US20070090026A1 - Continuous biomolecule separation in a nanofilter - Google Patents

Continuous biomolecule separation in a nanofilter Download PDF

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US20070090026A1
US20070090026A1 US11/543,252 US54325206A US2007090026A1 US 20070090026 A1 US20070090026 A1 US 20070090026A1 US 54325206 A US54325206 A US 54325206A US 2007090026 A1 US2007090026 A1 US 2007090026A1
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sorter
channels
fluid
rows
separation
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Jongyoon Han
Jianping Fu
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Massachusetts Institute of Technology
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis, ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/027Nanofiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502746Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502761Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0647Handling flowable solids, e.g. microscopic beads, cells, particles
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0896Nanoscaled
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/08Regulating or influencing the flow resistance
    • B01L2400/084Passive control of flow resistance
    • B01L2400/086Passive control of flow resistance using baffles or other fixed flow obstructions
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/02Investigating particle size or size distribution
    • G01N2015/0288Sorting the particles

Abstract

This invention provides a method and an apparatus for quickly continuously fractionating biomolecules, such as DNAs, proteins and carbohydrates by taking advantage of differential bidirectional transport of biomolecules with varying physico-chemical characteristics, for example size, charge, hydrophobicity, or combinations thereof, through periodic arrays of microfabricated nanofilters. The passage of biomolecules through the nanofilter is a function of both steric and electrostatic interactions between charged macromolecules and charged nanofilter walls, Continuous-flow separation through the devices of this invention are applicable for molecules varying in terms of any molecular properties (e.g., size, charge density or hydrophobicity) that can lead to differential transport across the nanofilters.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This Application claims the benefit of U.S. Provisional Application Ser. No. 60/723,926, filed Oct. 6, 2005, which is hereby incorporated in its entirety.
  • GOVERNMENT INTEREST STATEMENT
  • This invention was made in whole or in part with government support under grant number CTS-0347348, awarded by the National Science Foundation. The government may have certain rights in the invention.
  • FIELD OF THE INVENTION
  • This invention is directed to sorting devices comprising nanoseparation matrices, an apparatus comprising the same, and methods of use thereof for high throughput molecular separations. BACKGROUND OF THE INVENTION
  • In systems biology, and in the application of biomarker detection and biosensing, the separation and identification of many proteins, small molecules, and carbohydrates from a cell or from complex biological samples, is necessary. Often, one needs to profile the concentrations of many different biomarkers, cytokines and other signaling molecules contained in serum, to determine or diagnose the current progress of a disease. However, these biomarkers (typically smaller than 30 kD) are present at relatively low concentrations (pM˜nM), while majority proteins (albumin and globulins, typically larger than 40 kD) are present at much higher concentrations (μM˜mM), which critically limits the detection of the smaller biomarkers.
  • Pre-fractionation and separation could eliminate background molecules to enhance the detection ability of the signaling molecules, but none of the conventional separation techniques is appropriate for this task. Gel electrophoresis is routinely used for separating proteins based on size, but they are generally slow and hard to automate, and require bulky equipment. Capillary Electrophoresis (CE) with a liquid sieving matrix is currently the fastest size-based separation technique for protein, but polymeric sieving matrix can interfere with downstream separation and detection processes, which limits the automation of the entire sample preparation process. While microfluidic biomolecule separation systems hold much promise for miniaturizing and automating biomolecule analysis processes, most adopt the same gel sieve material in their separation, with all inherent limitations of the conventional techniques.
  • Micro/nanofluidic molecular sieving structures fabricated with semiconductor technology have been used to separate biomolecules as well, with much greater speed than their conventional counterparts, though to date the systems have only successfully been used for large biomolecule separation such as viral DNA based on size.
  • Thus, a high-throughput biomolecular sorter, which can be automated, and can be readily incorporated into downstream analysis modules is desirable, yet is currently not readily accomplished.
  • SUMMARY OF THE INVENTION
  • In one embodiment, this invention provides a biomolecular sorter comprising:
      • a) a substrate;
      • b) a plurality of obstacles arranged at regular intervals in a plurality of rows, columns or combinations thereof on a surface of said substrate;
      • c) a sample inlet to said sorter;
      • d) at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows; and
      • e) at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows;
        wherein said obstacles are so arranged as to form gaps between said obstacles, with horizontal gaps being of a width less than vertical gaps between said obstacles.
  • According to this aspect of the invention, and in one embodiment, the horizontal gaps are from about 10-5000 nm, and in another embodiment, the vertical gaps are from about 0.1-10 μm. In another embodiment, the obstacles in the rows are laterally shifted with respect to each row.
  • In one embodiment, the gaps form channels for fluid conductance, when fluid is introduced in said sorter. In another embodiment, microfluidic channels are in fluid communication with the channels. In one embodiment, channels comprise sample loading ports, and in another embodiment, the channels comprise sample collection ports.
  • In another embodiment, the microfluidic channels are in fluid communication with a reservoir. In one embodiment, voltage is applied to said reservoir, which in one embodiment is less than 1000 V. In another embodiment, pressure is applied to said reservoir.
  • In one embodiment, the electrostatic force field or hydrodynamic force field is applied in pulse-field operation mode, or in another embodiment, in continuous-field operation mode.
  • In another embodiment, this invention provides a method of sorting a fluid mixture comprising a plurality of biopolymers, which vary in terms of the physico-chemical characteristics of each of said plurality of biopolymers, said method comprising the steps of:
      • a) loading a fluid mixture comprising a plurality of biopolymers in a biomolecular sorter comprising:
        • i. a substrate;
        • ii. a plurality of obstacles arranged at regular intervals in a plurality of rows, columns or combinations thereof on a surface of said substrate, wherein said obstacles are so arranged as to form gaps between said obstacles, with horizontal gaps being of a width less than vertical gaps between said obstacles, and said gaps form channels for fluid conductance, when fluid is introduced in said sorter;
        • iii. a sample inlet to said sorter;
        • iv. microfluidic channels in fluid communication with said channels;
        • v. sample collection ports in fluid communication with said channels;
        • vi. at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows;
        • vii. at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows; and
      • b) applying said electrostatic force field or hydrodynamic force field parallel in direction to said rows, and said electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows, whereby applying said force fields allows for size-based separation of said plurality of biopolymers through said channels; and
      • c) collecting separated biopolymers obtained in (b) from said sample collection ports.
  • According to this aspect of the invention, and in one embodiment, the fluid mixture comprises a cell lysate or tissue homogenate, or in another embodiment, the fluid mixture comprises a large sample of deoxyribonucleic acids (DNA), proteins, or a combination thereof. In another embodiment, the fluid mixture comprises a buffered solution. In another embodiment, the method further comprises the step of sorting a sample of said mixture two or more times, wherein the pH or ionic strength of said buffered solution is varied at the time of said sorting. In another embodiment, the physico-chemical characteristics comprise size, charge, hydrophobicity, hydrophilicity, or a combination thereof.
  • In one embodiment, the electrostatic force field parallel in direction to the rows provides an electroosmotic driving force for the fluid. According to this aspect of the invention and in one embodiment, the fluid has an ionic strength of about 1-300 mM.
  • In one embodiment, the sorting is size-based. According to this aspect of the invention and in one embodiment, greater separation of the biopolymers is achieved with increasing voltage. In one embodiment, the voltage applied is at least 60V, or in another embodiment, at least 70V, or in another embodiment, at least 100V, or in another embodiment, at least 150V.
