Cell Adhesion and Growth to Peptide-Patterned Supported Lipid

Mar 3, 2007 - Integrating top-down and self-assembly in the fabrication of peptide and protein-based biomedical materials. Katherine H. Smith , Esther...
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Langmuir 2007, 23, 3849-3856

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Cell Adhesion and Growth to Peptide-Patterned Supported Lipid Membranes Dimitrios Stroumpoulis, Haining Zhang, Leticia Rubalcava, Jill Gliem, and Matthew Tirrell* Department of Chemical Engineering and Materials Research Laboratory, UniVersity of California, Santa Barbara, California 93106 ReceiVed August 10, 2006. In Final Form: January 18, 2007 Lipid vesicles displaying RGD peptide amphiphiles were fused with glass coverslips to control the ability of these surfaces to support cell adhesion and growth. Cell adhesion was prevented on phosphatidylcholine bilayers in the absence of RGD, whereas cells adhered and grew in the presence of accessible RGD amphiphiles. This specific interaction between cells and RGD peptides was further explored in a concentration-dependent fashion by creating surface composition arrays using microfluidics. For the range of concentrations studied adhesion and growth were favored by increased peptide concentration, but this concentration dependence was found to diminish in the higher concentration regions of the array. Developing peptide composition gradients in a membrane environment is demonstrated as an effective method to screen biological probes for cell adhesion and growth.

Introduction The communication of cells with their natural environment in vertebrate tissues is known to regulate a number of critical responses such as cell survival, adhesion, migration, proliferation, differentiation, and death.1-3 The extracellular space is composed of cells, soluble factors, and an intricate network of macromolecules constituting the extracellular matrix (ECM). The ECM components are largely produced locally by cells and include glycoproteins such as collagen, fibronectin, vitronectin, laminin, and fibrinogen. Engineering materials that are capable of supporting cell and tissue growth is a challenging task that involves identifying and incorporating biological signals into the material surfaces4 or scaffolds.5 One approach toward bioactivity in materials is to mimic the function of the ECM by displaying adhesion-promoting oligopeptides.6,7 The use of small peptides instead of the parent glycoprotein has several advantages, some of which are higher selectivity, enhanced control over cell adhesion and phenotype, lower cost, and improved stability.6,8 Effectively arranging those biologically active ligands with respect to a specific cell response is an optimization problem, where the tuning parameter is the presentation of the peptide ligands. A simple example of the above problem in two dimensions is the tuning of the GRGDSP to PHSRN peptide ratio in biomimetic membranes to maximize cell spreading, a response * Author to whom correspondence should be addressed (e-mail [email protected]). (1) Alberts, B. Molecular Biology of the Cell, 4th ed.; Garland Science: New York, 2002; pp xxxiv, 1463, [86]. (2) Freshney, R. I. Culture of Animal Cells: A Manual of Basic Technique, 4th ed.; Wiley-Liss: New York, 2000; pp xxvi, 577, [12] of plates. (3) Giancotti, F. G.; Ruoslahti, E. Transduction-integrin signaling. Science 1999, 285, 1028-1032. (4) Dillow, A. K.; Tirrell, M. Targeted cellular adhesion at biomaterial interfaces. Curr. Opin. Solid State Mater. Sci. 1998, 3, 252-259. (5) Stevens, M. M.; George, J. H. Exploring and engineering the cell surface interface. Science 2005, 310, 1135-1138. (6) Hubbell, J. A. Bioactive biomaterials. Curr. Opin. Biotechnol. 1999, 10, 123-129. (7) Tirrell, M.; Kokkoli, E.; Biesalski, M. The role of surface science in bioengineered materials. Surf. Sci. 2002, 500, 61-83. (8) Lebaron, R. G.; Athanasiou, K. A. Extracellular matrix cell adhesion peptides: functional applications in orthopedic materials. Tissue Eng. 2000, 6, 85-103.

