WO2001013088A1 - Procede et appareil d'etirage de polymeres - Google Patents

Procede et appareil d'etirage de polymeres Download PDF

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WO2001013088A1
WO2001013088A1 PCT/US2000/022253 US0022253W WO0113088A1 WO 2001013088 A1 WO2001013088 A1 WO 2001013088A1 US 0022253 W US0022253 W US 0022253W WO 0113088 A1 WO0113088 A1 WO 0113088A1
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channel
polymer
integrated apparatus
along
elongation structure
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PCT/US2000/022253
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Eugene Y. Chan
Lance C. Gleich
Parris S. Wellman
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U.S. Genomics, Inc.
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Priority to CA002381361A priority Critical patent/CA2381361A1/fr
Priority to IL14808800A priority patent/IL148088A0/xx
Priority to EP00955515A priority patent/EP1210578A4/fr
Priority to AU67711/00A priority patent/AU6771100A/en
Priority to JP2001517140A priority patent/JP2003507026A/ja
Publication of WO2001013088A1 publication Critical patent/WO2001013088A1/fr

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    • 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
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/431—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/432—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction with means for dividing the material flow into separate sub-flows and for repositioning and recombining these sub-flows; Cross-mixing, e.g. conducting the outer layer of the material nearer to the axis of the tube or vice-versa
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/433—Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
    • 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
    • B01L3/502746—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 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • 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
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0694—Creating chemical gradients in a fluid
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • 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
    • B01L2300/0883—Serpentine channels
    • 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
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • 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
    • B01L3/502769—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 characterised by multiphase flow arrangements
    • B01L3/502776—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 characterised by multiphase flow arrangements specially adapted for focusing or laminating flows
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/44—Resins; Plastics; Rubber; Leather
    • G01N33/442—Resins; Plastics

Definitions

  • the present invention relates to the general field of polymer characterization. More particularly, the invention relates to the use of structures to stretch a polymer or to select a polymer on the basis of length in a chip.
  • Macromolecules are involved in diverse and essential functions in living systems. The ability to decipher the functions, dynamics, and interactions of macromolecules is dependent upon an understanding of their chemical and three-dimensional structures. These three aspects - chemical and three-dimensional structures and dynamics - are interrelated. For example, the chemical composition of a protein, and more particularly the linear arrangement of amino acids, explicitly determines the three-dimensional structure into which the polypeptide chain folds after biosynthesis (Kim & Baldwin (1990) Ann. Rev. Biochem. 59: 631-660), which in turn determines the interactions that the protein will have with other macromolecules, and the relative mobilities of domains that allow the protein to function properly.
  • Bio macromolecules are either polymers or complexes of polymers. Different types of macromolecules are composed of different types of monomers, i.e., twenty amino acids in the case of proteins and four major nucleobases in the case of nucleic acids. A wealth of information can be obtained from a determination of the linear, or primary, sequence of the monomers in a polymer chain. For example, by determining the primary sequence of a nucleic acid, it is possible to determine the primary sequences of proteins encoded by the nucleic acid, to generate expression maps for the determination of mRNA expression patterns, to determine protein expression patterns, and to understand how mutations in genes correspond to a disease state.
  • the characteristic pattern of distribution of specific nucleobase sequences along a particular DNA polymer can be used to unequivocally identify the DNA, as in forensic analysis.
  • fast, accurate and inexpensive methods of characterizing polymers, and particularly nucleic acids are being developed as a result of the endeavor of the Human Genome Project to sequence the human genome.
  • a challenge to the characterization of the linear sequence of monomers in a polymer chain has come from the natural tendency of polymers in most media to adopt unpredictable, coiled conformations.
  • the average amount of such coiling is dependent on the interaction of the polymer with the surrounding solution, the rigidity of the polymer, and the energy of interaction of the polymer with itself. In most cases, the coiling is quite significant.
  • a ⁇ -phage DNA theoretically 16 ⁇ m long when stretched out so that the DNA is in the B conformation, has a random coil diameter of approximately 1 ⁇ m (Smith et al. (1989) Science 243:203-206).
  • DNA and many other biopolymers can be modeled as uniform elastic rods in a worm-like chain in order to determine their random coil properties (Austin et al. (1997) Physics Today 50(2):32-38).
  • One relevant parameter is the persistence length, P, the length over which directionality is maintained, which is given by:
  • K is the elastic bending modulus (Houseal et al. (1989) Biophys. J. 56:507-516), k B is the Boltzmann constant, and T is temperature (Austin et al. (1997) Physics Today 50(2):32- 38).
  • F s k B T/P (3) where all of the parameters are defined as above. Below F s , the relationship between the force applied and the amount of stretching is roughly linear; above F s , applying more force results in little change in the stretching (Smith et al. (1992) Science 258: 1122-1126; Bustamante (1994) Science 265:1599-1600). Hence, full stretching is essentially attained by applying F s .
  • the force required to stretch it from its coiled conformation to its full length, which stretched conformation retains the B conformation is about 0.1 pN.
  • Such a small force could, in principle, be obtained from virtually any source, including shear forces, electrical forces, and gravitational forces.
  • the length of S-DNA is less consistent than that of B- DNA stretched to its natural length and is more dependent on the exact force applied (Cluzel et al. (1996) Science 271 :792-794), varying linearly with applied force from 1.7 to 2.1 times the length of B-DNA. Since it may not be possible to know the exact force applied, it is desirable to avoid stretching DNA into its S-form. Therefore, a force having a range of about two orders of magnitude, from about 0.1 pN to 25 pN, is capable of consistent and predictable stretching of DNA to its fully extended B-form.
  • ⁇ is the viscosity of the solvent and the other parameters are as defined above.
  • the relaxation time is about 6 seconds, which can be increased to 20 seconds in a solution with a viscosity of 220 cp (Smith et al. (1999) Science 283:1724-1727) or by running the DNA in a confined space to lengthen P and change the viscous drag (Bakajin et al. (1998) Phys. Rev. Let. 80:2737-2740).
  • Relaxation time is also a function of the extent of stretching (Hatfield & Quake (1999) Phys. Rev. Let. 82:3548-3551), so the values calculated above are a lower bound on the actual relaxation time.
  • the polymer must be stretched out on a shorter time scale.
  • the appropriate time scale for stretching is the reciprocal of the strain rate.
  • a receding meniscus to align the polymer, a process sometimes referred to as "molecular combing.”
  • a second fluid is introduced that is substantially immiscible with the first, forming a meniscus at the interface.
  • the original fluid is then gradually removed by mechanical, thermal, electrical, or chemical means or simply by evaporation and is replaced by the new fluid.
  • the polymer is aligned perpendicular to the interface by surface tension and therefore, becomes stretched.
  • D 2.2 nm for double-stranded DNA
  • ⁇ surface tension
  • An alternative way to manipulate DNA immobilized at one end involves the use of an optical trap.
  • a laser beam (“optical tweezers”) imparts momentum to a DNA molecule through emitted photons.
  • optical tweezers By shifting the position of the photons, i.e., moving the beam, an extremely precise change can be induced in the direction of travel of the DNA (U.S. Pat. No. 5,079,169; Chu (1991) Science 253:861-866).
  • a DNA molecule can be stretched using optical tweezers.
  • the technique offers the advantage of being able to vary the force used for stretching and has been used to verify reptation theory (Perkins et al. (1994) Science 264:819-822).
  • the laser can only hold one molecule in place at a time and has to be realigned for each subsequent molecule, making it unattractive for high-throughput analyses.
  • a third method of stretching DNA involves electrophoresis of either a DNA immobilized at one end to move the unattached end of the molecule away from the fixed end and subsequently attaching the fixed end to a surface with avidin, or a DNA unattached at both ends and then attaching both ends to a surface with avidin (Kabata et al. (1993) Science 262:1561-1563; Zimmerman & Cox (1994) Nucl. Acids Res. 22:492-497). No attempt was made to characterize the quality of the stretching using this technique. Furthermore, this technique shares the disadvantages of the previously-mentioned techniques (with respect to post-immobilization processing). DNA has also been stretched by electrophoresis without fixing one end of the molecule.
  • DNA has been elongated by electrophoresis both in a gel and in solution, using electrical forces to move the DNA in position for reading (U.S. Pat. No. 5,538,898).
  • electrophoresis both in a gel and in solution, using electrical forces to move the DNA in position for reading.
  • no data were given to determine the quality of the stretching of large polymers, and the technique is limited to analyzing approximately 3 megabases at a time.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a tapered channel, said tapered channel decreasing linearly in width from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end; whereby when said at least one polymer in said fluid sample moves along said tapered channel, a shear force is applied to said at least one polymer.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus comprising: (a) at least one polymer in a fluid sample; and (b) an elongation structure for stretching said at least one polymer, wherein said elongation structure comprises a tapered channel, said tapered channel decreasing linearly in width from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end, whereby when said at least one polymer in said fluid sample moves along said tapered channel, a shear force is applied to said at least one polymer.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a tapered channel, said tapered channel decreasing in width at a greater than linear rate from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end; whereby when said at least one polymer in said fluid sample moves along said tapered channel, a shear force is applied to said at least one polymer.
