U.S. patent application number 13/644764 was filed with the patent office on 2013-01-31 for control of electrolyte solution in nanofluidic channels.
This patent application is currently assigned to STC.UNM. The applicant listed for this patent is Sang M. Han, Cornelius F. Ivory, Youn-Jin Oh. Invention is credited to Sang M. Han, Cornelius F. Ivory, Youn-Jin Oh.
Application Number | 20130026030 13/644764 |
Document ID | / |
Family ID | 42119939 |
Filed Date | 2013-01-31 |
United States Patent
Application |
20130026030 |
Kind Code |
A1 |
Ivory; Cornelius F. ; et
al. |
January 31, 2013 |
CONTROL OF ELECTROLYTE SOLUTION IN NANOFLUIDIC CHANNELS
Abstract
Various embodiments provide an exemplary lab-on-a-chip (LOC)
system that serves as an analytical tool and/or as a separation
medium for an electrolyte solution including various charged
molecular species. The LOC system can include an integrated
nanofluidic FET device in combination with suitable analysis
systems. By applying and controlling a longitudinal electric field
and a transverse electric potential, the flow and the pH of the
electrolyte solution in the nanofluidic channels can be
controlled.
Inventors: |
Ivory; Cornelius F.;
(Pullman, WA) ; Han; Sang M.; (Albuquerque,
NM) ; Oh; Youn-Jin; (Albuquerque, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ivory; Cornelius F.
Han; Sang M.
Oh; Youn-Jin |
Pullman
Albuquerque
Albuquerque |
WA
NM
NM |
US
US
US |
|
|
Assignee: |
STC.UNM
Albuquerque
NM
|
Family ID: |
42119939 |
Appl. No.: |
13/644764 |
Filed: |
October 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12721860 |
Mar 11, 2010 |
8303789 |
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13644764 |
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PCT/US09/61314 |
Oct 20, 2009 |
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12721860 |
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61106648 |
Oct 20, 2008 |
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Current U.S.
Class: |
204/242 ;
204/601; 204/603 |
Current CPC
Class: |
G01N 27/4473 20130101;
G01N 27/414 20130101; G01N 27/44795 20130101; G01N 33/6803
20130101 |
Class at
Publication: |
204/242 ;
204/601; 204/603 |
International
Class: |
G01N 27/453 20060101
G01N027/453; C07K 1/26 20060101 C07K001/26; G01N 27/414 20060101
G01N027/414 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under
Contract No. CTS-0404124 awarded by the National Science
Foundation. The U.S. Government has certain rights in this
invention.
Claims
1. A system for controlling an electrolyte solution, comprising: a
nanofluidic channel array disposed in a substrate, wherein the
nanofluidic channel array comprises a plurality of nanofluidic
channels and an electrically insulating surface layer for
insulating an electrolyte solution within each nanofluidic channel
from the substrate; a power supply sufficient to apply an electric
potential along a length of each nanofluidic channel such that the
electric potential forms a longitudinal electric field along each
nanofluidic channel; a multi-gate nanofluidic
field-effect-transistor (FET) comprising a plurality of FET gates
within the substrate, wherein the plurality of FET gates are spaced
along a length direction of the plurality of nanofluidic channels
and the multi-gate FET; and a leakage current path between the
plurality of FET gates and an exposed surface of the insulating
surface layer, such that a leakage current generated by the
multi-gate FET is sufficient to change at least one of a direction
of an electroosmotic (EO) flow, a speed of an EO flow, and a pH
value of an electrolyte solution within the nanofluidic channel
array.
2. The system of claim 1, wherein the electrically insulating
surface layer comprises SiO.sub.2 and a density of a leakage
current generated by the multi-gate FET is larger in magnitude with
a negative gate electric potential V.sub.G than with a positive
gate electric potential V.sub.G.
3. The system of claim 1, wherein the multi-gate FET is sized to
generate a gate potential sufficient to electrolyze water near each
FET gate.
4. The system of claim 1, further comprising an IR spectroscopy
system aligned with the substrate such that the nanofluidic channel
array is disposed along a direction of IR propagation from an IR
source of the IR spectroscopy system.
5. The system of claim 4, wherein the wherein IR spectroscopy
system comprises multiple internal reflection Fourier transform
infrared spectroscopy (MIR-FTIRS).
6. The system of claim 1, wherein the substrate is a
double-side-polished Si(100) substrate comprising an edge and the
IR spectroscopy system is positioned such that an IR beam output by
the IR spectroscopy system is directed onto the edge.
7. The system of claim 1, wherein the nanofluidic channel is
insulated from the substrate by an SiO.sub.2 electrically
insulating surface layer having a thickness ranging from about 50
nm to about 500 nm.
8. The system of claim 1, wherein each FET gate comprises a doped
layer having a thickness ranging from about 0.5 .mu.m to about 5
.mu.m.
9. The system of claim 1, further comprising an optically
transparent cover attached to a surface of the substrate which
seals the nanochannel array.
10. The system of claim 9, further comprising: at least one end
well in fluidic communication with the nanofluidic channel array;
and at least one hole through the optically transparent cover,
wherein the hole through the optically transparent cover exposes
the end well.
11. The system of claim 1, wherein the nanofluidic array comprises
between about 2 and about 10.sup.8 nanofluidic channels.
12. The system of claim 1, wherein each nanofluidic channel has a
length of between about 100 micrometers and about 10 centimeters
and a width of less than about 1000 nm.
13. The system of claim 1, wherein the plurality of FET gates are a
doped layer within the substrate and at least a portion of each FET
gate underlies each nanofluidic channel of the nanofluidic channel
array.
14. The system of claim 1, wherein each FET gate surrounds each
nanofluidic channel.
15. The system of claim 1, further comprising: at least one
substrate beveled edge; and a spectroscopy system aligned with the
substrate such that a beam output by the spectroscopy system is
directed onto the substrate beveled edge.
16. The system of claim 1, further comprising: the leakage current
generated by the multi-gate FET is sufficient to induce a first
change of a pH value of an electrolyte solution within the
nanofluidic channel array upon application of a gate electric
potential V.sub.G; and the leakage current generated by the
multi-gate FET is sufficient to induce a second change of a pH
value of the electrolyte solution within the nanofluidic channel
array upon prolonged application of the gate electric potential
V.sub.G due to water electrolysis caused by the leakage
current.
17. The system of claim 1, wherein a length direction of the
plurality of FETs is generally perpendicular to the length
direction of the plurality of nanofluidic channels.
18. The system of claim 1, wherein the system is a lab-on-a-chip
(LOC) system.
19. A lab-on-a-chip system, comprising: a substrate comprising at
least one beveled edge; a nanofluidic channel array disposed in the
substrate, wherein the nanofluidic channel array comprises a
plurality of nanofluidic channels and each nanofluidic channel
comprises an electrically insulating surface layer for electrically
insulating an electrolyte solution within each nanofluidic channel
from the substrate; a power supply sufficient to apply an electric
potential to a length of each nanofluidic channel such that the
electric potential forms a longitudinal electric field along each
nanofluidic channel; a multi-gate nanofluidic
field-effect-transistor (FET) comprising a plurality of FET gates
within the substrate, wherein: a length direction of the plurality
of FET gates is generally perpendicular to a length direction of
the plurality of nanofluidic channels; a leakage current path
between the plurality of FET gates and an exposed surface of the
insulating surface layer, such that a leakage current generated by
the multi-gate FET is sufficient to flow through the leakage path
to an electrolyte solution within the nanofluidic channel array;
and an IR spectroscopy system aligned with the substrate such that
the nanofluidic channel array is disposed along a direction of IR
propagation from an IR source of the IR spectroscopy system, and
further aligned such that an IR beam output by the IR spectroscopy
system is directed onto the beveled edge of the substrate.
20. The lab-on-a-chip system of claim 19, wherein the multi-gate
FET is sized to generate a leakage current which is sufficient to
change at least one of a direction of an electroosmotic (EO) flow
and a speed of an EO flow of an electrolyte solution within the
nanofluidic channel array.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/721,860 filed Mar. 11, 2010 which is a
continuation-in-part of PCT/US09/61314, entitled "High Resolution
Focusing and Separation of Proteins in Nanofluidic Channels," filed
Oct. 20, 2009, the complete disclosure of which is incorporated
herein by reference, which claims priority from U.S. Provisional
Patent Application Ser. No. 61/106,648, filed Oct. 20, 2008, which
is hereby incorporated by reference in its entirety.
DESCRIPTION OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates generally to electrolyte solutions in
nanofluidic channels and, more particularly, to systems and methods
for controlling a flow and pH of the electrolyte solutions in
nanofluidic channels.
[0005] 2. Background of the Invention
[0006] In order to characterize and understand protein function and
regulation, proteins must be first separated and then detected. The
most common technique for protein separations is gel
electrophoresis. Today, 1-D and 2-D polyacrylamide gel
electrophoresis (PAGE) setup is commercially available and widely
used as a standard technique. Despite its widespread use, however,
the PAGE technique has its own limitations, such as requiring a
large amount of sample, low reproducibility, breakdown under high
electric field, and low dynamic range.
