AU4569699A - Creación de Inventos y Patentes con Inteligencia Artificial

AU4569699A – A microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same
– Google Patents

AU4569699A – A microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same
– Google Patents
A microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same

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Publication number
AU4569699A

AU4569699A
AU45696/99A
AU4569699A
AU4569699A
AU 4569699 A
AU4569699 A
AU 4569699A
AU 45696/99 A
AU45696/99 A
AU 45696/99A
AU 4569699 A
AU4569699 A
AU 4569699A
AU 4569699 A
AU4569699 A
AU 4569699A
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Australia
Prior art keywords
focusing
fluid
sample
channels
channel
Prior art date
1998-06-16
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Granted

Application number
AU45696/99A
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AU744627B2
(en

Inventor
Stephen C. Jacobson
J. Michael Ramsey
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UT Battelle LLC

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UT Battelle LLC
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1998-06-16
Filing date
1999-06-16
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2000-01-05

1999-06-16
Application filed by UT Battelle LLC
filed
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UT Battelle LLC

2000-01-05
Publication of AU4569699A
publication
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patent/AU4569699A/en

2002-02-28
Application granted
granted
Critical

2002-02-28
Publication of AU744627B2
publication
Critical
patent/AU744627B2/en

2019-06-16
Anticipated expiration
legal-status
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Status
Ceased
legal-status
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Current

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Classifications

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING

B01F33/00—Other mixers; Mixing plants; Combinations of mixers

B01F33/30—Micromixers

B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions

B01F33/3011—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING

B01F33/00—Other mixers; Mixing plants; Combinations of mixers

B01F33/30—Micromixers

B01F33/3031—Micromixers using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids

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

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials

G01N15/10—Investigating individual particles

G01N15/14—Electro-optical investigation, e.g. flow cytometers

G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means

G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis

G01N27/416—Systems

G01N27/447—Systems using electrophoresis

G01N27/44704—Details; Accessories

G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones

G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means

G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis

G01N27/416—Systems

G01N27/447—Systems using electrophoresis

G01N27/44756—Apparatus specially adapted therefor

G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means

G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis

G01N27/416—Systems

G01N27/447—Systems using electrophoresis

G01N27/44756—Apparatus specially adapted therefor

G01N27/44791—Microapparatus

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING

B01F33/00—Other mixers; Mixing plants; Combinations of mixers

B01F33/30—Micromixers

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

B01L2300/00—Additional constructional details

B01L2300/08—Geometry, shape and general structure

B01L2300/0809—Geometry, shape and general structure rectangular shaped

B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions

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

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

B01L2400/00—Moving or stopping fluids

B01L2400/04—Moving fluids with specific forces or mechanical means

B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces

B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

B01L2400/00—Moving or stopping fluids

B01L2400/04—Moving fluids with specific forces or mechanical means

B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces

B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic

B01L2400/0418—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electro-osmotic flow [EOF]

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

B01L2400/00—Moving or stopping fluids

B01L2400/04—Moving fluids with specific forces or mechanical means

B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces

B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic

B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow

B—PERFORMING OPERATIONS; TRANSPORTING

B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL

B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

B01L2400/00—Moving or stopping fluids

B01L2400/04—Moving fluids with specific forces or mechanical means

B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure

B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

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

B01L3/502738—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 integrated valves

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials

G01N15/10—Investigating individual particles

G01N15/14—Electro-optical investigation, e.g. flow cytometers

G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals

G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials

G01N15/10—Investigating individual particles

G01N15/14—Electro-optical investigation, e.g. flow cytometers

G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals

G01N15/1463—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals using image analysis for extracting features of the particle

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials

G01N15/10—Investigating individual particles

G01N15/14—Electro-optical investigation, e.g. flow cytometers

G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials

G01N15/10—Investigating individual particles

G01N15/14—Electro-optical investigation, e.g. flow cytometers

G01N2015/149—Sorting the particles

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation

G01N30/02—Column chromatography

G01N30/60—Construction of the column

G01N30/6095—Micromachined or nanomachined, e.g. micro- or nanosize

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S366/00—Agitating

Y10S366/01—Micromixers: continuous laminar flow with laminar boundary mixing in the linear direction parallel to the fluid propagation with or without conduit geometry influences from the pathway

