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`05/09/03
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`"Express Mail" mailing label number EV339773018US.
`Date of Deposit May 9, 2003
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`Case No. 7314-
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`To the Commissioner for Patents:
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`PATENT APPLICATION TRANSMITTAL LETTER
`
`Transmitted herewith for filing is the patent application of: DEVICE AND METHOD FOR PRESSURE-DRIVEN PLUG TRANSPORT for
`
`: Rustem F. lsmagilov, Joshua David Tice, and Helen Song. Enclosed are:
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`K4
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`fl sheet(s) of drawings, 1_1_7 pages of application (including title page), and the following Appendices : __
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`Declaration.
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`Power of Attorney.
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`Applicant claims small entity status. See 37 CFR 1.27.
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`Assignment transmittal letter and Assignment of the invention to :
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`return gost card.
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`‘.3
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`Claims as; Filed
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`Col. 1
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`Col. 2
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`Basic Fee
`Total Claims
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`.
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`1
`4-3
`lndep. Claims
`Multi le Deendent Claims Present
`*If the difference in col. 1 is less than zero,
`enter "0" in col. 2.
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`Other Than
`Small Entity
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`35
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`_
`Small Entity_
`Fee
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`$333.001
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`+$140=»
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`
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`Total
`EEG
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`Please charge my Deposit Account No. 23-1925 in the amount of $2
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`. A duplicate copy of this sheet is enclosed.
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`Check in the amount of $2 750.00 to cover the Filing Fee is enclosed.
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`The Commissioner is hereby authorized to charge payment of the following fees associated with this communication or credit
`any overpayment to Deposit Account No. 23-1925. A duplicate copy of this sheet is enclosed.
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`_lZ
`Any additional filing fees required under 37 CFR § 1.16.
`E
`Any patent application processing fees under 37 CFR §1.17.
`/[ffhe Commissioner is hereby authorized to charge payment of the following fees during the pendency of this application or
`credit any overpayment to Deposit Account No. 23-1925. A duplicate copy of this sheet is enclosed.
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`Cl
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`CI
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`'i’)’
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`-70
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`Date
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`Any filing fees under 37 CFR § 1.16 for presentation of extra claims.
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`Any patent application processing fees under 37 CFR § 1.17.
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`The issue fee set in 37 CFR § 1.18 at or before mailing of the Notice of Allowance, pursuant to 37 CFR § 1.311(b).
`
`K. Shannon Mrksich
`BRINKS HOFER GILSON & LIONE
`Registration No. 36,675
`Customer No. 00757 - Brinks Hofer Gilson Lione
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`-
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`Rev. Oct-O1
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`\\Bhg|main3\c0mmon\nquevada\UCTech\7814-84\7814—84 Transmittal Letter 5-8-03.doc
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`31:33 ’4':ll=?:
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`“Express Mail” Mailing Label Number EV3397730l8US
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`Date of Deposit: May 9, 2003
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`Our Case No.7814—84
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`PROVISIONAL APPLICATION FOR UNITED STATES LETTERS PATENT
`
`INVENTOR:
`
`Rustem F. Ismagilov
`1700 E. 56th St, Apt. 2804
`Chicago, Illinois 60637
`
`Joshua David Tice
`10 Smith Road
`
`Webster, NY 14580
`
`I
`Helen Song
`1217 E. 52nd Street Apt #
`Chicago, IL 60615
`
`TITLE:
`
`DEVICE AND METHOD FOR
`PRESSURE-DRIVEN PLUG
`TRANSPORT
`
`ATTORNEY:
`
`SHANNON MRKSICH
`
`Reg. No. 36,675
`BRINKS HOFER GILSON & LIONE
`P.O. BOX 10395
`
`CHICAGO, ILLINOIS 60610
`
`(312) 321-4200
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`2
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`

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`;:§l..‘il;iI‘;l"“'r.