  • In one embodiment, the fluid has an ionic strength of at least 100 mM. In one embodiment, the fluid has an ionic strength of at least 125 mM, or in another embodiment, at least 150 mM.
  • In one embodiment, the sorting is charge-based. According to this aspect of the invention and in one embodiment, greater resolution of said biopolymers is achieved when the applied voltage is greater than 40 V.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:
  • FIG. 1 schematically depicts negatively charged biomolecules assuming bidirectional motion in the ANA under the influence of two orthogonal electric fields Ex and Ey. On the left, cross-sections of the nanochannels are shown (lighter zones highlight the source of separation), whereas on the right different migration trajectories of biomolecules are presented in the top view of the ANA. Nanofilters (1-20) (with width ws, length ls, and depth ds) arranged in rows are separated by deep channels (1-10) (with width ld and depth dd). Rectangular pillars (1-30) (with width wp and length ls) between nanofilters serve as supporting structures to prevent collapse of the top ceiling. The Debye-length λD is the thickness of the electrical double layer, θ is the deflection angle, and L is the mean characteristic drift distance between two consecutive nanofilter crossings. a, Ogston sieving. Compared to larger DNA or larger native protein molecules, smaller ones are preferred for passage through the nanofilter due to their greater retained configurational freedom, resulting in a greater nanofilter jump passage rate Px. b, Entropic trapping. Longer DNA molecules have larger surface area contacting the nanofilter threshold, resulting in a greater probability for hernia formation and thus a greater nanofilter passage rate Px. c, Electrostatic governed separation by sample net charge for native proteins at low ionic strength. Electrostatic repulsion from the 55-nm-high channels is smaller for less negatively charged (green) than for more negatively charged native proteins (red), resulting in a greater passage rate Px for less negatively charged proteins.
  • FIG. 2A depicts the structures of an embodiment of the microfabricated two-dimensional nanofilter based device illustrating the sieving matrix integrated with the microfluidic channels. Scanning electron microscopy images show details of different device regions (clockwise from top right: sample injection channels, sample collection channels, and minimal sorting unit). The separation chamber (consisted of rows of nanofilters) is 5 mm×5 mm, and the nanofilter has width (Ws) and length (Ls) both of 1 μm. The many microfluidic channels (2-30) connecting to buffer reservoirs (2-20) produce electrostatic force field or hydrodynamic force field over the sieving matrix by acting as electric-current injectors or fluidic flow injectors, which enables separation of materials introduced into the sample reservoir (2-10). The inset shows a photograph of the thumbnail-sized device. The rectangular ANA is 5 mm×5 mm, and nanofilters (ws=1 μm, ls=1 μm and ds=55 nm) are spaced by 1 μm×1 μm square-shaped silicon pillars. Deep channels are 1 μm wide and 300 nm deep. Injection channels connecting sample reservoir inject biomolecule samples as a 30 μm wide stream. Injection channels are 1 mm from the top left corner. The red rectangle highlights the area in which the fluorescence photographs in FIG. 5 were taken.
  • FIG. 2B depicts the operation of an embodiment of the chip involving application of horizontal and vertical force fields across the entire nanofilter matrix. The four sides of the separation chamber are connected to the fluid reservoirs with microfluidic channels (left: reservoir port 1, 2, 3; right: reservoir port 4, 5; top: reservoir port 6, 7; bottom: reservoir port 8, 9, 10). Different voltages or pressures can be applied at the fluid reservoirs to generate the two-dimensional force field pattern.
  • FIG. 3 shows fluorescence images of continuous field separation of SDS-protein complexes inside an embodiment of the 2-D nanofilter based matrix. Band assignment for SDS-protein complexes: (1) low density human lipoprotein (MW: 179 kDa); (2) lectin phytohemagglutinin-L (MW: 120 kDa); (3) cholera toxin subunit B (MW: 11.4 kDa). (A) No separation could be observed if only horizontal field is applied (Eh=300 V/cm). (B&C) The three SDS-proteins species are resolved with both horizontal and vertical fields applied (Eh=200 V/cm, Ev=100 V/cm). Separated molecules are collected in different channels and routed to different reservoirs.
  • FIG. 4 shows fluorescence images of pulse field separation of SDS complexes inside an embodiment of the nanofilter matrix. FIG. 4 has the same band assignment as FIG. 3. In all the experiments, the horizontal and vertical electric fields are 200 V/cm (Eh) and 300 V/cm (Ev), respectively. Different durations are applied in different tests: (A) ΔTh/ΔTv=200 ms/200 ms; (B) ΔTh/ΔTv=200 ms/1000 ms; (C) ΔTh/ΔTv=100 ms/2000 ms;
  • FIG. 5 demonstrates Ogston sieving of PCR markers through the device. Fluorescent photographs of the PCR marker stream pattern were taken in the area highlighted by the red rectangle in FIG. 2. For a, Ex: floated, Ey=25 V/cm; for b, Ex=35 V/cm, Ey=25 V/cm; for c, Ex=60 V/cm, Ey=25 V/cm; for d, Ex=35 V/cm, Ey=12.5 V/cm; for e, Ex=35 V/cm, Ey=50 V/cm; for f, Ex=35 V/cm, Ey=75 V/cm. Band assignment: (1) 50-bp; (2) 150-bp; (3) 300-bp; (4) 500-bp; (5) 766-bp. Fluorescence intensity profiles (of arbitrary units) were measured at the ANA bottom edge. The bars underneath the peaks are centered at the means and label the stream widths (±s.d.).
  • FIG. 6 demonstrates entropic trapping of a λ DNA-Hind III digest. Fluorescent photographs show separation of the λ DNA-Hind III digest under different electric filed conditions. For a, b, f, Ex=185 V/cm, Ey=100 V/cm; for c, Ex=50 V/cm, Ey=100 V/cm; for d, Ex=145 V/cm, Ey=100 V/cm; for e, Ex=170 V/cm, Ey=100 V/cm. Band assignment: (1) 2.322-kbp; (2) 4.361-kbp; (3) 6.557-kbp; (4) 9.416-kbp; (5) 23.130-kbp. Fluorescence intensity profiles were measured at the ANA bottom edge. The bars underneath the peaks are centered at the means and label the stream widths (±s.d.). For g: Observation of the threshold horizontal field Es,c. A composite fluorescence photograph showing confining of λ DNA-Hind III digest in the initial injection deep channels with Ex=15 V/cm and Ey=25 V/cm and composite fluorescence photograph showing DNA molecules starting to jump across the nanofilter with Ex=50 V/cm and Ey=25 V/cm.
  • FIG. 7 demonstrates a continuous-flow separation of SDS-protein complexes through an embodiment of the device. a, Composite fluorescent photograph showing separation of cholera toxin subunit B (band 1) and β-galactosidase (band 2) with Ex=75 V/cm and Ey=50 V/cm. The three insets are electropherograms scanned at 1 mm, 3 mm, and 5 mm from the injection point, respectively. b, Measured deflection angle θ (top) of cholera toxin subunit B (˜) and β-galactosidase ()) as a function of Ex when Ey=50 V/cm. The bottom shows the corresponding separation resolutions. The ±s.d. of θ are indicated as error bars (drawn if larger than the symbol).