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

Y10T137/00—Fluid handling

Y10T137/206—Flow affected by fluid contact, energy field or coanda effect [e.g., pure fluid device or system]

Y10T137/2082—Utilizing particular fluid

Abstract

A microfabricated device and method for electrokinetic transport of a liquid phase biological or chemical material is described. In accordance with one aspect of the present invention there is provided a microchip that is adapted for the simultaneous spatial confinement of electrokinetically driven fluidic material streams on a substrate. The apparatus includes a focusing chamber formed in a surface of the substrate and in fluid communication with two sample fluid channels and three focusing fluid channels. The device further includes electromotive means operatively connected to the sources of the sample fluid and the source of focusing fluid for electrokinetically driving the respective streams of the sample and focusing fluids through the respective channels into the focusing chamber such that the focusing fluid streams spatially confine the first and second sample fluid streams within the focusing chamber. In accordance with another aspect of this invention, there is provided a cytometry method for analyzing microscopic particles in a fluid medium on a microchip by utilizing the focusing function of the microchip. In the disclosed cytometry process the width of the fluid stream is narrowed in the focusing chamber. The microscopic particles in the focused sample fluid are then detected and/or measured using light scattering or other techniques.

Description

WO 99/66318 PCT/US99/13542 A MICROFABRICATED DEVICE AND METHOD FOR MULTIPLEXED ELECTROKINETIC FOCUSING OF FLUID STREAMS AND A TRANSPORT CYTOMETRY METHOD USING SAME 5 FIELD OF THE INVENTION 10 This invention relates to microfabricated designs for the electrokinetic manipulation of fluidic chemical and biological materials. More specifically, this invention provides a microchip device for spatially confining two or more sample material streams simultaneously by way of electrokinetic forces and a demonstration of transport cytometry. 15 BACKGROUND OF THE INVENTION In order to facilitate the development of the biological and chemical sciences, fluidic microchip technologies are increasingly utilized to perform traditional chemical laboratory functions within a controlled microfabricated 20 environment. Microfabricated chemical instrumentation, also know as lab-on-a chip technology, requires the development of a plurality of microfabricated functional elements or unit processes cooperatively linked on the microchip to perform small volume chemical and biochemical measurements. Presently, these «on-chip» laboratories facilitate the precise transport and -*1 – WO 99/66318 PCT/US99/13542 analysis of fluidic chemical and biological materials. Microchips are characterized by reduced analysis time and reagent consumption, ease of automation, and valveless fluid control of sub-nanoliter volumes. A variety of electrically driven separations have been performed within microchannel 5 networks. Microchips have also been developed for controlling chemical reactions, including arrays for solid-phase chemistry, reaction wells for polymerase chain reactions, channels with immobilized enzymes for transport injection analysis, and manifolds for homogenous enzyme assays. The ability to design and machine channel manifolds with low-volume 10 connections renders microchips suitable for combining several steps of an analytical process on one device. Microchips that combine chemical reactions with the speed of microscale CE analysis have been demonstrated for pre- and post-separation reactions, for DNA restriction digests with fragment sizing, and for cell lysis, multiplex PCR amplification and electrophoretic sizing. 15 Electrokinetic techniques, i.e., electroosmotically induced fluid transport and/or electrophoretic migration of ions, are the preferred methods of manipulating biological and chemical materials on microchip devices. The mixing of two or more liquid-phase materials or the dispensing of a reagent material on a microchip is accomplished by controlling the electric potentials 20 applied to the various reservoirs to electrokinetically drive the materials housed therein through the channels of the microchip. Electrophoresis transports charged species, whereas electroosmosis imparts a velocity to all ions and neutral species. Under conditions where both electroosmosis and electrophoresis are operative, the net velocity of an ion will be the vector sum of the electroosmotic and 25 electrophoretic velocities. Electrokinetic transport mechanisms are highly effective for demonstrating a number of highly useful experiments as identified above. Several applications require the ability to spatially confine a sample material stream with consistent reproducibility. This spatial confinement or -2- WO 99/66318 PCT/US99/13542 «electrokinetic focusing» refers to the use of electrokinetic transport to confine spatially the transport of both fluids and ions. An example of such focusing is disclosed in related co-pending Application Serial No. 08/721,264, filed September 26, 1996, which describes and shows a microfabricated device and 5 method for spatially confining a sample stream. More recently, a need has arisen for an improved microchip wherein a plurality of microfluidic sample streams can be electrokinetically focused simultaneously and a need has been identified for efficiently analyzing microscopic particles in a fluid stream. 