette provided herein.

Referring toFIG. 5, which illustrates one embodiment of the lid of the cassette provided herein, the lid510may correspond to 12 assay channels502, each portion of the lid510corresponding to an assay channel502comprising first and second buffer reservoir ports550,552, first and second barrier ports554,555, a sample port560and a sample extraction port562.FIG. 5Aillustrates a side elevation cross-sectional view taken along the line T-T of the tray ofFIG. 5, which depicts a portion of the lid corresponding to a single assay channel (upper), portions V and W of which are enlarged (below) for clarity.FIG. 5Billustrates the enlarged views (V and W) shown inFIG. 5A, wherein the portion of the lid corresponding to an assay channel is disposed above a gel580comprising a sample well582and an extraction well583.FIGS. 5A and 5Billustrate one example of a sample port560comprising of a tip landing zone564(located in an enlarged area of the sample port) and a smaller area566of the sample port560, the smaller area566corresponding substantially with a desired location in the sample port560, which corresponds with a location above a sample well582in a gel580disposed therebelow. The surface area of the tip landing zone564that does not overlap with the smaller area566may also be referred to herein as a “shelf”. In certain embodiments, the area of the sample port560at the inner surface512of the lid is significantly less than the area of the sample port560at the outer surface514of the lid because the enlarged area accommodates tip splay, whereas the smaller area566of the sample port at the inner surface512can be reduced up to an area slightly larger than the projection area of the pipette tip.

FIGS. 5A and 5Balso illustrates one example of tip registration features568,569comprising a sloped surface for receiving a pipette tip. The sloped surface may be planar and/or curved, and at least partially defines the sample port orifice from the outer surface of the lid to the inner surface thereof. Since a purpose of the tip registration feature568,569is to ensure consistent (and preferably perpendicular) transit of the pipette tip into the cassette, the slope or draft angle of the registration feature(s)568,569preferably match(es) the draft angle of a pipette tip (e.g., 8°). In a preferred embodiment, tip registration features,569are present on a medial or front wall portion of the sample port560, i.e., a sample port wall more proximate to the opposing side of the assay channel.

Referring further toFIGS. 5A and 5B, in operation, a pipette tip570comprising a sample would be lowered down (i.e., along a z-axis relative to the plane of the lid) by a liquid handling device into the tip landing zone564of a sample port560. The tip570would then be moved laterally by the liquid handling device (i.e., along an x- and/or y-axis relative to the plane of the lid510) until it contacts (i.e., registers) a registration feature569. If the registration feature569is aligned with the smaller area566of the sample port560and the well582disposed in the gel580below, following registration, the contents of the tip570would be dispensed and the tip570would be removed from the sample port560by the liquid handling device.

FIGS. 5A and 5Balso illustrate one example of a sample extraction port562comprising a second tip landing zone565and a smaller area of the sample extraction port, the smaller area of the sample extraction port567corresponding with a desired location in the sample extraction port562, which corresponds with a location above a well583in a gel580disposed therebelow where a sample that has been subjected to electrophoresis is anticipated to be located. In operation, a sample that has been subjected to electrophoresis may be extracted from a gel580in the cassette by lowering a pipette tip571down (i.e., along a z-axis relative to the plane of the lid) into the second tip landing zone565of the sample extraction port562. The tip571would then be moved laterally by the liquid handling device (i.e., along an x- and/or y-axis relative to the plane of the lid) until it contacts (i.e., registers) a registration feature569a. If the registration feature569ais aligned with the smaller area567of the sample extraction port565and the well583disposed in the gel580below, following registration, the tip571would be lowered further into the extraction well582, the contents of the extraction well583would be aspirated and the tip571would be removed from the gel extraction well582and sample extraction port562by the liquid handling device.

In general, because the pipette tip has been positioned at a desired location corresponding to the extraction well, the pipette tip may be lowered into the extraction well without worry that the tip might puncture a wall defining the well, which may clog the tip and interfere with extraction of the sample and/or subsequent tip operations.

Referring toFIG. 6, which illustrates one embodiment of a lid provided herein (left), a lid610may be configured to correspond to a plurality of assay channels602, each portion of the lid corresponding to an assay channel602comprising first and second buffer reservoir ports650,652that are integral with first and second barrier ports654,655and further comprising a plurality of sample ports660, each of the sample ports comprising a tip landing zone674, one or more registration features664and desired locations690corresponding to a sample well in a gel disposed in a cassette comprising the lid610.