  • the present invention is also useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus comprising: (a) at least one polymer in a fluid sample; and (b) an elongation structure for stretching said at least one polymer, wherein said elongation structure comprises a tapered channel, said tapered channel decreasing in width at a greater than linear rate from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end; whereby when said at least one polymer in said fluid sample moves along said tapered channel, a shear force is applied to said at least one polymer.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a tapered channel, said tapered channel decreasing in width from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end; whereby when said at least one polymer in said fluid sample moves along said tapered channel, a shear force is applied to said at least one polymer, wherein said shear force produces a shear rate that is constant.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus comprising: (a) at least one polymer in a fluid sample; and (b) an elongation structure for stretching said at least one polymer, wherein said elongation structure comprises a tapered channel, said tapered channel decreasing in width from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end; whereby when said at least one polymer in said fluid sample moves along said tapered channel, a shear force is applied to said at least one polymer, wherein said shear force produces a shear rate that is constant.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a central channel for holding fluid and a plurality of side channels for holding fluid connected to said central channel; and wherein said at least one polymer, when present, moves along said central channel in an elongation direction.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising: (a) an elongation structure; (b) a delivery channel leading into and out of said elongation structure for delivering said at least one polymer sample in said fluid to said elongation structure; and (c) means for causing said at least one polymer in said fluid sample, when present, to move within said elongation structure, wherein said elongation structure comprises a central channel for holding fluid and a plurality of side channels for holding fluid connected to said central channel; and wherein, when said at least one polymer is present, said means for causing causes said at least one polymer to move along said central channel in an elongation direction.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching DNA in a fluid sample
  • an integrated apparatus for stretching DNA in a fluid sample comprising: (a) an elongation structure; (b) means for delivering said DNA in said fluid sample to said elongation structure; and (c) means for causing said DNA in said fluid sample, when present, to move within said elongation structure, wherein said elongation structure comprises a central channel for holding fluid and a plurality of side channels for holding fluid connected to said central channel; and wherein, when said DNA is present, said means for causing causes said DNA to move along said central channel in an elongation direction.
  • the present invention relates to an integrated apparatus comprising: (a) at least one polymer in a fluid sample; (b) an elongation structure for stretching said at least one polymer, wherein said elongation structure comprises a central channel for holding fluid and a plurality of side channels for holding fluid connected to said central channel.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a channel with at least one bend, and wherein said at least one polymer, when present, moves along said channel.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching DNA in a fluid sample comprising: (a) an elongation structure; and (b) means for delivering said DNA in said fluid sample to said elongation structure, wherein said elongation structure comprises a channel with at least one bend, and wherein said DNA, when present, moves along said channel.
  • the present invention relates to an integrated apparatus comprising: (a) at least one polymer in a fluid sample; and (b) an elongation structure for stretching said at least one polymer, wherein said elongation structure comprises a channel with at least one bend.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a tapered channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to motion of said at least one polymer.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a central channel along which said at least one polymer, when present, moves in a flow direction and a plurality of side channels connected to said central channel, and wherein said central channel further comprises a plurality of obstacles to motion of said at least one polymer.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a channel with at least one bend along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to motion of said at least one polymer.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of posts, at least one of said posts having a non-quadrilateral polygonal cross sectional shape.
  • the fifteenth, sixteenth and seventeenth embodiments of the present invention are useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to motion of said at least one polymer, said plurality of obstacles being positioned as a series of rows, each said row positioned perpendicular to said flow direction, and each successive row offset from a previous row, whereby at least a portion not equal to a multiple of 1/2 of one of said obstacles overlaps an extension of a gap formed by two adjacent obstacles in said previous row along said flow direction.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus comprising: (a) at least one polymer in a fluid sample, every said polymer having a diameter greater than or equal to a minimum diameter; and (b) an elongation structure for stretching said at least one polymer, wherein said elongation structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to motion of said at least one polymer, said plurality of obstacles positioned as a series of rows, each said row positioned perpendicular to said flow direction, and each adjacent pair of obstacles in each of said series of rows is separated by a distance greater than 50 times said minimum diameter.
  • This embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to motion of said at least one polymer, said plurality of obstacles decreasing in size along said flow direction.
  • This embodiment of the present invention is also useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching DNA comprising an elongation structure, wherein said elongation structure comprises a tapered central channel, said tapered central channel comprising a first end and a second end, and wherein said DNA, when present, moves along said tapered central channel in a direction from said first end to said second end, wherein said elongation further comprises a plurality of side channels connected to said tapered central channel, wherein said tapered central channel comprises at least one bend; and wherein said tapered central channel comprises a plurality of obstacles to motion of said DNA.
  • the present invention relates to an integrated apparatus for stretching DNA comprising an elongation structure, said elongation structure comprising: (a) a first tapered channel, said first tapered channel comprising a first end, a second end, and a plurality of posts between said first end and said second end in a staggered arrangement comprising a number of rows between 12 and 15, said first tapered channel decreasing in width at an angle of 26.6°, said angle being defined at said first end with respect to a constant-width channel, said first end having a width between 0.5 and 5 ⁇ m, said posts having a cross sectional area equal to 1.5 ⁇ m 2 and separated by a gap equal to 0.5 ⁇ m; and (b) a second tapered channel, said second tapered channel connected to said first tapered channel at said second end and decreasing in width such that a shear force producing a constant shear rate is applied to said DNA, when present, to a width between 0.5 and 5 ⁇ m, said second tapered channel having a length
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a tapered channel with a first end and a second end; and (b) moving said at least one polymer along said tapered channel from said first end to said second end, whereby said tapered channel causes a shear force that produces a constant shear rate to be applied to said at least one polymer as said at least one polymer moves along said tapered channel.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a linearly tapered channel with a first end and a second end; and (b) moving said at least one polymer along said tapered channel from said first end to said second end.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a tapered channel with a first end and a second end, said tapered channel decreasing at a greater than linear rate from said first end to said second end; and (b) moving said at least one polymer along said tapered channel from said first end to said second end.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a central channel holding fluid and a plurality of side channels holding fluid connected to said central channel, said central channel comprising a first end and a second end; and (b) moving said at least one polymer along said central channel from said first end to said second end.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a channel with at least one bend, said channel comprising a first end and a second end; and (b) moving said at least one polymer along said channel from said first end to said second end.
  • the method encompassed by this embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a channel and a plurality of obstacles to motion of said at least one polymer within said channel, said central channel comprising a first end and a second end; and (b) moving said at least one polymer along said channel from said first end to said second end, wherein said plurality of obstacles to motion decrease in size along a direction from said first end to said second end.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising a channel and a plurality of obstacles to motion of said at least one polymer within said channel, said central channel comprising a first end and a second end; and (b) moving said at least one polymer along said channel from said first end to said second end, wherein at least one of said obstacles has a non-quadrilateral polygonal cross-sectional shape.
  • the method encompassed by this embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to a method for stretching at least one polymer comprising the steps of: (a) delivering said at least one polymer to an elongation structure, said elongation structure comprising: (i) a tapered central channel with at least one bend, said tapered central channel comprising a first end and a second end; (ii) a plurality of side channels connected to said tapered central channel; and (hi) a plurality of obstacles to motion of said at least one polymer within said tapered central channel; and (b) moving said at least one polymer along said central channel from said first end to said second end.
  • the method encompassed by this embodiment of the present invention is useful for stretching polymers, particularly DNA, for further analysis.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, wherein said elongation structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises at least one step that decreases the depth, z, of the channel from a first end to a second end.
  • the present invention relates to an integrated apparatus comprising an elongation structure comprising a channel, said channel comprising at least one step that decreases the depth, z, of said channel from a first end to a second end, said channel comprising at least one polymer in a fluid sample, said channel being configured such that a shear force is applied to said at least one polymer as it moves in a direction from said first end to said second end.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, said elongation structure comprising: (a) a first channel, said first channel comprising a first end and a second end; and (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, along which said at least one polymer, when present, moves in a flow direction, and wherein said first channel decreases in width from said first end to said second end at a rate different from the rate at which said second channel decreases in width from said third end to said fourth end.
  • the present invention relates to an integrated apparatus for stretching at least one polymer in a fluid sample comprising an elongation structure, said elongation structure comprising: (a) a first channel having a width equal to 10 ⁇ m and a depth equal to 1 ⁇ m, said first channel comprising a first end, a second end, and a plurality of posts between said first end and said second end in a staggered arrangement comprising between at least 12 to 15 rows, said plurality of posts terminating at said second end and each post in said plurality of posts having a cross-sectional area of between 1 ⁇ m 2 and 25 ⁇ m 2 ; and (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width at a rate of 1/x 2 from said third end to said fourth end, said total width decreasing from 10 ⁇ m to 1 ⁇ m, wherein x is the distance along the length of said second
  • the present invention relates to an integrated apparatus for selectively stretching at least one polymer in a fluid sample on the basis of length, comprising an elongation structure, wherein said elongation structure comprises: (a) a first channel, said first channel comprising a first end, a second end, and a plurality of posts in a staggered arrangement between said first end and said second end, each post in said plurality of posts being situated at a distance no less than L from said second end; and (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end, along which said at least one polymer, when present, moves in a flow direction.