10 SUMMARY OF THE INVENTION In accordance with one aspect of the present invention there is provided a fluidic microchip that is adapted for the simultaneous spatial confinement of electrokinetically driven fluidic material streams on a substrate. The apparatus 15 includes a focusing chamber formed in a surface of the substrate and two sample channels formed in the surface of said substrate for carrying sample fluid streams therethrough. The device also includes three focusing channels formed in the surface of said substrate for carrying focusing fluid streams therethrough. The sample channels have respective first ends in fluid communication with a source 20 of a sample fluid and respective second ends in fluid communication with the focusing chamber. The focusing channels are formed such that one sample channel is positioned between the first and second focusing channels and the second sample channel is positioned between the second and third focusing channels. The focusing channels have respective first ends in fluid 25 communication with a source of focusing fluid and respective second ends in fluid communication with the focusing chamber. The apparatus further includes electromotive means operatively connected to the sources of the sample fluid and the source of focusing fluid for electrokinetically driving the respective streams of the sample and focusing fluids through the respective channels into said -3- WO 99/66318 PCTIUS99/13542 focusing chamber such that the focusing fluid streams spatially confine the first and second sample fluid streams within the focusing chamber. In accordance with another aspect of this invention, there is provided a method for analyzing microscopic particles in a fluid stream on a microchip. 5 The method includes the step of conducting a stream of a sample fluid containing the microscopic particles through a sample fluid channel to a focusing chamber. In the process the width of the sample fluid stream is narrowed in the focusing chamber by electrokinetically transporting a focusing fluid into the focusing chamber on opposite sides of the sample fluid stream. The process includes the 10 further step of detecting the microscopic particles in the focused sample fluid at a preselected detection zone on the microchip. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, and the following detailed description, will be 15 better understood when read in conjunction with the attached drawings, in which: Figure 1 is a schematic diagram of a fluidic microchip for spatially focusing sample material streams in accordance with the present invention; Figure 2A is a CCD image of a focusing chamber in the fluidic microchip device shown in Figure 1; 20 Figure 2B is a CCD image of the simultaneous focusing of two fluidic materials in the focusing chamber of Figure 2A; Figure 3 is a schematic diagram of a second embodiment of a fluidic microchip in accordance with the present invention; Figure 4 is a schematic diagram of a microchip in accordance with the 25 present invention that is used for transport cytometry of microscopic particles; Figure 5 is a schematic representation of the operation of a microchip according to the present showing the use of such a microchip for transport cytometry of microscopic latex particles in a fluid stream; and Figure 6 is a graph of the arrival time distribution of the microscopic latex -4- WO 99/66318 PCT/US99/13542 particles shown in Figure 5 as detected at the observation zone following electrokinetic focusing and light scattering detection. DETAILED DESCRIPTION 5 The fluidic microchip according to this invention will be described in connection with two preferred embodiments thereof. In one embodiment, a focusing chamber is provided on the microchip in which two sample material streams are laterally confined or focused using a focusing fluid provided through three focusing channels. In the second embodiment, the microchip according to 10 this invention includes collection channels for receiving the focused sample material streams and a buffer channel for providing a buffer fluid to selectively direct the transport of the focused sample material streams into the collection channels. A method of analyzing a fluidic sample material using a microchip according to this invention is also described in connection with a working 15 example of transport cytometry. MULTIPLEXED ELECTROKINETIC FOCUSING A device embodying this invention uses electrokinetic forces to drive streams of fluidic materials through microfabricated channels. The 20 microfabricated device includes multiple sample and focusing channels in fluidic communication with a focusing chamber for spatially confining the streams of fluidic materials traveling therethrough simultaneously. The focusing chamber is formed at the confluence of the sample and focusing channels that are formed in the substrate of the microchip. The sample and focusing fluid streams are 25 electrokinetically manipulated such that the widths of the sample fluid streams can be substantially narrowed. Focusing of the sample fluid streams is achieved using electrophoresis, electroosmosis, or a combination thereof. The focusing or spatial confinement is achieved by using a fluidic buffer material that is passed to the focusing chamber through the focusing channels. The spatial confinement of -5- WO 99/66318 PCT/US99/13542 the sample fluid stream passing through the focusing chamber is caused by having a greater electrokinetic field strength in the focusing fluid channels