Fabrication of Low-Cost Paper-Based Microfluidic Devices by Embossing or Cut-and-Stack Methods

This communication describes the use of embossing, and “cut-and-stack” methods of assembly, to generate microfluidic devices from omniphobic paper, and demonstrates that fluid flowing through these devices behaves similarly to fluid in an open-channel microfluidic device. The porosity of the paper to gasses allows processes not possible in devices made using PDMS or other non-porous materials. Droplet generators and phase separators, for example, could be made by embossing “T”-shaped channels on paper. Vertical stacking of embossed or cut layers of omniphobic paper generated three-dimensional systems of microchannels. The gas permeability of the paper allowed fluid in the microchannel to contact and exchange with environmental or directed gases. An aqueous stream of water containing a pH-indicator, as one demonstration, changed color upon exposure to air containing HCl or NH 3 gases.

The most widely used technology for the formation of microfluidic systemsfabrication using polydimethylsiloxane (PDMS) and soft-lithography-is still too expensive and/or technically demanding for applications requiring low cost (e.g., human and veterinary medicine in resource-poor settings, 1 food testing, 2 or environmental monitoring 3 ).To circumvent the issues of cost and availability, we, [4][5][6] and others, [7][8][9] have developed so-called "paper microfluidics" (a methodology in which fluid flow in paper is driven by capillary wetting, and directed by channel boundaries of hydrophobic wax 10 or polymer patterned in the paper by printing, 7 photolithography, 11 or other methods 12 ).Although wicking-driven paper devices are clearly attractive for their low cost, simplicity, and ability to generate 3D microfluidic systems, the cellulose fibers that partially fill the channels introduce complexity into the flow of liquids-a problem that is largely absent in open-channel PDMS-based microfluidic devices.These wicking-based devices also present a large area of potentially adsorptive surface (which may be useful or detrimental, depending on the application); the performance of wicking devices may also be influenced by environmental factors such as humidity.
This paper is one of several introducing a new technology that, among other things, enables paper to be used as the basis for pressure-driven open-channel microfluidic systems. 13,14 he key element in this technology is "omniphobic paper": that is, paper whose surface has been modified by treatment with a highly fluorinated alkylsilane (and which we therefore call "R F " paper 14 ).The static contact angle (θs) of water on R F paper is high, θs = 135°-155° (greater than water on Teflon, θs = 120°).R F paper is also able to resist wetting by many organic liquids: liquids with surface tensions as low as 27 mN/m (hexadecane) do not spread on R F paper. 14Paper can be readily formed into a variety of complex 3D shapes by embossing, engraving, cutting, folding, stacking, molding, or bending.
The surface modification of these paper structures by silanization (or other chemistry) enables them to be used as microfluidic devices.Combination of the shaped, surfacemodified paper with other materials (polymer tapes, hydrophilic paper and thread, metal films, etc.) generates a wide range of functions and devices having low weight and low cost of materials. 13Shaped, R F paper also enables new functions, because it has properties not readily encountered in conventional materials (for example, it is simultaneously inexpensive, lightweight, highly hydrophobic, highly gas permeable, easily burned, and thermally/electrically insulating).
We have described one versatile technique that involves cutting channels in cardstock paper using a programmable knife, 13 making these engraved structures omniphobic by silanizing them using a fluorinated alkyltrichlorosilane (R F SiCl3) in the vapor phase, and sealing the channels with pre-cut transparent tape. 14This paper describes a related method for fabricating simple open-channel microfluidic systems on Whatman ® #1 filter paper, based on embossing using reusable plastic molds or cutting and stacking appropriate structures into the filter paper followed by the silanization step previously mentioned.The former technique yields smaller channels, and is easier to carry out; The latter is more useful for larger features, and parallel production of large number of devices (by reel-to-reel embossing and processing).