  • FIG. 8 demonstrates crossover of Ogston sieving and entropic trapping of DNA molecules in an embodiment of the device. a, Stream deflection angle θ as a function of DNA length. For the left side of Ogston sieving, Ey is fixed at 25 V/cm, and Ex: 10 V/cm (▪), 35 V/cm (●), 60 V/cm (▴), 85 V/cm (▾). For the right side of entropic trapping, Ey=100 V/cm and Ex: 50 V/cm (˜), Ex: 80 V/cm (O), Ex: 110 V/cm (Δ), Ex: 145 V/cm (V), Ex: 170 V/cm (⋄), Ex: 185 V/cm (⋆). The ±s.d. of θ derived from the stream half-width are all less than 1°, so statistical error bars for θ are not plotted. b, Dependence of the effective peak capacity nc on Ex. For Ogston sieving (solid symbols), Ey=25 V/cm; for entropic trapping (open symbols), Ey=100 V/cm.
  • FIG. 9 demonstrates the tan θ of different streams as a function of Ex/Ey at a fixed Ey=25 V/cm (50-bp (˜), 150-bp (O), 300-bp (Δ), 500-bp (∇), 766-bp (⋄)). The ±s.d. of θ derived from the stream half-width are ail less than 1°, so statistical error bars for tan θ are not plotted. The colored solid lines are theoretical curves calculated from Eq. (7-2). The best fitting constant a has a mean about 177.5 and a ±s.d. about 12%.
  • FIG. 10 demonstrates another embodiment of size-based separation of proteins. (a) Fluorescence images showing three size-based separated streams of the proteins fibrinogen (MW 340 kDa) and lectin (MW 49 kDa), which are fluorescent in the green range (FITC filter set, left) and the orange-fluorescent B-phycoerythrin with MW 240 kDa (Texas Red® filter set, right). These measurements were performed at TBE 5× with Ex=100 V/cm and Ey=50 V/cm. (b) Fluorescence intensity as a function of the distance along the dashed lines (after 30% of the total ANA length in y-direction) presented in (a), showing the separation distance between the streams. The black line was deduced from the FITC and the grey line from the Texas Red® image. A Gaussian fluorescence intensity distribution is observed for all three proteins.
  • FIG. 11 demonstrates another embodiment of size-based separation of proteins and other biomolecules. (a) Separation distance between the streams of fibrinogen and B-phycoerythrin as a function of the ratio Ex/Ey at TBE 5×. For a chosen Ey a maximal separation distance is observed for a specific Ex, and the maximal separation distance increases with decreasing Ey, because the Peclet number decreases. (b) Standard deviation σ (half the stream width) of B-phycoerythrin versus Ex/Ey. Band broadening is observed with an increasing ratio of Ex/Ey and with a decreasing Ey.
  • FIG. 12 demonstrates another embodiment of charge-based separation. (a) Fluorescence images presenting charge-based separation of streptavidin (pI=5-6) and lectin (pI=8.0-8.8) at TBE 0.05× in the image on the left, whereas the right image shows that the MW difference of 3.8 kDa between these two proteins can not be resolved on this ANA at TBE 5×. These results confirm the separation obtained at TBE 0.05× is charge-based. (b) Fluorescence intensity (black: TBE 0.05×, grey: TBE 5×) versus distance along the dashed lines of the images in (a), after 5% of the total length of the ANA. At TBE 0.05× the fields were Ex=250 V/cm and Ey=75 V/cm and at TBE 5×Ex=150 V/cm and Ey=75 V/cm.
  • FIG. 13 demonstrates another embodiment of charged based separation of biomolecules. (a) Fluorescence images showing three charge-based separated streams corresponding to the proteins B-phycoerythrin (pI=4.2-4.4), fibrinogen (pI=5.5), and lectin (pI=8.0-8.8) observed with the FITC filter set (left) and Texas Red® filter set (right). The buffer was TBE 0.05×, pH=9.6 and the electric fields were Ex=250 V/cm and Ey=75 V/cm. (b) Fluorescence intensity (black: FITC filter set, grey: Texas Red® filter set) as a function of the distance along the dashed lines presented in (a), showing that a good separation is already obtained after 13% of the total length of the ANA. White dots are adsorbed proteins at the walls of the channels.
  • FIG. 14 demonstrates the resolution Rs of an embodiment of a charge-based separation between the streams B-phycoerythrin—lectin and B-phycoerythrin—fibrinogen as a function of Ex at a fixed electric field Ey=75 V/cm. An increasing resolution is obtained with increasing Ex, because more sieving events occur as the number of nanofilters in increased. The measurements were made at TBE 0.05× and evaluated at the same y-position as done in FIG. 5 after 13% of the total length of the ANA. The x-position of the center of the stream and its width were obtained by Gaussian fits. The connecting lines are for guidance only.
  • It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.
  • DETAILED DESCRIPTION OF THE PRESENT INVENTION
  • In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.
  • In one embodiment of this invention, there is provided a unique continuous-flow separation of small biomolecules such as native proteins, protein complexes and double-stranded DNA molecules (dsDNA) in a microfabricated chip, based on differential bidirectional transport of biomolecules of different sizes through periodic arrays of microfabricated nanofilters, which in one embodiment, comprises the use of a microfabricated molecular sieving chip that can size-fractionate small biomolecules without using gel sieving matrices, with a separation efficiency comparable to current means such as capillary gel electrophoresis.
  • In one embodiment, this invention provides a biomolecular sorter comprising:
      • a) a substrate;
      • b) a plurality of obstacles arranged at regular intervals in a plurality of rows, columns or combinations thereof on a surface of said substrate;
      • c) a sample inlet to said sorter;
      • d) at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows; and
      • e) at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows;
        wherein said obstacles are so arranged as to fonn gaps between said obstacles, with horizontal gaps being of a width less than vertical gaps between said obstacles.
  • In one embodiment, the substrate and/or other components of the sorter can be made from a wide variety of materials including, but not limited to, silicon, silicon dioxide, silicon nitride, glass and fused silica, gallium arsenide, indium phosphide, III-V materials, PDMS, silicone rubber, aluminum, ceramics, polyimide, quartz, plastics, resins and polymers including polymethylmethacrylate, acrylics, polyethylene, polyethylene terepthalate, polycarbonate, polystyrene and other styrene copolymers, polypropylene, polytetrafluoroethylene, superalloys, zircaloy, steel, gold, silver, copper, tungsten, molybdeumn, tantalum, KOVAR, KEVLAR, KAPTON, MYLAR, teflon, brass, sapphire, etc., or a combination thereof High quality glasses such as high melting borosilicate or fused silicas may be used, in some embodiments, for their UV transmission properties when any of the sample manipulation and/or detection steps require light based technologies. In addition, as outlined herein, portions of the internal and/or external surfaces of the device may be coated with a variety of coatings as needed, to facilitate the manipulation or detection technique performed.
  • In one embodiment, the obstacles comprise the same materials as the substrate, or in another embodiment, are comprised of a suitable material which prevents adhesion to the obstacles. In one embodiment, the channels formed via the positioning of the obstacles may similarly comprise the same materials, or may otherwise be treated, as will be appreciated by one skilled in the art, to facilitate sorting.
