U.S. patent application number 11/329639 was filed with the patent office on 2006-07-27 for fluid processing device comprising surface tension controlled valve.
This patent application is currently assigned to Applera Corporation. Invention is credited to Sergey V. Ermakov.
Application Number | 20060165565 11/329639 |
Document ID | / |
Family ID | 42539388 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060165565 |
Kind Code |
A1 |
Ermakov; Sergey V. |
July 27, 2006 |
Fluid processing device comprising surface tension controlled
valve
Abstract
A fluid processing device adapted to produce different oligomers
in a plurality of respective reaction sites and methods of using
the same are provided. The fluid processing device can comprise a
first manifold for delivering reactants to the plurality of
reaction sites, and a second manifold for removing waste from, and
optionally delivering wash fluid to, the plurality of reaction
sites. Surface tension controlled valves can be disposed in fluid
communication with the first manifold, the second manifold, or
both, and can selectively allow reactants and/or fluids into the
reaction sites. A method of making oligonucleotides is also
provided.
Inventors: |
Ermakov; Sergey V.;
(Hayward, CA) |
Correspondence
Address: |
KILYK & BOWERSOX, P.L.L.C.
3603 CHAIN BRIDGE ROAD
SUITE E
FAIRFAX
VA
22030
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
42539388 |
Appl. No.: |
11/329639 |
Filed: |
January 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11092180 |
Mar 29, 2005 |
|
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11329639 |
Jan 11, 2006 |
|
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60642828 |
Jan 11, 2005 |
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Current U.S.
Class: |
422/130 |
Current CPC
Class: |
B01L 2300/168 20130101;
B01L 2400/0415 20130101; B01L 2200/10 20130101; C40B 50/14
20130101; B01J 19/0046 20130101; B01L 3/502738 20130101; Y10T
436/115831 20150115; B01L 2400/0427 20130101; B01L 2400/06
20130101; B01J 2219/00675 20130101; B01J 2219/00389 20130101; B01L
2200/0605 20130101; B01J 2219/0045 20130101; B01J 2219/00695
20130101; B01L 7/52 20130101; B01L 2400/0409 20130101; B01L
2400/0448 20130101; B82Y 30/00 20130101; B01L 2300/0816 20130101;
Y10T 436/12 20150115; B01L 2400/0688 20130101; F15D 1/00 20130101;
B01L 2400/0406 20130101; C40B 60/14 20130101; B01J 2219/00441
20130101; B01J 2219/00722 20130101; B01L 2400/0487 20130101; B01L
2300/0864 20130101; Y10T 137/0391 20150401; B01L 3/502792 20130101;
B01J 2219/00367 20130101; B01J 2219/00369 20130101; Y10T 436/2575
20150115; B01J 2219/00439 20130101; Y10T 137/206 20150401; Y10T
137/2224 20150401; B01J 2219/00448 20130101; C40B 40/06 20130101;
F15D 1/06 20130101; B01L 3/50273 20130101; B01L 3/502784
20130101 |
Class at
Publication: |
422/130 |
International
Class: |
B01J 19/00 20060101
B01J019/00 |
Claims
1. A method comprising: introducing a first nucleic acid monomer
into a first fluid distribution manifold of a fluid processing
device; opening at least one surface tension controlled valve in
fluid communication with both the first fluid distribution manifold
and at least one respective reaction site, to form an open surface
tension controlled valve; moving the first nucleic acid monomer
from the first manifold, through the at least one open surface
tension controlled valve, and into the at least one respective
reaction site; and attaching the first nucleic acid monomer to a
first structure in the at least one respective reaction site to
form an extended structure.
2. The method of claim 1, wherein the first nucleic acid monomer is
a first protected nucleic acid monomer, the extended structure is a
protected extended structure, and the method further comprises:
washing the at least one respective reaction site subsequent to the
attaching; closing the at least one surface tension controlled
valve; introducing a deprotecting agent into the first manifold
then opening the at least one surface tension controlled valve to
form at least one reopened surface tension controlled valve; moving
the deprotecting agent from the first manifold, through the at
least one reopened surface tension controlled valve, and into the
at least one respective reaction site; and deprotecting the
protected extended structure to form a deprotected extended
structure.
3. The method of claim 2, further comprising: introducing a wash
reagent into a second manifold in fluid communication with the at
least one respective reaction site; moving the wash reagent from
the second manifold into the at least one respective reaction site;
and removing the wash reagent from the at least one respective
reaction site to form a washed and deprotected extended
structure.
4. The method of claim 1, wherein the first structure is supported
by a support and the method further comprises cleaving the extended
structure from the support to form a cleaved structure.
5. The method of claim 4, further comprising moving the cleaved
structure from the at least one respective reaction site into a
second reaction site that is in fluid communication with the at
least one respective reaction site.
6. The method of claim 1, wherein the extended structure comprises
a dimethyltrityl-protected phosphoramidite monomer.
7. The method of claim 1, wherein opening the at least one surface
tension controlled valve comprises directing electromagnetic
radiation toward the at least one surface tension controlled
valve.
8. The method of claim 1, wherein opening at least one surface
tension controlled valve comprises reflecting electromagnetic
radiation emitted from an electromagnetic radiation source toward
the at least one surface tension controlled valve.
9. The method of claim 8, wherein the reflecting comprises
individually controlling movement of a plurality of mirrors.
10. The method of claim 1, wherein the at least one surface tension
controlled valve comprises a plurality of surface tension
controlled valves, and the at least one respective reaction site
comprises a plurality of respective reaction sites.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/092,180 filed on Mar. 29, 2005, and claims
the benefit under 35 U.S.C. .sctn. 119(e) of Provisional
Application No. 60/642,828 filed on Jan. 11, 2005, each of which is
incorporated herein by reference in its entirety.
FIELD
[0002] The present teachings relate to valves in fluid processing
devices, and methods for using the same.
INTRODUCTION
[0003] One of the challenges encountered in fluid processing
devices, particularly devices designed for high throughput
operations, is how to effectively control fluid flow. A need exists
to individually and independently control fluid flow in thousands
of micro-channels without resorting to the fabrication of
sophisticated valving systems that can make microfluidic devices
very expensive.
SUMMARY
[0004] According to various embodiments, a device and method for
controlling fluid flow in a microfluidic system is provided. In
some embodiments, a surface tension controlled valving system for
biological fluid is provided that can comprise a channel connected
to an internal volume. The internal volume can be bound by an
insulating layer resistant to the flow of the biological liquid,
wherein the channel can be non-resistant to the flow of the
biological liquid. A photoconductive material can be coupled to the
insulating layer. An electrode can be coupled to the
photoconductive material and can be configured to electrically
couple with the insulating layer through the photoconductive
material. A power source can be electrically coupled to the
electrode. The power source can be configured to provide an
electrical potential difference between the photoconductive
material and the biological fluid. The photoconductive material can
be activatable by light directed thereat, to provide an electrical
potential difference between the insulating layer and the
biological fluid. The electrical potential difference can be
configured to reduce the resistance, of the insulating layer, to
the flow of the biological liquid.
[0005] According to various embodiments, a device for biological
fluid handling is provided that can comprise a valve configured for
light activation. The device can comprise a channel connected to an
internal volume. The internal volume can be bound by an insulating
layer resistant to the flow of the biological liquid, wherein the
channel can be non-resistant to the flow of the biological liquid.
A photoconductive material can be coupled to the insulating layer.
An electrode can be coupled to the photoconductive material and
configured to electrically couple with the insulating layer through
the photoconductive material. A power source can be electrically
coupled to the electrode. The power source can be configured to
provide an electrical potential difference between the
photoconductive material and the biological fluid. A light source
can be adapted to activate the photoconductive material thereby
providing the electrical potential difference between the
insulating layer and the biological fluid. The electrical potential
difference can be configured to reduce the resistance of the
insulating layer to the flow of the biological liquid.
[0006] According to various embodiments, a device for biological
fluid handling is provided that can comprise means for providing
the biological fluid to a valving means. The means for providing
the biological fluid can be non-resistant to the flow of the
biological liquid. The valving means can be resistant to the flow
of the biological liquid. The device can comprise a means for
electrowetting the valving means to reduce the resistance of the
valving means to the flow of the biological liquid. The device can
comprise a means for optically activating the means for
electrowetting.
[0007] According to various embodiments, a fluid processing device
is provided that can comprise a plurality of reaction sites. The
fluid processing device can comprise a first fluid transport
manifold in fluid communication with each of the plurality of
reaction sites. The fluid processing device can comprise a second
fluid transport manifold in fluid communication with each of the
plurality of reaction sites. The fluid processing device can
comprise a plurality of surface tension controlled valves disposed
between the first manifold and at least one respective reaction
site of the plurality of reaction sites. Each surface tension
controlled valve can be in fluid communication with the first
manifold and the at least one respective reaction site.
[0008] According to various embodiments, a method is provided for
synthesizing oligonucleotides or other chemical structures, from
component building blocks, the method can comprise introducing a
first monomer into a first fluid distribution manifold of a fluid
processing device, opening at least one surface tension control
valve in fluid communication with both the first fluid distribution
manifold and at least one respective reaction site, to form an open
surface tension control valve; moving the first monomer from the
first manifold, through the at least one open surface tension
control valve, and into the at least one respective reaction site,
and attaching the first monomer to a first structure in the at
least one respective reaction site to form an extended
structure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Various embodiments of the present teachings are exemplified
by the accompanying drawings which are incorporated in and
constitute a part of this specification. The teachings are not
limited to the embodiments depicted, and can include equivalent
structures and methods as set forth in the following description
and as would be known or recognized by those of ordinary skill in
the art given the present teachings. In the drawings:
[0010] FIG. 1 is a cross-sectional view of a channel and
illustrates connecting two reservoirs;
[0011] FIGS. 2A and 2B are cross-sectional views illustrating the
functioning of surface tension controlled valves;
[0012] FIG. 3 is a side view of a surface tension controlled valve
and illustrates movement of a liquid by electrowetting and
illustrating in phantom the effect that actuation of a surface
tension control valve can have on the shape of a drop of water;
[0013] FIG. 4A illustrates a surface tension controlled valve
closed to the flow of a liquid;
[0014] FIG. 4B illustrates a surface tension controlled valve
permitting the flow of a liquid;
[0015] FIG. 5A-5C illustrates movement of a liquid by
opto-electrowetting. The figure illustrates in phantom the effect
that actuation of a surface tension control valve can have on the
shape of a drop of water;
[0016] FIG. 6A-6C illustrates moving a liquid through a channel via
opto-electrowetting;
[0017] FIG. 7A-7D are cross-sectional views illustrating the
operation of a light-activated surface tension controlled
valve;
[0018] FIG. 8A is a cross-sectional view through a portion of a
device according to various embodiments and showing a
light-activated surface tension control valve in a closed
state;
[0019] FIG. 8B is the same cross-sectional view as shown in FIG.