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`DEVICE AND METHOD FOR PRESSURE—DRIVEN PLUG I
`TRANSPORT AND REACTION
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`BACKGROUND
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`Nonlinear dynamics, in conjunction with microfluidics, play a central role in
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`the design of the devices and the methods according to the invention. Microfluidics
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`deals with the transport of fluids through networks of channels, typically having
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`micrometer dimensions. Microfluidic systems (sometimes called labs-on-a-chip) find
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`applications in microscale chemical and biological analysis (micro—total—analysis
`systems). The main advantages of microfluidic systems are high speed and low
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`consumption of reagents. They are thus very promising for medical diagnostics and
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`high—throughput screening. Highly parallel arrays of microfluidic systems are used
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`for the synthesis of macroscopic quantities of chemical and biological compounds,
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`e. g. , the destruction of chemical warfare agents and pharmaceuticals synthesis. Their
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`advantage is improved control over mass and heat transport.
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`Microfluidic systems generally require means of pumping fluids through the
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`channels. In the two most common methods, the fluids are either driven by pressure
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`or driven by electroosmotic flow (EOF). Flows driven by EOF are attractive because
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`they can be easily controlled even in complicated networks. -EOF-driven flows have
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`flat, plug-like velocity profile, that is, the velocity of the fluid is the same near the
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`walls and in the middle of the channel. Thus, if small volumes of multiple analytes
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`are injected sequentially into a channel, these plugs are transported as non-
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`overlapping plugs (low dispersion), in which case the dispersion comes mostly from
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`the diffusion between plugs. A main disadvantage of EOF is that it is generated by
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`the motion of the double layer at the charged surfaces of the channel walls. EOF can
`therefore be highly sensitive to surface contamination by charged impurities. (This
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`may not be an issue when using channels with negative surface charges in DNA
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`analysis and manipulation because DNA is uniformly negatively charged and does not
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`adsorb to the walls. However, this can be a serious limitation in applications that
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`involve proteins that are often charged and tend to adsorb on charged surfaces. In
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`addition, high voltages are often undesirable, or sources of high voltages such as
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`portable analyzers may not be available.
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`Flows driven by pressure are typically significantly less sensitive to surface
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`chemistry than EOF. The main disadvantage of pressure—driven flows is that they
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`normally have a parabolic flow profile instead of the flat profile of EOF. Solutes in
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`the middle of the channel move much faster (about twice the average velocity of the
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`flow) than solutes near the walls of the channels. A parabolic velocity profile
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`normally leads to high dispersion in pressure—driven flows; a plug of solute injected
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`into a channel is immediately distorted and stretched along the channel. This
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`distortion is somewhat reduced by solute transport via diffusion from the middle of
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`the channel towards the walls and back. But the distortion is made worse by diffusion
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`along the channel (the overall dispersion is known as Taylor dispersion).
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`Taylor dispersion broadens and dilutes sample plugs. Some of the sample is
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`frequently left behind the plug as a tail. Overlap of these tails usually leads to cross-
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`contamination of samples in different plugs. Thus, samples are often introduced into
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`the channels individually, separated by buffer washes. On the other hand,
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`interleaving samples with long buffer plugs, or washing the system with buffer
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`between samples, reduces the throughput of the system.
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`In EOF, flow transport is essentially linear, that is, if two reactants are
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`introduced into a plug and transported by EOF, their residence time (and reaction
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`time) can be calculated simply by dividing the distance traveled in the channel by the
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`velocity. This linear transport allows precise control of residence times through a
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`proper adjustment of the channel lengths and flow rates. In contrast, dispersion in
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`pressure—driven flow typically creates a broad range of residence times for a plug
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`traveling in such flows, and this diminishes time control.
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`The issue of time control is important. Many chemical and biochemical
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`processes occur on particular time scales, and measurement of reaction times can be
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`indicative of concentrations of reagents or their reactivity. Stopped—flow type
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`instruments are typically used to perform these measurements. These instruments rely
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`on turbulent flow to mix the reagents and transport them with minimal dispersion.
