CERTIFICATE OF EFS FILING
`I hereby certify that this correspondence is being electronically transmitted to the United States Patent and Trademark
`Office, Commissioner for Patents, via the EFS on the below date:
`
`Date: February 9 2011
`
`Name: Jacob C. Bachman (Reg. No. 61 906)
`
`BRINKS
`HOFER
`GILSON
`&LIONE
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Signature: /Jacob C. Bachman/
`
`In re Appln. of:
`
`Rustem F. lsmagilov, Joshua David Tice, Cory John Gerdts, Bo Zheng
`
`For:
`
`DEVICE AND METHOD FOR PRESSURE-DRIVEN PLUG TRANSPORT
`
`Attorney Docket No.: 7814-305
`
`UTILITY PATENT APPLICATION TRANSMITTAL
`
`Commissioner for Patents
`PO Box 1450
`Alexandria, VA 22313-1450
`
`1. TRANSMITTED HEREWITH: New application under 37 CFR §1.53(b), which is a:
`[8] Continuation,
`D Divisional, or
`D Continuation-in-Part (CIP)
`Under 37 CFR §1.53(b) of prior application no. 12/777,099.
`Prior application information: Examiner: __ Art Unit: __
`D Maintenance of copendency of prior application: A request for extension of time and the appropriate fee
`have been filed in the pending prior application (or are being filed in the prior application concurrently
`herewith) to extend the period for response until __ .
`D Certified copy of priority document(s) has been filed in prior application no. __ .
`For Continuation or Divisional Applications only: The entire disclosure of the prior application, from which an oath or
`declaration is supplied as indicated below, is considered a part of the disclosure of the accompanying continuation or
`divisional application and is hereby incorporated by reference.
`
`2. ATTACHMENTS: The following application elements and other papers are attached:
`[8] Application Data Sheet. See 37 CFR § 1. 76.
`[8] Title page
`[8] Specification, including claims and Abstract (122 pages)
`[8] Drawings (64 sheet(s))
`D Appendices: __
`[8] Declaration or D Combined Declaration and Power of Attorney( __ pages):
`D newly-executed (original or copy)
`[8] copy from a prior application (37 CFR §1.63( d))
`D This application is filed by fewer than all the inventors named in the prior application, 37 CFR §1.53(d)(4).
`Please DELETE the following inventors(s) named in prior nonprovisional application no. __ :
`D English Translation Document:
`D is attached or D has been filed in prior application no. __ .
`D Preliminary Amendment (Note: Related application data required under 37 CFR §1.78, if any,
`appears in the Amendments to the Specification section of the Preliminary Amendment, including incorporations by
`reference.)
`D Petition to Suspend Prosecution for the Time Necessary to File an Amendment (New Application Filed
`Concurrently).
`D Information Disclosure Statement, including Form PT0-1449 ( __ sheets) and copies of references cited, if
`required.
`D Assignment to: __ :
`D with accompanying Assignment Recordation Cover Sheet, is attached.
`D was previously recorded on __ at Reel __ , Frame __ .
`
`Page 1 of2
`
`B R! N K S
`
`1
`
`

`
`UTILITY PATENT APPLICATION TRANSMITTAL, cant
`
`Docket No. 7814-305
`
`D Power of Attorney ( __ pages; D by inventor D by __ ).
`D The power appears in the original papers in the prior application.
`D The power doesn't appear in the original papers in the prior application, but was filed on __ .
`D A new power has been executed and is attached.O The power of attorney in the prior application is to:
`__ (Reg. No. __ ).
`D Nonpublication Request under 35 USC §122(b)(2)(B)(i).
`D Other:
`3. SMALL ENTITY STATUS:
`D Applicant is small entity (per 37 CFR §1.27).
`D A small entity statement was filed in prior application no. __ and such status is still proper and desired.
`D Small entity status is no longer desired.
