WO2004035212A1 - Systeme de manipulation de molecules a chaine longue a micro-canaux - Google Patents

Systeme de manipulation de molecules a chaine longue a micro-canaux Download PDF

Info

Publication number
WO2004035212A1
WO2004035212A1 PCT/US2003/033067 US0333067W WO2004035212A1 WO 2004035212 A1 WO2004035212 A1 WO 2004035212A1 US 0333067 W US0333067 W US 0333067W WO 2004035212 A1 WO2004035212 A1 WO 2004035212A1
Authority
WO
WIPO (PCT)
Prior art keywords
micro
channel
molecules
polymeric
polymeric molecule
Prior art date
Application number
PCT/US2003/033067
Other languages
English (en)
Inventor
David Charles Schwartz
Eileen T. Dimalanta
Juan J. De Pablo
Original Assignee
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Priority to AU2003301262A priority Critical patent/AU2003301262A1/en
Publication of WO2004035212A1 publication Critical patent/WO2004035212A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0663—Stretching or orienting elongated molecules or particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size

Definitions

  • the present invention relates to methods of manipulating molecules and, in particular, to a fluid transport system useful for straightening, aligning, and fixing long chain polymers such as DNA.
  • nucleic acid molecules e.g. DNA
  • sequencing of such molecules may be aided by optical techniques in which long portions of such molecules are straightened and fixed to a substrate for microscopic analysis.
  • the fixed molecule may be analyzed by creating "landmarks" on the molecule by attaching fluorescent markers to specific locations or by cutting it with restriction enzymes to form visible breaks at specific locations.
  • the order and relative separation of the landmarks is preserved because the molecule remains fixed, and may be used to produce an optical map of the molecule.
  • the optical map provides a framework on which other sequence information may be assembled.
  • the landmarks allow optical maps of fragments of long molecules to be assembled into the entire molecule by the process of matching fragments with similarly spaced landmarks.
  • the present invention provides a method for straightening, aligning, separating, and/or sorting polymeric molecules using well-controlled laminar flow in a micro-channel.
  • the molecules may be fixed to a substrate after straightening, aligning, separating and/or sorting, or may be analyzed suspended in a carrier liquid during continuous flow or using a hovering technique in which laminar flow is periodically reversed.
  • the present invention also provides an improved method of fabricating micro-channels and a simple mechanism for producing the necessary laminar flow.
  • one aspect of the present invention provides a method for elongating, separating, and/or aligning polymeric molecules comprising the steps of passing a liquid and polymeric molecule through a micro-channel sized to provide laminar flow of the liquid along a micro-channel length.
  • the flow of liquid is controlled to cause elongation of the polymeric molecule within the laminar flow without significant shearing.
  • the straightening is performed without mechanical "carding" in which microscopic posts either machined in silicon or formed of the microscopic structure of a gel or the like.
  • the elongated molecule may be viewed through a transparent wall of the micro-channel as supported in the carrier liquid, and/or may be manipulated through the introduction of a reactant such as an enzyme or other molecule possibly with stimulation by radiation, for example, with a focused laser.
  • a reactant such as an enzyme or other molecule possibly with stimulation by radiation, for example, with a focused laser.
  • the laminar flow may be periodically reversed to cause the polymeric molecule to hover in the elongated state.
  • One wall of the micro-channel may provide electrostatic attraction to the polymeric molecule, and the method may include the step of adsorbing the polymeric molecule to the first wall of the micro-channel in the elongated form.
  • fixation of the molecule for further analysis, manipulation, and/or storage.
  • the fixation may be affected by random encounters between the ends of the polymeric molecule and a wall of the micro-channel or by an external force such as an applied electrostatic or centrifugal acceleration.
  • Another aspect of the invention is that it may provide for a separating of polymeric molecules of differing molecular weights by controlling the laminar flow of the liquid holding the polymeric molecules.
  • This sorting may make use of the varying speeds in the laminar flow of elongated molecules as a function of their length and/or weight, or a functional dependence between the unfolding of the molecules and flow velocity and/or differences in diffusion rate of the ends of the molecule as a function of molecular length.