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`Turbulent flow normally occurs in tubes with large diameter and at high flow rates.
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`Thus stopped—flow instruments tend to use large volumes of reagents (on the order of
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`ml/s). A microfluidic analog of stopped—flow, which consumes smaller volumes of
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`reagents (typically p.L/min), could be useful as a scientific instrument, e. g., as a
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`diagnostic instrument. So far, microfluidic devices have not be able to compete with
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`stopped-flow type instruments because EOF is usually very slow (although with less
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`dispersion) while pressure—driven flows suffer from dispersion.
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`In addition, mixing in microfluidic systems is often slow regardless of the
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`method used to drive the fluid because flow is laminar in these systems (as opposed to
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`turbulent in larger systems). Mixing in laminar flows relies on diffusion and is
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`especially slow for larger molecules such as DNA and proteins.
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`In addition, particulates present handling difficulty in microfluidic systems.
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`While suspensions of cells in aqueous buffers can be relatively easy to handle because
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`cells are isodense with these buffers, particulates that are not isodense with the fluid
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`tend to settle at the bottom of the channel, thus eventually blocking the channel.
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`Therefore, samples for analysis often require filtration to remove particulates.
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`SUMMARY ACCORDING TO THE INVENTION
`
`In accordance with the invention, a method of conducting a reaction within a
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`substrate is provided that comprises introducing a carrier-fluid into a first charmel of
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`the substrate; introducing at least two different plug-fluids into the first channel; and
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`applying pressure to the first channel to induce a fluid flow in the substrate to form
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`substantially identical plugs comprising a mixture of plug-fluids. The plug-fluids are
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`immiscible with the carrier-fluid. During plug formation, the cross—section of the plug
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`is substantially similar to the cross—section of the first channel, so that the plug is
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`substantially in contact with all walls of the first channel. After plug formation, the
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`cross—section of the plug may be smaller than the cross—section of the channel. A thin
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`layer of carrier-fluid typically exists between the wall of the channel and the plug,
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`although in some cases this layer disappears. In general, each plug is substantially
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`similar in size when initially formed in the channel.
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`In addition, the capillary number
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`of the plug in the channel is low, typically less than 1, preferably 5 about 0.2, more
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`preferably _<_ about 0.1.
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`When plugs are formed from more than one plug—fluid, the fluids are rapidly
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`mixed. Mixing inside plugs is further enhanced when the channels are not straight
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`(i. e., when chaotic flows are generated). Aperiodic channel designs are preferred to
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`induce rapid mixing within plugs.
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`In other cases, mixing can be slowed down or
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`controlled such as by using winding channels, varying the fluid viscosities, varying
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`the plug-fluid composition, and twirling, which can also be controlled.
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`The device of the present invention can be used to merge one or more plug
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`fluids. The plug—fluids are introduced either through a single inlet or from multiple
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`inlets. When the plug—fluids are introduced through a single inlet, they are preferably
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`mixed just upstream of the inlet, so that substantial mixing does not occur prior to
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`introduction into the first channel. W’hen plugs fluids are introduced through multiple
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`inlets, one or more physical properties (such as the viscosity, plug dimensions, surface
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`tension at the interface between the plug fluids and the carrier-fluid, or the surface
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`tension at the interface between the plug fluids and the walls of the channel) of the
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`plug-fluids are adjusted so that plugs composed of different plug—fluids merge into a
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`series of plugs prior to the outlet (that is, a series of plugs are formed which are
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`composed ofa mixture of plug—fluids). Alternatively, the plug—fluids can be
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`introduced into separate channels to form plugs composed of single plug—fluids.
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`These channels are then merged into a single merged channel. The continuous fluid
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`flow within the substrate forms merged plugs in the single merged channel.
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`The device of the present invention can be used to split plugs into two or more
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`channels.