8A, but wherein the light-activated surface tension control valve
is in an open state;
[0020] FIG. 8C is the same cross-sectional view as shown in FIG.
8A, but wherein the light-activated surface tension control valve
is in an open state and liquid has passed through the valve;
[0021] FIG. 8D is the same cross-sectional view as shown in FIG.
8A, but wherein the valve is in a closed state after liquid has
passed through the valve;
[0022] FIG. 9 is a top plan view of a portion of a fluid processing
device according to various embodiments;
[0023] FIG. 10 is a top plan close-up view of a portion of a fluid
processing device according to various embodiments;
[0024] FIG. 11 is a perspective view of a system for processing a
fluid processing device according to various embodiments;
[0025] FIG. 12 is a perspective view of a system for processing a
fluid processing device according to various embodiments; and
[0026] FIG. 13 is a perspective view of yet another system for
processing a fluid processing device according to various
embodiments.
[0027] It is to be understood that both the foregoing general
description, figures, and the following detailed description are
exemplary and explanatory only.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0028] Reference will now be made to various exemplary embodiments,
examples of which are illustrated in the accompanying drawings.
Wherever possible, the same reference numbers are used in the
drawings and the description to refer to the same or like
parts.
[0029] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities,
percentages or proportions, and other numerical values used in the
specification and claims, are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least each numerical parameter
should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding
techniques.
[0030] 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,
can inherently contain 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 subranges subsumed therein. For
example, a range of "less than 10" includes any and all subranges
between (and including) the minimum value of zero and the maximum
value of 10, that is, any and all subranges 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.
[0031] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" can
include plural referents unless expressly and unequivocally limited
to one referent. Thus, for example, reference to "a channel
species" can include two or more different channels. As used
herein, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0032] According to various embodiments, surface tension controlled
valves can be operable with any biological liquid that can be
manipulated by electrowetting forces. The term "biological liquid"
as used herein refers to liquid with biomolecules, for example
nucleic acids, peptides, enzymes, cells, etc. Biological liquids
that are electrolytic can be used in the surface tension controlled
valves according the present teachings. The term "electrolytic" can
refer to a liquid containing substances dissolved therein, such as
ionic salts, that can enable the liquid to conduct an electric
current. By way of non-limiting example, biological liquids that
can be used in the surface tension controlled valves according to
the present teachings can include aqueous liquids, such as water
and buffered saline, as well as non-aqueous fluids such as
dimethylsulfoxide and other non-aqueous solvents. The biological
liquids can include ionic liquids that can be used in surface
tension controlled valves.
[0033] The term "ionic liquids" as used herein refers to salts that
are liquid over a wide temperature range, including room
temperature. The biological liquid can include various substances,
particulate and otherwise. Such substances can include, for
example, surfactants, including anionic, nonionic, cationic, and
amphoteric surfactants. The composition of the liquid, including
the presence of surfactants, biomolecules, and other substances,
can influence the surface wetting, and thus the contact angle, of
the liquid.
[0034] The term "reflective material" as used herein refers to any
material that can reflect a predetermined wavelength of light.
Reflective materials can be a coating, a distinct layer, or a
various components described herein can themselves act as a
reflective materials. Some exemplary reflective materials comprise,
for example, insulators, such as SiO.sub.2, TiN, SiON;
semiconductor materials, such as silicon, germanium, silicon
germanium, and compound semiconductors; polymers, such as
Teflon.RTM., Teflon.RTM. AF; an organic-inorganic hybrid material
as disclosed above, or any other reflective material that will be
known to one of ordinary skill in the art.
[0035] The term "device" as used herein refers to a device that can
be used in any number of biological processes involving
microfluidics, e.g. microscale amounts, of fluid or larger scale.
Generally, microfluidics can involve handling volumes of one
microliter or less. Features contained in microfluidic devices
typically have millimeter to submicrometer dimensions, and can be
adapted to the specific use of the microfluidic device.
[0036] The term "contact angle" as used herein describes the angle
formed as a result of contact between a fluid and a solid surface.
It reflects the interfacial affinity between the fluid and the
solid surface, i.e., the wettability of the surface with respect to
the fluid. The contact angle .THETA. is inversely correlated with
interfacial affinity. When the fluid is in direct contact with the
solid surface, the contact angle is at least 0.degree. but less
than 180.degree.. A contact angle of 180.degree. or greater
indicates that the fluid is not in direct contact with the solid
surface. In such a case, the fluid may directly contact the surface
through an interposing fluid, or may be levitated from the solid
surface. By way of illustration, a highly hydrophilic surface can
form a low angle, e.g., 10, with respect to water droplets.
Similarly, a highly hydrophobic surface can form a high contact
angle, such as 179.degree., with respect to water.
[0037] According to various embodiments, a surface tension
controlled valving system for biological fluid is provided that can
comprise a channel connected to an internal volume for the valving
system, the internal volume can be bound by an insulating layer
resistant to the flow of the biological liquid and the channel
cannot be resistant to the flow of the biological liquid. In
various embodiments, the biological liquid can be an
electrolyte.
[0038] According to various embodiments, a photoconductive material
can be coupled to the insulating layer, an electrode can be coupled
to the photoconductive material and configured to electrically
couple with the insulating layer through the photoconductive
material, and a power source can be electrically coupled to the
electrode. In various embodiments, the power source can be
configured to provide an electrical potential difference between
the photoconductive material and the biological fluid, wherein the
photoconductive material is activatable by directed light. The
photoconductive material can provide an electrical potential
difference between the insulating layer and the biological
fluid.
[0039] According to various embodiments, the system can comprise a
conductive layer between the insulating layer and the
photoconductive material. An insulating material can be hydrophobic
and can be resistant to the flow of a biological liquid. The
resistance of insulating material to the flow of the biological
liquid can also be lowered by using an insulating material that is
hydrophilic when activated.
[0040] According to various embodiments, the channel can be
connected to a first reservoir. A valve can control the flow of the
biological liquid from the first reservoir to a second reservoir.
The reservoir can comprise wells or channels.
[0041] According to various embodiments, a fluid processing device
for biological fluid handling, is provided that can comprise a
valve configured for light activation, a channel connected to an
internal volume of the valve, wherein the internal volume can be
bound by an insulating layer resistant to the flow of the
biological liquid. In various embodiments, the device can comprise
a channel that may not be resistant to the flow of the biological
liquid, a photoconductive material electrically coupled to the
insulating layer, an electrode coupled to the photoconductive
material and configured to electrically couple with the insulating
layer through the photoconductive material, and a power source
electrically coupled to the electrode. In various embodiments, the
power source can be electrically coupled to the photoconductive
material and can be configured to provide an electrical potential
difference across the insulating material. In various embodiments,
a light source can be configured to activate the photoconductive
material thereby providing the electrical potential difference
across the insulating layer, wherein the electrical potential
difference can be configured to reduce the resistance of the
insulating layer to the flow of the biological liquid.
[0042] According to various embodiments, the light source can be a
collimated light source. The collimated light source can comprise
lasers, lamps, and/or light emitting diodes. In various
embodiments, the light source can be directed over a portion of the
photoconductive material by an array of microfabricated mirrors. In
other embodiments, the light source can be a laser beam, and the
laser beam can be directed over a portion of the photoconductive
material by a galvo-mirror.
[0043] According to various embodiments, the light source can be
configured to direct a beam of light through the insulating layer
and through the biological liquid to reach the photoconductive
material. The light source can be configured to direct a beam of
light to the photoconductive material substantially axially.
[0044] According to various embodiments, the channel of the device
can be configured to provide a waveguide for the light. In various
embodiments, the walls of the channel can be the waveguide. In
other embodiments, the channel can be the waveguide.
[0045] According to various embodiments, a fluid processing device
for biological fluid handling is provided that can comprise a means
for providing the biological fluid to a valving means. The means
for providing the biological fluid can allow the flow of the
biological liquid. The fluid processing device can comprise a
valving means that can be resistant to the flow of the biological
liquid. The fluid processing device can comprise a means for
electrowetting the valving means to reduce the resistance of the
valving means to the flow of the biological liquid. The fluid
processing device and means for optically activating the means for
electrowetting. In various embodiments, the means for optically
activating can comprise a means for selectively positioning light
onto a portion of the valving means.
[0046] According to various embodiments, a fluid processing device
is provided that can comprise a plurality of reaction sites, a
first fluid transport manifold in fluid communication with each of
the plurality of the reaction sites, a second fluid transport
manifold in fluid communication with each of the plurality of
sites, a plurality of surface tension controlled valves, or a
combination thereof. In various embodiments, at least one of the
plurality of surface tension controlled valves can be disposed
between the first manifold and at least one respective reaction
site of the plurality of reaction sites. Each surface tension
controlled valve can be in fluid communication with the first
manifold and the at least one respective reaction site. In various
other embodiments, at least one of the plurality of surface tension
controlled valves can comprise a light-actuated valve. In various
embodiments, the system can comprise a plurality of respective
different sources of nucleic acid base. The system can comprise a
loading device for individually loading the different nucleic acid
bases from the plurality of respective different sources into the
first manifold.