In general, the configuration of the sample ports provided in the lids of the cassettes provided herein facilitates standardization of the position of multiple mandrel engaged pipette tips by providing tip registration features against which said tips may register prior to descending into a pre-formed well in a gel disposed therebelow.

In the embodiment shown inFIG. 6, each portion of the lid610corresponding to an assay channel602comprises a rectangular sample port660comprising two desired locations690corresponding to two wells in a gel that may be disposed therebelow. These two desired locations690are illustrated as circles inFIG. 6, but may be otherwise shaped (e.g., rectangular or square). Two tip landing zones674are provided in each sample port690, each being located in a distal portion of the sample port660, opposite the desired locations690. The registration features664in each sample port664are defined by walls of the sample port660. An extension666projecting from the sample port wall opposite the tip landing zone674into the sample port660is provided to prohibit cross contamination between wells in the event of overflow. In operation, a pipette tip comprising a sample would be lowered a pre-determined distance (i.e., along a z-axis relative to the plane of the lid610) by a liquid handling device into a tip landing zone674of a sample port660. The tip would then be moved laterally by the liquid handling device along a first axis (for example, along an x-axis relative to the plane of the lid610) until it contacts (i.e., registers) a first registration feature664(i.e., a wall of the sample port660). The tip would then be moved along a second axis (for example, along a y-axis relative to the plane of the lid610) until it contacts (i.e., registers) a second registration feature664a(i.e., a second wall of the sample port). In one embodiment, if the second registration feature664ais aligned with a desired location690, following registration with the second registration feature664a, the contents of the tip would be dispensed and the tip would be removed from the sample port660by the liquid handling device. In one embodiment, if the second registration feature664ais aligned with a desired location690, following registration with the second registration feature664a, the tip would be lowered a pre-determined distance (i.e., along a z-axis relative to the plane of the lid610) by a liquid handling device into a well disposed in a gel therebelow, the contents of the tip would be dispensed in the well and the tip would be removed from the sample port660by the liquid handling device.

In general, dispensing of the sample may be done above a well disposed below a sample port or, preferably, in a well disposed below a sample port. Because the pipette tip has been positioned at a desired location corresponding to the well, the pipette tip may be lowered into the well without worry that the tip might pucture a wall defining the well, which may clog the tip and interfere with dispensing and/or subsequent tip operations.

In general, additional tip registration features and additional desired locations corresponding to sample wells may be added to the sample ports of various embodiments of the cassette provided herein. Thereby allowing a single tip landing zone to correspond with a plurality of tip registration features and desired locations in a sample port.

For example, referring toFIG. 7, which illustrates one embodiment of a sample port in a lid provided herein, each sample port760may comprise a plurality desired locations790corresponding to a plurality of sample wells782in a gel disposed in a cassette comprising the lid. In this embodiment, a plurality of registration features comprising projections764,764aextend from a second wall712aof the sample port, a first wall being adjacent to the plurality of desired locations712. In operation, a pipette tip770comprising a sample would be lowered down a pre-determined distance (i.e., along a z-axis relative to the plane of the lid) by a liquid handling device into the tip landing zone774of the sample port760. The tip770would then be moved laterally by the liquid handling device along a first axis (for example, along an x-axis relative to the plane of the lid, as illustrated inFIG. 8) until it contacts (i.e., registers) a first registration feature712(i.e., the first wall of the sample port). The tip would then be moved along a second axis (for example, along a y-axis relative to the plane of the lid) a pre-determined distance (e.g., a distance equal to the radius of a tip splay, referred to herein as a tip splay zone775, which encompasses an area of maximum predicted tip splay) and then it is moved along the first axis towards a projection registration feature764(e.g., the longest projection extending from the second wall712a). InFIG. 7, the trajectory of the tip is illustrated by arrows.

In general, the process of movement in the x- and y-axes until a tip registers with a registration feature may be repeated by registering a tip multiple times with a plurality of registration features until the tip registers with a registration feature that is aligned with a desired location at which point, the contents of the tip would be dispensed and the tip would be removed from the sample port by the liquid handling device. InFIG. 7, the registration feature aligned with the desired location790is the second wall712aof the sample port760.