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`Using the above devices and techniques, a variety of reactions can be
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`conducted, including polymerizations, crystallizations (including small molecule and
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`proteins), nanoparticle synthesis, formation of unstable intermediates, enzyme-
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`catalyzed reactions and assays, protein—protein binding, etc. More than one reaction
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`can be conducted, either simultaneously or sequentially. '
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`Further, the present invention also provides a device comprising one or more
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`substrates in accordance with the present invention.
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`The devices and methods according to the invention include various non-
`
`limiting embodiments or modifications several of which are discussed in details
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`below.
`
`BRIEF DESCRIPTION OF THE DRAWINGS AND PHOTOGRAPHS
`
`FIG. 1A is a schematic diagram of a basic channel design that may be used to
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`induce rapid mixing in plugs. FIG. 1B(1)-(4) are schematic diagrams depicting a
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`series of periodic variations of the basic channel design. FIG. lC(1)-(4) are schematic
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`diagrams depicting a series of aperiodic combinations resulting from a sequence of
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`alternating elements taken from a basic design element shown in FIG. IA and an
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`element from the periodic variation series shown in FIGS. lOB(l)—(4).
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`FIG. 2A is a schematic diagram contrasting laminar flow transport and plug
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`transport in a channel. FIG. 2B(l) shows a photograph (right side, top portion)
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`illustrating rapid mixing inside plugs moving through winding channels. FIG. 2B(2)
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`shows a photograph (right side, lower portion) showing that winding channels do not
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`accelerate mixing in a laminar flow in the absence of PFD.
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`FIG. 3 shows photographs (right side) and schematic diagrams (left side) that
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`depict a stream of plugs from an aqueous plug—fluid and an oil (carrier-fluid) in
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`curved channels at flow rates of0.5 }.l.L/Il’ll1’l and 1.0 uL/min.
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`FIG. 4 shows a photograph (lower portion) and a schematic diagram (upper
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`portion) that illustrate plug formation through the injection of oil and multiple plug-
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`fluids.
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`FIG. 5 is a schematic diagram that illustrates a two—step reaction in which
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`plugs are formed through the injection of oil and multiple plug-fluids using a
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`combination of different geometries for controlling reactions and mixing.
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`FIG. 6 is a schematic representation of part of a microfluidic network that uses
`multiple inlets and that allows for both splitting and merging of plugs. This schematic
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`diagram shows two reactions that are conducted simultaneously. A third reaction
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`(between the first two reaction mixtures) is conducted using precise time delay.
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`FIG. 7(a)—(b) show microphotographs (10 us exposure) illustrating rapid
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`mixing inside plugs (a) and negligible mixing in a laminar flow (b) moving through
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`winding channels at the same total flow velocity. FIG. 7(c) shows a false-color
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`microphotograph (2 s exposure, individual plugs are invisible) showing timc—averaged
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`fluorescence arising from rapid mixing inside plugs of solutions of Fluo—4 and CaCl2.
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`FIG. 7(d) shows a plot of the relative normalized intensity (I) of fluorescence obtained
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`from images such as shown in (c) as ii function of distance (left) traveled by the plugs
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`and of time required to travel that distance (right) at a given flow rate. FIG.
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`7(e) shows a false-color microphotograph (2 s exposure) of the weak fluorescence
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`arising from negligible mixing in a laminar flow ofthe solutions used in (c).
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`FIG. 8 shows photographs (right side) and schematics (left side) that illustrate
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`fast mixing at flow rates of about 0.5 ILL/min and about 1.0 p.L/min using 90°—step
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`channels.
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`FIG. 9 shows schematics (left side) and photographs (right side) illustrates
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`fast mixing at flow rates of about 1.0 ILL/H1111 and about 0.5 ILL/min using 135°—step
`)
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`channels.
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`FIG. 10a) is a schematic diagram depicting three—dimensional confocal
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`visualization of chaotic flows in plugs. FIG. 10b) is a plot showing a sequence
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`preferably used for visualization of a three—dimensional flow.