  • In one embodiment, the invention provides for a microchip comprising the biomolecular sorter or sorters of this invention. In one embodiment, the microchip may be made of a wide variety of materials and can be configured in a large number of ways, as described and exemplified herein, in some embodiments, and other embodiments will be apparent to one of skill in the art. The composition of the substrate will depend on a variety of factors, including the techniques used to create the device, the use of the device, the composition of the sample, the molecules to be sorted, the type of analysis conducted following molecular sorting, the size of internal structures, the presence or absence of electronic components, and the technique used to move fluid, etc. In some embodiments, the devices of the invention will be sterilizable as well, in some embodiments, this is not required. In some embodiments, the devices are disposable or, in another embodiment, re-usable.
  • Microfluidic chips used in the methods and devices of this invention may be fabricated using a variety of techniques, including, but not limited to, hot embossing, such as described in H. Becker, et al., Sensors and Materials, 11, 297, (1999), hereby incorporated by reference, molding of elastomers, such as described in D. C. Duffy, et. al., Anal. Chem., 70, 4974, (1998), hereby incorporated by reference, injection molding, LIGA, soft lithography, silicon fabrication and related thin film processing techniques, as known in the art, photolithography and reactive ion etching techniques, as exemplified herein. In one embodiment, glass etching and diffusion bonding of fused silica substrates may be used to prepare microfluidic chips.
  • The arrangement of the obstacles in the sorter forms an array or rows and columns, such that the regions between obstacles in respective rows and columns are continuous, forming channels. For example, as described in FIG. 1, each row, which comprises a “thick region” (1-10) forming a channel for conveying fluid comprising molecules to be sorted, as described herein. The “thin regions” (1-20) provide steric hindrance such that larger molecules do not pass readily through these regions. In one embodiment, the regions are aligned, such that they form parallel columns, which serve as channels for conveying fluid comprising molecules to be sorted, which are small enough in size to pass through the regions. In one embodiment, the regions are staggered, such that they form staggered columns, serving as channels as described. Molecules, in turn, in a size restricted manner, transit through the thick and thin regions, partly in a direction which is in parallel to and partly in a direction which is perpendicular to the original plane of entry in the sorter.
  • In one embodiment, the thin regions or columns of this invention comprise nanofluidic channels, which have a width ranging from about 10-5000 nm. In one embodiment, the thick regions comprise microfluidic channels which have a width ranging from about 0.5-500 micron.
  • In one embodiment, the microfluidic or nanofluidic channels used in the devices and/or methods of this invention, which convey fluid, may be constructed from a material which renders it transparent or semitransparent, in order to image the solutions being sorted, or in another embodiment, to ascertain the progress of the sorting, etc. In some embodiments, the materials firther have low conductivity and high chemical resistance to buffer solutions and/or mild organics. In other embodiments, the material is of a machinable or moldable polymeric material, and may comprise insulators, ceramics, metals or insulator-coated metals. In other embodiments, the channel may be constructed from a polymer material that is resistant to alkaline aqueous solutions and mild organics. In another embodiment, the channel comprises at least one surface which is transparent or semi-transparent, such that, in one embodiment, imaging of the sorter is possible.
  • In one embodiment, the term “minimal sorting unit” as described herein, refers to the arrangement of obstacles on the substrate, forming rows and columns, as described hereinabove, which in turn serve as channels for the conductance of the fluid mixture comprising the molecules to be sorted. In one embodiment of this invention, the sorter may further comprise microfluidic channels in fluid communication with the minimal sorting unit. In one embodiment, the latter channels are in fluid communication with a sample reservoir, or in another embodiment, buffer reservoir, or in another embodiment, inlet port, or in another embodiment, outlet port. According to this aspect of the invention, the microfluidic channels may serve as a conduit for conveying material into and out of the minimal sorting unit.
  • In one embodiment, a device of this invention may comprise an array comprising one or more minimal sorting units. Such arrays may be referred to herein, in other embodiments, as “anisotropic nanofilter arrays (ANAs)”.
  • In one embodiment, the inlet may comprise an area of the chip in fluidic communication with one or more microfluidic channels, in one embodiment, and/or a sample reservoir, in another embodiment. Inlets and outlets may be fabricated in a wide variety of ways, depending upon, in one embodiment, on the substrate material utilized, and/or in another embodiment, the dimensions used. In one embodiment inlets and/or outlets are formed using conventional tubing, which prevents sample leakage, when fluid is applied to the device, under pressure. In one embodiment inlets and/or outlets are formed of a material which withstands application of voltage, even high voltage, to the device. In one embodiment, the inlet may further comprise a means of applying a constant pressure, to generate pressure-driven flow in the device.
  • The sorters of this invention, may be referred to in some embodiments, as a “device” or “apparatus”, and will comprise at least the elements as described herein. In one embodiment, the devices of this invention comprise at least one microchannel and at least one nanochannel. In one embodiment, the terms “nanochannel” and “nanofilter” are used herein interchangeably, and refer to a size-selective construction on the sorting device, as a result of the arrangement of the obstacles and construction of the device, such that size-dependent separation is accomplished, as described. In one embodiment, the device is formed using the technology of microfabrication and nanofabrication, for formation of the respective channels.
  • Microfabrication technology, or microtechnology or MEMS, in one embodiment, applies the tools and processes of semiconductor fabrication to the formation of, for example, physical structures. Microfabrication technology allows one, in one embodiment, to precisely design features (e.g., reservoirs, wells, channels) with dimensions in the range of <1 mm to several centimeters on chips made, in other embodiments, of silicon, glass, or plastics. Such technology may be used to construct the microchannels of the sorter, in one embodiment.
  • In another embodiment, NEMS or nanotechnology is used to construct the nanochannels. In one embodiment, the nanochannels can be fabricated with nanoimprint lithography (NIL), as described in Z. N. Yu, P. Deshpande, W. Wu, J. Wang and S. Y. Chou, Appl. Phys. Lett. 77 (7), 927 (2000); S. Y. Chou, P. R. Krauss, and P. J. Renstrom, Appl. Phys. Lett. 67 (21), 3114 (1995); Stephen Y. Chou, Peter R. Krauss and Preston J. Renstrom, Science 272, 85 (1996) and U.S. Pat. No. 5,772,905 hereby incorporated herein, in their entirety, by reference. In one embodiment, the nanochannels and/or microchannels can be formed by photolithography and reactive ion etching (RIE) techniques, with nanofilter gap thickness (ds) as thin as 10 nm. In one embodiment, the formation of the device may employ nanoimprint lithography, interference lithography, self-assembled copolymer pattern transfer, spin coating, electron beam lithography, focused ion beam milling, wet-etching, plasma-enhanced chemical vapor deposition, electron beam evaporation, sputter deposition, and combinations thereof. Alternatively, other conventional methods can be used to form the nanochannels and/or microchannels.
  • In one embodiment, the sorter comprising nanochannels and microchannels are formed as exemplified hereinbelow in Example 1.
  • In one embodiment, a series of reactive ion etchings are conducted, after which nanochannels are patterned with standard lithography tools. In one embodiment, the etchings are conducted with a particular geometry, which, in another embodiment, determines the interface between the microchannels, and/or nanochannels. In one embodiment, etchings, which create the microchannels, are performed perpendicular to the plane in which etchings for the nanofilters were created.
  • In another embodiment, electrical insulation of the device is accomplished. In one embodiment, such insulation is accomplished via thermal oxidation of the device. In another embodiment, a surface of the device, which in another embodiment is the bottom surface, may be affixed to a substrate, such as, for example, and in one embodiment, a Pyrex wafer. In one embodiment, the wafer may be affixed using anodic bonding techniques.