[0007] To overcome these limitations of the conventional PAGE
technique, a number of new separation platforms have emerged using
microfluidic and nanofluidic channels. For example,
micro/nanofluidic devices fabricated using conventional
semiconductor manufacturing methods potentially use smaller sample
amounts, lower electrical field, shorter analysis time, and higher
throughput than the PAGE technique. It is desirable to provide an
analytical tool and separation medium to control bio-separation,
detection, and chemical analysis using nanofluidic devices.
Specifically, there is a need to overcome these and other problems
of the prior art and to provide systems and methods for controlling
a flow and/or a pH value of an electrolyte solution that contains
charged species in nanofluidic devices.
SUMMARY OF THE INVENTION
[0008] According to various embodiments, the present teachings
include a method for controlling an electrolyte solution in a
nanofluidic channel. Specifically, a plurality of nanofluidic
channels in a substrate can be provided with each nanofluidic
channel including an insulating surface layer, such that an
electrolyte solution in the nanofluidic channel can be insulated
from the substrate. A multi-gate nanofluidic
field-effect-transistor (FET) can be configured to have a plurality
of gates in the substrate. The plurality of gates can be spaced
along a length direction of the nanofluidic channels with each gate
surrounding the nanofluidic channel perpendicularly to the length
direction of the nanofluidic channels. In this method, an electric
potential V.sub.EO can be applied to a length of the electrolyte
solution in the nanofluidic channels to generate an electroosmotic
(EO) flow of the electrolyte solution along the nanofluidic
channels. The electrolyte solution can include a plurality of
charged species. A gate electric potential V.sub.G can then be
applied to each FET gate to generate a leakage current to change at
least one of a direction and a speed of the EO flow and a pH value
of the electrolyte solution.
[0009] According to various embodiments, the present teachings also
include a lab-on-a-chip system. The lab-on-a-chip system can
include a nanofluidic array disposed in a substrate. The
nanofluidic array can include a plurality of nanofluidic channels
with each nanofluidic channel including an insulating surface layer
such that an electrolyte solution in the nanofluidic channel can be
insulated from the substrate. A power supply can be included for
applying an electric potential to a length of the protein mixture
solution to form a longitudinal electric field along each
nanofluidic channel. A multi-gate nanofluidic
field-effect-transistor (FET) can also be included having a
plurality of gates in the substrate. The plurality of gates can be
spaced along a length direction of the nanofluidic channels with
each gate in the substrate surrounding the nanofluidic channel. The
lab-on-a-chip system can further include an IR spectroscopy system
configured such that the nanochannel array can be disposed along
the direction of IR propagation from an IR source of the IR
spectroscopy system.
[0010] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
[0012] FIG. 1 depicts an exemplary method for focusing and/or
separating proteins in accordance with various embodiments of the
present teachings.
[0013] FIGS. 2A-2B depict portions of an exemplary nanofluidic
device in accordance with various embodiments of the present
teachings.
[0014] FIG. 2C depicts an exemplary nanofluidic system in
accordance with various embodiments of the present teachings.
[0015] FIG. 2D depicts an exemplary nanofluidic channel during
protein focusing in accordance with various embodiments of the
present teachings.
[0016] FIG. 2E depicts another exemplary nanofluidic system in
accordance with various embodiments of the present teachings.
[0017] FIG. 3A depicts an exemplary experimental setup including an
integrated nanofluidic FET in combination with exemplary analysis
systems in accordance with various embodiments of the present
teachings.
[0018] FIG. 3B depicts a cross-sectional view of a portion of an
exemplary nanochannel array in accordance with various embodiments
of the present teachings.
[0019] FIG. 4 depicts an exemplary measurement of leakage current
density in accordance with various embodiments of the present
teachings.
[0020] FIGS. 5A-5C depict exemplary models for electroosmotic (EO)
flow and pH shift in nanochannels in accordance with various
embodiments of the present teachings.
DESCRIPTION OF THE EMBODIMENTS
[0021] Reference will now be made in detail to exemplary
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. Wherever possible, the same reference
numbers will be used throughout the drawings to refer to the same
or like parts. In the following description, reference is made to
the accompanying drawings that form a part thereof, and in which is
shown by way of illustration specific exemplary embodiments in
which the invention may be practiced. These embodiments are
described in sufficient detail to enable those skilled in the art
to practice the invention and it is to be understood that other
embodiments may be utilized and that changes may be made without
departing from the scope of the invention. The following
description is, therefore, merely exemplary.
[0022] Exemplary embodiments provide systems and methods for
focusing and/or separating proteins using nanofluidic channels
and/or arrays of nanofluidic channels. The disclosed nanofluidic
apparatus, systems and methods can provide a versatile platform to
separate proteins, for example low-abundance proteins, with high
resolution, using separation techniques including for example,
isoelectric focusing (IEF), dynamic field gradient focusing (DFGF)
and/or a combination thereof. In embodiments, a control scheme
using multi-gate nanofluidic field-effect-transistors (FETs) can be
combined with the disclosed nanofluidic technique.
[0023] In embodiments, a stable pH gradient can be established in
nanofluidic channels without the use of ampholytes upon an
application of a longitudinal electric field to the protein mixture
solution in the nanofluidic channels, thereby allowing for
isoelectric focusing (IEF). In embodiments, the balance between
electroosmosis and electrophoresis (e.g., electrophoretic mobility
vs. counter flow buffer) can also be controlled dynamically in
nanofliuidic channels to achieve dynamic field gradient focusing
(DFGF). In embodiments, IEF and DFGF can work simultaneously in the
same system to concentrate, focus and/or separate proteins.
[0024] Various embodiments therefore allow high resolution IEF
and/or DFGF and separation of proteins using the nanochannel array,
in combination with electroosmosis, electrophoresis, pH gradient,
protein-wall interactions, and different mobility of proteins. As
compared with conventional techniques, the disclosed systems and
methods do not use ampholytes to build up the pH gradient, do not
use multi buffer ionic species to induce diffusion potential, and
do not use surface treatment to enhance isoelectric focusing. The
disclosed systems and methods, however, can use low electric
potential to achieve isoelectric focusing of proteins. The low
electric potential for IEF can be, for example, about 5 V or less
or in embodiments, about 3 V or less.
[0025] FIG. 1 depicts an exemplary method 100 for focusing and/or
separating proteins in accordance with various embodiments of the
present teachings. FIGS. 2A-2E depict a schematic of the exemplary
nanofluidic device and system to conduct high-resolution focusing
and separation of proteins within nanochannels in accordance with
various embodiments of the present teachings.
[0026] Specifically, FIG. 2A depicts a schematic top view of a
portion of an exemplary nanofluidic device 200; FIG. 2B depicts a
close-up schematic of an exemplary nanofluidic array 220 of the
device 200; FIG. 2C depicts an exemplary system 220C for focusing
and/or separating proteins using the device 200; FIG. 2D depicts a
cross-sectional schematic of an exemplary nanochannel when used to
focus/separate proteins; and FIG. 2E depicts another exemplary
system 200E using a control scheme of multi-gate nanofluidic FETs
(field-effect-transistors) in accordance with various embodiments
of the present teachings.
[0027] Note that although the method 100 will be described in
reference to FIGS. 2A-2E for illustrative purposes, the process of
method 100 is not limited to the structures shown in FIGS. 2A-2E.
In addition, while the method 100 of FIG. 1 is illustrated and
described below as a series of acts or events, it will be
appreciated that the present teachings are not limited by the
illustrated ordering of such acts or events. For example, some acts
may occur in different orders and/or concurrently with other acts
or events apart from those illustrated and/or described herein.
Also, not all illustrated steps may be required to implement a
methodology in accordance with one or more aspects or embodiments
of the present invention. Further, one or more of the acts depicted
herein may be carried out in one or more separate acts and/or
phases.
[0028] At 110 of FIG. 1, a nanofluidic device 200 can be provided
as shown in FIGS. 2A-2E. The device 200 can include one or more
nanochannel arrays 220 formed in a substrate 210 (see FIGS. 2C-2E).
In embodiments, the one or more nanochannel arrays 220 can be
configured to be parallel. The substrate 210 can be made of any
suitable substrate material including for example silicon, a III-V
substrate, ceramic, glass, plastic, etc. In embodiments, the
substrate material can be a semiconducting material including
silicon and/or germanium.
[0029] Each array 220 can include a plurality of nanochannels 222
(or nanofluidic channels). In embodiments, each array 220 can have
a desired number of nanofluidic channels 222, for example, about 2
to about 10.sup.8. In embodiments, the nanofluidic channels 222 can
be configured to be substantially parallel. In certain embodiments,
each array 220 can include from about 120 to about 180 parallel
nanochannels, although other number of parallel channels can also
be used for the disclosed nanofluidic device.
[0030] In embodiments, the nanochannel 222 can have at least one
minor dimension, for example, depth of about 1000 nanometers or
less, in embodiments, of about 500 nanometers or less. In an
exemplary embodiment, the nanochannel 222 can have at least one
minor dimension, for example, width of about 1000 nanometers or
less, in embodiments, ranging from about 15 nanometers to about 100
nanometers. In embodiments, the nanochannel 222 can have one of the
width and the depth of about 1000 nm or less. In embodiments, the
nanochannel 222 can have a length of at least about 100
micrometers, for example, ranging from about 100 micrometers to
about 2 centimeters or to about 10 centimeters.