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`FIG. 11 shows a schematic diagram of a channel geometry designed to
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`implement and visualize the baker’s transformation of plugs flowing through
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`microfluidic channels.
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`FIG. 12 shows photographs depicting the merging of plugs (top) and splitting
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`of plugs (bottom) that flow in separate channels or channel branches that are
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`perpendicular.
`FIG. 13 shows UV-VIS spectra of CdS nanoparticles formed by rapid mixing
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`in plugs (spectrum with a sharp absorption peak) and by conventional mixing of
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`solutions.
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`FIG. 14 shows schematic diagrams (left side) and photographs (right side) that
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`illustrate the synthesis of CdS nanoparticles in PDMS microfluidic channels in single-
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`phase aqueous laminar flow (FIG. 14A) and in aqueous plugs that are surrounded by
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`water-immiscible perfluorodecaline (FIG. 14B).
`
`FIG. 15 shows schematic representations of the synthesis of CdS nanoparticles
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`inside plugs.
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`FIG. 16 is a schematic illustration of a microfluidic device according to the
`
`invention that illustrates the trapping of plugs.
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`FIG. 17 is a schematic of a microfluidic method for forming plugs with
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`variable compositions for protein crystallization.
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`FIG. 18 is a schematic illustration of a method for controlling heterogeneous
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`nucleation by varying the surface chemistry at the interface of an aqueous plug-fluid
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`and a carrier—fluid.
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`FIG. 19 is a schematic diagram that illustrates a method of separating
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`nucleation and growth using a microfluidic network according to the present
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`invention.
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`FIG. 20 show schematic diagrams that illustrate two methods that provide a
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`precise and reproducible degree of control over mixing and that can be used to
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`determine the effect of mixing on protein crystallization.
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`FIG. 21 is a reaction diagram illustrating an unstable point in the chlorite-
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`thiosulfate reaction.
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`FIG. 22A—D are schematic diagrams that show various examples of geometries
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`of microfluidic channels according to the invention for obtaining kinetic information
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`from single optical images.
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`FIG. 23 shows a schematic of a microfluidic network (left side) and a table of
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`parameters for a network having channel heights of 15 and 2 um.
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`FIG. 24 shows a reaction scheme that depicts examples of fluorinated
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`surfactants that fonn monolayers that are:
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`(a) resistant to protein adsorption; (b)
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`positively charged; and (c) negatively charged. FIG. 24b shows a chemical structure
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`of neutral surfactants charged by interactions with water by protonation of an amine
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`or a guanidinium group. FIG. 24c shows a chemical structure of neutral surfactants
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`charged by interactions with water deprotonation of a carboxylic acid group.
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`FIG. 25 are schematic diagrams of microfluidic network (left side of a), b),
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`and c)) that can be used for controlling the concentrations of aqueous solutions inside
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`the plugs, as well as photographs (right side of a), b), and c)) showing the formation
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`of plugs with different concentrations of the aqueous streams.
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`FIG. 26 are schematic diagrams of microfluidic network (left side of a) and b))
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`and photographs (right side of a) and b)) of the plug-forming region of the network in
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`which the aqueous streams were dyed with red and green food dyes to show their flow
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`patterns.
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`FIG. 27 are photographs and plots showing the effects of initial conditions on
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`mixing by recirculating flow inside plugs moving through straight microchannels.
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`FIG. 27al) is a schematic diagram showing that recirculating flow (shown by black
`
`arrows) efficiently mixed solutions of reagents that were initially localized in the front
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`and back halves of the plug. FIG. 27a2) is a schematic diagram showing that
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`recirculating flow (shown by black arrows) did not efficiently mix solutions of
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`reagents that were initially localized in the left and right halves of the plugs. FIG.