  • the present invention relates to an integrated apparatus for stretching a plurality of polymers having varying lengths in a fluid sample, comprising an elongation structure, wherein said elongation structure comprises: (a) a first channel, said first channel comprising a first end and a second end; (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end; and (c) a plurality of posts in a staggered arrangement in said first channel and said second channel, along which said plurality of polymers, when present, move in a flow direction.
  • the present invention relates to a method for stretching at least one polymer, comprising moving said at least one polymer along an elongation structure, said elongation structure comprising a first channel, said first channel comprising a first end and a second end, and a second channel, said second channel comprising a third end and a fourth end, said third end connected to said first channel at said second end, wherein said first channel decreases in width from said first end to said second end at a rate different from the rate at which said second channel decreases in width from said third end to said fourth end.
  • the present invention relates to a method for stretching at least one polymer having a length greater than or equal to L in a fluid sample comprising moving said at least one polymer along an elongation structure, said elongation structure comprising a first channel, said first channel comprising a first end, a second end, and a plurality of posts in a staggered arrangement between said first end and said second end, each post in said plurality of posts being situated at a distance L from said second end, and a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end, wherein a polymer having a length greater than or equal to L is stretched and a polymer having a length less than L is not stretched.
  • the present invention relates to a method for stretching a plurality of polymers having varying lengths in a fluid sample comprising moving said plurality of polymers along an elongation structure, said elongation structure comprising: (a) a first channel, said first channel comprising a first end and a second end; (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width
  • the present invention relates to a method for stretching at least one polymer, comprising moving said at least one polymer along an elongation structure, said elongation structure comprising: (a) a first channel having a width equal to
  • said first channel comprising a first end, a second end, and a plurality of posts between said first end and said second end in a staggered arrangement comprising between at least 12 to 15 rows, said plurality of posts terminating at said second end and each post in said plurality of posts having a cross-sectional area of between 1 ⁇ m 2 and 25 ⁇ m 2 ; and (b) a second channel, said second channel comprising a
  • FIG. 1 shows examples of various structures that fall within the scope of the invention.
  • FIG. 2(a-m) shows (a) several embodiments of stretching structures involving funnels, posts, branches, and serial structures; (b) an enlarged example of two-funnel structures with posts in serial; (c) several embodiments of complex post anangements and branched structures; (d) an embodiment of a structure containing serial and parallel structures; (e) an asymmetric branched structure; (f) an structure having a combination of small obstacles 0 which define small gaps; (g) a structure having a combination of polygons, bars, and posts; (h) an asymmetric bent structure; (i) an enlarged view of a branched structure having posts; (j) a large funnel structure with support posts; (k) a funnel structure with posts; (1) funnel structures with a linear increase in flow rate with and without posts; and (m) a summary of some of the funnel structures encompassed by the present invention.
  • FIG. 3 shows an embodiment of the shear-stretching regime using a constantly-tapered channel.
  • FIG. 4 shows an embodiment of the shear-stretching regime in which the shear rate drastically increases as flow proceeds down the length of the channel.
  • FIG. 5 shows an embodiment of the shear-stretching regime using a tapered channel designed to impart a constant shear force.
  • FIG. 6 shows an embodiment of the shear-stretching regime in which the shear comes from the addition of fluid from side channels.
  • FIG. 7 shows how shear force is imparted in a narrowing channel, with local components of rotational and extensional force nearly equal; (b) shows how shear force is imparted when addition of fluid creates the force, with extensional force exceeding the rotational force.
  • FIG. 8 shows an embodiment of the shear-stretching regime in which shear comes from both a narrowing channel and the presence of side channels.
  • FIG. 9 shows the "racetrack effect” of fluid on the outside of a bend taking longer to pass around the corner than fluid on the inside; (b) shows how the "racetrack effect” can lead to the uncoiling of a polymer in a bend.
  • FIG. 10 displays an embodiment of the tortuosity regime, in which the channels follow a sine wave shape.
  • FIG. 11 displays an embodiment of the tortuosity regime in which the channels follow a zig-zag shape.
  • FIG. 12 displays an embodiment of the tortuosity regime in which the channels follow right angles in a "snake" shape.
  • FIG. 13 shows how a tortuous channel can be used for multiple detection of the same polymer as it travels down a channel.
  • FIG. 14 shows how a polymer can stretch in an embodiment of the obstacle field regime with gradated sizing of obstacles.
  • FIG 15 shows the coordinate frame for an elongation structure.
  • FIG. 16 (a) shows an embodiment of the obstacle field regime with square-grid alignment of circular obstacles; (b) shows an embodiment of the obstacle field regime with an offset- grid alignment of circular obstacles.
  • FIG. 17 shows an embodiment of the obstacle field regime with close spacing of rectangular obstacles of an exaggerated aspect ratio.
  • FIG. 18 shows an embodiment of the obstacle field regime with close spacing of circular obstacles.
  • FIG. 19 shows an embodiment of the obstacle field regime with three gradated sizes of circular obstacles.
  • FIG. 20 shows a configuration for consistent unraveling, delivery, and stretching of DNA of varying sizes.
  • FIG. 21 shows a configuration of a preferred embodiment of a structure for stretching DNA that combines a post field, a funnel that tapers as 1/x 2 , wherein x is the distance along the length of the funnel, and a step that reduces the channel depth.
  • FIG. 22 shows a schematic of a molecular size sorting device, wherein signals of molecules of length L or greater can be easily distinguished from signals of molecules of length less than L.
  • FIG. 23 shows a schematic of a device that stretches molecules of all lengths, such that signals from all of them are uniformly detected.
  • FIG. 24 shows a sensitive optical apparatus that utilizes confocal fluorescence illumination and detection.
  • FIG. 25 demonstrates one embodiment of the overall polymer analysis system.
  • FIG. 26 shows DNA in various stretching states in the entrance to a constant-shear channel.
  • FIG. 27 (a-g) show a 50 kb DNA being stretched out in a tapered channel.
  • FIG. 28 shows a DNA measured at 537 kb stretched out in a channel.
  • FIG. 29 shows a histogram displaying experimentally determined DNA lengths.
  • FIG. 30 shows histograms of experimentally determined lengths of phage lambda DNA from the structure of FIG. 20 (a) without posts, and (b) with posts.
  • the present invention provides structures that allow polymers of any length, including nucleic acids containing entire genomes, to be stretched into a long, linear conformation for further analysis.
  • Polymers are loaded into a device and run through the structures, propelled by, inter alia, physical, electrical or chemical forces. Stretching is achieved by, e.g., applying shear forces as the polymer passes through the structures, placing obstacles in the path of the polymer, or a combination thereof. Because the forces are applied continuously, it is possible to stretch out polymers to a length that is equal to or greater than the active area of the apparatus, i.e., where information about the polymer is collected as the polymer is analyzed.
  • Extended polymers or ensembles of polymers are characterized. Extended, labeled polymers are moved past at least one station, at which labeled units of the polymers interact with the station to produce an object-dependent impulse.
  • moved past refers to embodiments in which the station is stationary and the extended polymer is in motion, the station is in motion and the extended polymer is stationary, and the station and extended polymer are both in motion.
  • the polymers may be used for characterizing any polymer, it is preferable that the polymers have a predominantly, though not necessarily exclusively, linear or single-chain arrangement.
  • examples of such polymers include biological polymers such as deoxyribonucleic acids, ribonucleic acids, polypeptides, and oligosaccharides.
  • the polymers may be heterogeneous in backbone composition, thereby containing any possible combination of individual monomer units linked together, e.g., peptide-nucleic acids (PNA), which have amino acids linked to nucleic acids.
  • PNA peptide-nucleic acids
  • the polymers are homogeneous in backbone composition and are, e.g., nucleic acids, polypeptides or oligosaccharides.
  • a nucleic acid as used herein is a biopolymer comprised of nucleotides, such as deoxyribose nucleic acid (DNA) or ribose nucleic acid (RNA).
  • a protein or polypeptide as used herein is a biopolymer comprised of amino acids.
  • the extended object is a double-stranded DNA molecule.
  • linked means two units are joined to each other by any physicochemical means. Any linkage known to those of ordinary skill in the art, covalent or non-covalent, is embraced. Natural linkages, e.g., amide, ester, and thioester linkages, which are those ordinarily found in nature to connect the individual units of a particular polymer, are most common. However, the individual units of a polymer stretched by the structures of the invention may be joined by synthetic or modified linkages.
  • a polymer is made up of a plurality of individual units, which are building blocks or monomers that are linked either directly or indirectly to other building blocks or monomers to form the polymer.
  • the polymer preferably comprises at least two chemically distinct linked monomers.
  • the at least two chemically distinct linked monomers may produce or be labeled to produce different signals.
  • Different types of polymers are composed of different monomers.
  • DNA is a biopolymer comprising a deoxyribose phosphate backbone to which are attached purines and pyrimidines such as adenine, cytosine, guanine, thymine, 5-methylcytosine, 2-aminopurine, hypoxantine, and other naturally and non- naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.
  • RNA is a biopolymer comprising a ribose phosphate backbone to which are attached purines and pyrimidines such as those described for DNA but wherein uracil is substituted for thymidine.
  • Deoxyribonucleotides may be joined to one another via an ester linkage through the 5' or 3' hydroxyl groups to form the DNA polymer. Ribonucleotides may be joined to one another via an ester linkage through the 5', 3' or 2' hydroxyl groups. Alternatively, DNA or RNA units having a 5', 3' or 2' amino group may be joined via an amide linkage to other units of the polymer.