  • In one embodiment, the fabrication may use a shaped sacrificial layer, which is sandwiched between permanent floor and ceiling layers, with the shape of the sacrificial layer defining a working gap. When the sacrificial layer is removed, the working gap becomes a fluid channel having the desired configuration. This approach, in one embodiment, allows a precise definition of the height, width and shape of interior working spaces, or fluid channels, in the structure of a fluidic device.
  • The sacrificial layer is formed on a substrate, shaped by a suitable lithographic process, for example, and is covered by a ceiling layer. Thereafter, the sacrificial layer may be removed with a wet chemical etch, leaving behind empty spaces between the floor and ceiling layers which form working gaps which may be used as flow channels, filters and/or reservoirs for the sorting device. In such a device, the vertical dimension, or height, of a working gap is determined by the thickness of the sacrificial layer film, which is made with, for example, chemical vapor deposition (CVD) techniques, and accordingly, this dimension can be very small.
  • The channels, chambers, and/or filters have dimensions on the order of microns, in the case of the microchannels and chambers/reservoirs, and nanometers, in the case of nanofilters/nanochannels. In some embodiments, structures with larger dimensions, such as on the order of millimeters, are used, and represent embodiments of this invention. In one embodiment, the width and/or length of the microfluidic chamber ranges from 100-1000 μm, and the depth of the microfluidic chamber ranges from 0.1-100 μm. In one embodiment, the width of the microchannel is between 0.1-1000 μm, or in another embodiment, between 1 and 150 μm, or in another embodiment, between 20 and 500 μm, or in another embodiment, between 25 and 750 μm, or in another embodiment, between 500 and 1000 μm. In one embodiment the depth of the microchannel is between 0.1-50 μm, or in another embodiment, between 0.5 and 5 μm, or in another embodiment, between 5 and 15 μm, or in another embodiment, between 10 and 25 μm, or in another embodiment between 15 and 50 μm.
  • In another embodiment, the width of the nanofilter is between 10 nm-500 μm, or in another embodiment, between 10 nm and 15 μm, or in another embodiment, between 20 nm and 25 μm, or in another embodiment, between 50 nm and 40 μm, or in another embodiment, between 50 nm and 50 μm. In another embodiment, the depth of the nanochannel is between 10-1000 nanometers, or in another embodiment, between 20 and 50 nanometers, or in another embodiment, between 20 and 75 nanometers, or in another embodiment, between 30 and 75 nanometers or in another embodiment, between 50 and 100 nanometers.
  • In one embodiment, the microchannels, which form the rows of the device and nanofilters are oriented perpendicularly, with respect to each other. In one embodiment, the term “perpendicular” or “perpendicularly” refers to an orientation of one channel being at a 90° angle with respect to the longitudinal axis of another channel, ±5 or in another embodiment, at a 90° angle of ±10°, or in another embodiment, at a 90° angle ±20°.
  • In one embodiment, the sorter of this invention may comprise a plurality of channels, including a plurality of microchannels, or a plurality of nanochannels, or a combination thereof. In one embodiment, the phrase “a plurality of channels refers to more than two channels, or, in another embodiment, more than 5, or, in other embodiments, more than 10, 96, 100, 384, 1,000, 1,536, 10,000, 100,000 or 1,000,000 channels.
  • In one embodiment, the surface of the microchannel and/or nanofilter may be functionalized to reduce or enhance adsorption of species of interest to the surface of the device. In another embodiment, the surface of the microchannel and/or nanofilter has been functionalized to enhance or reduce the operation efficiency of the device.
  • In one embodiment, the device is further modified to contain an active agent in the microchannel. For example, and in one embodiment, the microchannel is coated with an enzyme at a region wherein molecules within the mixture will separate in a size-restricted manner, according to the methods of this invention. According to this aspect, the enzyme, such as, a protease, may come into contact with concentrated proteins, and digest them, which in another embodiment, allows for further sorting of the digested species. The digestion products may, in another embodiment, be conveyed to a peptide analysis module, downstream of the sorting device. The amino acid sequences of the digestion products may be determined and assembled to generate a sequence of the polypeptide. Prior to delivery to a peptide analysis module, the peptide may be conveyed to an interfacing module, which in turn, may perform one or more additional steps of separating, concentrating, and or focusing.
  • In another embodiment, the microchannel may be coated with a label, which in one embodiment is tagged, in order to identify a particular protein or peptide, or other molecule containing the recognized epitope, which may be a means of sensitive detection of a molecule in a large mixture, present at low concentration.
  • For example, in some embodiments, reagents may be incorporated in the buffers used in the methods and devices of this invention, to enable chemiluminescence detection. In some embodiments the method of detecting the labeled material includes, but is not limited to, optical absorbance, refractive index, fluorescence, phosphorescence, chemiluminescence, electrochemiluminescence, electrochemical detection, voltometry or conductivity. In some embodiments, detection occurs using laser-induced fluorescence, as is known in the art.
  • In some embodiments, the labels may include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, fluorescamine, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, 1,1′-[1,3-propanediylbis[(dimethylimino-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-,tetraioide, which is sold under the name YOYO-1, Cy and Alexa dyes, and others described in the 9th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. Labels may be added to ‘label’ the desired molecule, prior to introduction into the sorters of this invention, in some embodiments, and in some embodiments the label is supplied in a microfluidic chamber. In some embodiments, the labels are attached covalently as is known in the art, or in other embodiments, via non-covalent attachment.
  • In some embodiments, photodiodes, confocal microscopes, CCD cameras, or photomultiplier tubes maybe used to image the labels thus incorporated, and may, in some embodiments, comprise the apparatus of the invention, representing in some embodiments, a “lab on a chip” mechanism, as described further, as well, hereinbelow.
  • In one embodiment, detection is accomplished using laser-induced fluorescence, as known in the art. In some embodiments, the apparatus may further comprise a light source, detector, and other optical components to direct light onto the microfluidic chamber/chip and thereby collect fluorescent radiation thus emitted. The light source may comprise a laser light source, such as, in some embodiments, a laser diode, or in other embodiments, a violet or a red laser diode. In other embodiments, VCSELs, VECSELs, or diode-pumped solid state lasers may be similarly used. In some embodiments, a Brewster's angle laser induced fluorescence detector may used. In some embodiments, one or more beam steering mirrors may be used to direct the beam to a desired location for detection.
  • In one embodiment, the buffered solution is flowed through the chamber at a relatively constant flow rate, which in one embodiment ranges from about 0.5-15 μl/minute. According to this aspect of the invention, pressure applied to the device will be such as to accommodate a relatively constant flow rate, as desired, as will be understood by one skilled in the art.
  • In one embodiment, any of various mechanisms may be employed to manipulate, transport, and/or move fluid within the device, to convey the fluid within the microfluidic chamber, as well as into or out of the chamber. In some embodiments, pressurized fluid flow is applied from a syringe, or, in another embodiment, other pressure source, attached to, in one embodiment, an inlet of a device of this invention.
  • In some embodiment, a pressure stop is positioned between two or more channels in an apparatus of this invention, such that the pressure-driven flow through a first microchamber does not influence the flow through a second microchamber, in some embodiments of this invention. According to this aspect of the invention, and in one embodiment, separation may be affected by the pressure applied for the sorting of the molecules within the given microfluidic chamber.