associated with proliferation.9 One method to determine the optimum ratio value is by conducting a series of experiments using surfaces coated with different peptide ratios.10 Alternatively, a parallel analysis method could be engineered to obtain the same level of information with a single experiment. Such screening platforms could potentially provide biomaterial engineers with a valuable tool to explore the relationship between the form of ligand presentation and cell function. Surface composition gradients of adhesive ECM proteins have been reported in the literature as useful platforms for understanding cellular adhesion11 and motility.12 Using peptide gradients to search for optimal compositions of peptide mixtures could add a useful tool to biomaterial design. A biologically inspired route to fabricate multifunctional peptide-displaying materials is self-assembly.13 Peptide-lipid conjugate molecules were introduced by Berndt and colleagues14 to combine biological activity with self-assembly character and have been shown to successfully produce supported biomimetic membranes.10,15,16 Supported planar bilayers (SPB) are a good platform from which to study molecular interactions at interfaces,17 because transmembrane proteins and peptides can be incorporated in a biologically relevant environment with precise control over their concentration, presentation, and lateral mobility. SPBs can be (9) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric control of cell life and death. Science 1997, 276, 1425-1428. (10) Ochsenhirt, S. E.; Kokkoli, E.; McCarthy, J. B.; Tirrell, M. Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement. Biomaterials 2006, 27, 3863-3874. (11) Plummer, S. T.; Wang, Q.; Bohn, P. W.; Stockton, R.; Schwartz, M. A. Electrochemically derived gradients of the extracellular matrix protein fibronectin on gold. Langmuir 2003, 19, 7528-7536. (12) Smith, J. T.; Tomfohr, J. K.; Wells, M. C.; Beebe, T. P.; Kepler, T. B.; Reichert, W. M. Measurement of cell migration on surface-bound fibronectin gradients. Langmuir 2004, 20, 8279-8286. (13) Tirrell, M. Modular materials by self-assembly. AIChE J. 2005, 51, 23862390. (14) Berndt, P.; Fields, G. B.; Tirrell, M. Synthetic lipidation of peptides and amino-acidssmonolayer structure and properties. J. Am. Chem. Soc. 1995, 117, 9515-9522. (15) Dori, Y.; Bianco-Peled, H.; Satija, S. K.; Fields, G. B.; McCarthy, J. B.; Tirrell, M. Ligand accessibility as means to control cell response to bioactive bilayer membranes. J. Biomed. Mater. Res. 2000, 50, 75-81. (16) Dillow, A. K.; Ochsenhirt, S. E.; McCarthy, J. B.; Fields, G. B.; Tirrell, M. Adhesion of alpha(5)beta(1) receptors to biomimetic substrates constructed from peptide amphiphiles. Biomaterials 2001, 22, 1493-1505. (17) Groves, J. T.; Dustin, M. L. Supported planar bilayers in studies on immune cell adhesion and communication. J. Immunol. Methods 2003, 278, 19-32.

10.1021/la062375p CCC: $37.00 © 2007 American Chemical Society Published on Web 03/03/2007

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Figure 1. Chemical structure of RGD peptide amphiphiles used: (C16)2-Glu-C2-GRGDSP and (C16)2-Glu-PEO-GRGDSP.