  • the polymers may be naturally-occurring or non-naturally occurring polymers.
  • Polymers can be isolated, e.g., from natural sources using biochemical purification techniques.
  • polymers may be synthesized, e.g., enzymatically by in vitro amplification using the polymerase chain reaction (PCR), by chemical synthesis, or by recombinant techniques.
  • PCR polymerase chain reaction
  • object-dependent impulse is a detectable physical quantity which transmits or conveys information about the structural characteristics of at least one unit- specific marker of an extended polymer.
  • a unit-specific marker as used herein, can either be a measurable intrinsic property of a particular type of individual unit of the extended polymer, e.g., the distinct absorption maxima of the naturally occurring nucleobases of o DNA (the polymer is intrinsically labeled), or a compound having a measurable property that is specifically associated with one or more individual units of a polymer (the polymer is extrinsically labeled).
  • a unit-specific marker of an extrinsically labeled polymer may be a particular fluorescent dye with which all nucleobases of a particular type, e.g., all thymine nucleobases, in a DNA strand are labeled.
  • a unit-specific marker of an 5 extrinsically labeled polymer may be a fluorescently labeled oligonucleotide of defined length and sequence that hybridizes to and therefore "marks" the complementary sequence present in a target DNA.
  • Unit-specific markers may further include, but are not limited to, sequence specific major or minor groove binders and intercalators, sequence-specific DNA or peptide binding proteins, sequence specific PNAs, etc.
  • the detectable physical quantity may be in any form that is capable of being measured.
  • the detectable physical quantity may be electromagnetic radiation, chemical conductance, radioactivity, etc.
  • the object-dependent impulse may arise from energy transfer, directed excitation, quenching, changes in conductance (resistance), or any other physical changes.
  • the object-dependent impulse arises from fluorescence resonance energy transfer ("FRET") between the unit-specific marker and the station, or the environment surrounding the station.
  • FRET fluorescence resonance energy transfer
  • the object-dependent impulse results from direct excitation in a confined or localized region, or epiillumination of a confocal volume or slit- based excitation is used.
  • Possible analyses of polymers include, but are not limited to: determination of polymer length, determination of polymer sequence, determination of polymer velocity, determination of the degree of identity of two polymers, determination of characteristic patterns of unit-specific markers of a polymer to produce a "fingerprint", and characterization of a heterogeneous population of polymers using a statistical distribution of unit-specific markers within a sample population.
  • channels are designed to provide a constant shear force on the polymer in a narrowing channel, causing it to remain in a stretched conformation.
  • a constant shear rate, or change in average velocity with distance in the channel is defined as S:
  • x is the distance down a substantially rectangular channel
  • u is the average fluid velocity in the x direction, which is computed from the overall fluid flow (Q) and the cross- sectional area, A, of the channel as follows:
  • L is the length of the channel wall, approximately the length of the channel in which the constant shear is maintained.
  • an aqueous channel with 1 ⁇ m depth, 1 mm length, and shear rate of 0.25/s gives a force of approximately 0.25 pN, adequate to stretch DNA, which the inventors have verified experimentally. Notably, this result confirms that the constant-shear channel not only maintains the extension of previously-stretched DNA, but also contributes to further stretching of DNA, or stretches DNA on its own.
  • the two general methods for achieving stretching have been combined.
  • Gradated arrays of obstacles that are posts have been placed in structures which also impart shear forces on passing molecules, ensuring not only the initial stretching of the polymer by the obstacles, but also the maintenance of stretching after the polymer has traversed the obstacles.
  • the structures for stretching DNA of the present invention comprise two components: a delivery region and a region of polymer elongation.
  • the delivery region is a wider channel that leads into and out of the region of polymer elongation.
  • the region of elongation comprises at least one of four main components: (1) funnels; (2) structures having branched channels; (3) channels with bends or curves; and (4) obstacles defining small gaps, wherein the obstacles can be, inter alia, posts or steps.
  • the invention encompasses combinations of the four main components and variations of the main components themselves.
  • a combination of two or more of the main component features can give rise to additional designs that work well to extend and stretch polymers, particularly DNA, in a controllable fashion.
  • several of the same design may be repeated in parallel or in series.
  • FIG. 1 Examples of structures (FIG. 1) that fall within the scope of the invention include, but are not limited to: i) funnels with a non-linear increase in fluid velocity; ii) funnels with a linear increase in fluid velocity; iii) funnels with obstacles defining small gaps as the region of DNA elongation; iv) funnels with a non-linear increase in fluid velocity and obstacles defining small gaps; v) funnels with a linear increase in fluid velocity and obstacles defining small gaps; vi) funnels with mixed obstacle sizes and gaps, including a gradient of obstacles sizes and gaps; vii) branched structures having regions of increased fluid velocity from converging channels; viii) branched structures having multiple regions of increased fluid velocity from multiple converging channels; ix) branched structures having obstacles defining small gaps; x) branched structures which have at least one funnel as one of the branches; xi) branched structures with mixed obstacle sizes and gaps, including a gradient of obstacle sizes and gaps; xii) structures which have
  • FIG. 2(a-l) Further examples of structures that fall within the scope of the invention are shown in FIG. 2(a-l). These include, several embodiments of stretching structures involving funnels, obstacles, branches, and serial structures; two funnel structures with posts in serial; embodiments of several complex post arrangements and branched structures; an asymmetric branched structure; a structure with a combination of small obstacles that define small gaps; a structure with a combination of polygonal, bar, and post obstacles; an asymmetric bent structure; a branched structure having posts; a large funnel structure with support posts; a funnel structure with posts; funnel structures with a linear increase in flow rate both with and without posts.
  • FIG. 2(m) is a summary of some of the possible funnel structures.
  • the elongation structures of the invention can have lengths of from 1 ⁇ m to 2 cm, preferably from 1 ⁇ m to 1 mm, widths of from 2 ⁇ m to 1 mm, and depths of from 0.1 ⁇ m to 10 ⁇ m.
  • Funnel structures are tapered channels that apply shear forces in a regular and continuous manner as the polymer flows down the channel.
  • the particular shear forces are defined by the type of channel structure and shape.
  • the channel is a tapered channel (FIG. 3) that begins at a given width and continuously decreases to a second width, creating an increasing shear force in the funnel portion of the channel defined by:
  • du/dx (-Q/H)(dW/dx)(l/W 2 ) (13)
  • the width decreases linearly so that dW/dx is constant; in this embodiment, the shear, du/dx, thus increases as W decreases.
  • the angle of the funnel as measured from the continuation of a straight wall is preferably between 1 ° and 75°, with a most preferred value of 26.6° for DNA in a low viscosity solution such as TE (10 mM TRIS, 1 raM EDTA) buffer, pH 8.0.
  • Starting widths for the linear funnel embodiment preferably range from 1 micron to 1 cm, with ending widths preferably in the range of 1 nm to 1 mm depending on the polymer in question, with most- preferred values of 50 microns and 5 microns, respectively, for DNA.
  • the channel could also be configured such that the width decreases at an increasing rate as fluid passes down the channel (FIG. 4), resulting in an increase in shear as the channel is traversed.
  • Such tapers offer especially good protection against natural relaxation of the polymer, since as time passes and the molecules move down the channel, they face increasing counter-forces to their tendency to recoil.
  • the increasing force taper allows some design flexibility; any polymer that will encounter shear forces large enough to cause the polymer to stretch in the taper and will not encounter shear forces large enough to cause the polymer to break in the taper can be successfully run through the taper and stretched. There is no need to find the ideal or threshold force for the polymer, only an effective range.
  • the width of the funnel, W decreases as V(ax n +b), where n is any real number greater than 1 , a is a nonzero real number, b is a real number, and x is distance along the length of the funnel (and the direction of polymer flow).
  • channels are designed such that the shear rate is constant, leading to a tapered channel such as that shown in FIG. 5.
  • the value of the constant shear rate required to achieve an adequate force to completely stretch the polymer over the course of the channel will vary based on the length of that channel (refer to Eq. (12)). Therefore, 0.01/s might be a reasonable shear rate in order to completely stretch a polymer in a very long, e.g. > 1 cm, channel, but might result in almost no polymer stretching in a very short, e.g. ⁇ 10 ⁇ m, channel.
  • Lengths of channels may vary significantly, with preferred values from 10 ⁇ m to 1 cm and the most preferred values in the range of 1 - 2 mm. In one embodiment, the channel is 1 mm long and the shear rate is 0.075/s.
  • the shear rate of the funnel can be determined by measuring the distance between two known points on a strand of DNA.
  • concatamers of ⁇ DNA are used as standards for shear force measurements.
  • a unique sequence on each concatamer is fluorescently tagged with a hybridization probe.
  • the interprobe distance on the concatamer is thus the length of a single ⁇ DNA molecule (48 kilobases).
  • the physical distance between the probes is determined using video microscopy or time-of-flight measurements.
  • the physical distance for ⁇ DNA in native solution is 14.1 ⁇ m. This value is compared with the actual measured physical distance. For instance, if the measured distance is 15.0 ⁇ m, then the shear rate can be calculated from the amount of stretching that the DNA has experienced in the stretching structures.