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`27b) shows a schematic diagram showing the inlet portions (left side) and
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`photographs of images showing measurements of various periods and lengths of
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`plugs. FIG. 27c1) shows a graph of the relative optical intensity of Fe(SCN)x(3"‘)+
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`complexes in plugs of varying lengths. FIG. 27c2) is the same as FIG. 7cl) except
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`that each plug traverses a distance of 1.3 mm.
`
`FIG. 28 is a schematic illustration of a plug showing the notation used to
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`identify different regions of the plugs relative to the direction of motion.
`
`FIG. 29a)-b) are plots of the periods and the lengths of plugs as a function of
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`total flow velocity (FIG. 29a)) and water fraction (FIG. 29b)).
`
`FIG. 30 shows photographs illustrating weak dependence of periods, length of
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`plugs, and flow patterns inside plugs on total flow velocity.
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`FIG. 31 are plots showing the distribution of periods and lengths of plugs
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`where the water fractions were 0.20, 0.40, and 0.73, respectively.
`
`FIG. 32 shows photographs (middle and right side) that show that plug traps
`
`are not required for crystal formation in a microfluidic network, as well as a diagram
`of the mierofluidic network (left side).
`FIG. 33a—d_(left side) are top views of microfluidic networks (left side) and
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`photographs (right side) that comprise channels having either uniform or nonuniform
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`dimension. FIG. 33a shows that merging of the plugs occurs infrequently in the T-
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`shaped channel shown in the photographs. FIG. 33b illustrates plug merging
`
`occurring between plugs arriving at different times at the Y-shaped junction
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`(magnified view shown). FIG. 33c depicts in-phase merging, i.e., plug merging upon
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`simultaneous arrival of at least two plugs at a junction, ofplugs of different sizes
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`generated using different oil/water ratios at the two pairs of inlets. FIG. 33d
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`illustrates defects (i.e. , plugs that fail to undergo merging when they would normally
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`merge under typical or ideal conditions) produced by fluctuations in the relative
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`velocity of the two incoming streams of plugs.
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`FIG. 34a—c show a schematic diagram (a, left side) and photographs (b, e) each
`
`of which depicts a channel network viewed from the top. FIG. 34a is a schematic
`
`diagram of the channel network used in the experiment. FIG. 34b is a photograph
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`showing the splitting of plugs into plugs of approximately one-half the size of the
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`‘.1l§iflI‘i*il4 -“Eli f}Z?“' ‘IE5
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`initial plugs. FIG. 34c is a photograph showing the asymmetric splitting of plugs
`
`which occurred when P; < P2.
`
`FIG. 35 shows a schematic diagram (a, left side) and photographs (b, c) that
`
`depicts the splitting of plugs using microfluidic networks without constrictions near
`
`the junction.
`
`FIG. 36 shows a photograph (right side) of lysozyme crystals grown in water
`
`plugs in the wells of the microfluidic channel, as well as a diagram (left side) of the
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`microfluidic network used in the crystallization.
`
`FIG. 37 is a schematic diagram that depicts a microfluidic device according to
`
`the invention that can be used to amplify a small chemical signal using an
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`autocatalytic (and possibly unstable) reaction mixture.
`
`FIG. 38 is a schematic diagram that illustrates a method for a multi-stage
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`chemical amplification which can be used to detect as few as a single molecule.
`
`FIG. 39 shows a diagram (left side) of the microfluidic network and a
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`photograph (right side) of water plugs attached to the PDMS wall.
`
`FIG. 40 is a schematic representation (left side) of a microfluidic network used
`
`to measure kinetics data for the reaction of RNase A using a fluorogenic substrate
`
`(on-chip enzyme kinetics), and plots that shows the kinetic data for the reaction
`
`between RNase A and a fluorogenic substrate.
`
`FIG. 41 shows a photograph (middle and right side) of the water droplet
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`region of the microfluidic network (T stands for time), as well as a diagram of the
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`microfluidic network (left side).