`4. FEE CALCULATION (AFTER ENTRY OF ANY PRELIMINARY AMENDMENT(S) IN ITEM #2 ABOVE):
`Small Entity
`Not a Small Entity
`Col. 1
`Col. 2
`Claims as Filed
`No. Filed
`No. Extra
`Rate
`Rate
`Fee
`$
`$
`$
`$
`
`For
`Basic Fee
`14-20
`Total Claims
`1-3
`Independent Claims
`Multiple Dependent Claims Present
`Utility Application Size Fee when filing via EFS
`(see MPEP 607 Filing Fee)
`
`Search Fee
`Examination Fee
`
`*If the difference in col. 1 is less than zero, enter "0" in col. 2.
`
`Fee
`82
`
`$
`x$26=
`$
`x$110=
`$
`+$195=
`$
`No. of pages __ X .75 =
`/50=
`- - - 100 =
`X $135=
`$
`+$270=
`$
`+$110=
`$
`Total
`$
`
`or
`or
`or
`or
`or
`
`or
`
`or
`or
`or
`
`x$52=
`x$220=
`+$390=
`No. of pages 186 X .75 =
`139.5- 100 = 39.5/50 =
`1 X $270=
`+$540=
`+$220=
`Total
`
`330
`
`$270
`$540
`$220
`$1,360
`
`5. FEE PAYMENT:
`D Payment by credit card in the amount of$ __ (Form PT0-2038 is attached).
`[8] Please charge Deposit Account No. 23-1925 in the amount of $1,360.
`[8] The Director is hereby authorized to charge payment of the following fees associated with this communication or
`credit any overpayment to Deposit Account No. 23-1925.
`[8] Any additional filing fees required under 37 CFR § 1.16.
`[8] Any patent application processing fees under 37 CFR §1.17.
`D The Director 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.
`D Any filing fees under 37 CFR § 1.16 for presentation of extra claims.
`D Any patent application processing fees under 37 CFR § 1.17.
`D 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).
`
`6. CORRESPONDENCE ADDRESS: Please recognize the correspondence address for this application as the address
`associated with the following Customer Number:
`Customer No.: 00757 - Brinks Hofer Gilson Lione
`
`7. PLEASE DIRECT all telephonic communications to: Jacob C. Bachman (tel: (312) 321-4200).
`
`Respectfully submitted,
`
`February 9, 2011
`Date
`
`/Jacob C. Bachman/
`Jacob C. Bachman (Reg. No. 61 ,906)
`
`B Rl N K S
`
`Page 2 of2
`
`2
`
`

`
`Our Case No.7814-305
`Client Ref No. RDT -800/US09
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`APPLICATION FOR UNITED STATES LETTERS PATENT
`
`INVENTOR:
`
`TITLE:
`
`ATTORNEYS:
`
`Rustern F. Isrnagilov
`1700 E. 56th St, Apt. 2804
`Chicago, Illinois 60637
`
`Joshua David Tice
`10 Smith Road
`Webster, NY 14580
`
`Cory John Gerdts
`5050 S. Lake Shore Drive, Apt. 310
`Chicago, IL 60615
`
`Bo Zheng
`1401 E. 55th St., Apt. 508N
`Chicago, IL 60615
`
`DEVICE AND METHOD FOR
`PRESSURE-DRIVEN PLUG
`TRANSPORT
`
`JACOB C. BACHMAN
`Reg. No. 61,906
`K SHANNON MRKSICH
`Reg. No. 36,675
`BRINKS HOFER GILSON & LIONE
`P.O. BOX 10395
`CHICAGO, ILLINOIS 60610
`(312) 321-4200
`
`3
`
`

`
`DEVICE AND METHOD FOR PRESSURE-DRIVEN PLUG
`TRANSPORT AND REACTION
`
`This application is a continuation of Application No. 12/777,099, filed May
`
`5
`
`5, 2010, which is a continuation of Application No. 10/765,718, filed January 26,
`
`2004, which is a continuation-in-part of Application No. 10/434,970, filed May 9,
`
`2003, which claims the benefit ofU.S. Provisional Application No. 60/394,544,
`
`filed July 8, 2002, and U.S. Provisional Application No. 60/379,927, filed May 9,
`
`2002, all of which are incorporated herein by reference.