Paper microfluidics, as a generic technology, would benefit from materials and systems that would preserve the advantages of paper while allowing fluids to flow in a pressure-driven open channel.These devices would find application in situations requiring the control of fluid flows afforded by open channels (for example, high-resolution capillary electrophoresis 15 ), applications that require manipulation of fluids containing suspended particles (such as blood, environmental slurries, multi-phase suspensions, and most unprocessed biological samples), 16,17 in analysis and/or manipulation of mixtures of compounds that would separate chromatographically in a wicking-based devices, 18 or the manipulation of complex chemical mixtures. 19,20 pen-channel microfluidic devices might also be useful in the synthesis of particles 21,22 and in processes, such as microfluidic shear separation, 23 where the fluidic-flow properties of a liquid are of interest.
The surfaces of the cellulose fibers that make up paper have a high density of hydroxyl and acetal groups, and are normally hydrophilic.Paper can be made hydrophobic by physisorption (coating the fibers or filling the voids with a hydrophobic material) 7,10 or chemisorptions (applying reactions that modify the OH groups, such as silanization, acylation, or epoxide and thiirane ring opening). 12Of these, we have chosen silanization because: i) the reaction occurs readily at room temperature when the hydroxyl groups are exposed to vapors of a silanizing agent and ii) there is a variety of commercially available organosilanes, with different terminal groups, that can be used to modify the surface of paper.
Exposure of Whatman #1 filter paper, for example, to vapors of many perfluorinated alkylsilanes, R F SiCl3, under vacuum renders it hydrophobic (static contact angle of water, θs >135°). 14The reaction of the hydroxyl groups of paper and alkyltrichlorosilanes, R F SiCl3, is rapid and requires no catalyst.This reagent does not react with acetal moieties, and as such, can be used for surface functionalization of paper without damaging the linkages that make up the backbone of the polymeric cellulose.

Results and Discussion
Fabrication of Dies for Embossing Paper.The polymeric dies used to emboss open channels on paper were designed using computer-aided design (CAD, Airlibre Inc.) and generated by 3D printing (StrataSys Dimension Elite) using acrylonitrile butadiene styrene (ABS) co-polymer. 24Paper releases readily from these ABS dies after embossing.We have used the dies over a hundred times with no damage or degradation in performance.Dies for embossing can also be generated via other processes such as thermoplastic casting, laser cutting, or selective etching. 25The supporting information (Figure S1 and S2) gives detailed schematics and pictures of the embossing dies we used.

Fabrication of Open-channel Microfluidic Devices by Embossing.
We fabricated the open-channel microfluidic devices by sandwiching a sheet of paper between two dies with complimentary shapes (see Figure 1a-b).Paper is compressed into a channel by applying pressure (~ 0.2 kg/cm 2 ).To increase moldability, we put a few drops of ethanol (~ 400 µL per device) on the paper to wet its surface and reduce its glass transition temperature. 26Wetting the paper with liquid ethanol makes the paper easier to emboss with less force and avoids tearing the paper on the edges.Once embossing was finished (~ 2 s), the paper was allowed to dry for ~ 30 s in an oven at 60 °C.
Assembly of the Microfluidic Devices.We covered the channels with transparent adhesive tape (Fellowes adhesive sheet, PET/EVA/LDPE) on which inlet and outlet holes had been cut (using a laser cutter or a punch, see Figure 1c).This process for fabrication of devices is rapid (>100 devices/h) and requires no special facilities or tools.We employed this two-step method to fabricate microfluidic devices and multiple-well-plates. Figure 1d and 1e show a 2D microfluidic device with a channel (2 mm width, 800 µm depth) fabricated by embossing.Fluid inlet tubes were supported with 2-mm thick PDMS slabs, which were connected to the device using a double-sided adhesive layer (3M Command TM medium picture hanging strips, 17201, www.command.com).Figure 1e shows the laminar flow of two aqueous dye solutions at a rate of ~ 1 mL/min along an embossed channel in a paper microfluidic device.The laminar flow was preserved for flow rates ranging between 0.05-2 mL/min without observing any leakage or delamination of the top adhesive layer.We fabricated other microfluidic designs ("T", "U", "Y", see Figure S4, S6, S7) and estimated the Reynolds numbers of water flowing through them to range from 45-1800 (at flow rates ranging from 0.05 mL/min to 2 mL/min, see Supporting Information).All devices showed laminar flow (expected for those Reynolds numbers 28 ), even when three dye solutions were introduced into the system (Figure S5, Movie_M1 and M2).
Imaging the Channels.To understand whether embossing had a significant influence on the structure of the paper, we compared images of different parts of the devices using scanning electron microscopy (SEM) (Figure 2).Figures 2 b-e shows that the fibers of the paper were stretched in the lateral walls of the channel, since these regions were where the paper was most strained during embossing.The fiber organization in the bottom of the channel is generally retained, i.e. there are no observable structural differences in fiber organization between the bottom and the non-embossed regions of the paper (Figure 2 c-e).
The depth profile of the embossed channel was also analyzed through contactless surface profilometry (Figure 2a).The roughness of the walls of the channel-where most stress was observed-was ~22 µm, while the roughness on the bottom of the channel was comparable to regions of the paper device that were not embossed (~73 µm).
Three-Dimensional Microfluidic Devices.We fabricated 3D microfluidic devices on R F paper using a two-layer approach (see Figure 3a).To connect the two layers of R F paper, we punched holes on the top layer, and through the connecting double-sided tape; these holes allowed the fluid to flow into or out of the bottom layer (Figure 3a, i to iv).To test this design, we passed solutions of dyes in water through the device (Figure 3b-d).As with the 2D microfluidic design, we observed that the fluids moved well across the channel at flow rates ranging from 0.05 mL/min to 2 mL/min (A video of this device in action is given in the supporting information, Movie_M4).We also ran a 3D microfluidic device using three aqueous solutions: Two on a Y-inlet leading to laminar flow, and the other solution in the cross-channel (Figure S7).We observed that, as the fluid moved along the channel, significant mixing was observed as a reflection of the influence of the channel architecture on the flows and eddies 29 (see Figure S6, S7, and Movie_M5).
Droplet Generators.1][32] When two streams of immiscible liquids, with low Reynolds number, meet in orthogonal channels in a T-junction (with the more rapidly flowing stream in the straight top channel), shear forces break up the more slowly flowing liquid into droplets.Embossed R F paper devices are omniphobic with a rough porous surface.
To generate droplets, we used a two-inlet "T" shaped device into which we introduced dyed water and dyed hexadecane (Figure 4).Neither the water nor the hexadecane wetted the omniphobic paper.Figure 4a shows a device, with inlets connected; Figure 4b shows the same device in use (Movie_M6).We used different flow rates to obtain monodisperse droplets of different sizes and shapes at frequencies between 0.2 and 10 Hz (Figure 4d-f).The coefficient of variation (CV) of the length of the droplets (the distance between the furthest downstream and upstream points along the interface of a fully detached plug) generated at a frequency of 1.25 Hz was 1.2%.As in other microfluidic devices, 33 droplet generation with our paper devices follows the simple scaling law (Eq.1). 34,35 (1 Where L is the length and w is the width of the droplet, Qwater and QHD are the flow rates of water and hexadecane (HD) respectively, and, α is a constant that depends on the geometry of the junction.A T-inlet device generated using a knife cutter showed similar performance. 13crofluidic Devices made by Cutting and Stacking Omniphobic Paper.We have demonstrated omniphobic paper-based open-channel microfluidic devices that were realized through embossing paper.We also demonstrated that similar devices could be realized by cutting and stacking different paper labels or different layers of paper connected using pre-cut double-sided tape.Figure 5a shows how to make microfluidic devices by stacking a paper label (white labels for laser printers, S-5044, www.uline.com)with the silhouette of the channel cut out on top of a non-patterned label.We made the cut-and-stack paper devices omniphobic by silanization with R F SiCl3, and sealed the channel with a layer of transparent adhesive tape, as described before.In comparison to embossing, the cutting and stacking technique has six advantages: i) No molds are needed.The cut patterns can be printed out using a regular printer, or drawn with a pen and a ruler.ii) The process has a higher success rate (~ 98% of devices work, as compared with ~ 85% of embossing).iii) Final construction consists of predictable, uniform, and tunable geometries that can be stack on top of others to configure a final device (Figure 5b).The patterned paper labels and tape serve as the side walls of the channel, enabling the depth of the channel to be readily manipulated, i.e., the depth of the channel is governed by the number of paper layers that are stacked to form the channel.iv) The flat surfaces generated in this procedure make the assembly of the devices rapid and easy, using manual procedures.v) The adhesive layers in these devices allows for the direct insertion and sealing of the tubing into the channel, without requiring external adaptors.(Figure 5b,c).vi) Thin microfluidic devices (~ 600-µm thick devices were fabricated using 150-µm thick tape and 220-µm thick paper labels) fabricated with this technique can withstand bending and conform to curved surfaces using the adhesive of the bottom label (Figure 5c).