[0031] The nanochannels 222 and their arrays 220 can be fabricated
using suitable semiconductor fabrication processes. For example,
the nanochannels 222 can be formed in the substrate 210 by a
lithography process, such as interferometric lithography (IL) and
an etching process, such as a plasma etching process of the
substrate 210. In embodiments, an electrically insulating layer
(see 325 of FIG. 3B) can be formed on substrate wall surfaces of
each nanochannel 222. The insulating layer can be formed of, for
example, SiO.sub.2, Si.sub.3N.sub.4, Al.sub.2O.sub.3, TiO.sub.2,
and/or a combination thereof. In an exemplary embodiment, a
thermally grown SiO.sub.2 layer having a thickness of, for example,
about 100 nm or less, can be used as an electrically insulating
layer between the substrate nanochannel walls and the fluid flowing
through each channel.
[0032] In embodiments, the device 200 can include a plurality of
end wells 228. The end wells 228 can be connected with each
nanochannel 222 and/or nanochannel array 220, wherein, for example,
liquid can pass through the nanochannels 222 from the end well 228
by capillary force. In embodiments, the end wells 228 can be used
as liquid or solution reservoirs for introducing and storing the
liquid or solution.
[0033] In embodiments, as shown in FIGS. 2D-2E, the nanochannels
222 of the nanofluidic device 200 can be sealed with an optically
transparent material 230, for example, a Pyrex cover, which can be
bonded onto the substrate 210, for example, by anodic bonding to
form the nanofluidic device 200. In embodiments, the optically
transparent material 230 can also include, for example, glass,
quartz, polydimethylsiloxane (PDMS), and/or plastic.
[0034] In various exemplary embodiments, the nanochannel 222 can
have a width on the order of a thickness of electric double layer
(EDL). Due to this dimensional comparability, the EDLs can overlap
in nanofluidic channels, giving rise to unique characteristics that
are not readily achievable in conventional microfluidic channels,
but can be exploited for biomolecular separations. There can be
many advantages provided by nanofluidic channels. For example,
electroosmosis (EO) can be a dominant mechanism of molecular
transport over electrophoresis (EP) and can be controlled by
modulating the .zeta.-potential with an externally applied electric
potential (V) to the nanochannel walls. In another example,
electrostatic interaction of charged biomolecules and nanochannel
walls can be more pronounced than in microfluidic channels and can
allow one to control the electrokinetic mobility of charged
molecules much more effectively than in microchannels.
Additionally, differently sized molecules, such as DNA and protein
molecules, can be separated using the nanoscale sieving structure,
where surface charges can be controlled.
[0035] At 120 of FIG. 1, a protein-containing solution or a protein
mixture solution can be introduced into the nanofluidic channels
222 and/or their arrays 220. For example, the protein mixture
solution can be introduced into one of the end wells 228 and can
further fill the nanofluidic channels 222 and/or their arrays 220
by capillary force or by electroosmosis.
[0036] In embodiments, prior to introducing the protein mixture
solution, both the end wells 228 and/or the nanochannels 222/arrays
220 can first be filled with a buffer solution, for example, by
capillary force. In embodiments, the buffer solution can be
selected according to the specification of particular proteins and
can be used to dilute proteins. For example, the buffer solution
can have a pH ranging from about 2 to about and an ionic strength
ranging from about 0.1 mM to about 100 mM. Depending on the
proteins used, other pH ranges and ionic strengths can also be
included in various embodiments.
[0037] The buffer solution can be equilibrated for a period of time
in order to reach equilibrium of materials, for example, between
the buffer solution and the nanochannel walls that include a
material of, for example, SiO.sub.2. Depending on the system, the
equilibrium time can be determined by IR absorbance spectra over
time, for example, measured by MIR-FTIRS setup as shown in FIG. 3A.
In an exemplary embodiment, after a period of time, if no
noticeable changes are detected by IR absorbance spectra, the
buffer solution in the nanochannels 222 or the nanoarrays 220 can
be assumed to be equilibrated. In embodiments, the equilibrium time
can range from about 10 minutes to about 30 minutes, although other
equilibrium time can be used depending on the material systems.
[0038] In embodiments, the substrate 210 can have beveled edges on
both ends that can allow IR access through the substrate and
perform multiple internal reflection Fourier transform infrared
(MIR-FTIR) spectroscopy to probe molecules in the nanochannels 222,
as shown in FIG. 3A. In embodiments, the IR technique used to
monitor the system equilibrium can include the IR technique as
described in U.S. Pat. No. 7,200,311, entitled "Surface Corrugation
on Internal Reflection Infrared Waveguide for Enhanced Detection
Sensitivity and Selectivity," which is hereby incorporated by
reference in its entirety.
[0039] In embodiments, the protein mixture solution can have a
protein concentration ranging from a high concentration on the
order of millimolar to a low concentration on the order of
attomolar. For example, the protein mixture solution can have a
protein concentration of about 1 millimolar or less. In
embodiments, the protein mixture buffer solution can have low
abundance proteins with concentration on the order of picomolar.
Such low abundance proteins can be concentrated, focused, separated
and/or analyzed by using the nanofluidic devices and systems as
described herein.
[0040] At 130 of FIG. 1, an electric potential can be applied to a
length of the exemplary protein mixture solution in the nanofluidic
channels 222 or the nanofluidic arrays 220 to generate a
longitudinal electric field along the nanofluidic channels. In
embodiments, any suitable power supply as known to one of ordinary
skill in the art can be used to apply the electric potential.
[0041] In embodiments, suitable electrodes (see 240 in FIG. 2E) can
be used to contact the protein mixture solution for the application
of electric potential. For example, any conductive material,
including any metal-containing material or other known electrodes
can be used to contact with the protein mixture solution in the
nanofluidic channels 222.
[0042] In embodiments, two electrodes (see 240 in FIG. 2E) can be
used to apply electric potential through the protein mixture
solution and can be spaced apart from each other to form the
longitudinal electric field there-between (see FIGS. 2C-2E). In
exemplary embodiments, the electrodes can contact the protein
mixture solution in the end wells 228 through connectors 245,
wherein the center-to-center spacing between the two end wells 228
can range from about 100 .mu.m to about 2 cm or to about 10 cm.
[0043] After the electrodes are configured to contact the protein
mixture solution that contains one or more proteins, the electric
potential can then be applied to generate a longitudinal electric
field. The longitudinal electric field can result in one or more
focused protein bands 205 as in FIG. 2D.
[0044] In an exemplary embodiment, as shown in FIGS. 2C-2E, a
negative bias (V.sub.EP) can be applied to one end well 228, and
the opposite side end well 228 can be grounded to induce an
electrokinetic flow. In embodiments, the longitudinal electric
field ( ) along the nanochannels 222 or the arrays 220 can induce
an electroosmotic (EO) flow typically with opposing electrophoresis
(EP) for negatively charged molecules such as protein molecules
that are negatively charged. Because water electrolysis occurs at
both electrodes as electric potential is applied, a pH gradient can
be established along the longitudinal electric field, which is
along the length of the nanochannels 222 or the arrays 220.
[0045] In various embodiments, as shown in FIG. 2E, a multi-gate
nanofluidic FET control scheme 250 can be used in combination with
the focusing/separating system of FIG. 2C. In an exemplary
embodiment, a plurality of gates or gate regions 260, for example,
formed by highly doped regions of the substrate material such as
Si, can be placed along the nanochannels. By locally applying
additional electric potentials to the channel walls that
encapsulate the protein mixture solution, local pH value and
electrical potential in the solution contained in the nanochannels
and adjacent to the gate regions 260 can then be dynamically
controlled. In this manner, varying electrical potentials, for
example, gradient DC potentials, can be applied to the gate regions
260 to dynamically control the pH gradient and electric field
gradient in real time along the nanochanneis. Further, in
embodiments, the multi-gate nanofluidic FET control scheme 250 can
allow an application and manipulation of the additional electric
potentials to simultaneously control the electrokinetic transport
of proteins (e.g., electroosmosis vs. electrophoresis) along the
nanofluidic channels.
[0046] In embodiments, the multi-gate nanofluidic FET control
scheme 250 can include the scheme described in U.S. patent
application Ser. No. 11/184,540, entitled "Nanofluidics for
Bioseparation and Analysis," which is hereby incorporated by
reference in its entirety.
[0047] The protein bands 205 can then be focused with each focused
band corresponding to at least one protein of the protein mixture
solution and therefore to focus and/or separate the proteins. In
embodiments, the focused protein band 205 (see FIG. 2D) can be
statically focused for a period of time along the longitudinal
electric field and then flow forward electrokinetically by
electroosmosis/electrophoresis. In embodiments, the focused protein
band 205 can be statically focused for a period of time, for
example, ranging from about 5 minutes to about 30 minutes. In
embodiments, the focused protein band 205 can have a high
resolution, i.e., a narrow width of, for example about 100 .mu.m or
less, such as about 5 .mu.m.