`
`10 BACKGROUND
`
`Nonlinear dynamics, in conjunction with microfluidics, play a central role in
`
`the design of the devices and the methods according to the invention. Microfluidics
`
`deals with the transport of fluids through networks of channels, typically having
`
`micrometer dimensions. Microfluidic systems (sometimes called labs-on-a-chip) find
`
`15
`
`applications in microscale chemical and biological analysis (micro-total-analysis
`
`systems). The main advantages ofmicrofluidic systems are high speed and low
`
`consumption of reagents. They are thus very promising for medical diagnostics and
`
`high-throughput screening. Highly parallel arrays of microfluidic systems are used
`
`for the synthesis of macroscopic quantities of chemical and biological compounds,
`
`20
`
`e.g., the destruction of chemical warfare agents and pharmaceuticals synthesis. Their
`
`advantage is improved control over mass and heat transport.
`
`Microfluidic systems generally require means of pumping fluids through the
`
`channels. In the two most common methods, the fluids are either driven by pressure
`
`or driven by electroosmotic flow (EOF). Flows driven by EOF are attractive because
`
`25
`
`they can be easily controlled even in complicated networks. EOF-driven flows have
`
`flat, plug-like velocity profile, that is, the velocity of the fluid is the same near the
`
`walls and in the middle of the channel. Thus, if small volumes of multiple anal ytes
`
`are injected sequentially into a channel, these plugs are transported as non(cid:173)
`
`overlapping plugs (low dispersion), in which case the dispersion comes mostly from
`
`30
`
`the diffusion between plugs. A main disadvantage of EOF is that it is generated by
`
`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
`
`- 1 -
`
`4
`
`

`
`may not be an issue when using channels with negative surface charges in DNA
`
`analysis and manipulation because DNA is uniformly negatively charged and does not
`
`adsorb to the walls. However, this can be a serious limitation in applications that
`
`involve proteins that are often charged and tend to adsorb on charged surfaces. In
`
`5
`
`addition, high voltages are often undesirable, or sources of high voltages such as
`
`portable analyzers may not be available.
`
`Flows driven by pressure are typically significantly less sensitive to surface
`
`chemistry than EOF. The main disadvantage of pressure-driven flows is that they
`
`normally have a parabolic flow profile instead of the flat profile ofEOF. Solutes in
`
`10
`
`the middle ofthe channel move much faster (about twice the average velocity ofthe
`
`flow) than solutes near the walls of the channels. A parabolic velocity profile
`
`normally leads to high dispersion in pressure-driven flows; a plug of solute injected
`
`into a channel is immediately distorted and stretched along the channel. This
`
`distortion is somewhat reduced by solute transport via diffusion from the middle of
`
`15
`
`the channel towards the walls and back. But the distortion is made worse by diffusion
`
`along the channel (the overall dispersion is known as Taylor dispersion).
`
`Taylor dispersion broadens and dilutes sample plugs. Some of the sample is
`
`frequently left behind the plug as a tail. Overlap of these tails usually leads to cross(cid:173)
`
`contamination of samples in different plugs. Thus, samples are often introduced into
`
`20
`
`the channels individually, separated by buffer washes. On the other hand,
`
`interleaving samples with long buffer plugs, or washing the system with buffer
`
`between samples, reduces the throughput of the system.
`
`In EOF, flow transport is essentially linear, that is, if two reactants are
`
`introduced into a plug and transported by EOF, their residence time (and reaction
`
`25
`
`time) can be calculated simply by dividing the distance traveled in the channel by the
`
`velocity. This linear transport allows precise control of residence times through a
`
`proper adjustment of the channel lengths and flow rates. In contrast, dispersion in
`
`pressure-driven flow typically creates a broad range of residence times for a plug
`
`traveling in such flows, and this diminishes time control.
