Rapid assembly of multilayer microfluidic structures via 3D-printed transfer molding and bonding

Abstract

A critical feature of state-of-the-art microfluidic technologies is the ability to fabricate multilayer structures without relying on the expensive equipment and facilities required by soft lithography-defined processes. Here, three-dimensional (3D) printed polymer molds are used to construct multilayer poly(dimethylsiloxane) (PDMS) devices by employing unique molding, bonding, alignment, and rapid assembly processes. Specifically, a novel single-layer, two-sided molding method is developed to realize two channel levels, non-planar membranes/valves, vertical interconnects (vias) between channel levels, and integrated inlet/outlet ports for fast linkages to external fluidic systems. As a demonstration, a single-layer membrane microvalve is constructed and tested by applying various gate pressures under parametric variation of source pressure, illustrating a high degree of flow rate control. In addition, multilayer structures are fabricated through an intralayer bonding procedure that uses custom 3D-printed stamps to selectively apply uncured liquid PDMS adhesive only to bonding interfaces without clogging fluidic channels. Using integrated alignment marks to accurately position both stamps and individual layers, this technique is demonstrated by rapidly assembling a six-layer microfluidic device. By combining the versatility of 3D printing while retaining the favorable mechanical and biological properties of PDMS, this work can potentially open up a new class of manufacturing techniques for multilayer microfluidic systems.

OPEN

Microsystems & Nanoengineering (2016) 2, 16063; doi:http://dx.doi.org/10.1038/micronano.2016.63

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ARTICLE

Rapid assembly of multilayer microuidic structures via 3D-printed transfer molding and bonding

Casey C. Glick1,2, Mitchell T. Srimongkol2,*, Aaron J. Schwartz2,*, William S. Zhuang2,*, Joseph C. Lin2,*, Roseanne H. Warren2,3,

Dennis R. Tekell2, Panitan A. Satamalee2 and Liwei Lin2

A critical feature of state-of-the-art microuidic technologies is the ability to fabricate multilayer structures without relying on the expensive equipment and facilities required by soft lithography-dened processes. Here, three-dimensional (3D) printed polymer molds are used to construct multilayer poly(dimethylsiloxane) (PDMS) devices by employing unique molding, bonding, alignment, and rapid assembly processes. Specically, a novel single-layer, two-sided molding method is developed to realize two channel levels, non-planar membranes/valves, vertical interconnects (vias) between channel levels, and integrated inlet/outlet ports for fast linkages to external uidic systems. As a demonstration, a single-layer membrane microvalve is constructed and tested by applying various gate pressures under parametric variation of source pressure, illustrating a high degree of ow rate control. In addition, multilayer structures are fabricated through an intralayer bonding procedure that uses custom 3D-printed stamps to selectively apply uncured liquid PDMS adhesive only to bonding interfaces without clogging uidic channels. Using integrated alignment marks to accurately position both stamps and individual layers, this technique is demonstrated by rapidly assembling a six-layer microuidic device. By combining the versatility of 3D printing while retaining the favorable mechanical and biological properties of PDMS, this work can potentially open up a new class of manufacturing techniques for multilayer microuidic systems.

Keywords: 3D printing; microuidics; PDMS

Microsystems & Nanoengineering (2016) 2, 16063; doi:http://dx.doi.org/10.1038/micronano.2016.63

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INTRODUCTIONMicrouidic devices for manipulating uids have rapidly advanced since the 1980s because of their unique ability to fabricate low-cost, high-throughput platforms, particularly for chemical and biological research and lab-on-a-chip technologies1,2. The most

far-reaching breakthrough in microuidics has been the development of soft lithography: using rigid micromachined molds to pattern elastomeric polymers3. Among polymeric materials, poly (dimethylsiloxane) (PDMS) is commonly used because of its numerous favorable properties, including its ease in manufacturing, reasonable cost, strength, transparency, and especially biocompatibility4.

Traditional methods for fabricating microuidic devices involve using photolithography to construct micromolds with very ne features; however, this process can be long and costly. The increasing demand for microuidics is particularly high for multilayered devices featuring more sophisticated structures and components (including valves, pumps, and other active control mechanisms). For example, soft lithography through micromachining processes is generally restricted to monolithic rectilinear features5, although rounded and fully circular channels are common in large-scale uidic systems.

