Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word “PUNCHCARD MICROFLUIDICS” using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world.

Competing interests: The authors of this manuscript have read the journal’s policy and have the following competing interests: The authors have filed a patent for the microfluidic platform device. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Funding: The project was funded by C-IDEA grant (National Institutes of Health grant RC4 TW008781-01). Manu Prakash acknowledges support from Spectrum Foundation (NIH CTSA UL1 TR000093), Coulter Foundation, Pew Foundation, Society for Science and the Public (SPARK) and Moore Foundation. George Korir acknowledges support from Howard Hughes Medical Institute International Student Predoctoral Fellowship, Stanford University’s Dean’s Doctoral Diversity Fellowship, Ric Weiland Fellowship and the His-Fong Ho Engineering Graduate Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Korir, Prakash. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Our current implementation is inspired by punch card programming as historically applied in a wide range of applications beginning with control of textile looms [ 17 ], early computing [ 18 ] and music replay [ 19 ]. Punch cards enable the use of a single actuating platform to execute multiple programs resulting in implementation of complex instruction sets that can yield radically different outcomes by simply switching the punch card tape. Such a platform offers the flexibility of achieving multiple results without the need to redesign the system for new tasks. We have harnessed this approach and implemented it to manipulate fluids in a low-cost platform.

Here we present a programmable multiplex microfluidic system based on punch card programming that is hand-crank powered, low-cost, robust, and can run complex biological and chemical protocols with limited chances of human-error. Moreover, our system is rugged, portable, hand-held (weighing approximately 100 grams and measuring approximately 2 inches in length, 1.5 inches wide and 1 inch high) and is self-contained. The system does not require any external pumps or other supportive equipment to run. Multiple protocols can be run in parallel (multiple assays on the same sample or single assay on multiple samples), manipulating fluids arbitrarily with nanoliter volume precision. Because the program is encoded in punch card tape, the protocols can be easily shared like baseball cards to repeat or modify existing assays.

To address some of the challenges brought about by specific constrains in low-resource settings, several novel approaches have been implemented, including a finger-actuated microfluidic pump device [ 9 ] and battery-powered implementation of pneumatic valves using solenoids [ 10 ]. While promising, such approaches are either limited in range of fluid manipulation or still utilize external solenoid valves that can significantly increase device costs and depend on electrical or battery power-source. Dipsticks and lateral flow assays and in general “paper microfluidics” have found greater success in low-resource point-of-care diagnostics [ 11 – 15 ]. Although often low-cost, portable and easy to use, they have limited capacity to run multiplex, complex or a wide range of protocols and are often not as quantitative as traditional microfluidic assays except when paired with specific external readers [ 16 ]. Furthermore, due to inherent design limitations of capillary flow in a porous medium, paper microfluidics often cannot take advantage of droplet-based assays that are highly sensitive due to further reduction in associated fluid volumes and discrete and isolated nature of trapped fluid samples inside droplets. To address all the challenges listed above, the ideal technology would therefore have the capacity to (a) run complex, programmable, multiplex assays while being self-contained, (b) be capable of handling large fluid volumes in applications where the biological sample has few targeted events, (c) operate with both single phase and multiphase microfluidics and (d) would not require specialized training or any other external equipment.

With the implementation of pneumatic micro-valves, it is now possible to run thousands of assays in parallel on the same microfluidic chip [ 4 , 5 ]. Although significant progress has been made in development and manufacturing of complex microfluidic chips, current external control systems that are often required remain bulky and expensive [ 2 , 6 ]. A few applications for microfluidic devices in educational settings have been explored before, primarily focused on micro-fabrication techniques [ 7 ] while others have focused on applying existing platforms to teach principles of fluid dynamics [ 8 ]. However a gap still exists for a robust platform that can carry out complex multiplex assays yet being easy to program and use as desired.

The capability for performing robust and inexpensive assays that are easy to replicate has applications beyond medical diagnostics. When coupled to the capacity to easily manipulate fluids in a programmable fashion that is easy to implement and run, one can envision new applications in science education settings worldwide. Hands-on introduction of chemistry and biology for school children can instill a life-long passion for science [ 3 ]. Although many current scientists admit to having been inspired by open-ended explorations utilizing chemistry kits widely available several decades ago, safety concerns and expensive reagents have made this exploration currently unavailable. Low-cost self-sufficient microfluidic technologies with enclosed chemicals and small-volume reagent reservoirs could potentially provide a wide-ranging solution to the problems mentioned above.