`
`30
`
`The issue of time control is important. Many chemical and biochemical
`
`processes occur on particular time scales, and measurement of reaction times can be
`
`indicative of concentrations of reagents or their reactivity. Stopped-flow type
`
`instruments are typically used to perform these measurements. These instruments rely
`
`on turbulent flow to mix the reagents and transport them with minimal dispersion.
`
`-2-
`
`5
`
`

`
`Turbulent flow normally occurs in tubes with large diameter and at high flow rates.
`
`Thus stopped-flow instruments tend to use large volumes of reagents (on the order of
`
`ml/s). A microfluidic analog of stopped-flow, which consumes smaller volumes of
`
`reagents (typically !J.L/min), could be useful as a scientific instrument, e.g., as a
`
`5
`
`diagnostic instrument. So far, microfluidic devices have not be able to compete with
`
`stopped-flow type instruments because EOF is usually very slow (although with less
`
`dispersion) while pressure-driven flows suffer from dispersion.
`
`In addition, mixing in microfluidic systems is often slow regardless of the
`
`method used to drive the fluid because flow is laminar in these systems (as opposed to
`
`10
`
`turbulent in larger systems). Mixing in laminar flows relies on diffusion and is
`
`especially slow for larger molecules such as DNA and proteins.
`
`In addition, particulates present handling difficulty in microfluidic systems.
`
`While suspensions of cells in aqueous buffers can be relatively easy to handle because
`
`cells are isodense with these buffers, particulates that are not isodense with the fluid
`
`15
`
`tend to settle at the bottom of the channel, thus eventually blocking the channel.
`
`Therefore, samples for analysis often require filtration to remove particulates.
`
`SUMMARY ACCORDING TO THE INVENTION
`
`In one aspect, a method includes the steps of providing a microfluidic system
`
`comprising one or more channels, and providing within the one or more channels a
`
`20
`
`carrier fluid and a plurality of plugs immiscible with the carrier fluid including a first
`
`plug and a second plug. The first plug includes a marker identifying a characteristic of
`
`the first plug when the first plug is formed. The second plug includes a marker
`
`identifying a characteristic of the second plug when the second plug is formed.
`
`In another aspect, the marker may be an optically detectable group, moiety or
`
`25
`
`compound.
`
`In another aspect, the characteristic includes any one or combination of
`
`concentration, identity of chemical species, or elapsed time since droplets were
`
`formed.
`
`In another aspect, the marker includes a dye. For example, the dye may be
`
`30
`
`fluorescein. The marker may also have a fluorescence intensity indicative of a
`
`concentration of the marker.
`
`- 3-
`
`6
`
`

`
`In yet another aspect, the characteristic includes a concentration of a
`
`component within the plug when it is formed. For example, the marker may have a
`
`fluorescence intensity indicative of a concentration of the marker and identifying the
`
`concentration of a component within the plug when it is formed.
`
`5
`
`In a further aspect, the characteristic of the first plug as applied to the first
`
`component includes a concentration of a first component within the first plug when it
`
`is formed and the characteristic of the second plug includes a concentration of the
`
`second component different from the first component within the second plug when it
`
`is formed. The marker may also have a fluorescence intensity indicative of a
`
`10
`
`concentration ofthe marker and identifying the concentration ofthe first component
`
`within the first plug when it is formed and/or the concentration of the second
`
`component within the second plug when it is formed.
`
`In another aspect, the marker is present in the first plug at a first concentration
`
`and is present in the second plug at a second concentration. The first concentration
`
`15 may be suitable for directly or indirectly detecting, analyzing, and/or characterizing a
`
`third concentration of at least the first component and the second concentration may
`
`be suitable for directly or indirectly detecting, analyzing, and/or characterizing a
`
`fourth concentration of the first component.
`
`In yet another aspect, the method further includes providing dispersion in a
`
`20
`
`pressure-driven flow of a continuous stream of a first plug fluid thereby generating a
`
`gradient in the first plug fluid, and fixing the first concentration of the marker within
`
`the first plug by the formation of the first plug and fixing the second concentration of
`
`the marker within the second plug by the formation of the second plug.
