(19)
(11)EP 2 464 455 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
05.11.2014 Bulletin 2014/45

(21)Application number: 10755260.6

(22)Date of filing:  29.07.2010
(51)International Patent Classification (IPC): 
B01L 3/00(2006.01)
G01N 27/447(2006.01)
(86)International application number:
PCT/US2010/043759
(87)International publication number:
WO 2011/019515 (17.02.2011 Gazette  2011/07)

(54)

PINCHING CHANNELS FOR FRACTIONATION OF FRAGMENTED SAMPLES

FOKUSSIERUNGKANÄLE ZUM TRENNEN VON FRAGMENTIERTEN PROBEN

CANAUX POUR CONFINER UN ÉCHANTILLON FRAGMENTÉ


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

(30)Priority: 26.07.2010 US 843557
02.12.2009 US 266030 P
12.08.2009 US 233392 P

(43)Date of publication of application:
20.06.2012 Bulletin 2012/25

(73)Proprietor: Caliper Life Sciences, Inc.
Alameda, CA 94501-1038 (US)

(72)Inventors:
  • MOLHO, Josh
    Oakland CA 94619 (US)
  • XU, Hui
    Palo Alto CA 94303 (US)
  • ZANINOVICH, Jorge, J.
    Walnut Creek CA 94596 (US)

(74)Representative: Kiddle, Simon John et al
Mewburn Ellis LLP 33 Gutter Lane
London EC2V 8AS
London EC2V 8AS (GB)


(56)References cited: : 
WO-A1-01/51918
US-A1- 2003 044 832
WO-A1-03/013703
US-B1- 6 506 609
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    TECHNICAL FIELD



    [0001] The present disclosure is in the field of devices and systems for separation and isolation of sample components and methods for their use. In particular, described herein are systems, devices, and methods for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components.

    CROSS-REFERENCE TO RELATED APPLICATIONS



    [0002] This application claims the benefit of U.S. Nonprovisional Application No. 12/843,557, filed July 26, 2010, U.S. Provisional Application No. 61/233,392 filed August 12, 2009, and U.S. Provisional Application No. 61/266,030 filed December 02, 2009.

    BACKGROUND OF THE INVENTION



    [0003] Separations-based analyses are a prominent part of biological research, allowing one to characterize different biological samples, reaction products and the like. Examples of some of the more prevalent separations-based analyses include electrophoretic separations of macromolecular species, e.g., proteins and nucleic acids. Electrophoresis, e.g., capillary electrophoresis, has been established as a highly effective method for separating macromolecular species in order that they might be further characterized. Protein and nucleic acid molecules are two major examples of molecular species that are routinely fractionated and characterized using electrophoretic systems.

    [0004] Both microfluidic and macrofluidic devices have been applied in separations-based analyses. Examples of novel microfluidic devices and methods for use in the separation of molecular, and particularly macromolecular species by electrophoretic means are described in United States Patent Nos. 5,958,694, 6,032,710, and 7,419,784, for example. In such devices, the sample containing the macromolecular species for which separation is desired is placed in one end of a separation channel located in a microfluidic substrate and a voltage gradient is applied along the length of the channel. As the sample components are electrophoretically transported along the length of the channel and through the separation (sieving) matrix disposed therein, those components are resolved. The separated components are then detected at a detection point along the length of the channel, typically near the terminus of the separation channel downstream from the point at which the sample was introduced. Following detection, the separated components are typically directed to a collection reservoir/well in the device (or to an external device such as a multiwell plate via a capillary pipettor, for example) for subsequent extraction or disposal.

    [0005] In many situations, it is desirable to extract selected fragments of interest, such as DNA fragments, following the separation of the fragments into bands in the separation matrix for further processing or analysis, e.g., restriction enzyme modification, T4 ligation, PCR amplification, mass spectroscopy, or polynucleotide kinase reactions. The typical process used by laboratory researchers for extracting and isolating selected DNA fragments of interest (and other desired nucleic acid and protein fragments) from a separation matrix (such as an agarose gel) involves excising the desired fragments from the separation matrix and then extracting and purifying the excised fragment(s). First, the separated fragments are stained and illuminated by shining ultraviolet (UV) light on the fragments to visualize the separated bands. A razor blade is then used to manually cut above and below each fragment of interest so that one or more slices of the sieving can be removed. Then the DNA is extracted from the removed slices using various solutions and heating to dissolve the sieving matrix. The DNA can be further purified by standard solid phase extraction (binding the DNA to a solid surface such as glass followed by washing and finally elution). The recovered DNA can then be used for further processing or analysis. This extraction process, however, is time consuming, laborious and potentially damaging to the DNA (e.g., nicking of the DNA can occur if the DNA is exposed to ultraviolet light too long while the fragments of interest are being illuminated for excision).

    [0006] Thus, in performing separations-based analyses, it would be desirable to be able to also isolate or extract one or more of the separated components in the device itself for further analysis or processing. The recovered or isolated fragments could then be used for a variety of different processes including, for example, the following: amplification using polymerase chain reaction (PCR); ligation reactions for cloning small to medium-sized strands of DNA into bacterial plasmids, bacteriophages, and small animal viruses to allow the production of pure DNA in sufficient quantities to allow its chemical analysis; adapter ligation used in high-throughput sequencing; reactions to dissolve a separated protein or nucleic acid component in a suitable matrix for further analysis by a mass spectrometer using, for example, Matrix-Assisted Laser Desorption Ionization (MALDI); binding reactions to bind a labeling agent to one or more separated protein or nucleic acid components for further analysis; or other similar post-detection processes. In addition, in the case of PCR samples, it is important to be able to separate smaller dimer and primer molecules from the main nucleic acid fragments in the sample and then isolate and collect the main nucleic acid fragments for further analysis or processing, while directing the smaller primer and dimer components to a waste reservoir/cell for removal and subsequent disposal.