The use of microfluidic technology, where small volumes of fluids are manipulated in carrying out miniaturized laboratory assays, has drawn considerable attention owing to inherent advantages that include minimized reagent consumption, miniaturized reaction volumes and the potential to yield robust and rapid results [ 1 ]. Application of microfluidics for robust multiplex diagnostic tests in extremely low-resource settings holds great promise but remains currently unfulfilled due to a variety of challenging factors including absence of electricity, lack of refrigeration for reagent storage, unavailable calibration services for devices over time, challenging operating conditions such as fluctuating temperatures and lack of skilled personnel [ 2 ]. There is an especially urgent need for multiplexed tests either to diagnose a disease caused by multiple agents, aid in the differential diagnosis of diseases that clinically present similarly or cases of co-morbidities due to a high disease burden in developing countries[ 2 ]. Therefore a successful implementation in these settings requires surmounting the above-mentioned challenges, using a platform that is completely self-contained and modular in nature, coupled with access to stable reagents that can be easily replenished.

The capacity to implement complex robust multiplex assays in resource poor settings devoid of skilled personnel, power sources and supportive infrastructure can revolutionize difficult to execute applications in global health, environmental monitoring and forensics, anywhere around the world. Combining microfluidics with programming using paper punch card tapes, here we present a novel integrated general-purpose fluidic platform to address specific challenges for resource-poor settings. Powered manually by a hand-crank, our device incorporates a single-layer microfluidic chip in a plug-and-play fashion and is programmed by a paper tape with punched holes as discrete instructions. In addition to the above-mentioned applications, we aspire to enable children to have access to “programmable chemistry kits” in science education settings globally.

Mode of Operation

Our system is comprised of a paper-based punch card tape, a polydimethylsiloxane (PDMS) based single layer microfluidic chip and a mechanical reader/actuator that couples fluidic channels to paper tape (Fig. 1A–F). The platform implements three key components required in general purpose microfluidic processors: embedded flow-controlled micro-pumps, normally-closed valves and a novel impact based droplet generator (Fig. 1B–D). The mechanical reader/actuator is powered by a hand crank (Fig. 1A, inset) and interfaces with the punch card tape to uniquely read the punched code and correspondingly execute pumping, valving and droplet generation in a microfluidic chip. Our modular design enables microfluidic chips to be inserted and removed from the device in a plug-and-play fashion.

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larger image TIFF original image Download: Fig 1. (A) Punch card programmable microfluidic system comprising of a paper punch card tape, plug-in microfluidic chip and a mechanical reader/actuator (inset depicts hand-crank powered device in action). The punched tape moves through the device while being read sequentially. (B, C, D) Schematic depiction of device operation including (B) pumping achieved by rotating gear teeth interacting with a collapsible channel, (C) on-demand droplet generator using an impact-based jet formation (D) normally-closed microfluidic valves based on cantilever pins pushed against a chip. (E) All components including paper tape, mechanical reader/actuator, microfluidic chip and a hole-puncher for encoding the paper tape. (F) Top-down micrograph of the device with 15 active channels filled with colored fluids. https://doi.org/10.1371/journal.pone.0115993.g001

Protocols are encoded, stored and executed using paper tapes (width 41 mm). Our current implementation consists of a series of 15 parallel lines on which holes (5mm diameter) can be punched to actuate 15 independent pumps, valves or droplet generators (see Fig. 1F and S2 Fig.). We demonstrate one-to-one correspondence between a hole and execution of pump/valve/droplet generator. The device has a bandwidth of 15 independent bits that can be set simultaneously. Other coding schemes can be easily implemented by mechanical re-configuration.