`
`In another aspect, at a first time, the first plug fluid includes a second plug
`
`25
`
`fluid and a third plug fluid in a first ratio and, at a second time, includes the second
`
`plug fluid and the third plug fluid in a second ratio. The second plug fluid includes
`
`the first marker at a third concentration and the third plug fluid includes the first
`
`marker at a fourth concentration. The method further includes fixing the first
`
`concentration by forming the first plug at the first time and fixing the second
`
`30
`
`concentration by forming the second plug at the second time.
`
`In another aspect, the first ratio is determined by a first flow rate of the second
`
`plug fluid and a second flow rate of the third plug fluid, and the second ratio is
`
`determined by a third flow rate of the second plug fluid and a fourth flow rate of the
`
`third plug fluid.
`
`-4-
`
`7
`
`

`
`In a further aspect, the first plug includes a second marker at a third
`
`concentration and the second plug includes the second marker at a fourth
`
`concentration.
`
`In yet another aspect, the method further includes the steps of providing within
`
`5
`
`the one or more channels a second plug fluid immiscible with the carrier fluid,
`
`merging the first plug with the second plug fluid thereby forming a first merged plug,
`
`and merging the second plug with the second plug fluid thereby forming a second
`
`merged plug. A product produced by the method includes the first plug, second plug,
`
`first merged plug, and/or second merged plug.
`
`10
`
`In another aspect, the second plug fluid comprises a third component capable
`
`of reacting in the presence of the first plug fluid. The method further includes the
`
`steps of measuring the reaction of the third component, if it occurs, in the first merged
`
`plug by measuring a property or characteristic of the first merged plug and directly or
`
`indirectly measuring the first concentration, and measuring the reaction of the third
`
`15
`
`component, if it occurs, in the second merged plug by measuring a property or
`
`characteristic of the second merged plug and directly or indirectly measuring the
`
`second concentration. The method may also include the steps of comparing the
`
`reactions of the third component in the first merged plug and the second merged plug.
`
`In a further aspect, the first concentration may be suitable for directly or
`
`20
`
`indirectly identifying or detecting a first chemical species including the first
`
`component, and the second concentration may be suitable for directly or indirectly
`
`identifying or detecting a second chemical species including the second component.
`
`In another aspect, a product includes a first and a second plug and is made by
`
`a process including the steps of providing a microfluidic system comprising one or
`
`25 more channels, and providing within the one or more channels a carrier fluid and a
`
`plurality of plugs immiscible with the carrier fluid including the first plug and the
`
`second plug. The first plug includes a marker identifying a characteristic of the first
`
`plug when the first plug is formed. The second plug includes a marker identifying a
`
`characteristic of the second plug when the second plug is formed.
`
`30
`
`In yet another aspect, an apparatus is constructed and adapted to provide a
`
`plurality of plugs immiscible with a carrier fluid including a first plug and a second
`
`plug. The first plug includes a marker identifying a characteristic of the first plug
`
`when the first plug is formed. The second plug includes a marker identifying a
`
`characteristic of the second plug when the second plug is formed.
`
`- 5 -
`
`8
`
`

`
`BRIEF DESCRIPTION OF THE DRAWINGS AND PHOTOGRAPHS
`
`FIG. 1A is a schematic diagram of a basic channel design that may be used to
`
`induce rapid mixing in plugs. FIG. 1B(1)-(4) are schematic diagrams depicting a
`
`5
`
`series of periodic variations of the basic channel design. FIG. 1C(1)-(4) are schematic
`
`diagrams depicting a series of aperiodic combinations resulting from a sequence of
`
`alternating elements taken from a basic design element shown in FIG. 1A and an
`
`element from the periodic variation series shown in FIGS. 10B(1)-(4).
`
`FIG. 2A is a schematic diagram contrasting laminar flow transport and plug
`
`10
`
`transport in a channel. FIG. 2B(l) shows a photograph (right side, top portion)
`
`illustrating rapid mixing inside plugs moving through winding channels. FIG. 2B(2)
`
`shows a photograph (right side, lower portion) showing that winding channels do not
`
`accelerate mixing in a laminar flow in the absence ofPFD.