In embodiments where tip registration features are projections764,764athat extend into the sample port760from a wall712aof the sample port760opposite the tip landing zone774, e.g., as shown inFIG. 7, each additional projection (e.g.,764a) would decrease in length, thereby allowing the previous projection (e.g.,764) to be used as a registration features in guiding the tip770from the relatively large landing zone774, to the relative small desired location790corresponding to a well782that may be disposed therebelow. In one embodiment, the difference in length of neighbouring projections764,764ais at least as large as the total position variability that can be attributed to tip splay. For example, the tip landing zone774encompasses an area that is large enough to accommodate predicted tip splay (i.e., a tip splay zone775) and the difference in length of subsequent registration features (i.e., projections764,764a) would be equal to or greater than the radius of the tip splay zone775.

It will be understood that various configurations of registration features for guiding a pipette tip to a desired location in a sample port are encompassed by the cassettes disclosed herein.

Referring toFIG. 9, an alternative embodiment of a lid910of a cassette provided herein is shown, the alternative embodiment comprising in each portion of the lid910corresponding to an assay channel902a first buffer reservoir port950that is integral with a first barrier port954, a second buffer reservoir port952that is integral with a second barrier port955, a sample port960and a sample extraction port962. The first and second barrier ports954,955are configured to receive first and second removable barriers (see detail A).

Referring toFIG. 10, an alternative embodiment of a lid1010of a cassette provided herein is shown, the alternative embodiment comprising in each portion of the lid1010corresponding to an assay channel1002a first buffer reservoir port1050that is integral with a first barrier port1054, a second buffer reservoir port1052that is integral with a second barrier port1055and a sample port1060. The sample ports1060are configured to comprise two desired locations1090,1090acorresponding to two wells in a gel that may be disposed therebelow.

Referring toFIG. 11, an alternative embodiment of a lid1110of a cassette provided herein is shown, the alternative embodiment comprising in each portion of the lid1110corresponding to an assay channel1102a first buffer reservoir port1150that is integral with a first barrier port1154, a second buffer reservoir port1152that is integral with a second barrier port1155, a first sample port1160, and a second sample port1160a. The first and second sample ports1160,1160aare each configured to comprise two desired locations1190,1190acorresponding to two wells in a gel that may be disposed therebelow.

Dimensions provided in the figures are non-limiting examples only. In one preferred embodiment, an assembled cassette has a height of about 11.3 cm, a width of about 10.9 cm and a maximum depth of about 2.3 cm.

Manufacturing

In a second aspect, a method for manufacturing one or more embodiments of the cassette provided herein is disclosed. In one embodiment, the method comprises forming at least part of the tray and lid portions of the cassette from an optically neutral material, such as, for example, a clear thermoplastic, such as acrylic (e.g., poly methyl methacrylate) or non-autofluorescing polycarbonate. Methods of manufacturing articles from thermoplastics known in the art may be used to form at least part of tray and lid portions of the cassette, such as, for example, machining, injection molding, laser cutting or three dimensional printing.

In one embodiment, the method further comprises positioning a first removable barrier between the first buffer reservoir and the media channel in each of the assay channels of the formed tray and a second removable barrier between the second buffer reservoir and the media channel in each of the assay channels of the formed tray. In general, it is not necessary that the first and second removable barriers create a fluid impervious seal between the media channel and an adjacent buffer reservoir. Rather, it is only necessary that the removable barriers create a fluid barrier that substantially impedes cross migration of dissimilar fluids when there is not a material hydrostatic pressure differential across the barrier. When cross migration is no longer an issue due to, for example, a phase change in one of the fluids, the removable barriers is are longer needed can be disabled, e.g., by removal of the removable barriers from the assay channels.

Referring toFIG. 12, which illustrates one embodiment of a barrier1256for use in manufacturing various embodiments of the cassette provided herein, the barrier1256may have an elongated comb shape, each tooth1258in the barrier1256being sized and configured to engage the width and depth of an assay channel in the tray of a cassette. In a preferred embodiment, the number of teeth1258in a barrier1256corresponds to the number of assay channels in a cassette and the teeth1258are spaced along a longitudinal axis of the barrier1256such that they may be engaged in the assay channels of the cassette when the barrier1256is introduced into the cassette.

Referring toFIG. 13, which illustrates a top plan view of one embodiment of a cassette provided herein, and13B, which illustrates a side view of the cassette shown inFIG. 13, in one embodiment, a first removable barrier1356is positioned between the first buffer reservoir1330and the media channel1340in each of the assay channels1302and a second removable barrier1356ais positioned between the second buffer reservoir1332and the media channel1340in each of the assay channels1302. In one embodiment, positioning of the first and second barriers1356,1356amay be done directly into a tray portion of a cassette. Referring toFIGS. 13 and 13A, in a preferred embodiment, positioning of the first and second barriers may be done via barrier ports1354in a lid1310of a cassette1300. In a preferred embodiment, the barrier ports1354may comprise one or more structures for engaging at least part of one or more of the first and second removable barriers.