  • the predicted shear on the DNA as measured by the velocity of the DNA and the dimensions of the channel (see Equation 10), is matched with the elongation of the DNA and its intrinsic non- linear stiffness.
  • Branched channels A second aspect of the invention used to stretch and elongate polymers is to create branched structures, which cause either changes in fluid flow rates or changes in polymer directionality (see below in Structures with bends or curves). Side channels feed more fluid into a main channel, resulting in a change in fluid velocity and hence causing polymer stretching.
  • a typical arrangement of branched channels is shown in FIG. 6. Side channels preferably have a combined cross-sectional area ranging from about 1% to 500% of the cross-sectional area of the main channel. Most preferably, side channels have a combined cross-sectional area of about 50% of the cross-sectional area of the main channel.
  • the side channels are present in a pattern that is repeated, which results in a dilution of the shear force at each individual entrance to the main channel and, hence, a closer approximation of a constant-shear situation.
  • This arrangement highlights the advantages and disadvantages of the side channels.
  • One disadvantage of this component of polymer elongation is that all of the force on the main channel fluid is dissipated in a relatively small region near the junction of the main channel and the side channels. Therefore, this configuration does not lead to a constant-force situation.
  • an advantage of this component of polymer elongation is that, because the additional fluid in the side channels is moving in the same direction as the fluid in the main channel, the force is not purely shear force, but has a substantial extensional flow component.
  • Pure shear which is the force exerted by a tapered funnel on a polymer, is a superposition of extensional forces and rotational forces as shown in FIG. 7(a).
  • the extensional force on a polymer accelerates it in the direction of the fluid flow, such that the portion of the polymer located in the region of extensional flow moves faster than the portion still located in a more stagnant region, stretching out the polymer.
  • the rotational force causes the polymer to spin or "tumble" in conformation, which can cause stretched portions of the polymer to fold up on themselves and recoil.
  • the polymer tends to accelerate away from the junction, which results in lower rotational forces, thus allowing for better stretching.
  • the channel dimensions may be modified and the flow rate increased in the same region of the chip.
  • a significant increase in the flow rate followed by a constant-shear section is one way not only to stretch out a polymer, but also to direct it away from the walls of the channel.
  • One arcangement embracing this embodiment of the invention is shown in FIG. 8.
  • additional flow is brought in only from one side of the main channel, thereby positioning a polymer traveling down the main channel toward one side. This positioning design could be used to ensure that a polymer is aligned to pass under a narrow detector in a broader channel.
  • the third aspect of the invention uses tortuosity to achieve stretching. As fluid flow encounters changes in its path, alignments ranging from a small bend to a right angle, the fluid on the outside of the curve or corner will take longer to go around the turn than the fluid on the inside of the curve or corner (FIG. 9(a)). This so- called “racetrack effect” can help stretch out polymers. Such a bend does not include a "T" junction. In a rectangular section of a channel, a polymer may flow such that it straddles more than one fluid flow line, and since the fluid in each line travels at the same velocity, it retains its configuration.
  • One of the gentler incarnations of the tortuosity regime is an embodiment where the configuration of the channel follows a sine wave pattern (FIG. 10).
  • the channel takes the form of a zig-zag shape (FIG. 11), or, in yet a further embodiment, even a "snake"-shape with only right-angle corners (FIG. 12), though this severe of a corner tends to cause stagnant flows and other undesirable fluid dynamics.
  • each bend preferably has an angle between 5 ° and 75°; for DNA a preferred value of every such angle is 26.6 (effectively a 53.4° angle where the zig-zag reverses).
  • Such zig-zag shapes may be periodic, in which the angle of the bends is always the same, or may comprise a pattern of differential bends.
  • the period of repetition for the zig-zags may vary from as little as 2 ⁇ m to 1 cm, with preferred values of 20-50 ⁇ m for DNA (1000 times the persistence length).
  • the amplitude to period ratios are preferably between 0.01 and 5.
  • the number of periods for any of these patterns may vary from 1 period to 500, with a preferred value of 10.
  • tortuous channels are used to create multiple detection possibilities.
  • a detector such as a position-dependent photomultiplier tube arranged in a 1 x 256 array
  • the tortuous channel can be aligned so that it repeatedly crosses the detection zone at defined locations.
  • the polymer being stretched is then observed at several locations, creating redundancy and error checking in the system.
  • Such an arcangement is shown in Fig. 13, with fluid traveling down channel 111 passing through detection zone 110 at six locations, 112-117.
  • the fourth aspect of structures which tend to cause stretching is the field of obstacles.
  • obstacles induce stretching both by reducing the available cross-sectional area of the channel (causing local strain on the molecules) and by acting as physical barriers which cannot be passed by large coils of polymer.
  • One example is a configuration of posts that work to actually stretch a polymer and is shown in FIG. 14.
  • the obstacles can vary in cross-sectional shape and in cross-sectional area.
  • cross-sectional shape and “cross-sectional area,” as used herein with reference to obstacles, and unless otherwise indicated, refer to the shape of the X-Y projection and the area of the X-Y plane of the obstacle, respectively, as shown in FIG. 15.
  • the obstacles comprise square posts, round posts, elliptical posts or posts with a rectangular cross-section of any aspect ratio (including extremely long "bars"); in other embodiments, the obstacles comprise posts with a cross-section shaped as a regular or irregular non-quadrilateral polygon.
  • the cross-sectional shape is triangular. In other preferred embodiments, these shapes are modified to have a concave edge on the edge that faces the direction from which the fluid is coming (such as a shallow U-shape).
  • posts having a cross-sectional shape wherein one dimension is longer than the other preferably have an aspect ratio of 2 to 20, more preferably of 2 to 5.
  • Each of these obstacles may be placed at any angle to the direction of flow.
  • the obstacles are aligned with either a flat surface perpendicular to the direction of the flow, or at a 45° angle to the flow, though if preferential positioning of the polymer molecules is desired, other angles which physically direct polymers toward a destination would be used.
  • obstacles wherein one dimension is longer than the other are placed with their longer dimension perpendicular to the flow direction.
  • Another factor in the layout of the obstacles is the grid on which they are placed. If placed on a repeating square matrix (FIG. 16(a)), certain fluid flow lines are almost unaffected by the obstacles, and unstretched or poorly stretched polymers may be able to track along these flow lines and make it through the obstacle field without being stretched.
  • each successive column is preferably offset to place the next obstacle where the gap in the previous column had been (FIG. 16(b)), forcing all flow lines to have curvature and inducing stretching on all passing molecules.
  • the offset may also be less than the full 50% of the repetition unit so that every other column is not in the same alignment as shown in the figures; every fourth or sixth column may have an identical alignment, for example, or there may never be a repetition of alignment, as long as the flow lines at some point are forced to curve around an obstacle.
  • the width of the passages between obstacles should not be smaller than the diameter of the stretched polymer, and is preferably not less than approximately 50 times the diameter of the stretched polymer in order to increase the probability that the polymer will be able to pass through the channel without becoming stuck in the obstacle field.
  • An example of inadequate passage width leading to polymers not getting through the obstacles is shown in FIG. 17.
  • the passages are preferably not as wide as the diameter of the coiled polymer, in which case the coil could pass through the obstacle field without having to stretch at all.
  • the preferred spacing of the obstacles is highly dependent on the polymer being analyzed.
  • the passage width is preferably between 100 nm and 800 nm, with a most preferred value equal to 500 nm.
  • gels may be used in place of obstacle fields, giving pore sizes (equivalent to passage width in the fields) of 1 nm to 1000 nm.
  • the total Y-Z cross-sectional area occupied by the obstacles most directly impacts the velocity gradients that occur in between the obstacles, and which encourage stretching.
  • a larger ratio of obstacle Y-Z cross-sectional area to total channel Y-Z cross-sectional area also known as the fill ratio, which when expressed as a percentage is given by 100 multiplied by the ratio of the total area of the posts to the total area of the channel
  • the fill ratio is preferably between 33% and 95%.
  • a post having a 1 ⁇ m 2 Y-Z cross-sectional area in a channel having a 3 ⁇ m 2 Y-Z cross-sectional area has a fill ratio of 33%
  • a 20 ⁇ m 2 post in a 21 ⁇ m 2 channel has a fill ratio of 95%.
  • the most preferred value for the fill ratio is between 50% and 80% for DNA.
  • differential passage widths are used in some embodiments of the invention. In some embodiments, this is accomplished by varying the size of the obstacles. In other embodiments, this is accomplished by varying the fill ratio. In still other embodiments, both obstacle size and fill ratio are varied. In such embodiments, polymers first encounter wide passages between obstacles and subsequently encounter passages of decreasing widths (FIG. 19), forcing them to gradually become more elongated in order to proceed down the smaller channels. In a preferced embodiment, passage widths are gradated from about 5 ⁇ m per passage to about 1 ⁇ m per passage in the flow direction.
  • ppst sizes are gradated from a cross-sectional area of about 10 ⁇ m 2 to about 1 ⁇ m 2 in the flow direction.
  • the obstacle cross-sectional area and passage width may be varied individually to achieve similar effects, i.e., the obstacle size may change and the passage size may remain constant, or the passage size may change and the obstacle size may remain constant.
  • all obstacles have the same cross-sectional area, but the fill ratio increases in the flow direction.