[0048] In embodiments, formation of the protein band 205 and
movement of the band 205 through the nanochannels 222 or the
nanoarrays 220 can be controlled by controlling the application of
the longitudinal electric potential and/or the electric potentials
that are applied to individual gates 260 of the multi-gate
nanofluidic FETs.
[0049] In one embodiment, the location of band formation can be
controlled along the longitudinal electric field by increasing or
decreasing the electric potential applied to the well 228 as shown
in FIG. 2C. For example, as observed in experiments, an increased
electric potential can push or advance the proteins move further
along the longitudinal electric field and can thus form a focused
band even further from its original spot.
[0050] In one embodiment, the length of time for forming the
focused protein band 205, i.e., from the time when the electric
potential is applied to the time when the band forms, can be
controlled by controlling the application of electric potential.
For example, an increased (or decreased) electric potential can
reduce (or increase) the band formation time. This is because the
movement of protein bands can be determined by the equilibrium
between electrophoresis and electroosmosis, wherein electroosmosis
can be much more dominant than other forces such as
electrophoresis, ion diffusion, and pH gradient effect in
nanochannels having a width of about 100 nm or less. The electric
potential modulation can therefore govern the solution flow by
electroosmosis in the early stage. That is, an increased (or
decreased) electric potential can enhance (or weaken) the
electroosmosis flow. In embodiments, as compared with conventional
separation techniques, the disclosed systems and methods for
focusing/separating proteins can form a focused band in high speed,
for example, of about 1 minute or shorter. In a particular example,
when focusing protein ovalbumin (OVA) using an electric potential
of about -40V, the focused band formation time can be as short as
about 10 seconds.
[0051] In one embodiment, the movement of proteins in the
nanochannels can be controlled, for example, by controlling the
amount and/or the direction of the applied electric potential. For
example, proteins can be moved repeatably and continuously in both
directions along the longitudinal electric field or along the
length between the two electrodes through which electric potential
can be applied. In an exemplary embodiment, proteins can move in an
opposite direction along the longitudinal electric field by
increasing or decreasing the electric potential applied. The
changed potential can break the electrokinetic equilibrium that is
previously formed by the previously applied electric potential. In
embodiments, the amount of electric potential can be changed in an
alternating fashion, in an increasing fashion or in a decreasing
fashion so as to control the flowing directions of the proteins
between the two electrodes for repeatable focusing and
separation.
[0052] In embodiments, the electric potential can be a
pre-determined electric potential depending on, for example,
molecular weights of proteins in the protein mixture solution. In
embodiments, a high molecular weight of proteins may require a high
electric potential for the mobility of proteins. In exemplary
embodiments, molecular weights of BSA (bovine serum albumin, MW
.about.66,000) and OVA (ovalbumin, MW .about.45,000) can be 4 times
smaller than those of RPE (r-phycoerythrin, MW .about.240,000) and
APC (allophycocyanin, MW .about.104,000). In embodiments, BSA and
OVA can then be highly controllable with a low-magnitude electric
potential, for example, as low as -5 V.sub.EP due to their low
molecular weights. In contrast, RPE and APC bands can appear at a
high-magnitude electric potential, for example, with
V.sub.EP>-60 in magnitude.
[0053] In embodiments, various proteins can be concentrated,
focused, separated and/or analyzed by using the systems and methods
as described in FIGS. 1-2. The separation mobility of proteins can
be determined by size (i.e., size exclusion) and/or molecular
weight of proteins, electrostatic interaction of charged species in
the protein mixture solution and electrodes (e.g., the applied
electric potential that determines pH gradient generation), etc.
Different electric mobilities of proteins can result in protein
separation with proteins having different band formation times and
band formation locations.
[0054] In embodiments, the systems and methods described herein can
be used to focus and/or separate proteins for certain protein
systems, for example, where proteins having same sign of charge but
having sizes slightly different yet in the same size range are
mixed in a solution. For example, APC and RPE are large proteins
with net negative charges but slightly different in size. In
another example, BSA and OVA are small proteins with net negative
charges but slightly different in size.
[0055] As disclosed, the focused bands of exemplary BSA and OVA in
the nanochannels and nanoarrays can be highly selective and very
narrow as compared with other methods using microchannels or
capillaries. Further, protein focusing and separation as disclosed
herein can be achieved by applying a very low electric potential,
for example, as low as 3.6 V. Note that, unlike conventional
methods, no additional ampholytes nor special buffer ions are used
to establish the pH gradient and to induce protein-focusing.
Furthermore, the high-resolution protein focusing of BSA and OVA in
nanochannels is repeatable.
[0056] In this manner, the band formation and separation of
proteins can be achieved by isoelectric focusing due to a
longitudinal pH gradient along the nanochannels created by water
electrolysis occurring on the electrodes 240 in the end wells 228,
in conjunction with DFGF due to the force balance of
electroosmosis/electrophoresis and ion concentration
polarization.
[0057] Various embodiments also include an exemplary lab-on-a-chip
(LOC) system that serves as an analytical tool and/or as a
separation medium for charged molecular species including, but not
limited to, proteins, charged dye molecules, or any other charged
molecules. The LOC system can include an integrated nanofluidic FET
device as disclosed herein in combination with suitable analysis
systems. The integrated nanofluidic FET device can include a
control scheme of multi-gate nanofluidic field-effect-transistors
(FETs), for example, as depicted in FIG. 2E. The LOC system can be
used to monitor the flow of an electrolyte solution that contains
various charged molecules using, for example, a confocal
microscopy. The LOC system can also be used to probe the charged
molecules in the electrolyte solution as well as the pH value of
the electrolyte solution using, for example, an infrared
spectroscopy.
[0058] FIG. 3A depicts an exemplary experimental setup including an
integrated nanofluidic FET in combination with exemplary analysis
systems in accordance with various embodiments of the present
teachings. As shown, the setup in FIG. 3A can include a nanofluidic
device, for example, the device shown in FIGS. 2A-2E having one or
more nanochannel arrays 220. The nanochannel arrays 220 can be
covered by an optically transparent material 230. Each nanochannel
array 220 can include a plurality of nanochannels 222 (see FIG. 2B)
and can be connected with end wells 228a-b (see FIG. 3A) on
opposite ends. In embodiments, channel-to-channel dimensional
uniformity can be provided in both transverse and longitudinal
directions in order to obtain efficient isolation and elution of
separated charged molecules.
[0059] FIG. 3B depicts a cross-sectional view of a portion of an
exemplary nanochannel array 220 with respect to the array shown in
FIG. 2B in accordance with various embodiments of the present
teachings. As shown, an electrically insulating layer 325, for
example, a layer of SiO.sub.2, Si.sub.3N.sub.4, Al.sub.2O.sub.3,
TiO.sub.2, and/or a combination thereof, can be formed on substrate
surfaces of each nanochannel 222, specifically, on the walls and
the bottom of each channel. In this case, the electrolyte solution
in the nanofluidic channel can be insulated from the substrate 210.
In embodiments, the insulating surface layer 325 can have a
thickness ranging from about 50 nm to about 500 nm although other
thicknesses can also be included for the insulating surface layer
325 in accordance with various embodiments.
[0060] In embodiments, the end wells 228a-b can have various
cross-sectional shapes including, but not limited to, a circle, a
polygon, a star, a rectangle, or a square, wherein the transparent
cover 230 can have corresponding holes drilled there-through in
order to facilitate an application of the electrolyte solution into
the end wells 228a-b. For example, square-shaped holes can be used
for an exemplary Pyrex slip cover and can also be used for the end
wells in order to insure an even introduction of the electrolyte
solution into each nanochannel 222.
[0061] In embodiments, the setup in FIG. 3A can also include a gate
contact 340, for example, an FET gate. The FET gate contact 340 can
be, for example, a gate metal that makes contact with each of the
FET gates or gate regions 260 (see FIG. 2E). In embodiments, the
dopants used for the gate regions 260 can include, for example,
boron. In an exemplary embodiment, the boron dopant diffusion can
be carried out having a dopant level on an order of, for example,
about 1.times.10.sup.20 cm.sup.-3. The gate regions 260 can have a
reduced contact resistance and can be used to efficiently control
the surface charge on the insulating surface layer 325 of the
nanochannel 222. In embodiments, the gate regions 260 can include a
diffusion layer having a diffusion depth ranging from about 0.5
.mu.m to about 5 .mu.m.
[0062] In embodiments, the FET gate contact 340 can be configured
connecting to each highly doped gate region 260 in the substrate
210, e.g., a silicon substrate. The gate region 260 can be formed
in a direction perpendicularly to the channel length and
surrounding the nanofluidic channel 222, while the nanochannel
array 220 can be fabricated along the direction of IR propagation
from an IR source of an IR spectroscopy. In embodiments, the IR
spectroscopy can include, for example, multiple internal reflection
Fourier transform infrared spectroscopy (MIR-FTIRS) shown by 360 in
FIG. 3A.