One method of increasing geometric complexity is 'multilevel soft lithography'6, in which the channels are non-planar and/or rounded7. Although rounded channels are benecial for some microuidic applications, few groups have developed appropriate

fabrication techniques (Supplementary Material S3.4)810 because multilevel soft lithography has historically required multiple photolithography steps11. Although grayscale lithography whereby resists are exposed to non-binary shades of graycan potentially generate rounded microuidic channels1214, the

process still requires multiple exposures to obtain larger aspect ratios15,16. Furthermore, although multilayer PDMS-manufacturing techniques have been demonstrated by several groups17,18, these

are even more time-consuming and labor-intensive, requiring multiple lithography steps and precision alignment, issues that are only partially addressed by dedicated PDMS-alignment tools19.

Three-dimensional (3D) printing offers a unique route for building multilevel and multilayer microuidic devices directly, or indirectly via molding processes. For example, various groups have used 3D printers to fabricate simple microuidic devices with truly 3D geometries, including microuidic devices without moving elements, such as resistors20 and modular components21, as well as those with movable components, such as capacitors, diodes, and transistors22. Currently, the eld of 3D-printed microuidics is limited by the following: (1) the available resolution of the printer20; (2) surface roughness23,24; and (3) material types25,26; however, 3D printing technologies are expected to rapidly advance and address these matters in the coming years. For further details on current 3D printer capabilities, including printer resolution and surface roughness, see reviews in Refs. 2732.

1Department of Physics, University of California, Berkeley, CA 94720, USA; 2Department of Mechanical Engineering, University of California, Berkeley, CA 94720, USA and

3Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA.

Correspondence: Casey C. Glick (mailto:[email protected]

Web End [email protected]) or Liwei Lin (mailto:[email protected]

Web End [email protected]) *These authors contributed equally to this work.

Received: 25 April 2016; revised: 21 July 2016; accepted: 21 July 2016

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Although direct 3D printing is a rapid process for prototyping, for making multiple copies of microuidic devices, 3D-printed transfer molding (PTM), in which polymer is poured into a 3D-printed mold, remains faster, cheaper, and more reliable. First pioneered by McDonald et al.33 using fused deposition modeling techniques, the technique has since been used with stereolithography34 and multijet printing23 as well as with wax printers35 and ofce-quality laser printers36,37. Although PTM does not allow the geometric exibility of fully 3D-printed microuidics, it possesses the following three notable advantages: (i) each mold can be used for multiple microuidic devices, reducing 3D printing times and costs, (ii) many 3D printers exhibit lower resolution for features requiring support materials, and (iii) the process is compatible with conventional microuidic fabrication materials, most notably PDMS34,35,3739. Because PDMS can be used to

transfer patterns with high delity, the resolution and surface nish of the mold dene the resulting quality of the resulting PDMS structure38,40, provided the mold features reasonable aspect ratios23.

In addition to patterning features externally, PTM has also been used to fabricate internal features, reducing certain monolithic constraints. Hwang et al.41,42 have developed printed molds that are enveloped by PDMS and then withdrawn after curing, relying on the exibility of PDMS to remove the components. Although similar to fugitive ink processes4345, fugitive ink molding has fewer geometric constraints but requires a printing step for every nal device, whereas solid internal structures must be designed for withdrawal but can be reused23,41,42. Chan et al.46 have

fabricated molds with overhangs in a basket weave pattern, which can be used to generate microuidic vias and valving in a single step, repairing demolding damage by thermally healing the PDMS, with the restriction that the vias be designed in parallel.

PTM is also increasingly integrated with other 3D printing processes to reduce the challenges of multicomponent assembly and to interface microuidic devices with external systems29.

Although some PTM devices can interface directly, whether, by punching holes (in a manner, similar to standard soft lithography processes)36, molding open wells33,47, and adding connectors during the curing process34,37,48, other PTM devices can interface

indirectly by attaching to 3D-printed components that do have the desired interfaces39. Similar to soft lithography, PTM devices are sealed with glass or other 3D-printed components to provide enclosed channels after molding (including, through plasma bonding33, mechanical pressure39, or tape39).

This work advances 3D PTM techniques from single-sided microuidics33,34,37 to multilayer microuidic manufacturing, using

the ease of 3D printing to create multiple molds with alignment structures to shape multiple layers of PDMS structures and quickly assemble them in the nal step. First, we discuss details specic to the ProJet 3000 3D printer, including resolution and surface treatments. Next, we examine the single-step double-sided molding method used to create PDMS components with complex geometries including vias, thin membranes, and rounded channels that are difcult to achieve using standard soft lithography, as well as integrated input/output marks that do not require positioning external components during PDMS curing. Finally, we demonstrate rapid assembly of multilayer microuidic devices including integrated alignment marks, which enable tactileas opposed to opticalalignment of layers to within the resolution of the 3D printer (including PDMSPDMS for multilayer assembly and moldPDMS for interfacing with other 3D-printed objects), and bonding techniques, including a specialized variant of adhesive bonding techniques introduced by Satyanarayana et al.49. Using custom 3D printing to selectively apply a thin layer of liquid PDMS adhesive to non-channel areas, layers were successfully bonded without the adhesive clogging narrow channels, obviating problems associated with mold surface roughness.

MATERIALS AND METHODS3D-printed moldsMicrouidic components were designed and converted from a positive to a negative mold shape using the computer-aided-design program SolidWorks. 3D printing of molds was achieved using a ProJet 3000 3D printer (3D Systems, Rock Hill, SC, USA)50.

During printing, the ProJet 3000 alternately deposited 3D Systems proprietary structural epoxy (VisiJetEX200 plastic material51,52) and sacricial wax support material (VisiJetS100

hydroxylated wax53) in 0.35 m layers. The wax was used as a temporary support for cavities and overhangs and was removed during post processing. The printer was capable of resolving extruded features as small as 50 m and intruded features as small as 100 m. For more information on chemical and material properties, see Supplementary Material S1.

Mold post processingFollowing printing, the molds were cleaned to remove the sacricial wax. First, the molds were baked in a VWR 1330 FM oven (VWR, Radnor, PA, USA) at 75 C for 45 min to melt the sacricial wax. The molds were then washed in a sequence of three cleaning baths for 10 min in each bath to remove leftover wax: warm Bayes mineral oil, Ajax dish detergent in water, and potable water. The baths were heated to 75 C to ensure that the wax did not solidify and were placed on a hotplate with a magnetic stir bar to enhance the removal of wax, oil, and soap, respectively. The molds were then dried by baking at 80 C for 60 min. After cleaning and drying, the 3D-printed molds were treated with an uorinated silane anti-adhesive agent, trichloro (1H,1H,2H,2H-peruorooctyl)silane gas (PFOTS, Sigma-Aldrich, St Louis, MO, USA) to make the surface hydrophobic and to facilitate the rapid removal of PDMS. Next, the molds and a 1.5-cm Outer Diameter (OD) glass vial containing 0.3 ml PFOTS agent were placed in a vacuum desiccator (104 torr) for 30 min for the vapor treatment. Shorter treatment times resulted in PDMS bonding to the mold, and longer treatment times caused a build-up of PFOTS, which inhibited complete curing of the PDMS near the surface (Supplementary Material S2.3)54,55.

PDMS moldingThe 3D-printed molds were placed on a foil-wrapped 3D-printed molding tray (Supplementary Material S2.1) shaped to substantially reduce PDMS waste. PDMS (Sylgard 184 Elastomer Kit, Dow Corning, Midlind, MI, USA) was prepared using the standard 10:1 base:curing agent ratio. The PDMS mixture was degassed in a vacuum chamber to 104 torr (Supplementary Material S7) for 10 min and poured on the 3D-printed molds. The lled molds were then returned to the vacuum chamber for 30 min to degas and increase PDMS conformity. For double-sided molding processes, this degassing also serves to load uncured PDMS between the upper and lower molds. Following degassing, the molds were baked in an 80 C oven for 50 min. The PDMS microuidic components were removed from the molds by rst cutting away excess PDMS using the edge of the mold as a guide and then by manually peeling the PDMS from the mold. This step was performed carefully to avoid damaging the higher aspect features; without structural features such as widened bases and llets, many devices lost at least one input/output port within 510 demolding events because of handling error (Supplementary Material S2.2). Provided that no features were broken during the demolding process and PDMS did not permanently bond to the mold, the molds were reused without an additional cleaning process. Approximately every 1020 moldings, mold hydrophobi-city was refreshed by repeating the PFOTS treatment, which was performed when PDMS began adhering excessively to the printed

