On-chip cellular force sensors are fabricated from cantilever poly(3,4-ethylene dioxythiophene) filaments that visibly deflect under forces exerted at individual pseudopod-filament adhesive contacts. The shape of the deflected filaments and their ∼3 nN/μm spring constants are predicted by cantilever rod theory. Pulling forces exerted by Dictyostelium discoideumcells at these contacts are observed to reach ∼20 nN without breaking the contact

Flanders, Bret N.

Law, Bruce M.

McBride, Sean P.

Moore-Nichols, David

Paneru, Govind

Thapa, Prem S.

English

Marshall University

Marshall UniversityMarshall Digital ScholarPhysics Faculty Research PhysicsFall 9-2-2011Forces at Individual Pseudopod-Filament AdhesiveContactsGovind PaneruPrem S. ThapaSean P. McBrideMarshall University, mcbrides@marshall.eduDavid Moore-NicholsBruce M. LawSee next page for additional authorsFollow this and additional works at: http://mds.marshall.edu/physics_facultyPart of the Physics CommonsThis Article is brought to you for free and open access by the Physics at Marshall Digital Scholar. It has been accepted for inclusion in Physics FacultyResearch by an authorized administrator of Marshall Digital Scholar. For more information, please contact zhangj@marshall.edu, martj@marshall.edu.Recommended CitationPaneru G, Thapa P S, McBride S P, Nichols D M, Law B M and Flanders B N 2011 Forces at individual pseudopod-filament adhesivecontacts. Appl. Phys. Lett. 99 093702AuthorsGovind Paneru, Prem S. Thapa, Sean P. McBride, David Moore-Nichols, Bruce M. Law, and Bret N. FlandersThis article is available at Marshall Digital Scholar: http://mds.marshall.edu/physics_faculty/56Forces at individual pseudopod-filament adhesive contactsGovind Paneru, Prem S. Thapa, Sean P. McBride, David Moore-Nichols, Bruce M. Law, and Bret N. Flanders  Citation: Applied Physics Letters 99, 093702 (2011); doi: 10.1063/1.3628454 View online: http://dx.doi.org/10.1063/1.3628454 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/99/9?ver=pdfcov Published by the AIP Publishing  Articles you may be interested in Direct observation of dynamic force propagation between focal adhesions of cells on microposts by atomic forcemicroscopy Appl. Phys. Lett. 99, 263703 (2011); 10.1063/1.3672225  Fabrication of three-dimensional structures for the assessment of cell mechanical interactions within cellmonolayers J. Vac. Sci. Technol. B 28, C6K1 (2010); 10.1116/1.3511435  Morphological observation and adhesive property measurement on human ovary carcinoma cells by atomic forcemicroscopy J. Vac. Sci. Technol. B 27, 1370 (2009); 10.1116/1.3066057  Modeling the soft backing layer thickness effect on adhesion of elastic microfiber arrays J. Appl. Phys. 104, 044301 (2008); 10.1063/1.2968249  Adhesive contact driven by electrostatic forces J. Appl. Phys. 99, 054906 (2006); 10.1063/1.2178854    This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:128.135.100.108 On: Sun, 28 Dec 2014 19:31:35Forces at individual pseudopod-filament adhesive contactsGovind Paneru,1 Prem S. Thapa,1 Sean P. McBride,1 David Moore-Nichols,2 Bruce M. Law,1and Bret N. Flanders1,a)1Department of Physics, Kansas State University, Manhattan, Kansas 66506-2601, USA2Imaging and Analytical Microscopy Lab, University of Kansas, Lawrence, Kansas 66045, USA(Received 1 June 2011; accepted 28 July 2011; published online 2 September 2011)On-chip cellular force sensors are fabricated from cantilever poly(3,4-ethylene dioxythiophene)filaments that visibly deflect under forces exerted at individual pseudopod-filament adhesivecontacts. The shape of the deflected filaments and their 3 nN/lm spring constants are predictedby cantilever rod theory. Pulling forces exerted by Dictyostelium discoideum cells at these contactsare observed to reach 20 nN without breaking the contact. VC 2011 American Institute of Physics.[doi:10.1063/1.3628454]The forces that cells exert on substrates at adhesive con-tacts are critical to basic processes such as migration and celldivision. Pseudopods are exploratory appendages that crawlingcells like Dictyostelium discoideum, leukocytes,1 and breastcancer cells2 extend to probe the anterior substrate surface. Ad-hesive contact between the tips of the pseudopods and the sub-strate occurs frequently. These are the first contacts that thecell makes with the anterior substrate region. In addition toforce-application, environmental sensing occurs at these con-tact sites, influencing whether the cell alters or persists in itsdirection-of-migration.3 Despite the importance of pseudopod-substrate adhesive contacts in force transmission and environ-mental sensing, there has been little characterization of pseu-dopod-substrate adhesion at the single contact-level.The most widely used technique for characterizing cel-lular forces is the deformable substrate method where cell-induced wrinkling or marker-displacement of the elastic sub-strate is observed.4 A key advantage of this approach is thatthe substrate displacements occur in the imaging plane of theoptical microscope, permitting direct visualization of theprocess. However, the forces at the discrete adhesive contactsites are not directly measured but are instead extracted by anon-trivial modeling effort that correlates the measured sub-strate displacement-field with the inferred force field andwith the discrete sites.5 Off-substrate forces may be directlymeasured with an atomic force microscope (AFM) with ex-quisite precision6; however, visualization during such meas-urements, which are usually made in a plane normal to theoptical imaging plane, can be a challenge. Methodology forthe simultaneous visualization and direct characterization offorces exerted by individual pseudopodia is needed.To this end, we have fabricated on-chip cantileverpoly(3,4-ethylene dioxythiophene) (PEDOT) filaments thatvisibly deflect under forces exerted at individual pseudopod-filament contacts. PEDOT was chosen for its biocompatibil-ity.7 A typical 3.2 lm long, 400 nm wide pseudopod isindicated by the arrow in Figure 1(a). Direct characterizationof an individual pseudopod requires a probe of comparabledimension. To produce such filaments, the simple polymer-ization technique directed electrochemical nanowire assem-bly (DENA) was employed.8 Briefly, a 3 ll aliquot ofaqueous solution containing 0.01 M 3,4-ethylene dioxythio-phene and 0.02 M poly(sodium styrene sulfonate) was de-posited across the 30 lm gap between a pair of tapered,lithographic Au electrodes. The filament in Figure 1(b) wasproduced by applying a 63.5 V 20 kHz square wave voltagesignal across the electrodes to induce filament growth fromthe right electrode at a rate of 5 lm/s. The voltage signalwas terminated when the filament reached the desired lengthof 14 lm. The scanning electron microscopy (SEM) basedimage in the inset shows its lengthwise-averaged width to be3206 30 nm. Comparison to Figure 1(a) shows filament andpseudopod widths to be comparable, as desired. These fila-ments are rigidly bonded to the on-chip electrode but not tothe glass substrate and, hence, are cantilever structures.Type KAx3 D. discoideum cells were grown at 24 C inPetri dishes containing HL-5 culturing medium.9 Prior totransfer to the chips, 1000 ll of the cell-medium suspensionwas centrifuged for 10 s at 1.34 103 g. The HL-5 super-natant was replaced with 1000 ll of 12 mM phosphatebuffer, followed by gentle shaking for 1 min. This processwas twice-repeated before suspending the cells in 300 ll ofphosphate buffer and starving them for 4-6 h. To preventevaporation of the cell medium, a 60 ll hybridization cham-ber (Grace Biolabs) was adhered to the filament-laden chip.Before sealing with a transparent lid, 10 ll volumes of cellsuspension and phosphate buffer were deposited in the cham-ber. Typical cell surface densities were 103 mm2. A wait-ing time of 20 min following cell deposition was requiredfor the cells to settle, to begin migrating, and for a single cellto randomly contact the filament.FIG. 1. (a) Scanning electron micrograph of fixed D. discoideum cell withextending pseudopods. Scale bar¼ 1 lm. (b) Optical micrograph of PEDOTfilament grown by the DENA technique. FG : function generator. Scalebar¼ 10 lm. Inset: a scanning electron micrograph of a filament. Scalebar¼ 1 lm.a)Electronic mail: bret.flanders@phys.ksu.edu.0003-6951/2011/99(9)/093702/3/$30.00 VC 2011 American Institute of Physics99, 093702-1APPLIED PHYSICS LETTERS 99, 093702 (2011) This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:128.135.100.108 On: Sun, 28 Dec 2014 19:31:35Figures 2(a)–2(f) constitute a series of bright-fieldimages (collected on a microscope of 0.75 numerical aper-ture) of four D. discoideum cells migrating randomly on aglass slide. One of these cells contacts the cantilever filamentin Figure 2(a). This cell deflects the filament by exertingpulling force on it in Figures 2(b)–2(e), and releases it in Fig-ure 2(f) (see online video of this event, shown at 3  theactual rate). The shape of the pseudopod evolves throughoutthis event. We have observed 102 such events. Clearly,these filaments are flexible enough to deflect visibly uponcontact by a foraging cell (yet stiff enough to resist visiblethermal motion). In the small deflection approximation, theshape of a cantilever rod of length L and radius r that is bentby a force FA applied to its free end is described by10dFðxÞ ¼ FA6EIx2ð3L xÞ; (1)where I¼pr4/4 is the area moment of inertia of the solid cy-lindrical rod, E is Young’s modulus of the rod-material, andx denotes position along the rod length with respect to thefixed end. This function was fitted to the deflected filamentprofiles in panels (b)–(e), as designated by the white dashedcurves overlaid upon these micrographs. As discussed below,no adjustable parameters were used in achieving these fits.Figure 3(a) shows a scanning electron micrograph of aD. discoideum cell that was fixed shortly (5 s) after establish-ing pseudopod-filament contact.11 An enlarged view of thecontact region is shown in Figure 3(b). The surface of thepseudopod-tip is butted against the left side of the filament.The pseudopod does not encompass the filament. The twoother pseudopod-filament contacts that were characterizedalso exhibited butt-joint contact-structure. Deflection by apulling-force (as illustrated in Figures 2(b)–2(e)) that isapplied at a simple butt-joint implies adhesive contactbetween the joined pseudopod and filament surfaces. As withbetter characterized adhesive contacts like focal adhesions12and actin foci,13 adhesion is likely due to numerous trans-membrane cellular adhesion molecules (of undeterminedtype) that bind the substrate surface.Knowledge of the radius, Young’s modulus, and lengthof a solid, cylindrical cantilever rod permits calculation of itstheoretical spring constant kTh in the small deflection approxi-mation: kTh¼ 3EI/L3.10 To assess how well this simple equa-tion predicts the spring constants of PEDOT filaments, wehave used an AFM to directly measure the kF values of severalfilaments and compared these values to the corresponding kThvalues. The AFM (MFP-3D, Asylum Research) was calibratedby pressing its cantilever (NP-0, Veeco) against a hard glasssurface to quantify the cantilever deflection-photodiode volt-age relationship. The spring constant kC of this cantilever wasdetermined by the thermal method.14 Hooke’s law then givesthe magnitude of the elastic force exerted by the cantilever FCfor deflection dC: FC¼ kCdC. To measure the spring constantof a PEDOT filament kF, the AFM cantilever was pressedagainst an individual filament by lowering the AFM head bydistance Dz, as depicted in Figure 4(a).15 This measurementdeflects the filament by distance dF and yields a dC vs Dz pro-file (solid line in Figure 4(b)). The opposing forces exerted bythe filament FF and cantilever FC are equal in magnitude(Newton’s 3rd law); hence, kFdF¼ kCdC, where dF is the(unknown) filament displacement and FF¼ kFdF. Dz is relatedto dC and dF by Dz¼ dCþ dF, giving kF ¼ kCðDz=dC  1Þ1.FIG. 2. Series of optical micrographs ofa cantilever PEDOT filament (a) in itsneutral position 7 s after contact initia-tion by the pseudopod. Scale bar¼ 10lm; (b)-(e) while being deflectedupwards by the cell at times 37 s, 46 s,54 s, and 65 s, respectively; and (f) attime 105 s when the filament is back inits neutral position after release by thecell (where a video of this event isshown at 3  the actual rate). The whitedotted curves on panels (b)-(e) representthe deflected filament shapes predictedby cantilever rod theory (enhancedonline) [URL: http://dx.doi.org/10.1063/1.3628454.1].FIG. 3. (a) Scanning electron micro-graph of fixed cell with a pseudopod indirect contact with PEDOT filament.Scale bar¼ 2 lm. (b) Enlarged view ofcontact region. Scale bar¼ 500 nm.093702-2 Paneru et al. Appl. Phys. Lett. 99, 093702 (2011) This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:128.135.100.108 On: Sun, 28 Dec 2014 19:31:35The effects of AFM cantilever tilt by angle h (11 for all casesin this study) and off-end loading of the filament areaccounted for by corrective factors, yielding16kF ¼ kCðDz=dC  1Þ1 L DLL 3cos2h: (2)DL is the distance from the filament tip to the loading pointas measured via an internal optical microscope in the AFM.The spring constants of six different PEDOT filaments kFwere obtained by substituting into Eq. (2) the correspondingkC, L, DL, and dC=Dz values given in Table I. Each filamentwas characterized three times with each of three differentcantilevers whose spring constants varied significantly. Theaverages of these nine determinations for each of the six fila-ments are reported in column kF.To calculate the kTh values, we approximate the PEDOTfilament shapes as cylinders having radii equal to the length-wise averaged radii of the filaments. These SEM determinedvalues are reported with their standard deviations in Table I.Also, we have taken E¼ 2.0 GPa, the average of two recentdeterminations (1.8 GPa (Ref. 17) and 2.26 GPa (Ref. 18)) ofthe PEDOT Young’s modulus. Figure 4(c) plots kTh vs kF. Thehorizontal error bars denote the standard error associated withthe kF determinations; the vertical error bars result from propa-gation of radial standard deviations and 60.03 lm length non-uniformities in the kTh calculations. The solid line, the best-fitto these points (constrained to pass through the origin), has anear-unity slope of 1.08. Hence, the correlation between kThand kF is strong, indicating that cantilever rod theory providesreasonable predictions of the filament spring constants. Whileit lies beyond the scope of this letter to do so here, the PEDOTfilaments have lengthwise radial variations of 10%–20%, sothe success of kTh¼ 3EI/L3 deserves further examination.Figure 4(d) shows the filament deflection-values(unfilled circles) corresponding to frames 2(a)–2(f) (exceptfor the point at 83 s whose image is not shown in Figure 2).SEM analysis of this filament revealed a 220 nm lengthwiseaveraged radius and 16.0 lm length. Hence, as demonstratedabove, cantilever rod theory (kF¼ 3EI/L3) indicates a springconstant kF of 2.76 0.7 nN/lm; the sizable uncertainty isexpected given the highly nonlinear functionality of kF. Con-version of these dF-values to FA-values via Hooke’s law(FA¼ dFkF) yields the filled circles in Figure 4(d). (The errorbars reflect the propagated uncertainties of dF and kF.) Asthese data and Figure 2(e) show, FA reaches 21 nN withoutbreaking contact. The measured force values of 8, 13, 18,and 21 nN reported in Figure 4(d) (along withI¼ 1.8 1027 m4 and E¼ 2.0 GPa) were used to calculatethe parameters FA/6EI in Eq. (1) to fully determine the shapefunctions (white dotted lines) shown in Figures 2(b)–2(e),respectively. The close agreement with the measured shapesconfirms the usefulness of cantilever rod theory for predict-ing the elastic properties of these PEDOT filaments. In futurestudies, we will employ this methodology for the simultane-ous visualization and measurement of forces exerted at sin-gle pseudopod-filament contacts to articulate the factors thatdictate adhesion strength and duration.This work was partially supported by National ScienceFoundation grants PHY-0646966 (B.N.F.) and DMR-0603144 (B.M.L.).1P. Friedl, S. Borgmann, and E. B. Brocker, J. Leukoc. Biol. 70, 491(2001).2A. Muller, B. Homey, H. Soto, N. F. Ge, D. Catron, M. E. Buchanan,T. McClanahan, E. Murphy, W. Yuan, S. N. Wagner, J. L. Barrera, A.Mohar, E. Verastegui, and A. Zlotnik, Nature 410(6824), 50 (2001).3N. Andrew and R. H. Insall, Nat. Cell Biol. 9(2), 193 (2007).4A. K. Harris, D. Stopak, and P. Wild, Science 208, 177 (1980).5U. S. Schwartz, N. Q. Balaban, D. Riveline, A. Bershadsky, B. Geiger, andS. A. Safran, Biophys. J. 83, 1380 (2002).6G. Pfister, C. M. Stroh, H. Perschinka, M. Kind, M. Knoflach, P. Hinter-dorfer, and G. Wick, J. Cell Sci. 118(8), 1587 (2005).7S. M. Richardson-Burns, J. L. Hendricks, and D. C. Martin, J. Neural Eng.4, L6 (2007).8P. S. Thapa, B. J. Ackerson, D. R. Grischkowsky, and B. N. Flanders,Nanotechnology 20, 235307 (2009).9D. J. Watts and J. M. Ashworth, Biochem. J. 119, 171 (1970).10L. D. Landau and E. M. Lifshitz, Theory of Elasticity (Butterworth-Heine-mann, Oxford, 2000).11J. A. Araujo, F. C. Te´ran, R. A. Oliveira, E. A. A. Nour, M. A. P. Montene-gro, J. R. Campos, and R. F. Vazoller, J. Electron Microsc. 52(4), 429 (2003).12B. Geiger, J. P. Spatz, and A. D. Bershadsky, Nat. Rev. Mol. Cell Biol.10, 21 (2009).13K. S. K. Uchida and S. Yumura, J. Cell Sci. 117, 1443 (2004).14J. L. Hutter and J. Bechhoefer, Rev. Sci. Instrum. 64, 1868 (1993).15S. Yang and T. Saif, Rev. Sci. Instrum. 76, 044301 (2005).16R. S. Gates and M. G. Reitsma, Rev. Sci. Instrum. 78, 086101 (2007).17H. Okuzaki and M. Ishihara, Macromol. Rapid Commun. 24, 261 (2003).18D. Tahk, H. H. Lee, and D. Y. Khang, Macromolecules 42, 7079 (2009).FIG. 4. (a) Schematic of AFM-based determination of the filament springconstant. The gray curve denotes the AFM cantilever while the black curvedenotes the filament. (b) AFM cantilever deflection magnitude dC versusvertical position of AFM head z for pressure against a rigid surface (dashedprofile) and a PEDOT filament (solid profile). (c) Plot of the theoreticalspring constants of PEDOT filaments versus their measured spring constants.The solid line is the best linear fit to the points. (d) Cell enforced deflection(unfilled circles) and force (filled circles) measured during the eventdepicted in Figure 2.TABLE I. Measured properties of six PEDOT filaments and their associatedspring constants (in units of nN/lm).r (lm) L (lm) DL (lm) dcDz kc kTh kFkF0.726 0.08 13.24 1.3 0.59 450 550 490 4300.646 0.06 15.55 2.2 0.49 270 210 170 1800.526 0.06 12.18 1.8 0.77 110 190 240 2500.616 0.12 12.38 0.5 0.57 310 340 380 3600.286 0.06 12.04 2.2 0.42 58 16 20 240.296 0.06 9.50 0.5 0.90 55 39 40 45093702-3 Paneru et al. Appl. Phys. Lett. 99, 093702 (2011) This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:128.135.100.108 On: Sun, 28 Dec 2014 19:31:35

Forces at Individual Pseudopod-Filament Adhesive Contacts

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Forces at Individual Pseudopod-Filament Adhesive Contacts

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