[0063] In embodiments, the substrate 210 with beveled edges can be
used as a nanofluidic IR waveguide mounted on a metal housing 365
of the MIR-FTIRS 360 with IR optics including a polarizer 362, and
IR mirrors 364, 366. The reflective IR optics can direct the IR
beam 306 onto one of the beveled edges of the substrate 210. The IR
beam 306 that enters the substrate 210, for example, the Si MIR
crystal, can make approximately 35 top reflections from the channel
bottom before the beam exits the opposite end of the substrate 210.
The IR signal leaving the second beveled substrate edge can be
collected by a detector 369, such as a HgCdTe detector. Due to
these multiple reflections, the exemplary Si MIR crystal can be
opaque to IR, for example, below a wavenumber of about 1500
cm.sup.-1. In embodiments, the exemplary LOC setup of FIG. 3A can
further include, for example, confocal fluorescence microscopy,
such as laser-scanning confocal fluorescence microscopy (LS-CFM)
shown as 380 in FIG. 3A, configured to monitor the electrolyte
solution and the charged species in the nanochannels 222 through
the optically transparent cover 230.
[0064] In embodiments, the substrate 210 can include a
double-side-polished Si(100) wafer in order to prevent the
scattering and loss of IR beam intensity during multiple internal
reflection in the MIR-FTIRS analysis system 360 as shown in FIG.
3A.
[0065] In embodiments, the LOC setup of FIG. 3A can be used, for
example, to probe wall-molecule interactions and their impact on
.zeta.-potential; to monitor FET flow control in the nanochannels
as well as the pH value of the electrolyte solution in response to
a transverse gate electric potential V.sub.G; and/or to probe the
effect of small, but measurable leakage current through the FET
gate during the FET flow control.
[0066] For example, the setup of FIG. 3A can be used as an
analytical tool using MIR-FTIRS 360 to probe the signature
vibrational modes of charged molecules flowing through the
nanochannels 222. The spectrum changes in observable vibrational
modes can further provide information about diffusion rate, flow
speed, and wall adsorption/desorption of molecules, along with a pH
shift in the nanochannels.
[0067] In embodiments, while maintaining a constant longitudinal
electrical field with V.sub.EO 330, a DC potential can be applied
to each FET gate 260 (see FIG. 2E) through the gate contact 340 to
provide the transverse gate bias V.sub.G 350 (also see 250 in FIG.
2E) and to control the surface charge of the insulating layer 325
on channel walls and channel bottoms. That is, the surface charge
of the insulating layer 325 and therefore .zeta.-potential can be
modulated by the applied gate potential V.sub.G during the FET flow
control. In particular, the modulation of .zeta.-potential, with
concomitant protonation or deprotonation of functional groups of
the insulating surface layer 325, for example, functional SiOH
groups on SiO.sub.2 surfaces, can govern the direction of the
electroosmotic (EO) flow generated by the longitudinal electrical
field V.sub.EO and solution pH in nanochannels.
[0068] In embodiments, the flow response to the gate bias during
the FET flow control can be virtually immediate and repeatable. In
addition, the observed flow response can be independent of the
position of charged molecules with respect to the gate position.
That is, the flow response can be identical, independent of whether
the charged molecules are fore or aft of the gate region.
[0069] In embodiments, depending on the sign, polarity, and
magnitude of gate bias V.sub.G 350, a pH shift, for example, close
to a whole pH unit, can be observed. In embodiments, the pH shift
of the electrolyte solution containing charged species can be
monitored by a pH indicator including, for example, fluorescein.
The molecular structure and therefore the characteristic IR
vibrational modes of fluorescein indicator can be strongly
dependent on the pH of the buffered electrolyte solution. The
solution pH in the nanochannels can therefore be monitored by the
IR absorbance result of the pH indicator. Due to the pH shift in
the nanochannels, isoelectric focusing of charged molecules
including, for example, low-abundance proteins, can be achieved
with multiple gates placed along the channels to create a
longitudinal pH gradient.
[0070] In embodiments, unusual or anomalous flow characteristics,
for example, multi-reversed EO flow and pH shift can be obtained,
when the FET flow control further contains prolonged application of
the gate bias V.sub.G 350. With the prolonged application of the
gate bias V.sub.G, the initial flow direction and speed and the
initial pH response can be reversed and can further be
multi-reversed.
[0071] In embodiments, the term "prolonged application of gate
electric potential V.sub.G" refers to a certain amount or a certain
level of an application time of the gate electric potential
V.sub.G, where leakage current through the gate dielectric can
cause water electrolysis near the gate region.
[0072] In embodiments, the leakage current that flows from the FET
gate 260, through the insulating surface layer 325, e.g., the
thermally grown SiO.sub.2, and to the electrolyte solution in the
nanochannels 222 can be measured. Specifically, the leakage current
can be measured, for example, from the gate voltage source equipped
with a current readout.
[0073] FIG. 4 depicts an exemplary relationship between a leakage
current density (J.sub.leak) and a gate bias V.sub.G that ranges
from about -30V to about +30V in accordance with various
embodiments of the present teachings.
[0074] In the illustrated example of FIG. 4, the leakage current
density (J.sub.leak) can be on the order of nAcm.sup.-2 for V.sub.G
within the range between about -6 V and about +8 V. However,
J.sub.leak can increase in magnitude up to about -1.4
.mu.Acm.sup.-2 for V.sub.G below -6 V, and can be up to about 0.05
.mu.Acm.sup.-2 for V.sub.G above about +10 V. Note the asymmetry in
J.sub.leak, where J.sub.leak can be significantly larger in
magnitude with a negative gate bias V.sub.G than with a positive
gate bias V.sub.G. That is, the exemplary SiO.sub.2 walls will not
be as leaky with a positive gate bias V.sub.G up to about +20 V,
whereas a V.sub.G of less than about -10 V can lead to a
significant leakage current.
[0075] In embodiments, the leakage current can cause water
electrolysis in the electrolyte solution to generate H.sub.3O.sup.+
or OH.sup.- ions that populate the region near the insulating
surface layer 325 surrounded by the gate regions 260 (see FIG. 2E).
The generation and accumulation of these H.sub.3O.sup.+ or OH.sup.-
ions can then reverse the initial flow direction and/or speed, as
well as the pH shift set by the gate bias V.sub.G.
[0076] The following reactions show the water electrolysis and
other side reactions that occur at the anode and cathode.
Anode (+):2H.sub.2O(l).fwdarw.O.sub.2(g)+4H.sup.+(aq)+4e.sup.-
(1)
4H.sup.+(aq)+4H.sub.2O(l).fwdarw.4H.sub.3O.sup.+(aq) (2)
H.sup.+(aq)+OH.sup.-(aq)H.sub.2O(aq) (3)
Cathode (-):2H.sub.2O(l)+2e.sup.-.fwdarw.H.sub.2(g)+2OH.sup.-(aq)
(4)
O.sub.2(g)+2H.sub.2O(l)+4e.sup.-.fwdarw.4OH.sup.-(aq) (5)
2H.sup.+(aq)+2e.sup.-.fwdarw.H.sub.2(g) (6)
[0077] In an exemplary embodiment, upon applying a positive gate
bias (V.sub.G>0V), the SiO.sub.2 surface layer bordering the
heavily doped Si gate can serve as an anode, where O.sub.2 and
H.sub.3O.sup.+ ions are generated. The H.sup.+ ions produced from
the anode can in turn reach equilibrium with H.sub.2O. Conversely,
when a negative bias V.sub.G is applied to the gate, the SiO.sub.2
wall surrounding the gate can serve as a cathode, and OH.sup.- ions
can be generated by decomposition of H.sub.2O. Note that the side
reaction of equation (6) entails that the cathode can deplete
H.sup.+ ions, that are generated from the anode and have diffused
to the cathode, while producing hydrogen gas (H.sub.2). This latter
reaction can result in a greater absolute magnitude for the rate of
increase in pH with a negative gate bias V.sub.G than the absolute
magnitude for the rate of decrease in pH with a positive gate bias
V.sub.G of equal magnitude.
[0078] In exemplary embodiments, the leakage current can flow
asymmetrically from the gate defined in the substrate, through the
insulating surface layer, to the electrolyte solution and vice
versa. In embodiments, the I-V characteristics of the leakage
current density can be independent of the buffer used for the
electrolyte solution. Examples of the buffers used can include
tris-glycine, propionate, and/or NaOH at varying pH values.
[0079] In embodiments, in response to FET surface charge (e.g.,
upon applying V.sub.G) and the leakage current (e.g., upon the
prolonged application of V.sub.G), pH shift can occur in the
nanochannels. In embodiments, the pH shift during FET flow control
can be monitored using a pH indicator, for example, fluorescein,
which can in turn be monitored by an IR spectroscopy. By probing
the IR characteristic vibrational modes of fluorescein, such as
vibrational peak position and absorbance intensities, pH values of
the electrolyte solution can be determined.
[0080] For example, IR spectra can show pH-dependent differences
due to the protonation/deprotonation of the fluorescein indicator.
The neutral fluorescein at low pHs can be a dianion due to the
protonation of carboxyl and OH groups of xanthene ring. Thus,
fluorescein can be significantly less symmetric. In contrast,
fluorescein can have a highly symmetric structure including a
xanthene moiety with two identical oxygens by the deprotonation at
high pHs.