`
`FIG. 42 shows a schematic diagram (left side) of a microfluidic network and a
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`photograph (right side) of the ink plug region of the microfluidic network in which the
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`gradients were formed by varying the flow rates.
`
`FIG. 43 shows a schematic diagram (left side) of a microfluidic network and a
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`photograph (right side) of lysozyme crystals formed in the microfluidic network using
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`gradients.
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`FIG. 44 are schematic illustrations showing how an initial gradient may be
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`p created by injecting a discrete aqueous sample of a reagent B into a flowing stream of
`water.
`
`FIG. 45a) shows a schematic of the microfluidic network used to demonstrate
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`that on-chip dilutions can be accomplished by varying the flow rates of the reagents.
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`The blue rectangle outlines the field of View for images shown in FIG. 45c)-d). FIG.
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`45b) shows a graph quantifying this dilution method by measuring fluorescence of a
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`solution of fluorescein diluted in plugs in the microchannel.
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`DETAILED DESCRIPTION ACCORDING TO THE INVENTION
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`The term “analysis” generally refers to a process or step involving physical,
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`chemical, biochemical, or biological analysis that includes characterization, testing,
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`measurement, optimization, separation, synthesis, addition, filtration, dissolution, or
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`mixing.
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`The term “analysis unit” refers to a part of or a location in a substrate or
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`channel wherein a chemical undergoes one or more types of analyses.
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`The term “carrier-fluid” refers to a fluid that is immiscible with a plug-fluid.
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`The carrier—fluid may comprise a substance having both polar and non—polar groups or
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`moieties.
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`The term “channel” refers to a conduit that is typically enclosed, although it
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`may be at least partially open, and that allows the passage through it of one or more
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`types of substances or mixtures, which may be homogeneous or heterogeneous,
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`including compounds, solvents, solutions, emulsions, or dispersions, any one of which
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`may be in the solid, liquid, or gaseous phase. A charmel can assume any form or
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`shape such as tubular or cylindrical, a uniform or variable (e.g., tapered) diameter
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`along its length, and one or more cross-sectional shapes along its length such as
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`rectangular, circular, or triangular. A channel is typically made of a suitable material
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`such as a polymer, metal, glass, composite, or other relatively inert materials. As
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`used herein, the term “channel” includes microchannels that are of dimensions
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`suitable for use in devices. A network of channels refers to a multiplicity of channels
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`that are typically connected or in communication with each other. A channel may be
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`connected to at least one other channel through another type of conduit such as a
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`valve.
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`The term “chemical” refers to a substance, compound, mixture, solution,
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`emulsion, dispersion, molecule, ion, dimer, macromolecule such as a polymer or
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`protein, biomolecule, precipitate, crystal, chemical moiety or group, particle,
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`IO
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`nanoparticle, reagent, reaction product, solvent, or fluid any one of which may exist in
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`the solid, liquid, or gaseous state, and which is typically the subject of an analysis.
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`The term “detection region” refers to a part of or a location in a substrate or
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`channel wherein a chemical is identified, measured, or sorted based on a
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`predetermined property or characteristic.
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`The term “device” refers to a device fabricated or manufactured using
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`techniques such as wet or dry etching and/or conventional lithographic techniques or a
`micromachining technology such as soft lithography. As used herein, the tenn
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`“devices” includes those that are called, known, or classified as microfabricated
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`devices. A device according to the invention may have dimensions between about 0.3
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`cm to about 15 (for 6 inch wafer) cm per side and between about 1 micrometer to
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`about 1 cm thick, but the dimensions of the device may also lie outside these ranges.
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`The term “discrimination region” refers to a part of or a location in a substrate
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`or channel wherein the flow of a fluid can change direction to enter at least one other
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`channel such as a branch channel.
