Patent Grant
10272427
Microfluidic devices are used extensively for the capture, detection, classification, or quantification of molecules, molecular complexes, viruses, cells and particulates in environmental or biological samples. These devices include one or more microchannels of sub-millimeter (mm, 1 mm=10−3 meters) cross section formed in a chip of an inert material, which direct flow of one or more fluids from one or more corresponding reservoirs to interact with each other in a reaction chamber or at a detector or both.
Microfluidics technology has recently emerged as a powerful means to manipulate fluids at a microscale and fully integrate many components and steps for complex yet very precise biochemical analyses. Some of the promising applications include the development of inexpensive diagnostic devices that can be deployed in low-resource settings especially to meet global health challenges such as the lack of facilities and personnel to carry out medical diagnostics. Current microfluidic devices fall into either droplet-based (multiphase) or continuous-flow (single phase) systems. To successfully manipulate these fluids, a number of control strategies have been proposed which often require use of pumps and valves, either integrated on chip or off-chip. Even for integrated on-chip systems, the valves are controlled by external macro-scale elements, such as computers, and require power sources; thus limiting the usefulness of microfluidic tools in resource poor and field settings.
A majority of commercially available systems depend actively on external pumps, vacuum and pressure sources, or depend passively on capillary filling. These solutions are often prohibitively expensive for field deployment. Research and development of self-contained micro-valves and micro-pumps that are integrated into the system still generate systems that fall short when it comes to reliability and commercial viability. The reasons for some of these challenges include the dominance of surface effects over volume effects at the microscale level—leading to increases in required driving pressure, large frictional forces that must be overcome and bubbles acting as capacitances which absorb the actuator-generated pressure. Such effects make it difficult to make micro-pumps that can prime themselves (e.g., see J. D. Zhan, Methods in Bioengineering, Biomicrofabrication and Biomicrofluidics). In addition, current systems are designed for single use. While fabricating the devices in a large batch may help reduce the costs, clean-room processes and material costs can quickly add to the cost and are typically not available near resource-poor deployments. Moreover, running a complex protocol often requires a very complex device design and/or highly skilled personnel to carefully run through multiple steps with a low error rate.
Complexity of current microfluidic systems (control systems, number of parts, fragility) renders them ineffective in harsh conditions and field settings, often encountered in global health and other applications.
Techniques are provided for microfluidic movement control. In some embodiments, techniques are provided for completely programmable fluidic manipulation without requiring any external control elements or electricity, using punch cards. For usage of a single microfluidic chip for many different purposes, in some embodiments, the same chip can be programmed arbitrarily using different punch cards.
In a first set of embodiments, an apparatus includes a support structure, multiple actuators, and a punch card guide. The support structure has a recess configured for receiving a chip with a plurality of microfluidic channels. Each actuator includes multiple teeth protruding outward from the actuator, and each actuator is positioned so that at least in some angle of rotation a tooth of the actuator extends into the recess sufficiently to compress a microfluidic channel in a chip placed in the recess. The punch card guide is connected to the support structure and is configured to guide a punch card with a plurality of punched features. The guide is configured so that, at least in some angle of rotation, a tooth of an actuator extends into the punch card guide sufficiently to engage a punched feature on a punch card disposed in the punch card guide.
In some embodiments of the first set, an actuator is rotated by manually advancing a punch card through the punch card guide when a tooth of the actuator is engaged with a punch feature on the punch card. In some embodiments of the first set, the apparatus includes a valve moveably connected to the support structure. The valve includes a pin configured to extend into the recess sufficiently to close a microfluidic channel in the chip placed in the recess when no tooth of the plurality of teeth on an actuator is in contact with the valve and to withdraw from the recess when a tooth of the actuator is in contact with the valve.
In a second set of embodiments, a microfluidic channel chip is made of a body structure having formed therein multiple microfluidic channels in an actuator interaction region, a reaction chamber and a reservoir. Each microfluidic channel in the actuator interaction region has at least one dimension in a size range from about 0.1 micron to about 1000 microns (1 micron, also called micrometer, μm, =10−6 meters) and is separated from an outside of the body structure by an outer flexible substrate. The reaction chamber is in fluid communication with a first end of each of the microfluidic channels in the actuator interaction region. The reservoir is configured to hold a supply of a fluid in fluid communication with a second end opposite the first end of at least one channel of the microfluidic channels in the actuator interaction region.
In a third set of embodiments, a punch card medium includes a sheet of a first material on which is formed a plurality of punch features. Each punch feature is configured to engage a tooth extending from an actuator of multiple actuators in an apparatus. A different second material is encapsulated in the sheet at a location of at least one punch feature and configured to be released by an encounter with the tooth of the actuator.
In a fourth set of embodiments, a system includes a chip and an apparatus. The chip includes multiple microfluidic channels in an actuator interaction region separated from an outside of the chip by an outer flexible substrate. The apparatus includes multiple actuators rotatably connected to a support structure that has a recess for receiving the chip. Each actuator includes multiple teeth protruding outward from the actuator. Each actuator is positioned in the support structure so that, at least in some angle of rotation, a tooth of the actuator extends sufficiently into the recess to compress a microfluidic channel in the actuator interaction region of the chip disposed in the recess.
In some embodiments of the fourth set, the support structure of the apparatus also includes a punch card guide for guiding a punch card to contact the plurality of actuators. The system further includes a punch card comprising a sheet of a first card material on which is formed multiple punch features. Each punch feature is configured to engage a tooth of an actuator of the plurality of actuators.
In a fifth set of embodiments, a kit includes a chip and a punch card. The chip includes multiple microfluidic channels in an actuator interaction region separated from an outside of the chip by an outer flexible substrate. The punch card includes a sheet of a first card material on which is formed a plurality of punch features. Each punch feature is configured to engage a tooth of an actuator of a plurality of actuators in an apparatus in which each actuator is configured to compress a corresponding microfluidic channel of the plurality of microfluidic channels in the actuator interaction region.
In a sixth set of embodiments, a method includes obtaining an apparatus comprising an actuator rotatably connected to a support structure. The actuator includes multiple teeth. One tooth on the actuator is configured to compress a microfluidic channel in a chip when another tooth on the actuator is engaged in a punch feature in a punch card. The method also includes receiving a chip and punch card. The chip includes a microfluidic channel separated from outside the chip by a flexible substrate in an actuator interaction region. The punch card includes a sheet with multiple punch features, each configured to engage a tooth of the actuator. The method further includes contacting a sample to at least one of a capsule on the punch card, or to a reservoir in fluid communication with the microfluidic channel in the chip. The method still further includes advancing the punch card through the apparatus in contact with the actuator to cause a first tooth of the actuator to compress the microfluidic channel when a second tooth of the actuator engages a punch feature on the punch card.
Still other aspects, features, and advantages of the invention are readily apparent from the following detailed description, simply by illustrating a number of particular embodiments and implementations, including the best mode contemplated for carrying out the invention. The invention is also capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
The present invention is illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar elements and in which:
A method and apparatus and article of manufacture are described for programmable microfluidics. In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to avoid unnecessarily obscuring the present invention.
Some embodiments are described below in the context of microfluidics with just manual power. However, the invention is not limited to this context and may be used with electrical, gravitational, geothermal or other sources of power. Furthermore, some embodiments are described with the chip above the actuators driven by the punch card; however, in other embodiments the chip with microfluidic channels is beside or below the actuators. Many illustrated embodiments are also described in the context of two phase flow with water based fluids flowing as droplets in oil as an immiscible carrier fluid. However, in other embodiments other immiscible fluids are used, or single phase fluid flow (also called “continuous flow”) is utilized.