[0081] In embodiments, a calibration curve can be determined or
used between the pH value and the IR spectrum result, which shows,
for example, intensity ratio of characteristic IR absorbance of the
pH indicator. In addition, the calibration curve can be
independently verified by, for example, laser absorbance
spectroscopy, using SNARF as a pH indicator.
[0082] In embodiments, the magnitude of native pH shift in the
nanochannel can depend on the initial value of the buffered
electrolyte solution and may or may not be a constant shift. In
embodiments, the pH shift can be an indirect indicator of the level
of electrolysis and therefore the level of H.sub.3O.sup.+ or OH--
production caused by electrolysis. In embodiments, a first shift of
the pH value of the electrolyte solution can occur upon applying a
gate electric potential V.sub.G; and a second shift of the pH value
of the electrolyte solution can occur upon a prolonged application
of the gate electric potential V.sub.G due to water electrolysis
caused by the leakage current. In embodiments, the pH shift in
nanochannels can be experimentally monitored by the MIR-FTIRS
results using a corresponding calibration curve.
[0083] For example, the total pH change (.DELTA.pH.sub.total) in
nanochannels can include two main contributions: (1) the initial
protonation/deprotonation of SiOH groups on SiO.sub.2 walls upon
gate biasing (.DELTA.pH.sub.surf), and (2) the generation of
OH.sup.- or H3O.sup.+ ions by water electrolysis
(.DELTA.pH.sub.elect) with prolonged gate biasing.
.DELTA.pH.sub.total=.DELTA.pH.sub.surf+.DELTA.pH.sub.elect
[0084] In order to isolate the impact of gate biasing (i.e.,
.DELTA.pH.sub.surf) from that of electrolysis occurring at the two
electrodes 240 driving electroosmosis (EO) (i.e.,
.DELTA.pH.sub.elect), the two electrodes 240, e.g., Pt wires, that
are inserted into the inlet and outlet wells can be grounded. In an
exemplary embodiment, sample IR absorbance spectra can be
collected, for example, every 90 seconds, while a DC potential
V.sub.G is applied to the gate, for example, varying from about
-10V to about 20 V. The observed characteristic peak intensity
ratio from IR absorbance can be converted to its corresponding pH
value using a corresponding pH calibration curve.
[0085] In this example, upon applying a positive gate bias V.sub.G
of about +10V, the pH value in nanochannels can be observed to
increase, for example, from pH 4.5 to pH 5.3 for about 10 minutes.
The positive gate bias can induce positive charges on an exemplary
SiO.sub.2 walls, by protonation of SiOH groups on channel walls.
The positively charged walls can in turn attract negatively charged
ions including OH.sup.-. The accumulation of OH.sup.- near the
walls can then increase the pH value. Thus, the pH increase can
qualitatively agree with the accumulation of negative charges and
with a reversed flow during FET control.
[0086] In embodiments, the initial pH increase can occur over a
long period, if the initial positive gate bias is high. For
example, when the gate bias is about +30 V, the initial pH increase
can occur over a short period of time of about 60 seconds, where
double-reversed flow can also be observed.
[0087] Following the initial increase in pH, the pH can decrease,
e.g., after about minutes with prolonged gate biasing at VG=+10V.
This decrease in pH can be due to the production of H.sub.3O.sup.+
ions by water electrolysis. In fact, the rate of decrease in pH can
be more pronounced as V.sub.G is increased from about +10V to about
+20 V. This increase in V.sub.G can cause an increase in leakage
current density as shown in FIG. 4. The increased leakage current
density can produce H.sub.3O.sup.+ ions at a faster rate and
subsequently can result in the increased rate of pH decrease.
[0088] The pH in the nanochannels can be continuously monitored
upon grounding the gate and then switching to a negative gate bias,
for example, to about -10 or to about -20 V. For example, V.sub.G
can be set to zero for about 10 minutes. Upon grounding the gate
region 260 through the gate contact 340, the pH can reach a steady
pH value, for example, at about 3.
[0089] When a negative gate bias, for example, of about -10 V, is
applied, the pH can decrease, e.g., in 3 minutes. This decrease in
pH can be caused by the accumulation of positively charged ions,
including H.sub.3O.sup.+, near the insulating channel walls due to
the deprotonation of SiOH groups on SiO.sub.2 walls. This pH
decrease can qualitatively agree with the accelerated
electroosmotic (EO) flow of the electrolyte solution during FET
control.
[0090] With prolonged negative gate biasing of about -10 V,
however, the pH of the buffered electrolyte solution can increase,
counteracting the initial decrease, due to the production of
OH.sup.- ions from the channel walls by water electrolysis. The
rate of increase in pH can be more pronounced as V.sub.G is
increased in magnitude from about -10 to about -20 V. The absolute
magnitude for this rate of increase can be approximately a factor
of two greater than the rate of decrease in pH with a positive gate
bias of equal magnitude. This can be because of the depletion of
H.sup.+ at the negatively biased gate electrode (cathode), in
addition to the asymmetrically larger leakage current with a
negative gate bias.
[0091] In this manner, the observed pH shift in response to the
polarity and magnitude of the gate bias can consistently reflect
the sign and magnitude of the leakage current.
[0092] FIGS. 5A-C depict exemplary models showing an EO flow and pH
shift over time in nanochannels in accordance with various
embodiments of the present teachings. For example, wall adsorption
of the charged molecules; electrolysis from leakage current and
from the electrodes that drive the electroosmosis; pH shift due to
surface charge manipulation and/or from the electrolysis; and
diffusion and reaction of H.sub.3O.sup.+ and OH.sup.- can be
considered in this model. FIGS. 5A-C also schematically describe
how the leakage current ultimately induces the double-reversed flow
during the FET flow control.
[0093] In the exemplary nanofluidic system as shown in FIGS. 5A-C,
the initial buffer solution pH can be about 4 and the SiO.sub.2
channel walls 325 can be negatively charged. Positively charged
counter ions 02 can be accumulated near the channel walls 325.
[0094] FIG. 5A shows that upon applying the longitudinal electric
field 502 pointing to the left (L) in the illustration, these
positively charged ions 02 (e.g., H.sub.3O.sup.+ and other positive
buffer ions) near the wall 325 can move from right (R) to left,
i.e., from anode to cathode, inducing an EO flow 505a.
[0095] In FIG. 5B, to reverse the direction of the E flow 505a, a
positive gate bias 350 (V.sub.G>0V) can be applied to the gate
to raise the .zeta.-potential. Positively charged ions 02 can be
repelled from the walls 325, whereas negatively charged ions 01,
e.g., OH.sup.- and other negative buffer ions, can be attracted to
the walls 325. Thus, the EO flow can be a reversed EO flow 505b. As
shown, this reversal in the direction of EO flow can be induced by
the surface charge control.
[0096] In FIG. 5C, with a continuing application of the positive
gate bias 350 (V.sub.G>0V), however, the leakage current through
the gate and the SiO.sub.2 walls 325 can cause water electrolysis
and can generate H.sub.3O.sup.+ ions or H.sup.+ ions 03 near the
surface of the channel walls 325. These positive ions 03 can
populate the solution-wall interface by displacing the negative
ions 01 that have previously accumulated at the interface. The
population of H.sub.3O.sup.+ ions or H.sup.+ ions 03 can then lead
to a double-reversed flow. That is, small, but measurable leakage
current through the FET gate during FET flow control can lead to
anomalous flow characteristics over time.
[0097] A similar phenomenon can occur with a negative gate bias
V.sub.G (not shown) that produces OH.sup.- near the gate region,
where the EO flow can be accelerated upon the application of the
negative V.sub.G. The prolonged application of the gate electric
potential V.sub.G can then reverse a direction of the EO flow. In
this case, multi-reversed flow can also be observed. The difference
can include that the accelerated pace of the exemplary
double-reversed flow due to the asymmetry in the magnitude of
leakage current depending on V.sub.G polarity as indicated in FIG.
4.
[0098] In embodiments, the disclosed systems and methods in FIGS.
3-5 can be used for focusing, separating and analyzing proteins
with low concentrations, wherein the electrolyte solution can be a
protein mixture solution. The focusing and separation of proteins
in the protein mixture solution can be controlled and performed by
an isoelectric focusing (IEF), a dynamic field gradient focusing
(DFGF) and/or a combination thereof using the systems and methods
as described in FIGS. 3-5.
EXAMPLES
Example 1
Nanofluidic Device and Characterizations
[0099] A nanofluidic separation matrix was fabricated and operated
as described in Lab on a Chip 2009, entitled "Effect of
Wall-Molecule Interactions on Electrokinetic Transport of Charged
Molecules in Nanofluidic Channels during FET Flow Control;" in Lab
on a Chip 2009, entitled "Impact of Leakage Current and
Electrolysis on FET Flow Control and pH Changes in Nanofluidic
Channels;" and in Lab on a Chip 2009, entitled "Experimentally and
Theoretically Observed Native pH Shifts in a Nanochannel array;"
which are hereby incorporated by reference in their entirety.
[0100] An integrated nanofluidic device was fabricated based on
semiconductor device fabrication techniques as described in Lab on
a Chip 2008, entitled "Monitoring FET Flow Control and Wall
Adsorption of Charged Fluorescent Dye Molecules in Nanochannels
Integrated into a Multiple Internal Reflection Infrared Waveguide,"
which is hereby incorporated by reference in its entirety.