  • molecules may be separated by the laminar flow acting on their different configurations, or by adsorbing the unfolded molecules to a substrate, or by optical systems that may be distinguished between folded and unfolded molecules.
  • the apparatus used in the present invention may include an optical mapping surface and an elastic channel material releasably adhered to the optical mapping surface to create the micro-channel between the elastic channel material and the optical mapping surface sized to provide flow of a liquid and the polymeric molecule along a micro-channel length.
  • micro-channel can be removed from the optical mapping surface, when molecules are mounted on the surface they may be analyzed with any type of microscope, including electron microscopes, and are not limited to analyses with light microscopes that might penetrate a transparent optical mapping surface.
  • the first wall of the micro-channel may be transparent, for example, glass.
  • At least one end of the micro-channel may provide a funnel section opening to a reservoir holding the liquid and polymeric molecules.
  • the reservoir may be at least one end well extending perpendicular to the length of the micro-channel and wherein the apparatus further includes a housing allowing the end well and micro-channel to be received by a centrifuge with the end well extending along a principal axis of centrifugal acceleration and the micro- channel extending substantially across the principal axis of centrifugal acceleration.
  • the micro-channel may include a region of varying cross section to promote a gradient in flow rate.
  • FIG. 1 is a perspective view of one embodiment of the present invention showing a micro-channel communicating between a staging reservoir, holding polymeric molecules in a carrier liquid, and a collecting reservoir; the micro-channel attached to the reservoirs by funnel portions reducing shear and promoting laminar flow in the micro-channel and showing the use of a syringe pump to draw liquid through the micro-channel;
  • Fig. 2 is a cross-sectional view of the channel along lines 2-2 of Fig. 1 showing the increasing velocity of the laminar flow in the micro-channel toward the center of the micro-channel and an elongated DNA molecule centered in the micro- channel by the laminar flow;
  • FIG. 3 is a fragmentary view similar to Fig. 2 showing: a diffusion radius of one end of a polymeric molecule prior to adsorption to a wall of the micro- channel; a polymeric molecule having a trailing end attached to the wall of the micro-channel; and a polymeric molecule having a leading end attached to the wall of the micro-channel prior to adsorption of the entire length of the polymeric molecule to the wall of the micro-channel;
  • Fig. 4 is a cross-sectional view of multiple micro-channels during a first step of manufacturing the micro-channels in which a mold is used with an elastic molding compound to form upper walls of the micro-channels;
  • Fig. 5 is a figure similar to Fig. 4 showing removal of the mold and attachment of the upper walls of the micro-channel to a glass optical mapping surface in a second step of manufacturing;
  • FIG. 6 is an elevational, cross-sectional view of an alternative embodiment of the present invention in which centrifugal acceleration acting on a fluid head in the staging reservoir causes laminar flow in the micro-channel;
  • Fig. 7 is a plan view of the embodiment of Fig. 6 showing multiple parallel micro-channels, each with staging wells and receiving wells;
  • Fig. 8 is a perspective view of the embodiments of Figs. 6 and 7 as placed in a standard centrifuge cup, the latter in partial cut-away;
  • Fig. 9 is a simplified diagram of rotation of the centrifuge cup of Fig. 8 showing the vectors of motion and centripetal acceleration;
  • Fig. 9 is a simplified diagram of rotation of the centrifuge cup of Fig. 8 showing the vectors of motion and centripetal acceleration;
  • FIG. 10 is a plan view of an alternative micro-channel design providing varying cross-sections and inversely varying flow velocity such as may be used to sort polymeric molecules by size along the length of the micro-channel;
  • Fig. 11 is a flow diagram of the process of using the present invention to straighten and fix polymeric molecules;
  • Fig. 12 is a figure similar to that of Fig. 2 showing a sorting of the elongated molecules by length and hence by molecular weight according to their different velocities within the laminar flow;
  • Fig. 13 is a schematic representation of a functional relationship between transition to an elongated configuration and laminar flow rate for molecules of different molecular weights serving as a basis for a second type of sorting in which molecules are selectively elongated;
  • Fig. 14 is a figure similar to that of Fig. 12 showing separation of the molecules according to velocity differences caused by elongation and unelongated configurations;