`
`FIG. 3 shows photographs (right side) and schematic diagrams (left side) that
`
`15
`
`depict a stream of plugs from an aqueous plug-fluid and an oil (carrier-fluid) in
`
`curved channels at flow rates of0.5 !J,L/min and 1.0 11L/min.
`
`FIG. 4 shows a photograph (lower portion) and a schematic diagram (upper
`
`portion) that illustrate plug formation through the injection of oil and multiple plug(cid:173)
`
`fluids.
`
`20
`
`FIG. 5 is a schematic diagram that illustrates a two-step reaction in which
`
`plugs are formed through the injection of oil and multiple plug-fluids using a
`
`combination of different geometries for controlling reactions and mixing.
`
`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
`
`25
`
`diagram shows two reactions that are conducted simultaneously. A third reaction
`
`(between the first two reaction mixtures) is conducted using precise time delay.
`
`FIG. 7(a)-(b) show microphotographs (10 !J.S exposure) illustrating rapid
`
`mixing inside plugs (a) and negligible mixing in a laminar flow (b) moving through
`
`winding channels at the same total flow velocity. FIG. 7(c) shows a false-color
`
`30 microphotograph (2 s exposure, individual plugs are invisible) showing time-averaged
`
`fluorescence arising from rapid mixing inside plugs of solutions ofFluo-4 and CaCh
`
`FIG. 7( d) shows a plot of the relative normalized intensity (I) of fluorescence obtained
`
`from images such as shown in (c) as a function of distance (left) traveled by the plugs
`
`- 6-
`
`9
`
`

`
`and of time required to travel that distance (right) at a given flow rate. FIG.
`
`7(e) shows a false-color microphotograph (2 s exposure) of the weak fluorescence
`
`arising from negligible mixing in a laminar flow of the solutions used in (c).
`
`FIG. 8 shows photographs (right side) and schematics (left side) that illustrate
`
`5
`
`fast mixing at flow rates of about 0.5 ~L/min and about 1.0 ~L/min using 90°-step
`
`channels.
`
`FIG. 9 shows schematics (left side) and photographs (right side) illustrates
`
`fast mixing at flow rates of about 1.0 ~L/min and about 0.5 ~L/min using 135°-step
`
`channels.
`
`10
`
`FIG. lOa) is a schematic diagram depicting three-dimensional confocal
`
`visualization of chaotic flows in plugs. FIG. 1 Ob) is a plot showing a sequence
`
`preferably used for visualization of a three-dimensional flow.
`
`FIG. 11 shows a schematic diagram of a channel geometry designed to
`
`implement and visualize the baker's transformation of plugs flowing through
`
`15 microfluidic channels.
`
`FIG. 12 shows photographs depicting the merging of plugs (top) and splitting
`
`of plugs (bottom) that flow in separate channels or channel branches that are
`
`perpendicular.
`
`FIG. 13 shows UV-VIS spectra ofCdS nanoparticles formed by rapid mixing
`
`20
`
`in plugs (spectrum with a sharp absorption peak) and by conventional mixing of
`
`solutions.
`
`FIG. 14 shows schematic diagrams (left side) and photographs (right side) that
`
`illustrate the synthesis of CdS nanoparticles in PDMS microfluidic channels in single(cid:173)
`
`phase aqueous laminar flow (FIG. 14A) and in aqueous plugs that are surrounded by
`
`25 water-immiscible perfluorodecaline (FIG. 14B).
`
`FIG. 15 shows schematic representations of the synthesis of CdS nanoparticles
`
`inside plugs.
`
`FIG. 16 is a schematic illustration of a microfluidic device according to the
`
`invention that illustrates the trapping of plugs.
`
`30
`
`FIG. 17 is a schematic of a microfluidic method for forming plugs with
`
`variable compositions for protein crystallization.
