WO2008039209A1

WO2008039209A1 - Microfluidic serial dilution circuit

Info

Publication number: WO2008039209A1
Application number: PCT/US2006/039733
Authority: WO
Inventors: Gerald F. Joyce; Richard A. Mathies; William H. Grover; Alison M. Skelley; Brian M. Paegel
Original assignee: The Scripps Research Institute; The Regents Of The University Of California
Priority date: 2006-09-27
Filing date: 2006-10-09
Publication date: 2008-04-03
Also published as: WO2008039207A1

Abstract

A system includes a microfluidic mixing loop. The loop is flushable while leaving a carryover fluid sample in a portion of the loop. The loop also provides for mixing the carryover fluid sample with other fluid in the loop. Three loop valves may be coupled to the mixing loop, wherein one of the valves is within the carryover portion. A pair of access channels coupled to either end of the carryover portion of the microfluidic mixing loop provide fluidic access via two access channel valves on either end of the carryover portion coupled respectively to the pair of access channels. The carryover portion of a mixing loop may be filled with a sample to be diluted. The non-carryover portion of the mixing loop is flushed with a diluent, and the remaining sample in the carryover portion is mixed with the diluent in the non-carryover portion of the mixing loop.

Description

Microfluidic Serial Dilution Circuit

Related Application

[0001] This application claims priority to United States Provisional

Application serial number 60/827,208 (entitled MICROFLUIDIC SERIAL DILUTION CIRCUIT, filed September 27, 2006) which is incorporated herein by reference.

Technical Field

[0002] This inventive subject matter generally relates to the field of microfluidic method and apparatus and more particularly serial dilution using microfluidic method and apparatus.

Background

[0003] Serial dilution is among the most fundamental and widely practiced laboratory techniques, with applications ranging from measuring detector response, to determining kinetic rate constants, to culturing cells. Serial dilution may be particularly important in directed evolution experiments in which a population of RNA molecules is made to undergo repeated rounds of selective amplification, hi order to evolve molecules with interesting properties, it may be necessary to propagate the population of RNAs through many logs of selective growth. This may be accomplished by serially diluting an aliquot of the reaction mixture into fresh growth medium at regular intervals. Performing serial dilutions by manual pipeting is a mundane and time-consuming task that has limited the execution of highly longitudinal experiments in molecular evolution.

Brief Description of the Drawings

[0004] FIG. IA is a schematic diagram of a microfluidic serial dilution circuit according to an example embodiment.

[0005] FIG. IB is a cross section of a portion of the circuit of FIG. IA. [0006] FIGs. 2A and 2B illustrate pumping program schematics for a flush operation and a mix operation with corresponding still pictures of epifluorescence fluid according to an example embodiment.

[0007] FIGs. 3A and 3B illustrate quantitative evaluations of serial dilution performed by the microfluidic serial dilution circuit of FIG. IA.

[0008] FIG. 4 illustrates dependence of carryover fraction on device geometry in accordance with an example embodiment.

[0009] FIG. 5 illustrates mixing reproducibility in accordance with an example embodiment.

[0010] FIGs. 6A and 6B illustrate mixing transients at variable valve actuation times according to an example embodiment.

Detailed Description

[0011] In the following description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments which may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the scope of the present invention. The following description of example embodiments is, therefore, not to be taken in a limited sense, and the scope of the present invention is defined by the appended claims.

[0012] In vitro evolution of RNA molecules relies on a method for executing many consecutive serial dilutions. A microfluidic circuit has been developed to provide such consecutive serial dilutions. The term "microfluidic" is used to describe fluidic channels that are on the order of microns or smaller, such as nanometers in width and/or depth. FIG. 1 is a schematic diagram of a microfluidic serial dilution circuit 100 according to an example embodiment. The circuit 100 includes a microfluidic channel loop 110 that has three two way valves 115, 120 and 125 arranged sequentially around the loop 110. The two way valves prevent or allow fluid to flow through the loop 110, such as by associated pneumatic control lines 130 - A, 135 - B and 140 - C respectively. The labels A, B and C allow for convenient reference to the valves and their control lines when describing programming the order of operation of the valves to facilitate flow through the loop 110.

[0013] An input fluidic access reservoir 145, and output fluidic access reservoir 150 (1.1-mm diameter in one embodiment) are labeled R; and R₀, respectively. The reservoirs are coupled to the loop 110 by access channels 155 and 160 via corresponding valves 165, 170 which are controlled by control lines 175, 180 respectively. In one embodiment, the reservoirs may be replaced by access ports to external reservoirs or sources and sinks of desired fluids or samples. [0014] In one embodiment, the five membrane valve deflection chambers are labeled A, B, C, I, and O on their respective pneumatic control lines. Valves A - 115, B - 120, and C - 125 are two-way valves and are continuous only when open. Input and output valves 1 -165 and O - 170 are bus valves, connecting Rj -145 and R₀ - 150 to the mixing loop 110. When open, I and O allow flow from Ri and R₀ to and from the mixing loop. Fluidic continuity is preserved within the mixing loop even when I and O are closed.

[0015] FIG. IB depicts a cross section of the device at a two-way valve junction, showing fluidic wafer 181 and a manifold wafer 182, a PDMS membrane 185, a fluidic channel 110 and discontinuity, and a corresponding valve displacement or deflection chamber 190.

[0016] FIGs. 2A and 2B illustrate pumping program schematics for a flush operation 210 and a mix operation 220 with corresponding epifluorescence still pictures of fluorescent fluid according to an example embodiment. The operations may also be referred to as programs. Still frames are 50-ms exposures. The circuit may be initially primed with fluorescein dye to illustrate operation of the circuit. The dye is used to correspond to a sample whose dilution is easily visually observable. Fluid flow paths are indicated with gray arrows overlaid on the circuit schematic. The flush operation 210 may be used for diluent flushing and carryover isolation. Carryover isolation corresponds to sample fluid that remains between valves O and I when the reservoirs are connected to the loop 110, and the valve C is closed,

[0017] Flushing may be accomplished by serially actuating I, A, B, and O while keeping C closed. Buffer may be pumped from Rj to R₀, clearing the right side of the mixing loop while isolating the carryover aliquot on the left side (frames 1-4). An example of an open valve can be seen in frame 2, in which B is open and the entire valve may be filled with the concentrated dye solution. The mix operation 220 may be used to mix the diluent and the isolated carryover by serially actuating A, B, and C while I and O are kept closed (frames 5-8). The output reservoir, Ro, was manually evacuated in the time between frame 7 and frame 8 for the purpose of visualizing the fully mixed sample.

[0018] A sequence of valve states defines a pumping program. A variable hold step interposed between states in the sequence may be the valve actuation time. Three pumping programs were written to manipulate fluid in the serial dilution circuit. The valve sequences of each pumping program are written showing only the open valves at each step, and the hold step is indicated by a comma after each state in the sequence. For example, the program (AB, B) starts with valves A and B open and valves C, I, and O closed. This state may be followed by a hold step, then valve A is closed leaving only B open. The mix pumping program is the valve state sequence (A, AB₅ B, BC, C, AC).

[0019] The flush pumping program is the valve state sequence (A, AB, B,

BO, IO, IA). The prune pumping program is the valve state sequence (I, ACI, AC, ABCO, BO, O). Looping a pumping program results in continuous pumping. Each pumping program requires two input parameters for operation: the valve actuation time (in milliseconds) and the length of time the program is iterated (in seconds). [0020] Fluidic manipulation protocols are described in the text using the format: program(valve actuation time,iteration time), with valve actuation times given in milliseconds and iteration times given in seconds. For example, tnix(80,60) indicates that the mix program is run with 80 ms valve actuation time, iterated for 60 s.

[0021] The microfluidic circuit 100 may be fabricated in a three-layer glass-

PDMS-glass device. In one embodiment, a 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, such as increasing or decreasing the distance between valves I and O, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which may be comprised of a diluent flush cycle followed by a mixing cycle, may be carried out with n pipeting, and a sample volume of 400 nL may be sufficient for conducting an arbitrary number of serial dilutions. Mixing may be precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s in one embodiment. The microfluidic circuit may be generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture. The microfluidic circuit helps automate the fluid handling associated with serial dilution.

[0022] The core strengths of microfluidic technology are integration, high throughput, and low- volume handling. Microfluidic analogs outperform conventional instrumentation with regard to speed, throughput, and reagent consumption by an order of magnitude or more, and allow integration of sample preparation and analysis in a single device. Precise manipulation of fluids in these devices may be achieved by electrokinetic control, microfabricated membrane valves, or various other approaches to microfluidic transport and control. The combination of highly ordered flow and precise manipulation allows one to carry out diverse synthetic and analytical methods with remarkable control. [0023] Despite the near universal need for the preparation of standard samples, little work has been done to miniaturize and to expedite this process. Approaches have included variously configured splitter channels and differential metering of multiple inputs into addressable microfabricated assay wells. Each of these approaches to serial dilution requires N independent outputs (splitter branches, end reactors, etc.) for N consecutive dilutions, making them unsuitable for executing an arbitrary number of dilutions. The ideal circuit would automate sample and diluent metering and mixing, while scaling to an arbitrary number of serial dilutions. A microfluidic mixing loop addresses mixing requirements by reducing effective diffusion lengths, while providing a compact geometry for manipulating nanoliter volumes.

[0024] A microfluidic serial dilution circuit that implements these advantageous mixing and scaling characteristics and incorporates sample metering elements has been designed, fabricated, and characterized. It may be compact and does not geometrically constrain the number of possible serial dilutions. Precise metering of the sample carryover fraction and rapid, reproducible mixing of the diluent with the carryover are achieved in the same structure. The device may be computer controlled via the pneumatic lines, and the preparation of successive serial dilutions may be fully automated. Because the circuit employs microfluidic pumping, serially diluted sample aliquots can easily be routed from the dilution circuit to other microfluidic components, such as a separation channel or microreactor.

[0025] FIGs. 3 A and 3B illustrate quantitative evaluations of serial dilution performed by the microfluidic serial dilution circuit 100 of FIG. IA. FIG. 3 A is a graph of fluorescence versus time for three consecutive serial dilutions of fluorescein dye solution (300 nM in TAE buffer) into TAE buffer that were monitored using confocal fluorescence microscopy. The detector position is indicated in the inset circuit schematic 310. The second and third dilutions are shown in the five-fold magnified inset 315. Serial dilutions were performed by executmgflush( 100,60) followed by /»0(100,120). FIG. 3B is a log plot of fluorescence versus dilution cycles of a standard curve for 10 μM fluorescein. It was constructed from the average fluorescence intensity of the sample concentrate, and the intensity obtained after each of four consecutive six-fold dilutions. Each data point represents the average of eight independent experiments. The log plot exhibits excellent linearity over the three detectable orders of magnitude (R = 0.999).

[0026] FIG. 4 illustrates dependence of carryover fraction on device geometry in accordance with an example embodiment. The carryover fraction was related to the arc subtended by valves I and O. The inset 410 indicates the angle measurement, θ. CF = -0.02 + 0.005 θ; R² = 0.998.

[0027] FIG. 5 is a graph of fluorescence versus time that illustrates mixing reproducibility in accordance with an example embodiment. A solution of fluorescein dye was diluted using a circuit with a carryover fraction of 0.12. Two separate devices were operated with identical pumping parameters :flush( 100,90), /«zx(100, 120). The five profiles are offset by 200 CPS for clarity. The start of the flush and mix programs is indicated by arrows. The inset contains an overlay of the five replicates and a sample fit of an exponentially damped sinusoid. Diluent flushing and mixing are highly reproducible, with mixing transients agreeing in fit within 1%.

[0028] FIGs. 6A and 6B illustrate mixing transients at variable valve actuation times according to an example embodiment. In FIG. 6 A, a graph of fluorescence versus time illustrates mixing transients that were generated with variable actuation times and aligned to time t = 0. FIG. 6B is a graph of valve actuation time and standard deviation as a function of time plotted as solid lines, sampling valve actuation times of 300, 200, 100, and 50 ms. The standard deviation window width may be the period of the oscillation for each transient. A dashed line 610 at σ_Wi_n = 300 CPS indicates the threshold for complete mixing. Mixing times (•) measured at different valve actuation times are plotted discretely with respect to the left axis. Mixing times determined by this method exhibited ~5% standard error. [0029] The above graphs and data were obtained from a circuit built substantially in accordance with the following process. The results are merely examples, and results from structure created using the following process may vary. The three-layer glass-PDMS-glass sandwich structure of circuit 100 may be fabricated using common semiconductor fabrication processes. Features on the fluidic and manifold glass wafer layers may be isotropically etched to a depth of 50 μm. The etched fluidic and manifold layers may be visually aligned and reversibly bonded to one another with an intervening optically transparent PDMS membrane (250 μm thick, Rogers Corporation, Carol Stream, IL). Visual alignment and reversible bonding may be performed in a laminar flow hood to minimize particulate contamination of the clean glass wafers and PDMS membrane. Nylon tubing barbs (1/16") may be affixed to the fluidic chip surface at five pneumatic access holes to interface pneumatic control line tubing with the device. Reservoirs and vacuum access holes may be drilled with 1.1-mm-diameter diamond-coated drill bits.

[0030] In one embodiment, the fluidic channels are approximately 300 μm wide, and valve deflection chambers are approximately 1 mm in diameter. Both layers are approximately 50 μm deep. Dimensions listed are after isotropic etching. Two-way valves A, B, and C control fluid flow in the loop. Bus valves I and O control fluidic access to the input and output reservoirs, Rj and R₀, respectively. The loop remains continuous when the bus valves are closed, but fluid flow from Rj and to R₀ is prevented.

[0031] Computer-controlled pneumatic actuation of the membrane valves may be accomplished using a TTL-driven vacuum solenoid valve array (HVOlO, Humphrey, Kalamazoo, MI). On TTL low, the solenoid directs atmospheric pressure output, and the associated membrane valve rests in the closed state. On TTL high, the solenoid switches to vacuum and causes the associated membrane valve to deflect open. The solenoid array may be driven by the digital output of a NI6715 data acquisition PCMCIA card and PC laptop with software written in house (Lab VIEW, National Instruments, Austin, TX). [0032] Flow in the channels may be visualized using a solution of fluorescein dye (10 μM in TAE) and a fiber-coupled epifluorescence microscope (488-nm laser excitation). Epifluorescence movies of the various pumping programs maybe acquired using a 12-bit CoolSnap FX CCD (10 fps, 50-ms exposure, 8 x 8 pixel binning, Roper Scientific, Tucson, AZ). The illumination area was ~1.2 cm diameter and the power density may be 1 mW/mm². [0033] Confocal fluorescence data may be acquired using an inverted microscope fabricated in house. Laser excitation from a frequency-doubled diode laser may be coupled into the optical detection train with a dichroic long-pass mirror (505DRLP, Omega Optical, Brattleboro, VT) and focused on the microfluidic channels with an infinite conjugate microscope objective (4OX 0.6 NA, Newport, Irvine, CA). Fluorescence was collected with the same objective, spectrally filtered with a bandpass filter (535DF60, Omega Optical), and focused with a 100-mm focal length achromatic lens on a 100-μm pinhole before impinging a photon counting PMT (H7827, Hamamatsu Corp., Japan). For all confocal fluorescence measurements, the detector was positioned in the fluidic channel region bounded by valves A and B.

[0034] Fluid handling characteristics of the device may be quantitated using confocal fluorescence microscopy. The input reservoir, Ri, may be spotted with fluorescein solution and the circuit may be run with/>rzme(200,30) to prime with dye. A syringe loaded with TAE buffer (the diluent) may be used to rinse away residual dye solution in Rj and to load diluent. This standard procedure may be used to prepare the circuit for each of the following device characterization studies. [0035] The intrinsic carryover fraction (CF) for each serial dilution circuit maybe determined. The average fluorescence signal of the concentrated dye maybe measured, then the circuit may be run withflu$h(l 00,60), and the average buffer background fluorescence signal may be measured. Finally, the circuit may be run with raά(100,120) to mix the carryover into the diluent. After mixing, the average fluorescence signal of the diluted dye may be measured. The ratio of the background-subtracted diluted dye signal to the dye concentrate signal is the CF. To demonstrate multiple serial dilutions of the same sample, a sample of 10 μM fluorescein may be diluted in TAE using a mixing loop with CF of 0.2. To increase dynamic range, an OD 1 neutral density filter (Newport) may be placed in line to measure the sample concentrate fluorescence intensity. Thereafter, the filter may be removed and the fluorescence intensity of each consecutive dilution may be measured as described above.

[0036] Fluidic handling reproducibility may be evaluated by performing replicate dilutions. For each replicate, the circuit may be prepared as described. Then the circuit may be run withflush(l 00,90), followed by mά(100,120). Mixing may be characterized by performing dilutions with variable valve actuation time during the mixing step. The circuit may be primed as described, and mix(x,500) was initiated, where x may be systematically varied from 300 ms to 50 ms. [0037] Serial dilution of an analyte can be automated and carried out on the nanoliter scale using an appropriately configured microfluidic mixing loop. In-line computer-controlled membrane valves allow precise fluidic manipulation, automation, and parallelization. Fluidic operations, such as diluent flushing, mixing, and priming can be accurately and precisely performed without manual intervention, and performed simultaneously in many parallel circuits. A quantitative description of device performance was developed using epifluorescence flow visualization and confocal fluorescence microscopy.

[0038] Epifluorescence visualization of the pumping programs flush and mix is presented and described above with respect to FIG. 2. Diluent is pumped into the circuit through I, then through A and B, and finally out of the circuit through O. A plug of material in the region bounded by valves I and O and containing C is preserved by flush. Frames 1 through 4 show TAE buffer (diluent) being pumping from Rj to R₀ around the right side of the fluorescein dye-primed circuit. A plug of fluorescein dye (carryover) is preserved on the left (frame 4). The carryover and diluent are mixed together in the mix operation by serially actuating valves A, B, and C while keeping valves I and O closed. Frames 5 through 8 show the carryover being mixed into the diluent as the fluid is cyclically pumped, and the fluorescence intensity in the loop homogenizes.

[0039] A flush operation coupled to a mix operation constitutes a microfluidic serial dilution. Sample in the loop can be serially diluted many times to bring about consecutive serial dilutions of the concentrated sample. This concept is presented and described above with reference to FIG. 3 A. The detector was positioned between valves A and B to observe three consecutive serial dilutions of fluorescein dye concentrate (300 nM). As the dye is cyclically pumped, the concentrated dye signal maybe acquired. NeXt₉TTw₁S¹A(IOO₅OO) and 7wό;(100,120) are run sequentially to perform the serial dilution. The measured fluorescence is reduced to background as the buffer diluent passes the detector during flush, then a mixing transient is observed during mix as the diluent and carryover mix. Once mixing is complete, the same program sequence may be repeated to generate multiple serial dilutions.

[0040] The construction of a complete series of standards based on a single

10 μM fluorescein standard solution is presented in FIG. 3B. The log of the fluorescence intensity after each serial dilution was plotted as a function of the serial dilution cycle number, which is expected to be linear with slope proportional to the log of the carryover fraction (CF) of the circuit.

[0041] The intrinsic CF for a circuit may be determined by the fraction of the mixing loop bounded by valves I and O containing valve C. This fraction linearly depends on the angle θ subtended by the arc between valves I and O (FIG. 4, inset 410). The CF of circuits with various θ was measured and plotted as a function of θ. Linear agreement of CF with θ is excellent (R² = 0.998). The error associated with each CF determination was 1.5%. Ouήngflush steps the carryover may still in contact with the flushing diluent stream, so carryover sample near the I and O valve boundaries may diffuse into the diluent stream. As the diluent flush time is increased, more sample diffuses out and the CF decreases. The dependence of CF on flush time was studied using fluorescein dye and buffer, and found to vary by 5% over the range of 30-300 s.

[0042] Microfluidic devices are characterized by the reproducibility of operations such as mixing and dilution because the flow regime is laminar. This concept is illustrated in FIG 5. Replicate observations of a serial dilution conducted on two different devices demonstrate not only the reproducibility of dilutions performed in the same circuit, but also of dilutions performed on different devices. The inset of FIG. 5 presents an overlay of the replicates. Given identical fluidic programming, the rate of diluent flushing and the oscillations in the mixing transient are reproduced exactly between replicates.

[0043] In order to study the reproducibility of the mixing transient quantitatively, a dampened sinusoid was fit to the data. The functional dependence of the damped sinusoid, Ae^~kt sm{ωt) + b, contained least-squares fit parameters A, k, ω, and b, corresponding to the amplitude, damping factor, frequency, and offset after dilution, respectively. Typical R² values ranged from 0.90 to 0.98. Parameter ω was fit with less than 0.3% least-squares error, and the frequency determined from fits of the five replicates agreed within 1%. A typical fit curve is shown in the FIG. 5 inset, offset from the overlay. Values of R² less than 0.95 are attributed to a relatively poor description of damping by the exponential term. Nonetheless, this procedure yielded excellent data on the transient frequency for the purpose of demonstrating the reproducibility of mixing.

[0044] The time required to mix the diluent plug into the carryover plug may be influenced by the pumping rate, or valve actuation time, during cyclic mixing. FIG. 6 presents the dependence of the mixing transient morphology on the valve actuation time. As the valve actuation time is decreased from 300 ms to 50 ms, the linear flow velocity increases, and the mixing transient may be compressed in time. As the two plugs are pumped through each other, mixing may be expedited by the establishment of more diffusion planes. The dependence of mixing time on valve actuation time can be determined qualitatively from FIG. 6A. At 50 s, for example, the fluorescence intensity may be still widely varying in the 300-ms case, while the signal has completely steadied in the 50-ms case.

[0045] A quantitative study of mixing time is presented in FIG. 6B. The standard deviation of an w-second-wide window, ^_n, was plotted as a function of time to measure signal variance. The window width, n, was normalized by setting it equal to the transient period, 2π/ω, determined by fitting a damped sinusoid to each transient (described above). The deviation predictably drops as mixing proceeds. When the carryover and diluent are completely mixed, the standard deviation of the signal may be limited by the shot noise of the detector, σ_bkgd. The mixing time is the time required for σ_Wj_n to reach 2 σbkgd- At this limit of detection, the observer is theoretically unable to differentiate between contributions to signal variance that arise systematically (as a result of incomplete mixing) versus those that arise randomly (as a result of shot noise).

[0046] An analysis of mixing time as a function of valve actuation time, plotted discretely in Figure 6B, reveals that mixing may_. be expedited as valve actuation time is decreased from 300 ms to 80 ms. The time required for complete mixing is minimized from >150 s to 22 s over the range of actuation times studied. Further decreasing the valve actuation time from 80 ms to 50 ms did not significantly affect the mixing time. This agrees with measurements of linear flow rate as a function of valve actuation time; valve actuation appears to be limiting at valve actuation times shorter than 80 ms. The flow rate over the range of 80- to 50- ms valve actuation times gradually becomes independent of valve actuation time. Additionally, at higher flow velocities, transverse diffusion is limiting and the mixing time cannot be decreased absent a mechanism for establishing new boundary layers, for example by promoting torsional flow.

[0047] Serial dilution is a common operation in chemical measurements.

The construction of a series of standard samples can be time consuming and expensive, requiring many fluid metering steps and expending potentially valuable sample. The circuit described here carries out serial dilutions in 400 nL, though this is not a limit for circuit size. In practice this circuit could be scaled down or up depending on the desired sample volume. Design constraints would include the valve dead volume and carryover channel volume. This microfluidic circuit can generate an entire standard curve with only the diluent as an input. The standards are prepared in nanoliter quantities, conserving reagent and allowing facile integration with on-chip analytical techniques. For example, on-chip capillary electrophoresis or liquid chromatography could be coupled to the output of this circuit, relying on integrated pumping for standard injection. Importantly, this device can execute rapid and automated serial dilutions on the time scale of replication of a population of evolving RNA molecules, opening new avenues of inquiry in molecular evolution.

[0048] While a circular loop with three in-line valves is shown, the number of valves and shape of the loop may be varied, yet still provide a pumping and mixing capability. Oval, race track, polygonal or serpentine loops may be used. In further embodiments, the structure may exceed micrometer dimensions. Many different types of valves may be used without departing from the scope of the invention. The placement of reservoirs within the loop is optional, as they may be placed in different locations depending on layout constraints. Pumping fluid is accomplished through use of the valves in one embodiment. Other pumping mechanisms may be used, such as differential pressure and electromagnetic mechanisms. While carryover fractions of 0.04 to 0.2 were described, other fractions may be achieved in further embodiments. The circuit may be used to enable the automated serial dilution of a population of evolving RNA molecules, but is more generally applicable to almost any microfluidic architecture that involves serial dilution coupled to chemical synthesis or analysis.

[0049] The Abstract is provided to comply with 37 C.F.R. § 1.72(Jo) to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

Claims

1. A system comprising: a microfluidic mixing loop; means for flushing the loop while leaving a carryover fluid sample in a portion of the loop; and means for mixing the carryover fluid sample with other fluid in the loop.

2. The system of claim 1 wherein the means for flushing the loop comprises serially actuated valves disposed along the microfluidic mixing loop.

3. The system of claim 2 wherein the valves comprise membrane deflection chambers.

4. The system of claim 2 wherein the means for flushing the loop comprises pneumatic lines coupled to the valves for controlling the valves to open and close, selectively moving fluid through the mixing loop.

5. The system of claim 1 wherein the means for flushing the loop comprises a pair of reservoirs selectively coupled to ends of the carryover fluid sample portion of the loop.

6. The system of claim 5 wherein the reservoirs are selectively coupled to the ends of the carryover fluid sample portion of the loop by valves.

7. A system comprising: a microfluidic mixing loop having a carryover portion; three loop valves coupled to the mixing loop, wherein one of the valves is within the carryover portion; a pair of access channels coupled to either end of the carryover portion of the microfluidic mixing loop; and two access channel valves on either end of the carryover portion coupled respectively to the pair of access channels.

8. The system of claim 7 wherein two access channel valves on either end of the carryover portion allow selective access to the access channels.

9. The system of claim 7 wherein the five valves coupled to the mixing loop are selectively actuable.

10. The system of claim 9 and further comprising controllable pneumatic channels coupled to control the five valves coupled to the mixing loop.

11. The system of claim 9 wherein the valve within the carryover portion of the micro fluidic mixing loop may selectively prevent fluid flow within the carryover portion while the other four valves are serially actuated to flush the remainder of the microfluidic mixing loop.

12. The system of claim 9 wherein the three loop valves coupled to the mixing loop are serially actuated to mix fluid in the carryover portion with diluent in the loop.

13. The system of claim 9 wherein the loop valves are two way valves and the access channel valves are bus valves.

14. A method comprising: filling a carryover portion of a mixing loop with a sample to be diluted; flushing the non-carryover portion of the mixing loop with a diluent; and mixing the remaining sample in the carryover portion with the diluent in the non-carryover portion of the mixing loop.

15. The method of claim 14 and further comprising repeating the flushing and mixing elements to obtain a desired diluted sample.

16. The method of claim 14 wherein the sample size is approximately 400 nL.

17. The method of claim 14 wherein mixing and flushing are performed by selectively actuating valves coupled to the microfluidic mixing loop.

18. The method of claim 17 wherein flushing is performed by selectively opening and closing valves to input and output access channels having diluent.

19. The method of claim 17 wherein the valves are serially actuated.

20. The method of claim 19 wherein the valves are controlled via a program specifying valve actuation time and valve iteration time.

21. A system comprising a microfluidic serial dilution circuit that can perform multiple serial dilutions.

22. A method comprising automated serial microfluidic dilution coupled to chemical synthesis or analysis.

23. The method of claim 1, wherein the mixing of the carryover fluid sample with other fluid in the loop is reproducible.