  • Fig. 15 is a simplified representation of two images, a first showing elongated and aligned molecules together with coiled molecules, and the second showing a separation of the aligned and elongated molecules using standard image processing techniques;
  • Fig. 16 is a schematic representation of a device for providing molecular hovering of molecules in a laminar flow for manipulation
  • Fig. 17 is a figure similar to that of Figs. 2, 12, and 14 showing a technique in which an electrostatic field promotes the affixing of a molecule to a wall of the micro-channel;
  • Fig. 18 is a figure similar to that of Fig. 17 showing a second method for promoting affixing of the molecules.
  • the apparatus 10 of the present invention provides a generally planar channel plate 12 into which a longitudinally extending micro-channel 14 is formed, flanked by a staging reservoir 16 and a collecting reservoir 18 positioned at longitudinal ends of the channel plate 12.
  • Junctions between the longitudinal ends of the micro-channel 14 and staging reservoir 16 and collecting reservoir 18 are tapered to create funnel sections with narrow ends attached to the micro-channel 14 and wide ends attached to one of the staging reservoir 16 or collecting reservoir 18.
  • the funnel sections 20 provide a smooth transition of fluid from the staging reservoir 16 through the micro-channel 14 to the collecting reservoir 18 thereby promoting laminar flow within the micro- channel 14 and reducing breakage of polymeric molecules as will be described.
  • One common wall of the staging reservoir 16, the collecting reservoir 18, and the micro-channel 14 is provided by an optical mapping substrate 22 attached to the channel plate 12.
  • the substrate 22 thus encloses the staging reservoir 16, the collecting reservoir 18, and the micro-channel 14.
  • the substrate 22, for example, may be a glass slide, treated, as will be described below
  • a sample introduction port 24 may be formed in the optical mapping substrate 22 at the staging reservoir 16 to allow the introduction of polymeric molecules 36 and a carrier liquid 26 to the staging reservoir.
  • the sample introduction port 24 may be used for pressure equalization when materials are drawn through the micro-channel 14 or for the attachment of a pump to pressurize the staging reservoir 16 to cause materials to flow through the micro-channel 14.
  • a sample extraction port 27 may be formed in the optical mapping substrate 22 at the collecting reservoir 18 for removal of material, pressure equalization, or as shown, the attachment of pump 28 to draw the materials through the micro-channel 14.
  • the ports 24 and 27 may be formed in the channel plate 12.
  • the pump 28 is a syringe pump providing precisely metered flow using an electromechanical actuator and control system as is well understood in the art.
  • the syringe pump draws carrier liquid 26 and polymeric molecules 36 from staging reservoir 16 through the micro-channel 14 in the collecting reservoir 18 at a controlled flow rate as may be set to provide the desired laminar flow within the micro-channel 14.
  • the laminar flow 30 of carrier liquid 26 and polymeric molecules 36 within the micro-channel is such as to provide flow 32 parallel to the longitudinal walls of the micro-channel 14 with greatest flow velocities toward the cross-sectional center of the micro-channel 14 thereby defining a flow velocity profile 34.
  • the flow rate of the pump 28 and the size of the micro- channel 14 is selected to provide flow velocity profile 34 that promotes straightening of the particular polymeric molecule 36 contained within the carrier liquid 26 with the polymeric molecule 36 roughly centered within the lumen of the micro-channel 14. These settings may be determined empirically by visual observation of the polymeric molecules 36 at different flow rates.
  • laminar flow may be distinguished from capillary flow in which the liquid is drawn along the surface of the micro-channel 14 walls by the hydrophilic nature of those walls and where the center leading flow velocity profile 34 is not obtained.
  • the velocity of flow 32 may range from 15 to 70 micrometers per second as measured across the lumen of the micro-channel 14.
  • one acceptable flow would be approximately 5x10 " nl/sec.
  • the cross-sectional width 38 of the micro-channel 14 is 50 micrometers and is preferably less than 100 micrometers. More generally, it is believed that the width 38 will be between one and one hundred times the straightened length 40 of the polymeric molecule 36.
  • the ends 42 of the polymeric molecule 36 are more mobile than the remainder of the polymeric molecule 36 and may be modeled as having an effective diffusion radius 44 during the time the polymeric molecule 36 is in transit in the micro-channel 14 and generally greater than the polymeric molecule 36 as a whole.
  • the average flow rate of the carrier liquid 26 for the flow velocity profile 34 and the width 38 of the micro-channel 14 is thus adjusted so that this effective diffusion radius 44 is equal to or greater than the width 38 of the micro-channel 14.