[0047] According to various embodiments, the first manifold can
comprise one or more nucleic acid bases selected from adenine,
cytosine, guanine, thymine, and uracil. In various embodiments, the
fluid device can comprise a dimethyltrityl-protected
phosphoramidite nucleotide monomer disposed in the first
manifold.
[0048] According to various embodiments, the fluid processing
device can comprise a planar substrate. The first manifold, the
second manifold, and the plurality of reaction sites can be formed
in the substrate.
[0049] According to various embodiments, at least one of the
plurality of surface tension controlled valves can comprise an
electrically-actuated or electrically-activated valve. In other
embodiments, at least one of the plurality of surface tension
controlled valves can comprise a temperature-actuated valve. In
various embodiments, the fluid processing device can comprise a
fluid communication directly between two adjacent reaction sites of
the plurality of reaction sites.
[0050] According to various embodiments, a system is provided that
can comprise a fluid processing device that can comprise a
plurality of reaction sites. The fluid processing device can
comprise a first fluid transport manifold in fluid communication
with each of the plurality of the reaction sites. The fluid
processing device can comprise a second fluid transport manifold in
fluid communication with each of the plurality of sites. The fluid
processing device can comprise a plurality of surface tension
controlled valves. In various embodiments, at least one of the
plurality of surface tension controlled valves can be disposed
between the first manifold and at least one respective reaction
site of the plurality of reaction sites. Each surface tension
controlled valve can be in fluid communication with the first
manifold and the at least one respective reaction site.
[0051] According to various embodiments, the system can comprise a
pressure differential source in fluid communication with one or
more of the first manifold and the second manifold.
[0052] According to various embodiments, the system can comprise an
electromagnetic radiation source. The electromagnetic source can be
adapted to emit electromagnetic radiation toward one or more of the
plurality of surface tension controlled valves. The electromagnetic
radiation source can comprise a laser. In various embodiments, the
system can comprise a reflective device adapted to reflect
electromagnetic radiation emitted from the electromagnetic
radiation source toward one or more of the plurality of surface
tension controlled valves. The reflective device can comprise a
plurality of individually moveable mirrors.
[0053] According to various embodiments, the system can comprise a
control unit operatively connected to the electromagnetic radiation
source. The control unit can be adapted to control the
electromagnetic radiation source. In various embodiments, the
system can comprise at least one focusing lens disposed along an
emission beam path between the electromagnetic radiation source and
at least one of the plurality of surface tension controlled
valves.
[0054] According to various embodiments, the fluid processing
device can comprise at least one fluid communication between at
least two of the plurality of reaction sites, and the at least one
fluid communication bypasses the first and second manifolds. In
various embodiments, the system can comprise a thermal cycling
block adapted to hold the fluid processing device such that at
least one of the plurality of reaction sites is in heat-transfer
communication with the thermal cycling block. In various other
embodiments, the system can comprise a rotatable platen comprising
a top surface, and a holder adapted to hold the fluid processing
device in or on the top surface.
[0055] According to various embodiments, the system can comprise a
pump adapted to connect to the first manifold and force liquid into
the first manifold.
[0056] According to various embodiments, the system can comprise a
fluid processing device comprising a heater in heat-transfer
communication with a temperature-actuated valve. The heater can
comprise a control unit operatively connected to it and adapted to
control the heater. The fluid processing device can comprise at
least one of a plurality of surface tension control valves and an
electricity source. The electricity source can be electrically
connected to the electrically-actuated valve.
[0057] According to various embodiments, a fluid processing device
is provided that can include a plurality of reaction sites, a first
manifold in fluid communication with each of the reaction sites, a
second manifold in fluid communication with each of the reaction
sites, and at least one surface tension controlled valve positioned
in at least one channel between the first manifold and at least one
of the reaction sites. The reaction sites can each comprise support
structures, for example beads, or an inner surface suitable for the
attachment of oligomers or oligomer precursors thereto. According
to various embodiments, the fluid processing device can comprise a
plurality of surface tension controlled valves each in fluid
communication with the first manifold and one or more of the
reaction sites.
[0058] According to various embodiments, the fluid processing
device can comprise reactants and/or reaction components capable of
producing an oligomer in at least one of the reaction sites, or a
system that includes sources of reactants and/or reaction
components.
[0059] According to various embodiments, a system is provided that
can comprise a fluid processing device as described herein, and an
electromagnetic radiation source capable of emitting
electromagnetic radiation and directing the radiation toward one or
more surface tension controlled valves in the device. Alternatively
or additionally, the system can comprise other valve-actuating
devices besides an electromagnetic radiation source. Exemplary
actuators can comprise heaters adapted to direct heat toward one or
more surface tension controlled valves, or an electrical source
adapted to supply an electrical signal to one or more surface
tension controlled valves. By controlling the one or more surface
tension controlled valves, the systems described herein can be used
in directing the flow of reaction components in an order useful for
carrying out an oligonucleotide synthesis reaction within one or
more of the plurality of reaction sites.
[0060] According to various embodiments, a system is provided that
can comprise an electromagnetic radiation source or other actuating
or activating source, a reflective device, a pump, and a
thermocycler. The system can be adapted so that the reflective
device can direct electromagnetic radiation emitted from the
electromagnetic radiation source toward the one or more surface
tension controlled valves to selectively open or close the
respective one or more surface tension controlled valves. The pump
can be adapted to add or remove materials from the channels and
reaction sites. The thermocycler can be adapted to control the
temperature of the reaction sites, for example, to promote an
isothermal or thermally cycled nucleic acid sequence amplification
and/or detection assay. The system can comprise one or more control
units to control the actuating source, to control the reflective
device, to control the pump, and/or to control the
thermocycler.
[0061] According to various embodiments, a method is provided that
can comprise introducing a first monomer into a first fluid
distribution manifold of a fluid processing device, opening at
least one surface tension controlled valve in fluid communication
with both the first fluid distribution manifold and at least one
respective reaction site to form an open surface tension controlled
valve, moving the first monomer from the first manifold through the
at least one open surface tension controlled valve and into the at
least one respective reaction site, and attaching the first monomer
to a first structure in the at least one respective reaction site
to form an extended structure, or a combination thereof.
[0062] According to various embodiments, the opening of at least
one surface tension controlled valve can comprise directing
electromagnetic radiation, or reflecting electromagnetic radiation
emitted from an electromagnetic radiation source, toward the at
least one surface tension controlled valve. The reflecting can
comprise individually controlling movement of a plurality of
mirrors.
[0063] According to various embodiments, the method can provide a
first protected monomer, and a protected extended structure, and
the method can comprise washing the at least one respective
reaction site subsequent to the attaching, closing the at least one
surface tension controlled valve, introducing a deprotecting agent
into the first manifold, opening the at least one surface tension
controlled valve to form at least one reopened surface tension
controlled valve, moving the deprotecting agent from the first
manifold, through the at least one reopened surface tension
controlled valve and into the at least one respective reaction
site, deprotecting the protected extended structure to form a
deprotected extended structure, or a combination thereof.
[0064] According to various embodiments, the method can comprise
introducing a wash reagent into a second manifold in fluid
communication with the at least one respective reaction site,
moving the wash reagent from the second manifold into the at least
one respective reaction site, and removing the wash reagent from
the at least one respective reaction site to form a washed and
deprotected extended structure. According to various embodiments,
the extended structure can comprise a dimethyltrityl-protected
phosphoramidite monomer.
[0065] According to various embodiments, in the method the first
structure can be supported by a support. The method can further
comprise cleaving the extended structure from the support to form a
cleaved structure. In various embodiments, the cleaved structure
can be moved from the at least one respective reaction site into a
second reaction site that is in fluid communication with the at
least one respective reaction site.
[0066] According to various embodiments, a method is provided for
synthesizing oligonucleotides or other chemical structures, from
component building blocks. The method can comprise, for example,
introducing a first monomer into a first fluid distribution
manifold of a fluid processing device; opening at least one surface
tension controlled valve in fluid communication with both the first
fluid distribution manifold and at least one respective reaction
site, to form an open surface tension controlled valve; moving the
first monomer from the first manifold, through the at least one
open surface tension controlled valve, and into the at least one
respective reaction site; and attaching the first monomer to a
first structure in the at least one respective reaction site to
form an extended structure, or a combination thereof. The first
monomer can be, for example, a nucleotide, a nucleotide base, a
nucleotide analog, a protected chemical building block, or another
monomeric building block, unit, or structure that can bond with and
extend off of a support or precursor structure. The first monomer
can be a protected first monomer, for example, a protected first
nucleic acid monomer, and the extended structure can be a protected
extended structure. The method can further comprise: washing the at
least one respective reaction site subsequent to the attaching;
closing the at least one surface tension controlled valve;
introducing a deprotecting agent into the first manifold; opening
the at least one surface tension controlled valve to form at least
one reopened surface tension controlled valve; moving the
deprotecting agent from the first manifold, through the at least
one reopened surface tension controlled valve, and into the at
least one respective reaction site; deprotecting the extended
protected structure to form a deprotected extended structure or a
combination thereof. An additional monomer can then be added to the
deprotected extended structure and the process can be repeated. The
at least one surface tension controlled valve can comprise a
plurality of surface tension controlled valves, and the at least
one respective reaction site comprises a plurality of respective
reaction sites.
[0067] According to various embodiments, surface tension controlled
valves can comprise channels. Channels can comprise any volume
through which a liquid can be transported. The channels can be made
of glass, and can optionally be transparent, or at least partially
transparent, when employed in light-actuated surface controlled
tension valves. The channels can be constructed of any material
suitable for containment of a given liquid, for example glass or a
polymeric material. The channels can be of any dimension suitable
for manipulating fluids in a desired manner. For example, according
to various embodiments, the length, width and depth of the channels
may range, independently, from about 0.1 .mu.m to about 10 cm. The
channels can range, for example, from about 10 .mu.m to about 1
cm.