  • the cross-sectional area of the posts can vary from 0.1 ⁇ m 2 to 1 mm 2 , preferably from 0.1 ⁇ m 2 to 10 ⁇ m 2 , more preferably from 1 ⁇ m 2 to 100 ⁇ m 2 , even more preferably from 1 ⁇ m 2 to 25 ⁇ m 2 , depending on the size of the polymer being stretched and the size of the channel used.
  • Such pre-alignment of polymers serves to decrease the possibility of entanglement and hence provides more predictable stretching.
  • Obstacles can also be fabricated into the depth or z-dimension of the structures, i.e., by introducing "steps" into the top and/or bottom of the channel to decrease the depth. Instead of having obstacles placed across a channel, as discussed above, the entire channel can change in depth, providing the same kind of barrier and shear forces around the barrier as obstacles placed along the width of the channel. Furthermore, changes in depth can be relatively inexpensive to implement, as controlling the depth of etching on the sub-micron scale is generally easier than trying to create feature sizes on the sub-micron scale using photolithography. Without being bound by any theory, a significant change in depth at a specific location in essence creates the same effect as a single row of posts, or as a funnel of infinitely short length, x.
  • the height change can be designed to occur in several steps along the length of the channel, instead of in one step at a single location.
  • a single-step configuration reduces the height of the channel by a factor of five.
  • a configuration having at least one step reduces the height of the channel from by about a factor of 2 to by about a factor of 100.
  • the steps vary in height from about 0.1 ⁇ m to about 0.9 ⁇ m.
  • the three general aspects of structures, shear-inducing (i.e. , tapered and branched channels), tortuous, and obstacle-filled, are used in combination.
  • the constant-shear tapered channel for example, is good not only at stretching in itself, but in maintaining stretching in polymers that have already been stretched by obstacle fields.
  • a channel with a tortuous contour can also shrink in width following a constant-shear pattern to capitalize on both effects.
  • a gradated obstacle field or alignment structure is used to pre- stretch the polymer, followed by a section of fine obstacles, tortuous patterns, or high shear area to complete the stretching, and a constant-shear or increasing shear section to maintain the stretching until the detection point is reached.
  • an especially effective structure is a combination of an obstacle field upstream of a tapered channel.
  • the obstacle field serves to uncoil the DNA from its random coil configuration, presenting one end of the molecule preferentially to the downstream structure(s). It is advantageous for the obstacle field to be in a wide region of the channel where the flow velocity is relatively low such that the drag force applied to a molecule that becomes folded around or otherwise retained by one of the obstacles is not sufficient to break the molecule. As the molecule winds through the obstacle field, one end will tend to lead the rest of the molecule and enter the tapered channel first. The molecule will then be further stretched by the shear force of the flow through the tapered channel.
  • an obstacle field, step or alignment structure is used to pre-stretch and align the polymer, followed by a section of constant or increasing shear or elongation to complete and maintain the stretching until the detection region is reached.
  • the obstacle field is matched with a tapered channel in a way that avoids contractile flow (i.e., decreasing velocity).
  • posts or steps are located in or terminate at a tapered portion of the channel.
  • the channel is a two-funnel structure, that is, it has two tandem regions with different degrees of tapering.
  • An example of a two-funnel structure is shown in FIG. 20.
  • the two-funnel structure further comprises a post field in the first tapered region. In the two-funnel configuration, stretching of the polymer is completed in the second tapered region (right-most channel region in FIG. 20). Pressure driven flow is the preferced driving force because of its simplicity and ease of application.
  • the structure has a first channel region with a constant width of about 10 ⁇ m and a height of about 1 ⁇ m in which is placed an obstacle field along the flow direction and leading into a second channel region that is a funnel whose width tapers as 1/x 2 , from a width of about 10 ⁇ m to about 1 ⁇ m, and whose height is reduced in a single step at the entrance to the funnel from about 1 ⁇ m to about 0.25 ⁇ m (FIG. 21).
  • the ratio of the initial channel width to the final channel width is preferably greater than 10, and the length of the funnel portion is preferably less than one-half the initial width.
  • the obstacle field preferably comprises at least between 12 and 15 rows of posts having a cross-sectional area substantially equal to 1 ⁇ m, wherein the rows have an increasing fill ratio in the flow direction.
  • the rows have an increasing AH ratio from 0% to 50% in the flow direction
  • the subsequent 12-15 rows have a constant fill ratio of 50%o, wherein the centers adjacent rows of the subsequent 12-15 rows are at a distance of about 2 ⁇ m (FIG. 21).
  • the rows have a continuously increasing fill ratio from 0% to 80% in the flow direction.
  • post fields can be used to produce non-random alignment of polymers and to effectively separate one end of the polymer chain from the random coil that is the equilibrium structure of the polymer in solution. If a post field is placed at a distance L from the mouth of a tapered channel, which can be of any shape desired to maintain or produce stretching, e.g. , straight, constant shear, or higher order polynomial, the resulting structure can also be used to select molecules by length. This process is illustrated in FIG. 22.
  • FIG. 22 shows a schematic view of a post field constructed according to the methods described below (see Methods of fabricating structures ' ), positioned before a funnel region of shear or elongational flow. Because the posts fill a portion of the channel, fluid moving through the channel will experience a decrease in velocity as it moves from the post region into the straight section of the channel. This decrease in velocity produces a contracting flow, i.e., the polymer will re-coil in the region of decreased fluid velocity. DNA molecules that travel along the channel and become hooked around a post will be stretched by the flow.
  • the molecule has a length equal to or longer than the distance L from the posts to the start of the tapered region, it will be released from the post field into the region of elongational flow, in effect spanning the region of decreased fluid velocity without recoiling, and will remain stretched, as shown schematically by DNA molecule 1 in FIG. 22. If the molecule is shorter than L, e.g., DNA molecule 2 in FIG. 22, then it will leave the posts while still in the contracting flow region of the channel, where it will contract rapidly into an equilibrium coil. Therefore, a molecule having a length greater than or equal to L will be stretched and a molecule having a length less than L will not be stretched. If a detector is positioned at the exit from the funnel, as shown in FIG.
  • the signals from coiled molecules (length less than L) and stretched molecules (length greater than or equal to L) will be distinguishable.
  • the detector were monitoring intercalator- stained DNA, contracted molecules would produce a short, intense pulse, whereas fully- stretched molecules would produce a long, less intense signal.
  • L the distance from the trailing end of the post field to the mouth of the tapered region, to a length that is substantially the same as the length of the molecules from which signal is to be detected.
  • Tethered stretching entails creating an unequal force distribution on one end of the molecule to create full extension in a flow profile. Tethered stretching is straightforward to create using obstacles defining small gaps.
  • Uniform stretching is more complex and involves extensive modeling of polymer dynamics. Uniform stretching is defined as creating a uniform tension over each unit of the DNA molecule. Structures which are designed to create uniform stretching include those with constant shear forces in the x-direction of the design such as funnels with non-linear increases in flow rates.
  • Polymer size considerations are such that they are scalable and some are universal. Structures can be increased in size, and the relative dimensions changed, in order to accommodate polymer molecules of different lengths. Sizes of interest range from several kilobases to at least megabases of DNA, although there is no upper limit on the length of polymer molecules that can be accommodated. One megabase of DNA has a length greater than 300 microns. Channel dimensions can be made up to several millimeters. In this manner, whole chromosomes (ranging in size from 50 - 250 megabases) can be handled and stretched.
  • the delivery channels leading to the regions of DNA elongation can include delivery channels which are parallel, radial, branched, interconnected, and closed loops. Delivery channels in the preferced embodiment are wide channels, i.e., 1 - 1000 microns, which lead to regions of DNA stretching and elongation.
  • the preferced method to fabricate the designed structures is by lithography, such as e-beam lithography, deep-uv lithography, photolithography, LIGA (acronym of the German words "Lithographie,"
  • Two and three dimensional structures are fabricated by these techniques. Further methods to create three dimensional defined channels include track- etching and molding techniques. Other methods to create nano-sized obstacles include methods that involve chemical means such as photodeposition of colloids, self-assembly of localized polymers, and cross- linked networks of polymers. For example, a non-linear funnel with localized deposition of agarose gel in the funnel can create an environment of controlled stretching.
  • Structures intended to stretch out the polymer are not the only ones which may be useful to place in a channel. Structures designed to position the polymer favorably in one part of a channel over another are useful in ensuring that the polymer is fed to a particular stretching structure or to a particular detection zone. Besides the adding of fluid to a single channel as mentioned above (see Branched channels) the positioning can also be accomplished by forcing flow lines closer together. Polymers driven by fluid flow (induced by any of the later-cited methods such as pressure differential and gravity) will principally follow the fluid flow lines (in electrophoresis for charged biopolymers, the polymer follows the field lines, which can be similarly modified). Random motion can cause portions of the chain to move to an adjacent flow line.
  • other shapes are used to help in positioning, such as cross-shaped obstacles, wedges, and obstacle fields with offsets that tend to direct larger channels at a particular side of the channel. While it might seem intuitive that a channel with a simple bend in it should have a positioning effect, the velocity gradients involved are actually quite small and the effect by itself is quite modest.
  • the effectiveness of the shear-inducing regimes is enhanced by increasing the viscosity of the solution.