Referring further toFIG. 13, in one embodiment, the method of manufacture further comprises introducing media into each media channel1340in the plurality of assay channels1302of the formed tray. In one embodiment, media may be introduced directly into the media channels1340of the formed tray. In one embodiment, media may be introduced into the media channels1340via one or more sample port1360, one or more sample extraction port1362, and/or one or more portion1354a,1355aof one or more barrier ports1354,1355, wherein the one or more portion1354a,1355aof one or more barrier ports1354,1355is on a side of the first and/or second barrier, which abuts one or more media channels1340.

In one embodiment, the method further comprises positioning a comb into the introduced media in each media channel. Referring toFIG. 14, which illustrates one embodiment of a comb suitable for use in one embodiment of the method of manufacture provided herein, the comb1484may have an elongated shape, each tooth1486in the comb1484being sized and configured to form a well in each media channel of a cassette upon gelification of the media. In a preferred embodiment, the number of teeth1486in a comb1484corresponds to the number of assay channels in a cassette and the teeth1486are spaced along a longitudinal axis of the comb1484such that they may be engaged in the assay channels of the cassette when the comb1484is introduced into the cassette. Positioning of the comb1484may be done at a location in the assay channels where gel wells are desired. In a preferred embodiment, a second comb may be positioned in the assay channels comprising media at a position where sample extraction wells are desired.

In one embodiment, an additional comb1584may be combined with comb1484for use in various embodiments of the method of manufacture provided herein, as shown inFIG. 15A. In operation, combs1484and1584may be used to create a well in the plurality of media channels in various embodiments of the cassette provided herein, wherein the well comprises a standard well portion for receiving a dispensed sample that is created by a tooth1486in comb1484and further comprises a shelf. In one embodiment, such a shelf in the media of a media channel may be provided as an alternative to a tip landing zone that is manufactured in the lid of the cassette.

In one embodiment, the method further comprises introducing buffer into each first and second buffer reservoir in each assay channel of the formed tray. In one embodiment, buffer may be introduced directly into the first and second buffer reservoirs of the formed tray. In one embodiment, buffer may be introduced into the first and second buffer reservoirs via one or more buffer reservoir port and/or one or more portion of one or more barrier ports, wherein the one or more portion of one or more barrier ports is on a side of the first and/or second barrier, which abuts one or more buffer reservoirs.

In one embodiment, the method further comprises removing the comb from the introduced media after gelification of the introduced media and removing the barrier after gelification of the media. In a preferred embodiment, the barrier is easily removable, at least because it is not configured to form a water-tight seal with the assay channels. In this embodiment, introduction of the media and buffer may be carried out substantially simultaneously without mixing of the media and buffer, at least because the partial pressures on either side of the barrier caused, at least in part, by the fluid on either side of the barrier are nearly equivalent and opposing in force, thereby inhibiting mixing of the media and buffer. Simultaneous media and buffer introduction may be advantageous, at least for example, because it may speed manufacturing time.

In one embodiment, the method further comprises engaging the formed lid on the side walls of the formed tray, thereby creating a space between the floor of the formed tray and the lid, the space being occupied at least partially by the introduced media and the introduced buffer. The step of engaging the lid on the tray may be completed prior to or following introduction of media and/or buffer and prior to or following positioning of barrier(s) and/or comb(s). In one preferred embodiment, lid and tray portions of the cassette may be integral. In this embodiment, the lid and tray portions would be engaged to form a cassette prior to introduction of media and buffer and prior to positioning of barrier(s) and comb(s).

In one embodiment, once lid and tray portions are formed, the method of manufacturing various embodiments of a cassette provided herein further comprises: establishing one or more temporary barriers between one or more buffer receiving portions of the cassette and a media receiving portion of the cassette; substantially simultaneously the filling buffer receiving portion(s) and the media receiving portion to minimize hydrostatic pressure differentials across the temporary barrier; and removing the temporary barrier once the hydrostatic pressure differential decreases below a threshold value. Such methods, as previously intimated, are preferably carried out simultaneously for multiple assay channels so that the benefits of parallel processing can be fully realized.