    [0007] Thus, it would be advantageous to provide improved devices, systems and methods for use in separating sample materials into different sample components or fragments and then isolating one or more of the sample components for further processing or analysis.

    SUMMARY OF THE INVENTION



    [0008] One aspect of the present invention is a device for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components. The device comprises first and second pinching channels, each pinching channel having a first end and a second end; a separation channel extending between the first and second pinching channels, the separation channel having a first end and a second end; a collection leg having a first end and a second end; a collection well disposed in the collection leg between the first and second ends of the collection leg; a waste leg having a first end and a second end; and a switching region having an inlet end and an outlet end. The second end of each of the first pinching channel, the second pinching channel, and the separation channel are in fluid communication with the inlet end of the switching region, and the first end of the collection leg and the first end of the waste leg are in fluid communication with the outlet end of the switching region.

    [0009] Another aspect of the present invention is a system for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components. The system comprises a first device, a detector in sensory communication with the device, a fluid direction system, and a processor operably coupled to the detector and the fluid direction system. The first device comprises first and second pinching channels, a separation channel extending between the first and second pinching channels, a collection leg including a collection well disposed between a first end and a second end of the collection leg, and a waste leg. The first and second pinching channels and the separation channel are in fluid communication with an inlet end of the switching region, and the collection leg and the waste leg are in fluid communication with an outlet end of the switching region.

    [0010] Yet another aspect of the present invention is a method for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components. The method comprises loading a sample material into a loading well in fluid communication with a separation channel of a device; separating the sample material into a plurality of separated components in the separation channel, the separated components forming a stream; transporting the stream of separated components into a switching region of the device; transporting first and second buffer streams into the switching region on either side of the component stream such that the first and second buffer streams constrain and elongate the component stream as it is transported through the switching region; directing a first portion of the stream of separated components, a first portion of the first buffer stream and a first portion of the second buffer stream out of the switching region and into a waste leg of the device; directing a second portion of the stream of separated components, a second portion of the first buffer stream and a second portion of the second buffer stream out of the switching region and into a collection leg of the device; directing a third portion of the stream of separated components, a third portion of the first buffer stream and a third portion of the second buffer stream out of the switching region and into the waste leg of the device; and collecting a separated component from a collection well disposed in the collection leg.

    [0011] The aforementioned and other features and advantages of the invention will become further apparent from the following detailed description of the presently preferred embodiments, read in conjunction with the accompanying drawings, which are not to scale. The detailed description and drawings are merely illustrative of the invention, rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof.

    BRIEF DESCRIPTION OF THE DRAWINGS



    [0012] 

    FIG. 1 is a schematic illustration of a device for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components, in accordance with the present invention;

    FIG. 2 is a schematic illustration of another device for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components, in accordance with the present invention; and

    FIG. 3 is a photograph of a portion of a device in accordance with the present invention, the device shown during operation;

    FIG. 4 is a schematic illustration of a portion of the device of FIG. 1 showing stream lines achieved using the pinching channels of the device;

    FIG. 5 is a schematic illustration of a portion of a device without pinching channels showing stream lines achieved without the benefit of the pinching channels of the device of FIG. 1; and

    FIG. 6 is a schematic illustration of the device of FIG. 1 including information regarding a feedback control mechanism.


    DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS



    [0013] One aspect of the present invention is a device for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components. One embodiment of the device, in accordance with the present invention, is illustrated in FIG. 1. The illustrated device comprises first and second pinching channels 1 and 2, a switching region 3, a loading well 4, a collection well 5, a collection leg 6, a waste leg 7, a separation channel 8, a sieving matrix 9, and reservoirs 10-12. In the present embodiment, reservoirs 10 and 11 are waste reservoirs, while reservoir 12 is a buffer reservoir.

    [0014] As seen in FIG. 1, pinching channels 1 and 2 extend from reservoir 12 to switching region 3. Separation channel 8 also extends from reservoir 12 to switching region 3 and is positioned between pinching channels 1 and 2. The three channels merge at the inlet end of switching region 3. By having pinching channels 1 and 2 join separation channel 8 just above the switching region, little diffusion of the sample into the buffer streams occurs. Separation channel 8 includes a loading well 4 that is open to the external environment to permit a sample to be deposited into the loading well manually using, for example, a pipettor, or a sample may be deposited by an automated sample transfer device.

    [0015] Legs 6 and 7 extend from the outlet end of switching region 3 to reservoirs 10 and 11, respectively. As seen in FIG. 1, collection well 5 is positioned in collection leg 6. The collection well may be positioned anywhere in the collection leg between switching region 3 and reservoir 10. Positioning the collection well nearer to the switching region than to the reservoir may permit collection of a narrower, i.e., more precisely selected, band of the separated sample material. A larger collection well may permit collection of a broader band. Collection well 5 is open to the external environment to permit removal of the isolated sample component(s) from the device. Collection well 5 may be circular as seen in FIG. 1 or may be any other shape that facilitates collecting the isolated sample component(s). For example, a free-form shape is shown in FIG. 2. While the collection well is shown in leg 6 in the illustrations, it may alternatively be positioned in leg 7 in another embodiment. In this alternative embodiment, leg 6 would then be the waste leg, and leg 7 would be the collection leg.

    [0016] Separation channel 8 may include a sieving matrix 9 to facilitate electrophoretic separation of a sample within the separation channel. The sieving matrix may be, for example, agarose or a cross-linked gel. Pinching channels 1 and 2, switching region 3, legs 6 and 7, and reservoirs 10-12 may also include the sieving matrix, although reservoirs 10-12 are not completely filled with the sieving matrix and additionally contain a buffer. Loading well 4 and collection well 5 do not include the sieving matrix in order to facilitate introduction and withdrawal of sample materials. In alternative embodiments, the device may include a sieving matrix in only some or none of pinching channels 1 and 2, switching region 3, legs 6 and 7, separation channel 8 and reservoirs 10-12.