[0068] According to various embodiments, surface tension controlled
valves can comprise reservoirs. Reservoirs can include any space
capable of containing a liquid and communicating with at least one
channel. The reservoir can be constructed of any material capable
of holding a liquid, for example, a glass or a polymer. The
reservoir can be of any shape, for example it can be spherical,
semi-spherical, or conical. The reservoir can be of any size
sufficient to hold a desired volume of liquid. For example, the
reservoir may range in size from about 1 nanoliter to about 1
liter. In various embodiments, the reservoir can be unassociated
with an electrode, i.e., the liquid in the reservoir itself can be
adapted to not manipulate a liquid by virtue of a significant
electrical potential difference being applied to that liquid.
[0069] According to various embodiments, at least one portion of a
surface of at least one channel can be coated with a material that
it is chemically resistant to the flow of liquid through the
channel. Suitable non-limiting examples of such materials that can
comprise polymer coatings (e.g., polyamides, polymethylacrylates
and their copolymers), BN and SiN, deposited in accordance with any
of the thin-film deposition techniques known to those of ordinary
skill in the art, and polymer films such as, e.g., Teflon.TM.
(trademark for polytetrafluoroethylene).
[0070] According to various embodiments, at least one layer of
insulation material can be formed above the electrode. The surface
tension controlled valve can have the insulation layer disposed
between the electrode and the internal volume making up the
channel. In various embodiments, the insulation layer can include
at least one layer of silicon oxide and at least one layer of
Teflon.TM. (trademark for polytetrafluoroethylene). The thicknesses
of the two layers can be selected to provide the desired degree of
insulation without, in the case of a light-actuated surface tension
controlled valve, overly impeding the transmission of light.
[0071] According to various embodiments, electrodes can be made
from any conductive material such as, for example, copper, gold,
platinum, and conducting polymers, including polymers that are
conducting per se, and conducting composites containing a
non-conducting polymer and a conducting material such as a metal or
a conducting polymer. A single electrode can be used in the surface
tension controlled valves disclosed herein, or multiple electrodes,
for example, an array of electrodes, can be used. In various
embodiments, the electrode can be transparent, for example, can be
formed of transparent indium tin oxide. This can permit the passage
of light in accordance with certain embodiments of the light
actuated valve, and also can permit visual inspection of the
operation of the valve. In various embodiments, for example, in the
case of a light-actuated surface tension controlled valve, the
electrode or array of electrodes can be in electrical contact with
a photoconductive material.
[0072] According to various embodiments, the photoconductive
material that can be used in the light actuated valves corresponds
to a material with a dark conductivity ranging from 10.sup.-5 to
10.sup.-12 .OMEGA..sup.-1-cm.sup.-1. The photoconductive material
can exhibit relatively low conductivity when dark, and relatively
high conductivity when illuminated by a light source. In various
embodiments, an example of a suitable photoconductive material can
comprise amorphous silicon, which has a dark conductivity of
approximately 10.sup.-8 .OMEGA..sup.-1-cm.sup.-1. In various
embodiments, light with a wavelength ranging from 400 nm to 1100 nm
can be used to illuminate at least portions of the amorphous
silicon. The light intensity for activating the light actuated
surface tension controlled valve can be low. For example, a light
intensity that can be suitable for switching amorphous silicon is
65 mW/cm.sup.2. The layer of photoconductive material can permit
optical control of an electrical potential difference across a
corresponding portion of the channel.
[0073] According to various embodiments, the power source can be
chosen from any source suitable for providing a sufficient
electrical potential difference across a liquid in a channel. For
example, the power source can be configured to provide an
alternating voltage source. The voltage and frequency
characteristics can be chosen according to the materials used in
the surface tension controlled valve and/or a device in which the
valve is situated. The magnitude of the AC voltage source can vary
according to the properties, for example, the thickness, of the
materials used to construct the surface tension controlled valve.
In various embodiments, the AC voltage source can supply an
electrical potential difference ranging from 10 volts to several
hundred volts, with a frequency ranging from 10 Hz to 500 kHz. In
various embodiments, the AC voltage source can be coupled to the
surface tension controlled valve with only two leads. In other
embodiments, the AC voltage source can be inductively coupled such
that no electrical leads are required.
[0074] Exemplary circuits, voltage sources, potential differences,
voltages, and materials are described, for example, in U.S. Pat.
No. 6,958,132, to Chiou et al., issued Oct. 25, 2005, and U.S.
Patent Application Publication No. 2003/0224528 A1, published Dec.
4, 2003, which are incorporated herein in their entireties by
reference.
[0075] According to various embodiments, the light actuated surface
tension controlled valves can employ a light source to illuminate
the photoconductive material associated with the valve. The light
source can be chosen based on any light capable of changing the
conductive properties of the photoconductive material. Suitable
light sources can comprise collimated light sources, and can be
chosen from, for example, lamps, for example arc lamps, lasers, and
light-emitting diodes (LEDs). In various embodiments, the light
source can comprise one or more light sources. For example, a
surface tension controlled valve and/or a device containing a
surface tension controlled valve can include a first light source
and a second light source. In various embodiments comprising more
than one light source, the light sources can be chosen from any
effective light source. The light source can be directed along at
least one axis of the surface tension controlled valve by at least
one mirror, for example, a computer-controlled array of
microfabricated mirrors. In various embodiments, when the light
source is a laser beam, the laser beam may be directed over the
surface of the photoconductive material with a computer-controlled
galvo-mirror.
[0076] According to various embodiments, the light from the light
source can be directed to the photosensitive material by the
channel itself. The channel can provide a waveguide to internally
reflect and propagate the light so that it reaches the
photosensitive material. The waveguide can direct a beam of light
to the photoconductive material substantially axially along the
length of the channel. In various embodiments, the channel can be
configured to provide a waveguide for the light. In various
embodiments, the walls of the channel can provide the waveguide by
internally reflecting and propagating the light within the channel
wall. The channel walls can be constructed of substantially
transparent material with the outer surfaces of the transparent
material coated with a reflective material. In various embodiments,
the channel itself can be the waveguide by internally reflecting
and propagating the light within the channel volume whether filled
or empty. The inner walls of the channel can be coated with a
reflective material.
[0077] According to various embodiments, the surface tension
controlled valves disclosed herein can be used in a variety of
applications. For example, the valves can be used to move one or
more droplets or combine two or more droplets in a device used for
biological synthesis, biological monitoring, or biological
screening. In various embodiments, the surface tension controlled
valves disclosed herein can be used in microdevices designed for
one or more of PCR, ligase chain reactions, antibody binding
reaction, oligonucleotide ligations assays, and hybridization
assays.
[0078] According to various embodiments, individual fluid control
in a fluid processing device 5, for example a microfluidic device,
is provided with a surface tension controlled valve. Referring to
FIG. 1, a surface tension controlled valve 25 can include channel
30, and a portion 20 of the channel can initially be resistant to
the flow of a liquid (e.g., is hydrophobic in the case of an
aqueous liquid) from the internal volume of surface tension
controlled valve 25. As illustrated in FIG. 1, a valve 25 can
control fluid flow through channel 30 between reservoir 10 and
reservoir 40. The surface tension of the liquid, in combination
with the resistance of the surface of at least a portion of channel
30 to the flow of the liquid, can prevent its flow from reservoir
40 to reservoir 10.
[0079] According to various embodiments, the surface tension
controlled valve can comprise a channel or conduit. The channel or
conduit can comprise an initially or normally hydrophobic surface.
The surface tension controlled valve can be adapted to change the
contact angle and wetting of a liquid disposed therein with respect
to the inner surface of channel 30. This change can trigger the
movement of a liquid through channel 30.
[0080] Surface tension controlled valves can exploit the fact that
under certain circumstances the surface tension of the liquid can
change, and that change in turn can trigger a movement of that
liquid. Examples of such circumstances can include applied electric
field (electric field), applied electric field and light
(opto-electrowetting), local increase in temperature, and the
like.
[0081] According to various embodiments, a fluid processing device
can comprise a surface tension controlled valve disposed in a valve
channel that is in fluid communication with a supply channel and a
reaction region. The surface tension controlled valve can comprise
a channel with an initially or normally hydrophobic surface. The
surface tension controlled valve can be adapted to change the
contact angle and wetting of a liquid disposed therein with respect
to the inner surface of the valve channel. This change can trigger
the movement of a liquid through the valve channel. Examples of the
mechanism that can be used to trigger the movement can include the
application of an electric field as with electrowetting, the
application of an electric field and light as with
optoelectrowetting, the application of a local increase in
temperature, and the like.
[0082] According to various embodiments, as illustrated in FIG. 2A,
a portion of a fluid processing device is shown comprising first
channel 40 separated from second channel 30 by surface tension
controlled valve 25. The pressure created by the surface tension in
the surface tension controlled valve 25 can be sufficient to
prevent liquid 50, for example, an aqueous biological sample, from
entering second channel 30. If the pressure difference across the
surface tension controlled valve exceeds a certain threshold
pressure, the resistance to the flow due to the hydrophobic
properties of the valve can be overcome, and the liquid can flow
through the valve. Likewise, if the pressure of the sample liquid
is maintained below the threshold pressure, the valve will hold
back the liquid sample and prevent flow into channel 30. According
to various embodiments, by changing the surface tension of the
valve from having a hydrophobic property to having a hydrophilic
property, liquid movement through the valve can be regulated, even
at pressure below the threshold pressure described above.
[0083] According to various embodiments, if the surface of a
channel is resistant to the flow of a liquid, for example, is
hydrophobic, some additional force or pressure can be required to
push the liquid through the hydrophobic part of the channel. With
reference to FIG. 2A, this principle can be used in hydrophobic
valves when, for instance, the first liquid 50 under certain
pressure P.sub.1 can flow through channel 40 but not channel 30
filled with second liquid 60 (the second liquid can include a gas
or be a gas) under pressure P.sub.2 and separated by surface 20
that is resistant to the flow of the first liquid (e.g., the
surface is hydrophobic in the case of an aqueous first liquid). If
the pressure difference across the valve exceeds a certain
threshold pressure, for example, .delta.P.sub.Treshold (where
P.sub.Threshold=P.sub.1-P.sub.2), the resistance of surface 20 to
the flow of first liquid 1 can be overcome and the first liquid can
flow into the channel 30 (FIG. 2B).