`
`FIG. 18 is a schematic illustration of a method for controlling heterogeneous
`
`nucleation by varying the surface chemistry at the interface of an aqueous plug-fluid
`
`and a carrier-fluid.
`
`- 7 -
`
`10
`
`

`
`FIG. 19 is a schematic diagram that illustrates a method of separating
`
`nucleation and growth using a microfluidic network according to the present
`
`invention.
`
`FIG. 20 show schematic diagrams that illustrate two methods that provide a
`
`5
`
`precise and reproducible degree of control over mixing and that can be used to
`
`determine the effect of mixing on protein crystallization.
`
`FIG. 21 is a reaction diagram illustrating an unstable point in the chlorite(cid:173)
`
`thiosulfate reaction.
`
`FIG. 22A-D are schematic diagrams that show various examples of geometries
`
`10
`
`of microfluidic channels according to the invention for obtaining kinetic information
`
`from single optical images.
`
`FIG. 23 shows a schematic of a microfluidic network (left side) and a table of
`
`parameters for a network having channel heights of 15 and 2 ~m.
`
`FIG. 24 shows a reaction scheme that depicts examples of fluorinated
`
`15
`
`surfactants that form monolayers that are: (a) resistant to protein adsorption; (b)
`
`positively charged; and (c) negatively charged. FIG. 24b shows a chemical structure
`
`of neutral surfactants charged by interactions with water by protonation of an amine
`
`or a guanidinium group. FIG. 24c shows a chemical structure of neutral surfactants
`
`charged by interactions with water deprotonation of a carboxylic acid group.
`
`20
`
`FIG. 25 are schematic diagrams of microfluidic network (left side of a), b),
`
`and c)) that can be used for controlling the concentrations of aqueous solutions inside
`
`the plugs, as well as photographs (right side of a), b), and c)) showing the formation
`
`of plugs with different concentrations ofthe aqueous streams.
`
`FIG. 26 are schematic diagrams of microfluidic network (left side of a) and b))
`
`25
`
`and photographs (right side of a) and b)) ofthe plug-forming region ofthe network in
`
`which the aqueous streams were dyed with red and green food dyes to show their flow
`
`patterns.
`
`FIG. 27 are photographs and plots showing the effects of initial conditions on
`
`mixing by recirculating flow inside plugs moving through straight microchannels.
`
`30
`
`FIG. 27a1) is a schematic diagram showing that recirculating flow (shown by black
`
`arrows) efficiently mixed solutions of reagents that were initially localized in the front
`
`and back halves of the plug. FIG. 27a2) is a schematic diagram showing that
`
`recirculating flow (shown by black arrows) did not efficiently mix solutions of
`
`reagents that were initially localized in the left and right halves of the plugs. FIG.
`
`- 8 -
`
`11
`
`

`
`27b) shows a schematic diagram showing the inlet portions (left side) and
`
`photographs of images showing measurements ofvarious periods and lengths of
`plugs. FIG. 27c1) shows a graph ofthe relative optical intensity ofFe(SCN)/3-xr(cid:173)
`complexes in plugs ofvarying lengths. FIG. 27c2) is the same as FIG. 7c1) except
`
`5
`
`that each plug traverses a distance of 1.3 mm.
`
`FIG. 28 is a schematic illustration of a plug showing the notation used to
`
`identify different regions of the plugs relative to the direction of motion.
`
`FIG. 29a)-b) are plots ofthe periods and the lengths of plugs as a function of
`
`total flow velocity (FIG. 29a)) and water fraction (FIG. 29b )).
`
`10
`
`FIG. 30 shows photographs illustrating weak dependence of periods, length of
`
`plugs, and flow patterns inside plugs on total flow velocity.
`
`FIG. 31 are plots showing the distribution of periods and lengths of plugs
`
`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
`
`15
`
`are not required for crystal formation in a microfluidic network, as well as a diagram
`
`ofthe microfluidic network (left side).
