U.S. patent application number 10/606158 was filed with the patent office on 2005-09-01 for microfluidic devices for electrophoretic analysis of materials.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Parce, J. Wallace.
Application Number | 20050189224 10/606158 |
Document ID | / |
Family ID | 24742502 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050189224 |
Kind Code |
A1 |
Parce, J. Wallace |
September 1, 2005 |
Microfluidic devices for electrophoretic analysis of materials
Abstract
The present invention provides a microfluidic system for fast,
accurate and low cost electrophoretic analysis of materials in the
fields of chemistry, biochemistry, biotechnology, molecular biology
and numerous other fields. Light from periodically spaced regions
along a channel in the microfluidic system are received by a
photodetector. The intensity of light received by the photodetector
is modulated by the movement of species bands through the channel
under electrophoretic forces. By Fourier analysis, the velocity of
each species band is determined and the identification of the
species is made by its electrophoretic mobility in the channel.
Inventors: |
Parce, J. Wallace; (Palo
Alto, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Technologies Corp.
Mountain View
CA
|
Family ID: |
24742502 |
Appl. No.: |
10/606158 |
Filed: |
June 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10606158 |
Jun 25, 2003 |
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09977056 |
Oct 12, 2001 |
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6590653 |
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09977056 |
Oct 12, 2001 |
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09378169 |
Aug 19, 1999 |
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6337740 |
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09378169 |
Aug 19, 1999 |
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09207864 |
Dec 8, 1998 |
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6233048 |
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09207864 |
Dec 8, 1998 |
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08941679 |
Sep 30, 1997 |
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5852495 |
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08941679 |
Sep 30, 1997 |
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08683080 |
Jul 16, 1996 |
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5699157 |
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Current U.S.
Class: |
204/450 ;
204/600 |
Current CPC
Class: |
G01N 27/44721 20130101;
G01N 27/44791 20130101; Y10S 366/02 20130101 |
Class at
Publication: |
204/450 ;
204/600 |
International
Class: |
G01N 027/26 |
Claims
What is claimed is:
1. A method of separating components of a plurality of subject
materials in a microfluidic system, the method comprising: flowing
a first fluid comprising a series of a plurality of subject
materials through a first microchannel to a separation channel by
applying pressure to the first fluid wherein the pressure is
applied to the first fluid by a pressure source external to the
first microchannel; and applying an electrophoretic force to the
first fluid to introduce individual ones of the plurality of
subject materials into the separation channel, and to separate each
of the plurality of subject materials into its respective sample
components.
2. The method of claim 1, wherein the first microchannel is present
in a body structure, wherein the pressure is applied to the fluid
by a micropump present in the body structure.
3. The method of claim 1, wherein at least one of the plurality of
subject materials comprises one or more of: a nucleic acid, a
chemical compound, a mixture of chemical compounds, a
polysaccharide, a peptide, a protein, and a biological extract.
4. The method of claim 1, wherein each of the plurality of subject
materials is separated into one or more components in the
separation channel by the application of an electrophoretic
force.
5. The method of claim 1, wherein each of the plurality of subject
materials comprises one or more fluorescent labeled species.
6. The method of claim 1, further comprising the step of detecting
the respective sample components of each of the plurality of
subject materials.
7. The method of claim 6, wherein the detecting is performed with a
detector mounted proximal to the separation channel.
8. The method of claim 7, the detector comprising one or more of: a
light source, a lens, an optical filter, a band pass filter, and a
photoreceptor.
9. The method of claim 6, wherein detecting the sample components
comprises analyzing signal frequency from the detector.
10. A device for separating components of a fluid, the device
comprising: a microfluidic device comprising a body structure
comprising at least first and second intersecting microchannels
fabricated therein; a pressure source fluidly coupled to the first
microchannel; and a voltage or current controller operably coupled
to the second microchannel; wherein the first microchannel is
fluidly coupled to at least one source of a plurality of different
subject materials.
11. The device of claim 10, wherein the pressure source is external
to the body structure.
12. The device of claim 10, wherein the pressure source is a
micropump in the body structure.
13. The device of claim 10, wherein the voltage or current
controller is coupled to the second microchannel via electrodes
positioned in one or more reservoir, which reservoir is fluidly
coupled to the second microchannel.
14. The device of claim 10, wherein the voltage or current
controller comprises a voltage controller that provides selectable
voltage levels.