[0084] According to various embodiments, FIG. 2B can illustrate the
same device as shown in FIG. 2A, however, the surface tension of
the surface tension controlled valve 25 has been changed to be made
hydrophilic, thus enabling liquid 60 to pass through the valve 25
and into the second conduit 30. Changing the surface tension of the
valve can be accomplished by a variety of mechanism as described
herein.
[0085] According to various embodiments, a number of techniques are
provided that can make the pressure difference across the valve
exceed a threshold pressure, thereby allowing the passage of a
liquid. One technique can use electric fields to effect fluid
movement by relying on the ability of electric fields to change the
contact angle of the fluid on a surface that is initially resistant
to the flow of a liquid. When an electric field gradient is applied
to a droplet on a fluid-transporting surface, different contact
angles can be formed between leading and receding surfaces of the
droplet with respect to the fluid transporting surface. This
imbalance in surface tension forces can produce a net force that
moves the droplet. For example, in the case of a polar liquid
droplet, such as a droplet of an aqueous liquid, the application of
an electric potential difference across the liquid-solid interface
reduces the contact angle, thereby effectively making the surface
more hydrophilic. In various embodiments, the electrical potential
difference effecting the hydrophilic-hydrophobic conversion can be
controlled by closing a circuit to at least one electrode arranged
on at least one side of a channel making up the surface tension
controlled valve.
[0086] According to various embodiments, the surface tension of a
liquid in a surface tension controlled valve can be altered by
applying an electric field. An exemplary embodiment is shown in
FIG. 3.
[0087] According to various embodiments, and as illustrated in FIG.
3, the surface tension controlled valve can comprise a layered
structure capable of changing the surface tension of a surface. The
layered structure can comprise a first electrode 70, that can
comprise an electrode containing layer, positioned adjacent to a
second insulating layer 80. When the structure is connected to a
power source 100 through electrical leads, a change in surface
tension can be effected by application of an electrical signal to
the electrical leads. As a result of such a signal, the valve can
change the overall shape of a droplet of liquid 90 from a first
shape having a greater contact angle .THETA., to a second shape
having a lesser contact angle, by creating a difference in
electrical potential between the liquid and the electrode. By
increasing or decreasing the power of the electrical signal, the
shape of the droplet can be changed to take any of a variety of
forms.
[0088] According to various embodiments, an electrode 70 can be
embedded below a surface of an insulation layer 80, and a droplet
of liquid 90 can be disposed in the channel. The droplet of liquid
90 can be a polar liquid. The droplet of liquid 90 can form a
contact angle .THETA. with the surface of the insulation layer 80.
A power source 100 can be configured to apply an electrical
potential difference between the liquid droplet 90 and the
electrode 70. When the circuit including the electrode, power
source, and liquid droplet 90 is closed and the electrical
potential difference is applied, different contact angles 0) are
formed between leading and receding surfaces of the droplet with
respect to the surface 80. This imbalance in surface tension forces
can produce a net force and moves the droplet to the position
indicated by the broken line.
[0089] According to various embodiments and as illustrated for
example in FIG. 3, the top side of the electrode can be insulated
from the liquid droplet by an insulation layer 80. In a
microfluidic device, each electrode (and potentially each surface
tension controlled valve) can contain an independent electrical
addressing/connection, which can be accomplished by, for example,
disposing a printed circuit at the bottom of the chip.
[0090] According to various embodiments, one aspect of a surface
tension controlled valve is illustrated in FIG. 4A. Reservoir 40
can contain liquid 50 that flows into, but not past, a portion of
channel 30 that can be resistant to the flow of the liquid. An
electrode 70 and insulator 80 can be positioned along one wall of
channel 30, which channel communicates with reservoir 10. A power
source and electro-wetting circuit 100 can be configured to apply
an electrical potential difference across at least that portion of
the channel 30 that can be resistant to the flow of liquid 50.
Absent the presence of the electrical potential difference, or any
other surface tension-breaking source, the liquid will not flow
past that portion of channel 30 because the liquid does not exceed
a certain threshold pressure necessary to break the surface tension
of the liquid.
[0091] According to various embodiments, FIG. 4B illustrates the
operation of a surface tension controlled valve when circuit 100 is
closed and an electrical potential difference exists between
electrode 70 and the liquid 50 in channel 30. The applied electric
field can change the contact angle of the edge of the liquid
leading into channel 30, thereby changing surface resistance to the
flow. When the power source generates an electrical potential
difference, the imbalance in surface tension between the leading
and receding edges of the liquid can produce a net force, which can
cause movement of the liquid. The liquid is then permitted to flow
through channel 30, past the portion of the channel initially
resistant to the flow of the liquid, and into reservoir 10. In
various embodiments, opening the circuit and shutting off the
electric field can stop the flow of the liquid through channel
30.
[0092] As illustrated in FIGS. 5A-5C, a layer of photoconductive
material 160 can be added between embedded electrode array 170 and
electro-wetting circuit 100. In various other embodiments, a
conductive layer (not shown) can be positioned between
photoconductive material 160 and insulator 80. In various other
embodiments and as shown in FIG. 5B, array 170 can be replaced by a
continuous layer of electrically conductive material 180, which in
turn, can be connected to power source 100. In other embodiments,
as shown in FIG. 5C, an additional electrode 70 can be positioned
between photo conductive material 160 and insulator 80.
[0093] A droplet 90 of a liquid can form a contact angle .THETA.
with the surface upon which it rests. Although the power source may
be providing a current, the electro-wetting circuit will not close
unless the photoconductive material is illuminated with light 120.
Only then will the circuit close, enabling an electrical current to
flow between electrode array 170, or conductive layer 70, and
liquid droplet 90.
[0094] According to various embodiments, the fluid processing
device can comprise a surface tension controlled valve that
comprises a layered structure. The layered structure can comprise a
photoconductive layer, an electrode-containing layer, and an
insulating layer. A power supply can be connected through leads to
a liquid bead. Applying current can change the shape of the liquid
droplet. In other words, when illuminated by light, the
photoconductive layer of the surface tension controlled valve can
change locally and significantly in conductivity and, as a result,
the surface of the insulating layer that contacts the liquid
droplet can be made hydrophilic or more hydrophilic. The contact
angle or wetting of the liquid with respect to the surface can thus
be changed and the liquid can accordingly be propagated in a
certain direction.
[0095] FIGS. 6A-6C illustrates a channel 130 created by an internal
volume between insulating layers 80. The topside 110 of channel 130
can be sufficiently transparent to allow light beam 120 to pass
through to photoconductive layer 160, that contains electrodes 70.
When illuminated by light beam 120, the conductivity of the
illuminated portion of photoconductive layer 160 can change
significantly, thus allowing circuit 100 to close between
electrodes 70 and liquid droplet 90. More specifically, the portion
of the photoconductive material that is illuminated by a beam of
light can be capable of transmitting a higher electric field
intensity than a portion of the photoconductive layer that is not
illuminated. The applied potential difference can make the surface
less resistant to the flow of the liquid droplet, for example, more
hydrophilic in the case of an aqueous liquid. The contact angle of
the liquid can change, and the liquid propagates along the
channel.
[0096] In an alternative embodiment, and as illustrated in FIG. 6B,
array 170 is replaced by a continuous layer of electrically
conductive material 180, which in turn is connected to a power
source. FIG. 6C illustrates yet another embodiment, where a
continuous layer of electrically conductive material 180 is
connected to a power source, and electrodes 70 are absent.
[0097] According to various embodiments, light beams can change the
electrical resistance of a photoconductive layer in a
light-activated valve, allowing electrical current to flow through
one or more electrodes. Electrical current flow changes the contact
angle of the liquid in parts of the liquid droplet allowing the
liquid droplet to move toward the light beams.
[0098] According to various embodiments, biological fluid-handling
can be provided based on the above described teachings for a valve
configured for light activation. A channel can be connected to
section 20 that can form an internal volume of the valve. The
internal volume of the valve can be bound by an insulating layer
resistant to the flow of the biological liquid. The channel is not
resistant to the flow of the biological liquid. The photoconductive
material can be coupled to the insulating layer. The electrode that
forms the electro-wetting circuit can be coupled to the
photoconductive material and configured to electrically couple with
the insulating layer through the photoconductive material when the
photoconductive material is activated by light. The power source
can be electrically coupled to the electrode. The power source can
be configured to provide an electrical potential difference across
the insulting layer capable of changing the wettability of the
insulting material. The light source can be configured to activate
the photoconductive material thereby providing the electrical
potential difference between the insulating layer and the
biological fluid. The amount of electrical potential difference can
be configured to reduce the resistance of the insulating layer to
the flow of the biological liquid.
[0099] According to various embodiments, light beam 120 can be
capable of movement, for example, can be directed along the length
of the channel 130. Such movement can be possible by the use of any
device capable of moving a beam of light such as, by way of
non-limiting example, a galvo-mirror known in the art of laser
etching or an array of microfabricated mirrors known in the art of
digital light projection. As the light beam is directed along the
length of channel 130, the illuminated portions of the
photoconductive material can close the circuit between the
respective electrode and liquid droplet 90. The contact angle of
leading edge 140 of the droplet can change to a different contact
angle from the receding edge 150, an imbalance in surface tension
can result, and the droplet can thus propagate in the direction of
the beam of light.