  • Inlets/outlets allow access to the chambers to which they are connected for the purpose, in one embodiment, of introducing or, in another embodiment, of removing fluids from the chambers on the microfluidic chip. In one embodiment, inlets allow access to the chamber to which they are connected for the purpose of introducing fluids to the microchamber, from a sample reservoir, or in another embodiment, from a sample stored in a conventional storage means, such as a tube. In another embodiment, the outlet allows access of fluid from the microfluidic chamber which has undergone pI-based sorting, according to the methods of this invention. According to this aspect of the invention, the outlet may allow for the removal and storage of the sorted material, or in another embodiment, its conveyance to an analytical module, which in one embodiment, may be coupled thereto.
  • The sorting devices of this invention are so constructed that passage of biomolecules through a nanofilter is sterically hindered via an Ogston sieving process, with passage being size-dependent. An injected main stream of biomolecules separates into different streams based on molecular size, in some embodiments, as a function of the arrangement of the nanofilter arrays and applied horizontal and vertical force fields (electrostatic force field or hydrodynamic force field with pulse-field operation mode or continuous-field operation mode).
  • In one embodiment, the sorter comprises at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows. In one embodiment, the force field is applied in parallel direction to the microchannels, which comprise the rows of the sorters, as described. The force field may be thus formed, via application of an appropriate stimulus to a reservoir, as described, which is in fluid communication with microfluidic channels, which in turn further convey the stimulus to the microchannels of the minimal sorting unit.
  • In one embodiment, the microfluidic channels connecting to buffer reservoirs produce electrostatic force field or hydrodynamic force field over the minimal sorting unit by acting as electric-current injectors or fluidic flow injectors, depending upon the field applied.
  • The sorter will also comprise at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to the columns, and perpendicular in direction to the rows such that the two fields, as applied are perpendicularly applied, with respect to each other.
  • As exemplified herein, in FIG. 3, the application of two fields, in this case electrostatic force fields were necessary for the separation of the labeled species.
  • In one embodiment, the electrostatic force field may be applied to the apparatus via the disposition of electrodes on a surface of the apparatus, in conjunction with its their connection to a means of applying voltage, wherein the electrodes are so positioned such that following application of voltage, an electric field is generated, which is parallel in direction to the rows comprising the microchannels of the minimal sorting unit In another embodiment, the electrodes are so positioned such that following application of voltage, an electric field is generated, which is perpendicular in direction to the rows comprising the microchannels of the minimal sorting unit, which may also be referred to herein as a vertical force field. In some embodiments, electrodes are formed on the interior or exterior surfaces of the chip and are in electrical communication with the microfluidic channels.
  • According to this aspect of the invention, and in one embodiment, a power supply is coupled to the electrodes, which rnay further comprise a DC-to-DC converter, a voltage-controlled resistor, and a feedback circuit to control the resistor and converter to regulate the voltage of the power supply.
  • In some embodiments of the present invention, a power module is coupled to an external power supply. In other embodiments, the power module is powered using a portable power supply, such as batteries, solar power, wind power, nuclear power, and the like.
  • In some embodiments of the present invention, the voltage delivered to the device provides a field strength of up to 3.5×104 V/m. In one embodiment, an electric field with strength of at least 100 V/m is applied, or in another embodiment, at least 200 V/m, or in another embodiment, at least 300 V/m.
  • In one embodiment, the electrode metal contacts can be integrated using standard integrated circuit fabrication technology to be in contact with a reservoir, or in one embodiment, at least one microchannel, or in another embodiment, a combination thereof, and oriented as such, to establish a directional electric field, as described. Alternating current (AC), direct current (DC), or both types of fields can be applied. The electrodes can be made of almost any metal, and in one embodiment, comprise thin Al/Au metal layers deposited on defined line paths. In one embodiment, at least one end of one electrode is in contact with buffer solution in the reservoir.
  • In another embodiment, the sorting device may contain at least two pairs of electrodes, each providing an electric field in a different direction.
  • In another embodiment, at least one of the force fields may be a hydrodynamic force field. In one embodiment, both force fields are hydrodynamic force fields, or in another embodiment, one force field is hydrodynamic and the other electrostatic, or in another embodiment, both are electrostatic.
  • In one embodiment, a hydrodynamic force field is established via provision of a pressure driven flow, which may originate, in one embodiment, in the reservoirs, which convey fluid to the microchannels, which in turn convey the fluid to the minimal sorting unity, thus in fact acting as fluidic flow injectors. In one embodiment, the phrases “pressure-driven flow” refers to flow that is driven by a pressure source exerted on the conveyance of fluid through a segment of a channel, external to the channel segment through which such flow is driven.
  • Examples of pressure sources include negative and positive pressure sources or pumps external to the channel segment in question, including electrokinetic pressure pumps, which in one embodiment, are connected to a reservoir, or microchannel of this invention, which does not comprise the minimal sorting unit.
  • In one embodiment, reference to the term “liquid flow” may encompass any or all of the characteristics of flow of fluid or other material through a passage, conduit, channel or across a surface. Such characteristics include without limitation the flow rate, flow volume, the conformation and accompanying dispersion profile of the flowing fluid or other material, as well as other more generalized characteristics of flow, e.g., laminar flow, creeping flow, turbulent flow, etc.
  • In one embodiment, hybrid flow may comprise pressure-based relay of the liquid sample into the channel network, followed by electrokinetic movement of materials, or in another embodiment, electrokinetic movement of the liquid followed by pressure-driven flow. It is to be understood that both may be employed in the creation of a force field for either or both directions, as described herein, and may be used in order to affect the sorting efficiency or quality desired, when sorting a mixture of molecules.
  • The sorters of this invention and/or devices comprising the same may be used to sort a fluid mixture comprising a plurality of polymers, or in some embodiments, biopolymers, for example, peptides, nucleic acids, glycoproteins, carbohydrates, etc.
  • In one embodiment, this invention provides a method of sorting a fluid mixture comprising a plurality of polymers varying in terms of the physico-chemical characteristics of each of said plurality of biopolymers, said method comprising the steps of:
      • a) loading a fluid mixture comprising a plurality of polymers in a molecular sorter comprising:
        • i. a substrate;
        • ii. a plurality of obstacles arranged at regular intervals in a plurality of rows, columns or combinations thereof on a surface of said substrate, wherein said obstacles are so arranged as to form gaps between said obstacles, with horizontal gaps being of a width less than vertical gaps between said obstacles, and said gaps form channels for fluid conductance, when fluid is introduced in said sorter;
        • iii. a sample inlet to said sorter;
        • iv. microfluidic channels in fluid communication with said channels;
        • viii. sample collection ports in fluid communication with said channels;
        • ix. at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows;
        • x. at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows; and
      • b) applying said electrostatic force field or hydrodynamic force field parallel in direction to said rows, and said electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows, whereby applying said force fields allows for size-based separation of said plurality of biopolymers through said channels; and
      • c) collecting separated biopolyrners obtained in (b) from said sample collection ports.
  • In one embodiment, the invention provides an apparatus for quickly continuously fractionating biomolecules, such as DNAs, proteins and carbohydrates by taking advantage of differential bidirectional transport of biomolecules of different sizes through periodic arrays of microfabricated nanofilters.