15. The device of claim 10, wherein the device comprises reservoirs
at termini of each of the at least two channels.
16. The device of claim 15, wherein the voltage or current
controller comprises a voltage controller that provides selectable
voltage levels to more than one of the reservoirs.
17. The device of claim 10, wherein the second microchannel
comprises reservoirs at termini of the second microchannel.
18. The device of claim 17, comprising one or more electrodes in
one or more of the reservoirs.
19. The device of claim 10, further comprising a detector mounted
proximal to the second microchannel.
20. The device of claim 19, comprising one or more of: a light
source, a lens, an optical filter, a band pass filter, and a
photoreceptor.
21. The device of claim 19, further comprising a frequency analyzer
coupled to the detector.
22. The device of claim 21, wherein the frequency analyzer breaks
the electrical signals into their component frequencies.
23. The device of claim 10, wherein the first microchannel is
fluidly coupled to sources of a plurality of different subject
materials through an electropipettor.
24. The device of claim 10, the first microchannel is fluidly
coupled to a plurality of reservoirs, each of which contains one of
the plurality of different subject materials.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/977,056 filed Oct. 12, 2001, which is a
continuation of U.S. patent application Ser. No. 09/378,169 filed
Aug. 19, 1999 (now U.S. Pat. No. 6,337,740), which is a
continuation of U.S. patent application Ser. No. 09/207,864 filed
Dec. 8, 1998 (now U.S. Pat. No. 6,233,048), which is a continuation
of U.S. patent application Ser. No. 08/941,679 filed Sep. 30, 1997
(now U.S. Pat. No. 5,852,495), which is a continuation of U.S.
patent application Ser. No. 08/683,080 filed Jul. 16, 1996 (now
U.S. Pat. No. 5,699,157).
BACKGROUND OF THE INVENTION
[0002] There has been a growing interest in the manufacture and use
of microfluidic systems for the acquisition of chemical and
biochemical information. Techniques commonly associated with the
semiconductor electronics industry, such as photolithography, wet
chemical etching, etc., are used in the fabrication of these
microfluidic systems. The term, "microfluidic", refers to system or
devices having channels and chambers are generally fabricated at
the micron or submicron scale, e.g., having at least one
cross-sectional dimension in the range of from about 0.1 .mu.m to
about 500 .mu.m. Early discussions of the use of planar chip
technology for the fabrication of microfluidic systems are provided
in Manz et al., Trends in Anal. Chem. (1990) 10(5): 144-149 and
Manz et al., Avd. in Chromatog. (1993) 33:1-66, which describe the
fabrication of such fluidic devices and particularly microcapillary
devices, in silicon and glass substrates.
[0003] Application of microfluidic systems are myriad. For example,
International Patent Appln. WO 96/04547, published Feb. 15, 1996,
describes the use of microfluidic systems for capillary
electrophoresis, liquid chromatography, flow injection analysis,
and chemical reaction and synthesis. U.S. Pat. No. 5,942,443
entitled "HIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROSCALE
FLUIDIC DEVICES", filed Jun. 28, 1996 by J. Wallace Parce et al.
and assigned to the present assignee, discloses wide ranging
applications of microfluidic systems in rapidly assaying compounds
for their effects on chemical, and preferably, biochemical systems.
The phase, "biochemical system," generally refers to a chemical
interaction which involves molecules of the type generally found
within living organisms. Such interactions include the full range
of catabolic and anabolic reactions which occur in living systems
including enzymatic, binding, signalling and other reactions.
Biochemical systems of particular interest include, e.g.,
receptor-ligand interactions, enzyme-substrate interactions,
cellular signalling pathways, transport reactions involving model
barrier systems (e.g., cells or membrane fractions) for
bioavailability screening, and a variety of other general
systems.
[0004] As disclosed in International Patent Appln. WO 96/04547 and
U.S. Pat. No. 5,942,443 noted above, one of the operations which is
suitable for microfluidic systems is capillary electrophoresis. In
capillary electrophoresis charged molecular species, such as
nucleic acids or proteins, for example, are separated in solution
by an electric field. With very small capillary tubes as separation
channels in a microfluidic system, resolution is enhanced because
band broadening due to thermal convection is minimized. The
requirement of only a small amount of sample material containing
the molecular species is a further advantage of capillary
electrophoresis in microfluidic systems.