[0100] According to various embodiments, similar approaches can be
used to construct light-actuated valve (FIGS. 7A-7D). In normal
conditions (for example, no light) the valve can be closed, because
the liquid in channel 40 has not exceeded a threshold pressure such
that it can pass the portion 20 of channel 30 that is resistant to
the flow of the liquid 50 (FIG. 7A). Portion 20 of channel 30 can
be a surface tension controlled valve. Portion 20 can include
multi-layers 25 that can include an insulator, an electrode, a
photosensitive layer, or a combination thereof, as described above.
When light beam 120 illuminates and activates the electro-wetting
circuit formed in the area where liquid 50 contacts surface 20, the
surface becomes less resistant to the flow of liquid 50, and the
liquid moves into channel 30 (FIG. 7B). The beam of light 120 then
shifts toward channel 30 followed by the liquid (FIG. 7C). Once
light 120 moves across and above surface 20, and part of surface 20
is not illuminated anymore, some liquid can break apart from the
liquid in channel 40, and after the light is switched off, that
liquid can be displaced into channel 30 (FIG. 7D).
[0101] According to various embodiments, a fluid processing device
is provided that can be used to manipulate the delivery of
reactants or reaction components to a reaction site to enable the
production of one or more compounds comprising multiple building
blocks, for example, one or more desired oligomers or one or more
desired oligonucleotides. Oligomers as defined herein can include
polymers of amino acids, polymers of sugars, polymers of nucleotide
bases, polymers of nucleotide analogs, and/or polymers of other
nucleotide monomeric units herein referred to as nucleotides.
[0102] According to various embodiments, the device described
herein can be useful in carrying out chemical compound synthesis
methods using building blocks, exemplified herein with
oligonucleotide synthesis methods. These methods can comprise, for
example, various oligonucleotide extension reactions, protecting
and/or deprotecting reactions, capping reactions, washing steps,
cleaving reactions, and the like. Exemplary oligonucleotide
synthesis reactions can include those described, for example, in
U.S. patent application Ser. No. 10/891,650, filed Jul. 15, 2004,
which is incorporated herein in its entirety by reference.
[0103] The term "nucleotide base", as used herein, refers to a
substituted or unsubstituted aromatic ring or substituted or
unsubstituted aromatic rings. In certain embodiments, the aromatic
ring or rings contain at least one nitrogen atom. In certain
embodiments, the nucleotide base is capable of forming Watson-Crick
and/or Hoogsteen hydrogen bonds with an appropriately complementary
nucleotide base. Exemplary nucleotide bases and analogs thereof
include, but are not limited to, naturally occurring nucleotide
bases, adenine, guanine, cytosine, 6 methyl-cytosine, uracil,
thymine, and analogs of the naturally occurring nucleotide bases,
e.g., 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,
7-deaza-8-azaadenine, N6 -.DELTA.2-isopentenyladenine (6iA), N6
-.DELTA.2-isopentenyl-2-methylthioadenine (2ms6iA),
N2-dimethylguanine (dmG), 7-methylguanine (7mG), inosine,
nebularine, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine,
pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine,
7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,
4-thiouracil, O6-methylguanine, N6-methyladenine, O4-methylthymine,
5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines
(see, e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT
published application WO 01/38584), ethenoadenine, indoles such as
nitroindole and 4-methylindole, and pyrroles such as nitropyrrole.
Certain exemplary nucleotide bases can be found, e.g., in Fasman,
1989, Practical Handbook of Biochemistry and Molecular Biology, pp.
385-394, CRC Press, Boca Raton, Fla., and the references cited
therein.
[0104] The term "nucleotide", as used herein, refers to a compound
comprising a nucleotide base linked to the C-1' carbon of a sugar,
such as ribose, arabinose, xylose, and pyranose, and sugar analogs
thereof. The term nucleotide can also encompasses nucleotide
analogs. The sugar may be substituted or unsubstituted. Substituted
ribose sugars include, but are not limited to, those riboses in
which one or more of the carbon atoms, for example the 2'-carbon
atom, is substituted with one or more of the same or different Cl,
F, --R, --OR, --NR2 or halogen groups, where each R is
independently H, C1-C6 alkyl, or C5-C14 aryl. Exemplary riboses
include, but are not limited to, 2'-(C1-C6)alkoxyribose,
2'-(C5-C14)aryloxyribose, 2',3'-didehydroribose,
2'-deoxy-3'-haloribose, 2'-deoxy-3'-fluororibose,
2'-deoxy-3'-chlororibose, 2'-deoxy-3'-aminoribose,
2'-deoxy-3'-(C1-C6)alkylribose, 2'-deoxy-3'-(C1-C6)alkoxyribose and
2'-deoxy-3'-(C5-C14)aryloxyribose, ribose, 2'-deoxyribose,
2',3'-dideoxyribose, 2'-haloribose, 2'-fluororibose,
2'-chlororibose, and 2'-alkylribose, e.g., 2'-O-methyl, 4'-anomeric
nucleotides, 1'-anomeric nucleotides, 2'-4'- and 3'-4'-linked and
other "locked" or "LNA", bicyclic sugar modifications (see, e.g.,
PCT published application nos. WO 98/22489, WO 98/39352; and WO
99/14226). Exemplary LNA sugar analogs within a polynucleotide
include, but are not limited to, the structures: ##STR1## where B
is any nucleotide base.
[0105] Modifications at the 2'- or 3'-position of ribose can
comprise hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy,
butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino,
alkylamino, fluoro, chloro, and bromo. Nucleotides include, but are
not limited to, the natural D optical isomer, as well as the L
optical isomer forms (see, e.g., Garbesi (1993) Nucl. Acids Res.
21:4159-65; Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata,
(1993) Nucleic Acids Symposium Ser. No. 29:69-70). When the
nucleotide base is purine, e.g. A or G, the ribose sugar is
attached to the N9-position of the nucleotide base. When the
nucleotide base is pyrimidine, e.g. C, T or U, the pentose sugar is
attached to the N1-position of the nucleotide base, except for
pseudouridines, in which the pentose sugar is attached to the C5
position of the uracil nucleotide base (see, e.g., Komberg and
Baker, (1992) DNA Replication, 2nd Ed., Freeman, San Francisco,
Calif.).
[0106] One or more of the pentose carbons of a nucleotide can be
substituted with a phosphate ester having the formula: ##STR2##
where .alpha. is an integer from 0 to 4. In certain embodiments, a
is 2 and the phosphate ester is attached to the 3'- or 5'-carbon of
the pentose. In certain embodiments, the nucleotides can be those
in which the nucleotide base is a purine, a 7-deazapurine, a
pyrimidine, or an analog thereof. "Nucleotide 5'-triphosphate"
refers to a nucleotide with a triphosphate ester group at the 5'
position, and are sometimes denoted as "NTP", or "dNTP" and "ddNTP"
to particularly point out the structural features of the ribose
sugar. The triphosphate ester group can comprise sulfur
substitutions for the various oxygens, for example,
-thio-nucleotide 5'-triphosphates. For a review of nucleotide
chemistry, see: Shabarova, Z. and Bogdanov, A. Advanced Organic
Chemistry of Nucleic Acids, VCH, New York, 1994.
[0107] The term "nucleotide analog", as used herein, refers to
embodiments in which the pentose sugar and/or the nucleotide base
and/or one or more of the phosphate esters of a nucleotide may be
replaced with its respective analog. In certain embodiments,
exemplary pentose sugar analogs are those described above. In
certain embodiments, the nucleotide analogs can comprise a
nucleotide base analog as described above. In certain embodiments,
exemplary phosphate ester analogs can comprise, but are not limited
to, alkylphosphonates, methylphosphonates, phosphoramidates,
phosphotriesters, phosphorothioates, phosphorodithioates,
phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,
phosphoroanilidates, phosphoroamidates, boronophosphates, and the
like, and may include associated counterions.
[0108] Also included within the definition of "nucleotide analog"
are nucleotide analog monomers that can be polymerized into
polynucleotide analogs in which the DNA/RNA phosphate ester and/or
sugar phosphate ester backbone is replaced with a different type of
intemucleotide linkage. Exemplary polynucleotide analogs can
comprise, but are not limited to, peptide nucleic acids, in which
the sugar phosphate backbone of the polynucleotide is replaced by a
peptide backbone. Also included are intercalating nucleic acids
(INAs, as described in Christensen and Pedersen, 2002), and AEGIS
bases (Eragen, U.S. Pat. No. 5,432,272).
[0109] As used herein, the terms "polynucleotide",
"oligonucleotide", and "nucleic acid" are used interchangeably and
mean single-stranded or double-stranded polymers of nucleotide
monomers, including 2'-deoxyribonucleotides (DNA) and
ribonucleotides (RNA) linked by intemucleotide phosphodiester bond
linkages, or intemucleotide analogs, and associated counter ions,
e.g., H+, NH4+, trialkylammonium, Mg2+, Na+ and the like. A nucleic
acid can be composed entirely of deoxyribonucleotides, entirely of
ribonucleotides, or chimeric mixtures thereof. The nucleotide
monomer units can comprise any of the nucleotides described herein,
including, but not limited to, naturally occurring nucleotides and
nucleotide analogs. Nucleic acids typically can range in size from
a few monomeric units, e.g. 5-40 when they are sometimes referred
to in the art as oligonucleotides, to several thousands of
monomeric nucleotide units. Unless denoted otherwise, whenever a
nucleic acid sequence is represented, it will be understood that
the nucleotides are in 5' to 3' order from left to right and that
"A" denotes deoxyadenosine or an analog thereof, "C" denotes
deoxycytidine or an analog thereof, "G" denotes deoxyguanosine or
an analog thereof, and "T" denotes thymidine or an analog thereof,
unless otherwise noted.
[0110] Nucleic acids can comprise genomic DNA, cDNA, hnRNA, mRNA,
rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained from
subcellular organelles such as mitochondria or chloroplasts, and
nucleic acid obtained from microorganisms or DNA or RNA viruses
that may be present on or in a biological sample.
[0111] Nucleic acids can be composed of a single type of sugar
moiety, e.g., as in the case of RNA and DNA, or mixtures of
different sugar moieties, e.g., as in the case of RNA/DNA chimeras.