  • the substrate 22 may be treated to establish a positive charge density on its surface contacting the carrier liquid 26.
  • the surface may be a derivative with silage compounds, for example, those discussed in U.S. Patent 5,720,928, hereby incorporated in its entirety by reference.
  • the micro-channels 14 and optionally the staging reservoir 16, and collecting reservoir 18 of the apparatus 10 may be constructed in silicon using conventional photolithographic techniques.
  • the micro-channels 14 (and optionally the staging reservoir 16 and collecting reservoir 18) are constructed using a molded elastomeric polymer.
  • a mold 45 providing a planar substrate 46 with upstanding ridges 48 defining the volume of the micro-channels 14 may be fabricated using conventional photolithography in which a light sensitive photoresist is applied to a silicon wafer that will form the mold 45.
  • the photoresist is hardened by selective optical exposure and the unhardened portions washed away so that the photoresist provides a mask in the regions of the upstanding ridges 48 (e.g., the regions of the micro-channels 14 and the staging reservoir 16 and collecting reservoir 18).
  • the silicon wafer is then etched to a depth of 7 to 8 micrometers defining the height of the micro-channel 14.
  • an elastomeric polymer preferably poly(dimethylsiloxane) (“PDMS”) is then poured over this mold 45 to create the channel plate 12.
  • the PDMS channel plate 12 is then peeled from the mold 45 and exposed to oxygen plasma to make it hydrophilic.
  • the channel plate 12 may then be adhered to the substrate 22 creating the micro-channels 14 and optionally the staging reservoir 16 and collecting reservoir 18.
  • the PDMS of the channel plate 12 will naturally adhere to glass in a releasable manner to produce a leak resistant seal.
  • the seal is strong enough to resist leakage of fluids filling the micro-channels for the pressures used in this process, yet weak enough to be reversible, and thus make the channel plate 12 and substrates 22 reusable.
  • the substrate 22 By treatment of the substrate 22, as described above, to impress upon it a positive charge, and lack of treatment of channel plate 12 or by a treatment that promotes a negative surface charge on the channel plate 12 preferential adsorption of the polymeric molecules 36 by the substrate 22 may be promoted.
  • Optical mapping of the fixed polymeric molecules 36 may then be done through the transparent glass substrate 22 by means of an inverted microscope objective 50 such as a Zeiss Axiovert 135M such as is readily commercially available. Before the optical mapping, the polymeric molecule may be treated with fluorescent markers or restriction enzymes as are understood in the art.
  • the substrate 22 may be removed from the channel plate 12 and the top surface of the substrate 22 may be imaged.
  • the removal of the channel plate 12 may also assist in further treatment of the fixed polymeric molecules 36, for example, with restriction enzymes and the like and the drying of these molecules to further promote adhesion.
  • the ability to separate the channel plate 12 and substrate 22 allows one or both of these elements to be reused if desired.
  • Fig. 6 in an alternative embodiment to that shown in Fig. 1, the height of the staging reservoir 16 and a collecting reservoir 18 may be increased and ports 24 and 27 provided through the channel plate 12 opposite the substrate 22.
  • the substrate 22 of the embodiment of Fig. 6 may be attached to a weighted carrier 54 that fits within the cup 56 of a standard swing bucket centrifuge 58 with the channel plate 12 supported to be level with the top of the cup 56 and the staging reservoir 16 and collecting reservoir 18 extending upward therefrom.
  • the weighted carrier 54 is constructed so that the combination of the channel plate 12, the substrate 22, and the weighted carrier 54, when in position in the cup 56, have a center of mass 57 below the pivot 55 about which the cup is free to rotate.
  • a single channel plate 12 may incorporate multiple staging reservoirs 16, collecting reservoirs 18, and intervening micro- channels 14. As the pressure head 52 drops with flow through the micro-channel 14, the flow rate through the micro-channel 14 will also decrease.
  • Control of this rate of decrease can be obtained by adjusting the relative diameter or cross-sectional area of staging reservoir 16 compared to collecting reservoir 18. For example, by making the collecting reservoirs 18 of bigger diameter than the staging reservoirs 16, the pressure head 52 decreases more slowly. By making the diameter of the reservoirs 16 and 18 large with respect to the flow rate or concentrating the polymeric molecules in the bottom of the staging reservoir 16, the molecules will pass through the micro-channel 14 only during the initial flow period providing more constant flow and transit time of the polymeric molecules 36 through the micro-channel 14. [0075] Referring now to Fig.