As used herein a microfluidic channel is a leak-resistant channel having at least one dimension in a size range from about 0.1 micron to about 1000 microns. For brevity, the term “microchannel” is used interchangeably with the term “microfluidic channel.” A microfluidic device is a material object in which is formed one or more microfluidic channels. The term “microfluidic chip” is used interchangeably with the term “microfluidic device.” For brevity, a microfluidic chip is herein often referred to simply as a “chip.” A microfluidic protocol refers to a sequence of fluid movements in the microfluidic channels of a chip, and is also called simply a protocol herein for brevity. Thus a protocol implements a method on a chip. Protocols include assays that determine the existence or amount of a substance in a sample.
A punch card is a sheet of one or more layers of one or more materials on which persistent features, such as dimples and holes, can be punched or pressed or embossed or carved or otherwise imposed on the sheet. In some embodiments the punch card is of limited width and length (e.g., length about twice the width). In other embodiments, the punch card is of limited width but is extended substantially in length (much larger than twice the width) and is called a punch card tape or punch tape. Any material can be used to make the punch card, such as punched paper formerly used to input computer instructions, laminated paper, other papers such as card stock paper, Manila paper, sandpaper, Kraft paper available from Georgia-Pacific in Atlanta, Ga. and other derivatives of Kraft paper such as sack kraft paper, spinning kraft paper and hunting cartridge paper, leather, metal such as aluminum foil or other thin, flexible variations, cloth such as millinery buckram or bookbinding buckram other cloths stiffened in ways such as soaking in a substance such as pyroxylin or starch, or various polymers, such as Mylar® available from Tekra Corporation, New Berlin, Wis., and other materials, alone or in some combination. The term “punch card” is meant to refer to all such embodiments described in this paragraph.
As used herein, the term “actuator” refers to a component that causes, or is able to cause, a fluid in a microchannel to move. While actuators that revolve around a shaft are primarily depicted, other actuators that rock back and forth may also be used in some embodiments, along with, or instead of, actuators that revolve through 360 degrees around a shaft. In either case, the actuators are said to rotate relative to an actuator support structure and, thus, to be rotatably connected to the support structure.
A microfluidic actuation apparatus is described which reads features on a punch card to sequence in an arbitrary fashion (program), or to power, or both, fluid flow in a microfluidic chip. This simple solution allows for arbitrarily complex protocols to be executed by a chip without any requirement of external pumps, valves or even electricity. Different protocols are executed in a single chip by using different punch cards. In some embodiments, different protocols employ different chips and corresponding punch cards.
Some embodiments include a microfluidic chip that includes inputs and an output with channels in predefined locations for various functions. One part of the channel location is an actuator interaction region where an actuator external to the chip compresses the microchannel to propel or retard the flow of fluid in the microchannel. Some actuators include teeth (also called spokes) on a gear wheel to make contact with the channels in the actuator interaction region at a predefined height so as to push the fluids in them in each instance when engaged. The actuator in these embodiments is rotated by advancing a stiff paper punch card containing holes punched in predefined positions, so as to determine when the gear teeth are engaged thereby programming the instances when the fluid in the microfluidic channels is propelled forward. In a multi-phase set of these embodiments, the chip embodies a microfluidic design incorporating the use of T-junctions, an oil line of a predetermined flow rate, and other lines with other media so as to generate droplets which can then be manipulated as desired, corresponding to the programmed punch card.
Some embodiments disclosed here include a microfluidic movement system which is completely programmable, but requires no electricity or any other external control system. Numerous applications exist in ultra-low-cost biomedical devices, point of care diagnostic devices, research tools for automated pipetting of fluids, drug screening devices and so forth. The system is general purpose and operates in both single phase and multi-phase microfluidic modes. The system can be used in environmental monitoring applications which require conducting sophisticated molecular tests in a field setting. One or more components of the disclosed system are amenable to on-demand manufacturing and three-dimensional (3D) printing.
Some embodiments are used to manipulate fluids in innumerable ways simply by programming the same chip with a different punch card; e.g., for a new protocol. Re-programming just involves using a different punch card. The technology does not require any batteries or any other source of electricity; and thus eliminates the need for electric power. The technology does not require any external pumps to drive the fluid; since the motion of the gear train drives fluid automatically into and through the system.
The use of punched cards for creating, editing and storing computer programs continued up until the mid-1980s. However, means to use programs on punch cards to drive microfluidic channels without intervening electronic controls has not been suggested or demonstrated before.
Current approaches toward microfluidics programmability include the use of valve-based systems, such as the use of multiple micromechanical valves, each valve being separately controlled by an off-chip solenoid valve (e.g., see E. C. Jensen, B. P. Bhat and R. A. Mathies, Lab Chip, 2010, 10, 685-691). Another approach has been the use of thin film metal flexures in polydimethylsiloxane (PDMS) microfluidic devices to electronically control the opening and closing of specific valves without the use of any pressure (e.g., see M.-P. Chang and M. M. Maharbiz, Lab Chip, 2009, 9, 1274-1281). By designing the location of the valves and channels, fluid flow can be programmed.
However, the valve based systems are often single phase systems. Maintaining sample integrity in continuous flow systems has been a major challenge due to the ease with which samples that are not meant to mix can come into contact. Multi-phase systems, such as droplet-based systems, solve this problem by containing solutions of interest as emulsions in immiscible carrier fluids, thereby avoiding direct contact with device walls or with other samples until desired through specific manipulations. Multiphase systems have therefore been employed to program fluid flow using means such as the manipulation of conductive droplets using electrical means in electrowetting-on-dielectric microfluidic devices (e.g., see M. J. Jebrail and A. R. Wheeler, Curr. Opin. Chem. Biol., 2010, 14, 574-581; and S.-K. Fan, T.-H. Hsieh and D.-Y. Lin, Lab Chip, 2009, 9, 1236-1242).
Further disadvantages arise in these current systems when it comes to the limitations arising from the difficulty in integrating and miniaturizing components for increased density. Moreover, the programming process is extremely challenging, requiring exceptional knowledge and skills in microfabrication and physics for each design. Moreover, in some instances, the valves require external control for each valve leading to the need of an external controller, such as a computer, with increased complexity and cost, and often require highly trained personnel to operate.
Other approaches for controlling microfluidics mechanically include using human input such as thumb-actuated devices for pumping fluids in various compartments for storing the fluids. This approach does not allow for arbitrary control since only a limited number of inputs (two) can be given. Moreover, programmability is not possible with the above-described scheme (e.g., see X. Qiu, J. A. Thompson, Z. Chen, C. Liu, D. Chen, S. Ramprasad, M. G. Mauk, S. Ongagna, C. Barber, W. R. Abrams, D. Malamud, P. L. A. M. Corstjens; and H. H. Bau, Biomed Microdevices, 2009 December; 11[6]: 1175-1186). Furthermore, the user must be trained to perform the functions and highly practiced to perform them adeptly. The thumb-driven approach is also limited in that reagent introduction is complex and limited. Furthermore, great limitations exist in the complexity of the programs that can be run.