formed on flat surfaces using the Langmuir-Blodgett (LB) technique or alternatively from vesicle solutions. The fusion of vesicles with solid substrates offers simplicity and enhanced bilayer deposition rates18 over the LB method, whereas it can also be used with convex and enclosed surfaces. Furthermore, vesicle fusion has been demonstrated to enable micropattern formation19 in SPBs. In this paper, peptide-lipid conjugate molecules (peptide amphiphiles) containing the RGD amino acid sequence were synthesized and assembled into vesicles. Supported biomimetic membranes were prepared by vesicle fusion, and their ability to promote cell adhesion and growth was evaluated. Furthermore, lithographic patterning and a microfluidic device were used to prepare RGD surface composition gradients and to explore the effect of various surface properties on cell response. Materials and Methods Peptide Amphiphile Synthesis. Peptides were purchased from SynPep (Dublin, CA). Dialkyl ester lipid tails (C16) were connected to the peptide headgroup by a L-glutamic acid linker and a carbon spacer, as described elsewhere.14,16 An alternative polyethylene oxide (PEO; 3,6,9-trioxaundecanedioic acid) spacer molecule was also synthesized and used. Briefly, PEO was reacted with N,Ndicyclohexylcarbodiimide (DCC) in chloroform and dimethylformamide (DMF) in the presence of N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (4-DMAP), and N-hydroxysuccinimide (NHS) in an ice bath. (C16)2Glu P-Tos was dissolved in chloroform and DMF and added drop by drop to the active intermediate solution. The product was washed with HCl/H2O and dried with anhydrous sodium sulfate. The peptide amphiphiles were produced by Fmoc synthesis and purified by HPLC (Shimadzu) on a reversed-phase C4 column, with gradients of acetonitrile in water containing 0.1% TFA. MALDI-TOF mass spectrometry was used to verify the identity of the molecules. The molecular structures of the peptide amphiphiles used in this study are shown in Figure 1. Vesicle Preparation. Small unilamellar vesicle (SUV) solutions were prepared by mixing one or more of the following: L-Rphosphatidylcholine from egg (egg-PC) purchased from Avanti Polar Lipids (Alabaster, AL), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (Texas Red DHPE) purchased from Molecular Probes (Eugene, OR), (C16)2-Glu-PEO-GRGDSP, or (C16)2-Glu-PEO-GRGESP. Ten milligrams, total weight of amphiphiles, was dissolved in chloroform, and a thin film was formed (18) Stroumpoulis, D.; Parra, A.; Tirrell, M. A kinetic study of vesicle fusion on SiO2 surfaces by ellipsometry. AIChE J. 2006, 52, 2931-2937. (19) Groves, J. T.; Boxer, S. G. Micropattern formation in supported lipid membranes. Acc. Chem. Res. 2002, 35, 149-157.

on the walls of a glass test tube by nitrogen flow induced evaporation. The sample was left overnight in a vacuum chamber to ensure complete removal of the solvent. The film was reconstituted in 6 mL of Millipore water (18 MΩ‚cm), briefly vortexed, and left to hydrate in a 37 °C water bath for 1 h. The multilamellar vesicle solution thus formed was extruded five times through a polycarbonate membrane filter (100 nm nominal pore size) in a temperatureeontrolled extruder at 50 °C. Dynamic light scattering was used to confirm the formation of vesicles with a monodisperse diameter distribution around 100 nm. The extruded solutions were kept at 5 °C and used within 2 weeks. Microfabrication. Patterned surfaces were prepared from optical borosilicate cover glasses (Fisherbrand) on the basis of a protocol from Groves et al.20 Briefly, a 1000 Å layer of chromium was deposited on the coverslips by electron beam evaporation. The surfaces were spin coated with AZ 5214-E IR (Clariant, Somerville, NJ) photoresist and exposed through a photomask (Photosciences, Torrence, CA) to UV light by contact lithography. The resist was developed in Shipley MF-701 and treated with oxygen plasma for 30 s, and the chromium was etched using chromium mask etchant (Transene, Danvers, MA). A thin, optically transparent layer of chromium (∼20 Å) was deposited on the back side of the coverslips for resistance to vesicle adsorption. The microfluidic devices were fabricated from polydimethylsiloxane (PDMS; Dow Corning Sylgard 182) using a protocol described by Dertinger et al.21 A 3000 dpi resolution printer (Graphic Traffic, Santa Barbara, CA) generated a transparency, which was used in contact photolithography with 50 µm thick SU-8 2050 photoresist (MicroChem, Newton, MA) to produce a negative “master” of patterned photoresist on a silicon wafer. Positive replicas were fabricated by molding PDMS against the master, whereas inlets and outlets for the vesicle solutions were made using a sharpened needle. Bilayer Formation. Cover glasses (Fisherbrand) for membrane deposition were cleaned via plasma oxidation (Harrick, NY). A spreading solution was prepared by supplementing phosphatebuffered saline (PBS) 7.2 (10×) with 140 mM NaCl.22 For homogeneous concentration bilayers, a 200 µL drop of spreading solution followed by a 100 µL drop of the desired vesicle solution (at a concentration of 1.66 mg/mL) was placed in the center of the coverslip that was affixed to the bottom of a sterile Petri dish. A second coverslip was placed on top in a sandwich arrangement, and (20) Groves, J. T.; Mahal, L. K.; Bertozzi, C. R. Control of cell adhesion and growth with micropatterned supported lipid membranes. Langmuir 2001, 17, 5129-5133. (21) Dertinger, S. K. W.; Chiu, D. T.; Jeon, N. L.; Whitesides, G. M. Generation of gradients having complex shapes using microfluidic networks. Anal. Chem. 2001, 73, 1240-1246. (22) Burridge, K. A.; Figa, M. A.; Wong, J. Y. Patterning adjacent supported lipid bilayers of desired composition to investigate receptor-ligand binding under shear flow. Langmuir 2004, 20, 10252-10259.