Figure 6 shows one type of 3D paper-based microfluidic device which has two channels crossing each other fabricated by stacking two layers of R F paper patterned with channels on top of a layer of non-patterned R F paper.The channels are 2 mm wide and ~ 80 mm long.This device has two polyethylene tubes attached to the bottom layer of R F paper to deliver gases, through the paper, into the channels (Figure 6a).Two aqueous solutions of pH indicator (yellow-Phenol Red; dark brown-Bromophenol Blue sodium salt) were introduced into the channels through the two inlets located on the top left part of the device (Figure 6b).When no gas is delivered into the channels, the color of the liquid at the outlets indicates that the device enables streams of fluid to cross one another multiple times without mixing (Figure 6c).When we deliver HCl (g) and NH3 (g) through polyethylene tubes attached to the back of the device, the gases diffused through the bottom R F paper layer in the channels containing the indicator solution.The gases dissolve in the solution flowing through the channel, change the pH of the solution, and are visualized by a color change in the indicator dyes (Figure 6d).The yellow-Phenol Red solution changes to purple under basic conditions, and the dark brown-Bromophenol Blue sodium salt solution changes to orange on exposure to acidic conditions.The high gas permeability of R F paper provides a simple way to introduce a gas to a liquid (to capture components of the gas or as part of a sensor) without having a large exposed surface of liquid.The gas impermeable tape that is used to connect different layers prevents exposure of large areas of the flowing fluid to permeating gases, allowing the delivery to be targeted or localized.
Microfluidic Displays.After demonstrating a working gas-permeable 3D microfluidic device made by the cut-and-stack approach, we fabricated another microfluidic device with three letters "G", "M", and "W" connected via bottom channels following the cutting and stacking process described previously (Figure 5 and 6).It has one inlet and one outlet on the top right part of "G" and "W", respectively, as shown in Figure 7a.The inlet and outlet in this device are both on the back of the device.We used Bromothymol Blue solution, a universal indicator (pH sensing range 6.0-7.6,displaying yellow, green, and blue in acidic, neutral, and alkaline solutions, respectively), to demonstrate the possibility to induce sequential changes in the pH of a solution flowing through a microchannel made of R F paper by taking advantage of the gas permeability of this paper device.The three letters displayed in Figure 7a were initially green with the solution flowing from "G" to "W" along the direction of the arrow.A vial containing a solution of 15% NH4OH (NH3 source) was connected to the back of the device via a 1/32" diameter Teflon tube.We inserted the end of the tube into a PDMS slab for support and attached the tube to the non-patterned bottom layer of the device using a ring of double-sided adhesive tape.The NH3 (g) source was connected to the "M" on the back of the device (bottom left of "M", as indicated by an arrow in Figure 7b), resulting in a colorimetric change of "M" and "W" from green to blue.The color of letter "G" remained green, indicating that NH3 did not diffuse into the letter "G" under fluid flow.