Moreover, most, if not all of the above listed current approaches also feature programs and chips that are tightly connected, making it practically impossible to have simple chips running complex programs. The needed supportive architecture for current complex programmable microfluidic devices includes expensive equipment, such as computers or electronic hand-held devices, and an external power supply for fluid handling and processing. All such characteristics make such systems expensive and practically prohibitive for field-based approaches in low-resource settings.
Programmability of microfluidic systems described here presents the capacity to run countless protocols through a single chip and actuator apparatus design. Illustrated embodiments present a way to program microfluidic systems using a simple chip design. Fluids can be manipulated arbitrarily by programming an external actuation apparatus that is coupled to the chip. Various embodiments use a programmable punch card coupled to a hand-powered gear train that serves as a pump to drive fluid in a microfluidic chip from outside the chip. This capacity, coupled with a droplet-based, two-phase system, also provides the possibility of reusing the same device for multiple complex protocols by simply changing the punch card code.
The illustrated embodiments provide solutions that require no electrical power, yet can perform highly complex, programmable and/or parallel fluid manipulations for various assays in simple reusable chips. The approach disclosed here embeds complexity in the punch card tape (physical program) that serves as both a program and storage register and power transmission.
Some embodiments comprise a device capable of being modified to run at very high speeds for materials processing at a high throughput. In some embodiments, the designs for microfluidic chips expand the number of programming functions available on chip. Still other embodiments comprise devices capable of being made completely disposable to allow for one time use, such as for infectious diseases related applications. Some embodiments are capable of replacing the common pipettes used in lab settings or provide the advantage of a much more precise mechanism, or some combination. Some embodiments also offer the advantage of sharing the punch card program between individuals.
New features of various embodiments include: (1) Coupling of a microfluidic chip to a punch card; (2) Actuation of fluids purely driven by interaction with a gear train eliminating any external pumps; and (3) Programmable microfluidics that can operate in harsh field settings including developing countries, hospitals, environmental monitoring, etc.
Thus, various embodiments provide a novel, elegant and a highly accurate way to manipulate fluids at microscopic length scales in a completely programmable fashion. Each protocol can be broken down into a set of instructions that are punched onto the tape and which are mechanically read by an actuator apparatus and transferred to compressions on microchannels in the microfluidic chip. This opens the possibility of lightweight, hand-held diagnostic devices that can operate in harsh settings without the need for electricity, while still providing complex fluidic manipulation of high precision useful for many applications.
An illustrated system comprises several parts: a microfluidic chip made using polydimethylsiloxane (PDMS) with various input and output channels in a predetermined configuration, a stiff paper or other material with parallel lines spaced apart running parallel to the long edge, and an actuator apparatus. The actuator apparatus includes a bedplate metal support structure on which actuators and the supporting pieces are fastened. Example actuators include gear wheels with a cylinder or shaft having rotatably connected metallic disks each with teeth that are spaced circumferentially, or cams that move one or more pins up and down under a restoring force such as gravity or shape memory material or an external spring. In some embodiments, the teeth are spaced equally around the circumference. In various embodiments, various numbers of teeth are included on each actuator. In various embodiments, the teeth are shaped for various purposes, such as to engage the punch feature, compress channels, close channels like a valve or puncture a capsule in a punch feature or some combination. In some embodiments, different actuators in different rows have different numbers and types of teeth or different sizes.
In an illustrated embodiment, a stiff paper card is punched with holes, or other features, such as bumps or depressions, on the lines, which are aligned to the teeth on an actuator (such as a cylinder with multiple gears) and about two millimeters apart in an illustrated embodiment. In other embodiments, other spacings between lines are used. The spacings between the actuators in several illustrated embodiments are created using toroid discs made of plastic that are one and a half millimeters thick and are completely replaceable and adjustable. It is anticipated that thinner spacings are used in some embodiments to provide more actuators and therefore more channels for a given area. The opposite would also be true that is, if thicker spacings are used, fewer actuators would fit and subsequently fewer channels would be actuated for a given area. More closely packed actuators and channels at some point eventually lead to more than one channel being perturbed or actuated by an actuator, which may or may not be desirable depending on the application. The point where more than a single channel is perturbed by the action of a single actuator also depends on the thickness of the tooth on the actuator. Thinner teeth result in a lower likelihood of neighboring channels being perturbed for a particular density.
An illustrated embodiment features actuators with thicknesses ranging from 0.5 mm to 1 mm, spaced 2 mm apart. The actuator thickness in this embodiment is such that it is slightly larger than the channel being actuated to ensure that channel collapse is substantively complete. The thickness of the teeth or pins can also be increased or decreased arbitrarily compared to the thickness of the actuator. The limits for thin teeth or pins is determined partly by the material being used and its interaction with the material of the microchip and the size of microchip channels and substrates; if too thin, the tooth or pin can cut though the material which may be undesirable for channels that need to be intact for correct functioning.
Upon encountering a punch feature on the punch card, a tooth of the actuator gets engaged and rotates the actuator, pressing another tooth of the actuator, or a pin motivated by the tooth, to compress a microchannel in the chip. The other actuators are not engaged (unless there is a feature on the punch card on the particular line for another actuator at the same distance on the punch card). As one of the teeth on the actuator engages the feature, another tooth on the actuator, e.g., a tooth 180 degrees from the tooth engaging the punch feature, compresses the microfluidic channel on the chip mounted on another side of the actuators. The microchannels are aligned with the actuators' teeth so that the microchannel can be compressed every time the punch card has a feature at the particular line and distance. The distance along the length of the punch card corresponds to a sequence step or time.
In some embodiments, the microfluidic channels include at least one microchannel with a carrier fluid flowing at a predetermined flow rate, e.g., because of regular compression by one or more actuators due to regular spacing of features on the punch card. This carrier fluid is immiscible with the fluids in other channels. The input channels are designed in such a way as to have a T-junction with a microchannel carrying the immiscible fluid. For example, the input channels carry a water-based fluid while the immiscible carrier fluid is oil. The design is such that droplets can be generated at the T-junction through the shearing of the fluid whenever the tooth of an actuator pushes the fluid forward in a pulsatile way.
Fluids in the various droplets are manipulated as desired through various designs in the microfluidic chip, including but not limited to ways of passively mixing the fluids in any order in a reaction chamber or laminar flow channel or merging various fluid droplets in the reaction chamber as desired for various applications.
In the illustrated embodiment, the chip is made of polydimethylsiloxane (PDMS), which is inert, flexible at certain thicknesses, and transparent so that fluid reactions can be observed visually. In some embodiments, material coatings are employed to avoid or reduce wear and tear, including polymer coating to match material stiffness/hardness. An example of such an approach is the coating of actuator teeth in a polymer or material that will match the microchip stiffness/hardness. In some embodiments, mechanisms are included to reduce friction, such as timed release of a friction-reducing agent; or the use of other materials for the microchip, the actuator or both, leading to reduced friction. The channels can be formed in various ways in the material of the chip, in one block or multiple blocks that are then glued or melted together, such as by machining each block of material, etching each block, using a mold for one or more blocks, or injection molding one or more blocks, or by 3D printing with the chip material.
One wall of the channel 110 is a flexible substrate 222 (such as a thin PDMS substrate about 200 microns thick) that flexes when pressure is applied, e.g., by one or more teeth (such as tooth 232) of actuator 230. The shape of the teeth 232 varies in various embodiments, as described in more detail below. The opposite wall of the channel 110 is an opposite substrate 224. In some embodiments, the opposite substrate 224 is stiff (such as a thick PDMS substrate about 10 mm thick) and does not flex under the pressures applied by the actuator 230. In some embodiments the opposite substrate 224 is also flexible and also flexes to the same or lesser degree under pressure.