    [0017] Switching region 3 may be a simple intersection of the channels entering and exiting the region, or the switching region may be extended to form a channel. Switching region 3 is shown as a narrowed "neck" in FIGS. 1 and 2 (i.e., the cross-sectional dimension [width] of the separation channel is shown as being greater than the cross-sectional dimension [width]of the switching region). Other geometries are possible. The narrowed switching region is desirable because it both increases the electric field and therefore the migration velocity within the region and helps to prevent bubbles being trapped in a separation matrix during filling of the device. However, as will be discussed further below, the switching region need not be substantially narrowed for the electric field to be increased within the separated sample in the switching region because the sample stream is "pinched," i.e., constrained or contained and thereby elongated, by buffer transported into the switching region from the two pinching channels, increasing the current density (and therefore the electric field) and creating a faster electrophoretic velocity in the sample material within the switching region. Pinching of the sample stream can be seen in FIG. 3, where the sample stream appears light and buffer streams appear dark at the switching region.

    [0018] Pinching of the sample can also be seen in FIG. 4, which models transport of sample materials through a portion of the device of FIG. 1. As can be seen by the stream lines illustrated at 20 in FIG. 4, the entire sample stream is directed away from collection leg 6 and collection well 5. Contrast FIG. 4 with FIG. 5, which models transport of sample materials through a portion of a device that does not include pinching channels. Stream lines 20 of FIG. 5 show that a portion of the sample stream strays into the collection leg and collection well of the device of FIG. 5, resulting in unwanted sample components being present in the collection well when pinching streams are not employed.

    [0019] Another embodiment of the device, in accordance with the present invention, is illustrated in FIG. 2. Note that throughout the figures, like elements share like reference numbers. The illustrated device comprises first and second pinching channels 1 and 2, a switching region 3, a loading well 4, a collection well 5, a collection leg 6, a waste leg 7, a separation channel 8, a sieving matrix 9, and reservoirs 10-14. In the embodiment illustrated in FIG. 2, reservoirs 10 and 11 are waste reservoirs, while reservoirs 12-14 are buffer reservoirs. Refer to the discussion above for descriptions of elements 1-12. The embodiment shown in FIG. 2 differs from the embodiment shown in FIGS. 1 and 4 in that the device of FIG. 2 includes three buffer reservoirs rather than a single buffer reservoir.

    [0020] In the embodiment of FIG. 1, pinching channels 1 and 2 share the same reservoir (reservoir 12) with separation channel 8. In this embodiment, the pinching ratio (the ratio of the electrical current in the separation channel to the current in the pinching channels) is controlled by the ratio of the pinching channel resistance to the separation channel resistance, resistance being a function of the geometry (e.g., width, depth, length) of the channel. Having a shared reservoir for the separation channel and the two pinching channels offers the benefit of minimizing the number of electrodes and power supplies required by the device, resulting in a highly compact device.

    [0021] In the embodiment of FIG. 2, each of channels 1, 2, and 8 has its own separate reservoir, reservoirs 13, 12, and 14, respectively. By having separate reservoirs for each of the pinching channels and the separation channel, the pinching ratio can be controlled independent of the geometry of the channels. This may be accomplished by, for example, applying different voltages at the different reservoirs or controlling the current ratio between the separation channel and pinching channels directly using external hardware such as a power supply. Note that increasing the pinching ratio will provide better containment of the sample within the switching region at the cost of more current/power needed.

    [0022] Either of the embodiments described above may include a detection region (not shown) within which the sample component(s) intended for collection are detected in order to switch the desired component(s) into the collection leg. Alternatively, the sample component(s) may be identified based on a known transit time through the device.

    [0023] The materials of the device are chosen for their suitability for electrophoretic separations and for their inertness to the components to be separated and isolated in the device. Materials suitable for the device include, but are not limited to, glass and other ceramics, quartz, silicon, and polymeric substrates, e.g., plastics.

    [0024] Another aspect of the present invention is a system for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components. The system comprises a device such as has been described above as well as instrumentation for controlling the device. For example, the system may comprise a detector positioned in sensory communication with a detection region of the device, a processor operably coupled to the detector and to a fluid direction system that is configured to control movement of one or more sample components from the separation channel into the collection well of the collection leg based on information received from the detector. As used herein, the phrase "in sensory communication" refers to positioning of a detector such that it is operably connected to the device, i.e., capable of receiving a detectable signal from the contents of the device. In the case of optical signals, this requires only that the detector be positioned to receive the optical signal. The system may be configured to simultaneously control multiple fluidic circuits (a single fluidic circuit being shown, e.g., in FIG. 1). In such a configuration, the fluid direction system may be configured to control the movement of one or more sample components in one fluidic circuit based on information received by the detector in a parallel circuit.

    [0025] Yet another aspect of the present invention is a method for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components. The method may be carried out using a device or system such as has been described above and illustrated in FIGS. 1 and 2. The description below refers to the illustrated embodiments of the device, but the method may be varied depending on the geometry of the device used, various changes and modifications to the device being both possible and foreseen.

    [0026] One or more buffers are loaded into the buffer reservoir(s). In an alternative embodiment of the method, the device may be supplied with buffer already loaded into the reservoir(s). A low-ionic strength buffer may be loaded into collection well 5 and a higher ionic strength buffer may be loaded into all other reservoirs and channels. A low ionic strength buffer may be preferred for the collection well if the isolated sample component(s) will later undergo amplification using PCR. A higher ionic strength buffer may be preferred for the reservoirs to provide additional buffering capacity. Current may be passing through these channels for extended periods, for example up to 60 minutes, and a higher buffer concentration may mitigate changes in pH within the device due to the passage of current through the device. In addition, field amplified sample stacking (FASS) will occur when a higher conductivity buffer is used in the separation channel and the input sample is loaded in a low conductivity buffer. FASS will increase the sensitivity and resolution of the separation.