[0005] Nonetheless, there is still room for improvement in
capillary electrophoresis. One of the goals of microfluidic systems
is high throughput. Presently capillary electrophoresis in
microfluidic systems is performed by the observation of separating
bands of species migrating in a separation channel under an
electric field. The electrophoretic mobility of a species is
determined by the time required from the entry of a test compound
material into the separation channel for a species band from the
test compound material to pass a detection point along the
separation channel. The operation is completed after the last
species band clears the detection point. See, for example, the
above-cited International Patent Appln. WO 96/04547. While these
operations are fast compared to macroscale electrophoretic methods,
the operations fall short of a highly automated microfluidic
system, such as disclosed in the above-mentioned U.S. Pat. No.
5,942,443, for example.
[0006] In contrast, the present invention solves or substantially
mitigates these problems. With the present invention, the
electrophoretic mobility of each species is determined as the
various species undergo electrophoresis in a microfluidic system.
Identification of each species can be made automatically.
SUMMARY OF THE INVENTION
[0007] The present invention provides for a microfluidic system for
high-speed electrophoretic analysis of subject materials for
applications in the fields of chemistry, biochemistry,
biotechnology, molecular biology and numerous other areas. The
system has a channel in a substrate, a light source and a
photoreceptor. The channel holds subject materials in solution in
an electric field so that the materials move through the channel
and separate into bands according to species. The light source
excites fluorescent light in the species bands and the
photoreceptor is arranged to receive the fluorescent light from the
bands. The system further has a means for masking the channel so
that the photoreceptor can receive the fluorescent light only at
periodically spaced regions along the channel. The system also has
an unit connected to analyze the modulation frequencies of light
intensity received by the photoreceptor so that velocities of the
bands along the channel are determined. This allows the materials
to be analyzed.
[0008] In accordance with the present invention, the microfluidic
system can also be arranged to operate with species bands which
absorb the light from the light source. The absorbance of light by
the species bands creates the modulation in light intensity which
allow the velocities of the bands along the channel to be
determined and the subject material to be analyzed.
[0009] The present invention also provides for a method of
performing high-speed electrophoretic analysis of subject
materials. The method comprises the steps of holding the subject
materials in solution in a channel of a microfluidic system;
subjecting the materials to an electric field so that the subject
materials move through the channel and separate into species bands;
directing light toward the channel; receiving light from
periodically spaced regions along the channel simultaneously; and
analyzing the frequencies of light intensity of the received light
so that velocities of the bands along the channel can be determined
for analysis of said materials. The determination of the velocity
of a species band determines the electrophoretic mobility of the
species and its identification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic illustration of one embodiment of a
microfluidic system;
[0011] FIG. 2A is a representation of the details of a portion of
the microfluidic system according to one embodiment of the present
invention; FIG. 2B is a detailed representation of a portion of the
separation channel of microfluidic system of FIG. 2A;
[0012] FIG. 3A represents an alternative arrangement of the portion
of the microfluidic system according to another embodiment of the
present invention; FIG. 3B is a detailed representation of a
portion of the separation channel of microfluidic system of FIG.
3A; and
[0013] FIG. 4 represents still another arrangement of portion of
the microfluidic system according to another embodiment of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0014] General Description of Microfluidic Systems
[0015] FIG. 1 discloses a representative diagram of an exemplary
microfluidic system 100 according to the present invention. As
shown, the overall device 100 is fabricated in a planar substrate
102. Suitable substrate materials are generally selected based upon
their compatibility with the conditions present in the particular
operation to be performed by the device. Such conditions can
include extremes of pH, temperature, salt concentration, and
application of electrical fields. Additionally, substrate materials
are also selected for their inertness to critical components of an
analysis or synthesis to be carried out by the system.
[0016] Useful substrate materials include, e.g., glass, quartz and
silicon, as well as polymeric substrates, e.g., plastics. In the
case of conductive or semiconductive substrates, there should be an
insulating layer on the substrate. This is particularly important
where the device incorporates electrical elements, e.g., electrical
fluid direction systems, sensors and the like, or uses
electroosmotic forces to move materials about the system, as
discussed below. In the case of polymeric substrates, the substrate
materials may be rigid, semi-rigid, or non-rigid, opaque,
semi-opaque or transparent, depending upon the use for which they
are intended. For example, devices which include an optical or
visual detection element, are generally fabricated, at least in
part, from transparent materials to allow, or at least, facilitate
that detection. Alternatively, transparent windows of, e.g., glass
or quartz, may be incorporated into the device for these types
detection elements. Additionally, the polymeric materials may have
linear or branched backbones, and may be crosslinked or
non-crosslinked. Examples of particularly preferred polymeric
materials include, e.g., polydimethylsiloxanes (PDMS),
polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone,
polycarbonate and the like.