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Figure 1 Illustration of fabrication process (top) and technical capabilities (bottom) of 3D-printed transfer molding for double-sided microuidic devices. Fabrication: (a) mold is 3D-printed from a CAD model, treated, tted using alignment marks, and (b) lled with PDMS and cured. Excess PDMS is cut away and the mold is removed. (c) The resulting PDMS component contains integrated inlets/outlets, membranes, and vias, and is (d) bonded to glass to create a device with enclosed channels of arbitrary cross-section. Technical capabilities: the multilevel microuidic device shown in cross-section in (e) and photographed in (f) is fabricated using double-sided molding techniques and exhibits numerous design elements, such as two-layer uid ow, multiple microuidic vias, integrated uid inlets/outlets, an elliptical 350-m domed membrane, and a Quake-style membrane value, as well as alignment marks for use in generating multilayer devices. 3D, three-dimensional;

PDMS, poly(dimethylsiloxane).

mold (Supplementary Figure S7). For further discussion of molding techniques, see Supplementary Material S2.

RESULTSFigure 1 illustrates the process ow for fabricating multisided

PDMS microuidic devices using 3D-printed molds. The component mold is fabricated via the 3D printing process (Figure 1a), and PDMS is applied (Figure 1c), cured, and released from the mold (Figure 1c) by means of the PDMS molding steps described in Section MATERIALS AND METHODS. Integrated uid inlets (diameter 0.55 mm) are easily incorporated into the component through the mold design, which further simplies fabrication by eliminating the need for an additional hole-punching step. The PDMS component is then bonded to glass to create a complete microuidic device with enclosed channels (Figure 1d).

This 3D PTM and bonding technique can be used to fabricate conventional microuidic devices such as those commonly produced by soft lithography methods (Figure 2), but with faster prototyping and simpler processing, easier fabrication of complex 3D geometries, the ability to fabricate circular channel cross-sections, and integrated uidic interfaces. Furthermore, novel techniques were developed to fabricate double-sided or multilayer microuidic devices that maintain the basic procedure of generating a Computer aided design (CAD) model, 3D-printed mold, and PDMS replica of the mold. These techniques, including alignment marks to precisely position molds (for example, in double-sided molding) or PDMS layers (for example, in multilayer assembly) as well as PDMSPDMS bonding using a 3D-printed stamp, enable the design, fabrication, and assembly of complex microuidic systems as shown in Figure 1f (Section 'Multisided and multilayer molding techniques').

Single-sided molding techniquesUsing 3D-printed molds, semicircular and fully circular channel geometries are easily fabricated (Figure 2); the high delity of PDMS transfer molding for features as small as 80 nm ensures that

mold roughness is reliably transferred to the resulting PDMS40.

Surface texture in ProJet 3000 multijet printing arises both from the interface between sequential rows of epoxy and from structural irregularities within a row. The interfacial texture resulted in a peak surface asperity of ~ 20 m, measured by surface prolometry (Supplementary Figure S2c). Structural macroroughness, measured by Walczak et al.24, was 0.70 m and0.56 m in the x and y directions, respectively, values comparable to those achieved in micromilling. Supplementary Figure S2b shows a PDMS component after release from the 3D-printed molds, with an enlarged view of the surface roughness. Although this value is comparable to microuidics fabricated directly by 3D printing, transfer-molded PDMS components can produce narrower channels because interior cavities have the tendency to reow during printing. In addition, surface roughness in 3D-printed devices is currently higher than in those fabricated by conventional soft lithography23. For further discussion, see Supplementary Material S1.2 (for surface roughness) and Supplementary Material S3.1 (for single-sided molding).