relative to the sample fluid channels. Referring now to the drawings wherein like reference numerals refer to 5 the same or similar components across the several views, and in particular to Figure 1, there is shown a microfabricated (microchip) device 10 in accordance with the present invention. Microchip device 10 is designed and fabricated from a solid substrate material, preferably glass. However, silicon may also be used because of the well-developed technology permitting its precise and efficient 10 fabrication. Other materials, including polymers, quartz, fused silica, sapphire, or plastics are also suitable as substrate materials. The surface of the microfabricated device 10 is covered and sealed by a cover plate. The fluid conduction channels on microchip device 10 are formed using micromachining methods known to those skilled in the art. The micromachining 15 methods available include film deposition processes such as spin coating and chemical vapor deposition, laser fabrication or photolithographic techniques such as UV or X-ray processes, or etching methods which may be performed by either wet chemical processes or plasma processes. Preferably, the microchannel configurations are transferred onto the substrate material using a positive 20 photoresist, photomask, and UV exposure. The channels are etched into the substrate in a dilute, stirred HF/NH 4 F bath. The microfabricated device 10 has sample channels 28 and 36, focusing channels 40, 32, and 42, and waste channels 48, 50, and 46 formed in the surface thereof. Sample reservoirs 14 and 18 are formed or positioned at the ends of the 25 sample channels 28 and 36, respectively, such that the sample channels are in fluid communication with the respective reservoirs. Focusing reservoirs 12, 16 and 20 are similarly associated with the focusing channels 40, 32, and 42. A waste reservoir 24 is formed or positioned at the end of the waste channel 46. In the embodiment shown and described herein, the sample channels 28 and 36, the -6- WO 99/66318 PCT/US99/13542 focusing channels 40, 32, and 42, and the waste channels 48, 50, and 46 each have a nominal width of 42 ptm at half-depth and a nominal depth of 8.6 pm. An electric voltage source (not shown) is connected to the various reservoirs. Electrical contact to the reservoirs is made using platinum wire to 5 operably link the device 10 with the source of electric potential. The electroosmotic mobility for this microchip is that of native glass. Referring now to Figure 2A, a focusing chamber 22, is formed in the surface of the microchip device 10 at the confluence of sample channels 28 and 36, the focusing channels 40, 32, and 42, and the waste channels 48 and 50. In 10 the embodiment shown, the focusing chamber has a width of about 360 pm, a length of about 71 pm, and a depth of about 8.6 [tm. The channel and focusing chamber dimensions were measured using a stylus-based surface profiler and the channel widths and lengths reported were measured at half-depth. In the microchip device 10 shown and described herein, the sample fluid 15 is continuously infused through the focusing chamber. The electric potentials at the reservoirs are adjusted to increase or diminish the degree of lateral focusing. The potential applied at each sample and focusing reservoir is controlled independently, and the waste reservoir is grounded. Small variations in channel lengths and widths require that the voltages applied to the reservoirs differ 20 slightly for balancing the field strengths to obtain a symmetric fluid transport in the focusing chamber. The sample field strength is defined as the electric field strength in the sample channel, and correspondingly, the focusing field strength is the electric field strength in the focusing channels. All sample stream profiles are measured at full width half maximum (fwhm) at the exit of the focusing chamber 25 (inlet of the waste channel). Assuming that the experimental conditions, e.g., relative conductivities of the buffers, applied voltages, electroosmotic transport, remain unchanged, the variability of the stream width over time remains essentially constant. In the microchip device 10 according to this invention, it is not necessary -7- WO 99/66318 PCT/US99/13542 for the sample materials to traverse a narrow focusing chamber and transport into one or more narrow waste channels as depicted in Figure 2A. Instead, the sample and focusing channels may terminate in a focusing chamber having a width that spans all of the sample and focusing channels. In such case the focusing chamber 5 can be several mm or more in length. Focusing is still achieved with such an open focusing chamber design. However, spatial confinement of the sample streams is improved by about 1.6 times in a device as shown in Figure 2A wherein the three channels converge into a single, narrower waste channel. The focused stream width is proportioned to the width of the exit of the focusing 10 chamber (inlet of the waste channel). For example, in the embodiment shown in Figures 2A and 2B, the fluid streams in focusing channels 40 and 32 and sample channel 28 converge into waste channel 48, and the fluid streams in focusing channels 32 and 42 and sample channel 36 converge into waste channel 50. The narrow waste channels 48 and 50 provide a physical barrier, i.e., transport 15 obstruction, which improves the confinement of the electrokinetic focusing. This applies to single and multiple sample electrokinetic focusing chambers. Thus, tighter lateral confinement is achieved with narrower