[0017] The system shown in FIG. 1 includes a series of channels
110, 112, 114 and 116 fabricated into the surface of the substrate
102. As discussed in the definition of "microfluidic," these
channels typically have very small cross sectional dimensions,
preferably in the range from about 0.1 .mu.m to about 100 .mu.m.
For the particular applications discussed below, channels with
depths of about 10 .mu.m and widths of about 60 .mu.m work
effectively, though deviations from these dimensions are also
possible.
[0018] Manufacturing of these channels and other microscale
elements into the surface of the substrate 102 may be carried out
by any number of microfabrication techniques that are well known in
the art. For example, lithographic techniques may be employed in
fabricating glass, quartz or silicon substrates, for example, with
methods well known in the semiconductor manufacturing industries.
Photolithographic masking, plasma or wet etching and other
semiconductor processing technologies define microscale elements in
and on substrate surfaces. Alternatively, micromachining methods,
such as laser drilling, micromilling and the like, may be employed.
Similarly, for polymeric substrates, well known manufacturing
techniques may also be used. These techniques include injection
molding techniques or stamp molding methods where large numbers of
substrates may be produced using, e.g., rolling stamps to produce
large sheets of microscale substrates, or polymer microcasting
techniques where the substrate is polymerized within a
microfabricated mold.
[0019] Besides the substrate 102, the microfluidic system includes
an additional planar element (not shown) which overlays the
channeled substrate 102 to enclose and fluidly seal the various
channels to form conduits. The planar cover element may be attached
to the substrate by a variety of means, including, e.g., thermal
bonding, adhesives or, in the case of glass, or semi-rigid and
non-rigid polymeric substrates, a natural adhesion between the two
components. The planar cover element may additionally be provided
with access ports and/or reservoirs for introducing the various
fluid elements needed for a particular screen.
[0020] The system 100 shown in FIG. 1 also includes reservoirs 104,
106 and 108, which are disposed and fluidly connected at the ends
of the channels 114, 116 and 110 respectively. As shown, sample
channel 112, is used to introduce a plurality of different subject
materials into the device. It should be noted that the term,
"subject materials," simply refers to the material, such as a
chemical or biological compound, of interest. Subject compounds may
include a wide variety of different compounds, including chemical
compounds, mixtures of chemical compounds, e.g., polysaccharides,
small organic or inorganic molecules, biological macromolecules,
e.g., peptides, proteins, nucleic acids, or extracts made from
biological materials, such as bacteria, plants, fungi, or animal
cells or tissues, naturally occurring or synthetic
compositions.
[0021] Many methods have been described for the transport and
direction of fluids, e.g., samples, analytes, buffers and reagents,
within microfluidic systems or devices. One method moves fluids
within microfabricated devices by mechanical micropumps and valves
within the device. See, published U.K. Patent Application No. 2 248
891 (Oct. 18, 1990), published European Patent Application No. 568
902 (May 2, 1992), U.S. Pat. No. 5,271,724 (Aug. 21, 1991) and U.S.
Pat. No. 5,277,556 (Jul. 3, 1991). See also, U.S. Pat. No.
5,171,132 (Dec. 21, 1990) to Miyazaki et al. Another method uses
acoustic energy to move fluid samples within devices by the effects
of acoustic streaming. See, published PCT Application No. 94/05414
to Northrup and White. A straightforward method applies external
pressure to move fluids within the device. See, e.g., the
discussion in U.S. Pat. No. 5,304,487 to Wilding et al.
[0022] While these methods could be used to transfer the test
compound materials to the separation channel for electrophoresis, a
preferable method uses electric fields to move fluid materials
through the channels of a microfluidic system. See, e.g., published
European Patent Application No. 376 611 (Dec. 30, 1988) to Kovacs,
Harrison et al., Anal. Chem. (1992) 64:1926-1932 and Manz et al. J.
Chromatog. (1992) 593:253-258, U.S. Pat. No. 5,126,022 to Soane.
Electrokinetic forces have the advantages of direct control, fast
response and simplicity. Furthermore, the use of electrokinetic
forces to move the subject materials about the channels of the
microfluidic system 100 is consistent with the use of
electrophoretic forces in the separation channel 110.
