Patent ID: 12194468

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Samples often include or are treated to release materials capable of interfering with the detection of an analyte (e.g., a targeted nucleic acid). To remove these interfering materials, samples can be treated with a target capture reagent that includes a magnetically-responsive solid support for immobilizing the analyte. See, e.g., U.S. Pat. Nos. 4,486,539, 4,751,177, 5,288,609, 5,780,224, 6,433,160, and 6,534,273. Suitable solid supports are paramagnetic particles (0.7-1.05 micron particles, Sera-Mag™ MG-CM, available from Seradyn, Inc., Indianapolis, IN, as Cat. No. 24152105-050450) with covalently bound oligo(dT)14. When the solid supports are brought into close proximity to a magnetic force, the solid supports are drawn out of suspension and aggregate adjacent a surface of a sample holding container, thereby isolating any immobilized analyte within the container. Non-immobilized materials in the sample can then be aspirated or otherwise separated from immobilized analyte. One or more wash steps may be performed to further purify the analyte.

Methods, systems, and apparatus for performing a procedure for isolating and separating an analyte of interest from other components of a sample are embodied in a magnetic separation station, an embodiment of which is shown inFIG.4. The magnetic separation station comprises a housing configured to receive a reaction receptacle which contains a sample material and a target capture reagent including magnetically-responsive solid supports adapted to directly or indirectly bind to an analyte of interest, such as a nucleic acid, that may be present in the sample. Details of an exemplary reaction receptacle and sample preparation procedures are described in more detail below.

The magnetic separation station includes magnets for attracting the magnetically-responsive solid supports to a side wall of the reaction receptacle and apparatus for selectively moving the magnets between a first position in which the magnets have substantially no effect on the magnetically-responsive solid supports contained within the reaction receptacle and a second position in which the magnets draw the magnetically-responsive solid supports to a side wall of the reaction receptacle. In particular embodiments, the apparatus for selectively moving the magnets is configured to effect a linear translation of the magnets between the first and the second positions. Such an apparatus may comprise a sled on which the magnets are carried and which may be actuated for linear translation by a belt or a threaded rod driven by a motor. The magnetic separation station further includes apparatus for aspirating fluids from a reaction receptacle held in the station, apparatus for dispensing fluids into the reaction receptacle, and apparatus for agitating the reaction receptacle to re-suspend magnetically-responsive solid supports and other materials following the aspirating and dispensing steps. The apparatus for aspirating fluids from the reaction receptacles may include aspirator tubes, and removable protective tips may be placed on the ends of the aspirator tubes and exchanged for new tips after each reaction receptacle is processed by the magnetic separation station to prevent contamination from one reaction receptacle to the next. The apparatus for effecting linear movement of the magnets may include tip removal elements adapted for removing the tips from the aspirator tubes after each receptacle is processed by the magnetic separation station.

A reaction receptacle containing sample material and a target capture reagent that includes magnetically-responsive solid supports is placed into the magnetic separation station, and the magnets are moved from the first, non-affecting position to the second position adjacent to the reaction receptacle. The magnets are held in the second position for a specified dwell time to draw magnetically-responsive solid supports to the side of the reaction receptacle. After the specified dwell time, with the magnets still in the second position, the fluid contents of the reaction receptacle are aspirated from the receptacle. A wash solution or other suspending fluid is then dispensed into the reaction receptacle, the magnets are moved back to the first position, and the reaction receptacle is agitated to rinse the magnetically-responsive solid supports from the reaction receptacle wall and re-suspend the magnetically-responsive solid supports. The magnets are then moved back to the second position to draw the magnetically-responsive solid supports to the walls of the reaction receptacle and out of suspension. This process of applying a magnetic force for a specified dwell time, aspirating fluid from the reaction receptacle, and re-suspending the magnetically-responsive solid supports may be repeated a specified number of times.

The magnetic separation station may be part of an instrument including various modules configured to receive one or more reaction receptacles within which is performed one or more steps of a multi-step analytical process, such as a nucleic acid test (NAT), or other chemical, biochemical or biological process. The instrument may further include a transfer apparatus configured to transfer reaction receptacles between the various modules, including transporting reaction receptacles into and out of the magnetic separation station. The instrument and each individual component, such as the magnetic separation station, is automated and may be controlled by an instrument control module including a microprocessor executing an instrument control program stored thereon.

Further details of the magnetic separation station are described below.

Other aspects of the invention are embodied in a magnetic receptacle holding station, an embodiment of which is shown inFIG.16. The receptacle holding station comprises a structure configured for receiving and temporarily holding a reaction receptacle in a stationary position. The receptacle holding station includes magnets configured so as to be positioned adjacent to the reaction receptacle when the receptacle is held within the receptacle holding station. A reaction receptacle containing sample material to which magnetically-responsive solid supports have been added can be placed into the receptacle holding station for a specified dwell time prior to moving the reaction receptacle into the magnetic separation station. The magnetically-responsive solid supports will be drawn to the side wall of the reaction receptacle to form an aggregate of solid supports prior to the receptacle being placed in the magnetic separation station, thus reducing at least the first magnetic dwell time required within the magnetic separation station.

Further details of the magnetic receptacle holding station are described below.

Multiple Receptacle Devices

As shown inFIG.1, a reaction receptacle in the form of a multiple receptacle device (“MRD”)160that can be used in conjunction with the magnetic separation station and the magnetic receptacle holding station of the present invention comprises a plurality of individual receptacles162, preferably five. Other types of receptacle devices can be used in conjunction with the magnetic separation and magnetic receptacle holding stations, including devices comprising a single, individual receptacle. In the illustrated embodiment, the receptacles162are in the form of cylindrical tubes with open top ends and closed bottom ends and are connected to one another by a connecting rib structure164which defines a downwardly facing shoulder extending longitudinally along either side of the MRD160. In the illustrated embodiment of the MRD160, all of the receptacles162are substantially identical in size and shape. In other embodiments, the MRD160may include receptacles of varying sizes and shapes, which can be configured for use with the magnetic separation and magnetic receptacle holding stations.

In one embodiment, the MRD160is formed from injection molded polypropylene, such as that manufactured by Flint Hills Resources as product number P5M6K-048.

An arcuate shield structure169is provided at one end of the MRD160. An MRD manipulating structure166extends from the shield structure169. The manipulating structure is adapted to be engaged by a transport mechanism for moving the MRD160between different locations or modules of an instrument. Exemplary transport mechanisms that are compatible with the MRD160are described in U.S. Pat. No. 6,335,166 and in U.S. Provisional Application No. 61/178,728 and corresponding non-provisional U.S. application Ser. No. 12/781,241. MRD manipulating structure166comprises a laterally extending plate168extending from shield structure169with a vertically extending piece167on the opposite end of the plate168. A gusset wall165extends downwardly from lateral plate168between shield structure169and vertical piece167.

As shown inFIG.3, the shield structure169and vertical piece167have mutually facing convex surfaces. The MRD160may be engaged by a transport mechanism and other components, by moving an engaging member laterally (in the direction “A”) into the space between the shield structure169and the vertical piece167. The convex surfaces of the shield structure169and vertical piece167provide for wider points of entry for an engaging member undergoing a lateral relative motion into the space.