  • the micro- channel 14 may be given a varying cross-sectional area so that for a given net flow rate 65 a series of different flow velocities VI through V3 will be created at different locations along the micro-channel 14. It is believed that these varying flow velocities may effect a spatial separation of polymeric molecules 36 according to their length. This length sorting may be desirable to separate shorter polymeric molecules 36 from overlapping with longer polymeric molecules or for analytic separation of polymeric molecules 36 by length such as currently is done with electrophoresis.
  • a solution typically of water and polymeric molecules, for example, DNA
  • the polymeric molecules 36 may be treated with a condensing agent such as spermine causing them to coil, thereby reducing their damage during transfer to the apparatus 10 described above.
  • the water which will act as the carrier liquid 26
  • polymeric molecules 36 are inserted into the staging reservoir 16.
  • the staging reservoir 16 they may be treated, for example, with a saline solution to decondense the molecules over a period, loosening their spermine-induced coiling.
  • the carrier liquid 26 and polymeric molecules 36 flow through the micro-channel 14 driven by a pump, centrifuge, or other method. During the flow, polymeric molecules 36 attach to the substrate 22 in straightened configuration.
  • Additional treatment of the fixed polymeric molecules 36 may be performed, as indicated by process block 74, by a variety of methods known in the art including but not limited to tagging with fluorescent materials or cutting by restriction enzymes. This step may include staining the polymeric molecules 36 with a fluorescent dye to provide accurate measurement of segments of the polymeric molecules 36.
  • optical mapping of the fixed and treated polymeric molecules 36 may be performed either through the transparent optical mapping substrate 22 or by removing channel plate 12. After optical mapping, the fixed polymeric molecules 36 may be stored.
  • the laminar fluid flow used in the present invention in contrast to radial or other capillary fluid flows, is believed to reduce the number of overlapping molecules.
  • the controlled laminar flow may also provide more consistent elongation or stretching of the polymeric molecules 36. Elongation may be controlled by controlling the laminar flow velocity.
  • each polymeric molecule 36 will travel along a fixed given flow line.
  • An important consequence of this is that long molecules of up to an inch in length are microscopically flat. This means they will stay in focus and are easily manipulated for subsequent actions including digestion by enzymes or treatment with highly focused light or laser illumination.
  • the flow lines in laminar flow in a rectilinear micro-channel are essentially parallel and thus the tendency of the molecules 36 to follow flow lines provides an alignment of the molecules along a common axis such as simplifies optical mapping by reducing or eliminating any crossing of the molecules 36 such as would, for example, create hot spots of florescence from two molecules 36 at the crossing juncture, or otherwise interfere with analyses of the molecules.
  • the alignment also allows for higher density of usable molecules when affixed to a substrate or analyzed in the carrier liquid 26.
  • This sorting would cause selective adsorption of the molecules 36 as a function of molecular weight and may be promoted by control of the carrier fluid flow rate and the length of the micro-channel 14 so as to increase the chance of adsorption of desired (or undesired) molecules 36 selectively.
  • the velocity of the molecule within the laminar flow 30 appears to have a molecular weight dependency so that the molecules 36 will sort themselves out, to some extent, by molecular weight over the length of the micro-channel 14.
  • elongated molecules 36" having greater length (and hence number of base pairs and molecular weight) may be separated axially from elongated molecules 36 and 36' having lesser length.
  • a second effect, believed to be present in the micro- channel 14, is a strong dependency between molecular weight and the velocity of laminar flow necessary to cause the molecules 36 to transition between a coiled configuration and an elongated configuration.
  • the transition from a random coil to a full elongation configuration is quite sharp as indicated by the vertical sections of the chart of Fig. 13.
  • a given flow rate 80 for example, molecules of molecular weight M 3 and M 4 as shown by those similarly named curves will be elongated while molecules with molecular weights Mi and M 2 will remain in a coiled configuration. Adjustment of the flow velocity allows arbitrary separations between these different molecular weights.