For the current reader/actuator implementation, we exploit the mechanism of a Kikkerland Music Box—a toy readily available in the market. Although many such mechanical music toys exist, we utilize the open architecture in the Kikkerland setup for quick prototyping. A completely 3D printed version of the reader/actuator was also implemented (see S1 Fig., inset) as an initial step in the rapid development of subsequent generations of our device. The reader/actuator consists of a gear train powered by a hand-crank coupled to a rotating rod that reels in the punch card tape with an approximate gear ratio of 1:6. To initialize the device, punch card tape is inserted in a slot comprised of thin metal sheets that act as guides toward two counter-rotating rods that are coupled to a series of gears. The plastic encasing of one of the rods provides additional friction enabling the turning rods to effectively reel in the punch card tape as the hand-crank is turned. The driven rod coupled to the gear train also consists of 15 gear discs that share the rod as an axle and have four asymmetric teeth positioned 90 degrees apart from each other (Fig. 1B–D). Plastic spacers between metal discs result in a 2 mm spacing between adjacent discs with gear teeth. The discs have the capacity to move independently and are only engaged when a punched hole appears in the paper tape. As the punch card tape is reeled in, the gear tooth that is closest to the hole eventually gets caught up in the hole and gets pushed in the direction of the actuation of punch card tape (+Y-axis). This action results in a rotation of the disc and therefore the other three gear teeth on the same disc also rotate in concert. For implementation of valves and droplet-generators, a secondary cantilever based array of pins is also coupled to the rotating gear train, enabling a single hole to actuate a vertical pin (Z-axis) to move up and down (see S1 Movie).

A microfluidic chip is coupled to the device in a plug-and-play fashion, held in place only by friction. Each chip consists of patterned single layer microfluidic channels on a thin membrane of PDMS (thickness 500 +/- 50 μm) and couples directly to the gear train from the reader/actuator. The microfluidic channels are self-aligned to gear teeth and pins for fluidic pumping or valving with each actuation instance. We mold single layer microfluidic channels using standard soft-lithography with channel height of 50 μm and width of 200 μm. This thin film is bonded to a thicker slab of PDMS that provides mechanical support, inlet and outlet channels and means to couple the device to the reader/actuator. Next, we describe and demonstrate pumping, enhanced mixing, valving and on-demand droplet generation.