An illustrated embodiment comprises a flexible substrate of a soft elastic PDMS layer made of a base-to-crosslinker ratio of 20:1, which is about 200 microns thick. This thin PDMS substrate was made using a spin-coater such as the P6700 spin-coater from Specialty Coating systems based in Indianapolis, Ind., with the settings of 250 rotations per minute angular velocity for 30 seconds, before being cured at an oven set to 80 degrees Celsius for at least 30 minutes. This thin substrate has an elastic modulus of about 1.1 MPa. This flexible substrate provides the outside wall of channels with cross sections from about 250 microns to about 500 microns wide and height h 112 about 50 microns to about 100 microns. In some embodiments, the outer flexible substrate comprises a layer of polydimethylsiloxane (PDMS) less than about 50 microns thick, which provides sufficient strength to prevent rupture and sufficient flexibility to be easily deformed.
The opposite substrate is a stiffer, thicker PDMS layer of about 10 mm thick made of a base-to-crosslinker ratio of 5:1, with an elastic modulus of about 3.5 MPa. Other materials that would work include wood, glass, polymers such as 75-80 RTV urethane mold rubber available from West Palm Beach, Fla., polymers such as Delrin® manufactured by DuPont in Wilmington, plastics, metal, foam, or any other material that would be thick enough to ensure that the PDMS microchannel layer makes contact with the gear pins.
The glass slide 210 provides a stiff substrate for attachment to the actuator apparatus support structure 240 that houses the actuators and punch card guide. In some embodiments, opposite the outer flexible substrate of each microfluidic channel in the actuator interaction region is a layer of polydimethylsiloxane (PDMS) greater than about 50 microns thick. In various other embodiments, the thickness is selected within a range from about 50 microns to about 200 microns.
The chip with channel 110 is inserted into a recess in the actuator apparatus support structure 240. In some embodiments, the chip 100 is mounted to a glass slide 210 which is used to manipulate the chip 100 into the recess of the support structure 240, e.g., by resting the glass slide on top of the support structure 240. In some embodiments, the chip is removably inserted into the recess so that the same apparatus can be used with different chips of the same or different arrangement of channels, reservoirs and reaction chambers. Thus, in some embodiments, the recess is configured for removably receiving the chip with the plurality of microfluidic channels. In some embodiments, the chip is not removably inserted into the recess; but, in contrast, is permanently fixed to the actuation apparatus. Thus, in some embodiments, the chip with the plurality of microfluidic channels is fixed within the recess.
Mounted to the support structure 240, through a connection not in the plane depicted, is a shaft 242 (e.g., a cylindrical piece of metal). Rotating around shaft 242 is actuator 230 with teeth (also called pins) such as teeth 232. As described in more detail below, the teeth 232 can assume various shapes to achieve various functions. Any material can be used to make the actuators and teeth. In the illustrated embodiments, the thin actuators, about 0.5 mm thick, were made of stainless steel, while thicker actuators, about 1 mm thick, were made of aluminum. In some embodiments, the actuator is fabricated using other metals, or plastics or other materials that have less frictional action on the chip, such as wood of various kinds that is smoothened, polished or coated with a resin such as 75-80 RTV urethane mold rubber available from West Palm Beach, Fla., polymers such as Delrin® manufactured by DuPont in Wilmington, Del., ceramic such as porcelain, glass, sea shells, bone, enamel, polished rocks and other similar hard yet smooth materials.
Also mounted to the support structure 240, through a connection not in the plane depicted, are opposite members of a pair of punch card guides 244a and 244b, respectively (collectively referenced as punch card guides 244). The guides 244 are configured to hold a punch card 270 so that the punch card 270 contacts or nearly contacts the actuator 230.
As the punch card moves in the direction 274 with a tooth of actuator 230 engaged in a hole 272 or other punch feature of the punch card 270, the actuator 230 rotates in the actuator rotation direction ω280. This movement applies pressure on flexible substrate 222 by a tooth along the actuator (e.g., on the opposite side of the actuator) and propels some fluid in the channel 110 into the downstream fluid direction 282 along the fluid channel. The portion of chip 100 with a flexible substrate 222 for interaction with the teeth 232 of actuators 230 is called the actuator interaction region. In some embodiments, the actuators operate in multiple-modes whereby the movement of a particular actuator results in the movement of one or more gears connected to the actuator, which may or may not move multiple other actuators. With this capacity, increased complexity and functionality can be achieved.
In some embodiments, one side of the punch card guide is provided by another component (e.g., punch card guide 244b is actuator 230 or shaft 242 or a cam without teeth, not shown, attached to the shaft 242). In some embodiments, manually pulling the punch card 270 provides the power to rotate the actuator 230 in direction ω280 and propel the fluid into direction 282. In some embodiments, the punch card is advanced by turning a crank attached to one or more rollers serving as punch card guide 244. In some embodiments, the punch card 270 is advanced by turning a crank attached to the shaft 242 that is permanently engaged with at least one actuator corresponding to a row on a punch card with punch features at regular spacing. In such embodiments, turning the crank advances the punch card, and other punch features in the punch card variously engage the other actuators rotatably connected to the shaft 242. Thus, in some embodiments, an actuator of the plurality of actuators is rotated by manually advancing a punch card through the punch card guide when a tooth of the actuator is engaged with a punch feature of the plurality of punched features on the punch card. In some such embodiments, no actuator of the plurality of actuators is rotated by an electric motor.
Thus, the actuation apparatus includes a support structure 240, a plurality of actuators 230, and a punch card guide 244.
In the illustrated embodiment, there are 15 actuators evenly distributed on shaft 342. Thus, in this embodiment, each actuator of the plurality of actuators is rotatably connected to a single shaft connected to the support structure. In various other embodiments, more or fewer actuators are evenly or unevenly distributed on one or more shafts or cylinders. For example, in various embodiments, the apparatus includes a number of actuators in a range from about 8 to about 32. In various embodiments, the number of actuators can be in the hundreds, thousands or even millions. An example of where multiple ones could be an advantage is in genomic sequencing or the running to large arrays of assays needing many systems in parallel. An advantage of evenly distributed actuators is simplicity of design for a variety of chips and simplicity of providing punch cards with punch features. An advantage of uneven distribution is that one or more actuators of different widths can be employed to propel different amounts of fluid through the microchannels of corresponding widths on the chip. In some embodiments, spacing between a pair of actuators of the plurality of actuators along the support structure is in a range from about 20 microns to about 2000 microns.
In the illustrated embodiment, the recess 312 is shaped so that the chip 100 fits inside in only one orientation to ensure that the chip 100 is properly disposed in the recess 312 so that the microchannels in an actuation interaction region of chip 100 align with the actuators 330. Thus, the recess is configured for removably receiving the chip with the plurality of microfluidic channels.
Thus, an actuation apparatus includes a support structure 240, 340 including a recess 312 configured for receiving a chip 100 with a plurality of microfluidic channels 110. The apparatus also includes a plurality of actuators 230, 330 rotatably connected to the support structure; and, each actuator includes a plurality of teeth 232, 332 protruding outward from the actuator. Each actuator of the plurality of actuators is positioned so that, at least in some angle of rotation, a tooth of the actuator extends into the recess 312 sufficiently to compress a microfluidic channel 110 in a chip 100 placed in the recess 312. The apparatus also includes a punch card guide 244, 344 connected to the support structure 240, 310, respectively, and configured to guide a punch card 270, 370, respectively, with a plurality of punched features 372 (such as holes 272) so that at least in some angle of rotation a tooth of an actuator of the plurality of actuators extends into the punch card guide sufficiently to engage a punched feature on a punch card disposed in the punch card guide.
