Design, Fabrication, and Application

The fabrication of microfluidic devices starts ordinarily with the design of the microchip and its microchannel structures. Nowadays, computer-aided design (CAD) software allows to draw and finally manufacture precise channel structures with errors less than a few nanometers. In this thesis, all fabricated microchannel structures have been designed via AutoCAD software (Autodesk Inc.). Also, 3D printed sample holders made of polylactide (PLA) or acrylnitril-butadien-styrol-copolymer (ABS) have been drawn using the 3D functions of AutoCAD. The devices were home-fabricated by the additive manufacturing technology of an Ultimaker 2 (Ultimaker B.V.) that is working via fused deposition modeling (FDM), visible in Figure 4.

Figure 4 | 3D AutoCAD design for a 3D printed sample holder that consists of two separate pieces and is made of PLA by the FDM method of an Ultimaker2. The sample holder is created to employ liquid microjets within a micro gear pump recycling setup which is mobile and able to be installed at synchrotron measurement stations.

Beyond the FDM method, other 3D printing technologies, like stereolithography (SLA) or selective laser sintering (SLS), have already been developed during the 1980s for various materials like plastics, resins, ceramics or metals.^1,2 Today, all these 3D printing technologies are based on CAD-data and represent versatile systems for rapid prototyping processes. During the last decade, 3D fabrication advanced extremely fast and to astonishing precision down to the micrometer scale. Nowadays, a complete microfluidic chip can be produced in one step.^3,4 Still, depending on the 3D printing process and the required mechanical and chemical properties of the

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chip material, the resolution can only be a few hundred micrometers for the channel size. As processes like photo- and soft lithography can achieve even nano-sized master devices as casting forms, they are still often preferred.^5-7

In this thesis, the microfluidic master devices are fabricated within a cleanroom via a contact mask aligner MJB4 (SÜSS MicroTec SE, see Figure 5). The aligner is working with UV light (λ = 365 nm) and high-resolution film photomasks, respectively chromium-glass masks to achieve a resolution of 1 µm produced by JD Photo Data. After getting the photomasks, the actual photolithography starts by spin-coating a silicon wafer with a photoresist. For the applications in this thesis, a 3 inch silicon wafer (Silicon Materials Inc.) was sufficient and spin-coated (Cee 200X, Brewer Science Inc.) with a thin layer of the commercially available negative photoresist SU-8 from MicroChem Corporation.⁸ The layer thickness of this epoxy-based resist is adjustable to 0.5-150 µm by varying the viscosity depending on the amount of solvent γ-butyrolactone for a certain spin-speed in a range of 1000-4000 rpm. The structures on the photomask were transferred to this layer of SU-8 by exposing through the transparent parts of the mask and hereby cross-linking the photoresist in the exposed areas. Upon exposure, cross-linking proceeds in a first step, the formation of a strong acid during the exposure, and is followed by a second step, the acid-catalyzed and thermally driven epoxy cross-linking during post exposure bake (PEB).⁹ The finally obtained master contains the inverted positive structure of the desired microchannel network and can be reused and replicated over many cycles, allowing rapid prototyping at low cost. By employing only one layer of photoresist the common and so-called 2D microchannel chip designs are available. However, if a more complex channel network with 3D fluid focusing was required, the previous steps of spin-coating and exposure were repeated to build up additional layers whereby a precise alignment of different photomasks to the substrate was necessary, as illustrated in Figure 5. In the following development step, the uncured photoresist was removed with 1-methoxy-2-propanyl acetate (mr-Dev 600, micro resist technology GmbH).

After the photolithographical process within a cleanroom, also the soft lithography is carried out in a dust-free environment,¹⁰ within a laminar flow box (ScanLaf, Mars Safety Class 2) from LaboGene^TM. Here, the master is casted with polydimethylsiloxane (PDMS) to form the actual microchip with its channel structures by replica molding. For the replication of the micro-structured master, a 10:1 mixture (monomer : curing agent) of PDMS (Sylgard 184 kit, Dow Corning Corp.) was poured onto the master and degassed as well as baked for 1.5 h at 75 °C.

During heating, the terminal vinyl groups (SiCH=CH2) of the dimethyl- siloxane oligomer basic

19 Figure 5 | Equipment for photolithography. (a) Mask aligner MJB4 (SÜSS MicroTec SE) within the cleanroom of the physical chemistry chair of the University of Bayreuth in order to fabricate Si-master devices as casting forms. (b) Layout of the high-resolution photomasks for multilayer master devices.

component and the hydrosilane groups (SiH) of the hydrogen-methylsiloxane cross-linker component, which also contains a platinum-catalyst, are reacting via hydrosilylation to create the cross-linked polymer network PDMS. After demolding, the PDMS replica was cut with a razor blade along predefined grooves into individual parts. Inlet ports for the later polyethylene (PE) tube connection were punched into the PDMS with an Integra^® Miltex^® biopsy punch (1 mm, Integra LifeSciences Corp.). The pattern surface of the resulting PDMS chip parts can easily be characterized via scanning electron microscopy (SEM) in order to determine the exact channel height and to identify defects that could disrupt the laminar microflow later on. Then, the hydrophobic surface of the PDMS chip halves were activated by air plasma treatment (MiniFlecto^®, plasma technology GmbH) that led to the generation of hydrophilic silanol groups.

These silanol groups can be used to initiate a condensation reaction resulting in a covalent

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bonding between the both PDMS chip halves.¹¹ Apart from a thin unstructured piece of PDMS acting as bottom part for a 2D microchip, also a glass slide could be used to seal the structured PDMS half. If a 3D microfluidic chip with a 3-dimensional fluid focusing channel design has been fabricated, two structured PDMS microchip parts are needed to create the channel network.

A small drop of ultrapure water (Milli-Q, Merck KGaA) was added to generate a thin film of water which enabled the alignment of the two individual parts prior to the final bonding. After bringing both parts in close contact, integrated orientation structures allowed to snap in and to align the microstructures automatically. If necessary, fine adjustments were carried out under a microscope. Removing the water in an oven at 35 °C for 12 h resulted in a permanent covalent bonding of the microfluidic chip. The fabricated PDMS microfluidic devices used in this thesis are based on quasi-two- and three-dimensional focusing channel networks which are illustrated in Figure 6 and developed for the investigation of particle separation phenomena.

Figure 6 | Comparison 2D and 3D microfluidic devices. (a) Lateral 2D focusing flow cross with three inlet and one outlet channels. (b) Surrounding 3D focusing cross also with three inlets and one outlet however the two side channels as well as the outlet channel are more than twice as high as the main inlet channel.

Microfluidic chips made of elastomer PDMS have many advantages like an easy fabricating process,^12-14 good temperature stability between -50 and 200 °C as well as an excellent transparency to visible light between 240 and 1100 nm for all optical methods of analysis.¹⁵ Its low toxicity and high gas permeability is well-suited for cell culturing and growth studies.¹⁶ Additionally, the elasticity of PDMS can be controlled by the ratio of the oligomer and crosslinker. PDMS is electrically insulating and allows the integration of electrodes in order to manipulate the fluid flow by electric fields.^17,18 However, this soft material has also significant drawbacks e.g. its chemical resistance only against aqueous solutions and a small number of polar organic solvents like ethanol, isopropanol or acetone.¹⁵ Due to the fact that PDMS is hydrocarbon-based, organic solvents with solubility in hydrocarbons are able to swell and deform PDMS resulting ultimately in a collapse of the microfluidic channel.¹⁹ By fabricating