  • the actual force imparted by the constriction of a channel is proportional to the viscosity of the solution.
  • the viscosity of the solution is increased by the addition of one or more viscosity-modifying components.
  • Glycerol (with a viscosity of nearly 900 cP at room temperature) can be added to an aqueous solution in concentrations as high as 70% (w/v) if it does not react chemically with the polymer.
  • Sugars, such as sucrose, xylose, and sorbitol may also be added. Water- .
  • soluble polymers such as polyethylene glycol
  • polyethylene glycol may also be added.
  • high molecular weight polyacrylamide, polyethylene oxide or long-chain length polysaccharides (even at concentrations as low as 0.01% by weight) can increase the viscosity of aqueous solutions without modifying the structure of the DNA being characterized.
  • the viscosity may also be modified by adding an amount of the polymer being characterized, but which will not be detected by the detection zones of the structures. For example, if FRET is being performed on an extrinsically labeled DNA molecule, then additional DNA molecules that are not extrinsically labeled may be added to the labeled polymer solution in order to increase the viscosity. In this way, only labeled molecules are detected and the unlabeled DNA serves only to modify the viscosity of the solution, but does not interfere with signal generation from the labeled molecules.
  • viscosity is increased by decreasing the temperature; pure water, for example, nearly doubles in viscosity as it approaches the freezing point.
  • a decrease in temperature is used to minimize Brownian motion and extend relaxation times.
  • an aqueous buffer solution such as IX TE solution (10 mM TRIS, 1 mM EDTA)
  • IX TE solution 10 mM TRIS, 1 mM EDTA
  • the driving force for moving the polymer through the structures can come from any means, including physical, electrical, thermal, or chemical forces.
  • the simplest driving force is allowing flow to be driven by capillary action as the first contact is made between the sample solution and the device. While the surface energies involved can provide a high velocity in the channel, control of the flow in this regime is limited.
  • One advantage of setting up a concentration gradient is to provide an extremely slow, steady flow rate. This is accomplished by creating a large excess of a species at one side of the structures and consuming the diffusing species after it induces fluid flow through the structures to the other side, with control based on the excess concentration. The polymer flows through the structures along with the fluid whose flow is induced by the migrating species.
  • a preferced embodiment directly controls the flow of the fluid.
  • a pressure head is established on the entrance side of the structures, encouraging the fluid to flow to the far side, opened to atmospheric pressure or maintained at reduced pressure.
  • the pressure head may come from any device imposing a physical force, such as a syringe pump.
  • syringe pumps dispense up to the 100 pL/s range, and desired flow rates in a device may be under 1 pL/s, meaning that it may be necessary to create a "bypass channel" with a large cross-sectional area, thus increasing the desired flow rate of the device and allowing control with off-the-shelf equipment, with the loss only of some volume of sample.
  • one end of the system in devices with a pressure drop of less than atmospheric pressure, one end of the system is pulled with a vacuum, literally sucking material to be stretched through the structures.
  • the pressure drop required to induce flow at a desired velocity is a function of the channel geometry (especially the minimum cross-sectional dimension) and that velocity, but is typically within an order of magnitude of 10 psi for 100 micron per second flow in a millimeter- long, micron-deep channel which is otherwise quite wide through most of the device.
  • a combination of a pressure head at a first end of the channel and a vacuum at a second end of the channel are used to propel a polymer from the first end to the second end.
  • the polymer is controlled through the fluid flow by setting up a temperature gradient on each side of the stretching zone. Natural convection then creates a fluid flow through the stretching zone. Since it is much harder to create and control temperature gradients on the micron scale on which these devices operate, this method, like the chemical potential method, is preferably used for very low fluid flow.
  • the flow of the polymer is controlled, for charged polymers such as DNA, by setting up an electric field which acts on the charges on the polymer and not necessarily on the surrounding fluid at all (if it is uncharged). The electric field is preferably established by the presence of two oppositely-charged electrodes in solution, but entire arrays of electrodes can be used to create more complicated or uniform field patterns.
  • the polymers then follow electric field lines instead of flow lines (in some instances an inconsequential change, depending on the physical layout of the chip and the charge density of the solution).
  • This can be damaging to stretching if the surrounding solution contains oppositely-charged objects which flow in the opposite direction (electro- kinetic flow), or surface charges on the wall of the channels causing flow of ions along the walls (electro-osmotic flow), either of which can induce fluid flow in that opposite direction and impart viscous forces on the polymer.
  • opposing viscous forces have negligible impact on the electrophoretic driving force, allowing the polymer to proceed through the structures and become stretched.
  • the electro-osmotic flow can be reversed to provide viscous forces which assist the electrophoretic stretching.
  • a field strength of 1000 to 2000 V/m results in usable polymer velocities in the 100 micron per second range.
  • the devices creating the driving force are generally physically separated from the stretching zone.
  • the electrodes are located several millimeters to multiple centimeters away from the stretching zone, with the power supply located even further away.
  • the syringe pump while advantageous to be as close to the stretching zone as possible to minimize the needed pressure drop, will tend to be placed outside of the device because of its bulk.
  • the substrate used is selected for compatibility with both the solutions and the conditions to be used in analysis, including but not limited to extremes of salt concentrations, acid or base concentration, temperature, electric fields, and transparence to wavelengths used for optical excitation or emission.
  • the substrate material may include those associated with the semiconductor industry, such as fused silica, quartz, silicon, or gallium arsenide, or inert polymers such as polymethylmetacrylate, polydimethylsiloxane, polytetrafluoroethylene, polycarbonate, or polyvinylchloride. Because of its transmissive properties across a wide range of wavelengths, quartz is a preferced embodiment.
  • quartz as a substrate with an aqueous solution means that the surface in contact with the solution has a positive charge.
  • a coating is applied to the surface to eliminate the interactions which lead to the charge.
  • the coating may be obtained commercially (capillary coatings by Supelco, Bellafonte PA), or it can be applied by the use of a silane with a functional group on one end. The silane end will bond effectively irreversibly with the glass, and the functional group can react further to make the desired coating.
  • a silane with polyethyleneoxide effectively prevents interaction between the polymer and the walls without further reaction, and a silane with an acrylamide group can participate in a polymerization reaction to create a polyacrylamide coating which not only does not interact with DNA, but also inhibits electro-osmotic flow during electrophoresis.
  • the channels may be constructed on the substrate by any number of techniques, many derived from the semiconductor industry, depending on the substrate selected. These techniques include, but are not limited to, photolithography, reactive ion etching, wet chemical etching, electron beam writing, laser or air ablation, LIGA, and injection molding. A variety of these techniques applied to polymer-handling chips have been discussed in the literature, including Harrison et al. (Analytical Chemistry 1992 (64) 1926-1932), Seiler et al. (Analytical Chemistry 1993 (65) 1481-1488), Woolley et al. (Proceedings of the National Academy of Sciences November 1994 (91) 11348-11352), and Jacobsen et al. (Analytical Chemistry 1995 (67) 2059-2063).
  • the velocity in a given planar height of the channel is substantially uniform in a rectangular channel. This is true when the height is significantly less than the width of the channel, such that the no-slip condition at the wall results in a viscosity-induced parabolic velocity profile that is significant in the height axis, leaving only a small boundary region of slower flow in the width axis.
  • An aspect (width/height) ratio of approximately 10 or greater is required for such embodiments, according to the lubrication theory approximation (Deen, Analysis of Transport Phenomena, New York: Oxford University Press, 1998. 275-278).
  • a small height assists in detection when using a microscope objective in an optical system.
  • Typical objectives may have a depth of focus of 500 nm to several microns, so while the depth of channel could be anywhere from 50 nm to 100 ⁇ m as long as the aspect ratio is kept above 10 to accommodate the polymer being analyzed, the preferred embodiments have channel depths of 200 nm to 1 ⁇ m such that all material passing by in a channel will be in focus and accurately observed.
  • the invention also encompasses embodiments where the channels are not planar, and are fabricated with three dimensional channel fabrication techniques.
  • constant shear is induced not only from side walls, but from a gradient in channel height.
  • combinations of structures have one force acting on one axis and the other force acting in the other.
  • an obstacle field spans the width of the channel as its height decreases in a tapered shape.
  • a tortuous, inward-spiral design in a single plane which also decreases in channel width is used to impart shear forces which feed at its center into a vertical exit from the device through a hole in the bottom of the material, with detection near the entrance to the hole.
  • gravity is used in some embodiments to help create velocity differentials in the fluid. (Notably, gravity alone is not adequate to stretch a polymer or move it significantly with a flow since the force on a 100 kD polymer is barely more than 10" 18 N; any effect of gravity will be felt by the molecule through viscous forces.) 6.
  • FIG. 24 A sensitive optical apparatus for detection is shown in FIG. 24.
  • the apparatus utilizes confocal fluorescence illumination and detection.
  • Confocal illumination allows a small optical volume (of the order of femtoliters) to be illuminated. Both Rayleigh and Raman scattering are minimized using a small probe volume.
  • the beam from a 1 mW argon ion laser is passed through a laser line filter (514 nm), directed to a dichroic mirror, through a lOOx 1.2 NA oil immersion objective, and to the sample.
  • the fluorescent tag on the DNA can be one of several dyes including Cy-3, tetramefhylrhodamine, rhodamine 6G, and Alexa 546.