In the illustrated embodiment, actuator apparatus 300 includes a manual crank 350 mechanically connected by gears 314 to roller 340 and shaft 342, to advance the punch card 270. Because at least some of the actuators 330 are rotatably connected to the shaft 342, the rotation of shaft 342 can assist in the advancing of punch card 270 without rotating the actuators 330. In other embodiments, the manual crank 350 is mechanically connected to the roller 340 or other rollers that are part of the punch card guide 244, but not to the shaft 342. Thus, in some embodiments, the punch card guide comprises a pair of rollers and a crank, and at least one roller of the pair of rollers is mechanically connected to the crank such that the punch card is advanced when the crank is rotated. In some embodiments, the crank is rotated manually. In some other embodiments, the manual crank is replaced by a motor. In still other embodiments, the manual crank and motor are omitted; and, the apparatus 300 is operated by an operator manually pulling on the punch card to advance the punch card.
In some embodiments, fluid in one microchannel, such as the immiscible fluid in microchannel 120, is propelled at each step and so an actuator corresponding to that microchannel rotates at every step, as indicated by a feature 372 (such as hole 272) at a regular interval along the punch card 270, 370 (the regular interval corresponding to the distance between teeth on the actuator). In some of these embodiments, the actuator for the immiscible fluid channel is fixed to the shaft 342 and mechanically connected to the crank 350 or other handle.
In some embodiments, a spring or other component is included to store the mechanical energy produced by turning the crank, and the card is advanced by the unwinding of the spring or mechanical release of the other component. In some embodiments, the rate of mechanical release is controlled so that the card is advanced at a rate prescribed for one or more protocols. In some embodiments the punch card is advanced by an electronic motor powered by power line or battery or solar panel or electric generator or some other electric power source or by a different kind of motor powered directly by water or wind or geothermal or other energy source.
The reservoir 412 is configured for holding a fluid used in the protocol, including the immiscible carrier fluid, such as oil, for a two-phase protocol. In some embodiments, the chip is single-use; the reservoir is supplied with the fluid and the reservoir is sealed. In other embodiments, the chip is reused or reusable, and the chip 410 includes a fill port 414. In some embodiments, a stopper 415 is placed in the fill port 414 after the reservoir 412 is filled to keep the fluid within the reservoir 412. In some embodiments, the orientation of the chip 410 in use is such that gravity keeps the fluid in the reservoir 412 without the stopper 415, and the stopper 415 is omitted. In some embodiments, described in more detail below, one or more fluids for the protocol that uses chip 410 are provided in a capsule of the punch tape, and the reservoir 412 is omitted for one or more of the microchannels 420 in chip 410. Instead, fluid is transferred from the punch card 430, such as optional reagent bearing upper punch card 440, in the actuator interaction region 422 of the microchannel 420. In some embodiments, the reservoir is used in conjunction with the transfer of material from the punch card 430, for example a powder from the punch card is added to water or other solvent in the reservoir 412. In some embodiments, port 414 is connected to an external source of fluid, such as tubing, or to an external reservoir. In some of these embodiments, the reservoir 412 is omitted.
In various embodiments, one or more microchannels 420 converge into a reaction chamber 424 where fluids propelled in one or more actuator interaction regions react to provide a result 426 (also called a product) of the protocol. For example, in some embodiments, the result 426 of the reaction is a temperature change or a color change or change in composition or some other physical manifestation. In some embodiments, that result travels through the outflow port 428, with or without the carrier fluid, if any, and is detected by a user, either manually (e.g., by visual appearance, taste, odor, or texture, or some combination) or with the aid of some appropriate detector, such as a thermometer. For example, a fluid or solid of a particular color visible at the outflow port 428 indicates one of two or more possible results of the protocol, each different result indicated by a different color. In some embodiments, the result is deposited on the punch card 430 that is brought into contact with the reaction chamber 424 or outflow 428 by the punch card guide 244. For example a stain on the punch card 430 of a particular color indicates one of two or more possible results of the protocol, each different result indicated by a different colored stain. In
Thus, a microfluidic channel chip includes a body structure having formed therein a plurality of microfluidic channels in an actuator interaction region, a reaction chamber and a reservoir. Each microfluidic channel in the actuator interaction region has at least one dimension in a size range from about 0.1 micron to about 1000 microns, and is separated from an outside of the body structure by an outer flexible substrate. The reaction chamber is in fluid communication with a first end of each of the plurality of microfluidic channels in the actuator interaction region. The reservoir is configured to hold a supply of a fluid in fluid communication with a second end opposite the first end of at least one channel of the plurality of microfluidic channels in the actuator interaction region.
Initial multi-phase flow experiments with the illustrated embodiment have demonstrated the successful generation of droplets ranging from five to 20 nanoliters.
Various actuator and tooth shapes are used in various embodiments.
The eight evenly distributed teeth provide 45 degree symmetry.
In such embodiments, the pin 1192 is a tooth of a compound or multi-moded actuator that includes both the original actuator 1130 and cantilever 1190 with pin 1192.
Various actuator shapes and other ways to mechanically drive the fluid were explored, such as the up and down motion of pins to produce different flow patterns and ways of manipulating fluids to a desired effect. This allows the actuator to be used both for pumping and/or valving. When used for valving, the actuator valves can be either in “always ON” or “always OFF” state, switched for example by pins contacting gears as depicted in
Various size actuators and teeth are used in various embodiments. Ranges of actuator widths for various embodiments were described above. In various embodiments, the diameter of actuators vary from a smaller diameter of about 3 mm, which translates to lesser material for the thick substrate (thickness will reduce by the same order of magnitude) and also a shorter distance between the microchannel and the punch card tape (which will make it easier to transfer material into and out of the chip). A larger diameter of about 16 mm makes it possible to get many more pulses with each cranking action as you can fit more gear teeth. In some embodiments, teeth with variations in height are used on the same actuator (e.g., alternating teeth with about a 30 micron height difference, such as the second, fourth, sixth and eighth teeth having a height of about 1500 microns from an axis of rotation while the 1st, 3rd, 5th and 7th teeth having a height of about 1520 microns). This enables having fluid in some channels propelled with less perturbation than others, leading to variation in fluid dispensed. As determined above, there is a regime in which the channel collapse on actuation is not complete yet at least some fluid is still propelled.
In some embodiments, the actuator diameter and teeth height is selected to match a microchannel height plus thickness of the flexible substrate to fully motivate the fluid in the microchannel. In some embodiments, the actuator diameter and teeth height is selected to only partially penetrate the microchannel to allow for some backflow and mixing. In some embodiments, the actuator diameter and teeth height is selected to exceed the microchannel height to compress the opposite substrate or compress other microchannels farther from the outermost microchannel. In some embodiments, teeth of different heights are used on the same actuator. In some embodiments, a width of the tooth of the actuator is in a range from about 10 microns to about 1000 microns; and, spacing between a pair of actuators of the plurality of actuators along the support structure is in a range from about 20 microns to about 2000 microns. In various embodiments, a tooth of an actuator of the plurality of actuators protrudes outward from the actuator by a distance in a range from about one micron to about 100 microns to partially or completely fill the height of a microchannel in the actuator interaction region.
An analytical model was constructed for single phase fluid experiments of an elastomer coupling to a mechanical gear reflecting the mode of operation of the illustrated device embodiment.
L=(g2−(g−h1)2)1/2 (1).
The total distance that the tooth travels in the channel 1010 is 2L.
V=2L×y=2xy(g2−(g−h1)2)1/2 (2)
From this model, fluid velocity was calculated and confirmed for different actuation positions. The model includes operating parameters such as rotational velocity of the gear, elastic modulus of the polymer, and geometrical coupling parameters. In one mode of operation, a pinch in a channel travels along the direction of the channel as shown in
Measurements were made for two phase embodiments as well.
In some embodiments, the device includes the capacity to multiplex through having multiple layers being actuated simultaneously.
In the illustrated arrangement, the tooth 1932 of actuator 1930 extends through the first microchannel, e.g., with an actuation height h1 greater than the channel height h. This causes the second flexible substrate 1922b to deflect into the second microchannel 1910b and squeeze a volume of fluid in that microchannel as well. The actuation height in the second microchannel 1910 is less than in the first layer, but enough to increase the pressure on, and at least slightly deform, the third flexible substrate 1922c into the third microchannel 1910c. This squeezes a volume of fluid in the third microchannel 1910c as well. With this capacity, multiple experiments can be carried out simultaneously with the possibility of either merging or not merging the various layers downstream in various embodiments. Thus, in some embodiments, the chip body structure further has formed therein a different microfluidic channel separated from at least one channel of the plurality of microfluidic channels in the actuator interaction region by a different second flexible substrate in an opposite substrate of the at least one channel opposite the flexible substrate.
Such correspondence has been measured.
Similarly,
Correspondence also occurs for single phase microfluidics.
Droplets of a single reagent are formed in chip 2300 at a channel step comprising two oil channels and a water-based reagent channel.
In some embodiments, the reagents are encapsulated in the punch card material and introduced into the device, as liquid or powder or solid. Several ways are illustrated for introducing reagents contained in paper tape into the chip. In a first embodiment, a needle pushes through the paper which has reagents embedded in it to enable access of the same. The tape then passes by an open fluidic channel to allow the reagents to diffuse into the channel as another tooth presses on the same site to make fluid contact without leakage out of the microchannel. Thus dried or semi-dried reagents can be supplied to the chip on demand (as per the protocol). The same mechanism that is used to introduce reagents into the chip can also be used to introduce biological samples and patient body fluids. Thus multiplexing can be achieved by collecting and running assays for multiple patients simultaneously by introducing samples in different code lanes. Furthermore, multiple tests can be run on multiple patient samples by changing the protocol code in the punch card containing the samples from a given patient.
In a different embodiment, reagents are enclosed in capsules on the punch card tape coupled to device inlets. The side of the capsule facing the chip inlet has a thin film and is flush with the rest of the punch card with the capsule projecting outward toward the microchannels on the chip. The chip inlet features a concave shape and size that matches that of the capsule projecting away from the punch card surface. Upon activation, a compressor such as a gear pin pushes on the capsule from the bottom, leading to the rupture of the thin film and the release of the reagents into the microchannel. In some embodiments, the inlet channel then also features valves to ensure that flow is unidirectional.
In various embodiments, reagent or result storage in the system can occur either within the microchip, or in an external reservoir coupled to the microchip, or within the punch card tape or some combination. Some embodiments use bubble-capsules which are pouches or capsules on the punch card in which the reagents are stored. The reagents or materials are in liquid, solid or gaseous form and are released in a timely fashion depending on the assay or application in question In some embodiments, lyophilized reagents are stored in the punch card tape. This can be achieved either as part of sections on the punch card, strips on the punch card, or specific pouches for which controlled or uncontrolled release is achieved.
In another approach, reagents in the punch card tape are introduced through a porous substrate. In this mode, the fluid introduction is by diffusion into the microfluidic chip via the porous substrate.
Transfer of media/material into the chip from the punch card can occur in a variety of ways, in various embodiments, such as introducing material into the microchip using a sharp object or, using holes that are of dimensions that are below the capillary length scale of the fluid, or through porous structures. In some embodiments, described above, bubble capsules are disposed on the punch card tape, and their contents released in specific instances. In some embodiments, communication between the punch card and the microchip is effected using non-rotational mechanisms such as vertical pins and gates.
As the two layer punch card 2576 advances in direction 2571, the separator 2546 causes the upper layer 2576a to pass between the actuator 2430 and the chip 2501, while the lower layer 2576b passes the actuator on a side opposite to the chip 2501. The punch features on the lower layer 2576b act as described above to engage one tooth of the actuator and rotate the actuator. This action presses another tooth of the actuator against the chip 2501. With the upper layer 2576a now disposed between the actuator 230 and the chip 2501, the tooth also presses a capsule, if present, in the upper layer against the import portion 2518 of the microchannel. The import portion is configured to pass material from the capsule 2578 into the microchannel 2510. The material forms a droplet in the microchannel 2510; and the droplet is propelled downstream in flow direction 2511 forced by compression of the microchannel 2510 by the same or different actuator. Thus, the punch card guide further comprises a separator configured to separate an upper layer of a punch card, if present, from a lower layer of the punch card and pass the upper layer of the punch card between the plurality of actuators and a chip disposed in the recess.
As shown in the side view, there is a punch feature synchronized with every capsule, to press the capsule against the import portion. In the illustrated embodiment, the punch feature of the lower layer is collocated just below the capsule; but, in other embodiments, the punch feature may lead or follow the capsule. In the illustrated embodiment, the capsules are disposed in the first two lines of the punch card, as evident by the grayscale features in the top view of the punch card 2576, and microchannels with import portions are confined to those microchannels that are aligned with these first two actuators. Thus, in some embodiments, a punch card medium includes a sheet of a first material on which is formed a plurality of punch features, each punch feature configured to engage a tooth extending from an actuator of a plurality of actuators in an apparatus; and, a different second material encapsulated in the sheet at a location of at least one punch feature and configured to be released by an encounter with the tooth of the actuator. In the illustrated embodiment, the sheet of first material comprises a first sheet of the first material and a second sheet of a third material, wherein the second material is disposed in the second sheet. In some of these embodiments, the third material is a plastic material, such as Mylar. In some embodiments, the third material is the first material, e.g., both are Mylar. In some embodiments, the third material is different from the first material, e.g., the first material is stiff paper and the third material is Mylar.
In some embodiments, the results in the reaction chamber is transferred to the punch card 2576. For example, one or more layers of the punch card are caused by the punch guide to contact the reaction chamber at an export portion 2519, as indicated by the dashed line labeled upper layer 2576c. The export portion 2519 is configured to pass material from the reaction chamber to the punch card, e.g., by diffusion, capillary action or gravity through a port or through a porous, permeable or semipermeable membrane. Results from assays deposited on the punch card can take any form, such as outputs from colorimetric assays, agglutinations from chemical reactions or other forms that may or may not be visual. This capacity enables the ease of results storage. In some embodiments, transmission with the option of further processing such as DNA extraction (and other processes) is made possible by sending the punch card to a processing center after the run.
Thus, in the illustrated embodiment, the interface to the microchannels includes a mechanism to rupture the capsule holding the material to be introduced and provide a way for the material to get to the microchannel. In the illustrated embodiment, a sharp needle or a pin with a hollow center is used. This is then connected to an import channel 2523 that splits into (is in fluid communication with) two other channels, one of which is a chamber that has an absorbent material 2524 that can wick liquids, and the other being the microchannel 2510 that is actuated by the tooth of a actuator to propel the absorbed/wicked material downstream for further processing. An example of the wicking material 2524 is cellulose fiber pads made by Millipore in Billerica, Mass. The needle that serves as capillary tube 2528 and that ruptures the capsule holding the material to be introduced, in various embodiments, features either a beveled edge or a square cut to allow for air to break the fluid interface and leading to capillary pinning of the liquid so that not all the liquid is wicked into the absorbent pad. The microchip interface also features a shape that will accommodate the capsule and be strong enough to withstand the pressure of the actuation pin.
Punched holes on the protocol layer result in the engagement of the actuator teeth which makes them rotate. This rotation also leads to the rotation of another tooth that interfaces with a lever arm upon which the input punch pin is connected and actuated via a seesaw mechanism. The mechanism is such that the downward motion of the lever arm leads to the upward motion of the input punch pin which then presses the material in the capsule against the import port. The import port features a needle (or pin with a hollow center) that ruptures this capsule to allow for the access of the material into the device. After actuation, the lever arm goes back to its default position in which the punch pin is lowered and thus disengaged from the import port.
This action is illustrated in perspective detail in the next three figures.
Configurations of capsules on the punch card are illustrated in the next four figures.
In some embodiments, the sample or reagent is collected on site and not prepackaged in a capsule. In some of these embodiments, the sample/reagent is collected into one or more open top features, called dimples. In some embodiments an absorbent material is included in the dimple to hold the sample or reagent.
Thus, in these illustrated embodiments, the punch card tape features two layers—one of which being the layer holding the material to be injected into the device; the other being the protocol layer for triggering the material insertion process. The two layers can be separated in the middle to allow for the top layer (carrying the material to be inserted) to interface with the microfluidic chip while the lower layer has punched holes in instances where actuation is desired. The spacing of the punched holes and the material encapsulation is such that it will ensure that there is perfect alignment between the input port, the material to be inserted and the actuator for the insertion.
The material to be inserted is encapsulated in a medium that allows for rupture on the side interfacing with the microchip interface and a firm substrate on the side interfacing with the actuator that will push it against the microchip interface. An example of such a medium is a blister pack that is used in packaging of medical tablets. Some embodiments use aluminum foil (or other materials such as polythene) on one side to allow for easy rupture and a polymer (such as polyvinyl chloride, polychlorotrifluoro ethylene, cyclic olefin copolymers or other firm substrates including metals, paper or plastics) on the other to provide a firm substrate against which the actuator can press. The blisters can be manufactured by a company such as Visipak from Fenton, Mo. Moreover, the encapsulated material in the blister pack (or other holding mechanism) is such that it can be detached from the punch card tape material layer in one embodiment, or stay in place in another implementation for multiple insertion instances. In various embodiments, the location of the encapsulated material is anywhere along the punch card tape to allow for the introduction of the material as desired.
The punch card features the upper layer with the encapsulated material and another lower layer with, for example, punch card holes for actuation purposes used for the system. The presence or absence of holes in this second layer determines when the material is introduced. In this embodiment, the presence of a hole results in the immediate or subsequent introduction of the material into the microchip,
Such embodiments provide advantages over current methods that utilize microfluidic techniques, which have to interface chemical supplies to microfluidic chips externally. This requires infrastructure outside the chip to load reagents in and out. With the capacity to load reagents via the same punch tape that loads protocol information, some embodiments omit one or amore or all of the external reservoirs. Such elimination makes the entire platform compact and truly hand-held.
Thus, the microchip that interfaces with the actuators is tilted at an angle that is between zero and ninety degrees. The tilting is such that for a four-toothed actuator, with the channels in the microchip aligned to the actuator, the tooth closest to the microchannel impinges on the microchannel; thereby inducing a valving mechanism. At the neutral/default actuator position (not engaged with a punch feature, as depicted in
A different approach is used to valve each channel individually.
Thus, a valve is moveably connected to the support structure. The valve includes a pin configured to extend into the recess sufficiently to close a microfluidic channel in the chip placed in the recess when no tooth of the plurality of teeth on an actuator is in contact with the valve and configured to withdraw from the recess when a tooth of the actuator is in contact with the valve. This valving mechanism uses pin-like projections or pins attached to individual cantilevers. The neutral position is such that the pins are aligned to microchannels and impinge upon them to obstruct flow. Complete closure of the channel with the pin is achieved through this action and, in some embodiments, the introduction of a step elevation in the microchannel at the point where the valving pin makes contact with the channel to reduce the channel height and makes it easier to seal the channel shut. The cantilevers are plucked upon the actuation of the actuator tooth with a punch feature on the punch card. The downward motion of the cantilever upon plucking causes the pin to move down and forward thereby lowering the pin and opening the channel to let out the fluid being pumped. Immediately after this action, the valve goes back to its default position thereby closing the channel.
With the above described action, the default is an valve on position leading to the closure of the microchannel. In some embodiments, a valve in the default position is off, e.g., through the use of a pulley mechanism, flexures, or coupling to other valves or structures.
In various embodiments, any method may be utilized to fabricate the component parts, such as 3D printing in part or as a single unit, machined, bought and inserted as part of the machining or 3D printed part (e.g. dowel pins from PIC designs based in Middlebury, Conn., or other vendors), ranging in diameter from 1 mm to 3 mm made of either 416 stainless steel, carbon steel or 303 stainless steel (the advantage being corrosion resistance and strength at small diameters) to act as valves 2900 and the fixed pivot 2912 shown in the diagram.
Method 3100 includes steps 3110 through 3141. In step 3110, an apparatus is obtained with a plurality of actuators rotatably connected to a support structure with a recess for receiving a chip and with a punch card guide. Each actuator includes a multiple teeth configured to reach into the recess at a first rotation angle of the actuator and reach into the punch card guide at a second rotation angle. In some embodiments, step 3110 includes fabricating the apparatus.
In step 3120, a chip is formed to fit into the recess. The chip includes microchannels configured for compression in an actuator interaction region reachable by teeth of the actuators, and one or more reservoirs upstream of the actuator interaction region, and a reaction chamber downstream of the actuator interaction region. In some embodiments, the chip includes an optional import portion in the actuator interaction region for importing material from a capsule pressed against the import portion. In some embodiments, step 3120 includes fabricating the chip.
In step 3129, fluid is added to one or more reservoirs in the chip. For example, an oil reservoir is filled with oil and one or more reagent reservoirs are filled with solutions of the corresponding reagents.
In step 3131, a punch card is formed with punch features (such as holes) spaced apart to engage teeth on at least two actuators of the plurality of actuators to rotate those actuators and thus propel fluid in the corresponding microchannels of the chip. In various embodiments, the punch card has multiple coding schemes to correspond to different steps of the process or purposes of the platform. For instance, one code corresponds to the engagement of gears to perform a certain logical operation, another code corresponds to opening and closing one or several valves, and another code corresponds to engaging a single phase in lieu of multiple-phase flow, and still other code corresponds to either the uptake of new material (either from reservoirs and or external sources) or the deposit of processed material/results for storage or additional processing. In some embodiments information is stored either in the form of assay products, color changes, numerical revelations corresponding to particular results, the outputs of a particular stage in the assay or other products. The information is also stored in the form of clinical samples or other samples introduced in the device for additional processing, use, recording keeping or other forms of manipulation.
In step 3139, one or more material filled capsules are embedded in the punch card, such as in an upper layer of the punch card.
In step 3141, the apparatus or chips or corresponding punch cards or some combination are distributed, such as in one or more kits. For example, in some embodiments, a kit includes a chip and a punch card. The chip comprising a plurality of microfluidic channels in an actuator interaction region separated from an outside of the chip by an outer flexible substrate. The punch card comprising a sheet of a first card material on which is formed a plurality of punch features, each punch feature configured to engage a tooth of an actuator of a plurality of actuators in an apparatus, wherein each actuator is configured to compress a corresponding microfluidic channel of the plurality of microfluidic channels in the actuator interaction region.
Method 3150 of
Thus, in some embodiments, a method includes obtaining an apparatus comprising an actuator rotatably connected to a support structure, wherein the actuator comprises a plurality of teeth, one tooth on the actuator configured to compress a microfluidic channel in a chip when another tooth on the actuator is engaged in a punch feature in a punch card. The method also includes receiving a chip and a punch card for the chip. The chip includes a microfluidic channel separated from outside the chip by a flexible substrate in an actuator interaction region. The punch card includes a sheet comprising a plurality of punch features configured to engage a tooth of the plurality of teeth of the actuator. The method further includes contacting a sample to at least one of a capsule on the punch card, or a reservoir in fluid communication with the microfluidic channel in the chip. The method also includes advancing the punch card through the apparatus in contact with the actuator to cause a first tooth of the actuator to compress the microfluidic channel when a second tooth of the actuator engages a punch feature on the punch card.
The system is rugged and portable (currently, the actuation apparatus is about the size of an apple), making it possible for one to operate it in extremely low-resource settings yet have the robustness of a laboratory with multiple equipment. In various embodiments, the chip is made of different materials (including rubber and various polymers such as PDMS). In some embodiments, the chip is disposable while in other embodiments the chips are non-disposable or reusable. In some embodiments a single actuation apparatus is used for either disposable or non-disposable chips.
Thus, presented above is a programmable microfluidic system where the program is encoded in a punch card. The punched card also serves as a storage location of both wet and dry reagents and biological samples including patient samples. Some embodiments also employ the same punched card as a way to indicate results and store them temporarily. The punch card therefore serves multiple purposes of programming information in a spatial and temporal fashion, introducing reagents and biological samples, indicating results and storing them. This multiple use of an information register (punch card) to carry both information and materials has never been demonstrated before to the knowledge of the inventors.
Different protocols are run on different lines, in some embodiments. The multiplexing of runs enables the simultaneous processing of multiple experiments on a single sample (e.g. snake venom screens, newborn screening assays for inborn errors of metabolism or genetic mutations, screening various pathogens simultaneously on a single sample) or multiple samples for a single process (e.g. massively parallel diagnosis of diseases or medical conditions for multiple patients), which can be particularly handy in triage conditions following disease outbreaks or other similar conditions where a quick result is needed for multiple patients.
The entire system can be put in an incubator or other controlled environments simplifying processes such as the culturing of pathogens in patients samples or other ways to amplify the signal or as steps in a protocol. Assays such as those for the diagnosis of infectious diseases (that require multiple steps, incubation, etc.) or the determination of minimum inhibitory concentration of an antibiotic, to both determine the resistance of a microbe to the antibiotic or test out new drugs, are some of the potential applications of embodiments of the device. Moreover, the illustrated embodiments can potentially be used for the screening of newborns for multiple disease conditions that are treatable but not evident clinically—which can lead to many lives being saved as such programs are often non-existent in low resource settings. Other applications include the parallel testing of many chemotherapeutic agents against a particular cancer to determine the most effective medication or combination of medications for a particular case; drug discovery; screening pregnant women for common health risk factors; quick determination of the right antivenom for snake bites by parallel processing; and determining the best approach in treating patients with comorbidities, especially those that are immune-compromised, through the parallel processing of biological samples.
A variety of applications also exist outside the biomedical realm. Potential applications outside of medicine include programmable messages or pictorial displays, hydrocarbon sampling tests in oil discovery, testing soils samples for agricultural purposes, parallel processing of water samples for various contaminants in the environment and others. Using various embodiments, a moving display of text, numbers, patterns or various images can be made by punching holes to program the fluid flow on the microfluidic chip. Multiple channels can then be simultaneously controlled in this way. The display can be on the chip itself enabling the transmission of messages as the fluid is driven by hand. By merging or layering the output from multiple device layers complex images, colors and patterns can be displayed. Reagents or other solutions can be introduced arbitrarily either in the tape or via reservoirs located either within the thick substrate of the device or external to it.
In an example embodiment, an assay provides for the parallel staining of cells with different fluorescent markers for disease diagnosis. For instance, parallel chambers are chemically functionalized to have chemical tags such as avidin bound to the surface of the device. A biological fluid sample such as blood or cerebro-spinal fluid is introduced after the surface markers of interest in the cell have been tagged with a molecule such as biotin that attaches the cell to the surface of the device chamber. With the cells immobilized, different stains are introduced in different chambers at the exact sequence required, all in parallel complete with all the rinsing, incubation and staining steps. The cells are then fixed and imaged to identify them. An example of such a protocol is the hematoxylin and eosin stain in which the cells are first rinsed with saline solution and then hematoxylin is introduced for 15 minutes. The excess dye is then rinsed off for 20 minutes followed by a counterstain with eosin for two minutes. After the final rinse, the cells are then fixed with 4% paraformaldehyde before imaging. While this is happening in one chamber, a gram stain is run in another chamber, while yet another chamber has markers specific to certain cells, such as CD45 for hematopoietic cells except red blood cells. Different device inputs carry different solutions, releasing them to the chamber of interest at specific times. Another approach is to have the reagents in droplet form, only merging with the specific cells at the exact instance needed. Through a compilation of the data from the parallel processes, diagnoses are made.
To reiterate and expand, other example embodiments include: (i) assays for the diagnosis of infectious diseases; (ii) assays for the determination of minimum inhibitory concentration of an antibiotic to both determine the resistance of a microbe to the antibiotic or test out new drugs; (iii) assays for the screening of newborns for multiple disease conditions that are treatable but not evident clinically which can lead to many lives being saved as such programs are often non-existent on low resource settings; (iv) the parallel testing of many chemotherapeutic agents against a particular cancer to determine the most effective medication or combination of medications for a particular case; (v) drug discovery protocols; (vi) screening pregnant women for common health risk factors; (vii) quick determination of the right antivenom for snake bites by parallel processing; and (viii) determining the best approach in treating patients with cormobidities especially those that are immune-compromised through the parallel processing of biological samples. Embodiments that are outside of medicine include: (i) programmable messages or pictorial displays; (ii) hydrocarbon sampling tests in oil discovery; (iii) protocols for testing soils samples for agricultural purposes; and (iv) parallel processing of water samples for various contaminants in the environment.
In the foregoing specification, embodiments have been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element or step modified by the article.
This application is a 371 national stage application of PCT Application No. PCT/US2013/058543, filed Sep. 6, 2013, and claims the benefit of U.S. Provisional Application No. 61/697,798, filed on Sep. 6, 2012; the entire contents of which are hereby incorporated by reference as if fully set forth herein.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2013/058543 | 9/6/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2014/039844 | 3/13/2014 | WO | A |
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