[0023] To provide such electrokinetic transport, the system 100
includes a voltage controller that is capable of applying
selectable voltage levels, simultaneously, to each of the
reservoirs, including ground. Such a voltage controller can be
implemented using multiple voltage dividers and multiple relays to
obtain the selectable voltage levels. Alternatively, multiple
independent voltage sources may be used. The voltage controller is
electrically connected to each of the reservoirs via an electrode
positioned or fabricated within each of the plurality of
reservoirs. See, for example, published International Patent
Application No. WO 96/04547 to Ramsey, which is incorporated herein
by reference in its entirety for all purposes.
[0024] Alternatively, rather than voltage, another electrical
parameter, such as current, may be used to control the flow of
fluids through the channels. A description of such alternate
electrical parametric control is found in U.S. Pat. No. 5,800,690,
entitled "VARIABLE CONTROL OF ELECTROOSMOTIC AND/OR ELECTROPHORETIC
FORCES WITHIN A FLUID-CONTAINING STRUCTURE VIA ELECTRICAL FORCES",
filed Jul. 3, 1996 by Calvin Y. H. Chow and J. Wallace Parce and
assigned to the present assignee. This application is incorporated
herein by reference in its entirety for all purposes.
[0025] Stated more precisely, electrokinetic forces may be
separated into electroosmotic forces and electrophoretic forces.
The fluid control systems used in the system of the present
invention employ electroosmotic force to move, direct and mix
fluids in the various channels and reaction chambers present on the
surface of the substrate 102. In brief, when an appropriate fluid
is placed in a channel or other fluid conduit having functional
groups present at the surface, those groups can ionize. For
example, where the surface of the channel includes hydroxyl
functional groups at the surface, protons can leave the surface of
the channel and enter the fluid. Under such conditions, the surface
possesses a net negative charge, whereas the fluid possesses an
excess of protons or positive charge, particularly localized near
the interface between the channel surface and the fluid.
[0026] By applying an electric field across the length of the
channel, cations flow toward the negative electrode. Movement of
the positively charged species in the fluid pulls the solvent with
them. The steady state velocity of this fluid movement is generally
given by the equation: 1 v = E 4
[0027] where v is the solvent velocity, .epsilon. is the dielectric
constant of the fluid, .xi. is the zeta potential of the surface, E
is the electric field strength, and .eta. is the solvent viscosity.
Thus, as can be easily seen from this equation, the solvent
velocity is directly proportional to the zeta potential and the
applied field.
[0028] Besides electroosmotic forces, there are also
electrophoretic forces which affect charged molecules as they move
through the system 100. In the transport of subject materials from
one point to another point in the system 100, it is often desirable
for the composition of the subject materials to remain unaffected
in the transport, i.e., that the subject materials are not
electrophoretically differentiated in the transport until desired.
To do so, the subject materials are transported in fluid slug
regions 120 of predetermined ionic concentrations. The regions are
separated by buffer regions of varying ionic concentrations and
represented by buffer regions 121 in FIG. 1. A related patent
application, U.S. Pat. No. 5,779,868, entitled "ELECTROPIPETTOR AND
COMPENSATION MEANS FOR ELECTROPHORETIC BIAS," filed Jun. 28, 1996
by J. Wallace Parce and Michael R. Knapp, and assigned to the
present assignee, explains various arrangements of slugs, and
buffer regions of high and low ionic concentrations in transporting
subject materials with electrokinetic forces. The application is
incorporated herein by reference in its entirety for all purposes.
The application also explains how the channel 112 may be fluidly
connected to a source of large numbers of separate subject
materials which are individually introduced into the sample channel
112 and subsequently into the separation channel 110 for
analysis.
[0029] Electrophoresis in Microfluidic System and Operation
[0030] As described in the above-cited International Patent Appln.
WO 96/04547 and the previously mentioned U.S. Pat. No. 5,942,443
entitled "HIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROSCALE
FLUIDIC DEVICES", the disclosures of which are incorporated herein
by reference for all purposes, the slugs 120 of subject materials,
separated by buffers 121, are moved through the sample channel 112
and into the separation channel 110. Each slug 120 is subjected to
an electric field in the channel 110 so that the constituent
species in each slug 120 separates into species bands 123, as shown
in FIG. 1.