Glass PDMS spin bonding To create fully enclosed and tightly sealed microuidic channels, a glass-bonding step is required56.

However, because of the surface roughness of the 3D-printed molds, it was difcult to achieve a tight seal when bonding PDMS to glass using standard techniques such as oxygen plasma and ozone surface treatments. For this reason, specialized bonding techniques were necessary to nalize the microuidic devices fabricated through the 3D PTM process. Although some surface roughness was reduced by performing a standard surface treatment (for example, in oxygen plasma) and then tightly clamping the two bonding surfaces together to mechanically compress the surface prole, this technique was unreliable and often led to broken glass during the curing stage (Supplementary Material S5.1).

A more reliable glass-bonding technique uses spin-coating uncured liquid PDMS (PDMS) as both bonding agent and ller.

PDMS spin-bonding overcomes drawbacks associated with

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Figure 2 Single-sided fabrication and bonding process ow. (a) 3D-printed mold is printed from CAD le, including integrated inlet/outlet ports and guideposts to assist the removal of PDMS. (b) Mold is lled with PDMS, degassed, and baked, and (c) cured PDMS is demolded. (d) Cured PDMS is bonded to glass using the PDMSglass PDMS spin-bonding technique to compensate for surface roughness. (e) Final conceptual image with enclosed channel and 20-gauge connector pins attached. (f) Photograph of glass-bonded device with colored uid. 3D, three-dimensional; PDMS, poly(dimethylsiloxane).

surface roughness from 3D printing and achieves a tight bond between the PDMS component and the glass slide (Figure 2d). First, PDMS was spin-coated on a microscope coverslip (22 22 0.1 mm3, Fisher Scientic, Hampton, NH, USA) at 1800 Rotations/revolutions per minute (RPM) to achieve a thickness of 15 m (Ref. 57). The PDMS-coated slide was placed face up on a TexWipe TechniCloth (Kernersville, NC, USA) resting on a hotplate at 95 C for 70 s to partially cure the PDMS, increasing its viscosity. The molded PDMS component was then placed bonding side down into the curing PDMS, and 2030N downward force was applied to the component via a thick non-bonding glass side (Fisher Scientic 75 25 1 mm3) for 90 s. This thicker glass slide was used to apply pressure to equalize the distribution of the downward load and ensure that the PDMS bonded fully to the glass slide throughout its area. Next, the pressure was released, and the PDMSglass bond was left to cure on the hotplate for an additional 5 min (Supplementary Material S5.2).

For double-sided microuidic components, this process was repeated on the reverse side, with care taken to not fracture the fragile glass slide that had already been positioned during the rst bonding step. Using a smaller glass coverslip for the upper surface, the upper glass slide could be bonded in a position that maintained access input and output holes. Although the PDMS spin-bonding method worked for taller channels, for channels smaller than 100 m in height, excess PDMS was sometimes forced into the channels, causing permanent blockages. This problem is mitigated using a 3D-printed stamp to selectively apply PDMS only to non-channel areas of the PDMS device (Section 'PDMS stamp bonding and multilayer rapid assembly'). Reliability of the bonding process can be further improved through the use of a dedicated bonding platform19 or through the use of a vacuum-bonding apparatus to provide consistent uniform pressure5861. For more details on spin bonding and potential improvements, see Supplementary Material S5.3.

Integrated uid inlets Figure 2f shows a single-layer microuidic device with integrated uid inlets (diameter 0.55 mm), incorporated during mold design. These inlets simplify fabrication by eliminating the need for an additional hole-punching step. Six 20-gauge stainless steel interconnectors (Instech SC20/15, Plymouth Meeting, PA, USA, outer diameter 0.91 mm and length 15 mm) were easily inserted into the inlet ports for the connections to external uidic pipes as shown in Figure 2f. Due to the tight seal of the steel couples against the 40% smaller inlet

ports, the inlets were leak-resistant to pressures above 4 atm, the pressure at which the PDMSglass bond delaminated when using untreated glass coverslips (Supplementary Material S5.2.2). 3D-printed guideposts at the corner of the mold assist with the removal of the PDMS without damaging narrow gauge inlets and outlets (Supplementary Material S2.2).

Multisided and multilayer molding techniquesThe increasing demand for microuidics is particularly high for multilayered devices62. Multilayered fabrication allows for the implementation of more sophisticated and useful internal structures (including, valves or pumps) as well as reducing geometrical constraints by enabling uidic detours and vias. Creating multilayer microuidic devices using conventional techniques requires at least two lithography steps and one PDMSPDMS bonding step (such as, for 'Quake' membrane valves)63, and can

require up to four lithography steps and three PDMSPDMS bonding steps (such as, for PDMS-based uidic transistors)64.

Multilayer construction is used largely to overcome the limits of traditional 2D microuidic systems and to provide uids with an extra degree of freedom. These 3D devices may be constructed either from PDMS components with features molded on more than one side (Figures 3 and 4), or with several layers of PDMS components (Figure 5). Although multilayer assembly is relatively common in traditional soft lithography (despite the aforementioned difculties), double-sided molding is rare. Here, double-sided molding is accomplished through the use of in-mold alignment marks and can be used in preparation for multilayer assembly (for example, when constructing PDMS-alignment marks) or as a nal device, in which case double-sided glass-bonding is performed to seal channels on both sides, leaving inlet/ outlet holes uncovered on the upper surface (Supplementary Material S3.2).

Alignment marks With 3D-printed molding, rapid assembly of multilayer microuidic devices is easily achievable through the use of integrated alignment marks. Alignment marks can be used on the 3D-printed molds of the PDMS components, enabling multiple fabrication steps by allowing for the precise positioning of each layer without the need for a microscope. Figure 3a shows the four primary varieties of alignment marks: (i) moldmold alignment marks, used for fabricating double-sided PDMS components; (ii) moldPDMS-alignment marks, used in stamp bonding; (iii) PDMS

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Figure 3 Double-sided molding techniques and results. Conceptual illustration of alignment marks: (a) moldmold, (b) moldPDMS, (c) PDMS PDMS, and (d) PDMS height limiters. (e and f) Microuidic devices with integrated uidic vias: (e) simple overpass and (f) repeated crossover with mixing. (g and h) Membranes (350-m thick) for uid storage or hydrodynamic capacitance: (g) domed membrane (h) sinusoidal membrane for increased exibility. (i) Fully circular channels fabricated by bonding two complementary components using integrated PDMS

PDMS alignment marks. PDMS, poly(dimethylsiloxane).

PDMS alignment marks, used for fabricating double-sided channels and assembling multilayer devices; and (iv) PDMS height limiters, used for controlling the ultimate thickness of a PDMS layer. For more detailed process ows and examples, see Supplementary Material S4.

Microuidic vias The ability to route uid in three dimensions is frequently desired in microuidics because it reduces geometrical constraints by enabling uidic detours65. However, because these microuidic vias are time-consuming and costly to fabricate (generally requiring a minimum of three lithography steps and one PDMSPDMS alignment/bonding step), vias are not incorporated into microuidic devices unless required for specic functionality.

With 3D-printed double-sided molding, however, uidic vias are far simpler to fabricate45,46. Using double-sided molding

techniques with columns that run from the bottom mold and t into the upper mold (Figure 3b), smaller moldmold alignment marks can be constructed that allow uid to ow between the top and bottom face of a single layer of PDMS (Figure 1a) or between layers of bonded PDMS (Figure 5b), thus enabling multilayer microuidic devices. For technical discussion, see Supplementary Material S3.2.1 for fabrication information and Supplementary Material S6.1 for a comparison of via manufacturing methods.

Thin membranes. Double-sided PDMS molding also enables the construction of integrated thin membranes. Nesting (but non-contact) mold features created between the top and bottom molds can be lled by PDMS during vacuum-degassing, forming thin membranes upon curing. Figures 3g and h depict hyphenation problems membranes (domed and sinusoidal, respectively) that can potentially be used as uidic reservoirs or capacitors. The sinusoidal corrugation lowers the effective spring constant of the membrane, allowing it to store more uid. This technique has been used to generate membranes down to 200 m thick, limited by surface interaction effects that interfered with PDMS curing23 and caused membrane rupture upon demolding. For further discussion on membrane uses, limitations, and design considerations, see Supplementary Material S3.2.2.

Double-sided channels In certain microuidic applications (such as, optouidic lithography), it is useful to have components completely surrounded by PDMS66 or channels that have 360 curvature67,68. Figure 3i shows an image of a

fully rounded microuidic channel fabricated using the 3D

PTM process that avoids some of the difculties of current techniques for generating fully rounded channels. For further fabrication results and motivations, see Supplementary Material S3.4.

Membrane valves Another common requirement in multilayer microuidics is membrane valves, which use pneumatic or hydraulic pressure in one uid layer to moderate uid ow in a secondary layer65. Commonly, these membrane (or 'Quake') valves are two-layer constructions that use multiple pneumatic inputs to control complex arrays of microuidic reactors, although some studies use multiple layers to implement valves with active control or integrated pressure gain69,70. Fully 3D-printed microuidic systems with valving mechanisms have also been developed22,7173.

In this work, we fabricated a membrane valve using a single-step double-sided microuidic molding technique by linking a detour via with a thin membrane in an upper layer (Figures 4a and b). The membrane was 350 m thick, and the lower channel was 500 m deep (for schematics, see Supplementary Figure S13). Although standard membrane valves require a photoresist reow step during manufacture to allow the bottom layer to close fully, we were able to implement a rounded lower channel directly from the CAD le. To characterize the closing behavior of membrane valve, we ran a series of pressure sweeps (Fluigent MFCSEZ, Villejuif, France) and measured (Fluigent FlowUnit L) the resulting source-drain ow rate (Q): gate pressure (PG) was increased smoothly and source pressure (PS) was increased parametrically. The valve began closing at 160 kPa, was fully closed by 220 kPa, and exhibited a nearly linear response during the transition (Figure 4f). Further, PS did not substantially affect the PG of the initial drop in Q. Finally, to demonstrate the response time of the membrane valve within the closing pressure window, we manually cycled the gate pressure at various speeds and compared the pressure and ow rate response curves (Figure 4g). Note that the vertical axes have been rescaled and shifted to illustrate the high degree of qualitative agreement between pressure and ow rate. The time-differential response curves (Figure 4h) illustrate the rapid response time of ow rate to changes in gate pressure.

PDMS stamp bonding and multilayer rapid assembly Double-sided PDMS molding can be completed by spin-bonding to glass; however, fully multilayer microuidic devices (created by 3D-printed molding or conventional soft lithography) require a

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Figure 4 'Quake'-style membrane valves generated by single-step double-sided molding procedure. (a) Conceptual and (b) cross-sectional photograph of the membrane valve. (c) Top-down photograph, (d) microscope image illustrating the active valve region, and (e) microscope images of valve under various PG. (f) Valve characteristic curves under parametric PS sweep. Further Q, PG time series analysis: (g) ow rate compared with varying gate pressures and (h) rates of change of ow rate and gate pressures.

PDMSPDMS bonding step. For 3D molded PDMS components, we have developed 3D-printed stamps to selectively apply PDMS as a more consistent bonding technique, as shown in Figure 5a. The stamps can easily be designed and printed from the original CAD drawings to selectively apply uncured PDMS only to non-channel areas of the PDMS component. These stamps can contain features that intrude (Figure 5c) or extrude (Figure 5d) from the plane of the stamp, which allows the stamp to be used on numerous PDMS topographies. Using PDMS stamp bonding, multilayered microuidic devices are easily assembled, allowing uid ow within or between the various layers (Figure 5b). In addition, previously discussed techniques (including, alignment marks, integrated inlets/outlets, and variable channels) can be used in conjunction with rapid assembly. Including the 40 min baking time, PDMS stamp bonding allows a six-layer microuidic device (Figure 5e) to be assembled and bonded in under an hour.