A label-receiving structure174having a flat label-receiving surface175is provided on an end of the MRD160opposite the shield structure169and MRD manipulating structure166. Human and/or machine-readable labels, such as scanable bar codes, can be placed on the surface175to provide identifying and instructional information on the MRD160.

The MRD160preferably includes tip holding structures176adjacent the open mouth of each respective receptacle162. Each tip holding structure176provides a cylindrical orifice within which is received a conduit, such as contact-limiting tip170, that is adapted to be placed onto the end of an aspirator tube860. The construction and function of the tip170will be described below. Each holding structure176can be constructed and arranged to frictionally receive a tip170in a manner that prevents the tip170from falling out of the holding structure176when the MRD160is inverted, but permits the tip170to be removed from the holding structure176when engaged by an aspirator tube860.

As shown inFIG.2, the tip170comprises a generally cylindrical structure having a peripheral rim flange177and an upper collar178of generally larger diameter than a lower portion179of the tip170. The tip170is preferably formed from conductive polypropylene. When the tip170is inserted into an orifice of a holding structure176, the flange177contacts the top of structure176and the collar178provides a snug but releasable interference fit between the tip170and the holding structure176. Alternatively, each holding structure176may be configured to loosely receive a tip170so that the tip170is more easily removed from the holding structure when engaged by an aspirator tube860.

An axially extending through-hole180passes through the tip170. Hole180includes an outwardly flared end181at the top of the tip170which facilitates insertion of a tubular probe (not shown) into the tip170. Two annular ridges183may be provided on the inner wall of hole180. Ridges183provide an interference friction fit between the tip170and a tubular probe inserted into the tip170.

The bottom end of the tip170preferably includes a beveled portion182. When tip170is used on the end of an aspirator tube860that is inserted to the bottom of a reaction receptacle, such as a receptacle162of an MRD160, the beveled portion182prevents a vacuum from forming between the end of the tip170and the bottom of the reaction receptacle.

Further details regarding the MRD160may be found in U.S. Pat. No. 6,086,827.

Specimen Preparation Procedure

For nucleic acid tests, it may be necessary to lyse or permeabilize cells to first release a targeted nucleic acid and make it available for hybridization with a detection probe. See, e.g., Clark et al., “Method for Extracting Nucleic Acids from a Wide Range of Organisms,” U.S. Pat. No. 5,786,208. If the cells are lysed, the contents of the resulting lysate may include, in addition to nucleic acids, organelles, proteins (including enzymes such as proteases and nucleases), carbohydrates, and lipids, which may necessitate further purification of the nucleic acids. Additionally, for pathogenic organisms, chemical or thermal inactivation of the organisms may be desirable. Cells may be lysed or permeabilized by a variety of means well known to those skilled in the art, including by chemical, mechanical (e.g., sonication) and/or thermal means.

Various methods for capturing nucleic acids using magnetically-responsive solid supports are known in the art and can be employed in the present invention. These methods may be specific or non-specific for the targeted nucleic acid. One such method is Solid Phase Reversible Immobilization, which is based on the selective immobilization of nucleic acids onto magnetic microsolid support having carboxyl group-coated surfaces. See U.S. Pat. No. 5,705,628. In another method, magnetic particles having poly(dT) sequences derivatized thereon bind to capture probes having 5′ poly(dA) tails and 3′ target binding sequences. See U.S. Pat. No. 6,534,273. Still another approach is based on the ChargeSwitch® Technology, which is a magnetic bead-based technology that provides a switchable surface that is charge dependent on the surrounding buffer pH to facilitate nucleic acid purification (Invitrogen Corporation, Carlsbad, Calif.; Cat. No. CS12000). In low pH conditions, the ChargeSwitch® Magnetic Beads have a positive charge that binds the negatively charged nucleic acid backbone. Proteins and other contaminants that are not bound can be washed away. By raising the pH to 8.5, the charge on the surface is neutralized and the bound nucleic acids are eluted.

For approaches involving capture probes, the capture probes may be specific or non-specific for the targeted nucleic acids. A specific capture probe includes a target binding region that is selected to bind to a target nucleic acid under a predetermined set of conditions and not to non-target nucleic acids. A non-specific capture probe does not discriminate between target and non-target nucleic acids under the conditions of use. Wobble capture probes are an example of a non-specific capture probe and may include at least one random or non-random poly(K) sequence, where “K” can represent a guanine, thymine or uracil base. See U.S. Patent Application Publication No. US 2008-0286775 A1. In addition to hydrogen bonding with cytosine, its pyrimidine complement, guanine will also hydrogen bond with thymine and uracil. Each “K” may also represent a degenerate nucleoside such as inosine or nebularine, a universal base such as 3-nitropyrrole, 5-nitroindole or 4-methylindone, or a pyrimidine or purine base analog such as dP or dK. The poly(K) sequence of a wobble capture probe is of sufficient length to non-specifically bind the target nucleic acid, and is preferably 6 to 25 bases in length.

Sample material is prepared for a magnetic separation procedure by dispensing a specified amount of a target capture reagent into each sample-holding receptacle of a receptacle device. Dispensing may be performed manually or by an automated, robotic pipetting apparatus—into each of the receptacles162of the MRD160. The target capture reagent includes a solid support material able to directly or indirectly bind to an analyte, such as through a capture probe, thereby immobilizing the analyte on the solid support comprises magnetically-responsive particles or beads. The amount dispensed into each receptacle162is typically in the range of 100-500 ΦL.

Magnetic Separation Stations

Turning toFIGS.4-5, a magnetic separation station800includes a module housing802having an upper section801and a lower section803. Mounting flanges805,806extend from the lower section803for mounting the magnetic separation station800to a support surface by means of suitable mechanical fasteners. Locator pins807and811extend from the bottom of lower section803of housing802. Pins807and811register with apertures (not shown) formed in the support surface to help to locate the magnetic separation station800on the support surface before the housing802is secured by fasteners.

A loading slot804extends through the front wall of the lower section803to allow a transport mechanism (not shown) to place a receptacle device, such as an MRD160, into and remove the receptacle device from the magnetic separation station800. A tapered slot extension821may be provided around a portion of the loading slot804to facilitate receptacle insertion through the slot804. A divider808separates the upper section801from the lower section803. A receptacle carrier unit820is disposed adjacent the loading slot804, below the divider808, for operatively supporting the receptacle disposed within the magnetic separation station800. For purposes of illustration, the receptacle carrier unit820is shown inFIG.5carrying an MRD160, but other receptacles, including single, individual receptacles and multiple receptacle devices having receptacles of varying shapes and sizes, may be used. Turning toFIG.7, the receptacle carrier unit820has a slot822for receiving the upper end of a receptacle device, such as an MRD160. In the illustrated embodiment, a lower fork plate824attaches to the bottom of the receptacle carrier unit820and supports the underside of the connecting rib structure164of the MRD160when slid into the carrier unit820(seeFIGS.8and9). A spring clip826is attached to the carrier unit820with its opposed prongs831,833extending into the slot822to releasably hold the receptacle within the carrier unit820.