Pumping Integrated microfluidic pumps remove the dependence of a fluid-handling platform from external, bulky syringe pumps or pressure sources. Several implementations for microfluidic pumps have utilized the deformable nature of PDMS channels including pneumatic Quake-valve pumps [4], braille display based devices [20], finger-actuated flows [9], passive capillary pressure pumps [21] and external motors driving pins that actuate fluidic cavities [22]. While effective, they often require expensive external components (solenoid valves) and/or bulky pressure sources and do not always provide independent multiplexed control. In our current platform, we have implemented 15 integrated and independently controlled micro-pumps. The net output flow-rate can be controlled as a function of actuation frequency and actuation height (Fig. 1B and F). Actuation frequency for a particular pump is dependent on the total number of punched holes on the paper tape engaged per unit time. The actuation height (h) is determined by how the microchip interfaces with the gear teeth. The power source for the device is a hand-crank. The basic principle of the pump is illustrated in Fig. 1B, where a rotating disc couples a gear-tooth to a completely collapsible microfluidic channel. As the disc rotates, the squeeze contact point linearly translates the collapse position along the channel (Fig. 2A). The width of the channels (200 μm) was further chosen to be less than the gear teeth width at the actuation area (~480 μm wide) to ensure that the channel collapse is complete with each pumping cycle. The gear tooth engages with a given channel once for a single hole. With such a wide area of actuation for the pump, multiple channels can also be tied to the same gear teeth (single channel on the tape) further multiplexing pumping. To demonstrate operation of multiple pumps simultaneously, six independent pumps were operated with a “zig-zag” pattern of punched holes in a paper tape (Fig. 2D), resulting in continuous programmable operation of all the micro-pumps with desired flow rate set by frequency of holes in individual channels on the tape. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Punch card controlled integrated multiplexed microfluidic pumps. (A) A series of images of a single channel coupled to the rotating gear disc during one pumping cycle. (B) To characterize the flow pattern with each actuation, fluorescent polystyrene beads (2 μm) were used in de-ionized water. (C) Pumping in each cycle revealed a characteristic asymmetric pulsatile oscillatory flow depicted above as a kymograph. The amplitude of directed unidirectional flow depends on actuation height (h) and the angular velocity (ω) from the hand-crank. (D) Top-down view of the microfluidic chip with simultaneous operation of six punch card controlled integrated micro-pumps. Net flow rate in a fluidic line is a function of exact pattern of punched hole (number of holes punched and spacing between the same, an example pattern depicted above). (E) Effective flow rate characterized as a function of h and ω, easily achieving typical values demonstrated by integrated micro-pumps. https://doi.org/10.1371/journal.pone.0115993.g002 The vertical position of the microfluidic chip with respect to the reader/actuator determines the degree of collapse and its interaction with the microfluidic channel. Thus we can quantify the fluid flow in the channels with two simple parameters: height of the chip above the actuator (h) and angular velocity of the gear disc under rotation (ω). To characterize the pumping, fluid flow was imaged using red fluorescent microspheres (size 2 μm) (Fig. 2B). Individual trajectories of the beads, depicted as kymographs reveal oscillatory dynamics of the fluid flow as a function of h and ω (see Fig. 2C and S4 Fig.). For the purpose of data collection, precise angular velocity (ω) was implemented using a motor driving the hand-crank. The oscillatory dynamics arise from the complete collapse and ensuing recoil of the channel as a result of the elasticity of PDMS, leading to a forward and backward flow. The asymmetry in forward and back flow is introduced due to preferential movement of the channel collapse point in the forward direction. The net effect of the back flow is limited with increasing actuation height (Fig. 2E). For angular velocity (ω) of 1.5 rotations per second and displacement height h = 450 μm, a net flow rate of ~30 nL/s can be easily accomplished. Below the critical value h = 50 μm, no net forward flow is observed in the channels due to insufficient coupling of the rotating gear disc with the PDMS channel. With increasing actuation height, the arc across which total channel collapse occurs with each actuation increases leading to increased fluid flow with each rotational stroke (Fig. 2E). To characterize the robustness and determine wear and tear of the device, a long-time experiment was run at an angular velocity of 1.5 rotations per second and actuation height h = 400 μm (net flow rate of ~10 nL/s). After 100 hours of continuous operation (~ half a million cycles), the channels were still functional although worn down from the friction between the gear teeth and PDMS. To solve this problem of wear-and-tear over prolonged use, we developed a simple solution of applying Scotch tape on the exterior parts of the PDMS (at the point where the chip interfaces with the gear teeth). We demonstrate that this wear can be completely eliminated by this very simple solution. A test run for 100 hours of continuous operation at an angular velocity of 1.5 rotations per second (equivalent to 540,000 repeated cycles) at an actuation height (~450 μm, equivalent to net flow rate ~30 nL/s) displays no visible sign of wear and tear on the PDMS.

Enhanced fluid mixing Fluid mixing is a significant challenge in integrated microfluidic devices due to lack of fluid inertia at low Reynolds numbers. Complex micro structures such as herringbone geometry [23] have been utilized to implement mixing in single-phase flow. These work by extending the interfacial boundary between two miscible fluids, thus increasing the effectiveness of diffusion across this interface thus enhancing mixing. Here, we present a simple strategy for fluid mixing on our platform that requires no special fabrication steps and is based on a simple pattern of encoded holes on a punch card tape. Because mixing is induced in a programmed manner, it is possible to effectively turn mixing ON and OFF in our devices. By exploiting the precise control of relative actuation time (controlled by distance between punched holes) of each of the pumps operating adjacent channels, we implement a simple mixing strategy in our devices (Fig. 3A). As a demonstration, we mixed six different water-based fluids simultaneously in a single output channel. The fluid was pumped through the device using the punch card tape at a flow rate of ~100 nL/s with an effective Reynolds number of 0.2. Even at such low Reynolds numbers, efficient mixing was achieved within 16 mm downstream of the inlet channels. Since all pumps can be independently controlled by 15-channel punch card tape, the diffusion boundary between nearby fluid streams (for instance, red and green streamlines) can be folded significantly by offsetting the moment of flow injection. As an example, we demonstrate mixing using a “zig-zag” pattern of punched holes (Fig. 3A, right inset). The time-varying flow rate induces interfacial boundaries amongst neighboring streamlines to significantly fold, enhancing diffusion (as depicted in the time series in Fig. 3C). In addition to the “zig-zig” pattern, two other punch tape patterns were implemented leading to different observed mixing levels (see S6 Fig.). Various other patterns can be explored to study mixing efficiencies. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Enhanced mixing is achieved using a zig-zag pattern of punched holes. (A) Photomicrograph of six punch card controlled pumps driven by a zig-zag pattern (right inset). Left inset depicts the same device run through a traditional syringe pump (at the same flow rate) to highlight the striking difference in fluid mixing at the end of the channel (200 μm wide). (B) Mixing is quantified by mean-shift clustering approach (see methods for details) comparing four regions in the micro-channel marked a, b, c, d along the outflow. Six identified clusters merge into two. (C) Photomicrographs from video data reveal the mechanism for mixing. Pulsatile nature of flow induces increased folding of neighboring flow lines (and hence net interface length) thus enhancing diffusion and mixing. https://doi.org/10.1371/journal.pone.0115993.g003 To confirm that the folding only arises due to offset and pulsatile nature of our punch card controlled integrated micro pumps, we ran an equivalent experiment using a traditional syringe pump replicating the exact flow rate and flow geometry, as generated by our integrated micro-pumps. The streamlines did not mix and the six colored fluids remained effectively separated (Fig. 3, left inset). We further quantify the extent and speed of mixing in the six fluid streamlines from the six colored fluids pumped using a mean shift clustering algorithm [24–26] implemented on images taken along different points (white boxes marked a, b, c, d) along the channel (labeled with food color in water). See supplementary material (S1 File) and S3 Fig. for detailed implementation of the algorithm. Prior to mixing, six clusters corresponding to the six fluid colors were identified (Fig. 3B) and as samples were analyzed along the fluid channel, the number finally reduced to two clusters within a travel distance of 14 mm along the outlet channel (Fig. 3E).