focusing chamber and waste channels. The multiplexed electrokinetic focusing effect provided by the microchip 20 device 10 as described herein was observed by using laser induced fluorescence (LIF) in connection with a charge-coupled device (CCD) for imaging. An argon ion laser beam (514.5 nm, 100 mW) was expanded to approximately a 5 mm diameter at the surface of the microfabricated device 10 using a lens. The fluorescence signal was collected using an optical microscope, filtered spectrally 25 (550 nm cut-on), and measured by the CCD. In the example operation, the buffer material used in focusing channels 40, 32, and 42 is 10 mM sodium tetraborate, and the sample channel material is rhodamine 6G (10 pM) in 10 mM buffer. The focusing effect on the sample fluid streams in sample channels 28 and 32 as they pass through the focusing chamber 22 is clearly observable in the CCD image of -8- WO 99/66318 PCT/US99/13542 Figure 2B. In Figure 2B the sample fluid streams 28 and 36 are simultaneously focused to an average width of about 8 im using the focusing microchip 10 of Figure 1 and 2A. The stream width is measured at the exit of the focusing 5 chamber. The sample channels 28 and 36 have a center-to-center spacing of 160 ptm. The average electric field strength in the sample channels (28 and 36) is 40 V/cm. The average field strength in the two outside focusing channels (40 and 42) is 400V/cm and the average field strength in the center focusing channel (32) is 750 V/cm. The field strength in the center focusing channel is almost twice the 10 magnitude of the field in the outer focusing channel because the center channel must aid in focusing the sample fluid streams in channels 28 and 36. The electric potentials at each focusing reservoir (12, 16, and 20) are individually controlled in order to tune the focusing effect of the focusing fluid and achieve a symmetrically focused sample stream. Such functionality permits greater 15 flexibility for studying transport dynamics but would not be necessary once a microchip design is finalized. Lateral spatial confinement (focusing) of the sample material stream from sample channel 28 occurs when the transport of focusing fluid into the focusing chamber 22 from each of the focusing channels 40 and 32 is greater than the 20 transport of sample fluid into focusing chamber 22 from sample channel 28. Likewise, lateral spatial confinement of the sample material stream in sample channel 36 occurs when the transport of focusing fluid into the focusing chamber 22 from each of the focusing channels 32 and 42 is greater than the transport of sample fluid into focusing chamber 22 from sample channel 36. If the focusing 25 flow is too large the sample is not electrokinetically transported from the sample reservoir to the focusing chamber. The focusing channel 32 in the middle of microchip 10 confines the sample streams in the two adjacent sample channels 28 and 36, thereby limiting the total number of focusing channels needed. The multiplexed focusing provided by a microchip fabricated in accordance with the -9- WO 99/66318 PCT/US99/13542 present invention can be extended to larger numbers of sample streams as necessary. Shown in Figure 3 is a second embodiment of a multiplexed, electrokinetic focusing device according to the present invention. A microchip 5 device 5 has a focusing chamber 22, focusing channels 40, 32, and 42, sample channels 28 and 36, and waste channels 48 and 50 which are formed on a substrate in the same manner as the embodiment shown in Figures 1 and 2A. However, the microchip device five also has two sample collection channels 75 and 77, as well as a buffer channel 80, which are interconnected with the waste 10 channels 48 and 50. Each of the collection channels is in fluidic communication with a collection reservoir (not shown) disposed at a remote end of the respective collection channel. In the microchip device 5, one or both of the sample streams of interest can be diverted to a collection channel after being probed at a location just downstream of the focusing chamber 22. In the embodiment shown in Figure 15 3, sample fluid from sample channel 28 is directed to waste channel 48 after exiting the focusing chamber. A buffer fluid is provided through the buffer channel 80 at a transport level sufficient to prevent the focused sample fluid stream from traveling into the sample collection channel 75. Sample fluid from sample channel 36 is directed to collection channel 77 after it exits the focusing 20 chamber 22. Such transport direction is achieved by lowering the electric potential applied to the sample collection reservoir that is in fluidic communication with sample collection channel 77. An alternative method of directing the focused sample fluid to the sample collection channel is to raise the electric potential at the waste reservoir for waste channel 50 relative to the 25 collection reservoir for the sample collection channel 77. The sample collection channels can be operated independently or in concert with each other. Transport CYTOMETRY Referring now to Figure 4, a method of performing transport cytometry is – 10- WO 99/66318 PCT/US99/13542 demonstrated using electrokinetic focusing of a sample fluid containing microscopic latex particles. A microfabricated device 7 includes a waste reservoir 96, focusing reservoirs 92 and 94, a sample reservoir 90, and a collection reservoir 98. A sample channel 100 is in fluid communication at one thereof with 5 the sample reservoir 90. Focusing channels 102 and 104 are in fluid