[0101] In this example, the separation platform of the nanofluidic
device had seven nanochannel arrays with each array having a width
of about 50 .mu.m and a length of about 14 mm formed on a
rectangular Si substrate. The substrate had a width of about 1 cm
and a length of about 5 cm. Each nanochannel array of the device
included approximately a hundred twenty parallel nanochannels. The
dimensions of each nanochannel were about 100 nm width.times.400 nm
depth.times.14 mm length. Nanochannels were fabricated using
interferometric lithography (IL) and plasma etching of Si. A
thermally grown SiO.sub.2 layer (.about.100 nm) was used as an
electrically insulating layer between Si nanochannel walls and the
fluid. The nanochannels were sealed with a Pyrex cover by anodic
bonding to form the nanofluidic device.
[0102] Optical transparency through the anodically bonded Pyrex
cover allowed access to laser-scanning confocal fluorescence
microscopy [LS-CFM, Zeiss Axioskop (Chester, Va.) with an LSM5
scanning head] from the top, while IR-transparency through the Si
substrate with beveled edges allowed access to multiple-internal
reflection Fourier transform infrared spectroscopy (MIR-FTIRS,
Nicolet 870 with a mid-IR HgCdTe detector).
Example 2
System Equilibrium
[0103] The solution reservoirs (i.e., end wells) and nanochannels
were first filled completely with a buffer solution by capillary
force. Approximately 30 minutes lapsed before the system reached
equilibrium between the buffer solution and SiO.sub.2 channel
walls. This was based on an observation that no noticeable changes
were detected in IR absorbance spectra after 30 minutes. A mixture
of proteins was then introduced to one of the wells (as an inlet),
and a platinum wire was inserted into each well as an electrode. An
electric potential (V) was applied to the protein-containing well,
while grounding the other well to create a longitudinal electric
field ( ) along the nanochannels and to induce an electroosmotic
(EO) flow typically with opposing electrophoresis (EP) for
negatively charged molecules and to create a longitudinal pH
gradient by electrolysis occurring at the electrodes.
Example 3
Buffers and Proteins
[0104] Sodium phosphate buffer was used in the exemplary
experiments with a buffer pH of 7.2 and an ionic strength of 10 mM.
All proteins were diluted to .about.0.02 .mu.g/mL in the buffer
solution in experiments.
[0105] Various exemplary proteins were used as examples.
Allophycocyanin (APC), r-phycoerythrin (RPE), bovine serum albumin
(BSA) conjugated with Alexa Fluor.RTM. 488, and ovalbumin (OVA)
conjugated with Alexa Fluor.RTM. 555 (see Table 1), were purchased
from Invitrogen Corporation (Carlsbad, Calif.) and were used as
examples for analyzing proteins using the disclosed systems and
methods. Green fluorescent proteins (GFPs) were purchased from
Upstate Biotechnology (Lake Placid, N.Y.) and were used to
represent small proteins with net positive charge. Molecular
weights, isoelectric points, net charges, and specifications on
fluorescence (i.e., absorbance, emission, and excitation) of the
exemplary proteins are summarized in Table 1.
TABLE-US-00001 TABLE 1 Bovine Oval- Serum bumin- Albumin- Alexa
R-phyco- Allophy- Alexa Fluor erythrin cocyanin GFP Fluor 488 555
Iso- 5.1~4.2 4.8~4.95 5.67 4.47~4.85 4.43 electric point Molecular
240,000 104,000 30,000 66,000 45,000 weight Abs(nm) 480, 546, 650
515 565 EM(nm) 578 660 509 519 519 EX(nm) 568-590 633 488 488 555
Net Negative Negative Positive Negative Negative charge
Example 4
Protein Focusing of BSA and OVA
[0106] Protein focusing was conducted with an exemplary mixture of
BSA (I.sub.P=4.47.about.4.85) and OVA (I.sub.P=4.43). BSA and OVA
are small proteins with sizes slightly different and are all with
net negative charges.
[0107] In this example, a time series of schematic images from the
BSA/OVA mixture was observed by LS-CFM in one of the seven
nanochannel arrays. The BSA/OVA mixture can include protein
BSA-Alexa Fluor.RTM. 488 conjugates with EM.sub.BSA=488 and protein
OVA-Alexa Fluor.RTM. 555 conjugates with EM.sub.OVA=567. A negative
potential, V.sub.EP5=.about.5, was applied to the inlet well (a
right well as shown in FIGS. 2C-2E) in which proteins were
introduced.
[0108] As observed, for about 36 minutes after the electric
potential was applied, proteins were not detected in the
nanochannels. This is because electroosmosis flow from left to
right was strongly dominant than electrophoresis in nanochannels.
In the 37.sup.th minute, however, OVA was appeared and focused near
the inlet well forming a very sharp band of .about.5 .mu.m in
width.
[0109] As also observed, BSA repeatably advanced ahead of OVA from
the inlet well in all experiments. The high-resolution focusing of
these two proteins continued for about 15 minutes. Then, two bands
flowed at 1 .mu.m/s by electrophoresis and became dispersed after
traversing 2 mm from the inlet well. Proteins may pass through
their isoelectric point due to electrophoresis.
Example 5
Repeatable High-Resolution Protein Focusing of BSA and OVA
[0110] The high-resolution protein focusing of BSA and OVA in
nanochannels was repeatable. The high-resolution focusing of these
two proteins was monitored and demonstrated once again by
increasing the electric potential (V.sub.EP). A time-series snap
shots of electrokinetic flow and high-resolution focusing of BSA
and OVA were also observed when the electric potential (V.sub.EP)
magnitude was increased from -5 to -10 V. The time-series snap
shots were taken every 2 minutes. Note that a portion of proteins
was randomly dispersed in nanochannels since they previously flowed
from right to left by electrophoresis (see Example 4). Upon
applying -10 V.sub.EP, BSA and OVA quickly flowed back towards the
inlet well at 7.3 .mu.m/s for 6 minutes. This flow was induced
because the increased electric potential breaks electrokinetic
equilibrium and makes the electroosmosis flow from left to right
more dominant than electrophoresis.
[0111] In this Example 5, the focused band position was 300 .mu.m
from the inlet well, which is farther than the distance observed
from the lower electric potential (V.sub.EP=-5) in Example 4. This
indicates that the isoelectric points of proteins moved farther
into the nanochannels as the pH gradient changed due to the higher
magnitude of electric potential (-10 V.sub.EP). After stationary
focusing of the two proteins for about 12 minutes, they flowed
farther into the nanochannels at 0.3 .mu.m/s for 12 minutes.
[0112] Upon continuous biasing, the two proteins started to flow
again from right to left and became dispersed after flowing
approximately 3 mm from the inlet well. This result was consistent
with the results shown in Example 4. However, a sharp band of OVA
and a portion of BSA were observed remaining in the nanochannels.
While not desiring to be bound by any particular theory, this may
have been caused by: (1) adsorption of proteins to the nanochannel
walls or (2) continuous protein focusing after most of the proteins
have moved out of their isoelectric point by electrophoresis.
Example 6
Protein Separations
[0113] To achieve clear band separation of proteins, a protein
mixture including BSA, OVA, RPE, APC, and GFP were used in various
combinations. As a result, in the range of electric potential from
-5 to -40 V.sub.EP, BSA and OVA always appeared and formed sharp
bands. In contrast, RPE, APC, and GFP did not form bands until the
electric potential was raised to -40 V.sub.EP. However, upon
applying -60 V.sub.EP RPE and APC both appeared forming sharp bands
near the inlet well, while GFP still did not appear in the
nanochannels.
[0114] GFP (MW .about.30,000) was not shown in the range of
electric field (V.sub.EP=-5.about.-60) as studied here, although
the molecular size was the smallest. This phenomenon is likely due
to the electrostatic charge interaction of charged GFP and the
cathode. Therefore, the pH value in the nanochannel was estimated
to be approximately 6 at which GFP was positively charged, while
positively charged GFP did not flow into the nanochannels as long
as the negative potential was applied to the inlet electrode (the
cathode).
[0115] In experiments, BSA was observed to have a higher mobility
than OVA, and this mobility difference was used for separation.
Various experiments also included focusing and separation of BSA
and OVA by modulating the magnitude of the electric potential
(V.sub.EP) from -20 to -40 V.
[0116] As observed in the particular experiment, band formations of
BSA and OVA were achieved in 5 minutes upon applying an electric
potential of about -40 V.sub.EP. This band formation occurred more
quickly than in the case of using a low electric potential
(V.sub.EP). These band formations continued for 8 minutes. When the
electric potential magnitude was lowered to -20 V.sub.EP, these
bands quickly moved from right to left (e.g., from the 10.sup.th
minute to the 18.sup.th minute) at 60 .mu.m/s. That is, the
low-magnitude (-20V) weakened the electroosmosis flow, inducing
bands of proteins to move from right to left. The protein bands
again moved back towards the inlet upon raising the electric
potential magnitude to -40 V.sub.EP, and again its direction
reversed upon lowering the magnitude to -20 V.sub.EP. This
observation was highly repeatable. The other significant phenomenon
observed meanwhile was a separation of BSA and OVA. Upon lowering
the electric potential magnitude to -20 V.sub.EP, BSA completely
moved back to the inlet, whereas OVA was still focused and remained
at the same position.