    [0027] A sample is deposited into the loading well, shown at 4 in FIGS. 1 and 2. The sample may be deposited manually using, for example, a pipettor, or may be deposited by an automated sample transfer device. A voltage is applied to buffer reservoir 12, and a different voltage is applied to waste reservoir 11 to electrophoretically separate the sample into a plurality of sample components in separation channel 8. Initially, no electrical connection is made to waste reservoir 10 in order to maintain zero current within collection leg 6, thereby directing the separated sample into waste leg 7 and waste reservoir 11 until a component of the sample desired for isolation and collection reaches switching region 3. Alternatively, the fluid direction system may control the voltage at waste reservoir 10 in order to maintain zero current. Note that the term "zero current" is defined herein as a current that is negligible as compared to the current in the other leg, such as a current that is less than 2% of the current in the other leg.

    [0028] At the same time that the sample is being separated in separation channel 8, buffer is being transported through pinching channels 1 and 2 as a result of a voltage difference between waste reservoir 11 and the buffer reservoir connected to each of the pinching channels (reservoir 12 in FIG. 1, and reservoirs 13 and 14 in FIG. 2). As the separated sample passes through switching region 3, the buffer streams from the two pinching channels "pinch" (i.e., constrain or contain) the separated sample as seen in FIGS. 3 and 4,thereby increasing the field and creating a faster electrophoretic velocity in the sample material within the switching region. Pinching of the sample prevents leakage of the sample into the non-target leg. Leakage can be seen in FIG. 5, which illustrates a device that does not include pinching channels. Initially, the non-target leg is collection leg 6. Waste leg 7 remains the target leg until the component(s) to be isolated enter switching region 3, and the sample stream is momentarily switched, i.e., diverted, into collection leg 6, which then becomes the target leg.

    [0029] Once the one or more components that are to be isolated and collected are within switching region 3, a voltage is applied to waste reservoir 10, and a zero current is maintained at waste reservoir 11. This switches the direction of the sample stream from waste leg 7 into collection leg 6. The redirection of the sample stream is timed to isolate only the desired component(s) of the sample. Once the one or more desired components are within collection leg 6 and positioned at the location of collection well 5, the sample stream is directed back into waste leg 7 and waste reservoir 11 by once again controlling zero current to waste reservoir 10 and resuming the original voltage to waste reservoir 11. Once zero current is imposed at the electrical connection to waste reservoir 10, transport into collection leg 6 stops, and the desired one or more components of the sample remain in place within collection leg 6 at the location of collection well 5. The sample component(s) may then be removed from collection well 5 manually using, for example, a pipettor, or may be withdrawn by an automated sample transfer device.

    [0030] As described above, the pinching ratio of the device illustrated in FIG. 1 is controlled by the ratio of the pinching channel resistance to the separation channel resistance, resistance being a function of the geometry of the channel. The pinching ratio of the device illustrated in FIG. 2 can be controlled independently of the geometry, for example by applying different voltages at the various reservoirs or controlling the current ratio between the separation channel and pinching channels directly using external hardware such as a power supply. Increasing the pinching ratio provides better confinement of the sample at the switching region at the cost of needing more current/power.

    [0031] As has been discussed previously, pinching the sample stream as it is transported into switching region 3 increases the field and creates a faster electrophoretic velocity in the sample material within the switching region as compared to that within the separation channel. The value of pinching can be readily understood when placed in context. When fractionating DNA, for example, the higher velocity decreases the number of base pairs per unit distance. That is, as the ratio of the velocity in the switching region to the velocity in the separation channel is increased, the number of base pairs within switching region 3 is decreased, in effect "stretching out" the separated sample and elongating the sample stream. This increases the precision of diverting a small band comprising the desired component(s) to collection leg 6 because the physical size of the switching intersection is in some ways equivalent to the thickness of a scalpel blade used to cut a slice containing one or more bands out of a gel. When cutting a gel, the thickness of the blade determines the minimum size slice that can be cut from the gel because it is difficult (practically impossible) to cut a slice that is thinner than the thickness of the blade. If the gel could be stretched, a more precise selection could be cut from the gel using the same size scalpel. By the same token, in the present method, increasing the pinching ratio will increase the resolution of the "cut" performed by the device. Other methods besides or in addition to increasing the pinching ratio may be used to increase the velocity at the switching region. Any method that increases either the electric field or species mobility will be effective. Such methods include (but are not limited to) the following: changing the depth of the channel at the switching region, changing or removing the sieving matrix near the switching region (so as to increase the mobility of species) or creating a step reduction in conductivity near the region.

    [0032] While the sample is being separated, it is desirable to maintain the same separation field within the separation channel regardless of whether sample is being sent to the left (collection leg 6) or the right (waste leg 7). The collection well (seen at 5 in FIGS. 1 and 2) may, for example, contain buffer of a different conductivity than the running buffer, i.e., the buffer being transported through separation channel 8. As mentioned above, buffers of different conductivity may be used within the device, with a higher conductivity buffer in one or more of the channels than is present in collection well 5. Thus, the resistance could be different in the two legs and the exact value of the resistance unknown. Such uncertainty in the channel resistances means that the fluid direction system cannot maintain the same electric field in separation channel 8 when the target leg is switched. To overcome this problem, voltage may be controlled at the switching region by reading the voltage at the leg that has zero current. See FIG. 6. That is, because zero current is maintained in one leg, e.g., in leg 6, the voltage in leg 6 is the same throughout the leg, all the way up to switching region 3; therefore, reading the voltage in leg 6 becomes a way of measuring the voltage in the switching region (Vi). By adjusting the voltage and/or current in the other leg, leg 7, a desired Vi may be maintained at the switching region.

    [0033] In this way, the voltage drop between the sample loading well (seen at 5 in FIGS. 1 and 2) and the switching region (seen at 3 in FIGS. 1 and 2) will remain substantially the same, even if the resistances of the two legs are unknown (and possibly unequal). This feedback control is enabled by the design of the device because the pinching channels allow for confinement of the sample at the switching region even when there is zero current on one of the legs, as illustrated in FIG. 6.