communication at first ends thereof with focusing reservoirs 92 and 94, respectively. A waste channel 106 is in fluid communication at one thereof with the waste reservoir 96 and a collection channel 108 is in fluid communication at one thereof with the collection reservoir 98. A focusing chamber 22 is formed at 10 the confluence of the sample channel 100 and the focusing channels 102 and 104. A short channel segment 135 is provided on the downstream side of focusing chamber 22 to provide a probe region where microscopic particles in the sample fluid are analyzed. The probe region can be positioned at the exit of the focusing chamber 22 or slightly downstream of the focusing chamber 22 to minimize the 15 scattering of the light by the channel walls. An imaging device 160 and image control processor 150 are provided for obtaining and analyzing observable information about the particles. The microchip 7 has uniform channel dimensions of 53.5 im wide and 15.8 pm deep. Particle detection is preferably performed with a light scattering 20 technique, but other techniques such as fluorescence, absorbence, or refractive index could be used independently or in concert. The imaging device 160 is preferably embodied as an argon ion laser operating at a wavelength of 514.5 nm and a power of about 10 mW. The beam from the imaging device is focused to a spot approximately 50 m on the probe region 135 located slightly downstream of 25 the focusing chamber 22. Focusing of the laser beam is accomplished with a focusing lens having a 200mm focal length. The light scattered by the particles in the sample fluid is collected using a 100x (0.7 NA) objective lens. The scattered light is spatially filtered and measured using a photomultiplier tube. The photomultiplier response is amplified and recorded by processing means 150, – 11 – WO 99/66318 PCTIUS99/13542 preferably a Macintosh computer with an ADC interface and running Labview 4.1, a proprietary software product of National Instruments. In a working example of the cytometry process of this invention, a buffer solution of 10 mM sodium tetraborate containing latex spheres 1.89 pm in diameter was used. 5 The testing was performed with continuous transport of the particles through the focusing chamber 22, and the relative potentials at the reservoirs were varied to increase or diminish the degree of focusing of the sample fluid stream so that the focused stream was approximately commensurate with the diameter of the spatial filter used in the detection apparatus. The electric potentials applied at 10 the sample reservoir 90 and focusing reservoirs 92 and 94 were controlled independently, whereas the sample collection reservoir 98 was grounded. No electric potential was applied to the waste reservoir 96, i.e., it was allowed to float. Small variations in channel lengths and widths required that the voltages applied to the focusing reservoirs differ slightly to balance the field strengths and 15 to obtain symmetric fluid transport into the focusing chamber. Referring now to Figure 5, the manner in which the cytometry process according to this invention is carried out will be described. The particles 120 are transported from the sample reservoir 90 along sample channel 100. The stream of carrier fluid is transported to and confined laterally in the focusing chamber 22 20 as described above. The particles 120 pass through the probe region 135 and are detected using light scattering, fluorescence, or both. Figure 6 shows the arrival time distribution of the particles 120 after being electrokinetically focused and detected in the probe region 135. The scattering intensity is nearly uniform because of the narrow distribution of particle sizes and the small variation of their 25 lateral position resulting from the electrokinetic focusing. By alternating the electric potentials applied to the waste reservoir 96 and the collection reservoir 98 by way of the control processor 150, the latex particles 120 can be directed to either the waste reservoir or the collection reservoir based upon fluorescent signature of the particles observed in the probe region 135. It – 12 – WO 99/66318 PCTIUS99/13542 will be appreciated that the latex particles used in the present example, can be substituted by other types of particles such as cells, bacteria, other polymeric particles, inorganic particles, environmentally collected particles, molecules, etc. A microfabricated device in accordance with the present invention has 5 been described. The device employs electrokinetic focusing for spatially confining at least two streams of sample materials passing therethrough. In this way, the microfabricated device is capable of processing a plurality of sample materials simultaneously. The device of the present invention is advantageous for use in various analytical procedures, including cellular analysis. The spatial 10 confinement of the sample fluid in the focusing chamber allows high efficiency, high sensitivity fluorescence detection in a small volume and the parallel arrangement allows high sample throughput. Also, these devices can be operated with any combination of electrokinetic transport and pressure-driven flow to achieve comparable results. 15 The terms and expressions which have been employed are used as terms of description and not of limitation. There is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized, however, that various modifications such as channel dimension, location, and arrangement are possible within the scope of the 20 invention as claimed. – 13 –