In certain embodiments, nucleic acids can be ribopolynucleotides
and 2'-deoxyribopolynucleotides according to the structural
formulae below: ##STR3## wherein each B is independently the base
moiety of a nucleotide, e.g., a purine, a 7-deazapurine, a
pyrimidine, or an analog nucleotide; each m defines the length of
the respective nucleic acid and can range from zero to thousands,
tens of thousands, or even more; each R is independently selected
from the group comprising hydrogen, halogen, --R'', --OR'', and
--NR''R'', where each R'' is independently (C1-C6) alkyl or
(C5-C14) aryl, or two adjacent Rs are taken together to form a bond
such that the ribose sugar is 2',3'-didehydroribose; and each R' is
independently hydroxyl or ##STR4## where .alpha. is zero, one or
two.
[0112] In certain embodiments of the ribopolynucleotides and
2'-deoxyribopolynucleotides illustrated above, the nucleotide bases
B can be covalently attached to the Cl ' carbon of the sugar moiety
as previously described.
[0113] The terms "nucleic acid", "polynucleotide", and
"oligonucleotide" can comprise nucleic acid analogs, polynucleotide
analogs, and oligonucleotide analogs. The terms "nucleic acid
analog", "polynucleotide analog" and "oligonucleotide analog" are
used interchangeably and, as used herein, refer to a nucleic acid
that contains at least one nucleotide analog and/or at least one
phosphate ester analog and/or at least one pentose sugar analog.
The definition of nucleic acid analogs can also comprise nucleic
acids in which the phosphate ester and/or sugar phosphate ester
linkages are replaced with other types of linkages, such as
N-(2-aminoethyl)-glycine amides and other amides (see, e.g.,
Nielsen et al., 1991, Science 254: 1497-1500; WO 92/20702; U.S.
Pat. No. 5,719,262; U.S. Pat. No. 5,698,685); morpholinos (see,
e.g., U.S. Pat. No. 5,698,685; U.S. Pat. No. 5,378,841; U.S. Pat.
No. 5,185,144); carbamates (see, e.g., Stirchak & Summerton,
1987, J. Org. Chem. 52: 4202); methylene(methylimino) (see, e.g.,
Vasseur et al., 1992, J. Am. Chem. Soc. 114: 4006);
3'-thioformacetals (see, e.g., Jones et al., 1993, J. Org. Chem.
58: 2983); sulfamates (see, e.g., U.S. Pat. No. 5,470,967);
2-aminoethylglycine, commonly referred to as PNA (see, e.g.,
Buchardt, WO 92/20702; Nielsen (1991) Science 254:1497-1500); and
others (see, e.g., U.S. Pat. No. 5,817,781; Frier & Altman,
1997, Nucl. Acids Res. 25:4429 and the references cited therein).
Phosphate ester analogs include, but are not limited to, (i) C1C4
alkylphosphonate, e.g. methylphosphonate; (ii) phosphoramidate;
(iii) C1C6 alkyl-phosphotriester; (iv) phosphorothioate; and (v)
phosphorodithioate.
[0114] The surface tension controlled valves that can be used
according to various embodiments are herein exemplified by an
implementation represented by a channel having an inner surface
that is hydrophobic in the absence of illuminating radiation.
According to various embodiments, the surface tension controlled
valve can exploit the fact that under certain circumstances the
contact angle for a liquid of interest, or its surface tension,
changes and that change can in-turn trigger a movement of the
liquid. Such circumstances can comprise an applied electric field
(electrowetting), an applied electric field and light
(optoelectrowetting), an applied local increase of temperature
(thermo capillary effect), and the like. The liquid can be a liquid
sample, for example, a biological sample in water or a biological
sample in an aqueous medium. If the liquid is a biological sample,
it can comprise, for example, any of the nucleotides, nucleotide
bases, and/or nucleotide analogs described herein.
[0115] Exemplary surface tension controlled valves can comprise the
microfluidic electrowetting control devices described in U.S.
Patent Application Publication No. US 2004/0231987 A1, published
Nov. 25, 2004; the electrostatic actuators for microfluidics
described in U.S. Patent Application Publication No. US
2002/0043463 A1, published Apr. 18, 2002; the micropump device as
described in U.S. Patent Application Publication No. US
2002/0114715 A1, published Aug. 22, 2002; the electrowetting
microfluidic control device described in U.S. Patent Application
Publication No. US 2003/0164295 A1, published Sep. 4, 2003; the
control devices described in U.S. Patent Application Publication
No. US 2002/0168671 A1, published Nov. 14, 2002; the optical
microfluidic devices described in U.S. Patent Application
Publication No. US 2003/0047688 A1; and the injecting devices as
described in U.S. Patent Application Publication No. US
2003/0082081 A1; all of which are incorporated herein in their
entireties by reference.
[0116] According to various embodiments, and as illustrated in
FIGS. 8A-8D, fluid processing device 400 can comprise first channel
40 separated from second channel 30 by light-activated surface
tension controlled valve 20. When an area 120 including valve 20 is
illuminated by beams of light, the contact angle of liquid in valve
20 changes and enables a liquid droplet 414 (FIG. 4D) to be
separated from liquid 50 present in first channel 40. Conduit 408
can function to relieve air pressure differences caused by the
movement of drop 414 into second channel 30. Conduit 408 can
include first open end 409 in fluid communication with the interior
of the valve 20, and second open end 411 in fluid communication
with second channel 30.
[0117] According to various embodiments and as depicted in FIGS.
8A-8D, a surface tension controlled valve separating a first
liquid-containing channel and a second channel can normally or
originally be in a closed state in the absence of illuminating
radiation. If a light beam illuminates the surface tension
controlled valve where the liquid contacts the valve, the valve
surface can be made hydrophilic, enabling the liquid to move into
the valve. If the beam of light is then moved towards the second
conduit, the beam can be followed by the liquid as the localized
surface tension of the valve is changed. Once the light beam moves
past the valve surface such that part of the valve is no longer
illuminated. The localized valve surface will again become
hydrophobic and will be closed. A volume of liquid can thus be
taken-up from and broken away from the liquid in the first channel.
Upon continued movement of the light beam followed by switching off
the light, the remaining liquid in the valve can be moved into the
second channel.
[0118] According to various embodiments, and as illustrated in FIG.
9, a fluid processing device 500 can comprise first manifold 503 in
fluid communication with supply conduit 504. A conduit can be a
channel. The first manifold 503 can be in fluid communication with
several feeder conduits 506. A plurality of reaction sites 510 can
be in fluid communication with respective feeder channel 506.
Surface tension controlled valves 508 can be disposed in one or
more of feeder channels 506 adjacent to each reaction site 510. A
second manifold 517 can be in fluid communication with conduit 516
and a plurality of feeder conduits 514. Each feeder conduit 514 can
be in fluid communication with a respective plurality of reaction
sites 510, for example, on opposite sides of the respective
reaction regions relative to the respective feeder channels 506. In
various embodiments, conduits can be channels.
[0119] Fluid processing device 500 can be disposed in or upon a
chip or card 502. The chip or card 502 can comprise glass, silicon,
plastic, polycarbonate, polypropylene, polymers of cyclic olefins,
copolymers, combinations thereof, or the like. The chip or card 502
can be molded with features and enclosed by one or more cover films
or layers. Reaction regions 510 can be any suitable shape, for
example, well-shaped.
[0120] The conduits and reaction sites can have any of a variety of
dimensions. At least one feature can have at least one dimension of
five mm or less, for example, one mm or less, or 500 microns or
less. Conduit depths and widths can be equivalent or different from
one another. Different channel aspect ratios can be used. According
to various embodiments, channels can be dimensioned to permit
manipulation of fluids by capillary action, and to promote or
induce capillary fluid flow. The conduits can have various
cross-sectional shapes, including, for example, a square
cross-section, a rectangular cross-section, a circular
cross-section, a U-shaped cross-section, a V-shaped cross-section,
or a combination thereof.
[0121] If a conduit has an inner surface that contains both
hydrophobic and hydrophilic portions, some additional force or
pressure can be required to push the liquid through the hydrophobic
part of the channel as compared movement through a hydrophilic
portion of the same channel.
[0122] According to various embodiments, and as illustrated in FIG.
10, a fluid processing device 600 can comprise substrate 601, and
first manifold 604 can comprise main conduit 606 and several feeder
conduits 620, 622, and 624, wherein each feeder conduit can
comprise a respective surface tension controlled valve. The feeder
conduits 620, 622, 624 can be in fluid communication with reaction
sites 614, 616, and 618, respectively. Reaction sites 616 and 618
can be fluidically connected to one another by a conduit 626 that
comprises a surface tension controlled valve. The conduit 626 can
be directly between the reaction sites 616 and 618. Similarly,
reaction sites 614 and 616 can be fluidically connected by a
conduit 628 having a surface tension controlled valve. Controlling
the opening and closing of one or more of the surface tension
controlled valves 620, 622, 624 can enable the selective production
of a different oligomer in each respective reaction site 614, 616,
618. A second manifold 605 comprising a main conduit 608 and
several feeder conduits 610, 612, 614 can be in fluid communication
with the reaction sites 614, 616, 618, respectively. The second
manifold 605 can be used to carry away reactants, non-reactive
reaction components, and/or wash fluids, from the reaction regions.
The second manifold 605 can alternatively, or additionally, be used
to supply the reaction sites 614, 616, 618 with one or more
reactants, non-reactive reaction components, and wash fluids.
Through combinations of supply and wash steps and surface tension
controlled valve opening and closing steps, different
oligonucleotides can simultaneously be synthesized in the different
reaction sites of the device, as described in more detail
below.
[0123] According to some embodiments, a plurality of sets of
different reaction sites can be provided, wherein each set
comprises, for example, at least a pair of reaction sites, for
example, similar to the reaction site pair 614, 618 shown in FIG.