    [0034] Feedback control is achieved using an electrical circuit that controls and applies voltages and currents to electrodes connected to reservoirs of the device. The electrical circuit may include semiconductor and/or electromechanical devices used as switches. The switches may be used in linear (proportional) and/or non linear (ON/OFF) modes of operation. The electrical circuit may be computer controlled so that the magnitude of the applied voltages and currents, and the timing of their application, are specified by a computer algorithm, which may also be driven by the signal from the detector. The computer system may additionally be used to display, process, analyze, and store information gathered as a result of the operation of the device.

    [0035] Using the device, fragments of a specific size may be isolated from an initial sample having a wide size distribution. For many applications, the goal is to isolate a band of narrow size distribution in the collection well. However, for other applications, a wider size distribution may be desired or tolerated in exchange for collection of more mass. As can be seen in FIG. 1, collection well 5 is not necessarily the terminus of the electrical circuit. Therefore, material entering collection well 5 will continue to electrophorese towards reservoir 10, eventually passing out of the collection well. In this way, the size of collection well 5 influences the maximum amount of material that can be collected since the smallest, fastest moving fragments will eventually leave the collection well as larger, slower fragments continue to enter.

    [0036] The amount of material that can be collected may be increased by increasing the size of the collection well, by making this well the terminus of the electrical circuit, or by using a higher conductivity (or higher viscosity, or increased sieving) buffer in collection well 5. This will be possible if the sieving matrices in the channels are cross-linked or gelled. In this case, the collection well will be defined as a "hole" in the matrix that can be filled with a different buffer. When the collection buffer (the buffer in the collection well) is a higher conductivity than the running buffer (the buffer in the collection leg), material entering the well will concentrate and slow down (i.e., become stacked). Thus, for example, if the collection buffer is twice the conductivity of the running buffer, then the capacity of the collection well will be doubled. Alternatively, other methods of stacking may be used, such as increasing the viscosity, increasing the sieving or decreasing the current density (by increasing the cross-sectional area at the collection well). The viscosity may be increased by adding agents such as glycerol to the collection buffer. The sieving may be increased by adding polymers (such as PDMA) that impede the progress of the separated molecules.


    Claims

    1. A device for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components, comprising:

    first (1) and second (2) pinching channels, each pinching channel having a first end and a second end;

    a separation channel (9) extending between the first and second pinching channels, the separation channel having a first end and a second end;

    a collection leg (6) having a first end and a second end;

    a waste leg (7) having a first end and a second end; and

    a switching region (3) having an inlet end and an outlet end;

    wherein the second end of each of the first pinching channel, the second pinching channel, and the separation channel are in fluid communication with the inlet end of the switching region, and wherein the first end of the collection leg and the first end of the waste leg are in fluid communication with the outlet end of the switching region,
    characterised in a collection well (5) disposed in the collection leg between the first and second ends of the collection leg.


     
    2. The device of claim 1 further comprising:

    a first buffer reservoir (12), a first waste reservoir (10), and a second waste reservoir (11), wherein the first end of the separation channel is in fluid communication with the first buffer reservoir, the second end of the collection leg is in fluid communication with the first waste reservoir, and the second end of the waste leg is in fluid communication with the second waste reservoir.


     
    3. The device of claim 2, wherein the first end of the first pinching channel and the first end of the second pinching channel are in fluid communication with the first buffer reservoir.
     
    4. The device of claim 2 further comprising:

    a second buffer reservoir (13) and a third buffer reservoir (14), wherein the first end of the first pinching channel is in fluid communication with the second buffer reservoir and the first end of the second pinching channel is in fluid communication with the third buffer reservoir.


     
    5. The device of claim 1 further comprising:

    a sieving matrix disposed in the separation channel; or

    a loading well (4) disposed in the separation channel; or

    a sieving matrix disposed in the collection leg.


     
    6. The device of claim 1, wherein a cross-sectional dimension of the separation channel is greater than a cross-sectional dimension of the switching region.
     
    7. A system for isolating one or more sample components of a sample material following separation of the sample material into a plurality of sample components, comprising:

    according to claim 1;

    a detector in sensory communication with the device;

    a fluid direction system; and

    a processor operably coupled to the detector and the fluid direction system.


     
    8. The system of claim 7, wherein the fluid direction system is configured to control movement of one or more sample components from the separation channel into one of the collection leg and the waste leg.
     
    9. The system of claim 7 further comprising:

    a second device comprising first and second pinching channels, a separation channel extending between the first and second pinching channels, a collection leg, a waste leg and a switching region , the collection leg including a collection well disposed in the collection leg between a first end and a second end of the collection leg, wherein the first and second pinching channels and the separation channel are in fluid communication with an inlet end of the switching region, and the collection leg and the waste leg are in fluid communication with an outlet end of the switching region.


     
    10. The system of claim 9, wherein the fluid direction system is configured to simultaneously control movement of one or more sample components in the first device and one or more sample components in the second device.
     
    11. The system of claim 10, wherein the fluid direction system is configured to control movement of the one or more sample components in the first device and the one or more sample components in the second device based on information received by the detector in a parallel circuit.
     
    12. A method of separating a sample material into a plurality of separated components and isolating one or more of the separated components in a device or a system according to any one of claims 1 to 11, the method comprising:

    loading a sample material into a loading well in fluid communication with a separation channel of a device;

    separating the sample material into a plurality of separated components in the separation channel, the separated components forming a stream;

    transporting the stream of separated components into a switching region of the device;

    transporting first and second buffer streams into the switching region on either side of the component stream such that the first and second buffer streams constrain and elongate the component stream as it is transported through the switching region;

    directing a first portion of the stream of separated components, a first portion of the first buffer stream, and a first portion of the second buffer stream out of the switching region and into a waste leg of the device;

    directing a second portion of the stream of separated components, a second portion of the first buffer stream and a second portion of the second buffer stream out of the switching region and into a collection leg of the device;

    directing a third portion of the stream of separated components, a third portion of the first buffer stream and a third portion of the second buffer stream out of the switching region and into the waste leg of the device; and

    collecting a separated component from a collection well disposed in the collection leg.


     
    13. The method of claim 12, wherein only one of the collection leg and the waste leg has zero current, the method further comprising:

    determining the voltage in the leg that has zero current, thereby determining a first voltage in the switching region; and

    adjusting one or both of the voltage and the current in the leg that does not have zero current, thereby producing a second voltage in the switching region.


     
    14. The method of claim 12 further comprising:

    stacking one or more of the plurality of separated components in the collection well.


     
    15. The method of claim 14, wherein the collection leg includes a first sieving matrix that is one of cross-linked or gelled.
     
    16. The method of claim 15:

    wherein the collection well does not include a sieving matrix, and wherein the collection leg includes a buffer having a first conductivity and the collection well includes a buffer having a second conductivity, the second conductivity being higher than the first conductivity or;

    wherein the collection leg includes a buffer having a first viscosity and the collection well includes a buffer having a second viscosity, the first viscosity being less than the second viscosity or;

    wherein the collection well includes a second sieving matrix, the second sieving matrix providing increased sieving over the first sieving matrix.


     


    Ansprüche

    1. Vorrichtung zum Isolieren einer oder mehrerer Probekomponenten eines Probematerials infolge Trennung des Probematerials in eine Vielzahl von Probekomponenten, umfassend:

    ein erster (1) und zweiter (2) Fokussierungskanal, wobei jeder Fokussierungskanal ein erstes Ende und ein zweites Ende aufweist;

    einen Trennkanal (9), der sich zwischen dem ersten und dem zweiten Fokussierungskanal erstreckt, wobei der Trennkanal ein erstes Ende und ein zweites Ende aufweist;

    einen Sammelstrang (6) mit einem ersten Ende und einem zweiten Ende;

    einen Abfallstrang (7) mit einem ersten Ende und einem zweiten Ende; und

    einen Schaltbereich (3) mit einem Einlassende und einem Auslassende;

    wobei das zweite Ende jedes des ersten Fokussierungskanals, des zweiten Fokussierungskanals und des Trennkanals in Fluidkommunikation mit dem Einlassende des Schaltbereichs stehen und wobei das erste Ende des Sammelstranges und das erste Ende des Abfallstranges in Fluidkommunikation mit dem Auslassende des Schaltbereichs stehen,

    dadurch gekennzeichnet, dass eine Sammelvertiefung (5) im Sammelstrang zwischen dem ersten und dem zweiten Ende des Sammelstrangs angeordnet ist.


     
    2. Vorrichtung nach Anspruch 1, ferner umfassend:

    einen ersten Pufferbehälter (12), einen ersten Abfallbehälter (10) und einen zweiten Abfallbehälter (11), wobei das erste Ende des Trennkanals in Fluidkommunikation mit dem ersten Pufferbehälter ist und wobei das zweite Ende des Sammelstrangs in Fluidkommunikation mit dem ersten Abfallbehälter ist und das zweite Ende des Abfallstrangs in Fluidkommunikation mit dem zweiten Abfallbehälter ist.


     
    3. Vorrichtung nach Anspruch 2, wobei das erste Ende des ersten Fokussierungskanals und das erste Ende des zweiten Fokussierungskanals in Fluidkommunikation mit dem ersten Pufferbehälter stehen.
     
    4. Vorrichtung nach Anspruch 2, ferner umfassend:

    einen zweiten Pufferbehälter (13) und einen dritten Pufferbehälter (14), wobei das erste Ende des ersten Fokussierungskanals in Fluidkommunikation mit dem zweiten Pufferbehälter ist und das erste Ende des zweiten Fokussierungskanals in Fluidkommunikation mit dem dritten Pufferbehälter ist.


     
    5. Vorrichtung nach Anspruch 1, ferner umfassend:

    eine Siebmatrix, die im Trennkanal angeordnet ist; oder

    eine Füllvertiefung (4), die im Trennkanal angeordnet ist; oder

    eine Siebmatrix, die im Sammelstrang angeordnet ist.


     
    6. Vorrichtung nach Anspruch 1, wobei eine Querschnittsabmessung des Trennkanals größer ist als eine Querschnittsabmessung des Schaltbereichs.
     
    7. System zum Isolieren eines oder mehrerer Probekomponenten eines Probematerials infolge Trennung des Probematerials in eine Vielzahl von Probekomponenten, umfassend:

    eine erste Vorrichtung nach Anspruch 1;

    einen Detektor in Sensorkommunikation mit der Vorrichtung;

    ein Fluidleitungssystem; und

    einen Prozessor, der mit dem Detektor und dem Fluidleitungssystem betätigbar verbunden ist.


     
    8. System nach Anspruch 7, wobei das Fluidleitungssystem konfiguriert ist, eine Bewegung von einer oder mehreren Probekomponenten vom Trennkanal in den Sammelstrang oder den Abfallstrang zu steuern.
     
    9. System nach Anspruch 7, ferner umfassend:

    eine zweite Vorrichtung mit einem ersten und einem zweiten Fokussierungskanal, einem Trennkanal, der sich zwischen dem ersten und dem zweiten Fokussierungskanal erstreckt, einen Sammelstrang, einen Abfallstrang und einen Schaltbereich, wobei der Sammelstrang eine Sammelvertiefung aufweist, die im Sammelstrang zwischen einem ersten Ende und einem zweiten Ende des Sammelstrangs angeordnet ist, wobei der erste und der zweite Fokussierungskanal und der Trennkanal mit einem Einlassende des Schaltbereichs in Fluidkommunikation sind, und wobei der Sammelstrang und der Abfallstrang mit einem Auslassende des Schaltbereichs in Fluidkommunikation sind.


     
    10. System nach Anspruch 9, wobei das Fluidleitungssystem konfiguriert ist, Bewegungen von einem oder mehreren Probekomponenten in der ersten Vorrichtung und von einem oder mehreren Probekomponenten in der zweiten Vorrichtung gleichzeitig zu steuern.
     
    11. System nach Anspruch 10, wobei das Fluidleitungssystem konfiguriert ist, von der einen oder mehreren Probekomponenten in der ersten Vorrichtung und von der einen oder mehreren Probekomponenten in der zweiten Vorrichtung basierend auf Informationen zu steuern, die vom Detektor in einem Parallelschaltkreis empfangen werden.
     
    12. Verfahren des Trennens eines Probematerials in eine Vielzahl von getrennten Komponenten und Isolieren eines oder mehrerer der getrennten Komponenten in einer Vorrichtung oder einem System nach einem der Ansprüche 1 bis 11, wobei das Verfahren Folgendes umfasst:

    Füllen eines Probematerials in eine Füllvertiefung, die in Fluidkommunikation mit einem Trennkanal einer Vorrichtung ist;

    Trennen des Probematerials in eine Vielzahl von getrennten Komponenten im Trennkanal, wobei die getrennten Komponenten einen Strom ausbilden;

    Befördern des Stroms von getrennten Komponenten in einen Schaltbereich der Vorrichtung;

    Befördern des ersten und des zweiten Pufferstromes in den Schaltbereich auf beiden Seiten des Komponentenstromes, sodass der erste und der zweite Pufferstrom den Komponentenstrom einschränken und verlängern, während er durch den Schaltbereich befördert wird;

    Leiten eines ersten Teils des Stroms von getrennten Komponenten, eines ersten Teils des ersten Pufferstroms und eines ersten Teils des zweiten Pufferstroms aus dem Schaltbereich heraus und in einen Abfallstrang der Vorrichtung hinein;

    Leiten eines zweiten Teils des Stroms von getrennten Komponenten, eines zweiten Teils des ersten Pufferstroms und eines zweiten Teils des zweiten Pufferstroms aus dem Schaltbereich heraus und in einen Abfallstrang der Vorrichtung hinein;

    Leiten eines dritten Teils des Stroms von getrennten Komponenten, eines dritten Teils des ersten Pufferstroms und eines dritten Teils des zweiten Pufferstroms aus dem Schaltbereich heraus und in einen Abfallstrang der Vorrichtung hinein; und

    Sammeln einer getrennten Komponente aus einer im Sammelstrang angeordneten Sammelvertiefung.


     
    13. Verfahren nach Anspruch 12, wobei nur eines des Sammelstrangs und des Abfallstrangs Nullstrom aufweist, wobei das Verfahren Folgendes umfasst:

    Bestimmen der Spannung im Strang, der Strom Null aufweist, dadurch Bestimmen einer ersten Spannung im Schaltbereich; und

    Einstellen eines oder beider der Spannung und des Stroms im Strang, der keinen Strom Null aufweist, dadurch Erzeugen einer zweiten Spannung im Schaltbereich.


     
    14. Verfahren nach Anspruch 12, ferner umfassend:

    Stapeln eines oder mehrerer der Vielzahl von getrennten Komponenten in der Sammelvertiefung.


     
    15. Verfahren nach Anspruch 14, wobei der Sammelstrang eine erste Siebmatrix aufweist, die entweder vernetzt oder geliert ist.
     
    16. Verfahren nach Anspruch 15,
    wobei die Sammelvertiefung keine Siebmatrix umfasst und wobei der Sammelstrang einen Puffer mit einer ersten Leitfähigkeit umfasst und die Sammelvertiefung einen Puffer mit einer zweiten Leitfähigkeit umfasst, wobei die zweite Leitfähigkeit größer ist als die erste Leitfähigkeit, oder
    wobei der Sammelstrang einen Puffer mit einer ersten Viskosität umfasst und die Sammelvertiefung einen Puffer mit einer zweiten Viskosität aufweist, wobei die erste Viskosität geringer ist als die zweite Viskosität, oder
    wobei die Sammelvertiefung eine zweite Siebmatrix umfasst, wobei die zweite Siebmatrix verstärktes Sieben im Vergleich zur ersten Siebmatrix bereitstellt.
     


    Revendications

    1. Dispositif pour isoler un ou plusieurs constituants échantillons d'une substance échantillon suite à la séparation de la substance échantillon en une pluralité de constituants échantillons, comprenant :

    des premier (1) et second (2) canaux de pincement, chaque canal de pincement ayant une première extrémité et une seconde extrémité ;

    un canal de séparation (9) s'étendant entre les premier et second canaux de pincement, le canal de séparation ayant une première extrémité et une seconde extrémité ;

    un pied de collecte (6) ayant une première extrémité et une seconde extrémité ;

    un pied de déchets (7) ayant une première extrémité et une seconde extrémité ; et

    une région de commutation (3) ayant une extrémité d'entrée et une extrémité de sortie ;

    dans lequel la seconde extrémité de chacun du premier canal de pincement, du second canal de pincement et du canal de séparation sont en communication fluidique avec l'extrémité d'entrée de la région de commutation, et dans lequel la première extrémité du pied de collecte et la première extrémité du pied de déchets sont en communication fluidique avec l'extrémité de sortie de la région de commutation,

    caractérisé en ce que

    un puits de collecte (5) disposé dans le pied de collecte entre les première et seconde extrémités du pied de collecte.


     
    2. Dispositif selon la revendication 1, comprenant en outre :

    un premier réservoir tampon (12), un premier réservoir à déchets (10) et un second réservoir à déchets (11), dans lequel la première extrémité du canal de séparation est en communication fluidique avec le premier réservoir tampon, la seconde extrémité du pied de collecte est en communication fluidique avec le premier réservoir à déchets, et la seconde extrémité du pied de déchets est en communication fluidique avec le second réservoir à déchets.


     
    3. Dispositif selon la revendication 2, dans lequel la première extrémité du premier canal de pincement et la première extrémité du second canal de pincement sont en communication fluidique avec le premier réservoir tampon.
     
    4. Dispositif selon la revendication 2, comprenant en outre :

    un deuxième réservoir tampon (13) et un troisième réservoir tampon (14), dans lequel la première extrémité du premier canal de pincement est en communication fluidique avec le deuxième réservoir tampon et la première extrémité du deuxième canal de pincement est en communication fluidique avec le troisième réservoir tampon.


     
    5. Dispositif selon la revendication 1, comprenant en outre :

    une matrice de tamisage disposée dans le canal de séparation ; ou

    un puits de chargement (4) disposé dans le canal de séparation ; ou

    une matrice de tamisage disposée dans le pied de collecte.


     
    6. Dispositif selon la revendication 1, dans lequel une dimension en coupe du canal de séparation est supérieure à une dimension en coupe de la région de commutation.
     
    7. Système pour isoler un ou plusieurs constituants échantillons d'une substance échantillon suite à la séparation de la substance échantillon en une pluralité de constituants échantillons, comprenant :

    un premier dispositif selon la revendication 1 ;

    un détecteur en communication sensorielle avec le dispositif ;

    un système de direction du fluide ; et

    un processeur couplé opérationnellement au détecteur et au système de direction du fluide.


     
    8. Système selon la revendication 7, dans lequel le système de direction du fluide est configuré pour contrôler le déplacement d'un ou plusieurs constituants échantillons du canal de séparation vers l'un du pied de collecte et du pied de déchets.
     
    9. Système selon la revendication 7, comprenant en outre :

    un second dispositif comprenant des premier et second canaux de pincement, un canal de séparation s'étendant entre les premier et second canaux de pincement, un pied de collecte, un pied de déchets et une région de commutation, le pied de collecte comportant un puits de collecte disposé dans le pied de collecte entre une première extrémité et une seconde extrémité du pied de collecte, dans lequel les premier et second canaux de pincement et le canal de séparation sont en communication fluidique avec une extrémité d'entrée de la région de commutation, et le pied de collecte et le pied de déchets sont en communication fluidique avec une extrémité de sortie de la région de commutation.


     
    10. Système selon la revendication 9, dans lequel le système de direction du fluide est configuré pour contrôler simultanément le déplacement d'un ou plusieurs constituant(s) échantillon(s) dans le premier dispositif et un ou plusieurs constituant(s) échantillon(s) dans le second dispositif.
     
    11. Système selon la revendication 10, dans lequel le système de direction du fluide est configuré pour contrôler le déplacement du ou des constituant(s) échantillon(s) dans le premier dispositif et du ou des constituant(s) échantillon(s) dans le second dispositif, sur la base des informations reçues par le détecteur dans un circuit parallèle.
     
    12. Procédé de séparation d'une substance échantillon en une pluralité de constituants séparés et d'isolation d'un ou plusieurs des constituants séparés dans un dispositif ou un système selon l'une quelconque des revendications 1 à 11, le procédé comprenant :

    le chargement d'une substance échantillon dans un puits de chargement en communication fluidique avec un canal de séparation d'un dispositif ;

    la séparation de la substance échantillon en une pluralité de constituants séparés dans le canal de séparation, les constituants séparés formant un courant ;

    le transport du courant de constituants séparés dans une région de commutation du dispositif ;

    le transport des premier et second courants de solution tampon dans la région de commutation de l'un ou l'autre côté du courant de constituants de telle sorte que les premier et second courants de solution tampon contraignent et allongent le courant de constituants lorsqu'il est transporté à travers la région de commutation ;

    la direction d'une première partie du courant de constituants séparés, d'une première partie du premier courant de solution tampon et d'une première partie du second courant de solution tampon hors de la région de commutation et dans un pied de déchets du dispositif ;

    la direction d'une deuxième partie du courant de constituants séparés, d'une deuxième partie du premier courant de solution tampon et d'une deuxième partie du second courant de solution tampon hors de la région de commutation et dans un pied de collecte du dispositif ;

    la direction d'une troisième partie du courant de constituants séparés, d'une troisième partie du premier courant de solution tampon et d'une troisième partie du second courant de solution tampon hors de la région de commutation et dans le pied de déchets du dispositif ; et

    la collecte d'un constituant séparé à partir d'un puits de collecte disposé dans le pied de collecte.


     
    13. Procédé selon la revendication 12, dans lequel un seul du pied de collecte et du pied de déchets a un courant nul, le procédé comprenant en outre :

    la détermination de la tension dans le pied ayant un courant nul, afin de déterminer une première tension dans la région de commutation ; et

    l'ajustement d'un ou des deux de la tension et du courant dans le pied dont le courant n'est pas nul, produisant ainsi une seconde tension dans la région de commutation.


     
    14. Procédé selon la revendication 12, comprenant en outre :

    l'empilement d'un ou plusieurs de la pluralité de constituants séparés dans le puits de collecte.


     
    15. Procédé selon la revendication 14, dans lequel le pied de collecte comporte une première matrice de tamisage qui est soit réticulée, soit gélifiée.
     
    16. Procédé selon la revendication 15 :

    dans lequel le puits de collecte ne comporte pas de matrice de tamisage, et dans lequel le pied de collecte comporte une première solution tampon ayant une première conductivité et le puits de collecte comporte une solution tampon ayant une seconde conductivité, la seconde conductivité étant supérieure à la première conductivité ou ;

    dans lequel le pied de collecte comporte une solution tampon ayant une première viscosité et le puits de collecte comporte une solution tampon ayant une seconde viscosité, la première viscosité étant inférieure à la seconde viscosité ou ;

    dans lequel le puits de collecte comporte une seconde matrice de tamisage, la seconde matrice de tamisage permettant un tamisage amélioré par rapport à la première matrice de tamisage.


     




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    Cited references

    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description