Method of Using Cassette in an Electrophoresis Assay

In general, methods of using various embodiments of a cassette provided herein comprise generally directing a pipette tip to a sample port, for example by targeting a tip landing zone in the sample port; contacting the tip with a tip registration feature in the sample port; allowing the pipette tip to be guided along the tip registration feature to a desired location in the sample port corresponding with a well in a gel disposed in the cassette therebelow; and dispensing the contents of the pipette tip. Such method is preferably carried out simultaneously in a plurality of assay channels in the cassette. In operation, a gantry arm of a suitable liquid handling device comprises an array of mandrels, each comprising a pipette tip. The gantry arm is moved such that each pipette tip is moved downward into a tip landing zone of each sample port and then laterally in the sample port until each pipette tip contacts a tip registration feature, preferably each tip registers against the same portion of tip registration feature in each sample port, thereby ensuring uniform and consistent pipette transit into respective sample ports.

In one embodiment, the method comprises positioning the cassette in a liquid handling device. Liquid handling devices suitable for transferring liquids by pipette are known in the art. In this embodiment, one or more method steps are carried out by a liquid handling device. The method further comprises inserting a plurality of pipette tips into tip landing zones of the plurality of sample ports in a cassette. In a preferred embodiment, the plurality of pipette tips held by mandrels of the liquid handling device correspond to the plurality of sample ports and the relative spacing of the plurality of sample ports. The method further comprises moving the plurality of pipette tips inserted in the tip landing zones in one or more axes towards one or more of the tip registration features, the one or more axes being co-planar with the plane of the cassette lid. For example, the plurality of pipette tips inserted in the tip landing zones may be moved in an x-axis towards a plurality of registration features. The method further comprises registering the plurality of pipette tips against the one or more tip registration tip features, at least one of the one or more tip registration features per sample port being adjacent to the desired location in each of the sample ports. Registration of the tips with corresponding registration features in a sample port ensures that the tips are in the desired locations of each sample port, even if one or more tip on the liquid handling mandrel is splayed. The method further comprises dispensing a plurality of samples from the plurality of pipette tips registered against the registration features adjacent to the desired location in each of the sample ports. This step facilitates introduction of a sample into a well in an assay channel without worry of inserting the tip into the media because the tip(s) is not lowered into the well, rather, the tip remains above the well.

In one embodiment, the method further comprising engaging the cassette comprising the plurality of dispensed samples with electrodes and a power supply, thereby creating one or more electric fields in the cassette, the one or more electric fields being sufficient to case migration of the plurality of samples through the gelified media in the plurality of assay channels. Methods of electrophoretically treating samples (e.g., DNA, protein etc.) and tool for use of same are known in the art. In one embodiment, a single voltage may be applied to an entire cassette. In one embodiment, individual voltages may be applied to one or more assay channel in various embodiments of the cassette provided herein.

In a preferred embodiment, the method further comprises extraction of a migrated sample from the cassette, the cassette comprising a plurality of sample extraction ports. In this embodiment, the plurality of migrated samples are extracted from the cassette following exposure to the electric field. In one embodiment, the method of guiding pipette tips to the samples to be extracted is similar to the method of guiding pipette tips to the desired location in the sample port for sample introduction. For example, in one embodiment, the extraction comprises: inserting a second plurality of pipette tips into second tip landing zones of the plurality of sample extraction ports in the cassette. The liquid handling device then moves the second plurality of pipette tips inserted in the second tip landing zones in one or more axes towards one or more of the second tip registration features, the one or more axes being co-planar with the plane of the cassette lid, until the second plurality of pipette tips are registered against the one or more second tip registration tip features, at least one of the one or more second tip registration features per sample extraction port being adjacent to a second desired location in each of the sample extraction ports. As described above, this step adjusts for any tip splay in the second plurality of pipette tips. The method further comprises moving the registered second plurality of pipette tips downward into the gelified media below the second desired location in each of the sample ports and aspirating the plurality of migrated samples from the gelified media below the second desired location in each of the sample ports, thereby extracting the migrated samples. The plurality of pipette tips comprising the extracted, migrated samples are then retracting from the gelified media and the sample ports of the cassette.

Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the purpose and scope of the invention as outlined in the claims appended hereto. Any examples provided herein are included solely for the purpose of illustrating the invention and are not intended to limit the invention in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the invention and are not intended to be drawn to scale or to limit the invention in any way. The disclosures of all prior art recited herein are incorporated herein by reference in their entirety.