  • The molecules for separation may be any which may be distinguished by the methods and via the devices of this invention. In one embodiment, a solution or buffered medium comprising the molecules may be used in the methods and for the devices of this invention. In one embodiment, such solutions or buffered media may comprise natural or synthetic compounds. In another embodiment, the solutions or buffered media may comprise supernatants or culture media, which in one embodiment, are harvested from cells, such as bacterial cultures, or in another embodiment, cultures of engineered cells, wherein in one embodiment, the cells express mutated proteins, or overexpress proteins, or other molecules of interest which may be thus applied. In another embodiment, the solutions or buffered media may comprise lysates or homogenates of cells or tissue, which in one embodiment, may be otherwise manipulated for example, wherein the lysates are subject to filtration, lipase or collagenase, etc., digestion, as will be understood by one skilled in the art, wherein a solution of desired molecules may be obtained and subjected to the methods of this invention.
  • It is to be understood that any complex mixture, comprising two or more molecules which differ in terms of their molecular size, charge, hydrophobicity, hydrophilicity, or any physical or chemical characteristic, or combinations thereof, whose separation is desired, may be used for the methods and in the sorters/devices of this invention, and represents an embodiment thereof.
  • In another embodiment, the solutions or buffered media for use according to the methods and for use in the devices of this invention may comprise any fluid, having molecules for separation with the described properties, for example, bodily fluids such as, in some embodiments, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, or in another embodiment, homogenates of solid tissues, as described, such as, for example, liver, spleen, bone marrow, lung, muscle, nervous system tissue, etc., and may be obtained from virtually any organism, including, for example mammals, rodents, bacteria, etc. In some embodiments, the solutions or buffered media may comprise environmental samples such as, for example, materials obtained from air, agricultural, water or soil sources, which are present in a fluid which can be subjected to the methods of this invention. In another embodiment, such samples may be biological warfare agent samples; research samples and may comprise, for example, glycoproteins, biotoxins, purified proteins, etc.
  • As will be appreciated by those in the art, virtually any experimental manipulation may have been done on the sample prior to its use in embodiments of the present invention. For example, a variety of manipulations may be performed to generate a liquid sample of sufficient quantity from a raw sample. In some embodiments, gas samples and aerosol samples are so processed to generate a liquid sample containing molecules whose separation may be accomplished according to the methods of this invention.
  • In one embodiment, the device is adapted such that analysis of a species of interest may be conducted, in one embodiment, in the sorter, or in another embodiment, downstream of the sorter. In one embodiment, analysis downstream of the sorter refers to removal of the sorted species from the device, and placement in an appropriate setting for analysis, or in another embodiment, construction of a conduit from the sorter, for example, from a collection port, which relays the sorted material to an appropriate setting for analysis. In one embodiment, such analysis may comprise signal acquisition, and in another embodiment, a data processor. In one embodiment, the signal can be a photon, electrical current/impedance measurement or change in measurements. It is to be understood that the sorting device of this invention may be useful in various analytical systems, including bioanalysis Microsystems, due to its simplicity, performance, robustness, and integrabilty to other separation and detection systems, and any integration of the device into such a system is to be considered as part of this invention.
  • For example, as demonstrated herein in FIG. 3 or 4, when a mixture of fluorescently-labeled low density human lipoprotein (MW: 179 kDa), lectin phytohemagglutinin-L (MW: 120 kDa) and cholera toxin subunit B (MW: 11.4 kDa) were imaged following continuous- or pulse-field separation, the SDS-protein complexes separated into three different streams which were collected in different channels and routed to different reservoirs.
  • In one embodiment, the sorters/devices of this invention may be imaged with a two-dimensional detector. Imaging of the sorters/devices or parts thereof, may be accomplished by presenting it to a suitable apparatus for the collection of emitted signals, such as, in some embodiments, optical elements for the collection of light from the microchannels.
  • In another embodiment, the device is coupled to a separation system, or in another embodiment, a detection system, or in another embodiment, an analysis system or in another embodiment, a combination thereof. In another embodiment, the device is coupled to an illumination source.
  • In one embodiment, the sorter may be disposable, and in another embodiment, may be individually packaged, and in another embodiment, have a sample loading capacity of 1-50,000 individual fluid samples. In one embodiment, the sorter can be encased in a suitable housing, such as plastic, to provide a convenient and commercially-ready cartridge or cassette. In one embodiment, the sorter will have suitable features on or in the housing for inserting, guiding, and aligning the device, such that, for example, a sample loading compartment is aligned with a reservoir in another device, which is to be coupled to the sorter. For example, the sorter may be equipped with insertion slots, tracks, or a combination thereof, or other adaptations for automation of the sorting process via a device of this invention.
  • The sorter may be so adapted, in one embodiment, for high throughput sorting and analysis of multiple samples, such as will be useful in proteomics applications, as will be appreciated by one skilled in the art.
  • In one embodiment of the present invention, the sorter is a part of a larger system, which includes an apparatus to excite molecules inside the channels and detect and collect the resulting signals. In one embodiment, a laser beam may be focused upon the device, using a focusing lens, in another embodiment. The generated light signal from the molecules inside the device may be collected by focusing/collection lens, and, in another embodiment, reflected off a dichroic mirror/band pass filter into optical path, which may, in another embodiment, be fed into a CCD (charge coupled device) camera.
  • In another embodiment, an exciting light source could be passed through a dichroic mirror/band pass filter box and focusing/collecting scheme from the top of the concentrator. Various optical components and devices can also be used in the system to detect optical signals, such as digital cameras, PMTs (photomultiplier tubes), and APDs (Avalanche photodiodes).
  • In another embodiment, the system may further include a data processor. In one embodiment, the data processor can be used to process the signals from a CCD, to a digital image of the concentrated species onto a display. In one embodiment, the data processor can also analyze the digital image to provide characterization information, such as size statistics, histograms, karyotypes, mapping, diagnostics information and display the information in suitable form for data readout.
  • In one embodiment, the steps of sorting for example, polypeptides obtained from a given cell, producing digestion products, and analyzing digestion products to determine protein sequence, can be performed in parallel and/or iteratively for a given sample, providing a proteome map of the cell from which the polypeptides were obtained. Proteome maps from multiple different cells can be compared to identify differentially expressed polypeptides in these cells, and in other embodiments, the cells may be subjected to various treatments, conditions, or extracted from various sources, with the proteome map thus generated reflecting differential protein expression as a result of the status of the cell.
  • In one embodiment, subsequent to separation via the methods and utilizing the devices of this invention, further analysis of the sorted materials is possible. Such analysis may be via direct coupling of the machinery necessary for such analysis to the outlet of a microchamber, as herein described, or in another embodiment, samples are processed separately.
  • In one embodiment such subsequent analysis, in addition, or in parallel to those already described, may comprise electrophoresis, chromatography, mass spectroscopy, sequencing (for example, for the identification of particular proteins or peptides), NMR and others, as will be appreciated by one skilled in the art.
  • In some embodiments, features of the present invention include: 1) arrays of nanofilters serve as the sieving media; 2) Ogston sieving mechanism within the nanofilter and the resulting differential bidirectional transport of biomolecules; 3) various operation methods of the nanofilter array chip (electrostatic force field based or hydrodynamic force field based with pulse-field operation mode or continuous-field operation mode).
  • In one embodiment, the electrostatic force field or hydrodynamic force field is applied in pulse-field operation mode, or in another embodiment, in continuous-field operation mode, as described and exemplified herein. In one embodiment voltage, pressure, timing or a combination thereof are varied, when sorting a sample. In one embodiment, the sample may be repetitively sorted, varying specific conditions with each sort, to further distinguish sorted species, for example, to obtain greater resolution in terms of size-dependent sorting, as a function of the timing, voltage, pressure, or other means, as will be appreciated by one skilled in the art, in the context of the devices and methods of this invention.