After the exposure of "M" to NH3 (g), we exposed the letter "W" (arrow in Figure 7c) to HCl (g) coming through the bottom paper layer in an analogous way to what was done for NH3 (g).A vial containing concentrated HCl (fuming HCl) was used as our acidic gas source.The acidic gas, HCl (g), neutralized the ammonium hydroxide present in the solution of indicator and split the fluid flowing through the "W" into two pH regions where the solution again turned yellow on the side of the microfluidic channel exposed to the acid while the other half remained blue (Figure 7c).We used this effect to demonstrate that the gas-permeability of a microchannel can be controlled by partially covering the width of the channel with a gasimpermeable layer, like tape, therefore limiting the ability for a gas to be introduced to the whole channel.A closer look at the inlet and outlet (Figure 7a,b) of each letter reveals a square dark spot in the background except for the inlet of letter "W" which has a triangular dark spot.This darkened spots are the channels that connect the letters except that for letter "W" the channel has been partially blocked with tape to allow gas diffusion to only occur through half of the channel (the unblocked triangle).When we attached the HCl inlet tube under this channel, only half of the channel changed color from blue to yellow.The acid (yellow) and basic (blue) solutions, as expected, did not mix under laminar flow.After the fluid flow was stopped, the yellow and blue solutions gradually mixed due to diffusion (Figure 7d). Figure 7e-h show a similar channel filled with a red aqueous solution and blue dye in hexadecane demonstrating the omniphobicity of the paper channel.
96-zone Plate.We fabricated 96-zone plates by mechanically embossing Whatman #5 chromatography paper between two ABS dies and rendering the surfaces omniphobic by silanization with R F SiCl3 (see Figure S2).Embossed wells with a diameter of 12 mm and a depth of 2 mm could hold aqueous solutions with a volume of up to 100 µL.We similarly fabricated well-plates of tunable depth and diameter through the cut-and-stack method.The depth of the well was controlled by the number of layers used to make the device, while the diameter of the wells was determined by the size of holes made on the paper (Figure S3).

Conclusions
A two-step process-embossing or cutting, treatment with perfluorinated R F SiCl3 as a vapor, followed by assembly-makes it possible to fabricate open channel microfluidic devices on paper rapidly.Dies for embossing were fabricated in ABS using a 3D printer; they could be used multiple times (over 1000) with no damage or degradation in performance.
Fluid flow behavior in these paper-based microfluidic devices is similar to that observed in PDMS-based open channel devices.We observed laminar flow, droplet generation through shearing in a T-device, and 3D flow in a multi-layer device made by placing different layers of embossed paper on top of each other and connected them using double-sided adhesive tape.By connecting different layers through pre-cut hole, and covering the top with a layer of transparent adhesive tape, we created 3D microfluidic devices in which fluids could past one or another in different layers without mixing.In principle, the gas-permeable devices reported here can be applied to any liquid-or solid-gas physisorption process or chemical reaction with a broad range of applications in analysis, environmental monitoring, infochemistry, particle synthesis, and many others.
The cut-and-stack method is ideal for fabrication of reconfigurable 3D devices since the depth of the channels can be controlled by stacking layers of paper or using paper with different thicknesses.Adhesive and bendable microfluidic devices (as thin as 600 µm) were fabricated using paper labels and tape.Since paper can be folded, cut-and-stack method can be combined with origami to build complex fluid transport systems or devices.Although embossing and the engraving techniques have their advantages, the versatility of the cut-andstack method allows one to build more robust devices in a reliable manner using simple tools.

Fabrication
of Low-cost Paper-based Microfluidic Devices by Embossing or Cut-and-Stack Methods.

TABLE OF CONTENTS Omniphobic "R F " paper is used to fabricate open-channel microfluidic systems by embossing or "cut-and-stack" methods.
Microfluidic devices made of R F paper are mechanically flexible and highly permeable to gases, which allows processes not possible in microfluidic devices made of PDMS or other non-porous polymers.Keywords: Microfluidics, Omniphobic paper, Embossing, Cut-and-stack, Paper-based Microfluidics.Martin M. Thuo, Ramses V. Martinez, Wenjie Lan, Xinyu Liu, Jabulani Barber, Manza B. J. Atkinson, Dineth Bandarage, Jean-Francis Bloch, and George M. Whitesides