`
`FIG. 33a-d (left side) are top views ofmicrofluidic networks (left side) and
`
`photographs (right side) that comprise channels having either uniform or nonuniform
`
`dimension. FIG. 33a shows that merging of the plugs occurs infrequently in the T-
`
`20
`
`shaped channel shown in the photographs. FIG. 33b illustrates plug merging
`
`occurring between plugs arriving at different times at the Y -shaped junction
`
`(magnified view shown). FIG. 33c depicts in-phase merging, i.e., plug merging upon
`
`simultaneous arrival of at least two plugs at a junction, of plugs of different sizes
`
`generated using different oil/water ratios at the two pairs of inlets. FIG. 33d
`
`25
`
`illustrates defects (i.e., plugs that fail to undergo merging when they would normally
`
`merge under typical or ideal conditions) produced by fluctuations in the relative
`
`velocity of the two incoming streams of plugs.
`
`FIG. 34a-c show a schematic diagram (a, left side) and photographs (b, c) each
`
`of which depicts a channel network viewed from the top. FIG. 34a is a schematic
`
`30
`
`diagram of the channel network used in the experiment. FIG. 34b is a photograph
`
`showing the splitting of plugs into plugs of approximately one-half the size of the
`
`initial plugs. FIG. 34c is a photograph showing the asymmetric splitting of plugs
`
`which occurred when P1 < P2.
`
`- 9-
`
`12
`
`

`
`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) oflysozyme crystals grown in water
`
`5
`
`plugs in the wells of the microfluidic channel, as well as a diagram (left side) of the
`
`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
`
`autocatalytic (and possibly unstable) reaction mixture.
`
`10
`
`FIG. 38 is a schematic diagram that illustrates a method for a multi-stage
`
`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
`
`photograph (right side) of water plugs attached to the PDMS wall.
`
`FIG. 40 is a schematic representation (left side) of a microfluidic network used
`
`15
`
`to measure kinetics data for the reaction ofRNase 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) ofthe water droplet
`
`region of the microfluidic network (T stands for time), as well as a diagram of the
`
`20 microfluidic network (left side).
`
`FIG. 42 shows a schematic diagram (left side) of a microfluidic network and a
`
`photograph (right side) of the ink plug region ofthe microfluidic network in which the
`
`gradients were formed by varying the flow rates.
`
`FIG. 43 shows a schematic diagram (left side) of a microfluidic network and a
`
`25
`
`photograph (right side) of lysozyme crystals formed in the microfluidic network using
`
`gradients.
`
`FIG. 44 are schematic illustrations showing how an initial gradient may be
`
`created by injecting a discrete aqueous sample of a reagent B into a flowing stream of
`
`water.
`
`30
`
`FIG. 45a) shows a schematic of the microfluidic network used to demonstrate
`
`that on-chip dilutions can be accomplished by varying the flow rates of the reagents.
`
`The blue rectangle outlines the field of view for images shown in FIG. 45c)-d). FIG.
`
`45b) shows a graph quantifying this dilution method by measuring fluorescence of a
`
`solution of fluorescein diluted in plugs in the microchannel.
`
`- 10-
`
`13
`
`

`
`FIG. 46 shows a microbatch protein crystallization analogue scheme using a
`
`with a substrate that includes capillary tubing.
`
`FIG. 47a) shows a lysozyme crystal grown attached to a capillary tube wall.
`
`FIG. 4 7b) shows a thaumatin crystal grown at the interface of protein solution
`
`5
`
`and oil.
`
`FIG. 48a) shows a schematic illustration of a process for direct screening of
`
`crystals in a capillary tube by x-ray diffraction.
`
`FIG. 48b) shows an x-ray diffraction pattern from a thaumatin crystal grown
`
`inside a capillary tube using a microbatch analogue method (no evaporation).
`
`10
`
`FIG. 49 shows a vapor-diffusion protein crystallization analogue scheme with
`
`a substrate that includes capillary tubing.
`
`FIG. 50a) shows vapor diffusion in droplets surrounded by FMS-121 inside a
`
`capillary right after the flow was