  • In another embodiment, the method further comprises the step of sorting a sample, wherein the pH or ionic strength of the buffered solution is varied at the time of sorting, as described.
  • In another embodiment, the method further comprises the step of sorting a sample, wherein the following parameters are modified:
  • 1) Cross-sectional shape (or vertical profile) of the thin/thick regions can be rectangular-shaped to trapezoidal-shaped. This is determined by the fabrication process of the nanofilter matrix.
  • 2) Different regions of the minimal sorting unit could have different nanofilters and different arrangement of the nanofilter arrays. (for example, different thin/thick channel thickness combination along the vertical direction of the minimal sorting unit).
  • 3) Surface potential (surface charge density) can be changed/modulated by applying external potential to the substrate, as a gate potential.
  • In one embodiment, the methods and/or devices of this invention provide separation capability of small biomolecules, such as SDS-protein complexes, small double stranded DNA molecules (100 bp-2 Kb), undenatured proteins and carbohydrates, for example in FIGS. 3 and 4 demonstrated continuous-flow separation of SDS-protein complexes in a 60 nm nanofilter array chip under the continuous field separation mode (FIG. 3) and pulse-field separation mode (FIG. 4), respectively. Since with the same fabrication techniques, the nanofilter thin region depth can be further reduced down to ˜10 nm; the present invention provides a means for size-based continuous-flow separation of small biomolecules such as proteins and carbohydrates by using an embodiment of the nanofilter array chip described herein. The invention, in one embodiment, thus provides a microfabricated molecular sieving chip that can size-fractionate small biomolecules such as SDS-protein complexes and small dsDNA molecules without using gel sieving matrices.
  • In another embodiment, the invention encompasses continuous-flow operation of the nanofilter array chip ideal for preparatory sample fractionation with increased sample throughput. The separation efficiency of the miniature nanofilter array chip is comparable to current state of the art systems (i.e. capillary gel electrophoresis) and because of their regular sieving structures, the nanofilter array chip can be further optimized based on the understanding about the sieving process during the passage of the molecules through the nanofilter.
  • In another embodiment, the devices of this invention can be used in continuous-flow separation of native proteins based on either the protein's charge or size, depending on the ionic strength of the buffer employed with the device. If the thickness of the electrical double layer, the Debye-length λD, is modulated to be comparable to the height of the nanochannel (e.g. under low ionic strength), similar sized biomolecules bearing different net charges will have different apparent diffusion coefficients through a nanochannel. Such charge-selective phenomena can be exploited with the devices of this invention, in some embodiments, in combination with active transport of biomolecules through the nanofluidic filter array, when operated at low ionic strength (1.3 mM).
  • For example, SiO2-surfaces in some embodiments of the devices of this invention, are negatively charged, leading to surface charge dominated transport through nanochannels, where in this embodiment, biomolecules with a lower negative net charge can jump through nanofilters with a greater probability than biomolecules bearing a higher negative net charge, resulting in distinct streams through the devices in this embodiment If the device according to this embodiment, is operated at high ionic strength (130 mM) where the Debye-length λD is negligible, size-based separation of native proteins is expected, because steric interactions between the biomolecules and the nanofilter walls are dominant. In some embodiments, probe biomolecules that are smaller than the nanofilters, are separated due to Ogston sieving.
  • In another embodiment, the device is filled with low ionic strength buffer so that the thickness of the electrical double layer λD is no longer negligible, as compared to the height of the shallow nanochannels h3. At these ionic strengths co-ions are excluded and counterions are enriched in nanometer-sized apertures, called the exclusion-enrichment effect. This charge-selectivity favors proteins with lower negative net charge for passage compared to biomolecules with higher negative net charge, resulting in a bigger drift in the x-direction with decreasing net negative charge as exemplified herein.
  • In another embodiment, the nanofilter array chip may be batch-fabricated in a cleanroom environment, is chemically and mechanically robust, and can be used over a long period without degradation of its characteristics. The chemical nature of nanofilter array surface can be tailored for a specific biomolecule to be analyzed. The nanofilter array chip allows the use of different buffer systems, enabling the integration of different biomolecule sensors and separation and reaction chambers in one single chip, without the concern of sieving matrix crosstalk and contamination. The separation resolution of the nanofilter array chip can be further improved by scaling down the nanofilter. Since sub-100 nm resolution photolithography is now routinely performed in the microelectronics industry, such nanofilter array chip can be easily manufactured in a commercial setting.
  • The highlighted features listed above, inter-alia, make the nanofilter array chip an ideal candidate as a separation scheme for a truly integrated proteomic sample-preparation microsystem that includes fully integrated multiple separation and purification steps.
  • In another embodiment, the sorters/devices of this invention and methods of use thereof allow for size fractionation of smaller proteins out of a complex biomolecule sample. In serum analysis of biomarkers, it is essential to fractionate smaller signaling molecules out of larger structural proteins such as albumin. Dialysis membrane fraction is not ideal for this application since they tend to lose smaller, low-abundance proteins, and such methods are ideally suited for use with the sorters/devices and/or methods of this invention.
  • In another embodiment, the sorters/devices of this invention and methods of use thereof allow for size fractionation of carbohydrates. There is no well-established separation technique for carbohydrate and sugar molecules, which limits the development of potentially important biosensing and diagnostic tools based on sugar molecules. The sorters/devices of this invention can be engineered to provide good separation efficiency for sugar molecules, which is very difficult to separate and analyze with current conventional techniques.
  • In another embodiment, the sorters/devices of this invention and methods of use thereof allow for size fractionation of nanoparticles, nanotubes and other nanotechnology tools. Nanotechnology requires nanoparticles or nanotubes that are uniform in size and shape, while the chemical synthesis processes for these nanoparticles often generate particles that are diverse in size and shape. The sorters/devices of this invention are useful for separating different nanoparticles, based on their size and shape, to obtain pure, well-characterized groups of nanoparticles.
  • In another embodiment the sorters/devicesofthis invention and methods of use thereof allow for DNA sequencing. The sequencing of DNA is performed by size-fractionation of a group of single stranded DNA using capillary gel electrophoresis with polymeric liquid sieving media. Since the separation efficiency and resolution of the sorters/devices of this invention is comparable to that of capillary gel electrophoresis, one could thus sequence the DNA without using liquid sieving media
  • In another embodiment, the sorters/devices of this invention and methods of use thereof allow for rapid, continuous fractionation of any biomolecule, for example, nucleic acid, proteins, carbohydrates, etc., by taking advantage of differential bidirectional transport of biomolecules with varying physico-chemical characteristics, for example size, charge, hydrophobicity, or combinations thereof, through periodic arrays of microfabricated nanofilters. The passage of biomolecules through the nanofilter is a function, in some embodiments, of steric or electrostatic interactions between charged macromolecules and charged nanofilter walls. Continuous-flow separation through the sorters/devices of this invention are applicable for molecules varying in terms of any molecular properties (e.g., size, charge density or hydrophobicity) that can lead to differential transport across the nanofilters. Embodiments of this invention include methods of utilization thereof for the separation of such biomolecules.
  • In some embodiments, the separation methods/devices/sorters of this invention will include varying voltage or ionic strength of the solutions utilized, which in turn may optimize separation of the biomolecules to specific streams, and/or optimization of the resolution or