Cell Adhesion and Growth the substrate was allowed to interact with the vesicles for 40 min. The Petri dish was then filled with Millipore water, and the top coverslip was removed. For bilayers with a concentration gradient of peptides, six feed solutions were prepared by mixing spreading solutions with vesicles containing 0/0, 0.625/0.0625, 1.25/0.125, 2.5/0.25, 5/0.5, and 10/1% mole of the RGD amphiphile/TR-DHPE, respectively, at 1:1 ratio and loaded to 3 mL plastic syringes (Becton, Dickinson and Co., Franklin Lakes, NJ). A clean coverslip was assembled with the PDMS microfluidic mold forming a reversible seal, and the device was plasma treated to oxidize the channels. The assembly was immediately placed on top of a thin PDMS layer inside a large Petri dish, and the Petri dish was filled with Millipore water. The PDMS channels were filled with water using a vacuum pump.23 Polyethylene tubing was attached to the syringes and inserted into the PDMS mold, whereas a microfluidic pump (World Precision Instruments, model SP200i) was used to inject each vesicle solution at a flow rate of 5 µL/min for 30 min. The channels were subsequently flushed with Millipore water, and the glass coverlsip was separated from the mold. In both cases, the Petri dishes containing the bilayer substrates were washed several times with Millipore water to remove excess vesicles and then with Dulbecco’s modified Eagle’s medium (DMEM) (1×) to a final volume of 7 mL. Cell Experiments. L-Cells (mouse fibroblast) were cultured in DMEM containing 5% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. NIH3T3 cells were cultured in DMEM containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Cells were grown in a 37 °C incubator with 5% CO2 atmosphere. Cells were washed with a PBS (1×) solution, trypsinized, and resuspended in DMEM with either 5% FBS (L-cells) or 10% FCS (NIH3T3) at a concentration of 1 000 000 cells/mL. An average of 20 000 cells/ cm2 was added per sample. Microscopy. Imaging of the membranes and cells was performed on a Nikon Eclipse TE-200 microscope using a 10× phase 1 DL objective. Fluorescence images were taken with the same objective using a 100 W mercury arc lamp. The supported membranes were fluorescently labeled with 1 mol % of Texas Red lipids. Images were recorded with a digital color CCD camera (Cannon model Coolpix 995). In the fluorescence recovery after photobleach (FRAP) experiments, an approximately 600 µm diameter circular area was photobleached for 2 min using the full spectrum of the mercury arc lamp and a 40× objective. Adhered and spread cells on the peptide gradient surface were analyzed using imaging software. Within each composition strip the number and area of adhered cells were calculated and used to obtain the fractional number and area of cells on the gradient.

Results and Discussion Strategies for designing amphiphilic molecules that can be successfully incorporated into bilayer self-assemblies have to consider the critical packing parameter (V/ao/lc) requirement.24 For molecules intended to be used in a single-component membrane, the value of this parameter has to be roughly equal to 1. However, when those molecules are intended to constitute a small percentage of a mixed-lipid membrane (as in this work) and depending on the mixing ratio and packing shape of the ambient lipids, the packing parameter requirement can be relaxed to a smaller value. Peptide amphiphiles designed to participate in membrane systems typically consist of bulky (double) tails and small headgroups. The presentation of a given peptide is an important parameter determining the peptide’s ability to specifically target receptor units located close to the membrane, especially when the active amino acid sequence is located close (23) Monahan, J.; Gewirth, A. A.; Nuzzo, R. G. A method for filling complex polymeric microfluidic devices and arrays. Anal. Chem. 2001, 73, 3193-3197. (24) Israelachvili, J. N. Intermolecular and Surface Forces, 8th ed.; Academic Press: London, U.K., 2000; pp xxi, 450.

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to the spacer group. Earlier studies10,16,25,26 have indeed shown that the peptide presentation can significantly influence its ability to engage integrins, either immobilized on a surface or present in a cell membrane. One method to control the presentation of a peptide is by adjusting the size of the spacer group. Whereas very short spacer groups can be hydrophobic (C2), larger groups must be hydrophilic if they are to assume an extended conformation in an aqueous environment (PEO). The ability of RGD peptide amphiphiles to induce fibroblast cell adhesion in PC-supported membranes was investigated. Mouse cells were incubated on four types of surfaces: glasssupported egg-PC bilayers or egg-PC bilayers containing 5 mol % of either (C16)2-Glu-C2-GRGDSP, (C16)2-Glu-PEO-GRGDSP, or (C16)2-Glu-PEO-GRGESP. In all cases, the presence of 1 mol % of TR-DHPE allowed for bilayer visualization. Only membranes containing the (C16)2-Glu-PEO-GRGDSP amphiphile promoted cell adhesion, whereas the other three types of surfaces effectively blocked it. The results obtained from a typical adhesion experiment are presented in Figure 2. Strong adhesion was observed within 4 h when supported membranes contained the (C16)2-Glu-PEO-GRGDSP molecule. On the contrary, for cells seeded on egg-PC membranes, (C16)2-Glu-C2-GRGDSP- or (C16)2-Glu-PEO-GRGESP-containing membranes did not adhere and tended to clump. In every case, a bilayer was present between the cells and the substrate, as evident from the uniform fluorescence in Figure 2 [results shown for egg-PC- and (C16)2-Glu-PEO-GRGDSPcontaining bilayers] and from FRAP experiments (results not shown). However, a fluid bilayer cannot possibly support the tensile forces exerted by the cells.20 The stretched morphology of the fibroblasts on the (C16)2-Glu-PEO-GRGDSP-containing bilayers can be explained by a scenario involving cells partially penetrating the membrane and adhering strongly to the substrate. The RGD motif likely initiates the cascade of events that enables cells to adhere and spread, through specific ligand-integrin interactions. However, the presence of the RGD amino acid sequence in the membrane does not ensure cell adhesion as seen by the inability of the shorter (C16)2-Glu-C2-GRGDSP amphiphile to support adhesion to egg-PC bilayers. It appears that the accessibility of the ligand is an equally important parameter. Effective control over the normal distance of the peptides from the ambient lipid molecules in the membrane can actuate the receptor engagement. Supported bilayers are typically separated from the solid substrate by a thin (∼10 Å) film of water27 and retain many of the properties of free membranes, including lateral fluidity. The fluidity is long range, with mobile components in both leaflets of the bilayer diffusing freely over the entire surface of the substrate. To control the spatial concentration of a planar bilayer, two levels of patterning are required. For the primary level of patterning that aims to restrict the bilayer fluidity, barriers to lateral diffusion can be imposed on the supported membrane and partition it into corrals with a well-defined geometry. A variety of techniques have been developed for this purpose, including mechanical scoring of the surface, microcontact printing, selective (25) Jensen, T. W.; Hu, B. H.; Delatore, S. M.; Garcia, A. S.; Messersmith, P. B.; Miller, W. M. Lipopeptides incorporated into supported phospholipid monolayers have high specific activity at low incorporation levels. J. Am. Chem. Soc. 2004, 126, 15223-15230. (26) Pakalns, T.; Haverstick, K. L.; Fields, G. B.; McCarthy, J. B.; Mooradian, D. L.; Tirrell, M. Cellular recognition of synthetic peptide amphiphiles in selfassembled monolayer films. Biomaterials 1999, 20, 2265-2279. (27) Koenig, B. W.; Kruger, S.; Orts, W. J.; Majkrzak, C. F.; Berk, N. F.; Silverton, J. V.; Gawrisch, K. Neutron reflectivity and atomic force microscopy studies of a lipid bilayer in water adsorbed to the surface of a silicon single crystal. Langmuir 1996, 12, 1343-1350.