Valves Successful and arbitrary manipulation of fluids to run a wide range of chemical and biological assays requires the use of micro-valves to enable programmable spatiotemporal control of fluid flow. Combined with integrated pumps, valves can easily facilitate complex flow control strategies, as have been previously demonstrated [4, 5] in multi-layer microfluidic valve structures. Numerous large-scale integration architectures based on valving schemes have also been described, including precise design rules for operation [27]. Most implementations of dynamic programmable valves involve external electrical solenoids, external pressure sources and expensive electronic control. The need for external control systems often limits the impact that valve based microfluidics could have in resource-poor settings. We successfully implemented multiple, independently controlled, punch card programmable micro-valves (Fig. 4). The valving mechanism comprises of independent pins (~400 μm diameter) projecting perpendicularly (along the Z-axis) from a series of cantilevers that are attached to the base plate of the reader/actuator (Fig. 4B). In the default setting, the valve pins are passively aligned to the microfluidic channels pushing against the PDMS and thus collapsing the channel at the point of contact (OFF setting, Fig. 4D and E). During actuation, the gear train teeth pluck the cantilever beams, opening the channel due to the downward and forward motion of the valve pin. The channel is closed due to elastic recoil of the cantilever and pressure from the pin (Fig. 4C and D). A single cycle of a valve can be implemented in approximately 0.5 s (Fig. 4D). The implementation described above needs to be actuated to be in the open state (closed being the default state) to let fluid flow. A reverse valve that is open in the default mode can be implemented by simply reversing the leverage point of the cantilever. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Integrated punch card controlled normally-closed valves. (A) Schematic of normally-closed valves depicting mechanism of operation. (B) 3D printed cantilever beam array with spaced pins (2 mm apart), utilized for implementing 15 independent normally-closed valves. (C, D) Micrograph from video of ten normally-closed valves under operation (side view), all of which are independently actuated based on the punch card tape. (E) Normally-closed valve in action, at a single instance of opening and closing depicting the time duration for a single cycle (0.54 seconds). The image depicts the entire region of PDMS deformation with a completely collapsed channel. https://doi.org/10.1371/journal.pone.0115993.g004 As a demonstration, we implemented simultaneous valving and pumping in our device. This capability was made possible by simply shortening the valve pin. The microfluidic chip was then lowered to a height that would allow for integrated pumping (Fig. 1B) and valving (Fig. 1C) with each hole on the punch card tape. The coupled action allowed the valve to be open only when the fluid was being pumped and closed as soon as this action was complete. Because 15 independent punch card tape channels are available on the current implementation, the same number of independent valves and pumps can be operated simultaneously.