As an alternative to the arrangement shown inFIG.7, the receptacle carrier unit820may comprise a single, injection molded part, which may include an integrally-formed ledge if configured for supporting an MRD160, and an integrally-formed plastic spring element for retaining a receptacle within the receptacle carrier unit820.

An orbital mixer assembly828is coupled to the carrier unit820for orbitally mixing the contents of an MRD, or other receptacle device, held by the receptacle carrier unit820. The orbital mixer assembly828includes a stepper motor830mounted on a motor mounting plate832, a drive pulley834having an eccentric pin836, an idler pulley838having an eccentric pin840, and a belt835connecting drive pulley834with idler pulley838. A suitable stepper motor includes a VEXTA, model number PK245-02A, available from Oriental Motors Ltd. of Tokyo, Japan, and suitable belts835include a timing belt, model number A 6G16-170012, available from SDP/SI of New Hyde Park, New York As shown inFIGS.5and7, eccentric pin836fits within a slot842formed longitudinally in the receptacle carrier unit820. Eccentric pin840fits within a circular aperture844formed in the opposite end of receptacle carrier unit820. As the motor830turns the drive pulley834, idler pulley838also rotates via belt835and the receptacle carrier unit820is moved in a horizontal orbital path by the eccentric pins836,840engaged with the apertures842,844, respectively, formed in the carrier unit820. The rotation shaft839of the idler pulley838preferably extends upwardly and has a transverse slot841formed therethrough. An optical slotted sensor843is disposed at the same level as the slot841and measures the frequency of the idler pulley838via the sensor beam intermittently directed through slot841as the shaft839rotates. A suitable sensor includes an Optek Technology, Inc., model number OPB980T11, sensor, available from Optek Technology, Inc. of Carrollton, Texas.

As an alternative to slot841and sensor843, the frequency of idler pulley838may be measured by means of an encoder (not shown) mounted on the top of shaft839.

Drive pulley834also includes a locator plate846. Locator plate846passes through slotted optical sensors847,848mounted to a sensor mounting bracket845extending from motor mounting plate832. Suitable sensors include Optek Technology, Inc., model number OPB980T11, sensors, available from Optek Technology, Inc. of Carrollton, Texas. Locator plate846has a plurality of circumferentially spaced axial openings formed therein which register with one or both sensors847,848to indicate a position of the orbital mixer assembly828, and thus a position of the receptacle carrier unit820.

As an alternative to locator plate and sensors847,848, the frequency and position of drive pulley834may be measured by means of an encoder (not shown) coupled to the pulley834.

A pivoting magnet moving apparatus810is attached inside the lower section803so as to be pivotable about point812. The magnet moving apparatus810carries permanent magnets814, which are positioned on either side of a slot815formed in the magnet moving apparatus810. The magnet moving apparatus810is constructed and arranged to move the magnets814between an operational position and a non-operational position with respect to a receptacle device carried in the receptacle carrier unit820. In the operational position, the magnets814are disposed adjacent the receptacle, e.g., the MRD160, and in sufficient proximity to the receptacle so that magnetically-responsive solid supports within each receptacle162are drawn out of suspension by the attraction of the magnetic fields of the magnets814. In the non-operational position, the magnets are disposed at a sufficient distance from the receptacles162so as to have no substantial effect on the contents of the receptacles162. In the present context, “no substantial effect” means that the magnetically-responsive solid supports are not drawn out of suspension by the attraction of the magnetic fields of the magnets814.

Preferably five magnets, one corresponding to each individual receptacle162of the MRD160, are held in an aligned arrangement on each side of the magnet moving apparatus810. The magnets are preferably made of neodymium-iron-boron (NdFeB), minimum grade n-35 and have preferred dimensions of 0.5 inch width, 0.3 inch height, and 0.3 inch depth. An electric actuator, generally represented at816, pivots the magnet moving apparatus810up and down, thereby moving the magnets814. As shown inFIG.5, actuator816preferably comprises a rotary stepper motor819which rotates a drive screw mechanism coupled to the magnet moving apparatus810to selectively raise and lower the magnet moving apparatus810. Motor819is preferably an HSI linear stepper actuator, model number 26841-05, available from Haydon Switch and Instrument, Inc. of Waterbury, Connecticut.

A sensor818, preferably an optical slotted sensor, is positioned inside the lower section803of the housing for indicating the down, or “home”, or non-operational, position of the magnet moving apparatus810. Another sensor817, also preferably an optical slotted sensor, is preferably provided to indicate the up, or operational, position of the magnet moving apparatus810. Suitable sensors include model number OPB980T11, available from Optek Technology, Inc. of Carrollton, Texas.

An alternate embodiment of a magnet moving apparatus for moving the magnets between operational and non-operational positions with respect to the receptacle is shown inFIGS.10and11. The magnet moving apparatus comprises magnet slide200which comprises a magnet sled202that is moved along a linear path by a drive system232.

More specifically, the magnet sled202includes a first wall204having a guide rod aperture206and a rectangular, U-shaped cutout208. The a magnet sled202further comprises a second wall214having a guide rod aperture216and a rectangular cutout218formed therein. A first magnet220is positioned between the first wall204and the second wall214on one side of the respective cutouts208,218and is supported by a first magnet backing plate224. Similarly, a second magnet222is disposed between the first wall204and the second wall214on an opposite side of the respective rectangular cutouts208,218and is backed by a magnet backing plate (not shown). Magnets220,222may be made from NdFeB, grade n-35 or grade n-40. As an alternative to single magnets220,222on opposite sides of the magnet sled202, individual magnets may be provided on each side of the sled202, one for each receptacle162. In one embodiment, the sled includes five magnets on each side, each magnet having a size of approximately 12 mm×12 mm×7.5 mm and being made from NdFeB, grade n-40. The number of magnets corresponds to the number of receptacles162comprising the MRD160.

Magnet sled202further includes a bottom plate226with a plurality of tip stripping elements in the form of stripping openings228formed therein. Operation of the tip stripping openings228will be described below. Finally, the magnet sled202includes a guide surface formed in part by a straight laterally extended edge230formed in the first wall204. A similar laterally extending straight edge is formed in the back wall214. Any of the first and second walls204,214, first and second magnet backing plates224, and bottom plate226may be integrally formed with each other. Suitable materials for the first and second walls204,214and bottom plate226include non-magnetically-responsive materials such as plastics and aluminum. Preferred material for the first and second magnet backing plates224include magnetically-responsive materials, such as steel, to increase magnetic flux flowing through the magnets.

The drive system232comprises a drive motor234having a drive pulley236and mounted on the outside of the lower housing803. A drive belt238is carried on the drive pulley236and an idler pulley248and extends through an opening813formed in the lower housing803. Opposite ends243,245of the drive belt238are attached to the magnet sled202by means of a coupling bracket240. Suitable belts are available from the Gates Corporation.