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`The term “downstream” refers to a position relative to an initial position which
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`is reached after the fluid flows past the initial point. In a circulating flow device,
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`downstream refers to a position farther along the flow path of the fluid before it
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`crosses the initial point again. “Upstream” refers to a point in the flow path of a fluid
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`that the fluid reaches or passes before it reaches or passes a given initial point in a
`substrate or device.
`T
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`The term “flow” means any movement of a solid or a fluid such as a liquid.
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`For example, the movement of plug—fluid, carrier-fluid, or a plug in a substrate, or
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`component of a substrate according to the invention, or in a substrate or component of
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`a substrate involving a method according to the invention, e.g., through channels of a
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`microfluidic substrate according to the invention, comprises a flow. The application
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`of any force may be used to provide a flow, including without limitation: pressure,
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`capillary action, electro—osmosis, electrophoresis, dielectrophoresis, optical tweezers,
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`and combinations thereof, without regard for any particular theory or mechanism of
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`action.
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`The term “immiscible” refers to the resistance to mixing of at least two phases
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`or fluids under a given condition or set of conditions (e. g., temperature and/or
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`pressure) such that the at least two phases or fluids persist or remain at least partially
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`separated even after the phases have undergone some type of mechanical or physical
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`agitation. Phases or fluids that are immiscible are typically physically and/or
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`chemically discernible, or they may be separated at least to a certain extent.
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`The term “inlet port” refers to an area of a substrate that receives plug-fluids.
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`The inlet port may contain an inlet channel, a well or reservoir, an opening, and other
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`features that facilitate the entry of chemicals into the substrate. A substrate may
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`contain more than one inlet port if desired. The inlet port can be in fluid
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`communication with a channel or separated from the channel by a valve.
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`The term “nanoparticles” refers to atomic, molecular or macromolecular
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`particles typically in the length scale of approximately 1 - 100 nanometer range.
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`Typically, the novel and differentiating properties and functions of nanoparticles are
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`observed or developed at a critical length scale of matter typically under 100 nm.
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`Nanoparticles may be used in constructing nanoscale structures and they may be
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`integrated into larger material components, systems and architectures. In some
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`particular cases, the critical length scale for novel properties and phenomena
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`involving nanoparticles may be under 1 nm (e.g., manipulation of atoms at
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`approximately 0.1 nm) or it may be larger than 100 nm (e. g., nanoparticle reinforced
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`polymers have the unique feature at approximately 200-300 nm as a function of the
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`local bridges or bonds between the nanoparticles and the polymer).
`The term “nucleationcomposition” refers to a substance or mixture that
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`includes one or more nuclei capable of growing into a crystal under conditions
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`suitable for crystal formation. A nucleation composition may, for example, be
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`induced to undergo crystallization by evaporation, changes in reagent concentration,
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`adding a substance such as a precipitant, seeding with a solid material, mechanical
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`agitation, or scratching of a surface in contact with the nucleation composition.
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`The term “outlet port” refers to an area of a substrate that collects or dispenses
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`the plug—fluid, carrier—fluid, plugs or reaction product. A substrate may contain more
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`than one outlet port if desired.
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`The term “particles” means any discrete form or unit of matter. The term
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`“particle” or “particles” includes atoms, molecules, ions, dimers, polymers, or
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`biomolecules.
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`The term “particulate” refers to a cluster or agglomeration of particles such as
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`atoms, molecules, ions, dimers, polymers, or biomolecules. Particulates may
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`comprise solid matter or be substantially solid, but they may also be porous or
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`partially hollow. They may contain a liquid or gas. In addition, particulates may be
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`homogeneous or heterogeneous, that is, they may comprise one or more substances or
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`materials.
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`“Plugs” in accordance with the present invention are fonned in a substrate
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`when a stream of at least one plug-fluid is introduced into the flow of a carrier-fluid in
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`which it is substantially immiscible. The flow of the fluids in the device is induced by
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`a driving force or stimulus that arises, directly or indirectly, from the presence or
`application of, for example, pressure, radiation, heat, vibration, sound