Example 7
Nanofluidic Lab-on-a-Chip System
[0117] Double-side-polished Si (100) wafers about 1 cm wide and
about 5 cm long were used as the substrate. A 3-mm wide boron doped
gate region was defined perpendicularly to the channel direction at
the center of the wafer. The dopant diffusion was carried out for
about 60 minutes at about 1050.degree. C. in an O.sub.2/N.sub.2
environment, which resulted in the formation of a diffusion layer
with a depth of about 1-1.2 .mu.m and a dopant level on the order
of about 1.times.10.sup.20 cm.sup.-3. An array of nanochannels was
fabricated along the direction of IR propagation, using
interferometric lithography (IL) and plasma etching.
[0118] Immediately after the etching, each channel was
approximately 200 nm wide and 450 nm deep. The nanochannel array
occupied a total area of about 3 mm wide by 16 mm long, which
contained up to about 8000 nanochannels. A thermal SiO.sub.2 layer
was grown up to about 100 nm, reducing the channel width to about
100 nm and the channel depth to about 400 nm.
Example 8
pH Shift in Nanochannels
[0119] A buffer solution was introduced into the nanochannels by
capillary force. Platinum (Pt) wires were used as electrodes
immersed in two solution wells on opposite ends of the channels. To
induce an electroosmotic flow along the channels, a positive
potential (V.sub.EO>0) was applied to the inlet, and the outlet
was grounded, generating a longitudinal electrical field.
[0120] After the electroosmotic flow was induced, a potential
(V.sub.G) was applied to the highly doped gate to modulate the
surface charge on channel walls and to conduct FET flow control.
V.sub.EO and V.sub.G shared a common ground to maintain the same
reference potential.
[0121] During the FET control, the flow of fluorescent dye
molecules in the nanochannels was monitored by LS-CFM. Alexa 488
maleimide was used as an example to visualize the FET flow control
with LS-CFM, because the fluorescence intensity of Alexa 488 was
strong and stable in a relatively wide pH range from about 4 to
about 9. The excitation and emission wavelengths of Alexa 488 were
about 488 nm and 519 nm, respectively.
[0122] In the FET flow control experiments, Alexa 488 was dissolved
in a pH 4 buffer, since SiO.sub.2 channel walls have an isoelectric
point (pKa) of about 3.7, where the net charge on the surface is
zero. Above the isoelectric point, the surface charge became
increasingly negative, as surface hydroxyl groups (SiOH) became
deprotonated. Conversely, the surface charge gradually turned off
or became further protonated as [SiOH.sub.2].sup.+, as pH decreased
below pKa. Therefore, the surface charge control and its impact on
FET flow control were relatively more pronounced near the
isoelectric point.
[0123] To monitor the pH shift, a buffer solution was injected with
a desired pH value ranging from about 2 to about 8 into the
nanochannels, and the system was allowed to equilibrate for
approximately 20 minutes, after which no noticeable change in IR
spectrum was observed. An IR background spectrum was taken with 2
cm.sup.-1 resolution averaged over 100 scans. Taking the background
only with the buffer solution minimized interference from
absorption bands of water, when sample spectra were taken with
fluorescein solution. The channels were then cleaned in DI water
and dried on a hot plate. A buffer solution of fluorescein
(C.sub.20H.sub.12O.sub.5) dye molecules with a known pH was then
injected into one of the two solution wells to fill the
nanochannels, and a series of sample IR spectra with the same
resolution and averaging were taken to monitor the characteristic
vibrational modes of fluorescein dye molecules that are sensitive
to the pH shift.
[0124] Fluorescein was a commonly used fluorescent dye molecule,
whose quantum yield was strongly affected by solution pH. The
absorption and emission wavelengths of fluorescein are about 494
and 521 nm, respectively. The variation in quantum yield for
fluorescein was monitored with FTIR spectroscopy to relate a pH
shift to the molecule's structural change. Depending on the pH of
buffer solutions, fluorescein can become a cation (pKa <2.08), a
neutral molecule (pKa=2.08.about.4.31), an anion
(pKa=4.31.about.6.43) or a dianion (pKa >2.08) by protonation or
deprotonation of carboxyl group and OH group on the molecule. Thus,
the pH in nanochannels was monitored by analyzing the molecular
structure of charged species using MIR-FTIRS.
[0125] Deuterated water (D.sub.2O, 99.9 atom % D, obtained from
Sigma-Aldrich) instead of H.sub.2O was used to avoid overlapping
between the vibrational modes of H.sub.2O [.nu.s(OH) at
3400.about.3000 cm.sup.-1 and .delta.s(HOH) at 1640 cm.sup.-1], and
those of fluorescein [.nu.s(CH.sub.x) and .nu.as(CH.sub.x) at
3000-2800 cm.sup.-1 and .nu.as(COO.sup.-) and .nu.s(C--C) at
1600-1580 cm.sup.-1].
[0126] Various buffers were used to monitor IR spectra of
fluorescein in different pH buffer solutions from pH 2 to 8.
Chloroacetic acid (pKa=2.83) buffer was used for pH 2 to 3, acetate
buffer (pKa=4.76) was used for pH 4-5, pyridine buffer (pKa=5.23)
was used for pH 6, phosphate buffer (pKa=7.2) was used for pH 7,
and tris-glycine buffer (pKa=8.02) was used for pH 8. The pH for
each buffer was adjusted with HCl or NaOH, and the ionic strength
of all buffer solutions was approximately adjusted to about 1-2 mM.
At this ionic strength, .lamda..sub.D (1/k) is approximately about
10 nm determined by unit charge, dielectric permittivity, vacuum
dielectric constant, Boltzmann's constant, temperature, valence
charge, and charge density.
Example 9
Reversed EO Flow in Nanochannels
[0127] Using LS-CFM, the FET flow control was monitored with Alexa
488 maleimide (1 mgmL.sup.-1) dissolved in a pH 4 buffer solution.
After filling the nanochannels only with the buffer solution, Alexa
488 was injected into the inlet well. To induce an EO flow in the
nanochannels, a V.sub.EO of about +6 V was applied to the
right-side well in FIG. 3A, where Alexa 488 was contained, while
the opposite well on the left side was grounded. The electroosmotic
flow of Alexa 488 moving from the right side (V.sub.EO=+6) to the
left side (grounded) was at a rate of about 3.2 .mu.ms.sup.-1.
[0128] With a constant longitudinal electrical field with V.sub.EO,
when a negative bias (V.sub.G=-30V) was applied to the gate to
lower the .zeta.-potential, the flow velocity of Alexa 488 was
increased to about 25 .mu.ms.sup.-1 moving from right to left at an
accelerated pace. The enhanced flow velocity upon applying the
negative V.sub.G was an order of magnitude greater than the EO flow
velocity of about 3.2 .mu.ms.sup.-1, which was induced by the
longitudinal electrical field but without V.sub.G. Conversely,
Alexa 488 rapidly reversed its flow when the V.sub.G of +30V was
applied to the gate to raise the .zeta.-potential.
[0129] Confocal images of Alexa 488 upon inducing an EO flow with
V.sub.EO of about +6V and subsequently applying a positive gate
bias V.sub.G(+30V) shows that after inducing the reverse flow with
the positive V.sub.G, the speed and direction of EO flow are not
maintained constantly over time. When the EO flow was moving from
right to left at 3.2 .mu.ms.sup.-1, upon applying the positive gate
bias (V.sub.G=+30V), Alexa 488 reversed its flow moving from left
to right for about 60 seconds and then started to reverse its flow
again moving to the left.
[0130] While the invention has been illustrated with respect to one
or more implementations, alterations and/or modifications can be
made to the illustrated examples without departing from the spirit
and scope of the appended claims. In addition, while a particular
feature of the invention may have been disclosed with respect to
only one of several implementations, such feature may be combined
with one or more other features of the other implementations as may
be desired and advantageous for any given or particular function.
Furthermore, to the extent that the terms "including", "includes",
"having", "has", "with", or variants thereof are used in either the
detailed description and the claims, such terms are intended to be
inclusive in a manner similar to the term "comprising." As used
herein, the term "one or more of" with respect to a listing of
items such as, for example, A and B, means A alone, B alone, or A
and B. The term "at least one of" is used to mean one or more of
the listed items can be selected.
[0131] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all sub-ranges subsumed therein. For example, a
range of "less than 10" can include any and all sub-ranges between
(and including) the minimum value of zero and the maximum value of
10, that is, any and all sub-ranges having a minimum value of equal
to or greater than zero and a maximum value of equal to or less
than 10, e.g., 1 to 5. In certain cases, the numerical values as
stated for the parameter can take on negative values. In this case,
the example value of range stated as "less than 10" can assume
values as defined earlier plus negative values, e.g. -1, -1.2,
-1.89, -2, -2.5, -3, -10, -20, -30, etc.
[0132] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *