FLUIDICS SYSTEM FOR SEQUENTIAL DELIVERY OF REAGENTS
The invention provides a passive fluidics circuit for directing different fluids to a common volume, such as a reaction chamber or flow cell, without intermixing or cross contamination. The direction and rate of flow through junctions, nodes and passages of the fluidics circuit are controlled by the states of upstream valves (e.g. opened or closed), differential fluid pressures at circuit inlets or upstream reservoirs, flow path resistances, and the like. Free diffusion or leakage of fluids from unselected inlets into the common outlet or other inlets at junctions or nodes is prevented by the flow of the selected inlet fluid, a portion of which sweeps by the inlets of unselected fluids and exits the fluidics circuit by waste ports, thereby creating a barrier against undesired intermixing with the outlet flow through leakage or diffusion. The invention is particularly advantageous in apparatus for performing sensitive multistep reactions, such as pH-based DNA sequencing reactions.
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This is a continuation of U.S. patent application Ser. No. 13/245,649, filed 26 Sep. 2011, which is a continuation of U.S. patent application Ser. No. 12/785,667, filed 24 May 2010, now U.S. Pat. No. 8,546,128, which is a continuation-in-part of U.S. patent applications Ser. Nos. 12/474,897 (which claims priority under U.S. provisional application Ser. Nos.: 61/205,626, filed Jan. 22, 2009; 61/198,222, filed Nov. 4, 2008; and, 61/196,953, filed Oct. 22, 2008) and 12/475,311, both filed 29 May 2009, and claims priority under U.S. provisional application Ser. No. 61/291,627 filed 31 Dec. 2009. Each of the foregoing applications is incorporated by reference in their entireties.
BACKGROUNDMany applications require the regulation of multiple fluid flows in a manner that minimizes intermixing or cross-contamination of the different fluids. Such applications include multi-step synthetic or analytical processes that are carried out in a common volume and that comprise successive cycles of reagent delivery using fluids from separate reservoirs. e.g. Margulies et al. Nature, 437: 376-380 (2005); Merrifield et al, U.S. Pat. No. 3,531,258; Caruthers et al, U.S. Pat. No. 5,132,418; Rothberg et al, U.S. patent publication 2009/0127589, and the like. Although fluidics systems are available for selectively switching multiple reagent solutions to a common chamber for processing, they suffer from several deficiencies, including but not limited to, the presence of large surface areas that can adsorb or retain reagents, large physical size which makes it difficult to use with miniaturized fluidics components, e.g. see Rothberg et al (cited above), less accessible surfaces including edges and/or corners which make complete purging and removal of successive reagents difficult or inefficient, and the use of moving parts which can wear out and lead to higher manufacturing and assembly costs, e.g. Hunkapiller, U.S. Pat. No. 4,558,845; Wittmann-Liebold et al, U.S. Pat. No. 4,008;736; Farnsworth et al, U.S. Pat. No. 5,082,788; Garwood et al, U.S. Pat. No. 5,313,984; or the like.
In view of the above, it would be advantageous to have available a device for regulating multiple fluid flows to a common volume for complex synthetic or analytical processes which overcame the deficiencies of current approaches.
SUMMARY OF THE INVENTIONThe present invention is directed to apparatus and methods for delivering multiple fluids to a common volume, such as for example, a passage or conduit to a reaction chamber or flow cell. The invention also includes applications of such apparatus and methods in multistep analytical or synthetic processes. The present invention is exemplified in a number of implementations and applications, some of which are summarized below and throughout the specification.
In one aspect, the invention provides a passive fluidics circuit for sequentially directing different fluids to a common volume, such as a reaction chamber or flow cell, without intermixing or cross contamination. As used herein, such sequential directing is sometimes referred to as “multiplexing” a plurality of fluid flows. The direction and rate of flow through junctions, nodes and passages of the fluidics circuit are controlled by the states of upstream valves (e.g. opened or closed), differential fluid pressures at circuit inlets or upstream reservoirs, flow path resistances, and the like. Free diffusion or leakage of fluids from unselected inlets into the common outlet or other inlets at junctions or nodes is prevented by the flow of the selected inlet fluid, a portion of which sweeps by the inlets of unselected fluids and exits the fluidics circuit by way of waste ports, thereby creating a barrier against undesired intermixing with the outlet flow through leakage or diffusion. In one aspect, the selected fluidic inlet provides a laminar flow of fluid through the fluidics node.
In another aspect, the invention provides a fluidics circuit for controlling a plurality of fluid flows, the fluidics circuit comprising: (a) a fluidics node having an outlet and a plurality of fluid inlets; and (b) at least one waste port in fluid communication with the fluidics node by one or more passages each having a fluid resistance, the fluid resistances of the passages being selected so that whenever a fluid flows solely through a single fluid inlet to form a flow in the fluidics node a portion of such fluid exits the fluidics node through the outlet and the remainder of such fluid exits the fluidics node through the one or more passages, such that any fluid entering the fluidics node from inlets without fluid flows (i.e. “unselected inlets”) is directed through the one or more passages to the one or more waste ports. In one embodiment, the plurality of fluid flows is controlled to provide a predetermined sequence of fluid flows through the outlet of the fluidics node. In another embodiment, such control is implemented by valves and differential pressures applied to the fluids of the flows upstream of the fluidics circuit.
In another aspect, the invention provides a fluidics circuit with no moving parts that sequentially directs multiple fluids to a common volume with no intermixing. Since the fluidics circuit comprises only a node and a plurality interconnected passages where fluid movement is controlled by remotely positioned valves, pumps, it can be readily miniaturized by conventional microfluidics techniques for applications where size and mass are critical factors. Furthermore, the use of the fluidics circuit for fluid switching without the use of impermeable barriers makes the circuit ideal for use in processes where a stable reference potential is required, such as in electrochemical processes.
The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of mechanical engineering, electronics, fluid mechanics, and materials science, which are within the skill of the art. Such conventional techniques include, but are not limited to, design and fabrication of fluidics and microfluidics devices, and the like. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.
The invention provides methods and apparatus for rapidly and cleanly switching flows of different fluids to a common outlet using a fluidics circuit. In one aspect, the fluidics circuit of the invention is combined with fluidic reservoirs, valves, pressure sources, pumps, control systems, and/or like components, to form a fluidics system for delivering separate fluid flows having predetermined rates and durations to a common volume, such as an outlet, chamber, flow cell, or the like. Such fluidics circuits are particularly useful in fluidics systems in apparatus for carrying out multi-step chemical, enzymatic, or electrochemical processes, such as described Margulies et al, Nature, 437: 376-380 (2005); Merrifield et al. U.S. Pat. No. 3,531,258; Brenner et al, Nature Biotechnology, 18: 630-634 (2000); Ronaghi et al, Science, 281: 363-365 (1998); Caruthers et al, U.S. Pat. No. 5,132,418; Namsaraev et al, U.S. patent publication 2005/0100939; Rothberg et al, U.S. patent publication 2009/0127589; and the like.
In one aspect, the fluidics circuit of the invention provides a junction where a flow of a selected fluid is split into at least two branches: one branch is directed to an outlet and from there to a flow cell or reaction chamber for use and the other branch is directed past the unselected fluid inlets and from there away from the outlet and to a waste port. In one embodiment, such flows are created by balancing the fluid resistance of the fluid outlet and that of the one or more passages between the fluid inlets and the waste port. Preferably, the flow rates, fluid viscosities, compositions, and geometries and sizes of the passages, chambers and nodes are selected so that fluid flow is laminar within the fluidics circuit. Guidance for making such design choices is readily available from conventional treatises on fluid dynamics, e.g. Acheson, Elementary Fluid Dynamics (Clarendon Press, 1990), and from free or commercially available software for modeling fluidics systems, e.g. SolidWorks from Dassault Systems (Concord. Mass.); Flowmaster from Flow Master USA, Inc. (Glenview, Ill.); and Open FOAM (open source code for computational fluid dynamics available on the world wide web, www.openefd.co.uk). Fluidic circuits and apparatus of the invention are particularly well suited for meso-scale and micro-scale fluidics systems, for example, fluidics systems having passage cross-sections in the range of tens of square microns to a few square millimeters, or having flow rates in the range of from a few nL/sec to a hundreds of μL/sec. The number of fluid flows controlled by fluidics circuits of the invention can vary widely. In one aspect, fluidics circuits of the invention control a plurality of flows in the range of from 2 to 12 different fluids, or in another aspect in the range of from 2 to 6 different fluids.
Fluidics CircuitsThe design and operation of one embodiment of the invention is partially illustrated in FIG. I1A. Four fluid inlets, or reagent inputs, (100, 102, 104, 106) are connected to fluidics node (108) and are in fluid communication with, and on an opposing surface to outlet (110). Valve (111) is shown open so that fluid passes through inlet (100) into fluidics node (108). A portion (124) of the fluid travels through a passage shown on the left, a portion (126) travels through a passage shown on the right, and a portion exits the fluidics node through outlet (110). Preferably the three fluid flows are laminar and the flow along the surface containing the fluid inlets exits the fluidic node in a period of time that is much less than the time it would take material from the unselected inlets (diffuse effluent (128)) to diffuse to the opposing surface of the fluidics node. In this way, intermixing of the different input reagents that exit through outlet (110) is avoided. In one mode of operation, reagent inputs are selected by opening the valve corresponding to such reagent and closing all the other valves. As illustrated in this embodiment, valve (111) is open and valves (113, 115, and 117) are closed. In the closed state, even though there is no flow in the unselected inlets, a volume (for example, 120) of the unselected fluid is in free diffusive contact with the selected fluid. The split laminar flow of the selected fluid to both outlet (110) and past the unselected inlets and to the waste ports prevents undesired mixing.
In one aspect of the invention, such an apparatus comprises a reaction vessel coupled to an electronic sensor for monitoring products in the reaction vessel: a fluidics system including a fluidics circuit of the invention for sequentially delivering a plurality of different electrolytes including a selected electrolyte to the reaction vessel: and a reference electrode in contact with the selected electrolyte for providing a reference voltage to the electronic sensor, the reference voltage being provided without the reference electrode contacting any unselected electrolytes.
Materials and Methods of FabricationAs mentioned above, fluidic circuits of the invention may be fabrication by a variety of methods and materials. Factors to be considered in selecting materials include degree of chemical inertness required, operating conditions, e.g. temperature, and the like, volume of reagents to be delivered, whether or not a reference voltage is required, manufacturability, and the like. For small scale fluid deliveries, microfluidic fabrication techniques are well-suited for making fluidics circuits of the invention, and guidance for such techniques is readily available to one of ordinary skill in the art, e.g. Malloy, Plastic Part Design for Injection Molding: An Introduction (Hanser Gardner Publications, 1994); Herold et at, Editors, Lab-on-a-Chip Technology (Vol. 1): Fabrication and Microfluidics (Caister Academic Press, 2009): and the like. For meso-scale and larger scale fluid deliveries conventional milling techniques may be used to fabricate parts that may be assembled into fluidic circuits of the invention. In one aspect, plastics such as polycarbonate, polymethyl methacrylate, and the like, may be used to fabricate fluidics circuits of the invention.
Applications in Electrochemical ProcessesFluidics circuits of the invention are useful in electrochemical processes where multiple reagents are delivered to one or more reactors that are monitored with electronic sensors requiring a reference electrode. Exposure of a reference electrode to multiple reagents can introduce undesirable noise into the signals detected by the electronic sensors. Circumstances where this occurs are in methods and apparatus for carrying out label-free DNA sequencing, and in particular, pH-based DNA sequencing. The concept of label-free DNA sequencing, including pH-based DNA sequencing, has been described in the literature, including the following references that are incorporated by reference: Rothberg et al, U.S. patent publication 2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86 (2008); Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006); and the like. Briefly, in pH-based DNA sequencing, base incorporations are determined by measuring hydrogen ions that are generated as natural byproducts of polymerase catalyzed extension reactions. DNA templates each having a primer and polymerase operably bound are loaded into reaction chambers (such as the microwells disclosed in Rothberg et al, cited above), after which repeated cycles of deoxynucleoside triphosphate (dNTP) addition and washing are carried out. Such templates are typically attached as clonal populations to a solid support, such as a microparticle, bead, or the like, and such clonal populations are loaded into reaction chambers. In each addition step of the cycle, the polymerase extends the primer by incorporating added dNTP only if the next base in the template is the complement of the added dNTP. If there is one complementary base, there is one incorporation, if two, there are two incorporations, if three, there are three incorporations, and so on. With each such incorporation there is a hydrogen ion released, and collectively a population of templates releasing hydrogen ions causing very slight changes the local pH of the reaction chamber which is detected by an electronic sensor.
When valve (723) is open, wash solution from the auxiliary wash reservoir 1 (722) passes through passage (729), through valve (723), to passage (734), and to junction (731), where the flow splits between passage (735) and passage (741). As with the design of the fluidics circuits described above, the lengths and cross-sections of passages (735) and (734), and the driving forces of the wash solution and reagent are selected so that when valve (723) is open (as shown) solely wash solution enters flow chamber 1 and reagent from the fluidics circuit is directed solely to waste reservoir (744). When valve (723) is closed, then no wash solution moves in passage (729) and there is no barrier to the flow of reagent from passage (730), to passage (735), to passage (741), and to flow chamber 1. Likewise, when valve (725) is open, wash solution from the auxiliary wash reservoir 2 (724) passes through passage (743), through valve (725), to passage (736), and to junction (745), where the flow splits between passage (737) and passage (747). As above, the lengths and cross-sections of passages (736) and (737), and the driving forces of the wash solution and reagent are selected so that when valve (725) is open solely wash solution enters flow chamber 2 and reagent from the fluidics circuit is directed solely to waste reservoir (744). When valve (725) is closed (as shown), then no wash solution moves in passage (743) and there is no barrier to the flow of reagent from passage (732), to passage (737), to passage (747), and to flow chamber 2.
While the present invention has been described with reference to several particular example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present invention. The present invention is applicable to a variety of sensor implementations and other subject matter, in addition to those discussed above.
Definitions“Microfluidics device” means an integrated system of one or more chambers, ports, and channels that are interconnected and in fluid communication and designed for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions, such as sample introduction, fluid and/or reagent driving means, temperature control, detection systems, data collection and/or integration systems, and the like. Microfluidics devices may further include valves, pumps, and specialized functional coatings on interior walls, e.g. to prevent adsorption of sample components or reactants, facilitate reagent movement by electroosmosis, or the like. Such devices are usually fabricated in or as a solid substrate, which may be glass, plastic, or other solid polymeric materials, and typically have a planar format for ease of detecting and monitoring sample and reagent movement, especially via optical or electrochemical methods. Features of a microfluidic device usually have cross-sectional dimensions of less than a few hundred square micrometers and passages typically have capillary dimensions, e.g. having maximal cross-sectional dimensions of from about 500 μm to about 0.1 μm. Microfluidics devices typically have volume capacities in the range of from 1 μm to a few nL, e.g. 10-100 nL. The fabrication and operation of microfluidics devices are well-known in the art as exemplified by the following references that are incorporated by reference: Ramsey, U.S. Pat. Nos. 6,00 1,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al. U.S. Pat. Nos. 5,126,022 and 6,054,034; Nelson et al, U.S. Pat. No. 6,613,525; Maher et al, U.S. Pat. No. 6,399,952: Ricco et al, International patent publication WO 02/24322: Bjornson et al, International patent publication WO 99/19717; Wilding et al, U.S. Pat. Nos. 5,587,128; 5,498,392; Sia et at, Electrophoresis, 24: 3563-3576 (2003); Unger et al. Science, 288: 113-116 (2000); Enzelberger et al, U.S. Pat. No. 6,960,437.
Claims
1. A method for controlling fluid flow in a fluidic circuit, the fluidics circuit including a first fluid inlet and a second fluid inlet in fluid communication with a node via respective first and second passages, a fluid outlet in fluid communication with the node, and at least one waste port in fluid communication with the node via the first and second passages, the method comprising:
- flowing first fluid into the fluidics circuit via the first fluid inlet, a first portion of the first fluid flowing through the first passage, through the node and then through the fluid outlet, a second portion of the first fluid flowing through the first passage, through the node, through the second passage past the second fluid inlet and then into the at least one waste port, and a third portion of the first fluid flowing to the at least one waste port without passing through the node.
2. The method of claim 1, wherein the first passage and the second passage provide distinct fluid pathways between the node and the at least one waste port.
3. The method of claim 1, wherein the second and third portions flow through a waste passage to the at least one waste port.
4. The method of claim 1, wherein flowing the first fluid into the fluidics circuit includes flowing the first fluid through the fluid inlet to an inlet passage and through a T-junction between the inlet passage and the first passage.
5. The method of claim 1, further comprising:
- flowing a wash solution into the fluidics circuit following flowing the first solution, the wash solution entering the fluidics circuit via a wash solution inlet in communication with the fluid outlet, the wash solution flowing through the fluid outlet into the node, through the first and second passages to the at least one waste port.
6. The method of claim 5, further comprising:
- flowing a second fluid into the fluidics circuit via the second fluid inlet following flowing the wash solution, a first portion of the second fluid flowing through the second passage, through the node and then through the fluid outlet, and a second portion of the second fluid flowing through the second passage, through the node, through the first passage past the first fluid inlet and then into the at least one waste port.
7. The method of claim 6, wherein a third portion of the second fluid flows through the second passage to the at least one waste port without passing through the node.
8. The method of claim 1, further comprising:
- flowing a wash solution into the fluidics circuit following flowing the first solution while continuing to flow the first fluid, the wash solution entering the fluidics circuit via a wash solution inlet in communication with the fluid outlet, the wash solution flowing through the fluid outlet into the node, through the first and second passages to the at least one waste port, the first fluid flowing through the first passage to the at least one waste port without entering the node.
9. A method for controlling fluid flow in a fluidic circuit, the method comprising:
- flowing a wash solution through a wash solution inlet into the fluidic circuit, the wash solution flowing through a fluid outlet into a node and through a plurality of passages to at least one waste port, the plurality of passages each providing a distinct fluid pathway between the node and the at least one waste port;
- initiating flow of a first fluid into the fluidic circuit via a first fluid inlet while fluid the wash solution, the first fluid flowing along a first passage of the plurality of passages to the at least one waste port without entering the node; and
- stopping flow of the wash solution, a first portion of the first fluid flowing through the first passage to the at least one waste port without passing through the node, a second portion of the first fluid flowing through the first passage, through the node and then through the fluid outlet, and a third portion of the first fluid flowing through the first passage, through the node, through a second passage of the plurality of passages past a second fluid inlet and then into the at least one waste port.
10. The method of claim 9, further comprising:
- initiating flow of the wash solution and stopping flow of the first fluid;
- initiating flow of a second fluid into the fluidic circuit via a second fluid inlet while flowing the wash solution, the second fluid flowing along the second passage of the plurality of passages to the at least one waste port without entering the node; and
- stopping flow of the wash solution, a first portion of the second fluid flowing through the second passage to the at least one waste port without passing through the node, a second portion of the second fluid flowing through the second passage, through the node and then through the fluid outlet, and a third portion of the first fluid flowing through the second passage, through the node, through the first passage past the first fluid inlet and then into the at least one waste port.
11. The method of claim 9, wherein the first fluid flows through the fluid inlet to a t-junction with the first passage.
12. A method for controlling fluid flow in a fluidic circuit, the fluidics circuit including a plurality of fluid passages including at least three fluid passages providing distinct fluid pathways between a node and at least one waste port, each of the plurality of fluid passages uniquely associated with a fluid inlet via an associated junction, the method comprising:
- flowing a fluid into the fluidics circuit via one fluid inlet, a first portion of the fluid flowing through the associated junction and the associated fluid passage to the at least one waste port, a second portion flowing through the associated junction and the associated passage, through the node and then through the fluid outlet, and a third portion of the fluid flowing through the junction and the associated passage, through the node, through the other fluid passages of the plurality of fluid passages past the associated fluid inlets and then into the at least one waste port.
13. The method of claim 12, wherein each fluid passage of the plurality of fluid passages provides distinct fluid pathways between the node and the at least one waste port.
14. The method of claim 12, wherein the second and third portions flow through a waste passage to the at least one waste port.
15. The method of claim 12, wherein flowing the fluid into the fluidics circuit includes flowing the fluid through the fluid inlet to an inlet passage and through the junction between the inlet passage and the first passage.
16. The method of claim 12, further comprising:
- flowing a wash solution into the fluidics circuit following flowing the fluid, the wash solution entering the fluidics circuit via a wash solution inlet in communication with the fluid outlet, the wash solution flowing through the fluid outlet into the node, through the first and second passages to the at least one waste port.
17. The method of claim 16, further comprising:
- flowing a second fluid into the fluidics circuit via the second fluid inlet following flowing the wash solution, a first portion of the second fluid flowing through the second passage, through the node and then through the fluid outlet, and a second portion of the second fluid flowing through the second passage, through the node, through the first passage past the first fluid inlet and then into the at least one waste port.
18. The method of claim 17, wherein a third portion of the second fluid flows through the second passage to the at least one waste port without passing through the node.
19. The method of claim 12, further comprising:
- flowing a wash solution into the fluidics circuit following flowing the first solution while continuing to flow the fluid, the wash solution entering the fluidics circuit via a wash solution inlet in communication with the fluid outlet, the wash solution flowing through the fluid outlet into the node, through the first and second passages to the at least one waste port, the fluid flowing through the first passage to the at least one waste port.
20. The method of claim 19, wherein, while flowing the wash solution into the fluidics circuit while continuing to flow the fluid, the fluid flows through the first passage to the at least one waste port without flowing through the node.
Type: Application
Filed: May 30, 2014
Publication Date: Sep 18, 2014
Applicant: LIFE TECHNOLOGIES CORPORATION (Carlsbad, CA)
Inventors: Jonathan SCHULTZ (Oxford, MA), David MARRAN (Durham, CT)
Application Number: 14/291,330
International Classification: B01L 3/00 (20060101);