  • intercalator dyes can be used such as TOTO-3 (Molecular Probes).
  • the fluorescence emission from the sample is passed through a dichroic, a narrow bandpass (e.g.. Omega Optical), focused onto a 100 ⁇ m pinhole, passed through an aspheric lens, and ultimately focused onto an avalanche photodiode in photon counting mode (EG&G Canada).
  • the output signal is collected by a multichannel scalar (EG&G) and analyzed using a Pentium III type computer.
  • the confocal apparatus is appropriate for quantitative applications involving time-of-flight. Such applications include measuring distances on the DNA, detecting tagged sequences, and determining degrees of stretching in the DNA. Single fluorescent molecules can be detected using the apparatus.
  • an apparatus using an intensified CCD (ICCD, Princeton Instruments) mounted on a microscope is appropriate.
  • a set of constant-shear channels with a design shear rate of 0.085 /s preceded by two rows of 1.5 micron obstacles on a 2 micron pitch were created in a 0.090 inch thick quartz substrate by photolithography and e-beam methods.
  • the substrate was first cleaned by placement in an RCA solution (5 parts deionized water to 1 part 30% ammonium hydroxide/30 % hydrogen peroxide, the latter two from Sigma Chemical Co., St. Louis, MO) heated to 80 °C for twenty minutes, and dried under a nitrogen stream.
  • Shipley S1813 photoresist diluted in a 2:1 ratio with type R thinner (Shipley, Newton, MA) was then spun onto the quartz surface at 3250 rpm for 45 seconds in a spin coater and cured at 90°C in an oven for 0.5 hours.
  • the coarse constant-shear pattern was then contact printed onto the surface by a 12 s exposure to a mercury lamp, e.g., in a contact aligner from Carl Zeiss, Germany, followed by a 30 s rinse under 351 developer (Shipley) diluted in a 5:1 ratio with deionized water, further rinses in deionized water, and drying under a nitrogen stream.
  • the substrate was exposed to a 40 minute etch by CHF 3 in a Reactive Ion Etch (RIE) machine.
  • RIE Reactive Ion Etch
  • a solution of polymethylmethacrylate (650 MW) diluted to 3% in chlorobenzene was spun onto the surface at 2000 rpm for 45 seconds in a spin coater.
  • the coating was cured for one hour in an oven at 180°C, and a 60 ⁇ layer of chrome was added in an evaporator.
  • An e- beam write was performed to make the fine structures, e.g., the rows of obstacles, followed by a chrome etch in the REI machine and a deionized water rinse.
  • the substrate was then immersed for 90 seconds in a 2:1 v/v solution of isopropyl alcohol :methyl-isobutyl ketone heated to 21 °C for developing, followed by another UV-ozone cleaning.
  • Another CHF 3 etch in the REI machine followed by a wash with RCA solution were then performed.
  • Cover slips (Fisher Scientific, Pittsburgh, PA) of dimensions 45 mm x 50 mm x 0.15 mm were rinsed with deionized water and dried under a nitrogen stream.
  • a slab of silicone with a hole where the chip is mounted was placed on a cover slip, which was then exposed to a 30 W plasma cleaner for 50 seconds in order to make the surface hydrophilic. The silicone slab was then removed and the cover slip was rinsed in deionized water and dried under nitrogen. The fully-prepared chip was then carefully mounted onto the cover slip.
  • the delivery system consists of a polymer supply 151, which is driven by a syringe pump 150 through a chip 152 (see above) where the polymer is stretched out and excited by a laser beam from laser 154 which is detected by optical detector 153 and analyzed by computer 155 that also controls the pump 150 and detector 153.
  • Coliphage T4 DNA (Sigma, St.
  • Emission above 520 nm was passed through the B2A filter set and captured by a silicon-intensified camera (Hammamatsu's C2400-08) or by a CCD camera.
  • the image from the camera was output to a computer through an image capture card (such as the PCI- 1408 from National Instruments, Austin, TX) and analyzed with image processing software, which was a custom-written routine that identified the DNA on the screen based on its brightness against background and counted pixels to determine polymer length.
  • FIG. 26 A DNA molecule of approximately 190 kb (63 microns) is shown stretching out in the constant- shear section of the chip in FIG. 27 (a-g). A DNA fully stretched out in the chip is shown in FIG. 28. This molecule was measured at 139 microns, or 535 kb.
  • the sample was excited with a 100 W Hg lamp and observed with a SIT camera (Hammatsu C2400-08).
  • the video signal from the camera was fed to a video capture card in a Pentium-class computer running custom Lab View software that determined the length of a piece of DNA in pixels based on its velocity and time spent in the region of interest. Lengths of less than 30 microns were considered to be fragments and were discarded automatically, which led to the obtaining of only ten data points in the approximately two minute run of sample.
  • the DNA were found to be 50.6 ⁇ m long, with a range between 42 and 62 ⁇ m.
  • a histogram is shown in FIG. 29. The length is somewhat shorter than the expected value of 71.1 ⁇ m for a stained 164 kbp T4 DNA, implying the stretching in this design was not fully complete.
  • FIG. 20 The apparatus shown in FIG. 20 was used to obtain the data shown in FIG. 30 (b).
  • the apparatus used to obtain the data in Fig. 30 (a) had the same channel boundaries as the apparatus used to obtain the data shown in FIG. 30 (b) (i.e., the ratio of the sizes of the two tapered regions of the two-funnel apparatus were identical), except that there were no posts present in the structure.
  • a fused silica wafer Hoya Corp., San Jose, CA
  • the wafer was diced into 1 cm by 2 cm chips using a dicing saw (e.g.
  • Double stranded lambda DNA (Promega, Madison, WI) having a uniform length of
  • the chip and cover slip were placed on the microscope stage of a fluorescence microscope (e.g., Microphot series from Nikon) equipped with a 100 X piano apo lens (e.g., 5 from Nikon, Carl Zeiss) and a filter set optimized for use with TOTO-3 (e.g., XF-47 from Omega Optical, Brattleboro, VT). Excitation was from a 633 nm HeNe laser (e.g., from Melles Griot) focused on two spots aligned on the same flow line within the microchannel.
  • a fluorescence microscope e.g., Microphot series from Nikon
  • a 100 X piano apo lens e.g., 5 from Nikon, Carl Zeiss
  • TOTO-3 e.g., XF-47 from Omega Optical, Brattleboro, VT.
  • Excitation was from a 633 nm HeNe laser (e.g., from Melles Griot) focused on two spots aligned on the same flow line within the
  • the sample was loaded at the entrance of the channels by capillary action and the flow sustained using a vacuum at the other end of the chip (created by a vacuum pump from, e.g., 0 Welch Vaccum, Skokie, IL).
  • a vacuum pump from, e.g., 0 Welch Vaccum, Skokie, IL.
  • emission above 650 nm was passed through the filter set and captured by a pair of confocal detectors aligned above the spot. Time of flight between the detectors was used to determine velocity, which was used along with residence time in a laser spot to calculate the lengths of the molecules.
  • the two-funnel apparatus with posts stretches DNA better than the same apparatus without posts. Furthermore, this apparatus stretches the polymers more uniformly and efficiently than the two-funnel structure without posts.

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne des structures et des procédés permettant à des polymères longueurs de quelconques, y compris des acides nucléiques contenant des génomes entiers, d'être étirés dans une configuration cylindrique, linéaire pour des analyses supplémentaires. La présente invention concerne également des structures et des procédés de sélection et d'étirage de polymères en fonction de leurs longueurs. Les polymères sont chargés dans un dispositif et circulent à travers les structures. L'étirage est, par exemple, achevé par des forces de cisaillement lorsque le polymère passe à travers les structures ou une combinaison de celles-ci, en plaçant des obstacles dans le chemin du polymère. Etant donné que de multiples molécules peuvent être étirées successivement, des capacités de criblage extrêmement hautes, à savoir le criblage de plus d'une molécule par seconde est achevé.
PCT/US2000/022253 1999-08-13 2000-08-11 Procede et appareil d'etirage de polymeres WO2001013088A1 (fr)

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Application Number Priority Date Filing Date Title
CA002381361A CA2381361A1 (fr) 1999-08-13 2000-08-11 Procede et appareil d'etirage de polymeres
IL14808800A IL148088A0 (en) 1999-08-13 2000-08-11 Methods and apparatuses for stretching polymers
EP00955515A EP1210578A4 (fr) 1999-08-13 2000-08-11 Procede et appareil d'etirage de polymeres
AU67711/00A AU6771100A (en) 1999-08-13 2000-08-11 Methods and apparatuses for stretching polymers
JP2001517140A JP2003507026A (ja) 1999-08-13 2000-08-11 ポリマーを引き伸ばす方法および装置

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US14902099P 1999-08-13 1999-08-13
US60/149,020 1999-08-13

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JP (1) JP2003507026A (fr)
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IL (1) IL148088A0 (fr)
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EP1210578A1 (fr) 2002-06-05
CA2381361A1 (fr) 2001-02-22
JP2003507026A (ja) 2003-02-25
EP1210578A4 (fr) 2007-02-21
CN1379857A (zh) 2002-11-13
IL148088A0 (en) 2002-09-12
AU6771100A (en) 2001-03-13

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