  • a sorting of molecules by length may be performed in a manner different from that described above with respect to Fig. 12.
  • the laminar flow 30 acts to separate the elongated molecules 36 from the coil molecules 36' making use of a difference between velocity of the molecules 36 and 36' in the laminar flow 30.
  • the velocity of the flow over the length of the micro-channel 14 may be varied by changing the cross sectional area of the micro-channel 14. Thus different velocities may be used to promote elongation and separation.
  • elongated and aligned molecules 36 and the coil molecules 36' also allow them to be readily distinguished visually. This visual difference may be exploited in an automated procedure using well known image processing techniques which produce masks 82, for example, produced using a derivative of the image values taken in the direction of the alignment, such as tends to favor aligned molecules 36 (which have high derivatives only at their ends) but to block coiled molecules 36' (which may have high derivatives) over most of their length.
  • a hovering or suspension of the molecule 36 within the carrier liquid 26 is that the molecule 36 is free of surface effects that can hinder molecular assemblies and enzymatic action.
  • One apparatus for producing such a hovering may provide for a pump 28 such as the type described above with respect to Fig. 1 feeding the micro-channel 14, the latter that terminates in the staging reservoir 16.
  • a microscope objective 50 may be positioned along the length of the micro-channel 14 where the hovering molecule 36 will be positioned.
  • the microscope may be an epifluoresence microscope having an oil immersion lens and may be coupled to a camera (not shown) for recoding of the information so obtained.
  • the pump 28 ceases and a second oscillating pump 86 is activated, the oscillating pump 86 communicating through a branch 88 with the micro-channel 14.
  • the oscillating pump 86 may include a diaphragm 90 that is reciprocated by a solenoid 92 connected to a sinusoidal power supply 94.
  • a flexing of the diaphragm 90 provides for a simple, low frequency alternation of the laminar flow 30 within the micro-channel 14, preferably at a rate of 0.5 to 3 Hertz.
  • This ability to stabilize the molecule 36 and maintain its elongation allows complex manipulation of the molecule 36 during observation through microscope objective 50. Such manipulation may include reactions with introduced reagents 95 with and without through activation, for example, by a laser 93.
  • the reagents 95 may be introduced using second syringe 96 communicating through an orifice with the micro-channel 14 at the site of hovering.
  • the reagents 95 may include, for example, restriction enzymes including those activated by the laser 92 and other reactants and DNA or RNA molecules placed near the hovering molecule to promote assemblies of molecules together. These techniques may also be applied to molecules suspended in the micro-channel 14 without hovering. [0094] Using this technique, in the case of optical mapping of nuclei acid molecules 36, as an example, the elongated, flowing nucleic acid molecules can be digested with restriction enzymes as they pass the microscope.
  • the carrier liquid 26 can contain or have introduced restriction enzymes and each of the reagents necessary for digesting the nucleic acid molecule flowing through the chamber except that the divalent cation (usually Mg 2 +), which is necessary for enzyme activity, is present in a reversibly chelated form. As such, the nucleic acid is protected from digestion until the divalent cations are liberated.
  • a light-inactivated chelator such as, for example, DM-nitrophen, as described below in U.S. Patent 6,610,256, the cations can be released within the viewing/manipulation chamber as the fluid passes through the microscope light source.
  • the nucleic acid molecule 36 first becomes subject to digestion as it passes through the viewing/manipulation chamber. Further, as digestion occurs, the flow maintains the order of the resulting restriction fragments, which are imaged and which, therefore, instantly produce restriction maps which have been generated in real time.
  • An example of such a photo-inactivated chelator is also described in U.S. Patent 6,610,256.
  • the molecules 36 suspended in carrier liquid 26 as molecules 36 pass through the micro-channel 14 they may be adsorbed to a wall of the micro-channel 14 as has been described previously through normal action of the polymer ends under the laminar flow. Polymer ends are favored to be on the periphery of the coil molecules 36 so these sections have a high probability of attaching to the charged surfaces of the micro-channel 14.
  • More precise control over the location of fixation of the molecules may be provided, for example, by using the hovering technique described above with respect to Fig. 16 and an external force for sedimentating molecules 36. As shown