To bond two PDMS components with PDMS stamp bonding, PDMS was rst applied to a TechniCloth wipe, and the stamp was dipped in the PDMS. For extruded stamps, a copy of the PDMS component was used as an applicator (that is, a template stamp used to transfer PDMS to the extruded stamp topographies). Next, the stamp was 'blotted' with a clean TechniCloth to remove excess PDMS. The stamp was then pressed lightly ( 5 10 N) into its complementary PDMS component to deposit a thin, uniform layer of PDMS on the 4 cm2 PDMS component. Finally, the two PDMS components were pressed together and clamped in place to prevent shifting during curing. Clamping was performed with a 1 " C-clamp and was judged to be sufciently secure when the

visual roughness disappeared (as PDMS lled the empty spaces resulting from the roughness). Finally, the devices were cured at 80 C for 40 min, resulting in a complete multilevel PDMS device. For a more extensive discussion of stamp-bonding techniques, see Supplementary Material S5.3. Note that these precision stamp-bonding techniques may prove useful for bonding disparate materials that would otherwise require harsh plasma treatments7476 or for the precision placement of cells or other biomaterials7.

DISCUSSIONIn this work, we presented a novel method for rapidly manufacturing elastomeric microuidic devices using 3D printed transfer molding (PTM). Although this process was limited by the resolution and surface roughness of the ProJet 3000 multijet printer, the technique was able to reliably produce enclosed channels as narrow as 100 m. In conjunction with a spin-coated PDMS glass-bonding technique, designed to counteract the effects of the molds surface roughness, this method can produce single-layer microuidics more exibly than those produced in standard soft lithography fabrication processes. In addition, the transfer-molded microuidic devices are enhanced by numerous design elements, not limited to the following: controllably nonrectilinear channels, integrated inlets and outlets, vias and thin membranes, and integrated alignment marks, techniques that can be applied more generally across the 3D printer across 3D printer models and methodologies.

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Figure 5 Rapid assembly of multilayer PDMS microuidic devices achieved with 3D-printed molding. (a) Use of a 3D-printed stamp to selectively apply uncured PDMS to non-channel areas. (b) Conceptual illustration of alignment and assembly of multilayer microuidic device with illustration of intralayer uid ow. (c) Intruded stamp topography. (d) Second layer of six-layer device with corresponding stamp, which exhibits extruded stamp topography. (eh) Rapid assembly of multilayer microuidic device: (e) CAD model, (f) stacked individual layers, (g) assembled and bonded, and (h) uid ow spiraling between layers and mixing. 3D, three-dimensional; PDMS, poly(dimethylsiloxane).

Furthermore, PTM techniques are far more versatile than merely replicating existing soft lithography; by incorporating newly developed alignment marks and PDMS stamp-bonding, this process can produce complex multisided and multilayer micro-uidic devices with ease. Single-step double-sided manufacturing (in which features are patterned on both sides of the PDMS components) enables features such as microuidic vias and membrane valves. Microuidic vias, which allow uids to ow in three dimensions, reduce geometrical constraints and were fabricated here with diameters as narrow as 550 m to hold 20-gauge catheter couples. Membranes (350 m) were used to produce microuidic valves with an actuation pressure of 200 kPa. Furthermore, by combining the double-sided manufacture method with novel custom 3D-printed stamps, rapid assembly of multilayer microuidic devices was demonstrated. The 3D-printed stamps can selectively apply a thin layer of PDMS adhesiveused to compensate for surface roughnessto non-channel areas, preventing the PDMS from clogging the nal microuidic device. Furthermore, because adhesive bonding techniques have been used to bond disparate materials, we expect the stamp-printing techniques introduced here to remain relevant past the point at which printers have sufcient resolution to mitigate roughness issues. In summary, the 3D PTM process allows the rapid fabrication of multilayered microuidic devices, combining the exibility and speed of emerging 3D printing technology with the well-known mechanical and biological properties of PDMS favored by microuidic researchers.

ACKNOWLEDGEMENTS

We thank Judy Kim, Caroline Su, Kyungna Kim, Ariana Moini, and Xining Zhang for their help with this project, as well as the other students in the Lin Lab M3B program.

In addition, we thank the Center for Interdisciplinary Biological Inspiration in Education and Research (CiBER) at UC Berkeley for use of their ProJet 3000 3D printer and to the NSF GRFP and UC Berkeley Sensors and Actuators Center (BSAC) for helping fund this project.

COMPETING INTERESTS

The authors declare no conict of interest.

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