10. Each set can be configured to carry out a different set of
syntheses, for example, to provide a set of reactants useful to
detect a respective target. Each set can comprise a set of
oligonucleotides that can be differentially detectable independent
from the other sets of reactants. For example, the plurality of
sets of reaction sites can comprise four sets of three reaction
sites, wherein each set of three reaction sites can carry out the
respective synthesis of a particular forward oligonucleotide
primer, a corresponding reverse oligonucleotide primer, and a
detectable oligonucleotide, for example, labeled with a fluorescent
reporter dye. All of the different oligonucleotides from the four
sets of three reaction sites can be combined in, for example, a
central reaction site, and a multiplexed detection assay can be
carried out on a sample. In such an example, the presence, absence,
and/or amount, of four different target sequences, can be
independently detectable in the central reaction site. The
independent detection can be based on different excitation,
emission, or both excitation and emission spectra, of the different
detectable oligonucleotides. Exemplary of multiplex reactions are
those described, for example, in co-pending U.S. Provisional Patent
Application No. 60/699,782, filed Jul. 15, 2005, which is
incorporated herein in its entirety by reference.
[0124] According to various embodiments, and as illustrated in FIG.
11, a fluid processing system 700 can comprise a processing device,
for example, computer 702. The computer can be electrically
connected, for example, through wires or through a wireless
connection, to a suitable electromagnetic radiation source 706 that
is capable of sufficiently illuminating a surface tension
controlled valve to cause a change in the hydrophobic/hydrophilic
properties of the valve. Electromagnetic radiation source 706 can
comprise a laser, an ultraviolet light source, an infrared source,
an incandescent bulb, a fluorescent bulb, a light-emitting diode
(LED), an array of LEDs, combinations thereof, and the like.
[0125] According to various embodiments, fluid processing system
700 can comprise an apparatus 704 that can direct the
electromagnetic radiation toward a plurality of separate surface
tension controlled valves incorporated in a fluid processing device
710, for example, in a card or chip. Apparatus 704 can include an
electromagnetic radiation reflective device such as one or more
mirrors. Apparatus 704 can comprise a plurality of
independently-moveable, computer controllable, micro-mirrors 705,
as shown. The fluid processing system 700 can further comprise one
or more lenses 708, for focusing the electromagnetic radiation
reflected by micro-mirrors 705 toward fluid processing device 710.
Pumps 712, and 714, can be fluidically connected to fluid
processing device 710, for example, to one or more manifolds in the
device, and operatively connected to computer 702. Operatively
connected can be defined as electrically connected, mechanically
connected, fluidically connected, combinations thereof, and the
like. Pumps 712, 714, can be used to control, at least in-part, the
flow of fluids to and/or from fluid processing device 710.
[0126] According to various embodiments, and as illustrated in FIG.
12, a fluid processing system 800 is provided that can comprise a
mirror 808, for example, a galvo-mirror, controlled by a computer
801. Mirror 808 can direct electromagnetic radiation from an
electromagnetic radiation source 802 through one or more lenses 804
toward light-activated surface tension controlled valves in a fluid
processing device 806.
[0127] According to various embodiments, and as illustrated in FIG.
13, a fluid processing system 900 can comprise a rotatable carousel
902 having a top surface. Disposed upon the top surface of carousel
902 can be a plurality of fluid processing devices 904. Each device
904 can comprise a first manifold, a second manifold, a plurality
of reaction regions, and a plurality of surface tension controlled
valves, as described above. Carousel 902 can rotate so as to
position each device 904 above a heater 906, and adjacent two or
more pumps or pumping blocks 908, 910. Pumping blocks 908 and 910
can be any suitable pumping devices for moving reagents, or can be
pumping systems capable of independently addressing and pumping a
number of different reagents present inside the block itself, or in
fluid communication with the blocks. Reagents that can be pumped
into and out of the devices by pumping blocks 908 and 910 can
comprise nucleotides, nucleosides, nucleotide analogs, adenine,
cytosine, guanine, thymine, uracil, protected versions thereof,
deprotecting reagents, acids, capping reagents, wash fluids, or
combinations thereof.
[0128] Detection block 912 can comprise an electromagnetic
radiation source and an imaging system can be disposed above
carousel 902. Detection block 912 can comprise an electromagnetic
radiation source capable of selectively opening one or more surface
tension controlled valves of an underlying device 904. Detection
block 912 can also comprise an imaging system capable of recording
images of, or viewing, tagged molecules, for example, fluorescently
tagged molecules. The imaging system can comprise, for example, an
analog camera, a film camera, a digital camera, a CCD, or a
combination thereof. Fluid processing system 900 can include drive
unit 905 and control unit 914. Control unit 914 can be operatively
connected to optical block 912, pumping blocks 908, 910, heater
906, carousel 902, and/or drive unit 905. Operatively connected can
be defined as electrically connected, mechanically connected,
fluidically connected, combinations thereof, and the like.
[0129] According to various embodiments, a method of synthesizing
oligomers, for example, oligonucleotides, is provided for which
traditional phosphoramidite or other appropriate chemistry can be
used. The method can be used to create a plurality of identical
oligomers in each reaction site or to create a different oligomer
in each respective reaction site. The method can comprise providing
a fluid processing device as described herein, for example, that
can comprise one or more reaction sites each including an inner
surface, a first manifold in fluid communication with the one or
more reaction sites, a second manifold in fluid communication with
the one or more reaction sites, and one or more surface tension
controlled valves disposed in the first manifold.
[0130] The method can comprise introducing a first protected
monomer into the first manifold, whereby the first protected
monomer can be selectively introduced, through the one or more
surface tension controlled valves, into the one or more reaction
sites, depending upon how many surface tension controlled valves
are activated to become open. The first protected monomer can then
be attached to a structure or precursor in each reaction site, or
can be attached directly to the inner surface of each reaction
site. The attachment forms an extended structure. Excess first
protected monomer can then be drawn out of the one or more reaction
sites and through the second manifold, for example, by using a
pumping block or device to create a negative pressure differential.
At the same time, or subsequently, a wash fluid from the first
manifold or second manifold can be forced into, or drawn through
and away from, the one or more reaction sites that had the first
protected monomer loaded therein. The wash fluid can be forced into
or drawn through and away from the one or more reaction sites by a
pumping block or pump connected to the first manifold, the second
manifold, or both manifolds.
[0131] In a subsequent step, according to various embodiments, a
deprotecting agent, for example, trichloroacetic acid, or the like,
can be introduced into the first manifold and the one or more
surface tension controlled valves can be opened, enabling the
deprotecting agent to pass through and enter the one or more
reaction sites. The deprotecting agent can be moved into the one or
more reaction sites by using positive pressure, negative pressure,
gravity, centrifuigal force, capillary action, or the like. By
contacting the first extended structure with the deprotecting agent
in the one or more reaction sites, a deprotected extended structure
can be formed in the one or more reaction sites. Excess
deprotecting fluid can then be forced out or drawn out of the one
or more reaction sites and through the second manifold, for
example, by using a pumping block to create a negative pressure
differential. At the same time, or subsequently, a wash fluid from
the first manifold or second manifold can be forced into or drawn
through and away from the one or more reaction sites that had the
deprotecting agent loaded therein. The wash fluid can be forced
into or drawn through and away from the one or more reaction sites
by a pumping block or pump connected to the first manifold, the
second manifold, or both manifolds.
[0132] The wash fluid can then be removed from the reaction site by
a pumping block, by air pressure, by centrifugal force, or the
like. According to various embodiments, the deprotecting agent and
the wash fluid can be removed together from one or more of the
reaction sites.
[0133] After removing the deprotecting agent, a second protected
monomer can then be introduced from the first manifold, through the
one or more surface tension controlled valves, and into one or more
of the reaction sites. The second monomer can then bond to the
deprotected extended structure, if present in the respective
reaction site, to thereby form a second extended oligomer structure
having at least two monomeric subunits.
[0134] The abovementioned method can be repeated multiple times
until a desired oligomer has been formed. Once a completed oligomer
has been formed, it can be cleaved from its attachment site in the
reaction site and collected, for example, through the first or
second manifold.
[0135] According to various embodiments of the method, and with
reference to the device shown in FIG. 9, first manifold 503 can be
used for delivering reagents into the plurality of reaction sites,
while second manifold 517 can be used for drawing out or purging
excess reagents from the reaction sites. Alternatively or
additionally, the second manifold can be used to deliver reagents
or wash fluid into one or more of reaction sites 510. Each surface
tension controlled valve can be controlled independently so a user
can independently select whether a particular reagent is able to
enter a reaction site from first manifold 503. In this way, a
different oligomer can be produced in each reaction site. In
oligonucleotide synthesis, this selective synthesis can be
accomplished by the selective introduction of monomers,
deprotecting agent, washing fluid, or a combination thereof.
[0136] According to various embodiments of the method, the fluid
processing device can be used to synthesize at least two different
oligonucleotide primers and an oligonucleotide probe (as described
above) in three different interconnected reaction regions, for
example, in the three different reaction sites of the device shown
in FIG. 10. In an exemplary embodiment, one primer can be formed in
reaction site 614, a second primer can be formed in reaction site
618, and a probe can be formed or preloaded into reaction site 616.
After formation of the primers, they can be cleaved from their
respective reaction sites and combined into reaction site 616
containing the probe. A nucleic acid sample can then be introduced
into the reaction site 616 along with suitable reagents for a PCR
reaction. Reaction site 616 can then be sealed with oil, a polymer,
or with mechanical valving, to prevent evaporation, and then the
contents of the site can be thermally cycled. In such embodiments,
the device can be used for reagent synthesis and PCR using the
reagent.
[0137] Those skilled in the art can appreciate from the foregoing
description that the present teachings can be implemented in a
variety of forms. Therefore, while these teachings have been
described in connection with embodiments thereof, the teachings
should not be so limited. Various changes and modifications can be
made without departing from the teachings herein. All references,
patents, patent applications, and patent application publications
cited herein are incorporated in their entireties by reference for
all purposes.
* * * * *