The coupling bracket240includes a top plate241disposed across the top of the second magnet222and having belt retaining slots within which opposite ends243,245of the drive belt238are inserted and secured. A retainer tab247bent transversely with respect to the top plate241is positioned within a conforming slot formed in the first wall204. A similar tab (not shown) is provided on the opposite end of the top plate241and extends within a conforming slot formed in the second wall214for securing the coupling bracket240to the magnet sled202.

The magnet sled202is disposed inside the lower housing803with the guide surface230supported on a guide ledge242extending along an inner surface of the lower housing803. The opposite side of the magnet sled202is supported by a guide rod212extending across the lower housing803and through the guide rod apertures206and216. A bushing (not shown) may be provided at either or both of the guide rod apertures206,216for securely and slidably supporting the magnet sled202.

Rotation of the drive pulley236by the drive motor234turns the drive belt238to thereby move the magnet sled202between a non-operational position, such as shown inFIGS.10and11, and an operational position whereby the magnet sled202is moved to the opposite side of the lower housing803. When the magnet sled202is moved to the operational position, the lower ends of the receptacles162pass through the rectangular cutouts208,218of the first wall204and second wall214, respectively, so as to be disposed between the first magnet220and the second magnet222.

A retracted position sensor244mounted to an inner surface of the lower housing803indicates when the magnet sled202is in a retracted, or non-operational, position. Similarly, an extended position sensor246, also mounted to an inner surface of the lower housing803, indicates when the magnet sled202is in an extended, or operational, position. Sensors244and246may comprise slotted optical sensors which detect the presence of a tab (not shown) projecting from a lower portion of the magnet slide202.

A further alternative embodiment of a magnet moving apparatus is shown inFIGS.12-15. The magnet moving apparatus ofFIGS.12-15comprises a magnet slide250including a magnet sled252and a drive system284which moves the magnet sled252between a non-operational position (as shown inFIG.12) and an operational position with respect to an receptacle.

More specifically, the magnet sled252comprises a first wall254including a screw follower256and a rectangular opening258. An extended flange260may be provided around the rectangular opening258. The magnet sled252further comprises a second wall262having a guide bushing264and a rectangular opening266. A first magnet268is disposed between the first wall254and the second wall262and is supported by a first magnet backing plate272. Similarly, a second magnet270is disposed between the first wall254and the second wall262on an opposite side of the rectangular openings258,266and is supported by a second magnet backing plate274. Again, as an alternative to single magnets268,270on opposite sides of the magnet sled252, five individual magnets having a size of approximately 12 mm×12 mm×8 mm and made from NdFeB, grade n-40 can be provided on each side of the sled252.

The magnet sled252further includes a bottom plate276in which a plurality of tip stripping openings278are formed, a guide surface280, and a retainer bracket282. Guide surface280may comprise two surfaces disposed on opposite sides of the retainer bracket282.

A drive system284includes a drive motor to a286mounted on the exterior of the lower housing803and having a drive pulley288. A threaded drive screw292extends across the lower housing803and is journaled at its opposite ends to the lower housing wall so as to be rotatable about its longitudinal axis. Threaded drive screw292further includes a pulley294located at one end thereof. The threaded drive screw292is operatively coupled to the drive motor286by means of a drive belt290carried on the drive pulley288of the drive motor286and the pulley294of the threaded drive screw292.

The threaded drive screw292extends through the screw follower256of the first wall254and the guide bushing264of the second wall262. The guide surface280on the bottom surface of the magnet sled252and located on the opposite side of the sled252from the screw follower256and guide bushing264slidably rests on a guide flange295extending along an inner wall of the lower housing803. A lower portion of the retainer bracket282extends beneath the guide flange295so that the guide flange is disposed between the guide surface280and the retainer bracket282.

Rotation of the drive pulley288by the drive motor286is transferred to the threaded drive screw292by means of the drive belt290. The rotating drive screw292engaged with the screw follower256causes linear translation of the magnet sled252in a longitudinal direction with respect to the drive screw292. Rotation of the drive screw292in one direction will cause left to right translation of the magnet sled252, and rotation of the screw292in the opposite direction will cause right to left translation of the magnet sled252. The retainer bracket282engaged with the underside of the guide flange295prevents the magnet sled252from tipping out of contact with the guide flange295due to friction between the drive screw292and the screw follower256.

When the magnet sled252is moved from the non-operational position, shown inFIG.12, to an operational position, the receptacle passes through the rectangular openings258,266and is disposed between the first magnet268and second magnet270. The extended flange260formed around the rectangular opening258of the first wall254will assist in guiding the receptacle through the opening258. A retracted position sensor296mounted to the inner wall of the lower housing803indicates when the magnet sled252is in a retracted, or non-operational, position, and an extended position sensor298, also mounted to the inner wall of the lower housing803, indicates when the magnet sled252is in an extended, or operational, position with respect to the receptacle. Sensors296and298may comprise optical sensors which detect the presence of a tab extending from a portion of the magnet sled252.

Returning toFIG.4, wash solution delivery tubes854connect to fittings856and extend through a top surface of the module housing802. Wash solution delivery tubes854extend through the divider808via fittings856, to form a wash solution delivery network.

As shown inFIGS.8and9, wash solution dispenser nozzles858extending from the fittings856are disposed within the divider808. Each nozzle is located above a respective receptacle162of the MRD160at a laterally off-center position with respect to the receptacle162. Each nozzle includes a laterally-directed lower portion859for directing the wash solution into the respective receptacle162from the off-center position. Suitable wash solutions are known to those skilled in the art, an example of which contains 10 mM Trizma base, 0.15 M LiCl, 1 mM EDTA, and 3.67 mM lithium lauryl sulfate (LLS), at pH 7.5. Dispensing fluids into the receptacles162in a direction having a lateral component can limit splashing as the fluid runs down the sides of the respective receptacles162. In addition, the laterally directed fluid can rinse away materials clinging to the sides of the respective receptacles162.

As shown inFIGS.4and5, aspirator tubes860, or probes, extend through a tube holder862, to which the tubes860are fixedly secured, and extend through openings861in the divider808. A tip sense printed circuit board (“PCB”)809(seeFIG.7) is attached by mechanical fasteners to the side of divider808, below openings861. Aspirator hoses864connected to the aspirator tubes860extend to a vacuum pump (not shown), with aspirated fluid drawn off into a fluid waste container carried (not shown). In one embodiment, each of the aspirator tubes860has a length of 12 inches with an inside diameter of 0.041 inches.

The tube holder862is attached to a drive screw866actuated by a lift motor868. A suitable lift motor includes the VEXTA, model number PK245-02A, available from Oriental Motors Ltd. of Tokyo, Japan, and a suitable drive screw includes the ZBX series threaded anti-backlash lead screw, available from Kerk Motion Products, Inc. of Hollis, New Hampshire. In the illustrated embodiment, the tube holder862is attached to a threaded sleeve863of the drive screw866. Rod865and slide rail867function as a guide for the tube holder862. Alternatively, a linear bearing (not shown) may be employed as a guide for the tube holder862. Z-axis sensors829,827(slotted optical sensors) cooperate with a tab extending from the tube holder862and/or the threaded sleeve863to indicate top and bottom of stroke positions of the aspirator tubes860. Suitable Z-axis sensors include Optek Technology, Inc., model number OPB980T11, sensors, available from Optek Technology, Inc. of Carrollton, Texas. Together, the tube holder862, lift motor868, and drive screw866comprising an embodiment of a moving mechanism for the tubes860.

Cables bring power and control signals to the magnetic separation station800, via one or more connectors (one such connector is shown at reference number870).

The magnet moving apparatus810,200,250is initially in a non-operational position (e.g., as shown in phantom inFIG.5and inFIGS.10and12), as verified by the retracted position sensor818,244,296, when the receptacle is inserted into the magnetic separation station800through the insert opening804and into the receptacle carrier unit820. When the magnet moving apparatus is in the non-operational position, the magnetic fields of the magnets will have no substantial effect on the magnetically-responsive solid supports contained in the receptacle. The orbital mixer assembly828moves the receptacle carrier unit820a portion of a complete orbit so as to move the receptacle carrier unit820and MRD160laterally, so that each of the tips170carried by the tip holding structures176of the MRD160is aligned with each of the aspiration tubes860, as shown inFIG.9. The position of the receptacle carrier unit820is verified, for example, by the locator plate846and one of the sensors847,848. Alternatively, the stepper motor830can be moved a known number of steps to place the receptacle carrier unit820in the desired position, and one of the sensors847,848can be omitted. Note that magnet moving apparatus cannot move to an operational position when the receptacle carrier unit820has been moved to this tip engagement position because the MRD160carried by unit820will interfere with movement of the magnet moving apparatus.

The tube holder862and aspirator tubes860are lowered by the lift motor868and drive screw866until each of the aspirator tubes860frictionally engages a conduit, e.g., a tip170, held in an associated carrying structure176on the MRD160.

As shown inFIG.6, the lower end of each aspirator tube860is characterized by a tapering, step construction, whereby the tube860has a first portion851along most of the extent of the tube, a second portion853having a diameter smaller than that of the first portion851, and a third portion855having a diameter smaller than that of the second portion853. The diameter of the third portion855is such as to permit the end of the tube860to be inserted into the flared portion181of the through-hole180of the tip170and to create an interference friction fit between the outer surface of third portion855and a portion of the inner wall of the through-hole180, such as the two annular ridges183(seeFIG.2) or alternatively, longitudinally-oriented ridges (not shown), that line the inner wall of hole180of tip170. An annular shoulder857is defined at the transition between second portion853and third portion855. The shoulder857limits the extent to which the tube860can be inserted into the tip170, so that the tip can be stripped off after use, as will be described below.

The tips170may be at least partially electrically conductive, so that the presence of a tip170on an aspirator tube860can be verified by the capacitance of a capacitor comprising the aspirator tubes860and tips170as one half of the capacitor and the surrounding hardware of the magnetic separation station800(e.g., the metal divider808) as the other half of the capacitor. A suitable resin for forming the tips170is available from Fiberfil® Engineered Plastics Inc. as product number PP-61/EC/P BK, which is a carbon filled conductive polypropylene. As determined by the tip sense PCB809, the capacitance will change when the tips170are engaged with the ends of the aspirator tubes860.

In addition to, or as an alternative to capacitive tip sensing, five optical slotted sensors (not shown) can be strategically positioned above the divider808to verify the presence of a tip170on the end of each aspirator tube860. Suitable “tip-present” sensors include Optek Technology, Inc., model number OPB930W51, sensors, available from Optek Technology, Inc. of Carrollton, Texas. A tip170on the end of an aspirator tube860will break the beam of an associated sensor to verify presence of the tip170. If, following a tip pick-up move, tip engagement is not verified by the tip present sensors for all five aspirator tubes860, an error signal will be generated. The MRD160may be aborted, and the aborted MRD160will be retrieved from the magnetic separation station800and ultimately discarded, or processing may continue in the receptacle(s)162corresponding to aspirator tube(s)860for which tip presence is successfully verified

After successful conduit engagement, the orbital mixer assembly828moves the receptacle carrier unit820back to a fluid transfer position shown inFIG.8as verified by the locator plate846and one or both of the sensors847,848.

The magnet moving apparatus810,200,250is then moved to the operational position (e.g., as shown inFIG.4) so that the magnets are disposed adjacent opposite sides of the receptacle, such as MRD160. With the contents of the receptacle subjected to the magnetic fields of the magnets, the magnetically-responsive solid supports having targeted nucleic acids immobilized thereon will be drawn to the sides of the individual receptacles162adjacent the magnets. The remaining material within the receptacles162should be substantially unaffected, thereby isolating the target nucleic acids. The magnet moving apparatus will remain in the operational position for an appropriate dwell time, as defined by the assay protocol and controlled by the assay manager program, to cause the magnetic solid supports to adhere to the sides of the respective receptacles162. In one embodiment, the distance between the opposed magnets on opposite sides of the magnet moving apparatus is about 12.4 mm and the diameter of each receptacle162of MRD160is 11.4 mm, which means there is a gap of 0-1 mm between the magnet and the side of the receptacle162when the magnet moving apparatus is in the operational position. When the magnet moving apparatus is moved to the non-operational position, there is a clearance of at least 30 mm between the magnets and the receptacles160.

The aspirator tubes860are then lowered into the receptacles162of the MRD160to aspirate the fluid contents of the individual receptacles162, while the magnetic solid supports remain in the receptacles162, aggregated along the sides thereof, adjacent the magnets. The tips170at the ends of the aspirator tubes860ensure that the contents of each receptacle162do not come into contact with the sides of the aspirator tubes860during the aspirating procedure. Because the tips170will be discarded before a subsequent MRD160is processed in the magnetic separation station800, the chance of cross-contamination by the aspirator tubes860is minimized.

The electrically conductive tips170can be used in a known manner for capacitive fluid level sensing within the receptacles162of the MRDs160. The aspirator tubes860and the conductive tips170comprise one half of a capacitor, the surrounding conductive structure within the magnetic separation station comprises the second half of the capacitor, and the fluid medium between the two halves of the capacitor constitutes the dielectric. Capacitance changes due to a change in the nature of the dielectric can be detected.

The capacitive circuitry of the aspirator tubes860can be arranged so that all five aspirator tubes860operate as a single gang level-sensing mechanism. When any of the aspirator tubes860and its associated tip170contacts fluid material within a receptacle162, capacitance of the system changes due to the change in the dielectric. If the Z-position of the aspirator tubes860at which the capacitance change occurs is too high, then a high fluid level in at least one receptacle162is indicated, thus implying an aspiration failure or overdispense. On the other hand, if the Z-position of an aspirator tube860at which the capacitance change occurs is correct, but one or more of the other tubes has not yet contacted the top of the fluid due to a low fluid level, a low fluid level will be indicated.

Alternatively, the aspirator tube capacitive circuitry can be arranged so that each of the five aspirator tubes860operates as an individual level sensing mechanism.

With five individual level sensing mechanisms, the capacitive level sensing circuitry can detect failed fluid aspiration in one or more of the receptacles162if the fluid level in one or more of the receptacles162is high. Individual capacitive level sensing circuitry can detect failed fluid dispensing into one or more of the receptacles162if the fluid level in one or more of the receptacles162is low. Furthermore, the capacitive level sensing circuitry can be used for volume verification to determine if the volume in each receptacle162is within a prescribed range. Volume verification can be performed by stopping the descent of the aspirator tubes860at a position above expected fluid levels, e.g. 110% of expected fluid levels, to make sure none of the receptacles162has a level that high, and then stopping the descent of the aspirator tubes860at a position below the expected fluid levels, e.g. 90% of expected fluid levels, to make sure that each of the receptacles162has a fluid level at least that high.

Following aspiration, the aspirator tubes860are raised, the magnet moving apparatus moves to the non-operational position, the receptacle carrier unit820is moved to the fluid dispense position (FIG.9), and a prescribed volume of wash solution is dispensed into each receptacle162of the MRD160through the wash solution dispenser nozzles858. To prevent hanging drops of wash solution on the wash solution dispenser nozzles858, a brief, post-dispensing air aspiration is preferred.

The orbital mixer assembly828then moves the receptacle carriers820in a horizontal orbital path at high frequency (in one embodiment, 14 HZ, accelerating from 0 to 14 HZ in 1 second) to mix the contents of the receptacle. Mixing by moving, or agitating, the MRD160in a horizontal plane is preferred so as to avoid splashing the fluid contents of the receptacle and to avoid the creation of aerosols. Following mixing, the orbital mixer assembly828stops the receptacle carrier unit820at the fluid transfer position.

To further purify the targeted nucleic acids, the magnet moving apparatus810,200,250is again moved to the operational position and maintained in the operational position for a prescribed dwell period. After magnetic dwell, the aspirator tubes860with the engaged tips170are lowered to the bottoms of the receptacles162of the MRD160to aspirate the test specimen fluid and wash solution in an aspiration procedure essentially the same as that described above.

One or more additional wash cycles, each comprising a dispense, mix, magnetic dwell, and aspirate sequence, may be performed as defined by the assay protocol. Those skilled in the art of NATs will be able to determine the appropriate magnetic dwell times, number of wash cycles, wash solutions, etc. for a desired target capture procedure.

Multiple magnetic separation stations800can be employed in an instrument to permit separation wash procedures to be performed on multiple MRDs160in parallel. The number of magnetic separation stations800will vary depending on the desired throughput of the instrument.

After the final wash step, the magnet moving apparatus810,200,250is moved to the non-operational position, and the MRD160is removed from the magnetic separation station800by a transport mechanism. Prior to removing the MRD160from the magnetic separation station800, and preferably prior to magnet retraction, a final residual volume check may be performed by lowering the aspirator tubes860and tips170to a position just above the bottom of each receptacle162to determine if any excess fluid volume remains in the receptacle162.

After the MRD160is removed from the magnetic separation station800, the tips170are stripped from the aspiration tubes860by the tip stripping openings228,278.

The magnet sled202of the magnet slide200shown inFIG.10and the magnet sled252of the magnet slide250shown inFIG.12both include tip stripping openings228,278, respectively, formed along a central portion of a lower surface thereof between the first and second magnets. The tip stripping openings, as shown inFIG.15, comprise keyhole shape openings having a first portion279and a second portion277, with the first portion279being larger than the second portion277. The number of tip stripping openings is equal to the number of aspirator tubes860, which, in the illustrated embodiment, is five.

To strip the conduits, or tips170, off of each of the aspirator tubes860, the magnet sled202,252is positioned beneath the aspirator tubes860so that the larger portions279of the tip stripping openings278are aligned with each of the aspirator tubes860. The aspirator tubes860, with the tips170disposed thereon, are lowered through the first portions279of the stripping openings, which are large enough to permit the tips170to pass therethrough. After the tips170have passed through the tip stripping openings, the magnet sled is moved slightly so that the aspirator tubes860are disposed within the second, smaller portions277of the stripping openings, which are large enough to accommodate the aspirator tubes860, but are smaller than the outside diameter of the rim flange177of the tips170. The aspirator tubes860are then raised, and the tips170engage the peripheral edges surrounding the second portion277of the stripping openings, thereby pulling the tips170off of the aspirator tubes860as the aspirator tubes860ascend. Preferably, the tip stripping openings are disposed at staggered vertical locations so that as the aspirator tubes860are raised in unison, the tips170encounter the peripheral edges of the stripping openings in a staggered manner. For example, each stripping opening may be at a different vertical position, so that as the aspirator tubes860are moved with respect to the stripping openings, the tips170are sequentially removed from the associated aspirator tubes860one at a time. One benefit of staggering the stripping openings is that it results in smaller forces being exerted on the moving mechanism defined by the tube holder862, lift motor868, and drive screw866.

Although operation of the magnetic separation station800was previously described primarily in conjunction with an MRD160, the inventive aspects of the magnetic separation station800is not limited to its use with an MRD160as other types of receptacles, including single receptacles and multiple receptacle devices, including receptacles of varying sizes, can be processed in a magnetic separation station embodying aspects of the present invention.

Magnetic Receptacle Holding Stations

The magnetic receptacle holding station and various components thereof are shown inFIGS.16-19. As shown inFIGS.16and17, a magnetic receptacle holding station300includes a base block310with a first wall330, second wall360, and third wall368secured to and, extending upwardly from the base block310, and a shroud370partially covering the first, second, and third walls330,360,368. A first receptacle slot356defined between first and second walls330,360and a second receptacle slot358defined between second and third walls360,368are each configured to receive an MRD160, or other receptacle, as shown inFIG.16. As shown inFIG.18—in which the second wall360, third wall368, and shroud370are omitted—the base block310includes mounting flanges312for securing the receptacle holding station300to a datum plate, outer wall grooves314,316for securing the first wall330and third wall368, respectively, and a center slot318within which is secured the second wall360. Base block310and walls330,360,368may be made from a suitable plastic, such as Delrin® acetal resin or PVC.

A grounding connector element322is secured to one of the mounting flanges312.

Referring toFIG.18, first wall330includes a magnet slot332formed therein along a lower portion thereof and a clip slot334formed therein along an upper portion thereof. A magnet subassembly338is mounted within the magnet slot332by mechanical fasteners, such as screws. A clip element352is mounted within the clip slot334. A hook access corner cutout354is provided in the upper front corner of the first wall330. Third wall368is substantially a mirror image of first wall330and includes a magnet slot within which a magnet subassembly is mounted and a clip slot within which a clip is mounted. In the illustrated embodiment, third wall368does not include a hook access cutout. Second wall360includes a magnet slot362within which a magnet subassembly364is mounted. A partial corner hook access cutout366is provided at the upper front portion of the second wall360.

Shroud370includes a top panel372, side panels374, and a back panel376(seeFIG.17). Shroud370may be formed from an suitable material, such as sheet metal. Shroud370is secured to the first and third walls330,368, and a grounding connector320is secured to one side374of the shroud370.

Referring toFIG.19, the magnet subassembly includes a plurality of magnets340(five in the illustrated embodiment), each being of a generally solid rectangular shape. An upper holder plate344is disposed within holder plate grooves342formed within the top surface of each of the magnets340. Upper holder plate344includes a separating projection346at each end thereof and between adjacent magnets340to hold each magnet within its respective position. Similarly, the magnet subassembly338includes a lower holder plate348which is received within holder plate grooves formed in the lower surfaces of the magnets340and which includes a separating projection350at opposite ends thereof and between the adjacent magnets340.

A receptacle, such as an MRD160, can be placed within the receptacle holding slot356or358and supported on the upper edges336of the opposed first and second walls or second and third walls. Clip352, which may comprise a resilient projection extending into the slot356, releasably secures the MRD160within the slot. The hook access cutouts354,366permit a manipulating hook (not shown) to be positioned alongside the manipulating structure166of the MRD160and to engage the manipulating structure166in the direction A as shown inFIG.3.

An MRD160containing a sample material and a target capture reagent including magnetically-responsive solid supports can be placed within one of the slots356,358of the magnetic receptacle holding station300, and retained therein for a specified dwell time while the magnetically-responsive solid supports are drawn out of solution by the magnets of the magnetic receptacle holding station300. After the specified dwell time, the MRD160is moved from the magnetic receptacle holding station300to the magnetic separation station800. By placing the MRDs160into the magnetic receptacle holding station300for a specified dwell time prior to moving the MRDs160into the magnetic separation station800, the amount of magnetic dwell time required in the magnetic separation station800can be reduced, thereby reducing the amount of time that each MRD160must spend in the magnetic separation station800and improving overall instrument throughput.

A magnetic receptacle holding station300shown inFIGS.16-18is for illustration purposes only. It should be recognized that a receptacle holding station embodying

aspects of the present invention may have less than or more than three upright walls and two receptacle holding slots defined between opposed walls.

Transport Mechanism

An embodiment of a transport mechanism suitable for moving a receptacle, such as the MRD160, into and out of the magnetic separation station800and the magnetic receptacle holding station300and between the magnetic separation station800and the magnetic receptacle holding station300will now be described.

As shown inFIG.20, a receptacle transfer apparatus in the form of a receptacle distributor400comprises a receptacle carrier assembly402which translates along a transport track assembly408in an “X” direction” under the power of an X-translation system (described below). The receptacle carrier assembly402includes a receptacle distribution head404configured to carry a reaction receptacle, such as an MRD160, supported on a carrier assembly carriage418constructed and arranged to effect Z-axis translation and Θ rotation of the distribution head404. In the illustrated embodiment, the track assembly408is linear (i.e., straight) and substantially horizontal, but in other embodiments, the track assembly is non-linear (i.e., at least partially curved) and/or non-horizontal (i.e., at least a portion of the track assembly is inclined or vertical).

In the illustrated embodiment, track assembly408comprises a generally “L” shaped channel424comprising a base portion434—oriented substantially horizontally in the illustrated embodiment—and an upright backing440extending in an upright manner—oriented substantially vertically in the illustrated embodiment—from one edge of the horizontal base434. A stiffening flange430extends upright from an edge of the base portion434opposite the upright backing440, and a stiffening flange442extends laterally from an upper edge of the upright backing440. A guide rail446is mounted to the upright backing440and extends in a parallel orientation with respect to the base portion434. A cable guide track432is mounted to the base portion434.

An X-translation system410comprises a drive, or transmission, belt448trained over a driven pulley412disposed on one side of the upright backing440at a distal end414of the channel424and over an idler pulley436disposed on the same side of the upright backing440at a proximal end428of the channel424and attached at opposite ends thereof to the carrier assembly carriage418. Driven pulley412is operatively coupled to a carrier translation motor (not shown) mounted to an opposite side of the upright backing440. A rotational encoder (not shown) is coupled to the drive motor.

The drive belt448is preferably equipped with a belt tensioner438. Belt tensioner438comprises a sliding pulley mount, on which is mounted the idler pulley436, and a spring. The pulley mount is slidably supported by the upright backing440, but can be selectively fixed with respect to the upright backing440by a fastener element to prevent sliding of the mount. The spring urges the pulley mount in belt-tightening direction when the pulley mount436is not fixed with respect to the upright backing440.

The distribution head404of the carrier assembly402is carried along the transport track assembly408by the carrier assembly carriage418. The carrier assembly carriage418engages the guide rail446, and translates along the transport track assembly408. Rubber bumpers444,406may be provided at opposite ends of the guide rail446to absorb contact by the carriage418. Movement of the carrier assembly carriage418along the guide rail446is effected by the drive belt448. When the carrier translation motor rotates the driven pulley412in a counter-clockwise fashion, the carrier assembly402is moved in a first X direction (to the left in the illustrated embodiment) towards the proximal end428of transport track assembly408. Similarly, when the carrier translation motor rotates driven pulley412in a clockwise fashion, the carrier assembly402translates in a second X direction (to the right in the illustrated embodiment) towards the distal end414of transport track408assembly.

Details of the distribution head402are shown inFIG.21. Distribution head402includes a distribution frame454that is supported for rotation about a vertical axis of rotation by the carrier assembly carriage418. A side panel488is attached to one side of the distribution head frame454. Side panel488may be transparent so that the interior of the distribution head402is visible. Distribution head402further includes a receptacle hook500configured to engage the manipulating structure166of an MRD160. Devices other than a hook for engaging the receptacle and enabling physical manipulation of the engaged receptacle may be substituted.

A hook actuator system456effects linear translation (in the R direction relative to the Z-axis and the Θ direction) of the receptacle hook500between an extended position, as shown inFIG.21, and a retracted position in which the MRD160is withdrawn into the distribution head402. The hook actuator system456includes a hook carriage480to which the receptacle hook500is attached. A drive belt486is attached to the hook carriage480by a screw and bracket indicated at490. Drive belt486is carried on a drive wheel462and idler wheels464,496,452,458. Idler wheels452and458are attached to a fixed idler wheel bracket460, and idler wheel496is attached to an upper portion of a door engagement bracket494exterior to panel488.

Door engagement bracket494may be provided for opening a door covering the loading slot804. The door, which may be a pivoting, sliding, or rotating door, will include an arm or other projection depending from a portion of the door. In one embodiment, the distribution head402is positioned with the lower end of the door engagement bracket494in contact with the arm, and a slight X and/or Θ movement of the distribution head402is effected to move the door from a closed to an open position. The door is preferably spring-biased in a closed position, so that that when the arm is released from contact with the door engagement bracket494, the door will spring back to the closed position.

Drive wheel462is attached to an output shaft of a drive motor(not shown) (preferably a stepper motor) which is mounted to an opposite side of the distribution frame454. A rotational encoder (not shown) is attached to the drive motor. Drive wheel462preferably has a diameter of 9.55 mm resulting in a resolution of 0.15 mm per full motor step. The encoder had a resolution of 200 counts/revolution (A-B signals) resulting in a quadrupled resolution of 800 counts/revolution.