[0031] When the slugs 120 of subject materials are placed in the
separation channel 110, the materials are subjected to an electric
field by creating a large potential difference between the
terminals in the reservoir 104 and 108. The species in the slugs
separate according to their electric charges and sizes of their
molecules. The species are subjected to electric fields in the
range of 200 volts/cm. In accordance with one aspect of the present
invention, the species are labeled with fluorescent label
materials, such as fluorescent intercalating agents, such as
ethidium bromide for polynucleotides, or fluorescein isothiocyanate
or fluorescamine for proteins, as is typically done with
conventional electrophoresis.
[0032] As shown in FIG. 2A, the arrangement has a light source 120,
a first lens 124, a mask 122, the separation channel 110, a second
lens 126, a filter 128, and a photoreceptor 130 connected to a
frequency analyzer unit 134. The light source 120 emits light at
wavelengths to energize the fluorescent labels of the species in
the separation channel 110. Lamps, lasers and light-emitting diodes
may be used for the source 120. The mask 122 is located between the
light source 120 and the separation channel 110 and blocks light
from reaching selected portions of the channel 110.
[0033] The projection of the mask 122 by the light source 120 onto
the separation channel 110 results in a series of alternating
illuminated and darkened regions which are equally spaced along the
channel 110. Each darkened region 140 has the same width as another
darkened region along the separation channel 110 and is
approximately the same width as the species bands 123 in the
separation channel 110, as shown in FIG. 2B. The illuminated
regions 142 along the separation channel 110 are also approximately
the same width as the darkened regions 140. For example, with a
separation column approximately 10 .mu.m deep and 60 .mu.m wide,
the illuminated and darkened regions 142 and 140 are approximately
50-500 .mu.m along the separation channel 110.
[0034] As each species band from the sample slugs travel through
the alternating darkened and illuminated regions 140 and 142
respectively, the species bands 123 are alternately fluorescent in
the illuminated regions 142 and unlit in the darkened regions 140.
As each species travels down the separating channel 110, the
species fluoresces off and on with a characteristic frequency
corresponding to its velocity along the channel 110. The velocity,
v, of the particular species is directly related to the
electrophoretic mobility, .mu..sub.ep, of that species:
v=.mu..sub.ep.multidot.E
[0035] where E is the electric field. Thus a plurality of different
species moving through the separation channel 110 fluoresces at a
plurality of frequencies, each corresponding to a particular
species.
[0036] The light from the separation channel 110 is focussed by the
lens 126 upon the photoreceptor 130. The light received by the
photoreceptor 130, which may be a photomultiplier tube, a
photodiode, a CCD array, and the like, is converted into electrical
signals which are, in turn, sent to the frequency analyzer unit
134. The frequency analyzer unit 134, by straightforward Fourier
analysis, breaks the electrical signals into their component
frequencies. These electrical signal frequencies are the same as
that of the modulated light intensities generated by the species
undergoing electrophoresis in the separation channel 110. The
frequency of light intensity is related to the electrophoretic
mobility of each species band. Hence, a computer unit with a
calibrated look-up table can automatically identify each species
according to its electrical signal frequency from the frequency
analyzer unit 134. The electrophoresis operation is entirely
automated.
[0037] Note that each species band 123 need not pass completely
through the separation channel 110. Identification occurs as soon
as a characteristic optical modulation frequency is generated after
the species passes through a predetermined number of alternating
darkened and illuminated regions in the channel 110. Thus
electrophoresis is performed in a matter of seconds.
[0038] As stated above, the mask 122 is arranged such that the
alternating darkened and illuminated regions are approximately the
same width along the separation channel 110 with respect to each
other and to the widest species band. This ensures the largest
possible variation between the maxima and minima of light intensity
from the fluorescent species bands passing through the mask
regions.
[0039] As symbolically shown in FIG. 2A, the photoreceptor 130 is
placed along an axis formed with the light source 120, the mask 122
and the lens 126. An alternative arrangement has the light source
120 and the mask 122 off the axis so that light from the source 120
directed toward the separation channel 110 is also directed away
from the photoreceptor 130. This arrangement allows the
photoreceptor 130 to be illuminated only by the fluorescent light
from the labeled species in the channel 110. Furthermore, to avoid
contamination of the optical signals received by the photoreceptor
130, a filter 128 may be used for the photoreceptor 130. The filter
128 is a band-pass filter transmitting light only at wavelengths
emitted by the fluorescent species, and blocking light at other
wavelengths, i.e., light from the source 120. Alternatively, the
filter 128 might be selective toward blocking light at the light
source wavelengths. Typically, the fluorescent label materials
fluoresce at longer wavelengths than those of the source 120. For
example, for polynucleotides labeled with ethidium bromide as
subject materials for electrophoresis, a light source emitting
light at 540 nm is used and the species bands fluoresce at 610 nm.