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Fluid Mechanics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Signal Processing (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Abstract

Un flux laminaire d'un liquide porteur et de molécules polymères passant dans des micro-canaux est utilisé pour ordonner, aligner, séparer et/ou trier les molécules polymères. Ces molécules polymères peuvent être analysées et/ou manipulées dans le liquide porteur ou fixées à une paroi du micro-canal pour un traitement et une analyse ultérieurs. Les micro-canaux peuvent être fabriqués au moyen d'un matériau de moulage élastique. Une forme d'exécution de micro-canal crée un écoulement de fluide au moyen d'une centrifugeuse de laboratoire standard.
PCT/US2003/033067 2002-10-18 2003-10-17 Systeme de manipulation de molecules a chaine longue a micro-canaux WO2004035212A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003301262A AU2003301262A1 (en) 2002-10-18 2003-10-17 Micro-channel long molecule manipulation system

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41988402P 2002-10-18 2002-10-18
US60/419,884 2002-10-18

Publications (1)

Publication Number Publication Date
WO2004035212A1 true WO2004035212A1 (fr) 2004-04-29

Family

ID=32108147

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/033067 WO2004035212A1 (fr) 2002-10-18 2003-10-17 Systeme de manipulation de molecules a chaine longue a micro-canaux

Country Status (2)

Country Link
AU (1) AU2003301262A1 (fr)
WO (1) WO2004035212A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018218A1 (fr) * 1993-02-01 1994-08-18 Seq, Ltd. Procedes et appareil de sequençage de l'adn
WO2000009757A1 (fr) * 1998-08-13 2000-02-24 U.S. Genomics, Inc. Polymeres caracterises optiquement
US6123819A (en) * 1997-11-12 2000-09-26 Protiveris, Inc. Nanoelectrode arrays
US20020037499A1 (en) * 2000-06-05 2002-03-28 California Institute Of Technology Integrated active flux microfluidic devices and methods
US20020081744A1 (en) * 1999-08-13 2002-06-27 Chan Eugene Y. Methods and apparatuses for characterization of single polymers
US6438279B1 (en) * 1999-01-07 2002-08-20 Cornell Research Foundation, Inc. Unitary microcapiliary and waveguide structure and method of fabrication
US20020137218A1 (en) * 1995-12-18 2002-09-26 Alec Mian Devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system
US20030165964A1 (en) * 2001-08-27 2003-09-04 Hannah Eric C. Electron induced fluorescent method for nucleic acid sequencing