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Figure 2. Fibroblasts (mouse L-cells) cultured on supported membranes after 4 h. Membranes in (A), (B), and (E) are 99% egg-PC and 1% Texas Red DHPE, whereas those in (C), (D), and (F) are 94% egg-PC, 5% (C16)2-Glu-PEO-GRGDSP, and 1% Texas Red DHPE. Pictures A and B and C and D were taken on the same surface spots, in brightfield and fluorescent mode, respectively. Picture F may not accurately portray a spreading cell on an RGD bilayer as cells may displace the lipids and make direct contact with the substrate.

Figure 3. Microfluidic device designs used for generating gradients of vesicle solutions. (A) Microfluidic network device:28 vertical channels are 50 µm wide, horizontal channels are 100 µm wide, and the length of each serpentine channel is 6 mm. (B) Six-inlet device: channels are 100 µm wide, injection ports are 1 mm in diameter, and the exit channels are 3 mm × 10 mm.

polymerization, and standard photolithography.19 Standard photolithography was chosen for depositing chromium in a grid pattern, because this method leads to the formation of a permanent barrier, allowing the substrate to be used multiple times. For the secondary level of patterning, the aim is to vary the membrane composition at different lateral positions to control the spatial density of the cell-targeting peptides. Photolithographic patterning, microcontact printing, direct pipetting, and microfluidic patterning are some of the techniques that have been proposed19 for developing surface composition patterns, with the latter method enabling modification of enclosed geometries. Two types of microfluidic devices were investigated with respect to promoting a secondary level of membrane patterning (Figure 3). The first device was developed by Whitesides et al.21,28 and was (28) Jeon, N. L.; Dertinger, S. K. W.; Chiu, D. T.; Choi, I. S.; Stroock, A. D.; Whitesides, G. M. Generation of solution and surface gradients using microfluidic systems. Langmuir 2000, 16, 8311-8316.

Figure 4. Fluorescent micrographs of Texas Red labeled vesicles under flow in a microfluidic network device (Figure 3A). (A) Mixing does not occur in the serpentine channels. (B) A very steep gradient is generated at the exit.

demonstrated to successfully produce various shape composition gradients of fluorescein isothiocynate (FITC) solutions. The second device is an extension of a converging flow configuration used by Boxer et al.29 to produce limited mixing of two vesicle solutions. Figure 4 depicts the TR concentration profile generated using a microfluidic network device with two inlets and eight outlets. The flow pattern inside the serpentine channels indicates that (29) Kam, L.; Boxer, S. G. Formation of supported lipid bilayer composition arrays by controlled mixing and surface capture. J. Am. Chem. Soc. 2000, 122, 12901-12902.

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Figure 5. Fluorescent micrographs of Texas Red labeled vesicles under flow (A) and bilayer gradient generated on a glass coverslip after removal of the elastomer channels (B), employing the six-inlet device (Figure 3B). The fluorescence intensity as a function of position is plotted for each case.

negligible mixing occurs between the converging streams. This is particularly evident at the exit of the serpentine channels, where the two streams completely separate (highlighted area, Figure 4A). Consequently, the resulting composition gradient extends over a very small distance and is characterized by a steep transition between the two extreme concentration values (Figure 4B). Furthermore, the gradient generated by this method is very similar to the gradient produced by a two-streamconverging flow. A key difference between the vesicle solutions used in this case and the FITC solutions that were originally used28 is the diffusion coefficient value; 100 nm size vesicles have an estimated diffusion coefficient that is equal to D ) 5 × 10-8 cm2/s, which is 2 orders of magnitude smaller than the diffusion coefficient of FITC. Therefore, the diffusive mixing rate between two vesicle solutions is 2 orders of magnitude smaller than the diffusive mixing rate of two FITC solutions, and a considerably longer contact time is required to achieve complete mixing. The contact time can be evaluated using mass transport principles28 and is approximately 100 s. For the length of each serpentine channel (6 mm), a flow velocity of