The hook actuator system456preferably includes a belt tensioner476for maintaining proper tension in the belt486. Belt tensioner476includes a pivoting idler wheel bracket474to which idler wheel464is attached and which is pivotally attached to the side panel488by a pivot screw478. A slot470is formed in an end of the pivoting idler wheel bracket474, and a position lock screw468extends through the slot470into the side panel488. A spring472is disposed between a portion of the pivoting idler wheel bracket474and the fixed idler wheel bracket460. Tension in the belt486can be adjusted by loosening the position lock screw468, thereby allowing the spring472to pivot the pivoting idler wheel bracket474and thus urge the idler wheel464upwardly to create the proper tension in the drive belt486. When proper tension is achieved in the drive belt486, the position lock screw468can thereafter be retightened.

The hook carriage480includes a rail channel484that translates along a hook carriage guide rail450attached to an upper portion of the distribution head frame454. The receptacle hook500is attached to an insulation mount498disposed between the rail channel484and the hook500to electrically isolate the hook500from the distribution head402to facilitate capacitive sensing of contact by the hook500with another structural element of, e.g., the magnetic separation station800or the receptacle holding station300.

Further details of the receptacle distributor can be found in U.S. Patent Application No. 61/178,728, the disclosure of which is incorporated by reference.

A procedure for separating or isolating an analyte of interest, such as a target nucleic acid, from other components of a sample is represented by process510shown inFIG.22. The process begins at step512with a specimen preparation procedure whereby sample specimen and a target capture reagent including magnetically-responsive solid supports are added to a receptacle device (a single receptacle or multiple receptacles, e.g., the MRD160). The sample specimen and target capture reagent may be added to the receptacle device by any means known in the art, including manual and automated means.

In step514, the receptacle device containing the sample specimen and target capture reagent is subjected to conditions sufficient to cause the analyte of interest to be immobilized on the magnetically-responsive solid support. The conditions may include incubation of the receptacle device and its contents at one or more prescribed temperatures for prescribed periods of time.

Procedures for immobilizing targeted nucleic acids on magnetically-responsive solid supports are exemplified in U.S. Pat. No. 6,534,273 and U.S. Patent Application Publication No. 2008-0286775.

In step516, a decision is made as to whether to (1) move the receptacle device to a magnetic receptacle holding station300prior to moving the receptacle device to the magnetic separation station800or (2) move the receptacle device directly into a magnetic separation station800.

If the decision is made to omit placing the receptacle device in the magnetic receptacle holding station300, then, in step526, the receptacle device is placed in the magnetic separation station800, preferably using a receptacle transport mechanism, such as receptacle distributor400described above.

In step530, with the receptacle device supported by the receptacle carrier unit820of the magnetic separation station800, the receptacle carrier unit820is positioned to align each aspirator tube860with a tip170carried on the receptacle device, and each aspirator tube860is lowered until it is inserted into and frictionally engages a tip170. In alternative embodiments, the tips are not carried on the receptacle device but are otherwise provided to each aspirator tube860.

In step532, magnets, which are initially in an inoperative position with respect to the receptacle device when the receptacle device is first placed into the magnetic separation station800in step526, are moved to an operative position with respect to the receptacle device to draw magnetically-responsive solid supports toward the side of the receptacle device. In step534, the receptacle device is held stationary in the receptacle carrier unit820with the magnets in an operative position for a specified dwell period (in one embodiment, 120 seconds) sufficient to draw a substantial portion of the magnetically-responsive solid supports to the side wall of the receptacle device and out of suspension.

In step536, after performing a procedure to verify the presence of a tip170on each aspirator tube860, the receptacle carrier unit820is moved to position each receptacle below an associated aspirator tube860, each aspirator tube860is lowered into the associated receptacle, and fluid is aspirated from the receptacle in step536, preferably while the magnets are maintained in the operative position with respect to the receptacle device.

In step538, the magnets are moved to a non-operative position with respect to the receptacle device so that the magnetically-responsive solid supports of the target capture reagent will not be influenced by the magnetic force of the magnets.

In step540, a wash solution is dispensed into each receptacle (e.g., 1 mL of wash buffer), and, in step542, the receptacle device is agitated to dislodge the magnetically-responsive solid supports from the walls of the receptacle device and to re-suspend the magnetically-responsive solid supports.

In step544, a decision is made as to whether additional wash steps must be performed. Depending on the procedure protocol, the wash procedure may be repeated one or more times. In one embodiment,2wash cycles are performed. If the wash procedure is to be repeated, the process returns to step532, and steps532through542are repeated. If no further wash steps are to be performed, the receptacle device is removed from the magnetic separation station800in step546.

In step548, the tip170is stripped from each aspirator tube860, and the magnetic separation station800is now ready to receive the next receptacle device to perform the magnetic separation wash process. If, at step516, the decision was made to place the receptacle device in the receptacle holding station300prior to moving it to the magnetic separation station800, in step518the receptacle device is placed in the receptacle holding station300, preferably using a receptacle transport mechanism, such as receptacle distributor400described above.

In step520, the receptacle device is allowed to sit in the magnetic receptacle holding station300for a specified dwell period (in one embodiment, 580 seconds) sufficient to draw a substantial portion of the magnetically-responsive solid supports to the walls of the receptacle device and out of suspension.

In step522, after the specified dwell period, and assuming the availability of a magnetic separation station800, the receptacle device is moved from the receptacle holding station300to the magnetic separation station800, using, for example, the receptacle distributor400described above. In one embodiment, the transfer from the receptacle holding station300to the magnetic separation station occurs in 4 seconds.

In step524, each aspirator tube860is engaged with a tip170as described above in connection with step530.

In step528, magnets, which are initially in an inoperative position with respect to the receptacle device when the receptacle device is first placed into the magnetic separation station800in step522, are moved to an operative position with respect to the receptacle device. As the magnetically-responsive solid supports of the target capture probe contained in the receptacle device have already been subjected to a magnetic force for a specified dwell period within the magnetic receptacle holding station300in step520, an initial magnetic dwell period within the magnetic separation station800can be substantially shortened, or omitted altogether. That is, if the contents of the receptacle device are not agitated, a substantial portion of the solid supports will remain aggregated to the side of the receptacle device while the receptacle device is transferred from the receptacle holding station to the magnetic separation station. In experiments, the inventors have determined that the initial magnetic dwell can be reduced by 180 seconds by use of the magnetic receptacle holding station300(initial dwell of 300 seconds without first placing the receptacle device in the magnetic receptacle holding station as compared to 120 second initial magnetic dwell when the receptacle device is first placed in the magnetic receptacle holding station for 580 seconds).

The process next proceeds to step536, and fluid is aspirated from the receptacle device while the magnetically-responsive solid supports are held to the walls of the receptacle device by the magnets. The process then proceeds through steps540through548as described above—returning, as desired, to step532to repeat steps532-542.

While the present invention has been described and shown in considerable detail with reference to certain illustrative embodiments, those skilled in the art will readily appreciate other embodiments of the present invention. Accordingly, the present invention is deemed to include all modifications and variations encompassed within the spirit and scope of the following appended claims.