Claims (12)

1. An apparatus for the simultaneous spatial confinement of electrokinetically driven material streams on a substrate, comprising: a focusing chamber formed in a surface of the substrate; 5 first and second sample channels formed in the surface of said substrate for carrying sample fluid streams therethrough, said sample channels having respective first ends in fluid communication with a source of a sample fluid and respective second ends in fluid communication with said focusing chamber; 10 first, second, and third focusing channels formed in the surface of said substrate for carrying focusing fluid streams therethrough, said focusing channels being formed such that said first sample channel is positioned between said first and second focusing channels and said second sample channel is positioned between said second and third focusing channels, said focusing 15 channels having respective first ends in fluid communication with a source of focusing fluid and respective second ends in fluid communication with said focusing chamber; and electromotive means operatively connected to the source of the sample fluids and the source of focusing fluid for electrokinetically driving the 20 respective streams of the sample and focusing fluids through the respective channels into said focusing chamber such that the focusing fluid streams spatially confine the first and second sample fluid streams within said focusing chamber.

2. An apparatus according to claim 1 comprising first and second waste 25 channels formed in the surface of said substrate, said waste channels having respective first ends in fluid communication with said focusing chamber and having respective second ends in fluid communication with a waste reservoir. – 14 – WO 99/66318 PCT/US99/13542

3. An apparatus according to claim 2 comprising: first and second sample collection channels formed in the substrate for carrying the respective streams of the sample fluid, said sample collection channels having respective first ends in fluid communication with corresponding 5 waste channels and having respective second ends in fluid communication with first and second collection reservoirs, respectively; and a buffer channel formed in the substrate for carrying a stream of buffer fluid, said buffer channel having a first end in fluid communication with a source of buffer fluid and a second end in fluid communication with said first and 10 second waste channels; and electromotive means operatively connected to the source of the buffer fluid, the collection reservoir, and the waste reservoir for electrokinetically driving the buffer fluid through the buffer channel to direct the streams of the sample fluid to corresponding waste channels, to corresponding collection 15 channels, or to a combination of a waste channel and a collection channel.

4. A method for analyzing microscopic particles in a fluid medium on a microchip comprising the steps of: conducting a stream of a sample fluid containing microscopic particles 20 through a sample fluid channel to a focusing chamber; narrowing the width of the sample fluid stream in the focusing chamber by electrokinetically transporting a focusing fluid into the focusing chamber on opposite sides of the sample fluid stream; and detecting the microscopic particles in the focused sample fluid at a 25 preselected detection zone.

5. A method as set forth in Claim 4 wherein the step of narrowing the width of the sample fluid stream comprises the step of adjusting the width of the sample fluid stream in the focusing chamber to correspond substantially to the detection – 15- WO 99/66318 PCT/US99/13542 probe region.

6. A method as set forth in Claim 4, wherein the step of analyzing the microscopic particles comprises the step of measuring a physical characteristic of 5 the microscopic particles.

7. A method as set forth in Claim 6 comprising the steps of: comparing the measured physical characteristic to a reference value for the physical characteristic; 10 diverting the sample fluid stream to a first reservoir when the measured physical characteristic has a first quantitative relationship to the reference value; and diverting the sample fluid stream to a second reservoir when the measured physical characteristic has a second quantitative relationship to the reference 15 value.

8. Apparatus for analyzing microscopic particles in a fluid medium on a microchip having a substrate, comprising: a focusing chamber formed in a surface of the substrate; 20 a sample channel formed in the surface of said substrate for carrying sample fluid stream therethrough, said sample channel having a first end in fluid communication with a source of a sample fluid containing microscopic particles; first and second focusing channels formed in the surface of said 25 substrate for carrying focusing fluid streams therethrough, said focusing channels being formed such that said sample channel is positioned between said first and second focusing channels, said focusing channels having respective first ends in fluid communication with a source of focusing fluid and respective second ends in fluid communication with said focusing chamber; – 16- WO 99/66318 PCT/US99/13542 electromotive means operatively connected to the source of the sample fluid and the source of focusing fluid for electrokinetically driving the respective streams of the sample and focusing fluids through the respective channels into said focusing chamber such that the focusing fluid streams spatially 5 confine the sample fluid stream within said focusing chamber to a width that corresponds substantially to the detection probe region; and means for detecting the microscopic particles in the spatially confined sample fluid at a preselected detection zone on the microchip. 10

9. An apparatus as set forth in Claim 8 comprising a waste channel formed in the surface of said substrate, said waste channel having a first end in fluid communication with said focusing chamber and a second end in fluid communication with a waste reservoir. 15

10. An apparatus as set forth in Claim 9 comprising a collection channel formed in the surface of said substrate, said collection channel having a first end in fluid communication with said focusing chamber and a second end in fluid communication with a collection reservoir. 20

11. An apparatus as set forth in Claim 10 wherein the means for detecting the microscopic particles comprises analyzing means for measuring a physical characteristic of the microscopic particles.

12. An apparatus as set forth in Claim 11 wherein the analyzing means 25 comprises: comparing means for comparing the measured physical characteristic to a reference value for the physical characteristic; and diverting means for diverting the sample fluid stream to a first reservoir when the measured physical characteristic has a first quantitative relationship to – 17- WO 99/66318 PCTIUS99/13542 the reference value and for diverting the sample fluid stream to a second reservoir when the measured physical characteristic has a second quantitative relationship to the reference value. 5 -18-

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1999-12-23

EP1093579A4
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EP1093579B1
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2010-09-01

DE69942725D1
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2010-10-14

AU744627B2
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2002-02-28

JP3885147B2
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2007-02-21

US6120666A
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2000-09-19

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