For proteins labelled with fluorescein, a light source at 490 nm
works with species bands fluorescing at 525 nm.
[0040] As described above, the mask 122 is projected onto the
separation channel 110. An alternative arrangement imposes the mask
122 onto the substrate itself so that a series of alternating
darkened and light regions are created along the channel 110. Such
an arrangement is illustrated in FIG. 3A. The light source 120
illuminates the species bands 122 in the separation channel 110
directly. On the side of the channel 110 toward the photoreceptor
120, a mask 150 of alternating darkened and transparent regions 154
and 152 respectively are placed on the substrate 152, as shown in
FIG. 3B. The dimensions and spacing of the regions 154 and 152 are
the same as the projection of the mask 122 in FIGS. 2A and 2B.
[0041] Still another arrangement projects the fluorescent species
bands 123 in the separation channel 110 unto a mask 160, as shown
in FIG. 4. After being collimated by a lens 164, light from the
source 120 illuminates the species bands 123. Since light
fluoresces from the bands 123 isotropically, the light is projected
toward the mask 160 through a focussing lens 165. Light from the
other side of the mask 160 is focused by the lens 126 onto the
photodetector 120. As explained above, the elements of FIG. 4
illustrate a general relationship with each other. The lens 165,
mask 160, lens 126, filter 128, and photoreceptor 130 need not be
aligned with source 120, lens 164 and channel 110.
[0042] The arrangements above analyze the subject materials
undergoing electrophoresis by the reception of fluorescent light
from the moving species bands 123. The present invention also
operates with the absorbance of light by the subject material. For
example, using the arrangement of FIG. 2A, the light source 120 is
selected to radiate light at wavelengths which are absorbed by the
subject material. For proteins, the light source 120 may operate at
wavelengths of 280 nm, for example. For polynucleotides, 260 nm is
a suitable wavelength for the light source 120. The lens 126,
filter 128 and photoreceptor are arranged to receive the light from
the source 120 through the mask 122 and channel 120. The light
source 120, lens 124, mask 122, channel 110, lens 126, filter 128
and photoreceptor 130 are optically aligned and the filter 128 is
selected to pass light of the wavelength of interest from the
source 120 to the photoreceptor 130. More typically for absorption
measurements, the filter 128 is placed next to the source.
[0043] Rather than light from the species bands 123, darkness from
the light-absorbing bands 123 moving in the channel 110 causes a
varying signal to be received by the photoreceptor 130. Fourier
analysis of the signal ultimately identifies the species in the
channel 110. Similarly, the embodiments of the present invention
illustrated in FIGS. 3A and 4 can be adapted to light absorbance by
the species bands 123, rather than light fluorescence.
[0044] In another embodiment of the present invention, the mask 122
is eliminated. For example, a coherent light source, such as a
laser, is used for the source 120 and a pair of slits are located
between the source 120 and the channel 110. The slits are parallel
to each other and perpendicular to the length of channel 110. By
interference between the light emanating from the two slits, the
light falls in intensities of alternating minima and maxima along
the channel 110, like the operation of the mask 122 described
previously. Light received from the periodically spaced locations
of maxima allow the determination of the velocities of moving
species bands 123 by the frequency analysis of the light intensity
modulating in time, as described previously. This arrangement
operates in either fluorescing or absorbing mode. Of course, other
arrangements with one or more light sources 120 may also create
light patterns of minima and maxima intensities along the channel
110 without a mask.
[0045] Speed and sensitivity of the present invention are much
enhanced over previous systems which perform electrophoresis by the
measurement of a species band past a detection point. The present
invention has a higher signal-to-noise ratio since the light
signals from the fluorescent bands 123 are averaged over time by
the movement of the light signals past the mask regions, in
contrast to a single observation at the detection point.
[0046] Of course, the present invention also has the other
advantages of microfluidic systems, such as speed, low cost due to
the low consumption of materials and the low use of skilled labor,
and accuracy. The microfluidic system 100 has little or no
contamination with high reproducibility of results.
[0047] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. All publications and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
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