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018218A1 (fr) * 1993-02-01 1994-08-18 Seq, Ltd. Procedes et appareil de sequençage de l'adn
US20020137218A1 (en) * 1995-12-18 2002-09-26 Alec Mian Devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system
US6123819A (en) * 1997-11-12 2000-09-26 Protiveris, Inc. Nanoelectrode arrays
WO2000009757A1 (fr) * 1998-08-13 2000-02-24 U.S. Genomics, Inc. Polymeres caracterises optiquement
US6438279B1 (en) * 1999-01-07 2002-08-20 Cornell Research Foundation, Inc. Unitary microcapiliary and waveguide structure and method of fabrication
US20020081744A1 (en) * 1999-08-13 2002-06-27 Chan Eugene Y. Methods and apparatuses for characterization of single polymers
US20020037499A1 (en) * 2000-06-05 2002-03-28 California Institute Of Technology Integrated active flux microfluidic devices and methods
US20030165964A1 (en) * 2001-08-27 2003-09-04 Hannah Eric C. Electron induced fluorescent method for nucleic acid sequencing

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHIH-MING HO: "Fluidics - the link between micro and nano sciences and technologies", PROCEEDINGS OF THE IEEE 14TH. ANNUAL INTERNATIONAL CONFERENCE ON MICROELECTRO MECHANICAL SYSTEMS. MEMS 2001. INTERLAKEN, SWITZERLAND, JAN. 21 - 25, 2001, IEEE INTERNATIONAL MICRO ELECTRO MECHANICAL SYSTEMS CONFERENCE, NEW YORK, NY: IEEE, US, vol. CONF. 14, 21 January 2001 (2001-01-21), pages 375 - 384, XP010534628, ISBN: 0-7803-5998-4 *
UNGER M A ET AL: "Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 288, 7 April 2000 (2000-04-07), pages 113 - 116, XP002192277, ISSN: 0036-8075 *

Also Published As

Publication number Publication date
AU2003301262A1 (en) 2004-05-04

Similar Documents

Publication Publication Date Title
US7775368B2 (en) Micro-channel long molecule manipulation system
US9290812B2 (en) Methods and compositions for separating rare cells from fluid samples
US6949355B2 (en) Methods, compositions, and automated systems for separating rare cells from fluid samples
US7166443B2 (en) Methods, compositions, and automated systems for separating rare cells from fluid samples
US7138269B2 (en) Microflow system for particle separation and analysis
US6875329B2 (en) Method for separating substances using a dielectrophoretic apparatus
EP1511570B1 (fr) Structures microfluidiques
US20030119177A1 (en) Sample chip
JP2002539918A (ja) 固体表面上の空間的に向けられた相互作用
US20060252054A1 (en) Methods and compositions for detecting non-hematopoietic cells from a blood sample
JP2003507026A (ja) ポリマーを引き伸ばす方法および装置
TW201818983A (zh) 用於分離或富集化細胞的方法及組合物
KR20110110209A (ko) 생물학적 분석들을 수행하기 위한 분석 장치 및 방법
WO2004067708A2 (fr) Dispositif et procede pour fragmenter de la matiere par cisaillement hydrodynamique
AU2010212376A1 (en) Microfluidic device for cell separation and uses thereof
US6783928B2 (en) Microstructures for cell proliferation assays and semen analysis
US7220592B2 (en) Particulate processing system
US8142708B2 (en) Micro fluidic system for single molecule imaging
WO2004035212A1 (fr) Systeme de manipulation de molecules a chaine longue a micro-canaux
US8585881B2 (en) Localized chemical microgradients
JP6143282B2 (ja) 核酸の伸長・固定デバイス、該デバイスを用いた核酸の伸長・固定方法、核酸固定部材、及び核酸の伸長・固定デバイスを用いて作製した核酸標本

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP