Cell Adhesion and Detachment on Gold Surfaces Modified With a Thiol Functioalized RGD Peptide

8122019 Cell Adhesion and Detachment on Gold Surfaces Modified With a Thiol Functioalized RGD Peptide

httpslidepdfcomreaderfullcell-adhesion-and-detachment-on-gold-surfaces-modified-with-a-thiol-functioalized 111

Cell adhesion and detachment on gold surfaces modi1047297edwith a thiol-functionalized RGD peptide

Sang-Hee Yoon ab1 Mohammad RK Mofrad a

a Molecular Cell Biomechanics Laboratory Department of Bioengineering University of California Berkeley CA 94720 USAb Department of Mechanical Engineering University of California Berkeley CA 94720 USA

a r t i c l e i n f o

Article history

Received 5 May 2011Accepted 26 May 2011Available online 3 August 2011

Keywords

Cell adhesionCell detachmentElectrochemistryGoldPolyethylene glycolThiol-functionalized arginine-glycine-aspartic acid (RGD) peptide

a b s t r a c t

The dynamic nature of cell adhesion and detachment is critically important to a variety of physiologicaland pathophysiological phenomena Much however still remains uncertain and controversial about themechanochemical players and processes involved in cellular adhesion and detachment This leads to theneed for quantitative characterization of the adhesion and detachment of anchorage-dependent cellsHere cell adhesion and detachment up to subcellular level are examined using gold surfaces modi1047297edwith a thiol-functionalized arginine-glycine-aspartic acid (RGD) peptide A thiol self-assembled mono-layer (SAM) on top of the gold surfaces is reductively desorbed with activation potential to spatiotem-porally manipulate both cell adhesion and detachment This method maintains cells of interest living andintact during experiments making it possible to quantify cell adhesion and detachment as close aspossible to in vivo conditions Experimental characterizations for NIH 3T3 1047297broblasts are carried out witha focus on the following issues the effect of the size and geometric shape of gold surfaces on celladhesion the effect of cell con1047298uency cell shape and activation potential magnitude on cell detach-ment changes in the material properties of cells during cell detachment The 1047297ndings of this studyshould lead to better understanding of cellular dynamics in anchorage-dependent cells

2011 Elsevier Ltd All rights reserved

1 Introduction

Cell adhesion and detachment processes are mediated bya complex biomolecules from both sides of the cellematrix inter-face 1047297tting together like pieces of a three-dimensional puzzleWhen cells adhere to extracellular matrix (ECM) componentsintegrins are activated the activated integrins bind target ligandsthe bound integrins cluster together by changing their conforma-tion [1e3] The cytoplasmic domain of the clustered integrinsinteracts with focal adhesion (FA) proteins (eg talin focal adhe-

sion kinase vinculin and paxillin) to form FAs and then binds toactin 1047297laments (Fig 1 top) [4e6] Likewise the cell detachment orde-adhesion essential to many cellular dynamic phenomena (egcell migration) results from a concerted process involving thismolecular machinery composed of a host of extracellular trans-membrane and cytoplasmic proteins

Cell adhesion and detachment have profound effects on thebehavior and function of anchorage-dependent cells For examplecell adhesion and detachment are controlling parameters ina variety of biological phenomena (eg embryonic developmentcancer metastasis wound healing etc) and any abnormality in celladhesion and detachment accompanies diverse pathophysiologicalconsequences [13] The quantitative characterization of thedynamics of cell adhesion and detachment is therefore essential forunderstanding a variety of pathophysiological phenomena

Despite signi1047297cant progress over the past decade in character-

izing biomolecules and signaling pathways for cell adhesion anddetachment the biophysical details of cell adhesion and detach-ment still remain illusive [78] Initially the cell spreading andmigration experiments were used to characterize cell adhesion anddetachment These multistep biological phenomena howeverprovided their ambiguous and complicated roles incell adhesionand detachment [9] and failed to quantify the dynamic nature of cell adhesion and detachment Thus a variety of other experi-mental methods which can manipulate cell adhesion or detach-ment has been developed The techniques for cell adhesionmanipulation were photolithography [1011] e-beam lithography[12] dip-pen lithography [13] nanoimprint lithography [14] micro

Corresponding author Tel thorn1 510 643 8165 fax thorn1 510 642 5835E-mail address mofradberkeleyedu (MRK Mofrad)URL httpbiomechanicsberkeleyedu

1 Present address Wyss Institute for Biologically Inspired Engineering HarvardUniversity Cambridge MA 02138 USA

Contents lists available at ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e w w w e l s e v i e r co m l o c a t e b i o m a t e r i a l s

0142-9612$ e see front matter 2011 Elsevier Ltd All rights reserved

doi101016jbiomaterials201105077

Biomaterials 32 (2011) 7286e7296



contact printing [1516] elastomeric stencil [17] ink-jet printing[18] optical tweezer [19] electrophoresis [2021] and switchablesurface [22] the techniques for cell detachment manipulation were

hydrodynamic shear force assay [23e

29] centrifugal assay

[30e33] and micropipette aspiration [34e36] These techniquessuccessfully manipulated either cell adhesion or detachment Theywere however designed not to characterize cell behavior during

adhesion and detachment but to achieve cell adhesion or

Fig 1 An assay for spatiotemporally controlled manipulation of cell adhesion and detachment(A) A schematic of the assay composed of identical gold surfaces a SiO2 insulator

layer and a Pyrex glass substrate This assay is surface-modi1047297ed with thiol-functionalized RGD peptide (for the gold surfaces) and PEG (for the Pyrex glass substrate) to spatio-

temporally control cell adhesion and detachment (B) Spatiotemporal manipulation of cell adhesion Before cell adhesion manipulation RGD peptide is bound to all gold surfaces via

thiol and PEG is coated on the Pyrex glass substrate On cell adhesion manipulation the RGD peptide on a target gold surface (on the right side) is detached by activating the target

gold surface with activation potential followed by cell loading The loaded cells adhere only to an inactivated gold surface (on the left side) (C) Spatiotemporal manipulation of cell

detachment Before cell detachment manipulation cells are loaded and then adhere to all gold surfaces modi1047297ed with a thiol-functionalized RGD On cell detachment manipulation

the cells or one part of the cell is detached from a target gold surface (on the right side) with activation potential which yields the reductive desorption of a gold-thiol SAM (For

interpretation of the references to colour in this 1047297gure legend the reader is referred to the web version of this article)

S-H Yoon MRK Mofrad Biomaterials 32 (2011) 7286 e7296 7287



detachment for next-step applications (eg cell patterning (orpositioning) co-culture etc) Furthermore these techniquesfeatured unintended mechanical stimuli (cell denaturization [19]cell electrolysis [2021] and cell rupture [23e36]) to cells of interestand considerably deformed the cells before experiments thusresulting in inaccurate measurements [37] Recently electro-chemical methods have been developed to characterize the celladhesion and detachment of live and intact cells Jiang et almanipulate cell detachment using the electrochemical desorptionof an EG3-terminated SAM showing the direction of polarization of attached mammalian cells determined their motility direction [38]Inaba et al [39] noninvasively harvested anchorage-dependentcells by means of the electrochemical desorption of a SAM of alkanethiol for tissue engineering applications Guillaume-Gentilet al [40] switched the biointerfacial properties of micro-patterned

domains through the spatiotemporally controlled dissolution andadsorption of polyelectrolyte coatings for co-culture of twodifferent cells Although these previous electrochemical methodscontrolled either cell adhesion or cell detachment their success incharacterizing cell adhesion or detachment was limited to quali-tative results

A new assay is proposed here to manipulate both cell adhesionand cell detachment at cellular and even subcellular levels thusoffering a platform to quantify the adhesion and detachmentbehavior of anchorage-dependent cells which are still living andintact during experiments This assay has the following features inquantifying cell adhesion and detachment First of all the assaycharacterizes the cell adhesion and detachment behavior of livingand intact cells If the cells of interest are killed or receive anymechanical stimulus during experiments the method will have

Fig 2 Microfabrication and surface modi1047297cation of the assay (AeD) Microfabrication process starting with a 4-inch Pyrex glass wafer (A) patterning a CrAu layer through

photoresist patterning CrAu layer deposition and CrAu layer lift-off (B) patterning SiO 2 insulator layer through SiO2 layer deposition photoresist patterning SiO2 layer dry-

etching and photoresist removal (C) patterning the second CrAu layer using the same process used for the 1047297rst CrAu layer (D) (E) Assay for cell adhesion manipulation each

gold surface of which is an equilateral triangle square regular pentagon regular hexagon or circle (F) Assay for cell detachment manipulation (G) Assay for subcellular detachment

manipulation (H) Surface modi1047297cation process The assay is incubated with a PEG solution to make its Pyrex glass substrate cell-resistive (left) and then incubated with

a synthesized thiol-functionalized RGD solution to modify the gold surface cell-resistive (right) Scale bars of (E) and (G) are 10 mm and that of (F) is 100 mm (For interpretation of

the references to colour in this 1047297

gure legend the reader is referred to the web version of this article)




Fig 3 Characterizations of the surface modi1047297cations of the assay and potentiodynamic electrochemical characterization of the reductive desorption of a gold-thiol SAM (A) Contact

angles measured from a Pyrex glass substrate before (left) and after (right) PEG modi1047297cation The contact angle is changed from 257 15 to 615 38 through PEG modi1047297cation

(B) Cell (NIH 3T3 1047297broblast) loading on a Pyrex glass substrate before (left) and after (right) PEG modi 1047297cation showing the Pyrex glass substrate is changed from cell-adhesive to




a strong likelihood of disturbed results Secondly by employing anRGD peptide as a cell adhesion motif this assay provides cells witha microenvironment that is as similar as possible to the real in vivo

microenvironment This is because a microenvironment is one of the most dominant factors in determining cell adhesion anddetachment Moreover our assay can quantify both cell adhesionand cell detachment These features make our method unique incharacterizing cell adhesion and detachment on gold surfacesmodi1047297ed with a thiol-functionalized RGD peptide In this paper wecharacterize cell adhesion and detachment to address dependenceof cell adhesion on the size and geometric shape of gold surfacesdependence of cell detachment on cell con1047298uency initial cellshapeand activation potential magnitude changes in the materialproperties of cells detached at a subcellular level

2 Materials and methods

21 Spatiotemporal manipulation of cell adhesion and detachment

The assay was composed of an array of identical gold surfaces a SiO 2 insulatorlayer and a Pyrex glass substrate (Fig1 bottom)The gold surfacespatternedon thePyrex glass substrate provided sites for cell adhesion and detachment the insulatorlayer between gold surfaces and Pyrex glass substrate was designed to prevent

electrical short circuits during experiments as well as to minimize the distancebetween two neighboring gold surfaces The Pyrex glass substrate and gold surfaceswere modi1047297ed with polyethylene glycol (PEG) and thiol-functionalized RGD peptiderespectively The PEG modi1047297cation on the Pyrex glass substrate was designed toachieve a cell-resistive surface where hydrated neutral PEG chains stericallyrepulsed cells the thiol-functionalized RGD peptide modi1047297cation was intended tomake the gold surfaces cell-adhesive by tethering an RGD peptide to a gold surfacevia thiol compound The thiol compound created a SAM on the gold surfacesfollowing the spontaneous chemisorption

R S H thorn AuR S Au thorn 1=2H2 (1)

where R is a substituent [41] The thiol-functionalized RGD peptide therefore offereda cell (strictly speaking integrin)-binding site which was almost same as in vivo

microenvironment for cell adhesion and detachmentThe spatiotemporal manipulation of cell adhesion was implemented by selec-

tively detaching the RGD peptide from the gold surfaces with activation potentialof 09 to 18 V which yielded the reductive desorption of a gold-thiol SAM [42]

following the electrochemical reaction

R S Au thorn Hthorn thorn eR S H thorn Au (2)

After surface modi1047297cation with PEG and thiol-functionalized RGD peptide thethiol-functionalized RGD peptide was detached by applying activation potential toa target gold surface followed by soni1047297cation in a cell culture media and cell loading(Fig 1B) The loaded cells adhered only to the inactivated gold surface on whicha thiol-functionalized RGD peptide was placed because anchorage-dependent cellshad substantially more af 1047297nity for cell adhesion to RGD peptide (on the inactivatedgold surface) than to bare gold (on the activated gold surface)

The spatiotemporal manipulation of cell detachment using our assay was thesame as that of cell adhesion except the order between gold surface activation andcell loading When cells were loaded into the surface-modi1047297ed assay the loadedcellsgrafted to the RGD peptide On cell detachment manipulation (Fig1C) thecells(or one part of the cell) were detached from the assay by applying activationpotential which breaked the chemical bonding between gold and thiol When thedetached cellssensed no mechanicalanchorage(focal adhesion) to the gold surfaces

they started to retract by liquefying their cytoskeleton and changing the length of actin 1047297laments

22 Assay microfabrication

The assay was fabricated on a 4-inch Pyrex glass wafer with a thickness of 500 mm (Fig2A)After cleaning it with a piranha solution of 11vv 96 sulfuric acid(H2SO4) and 30 hydrogen peroxide (H2O2) for 10 min 1 mm-thick LOR resist (LOR 10A MicroChem Corp) was spin-coated at 4000 rpm for 40 s followed by soft

baking at 170 C for 5 min A 2 mm-thick positive photoresist (S1818 Rohm and HaasCorp) was spin-coated on the LOR resist at 4000 rpm for 40 s for double-layer resiststacking followed by soft baking at 110 C for 1 min An optical lithography wasmade to patternthe double-layerresist before e-beam evaporationprocess Thenextwasa depositionof 5 nm-thick chromium (Cr) adhesion layer and100 nm-thick gold(Au) layer on the wafer The CrAu-deposited wafer was immersed in an organicsolvent mixture (BAKER PRS-3000 Stripper Mallinckrodt Baker Inc) at 80 C for 4 hto lift off the double-layer resist (Fig 2B) Next a 2500 Aring-thick SiO2 insulator layerwas deposited by plasma-enhanced chemical vapor deposition (PECVD) process

This insulator layer was dry-etched to pattern through-holes for electrical inter-connection between 1047297rst and second CrAu layers (Fig 2C) Finally the second CrAulayer was deposited and patterned by using the same method for the 1047297rst one(Fig 2D) We fabricated three kinds of assays assay for cell adhesion manipulation(Fig 2E) where each gold surface has the same geometric shape (eg equilateraltriangle square regularpentagon regularhexagonor circle) andthe same size (eg9 mm2 25 mm2 64 mm2100 mm2 225 mm2 400 mm2 625 mm2 or 900 mm2) assay forcell detachment manipulation (Fig 2F) where each gold surface is 500 mm in lengthand 500 mm in width assay for subcellular detachment manipulation where eachgold line is 10 mm inwidth and 3 mm in distance between twoneighboring gold lines(Fig 2G) The microfabricated assay was wire-bonded in a chip carrier (Fig 2F)

23 PEG modi 1047297cation on Pyrex glass substrate

Before PEG modi1047297cation the microfabricated assay was cleaned with an oxygenplasma chamber (PM-100 Plasma Treatment System March Plasma Systems Inc) at100 W for 30 s The assay was then incubated with 2 vv m-PEG silane (Cl-PEG

silane Gelest) and 1 vv hydrochloric acid (HCl Fisher Scienti1047297c) dissolved inanhydrous toluene (Fisher Scienti1047297c) for 2 h (Fig 2H left) This Process was carriedout in a glove box under a nitrogen purge to avoid atmospheric moisture Theincubated assay was rinsed in fresh toluene and ethanol dried with nitrogen andcured at 120 C for 2 h The surface-modi1047297ed assay was stored in a vacuum desic-cator until the next surface modi1047297cation

24 Thiol-functionalized RGD peptide modi 1047297cation on gold surface

The gold surfaces of the assay were modi1047297ed with a thiol-functionalized RGDpeptide whose solution was synthesized by chemically combining cyclo (Arg-Gly-Asp-D-Phe-Lys) (c (RGDfK) C27H41N9O7 Peptides International Inc) with dithio-bis(succinimidylundecanoate) (C30H48N2O8S2 Dojindo Molecular Technologies Inc)as follows The c (RGDfK) was dissolved in dimethoxysulfoxide (DMSO Sigma-eAldrich)to get 1 mM aliquot andstored at 20 CThis reactionwas made in a glovebox under a nitrogen purge to protect the c (RGDfK) from exposure to atmosphericmoisture The maximum storage period of this solution was limited to 15 days

because this peptide easily lost its characteristics (eg anchor for avb3 integrin) Thedithiobis(succinimidylundecanoate) was also stored in 1 mM aliquot in DMSOat 20 CThis preparation wasalso done in moisture-freeenvironmentBefore goldsurface modi1047297cation both aliquots were warmed to room temperature in a desic-cator The c (RGDfK) aliquot was mixed with 1 vv triethylamine (Fisher Scienti1047297c)for 5 min to make all primary amines of a lysine amino acid unprotonated The samevolume of the dithiobis(succinimidylundecanoate) was added to the c (RGDfK)aliquot and then mixed well using a vortex mixer for 4 h to synthesize thiol-functionalized RGD peptide solution For the gold surface modi1047297cation (Fig 2Hright) the PEG-modi1047297ed assay was incubated with this solution for 1 h at roomtemperature to promote a spontaneous chemisorption between thiol and goldfollowed by soni1047297cationin DMSO for 3 min rinse in ethanol and phosphate bufferedsaline (PBS SigmaeAldrich) to eliminate an unbound thiol-functionalized RGDpeptide from gold surfaces The thiol made a SAM on the gold surfaces therebytethering an RGD peptide to the gold surfaces

25 Contact angle measurement

The contact angles of PEG-modi1047297ed Pyrex glass substrate and thiol-functionalized RGD peptide-modi1047297ed gold surface were measured with a contactangle measurement system goniometer (KRUumlSS582 KRUumlSS) A sessile drop modewas used to estimate the wetting properties of the above two surfaces The contactangles were averaged from 10 measurements The contact angle of PEG-modi1047297edPyrex glass substrate was compared to that of pure Pyrex glass substrate and thecontact angle of thiol-functionalized RGD peptide-modi1047297ed gold surface wascompared to those of bare gold surface and thiol-modi1047297ed gold surface

cell-resistive (C) Contact angles measured from bare gold (673 25 left) thiol-modi1047297ed gold (533 13 center) and thiol-functionalized RGD peptide-modi1047297ed gold

(246 28 right) (D) XPS survey spectrum of the gold surface modi1047297ed with a thiol-functionalized RGD peptide Detected are a gold peak from a gold surface a sulfur peak from

thiol a nitrogen peak from the amine group of an RGD peptide and carbon and oxygen peaks from the carboxylic acid group of an RGD peptide (E) Experimental setup for

potentiodynamic electrochemical characterization where the gold surface of the assay a platinum electrode and an AgAgCl electrode work as working counter and reference

electrodes respectively (F) Cyclic voltammetry measured from the gold surface modi1047297ed with a thiol-functionalized RGD peptide indicating the reductive desorption of the gold-

thiol SAM starts and 1047297nishes at 09 V and 155 V respectively and gets maximized at 14 V (For interpretation of the references to colour in this 1047297gure legend the reader is

referred to the web version of this article)




26 X-ray photoelectron spectroscopy (XPS) sample preparation and

characterization

An XPS survey scan was used to con1047297rm the existence of an RGD peptide linkedto gold surfaces via thiol after thiol-functionalized RGD peptide modi1047297cation AnXPS sample was prepared on a 4-inch silicon wafer e-beam evaporated with 5 nmCr adhesion layer and 50 nm Au layer This wafer was immersed for 2 h in theprepared thiol-functionalized RGD peptide solution A bare gold sample withoutany surface modi1047297cation was run as a control experiment The XPS analysis was

carried out with a customized ESCA (Omicron Nano Technology) at 1 108

Torrand all measured spectra were referenced to the position of the Au 4 f peaks Thescans were collected over a range of 20 eV around the peaks of interest with a passenergy of 235 eV

27 Potentiodynamic electrochemical characterization of reductive desorption of

gold-thiol SAM

A silicon wafer e-beam evaporated with 5 nm-thick Cr and 50 nm-thick Auwas modi1047297ed with a thiol-functionalized RGD peptide to prepare a cyclic vol-tammetry sample This thiol-functionalized RGD peptide-modi1047297ed gold surfacewas used as a working electrode while a platinum electrode and an AgAgClelectrode were used as counter electrode and reference electrode respectively(see Fig 3E) A voltage supplied by a DC power source (BampK Precision Corpo-ration) was applied between the gold-thiol SAM (or AgAgCl electrode) and theplatinum electrode The cyclic voltammetry was carried out in the Dubecco rsquos

phosphate buffered saline (DPBS (pH 74) Sigmae

Aldrich) solution with anEGampG potentiostat model 362 (AMETEK Princeton Applied Research) A scanstarted cathodically from 0 V to 2 V then annodically back to 0 V at a scan rateof 50 mV s1

28 Cell culture

The NIH 3T3 mouse embryonic 1047297broblast cell (NIH 3T3 1047297broblast) was culturedin Dulbeccorsquos modi1047297ed eagle medium (DMEM GIBCO) supplemented with 10fetal bovineserum (FBS GIBCO) and 1 Penicillin-Streptomycin (GIBCO) at 37 Cin a humidi1047297ed atmosphere of 5 CO2 The cell was passaged every 4 day as followsThe cell was washed 1 time in 1 PBS and trypsinized with05 Trypsin-EDTAsolution (SigmaeAldrich) After centrifuging the cell it was inoculated into a newPetri dish The NIH 3T3 1047297broblast with a passage number of 5e20 was used in theexperimental studies Before each experiment the surface-modi1047297ed assay wassterilized with 70 ethanol washed twice with 1 PBS and placed in a Petri dish

containing5 ml cell culture mediumwith a cell suspension of about 1 10

6

cellsmlFor subcellular detachment experiments the cell suspension concentration waschanged into 1 104 cellsml After 1 h unadhered NIH 3T3 1047297broblast was removedby additional wash in PBS followed by culture medium replacement All experi-ments were carried out after 24 h of cell loading in a self-designed chamber witha humidi1047297ed atmosphere of 5 CO2 and at 37 C

29 Immuno 1047298uorescence microscopy

Cells were 1047297xed with 4 formaldehyde solution (Fisher Scienti1047297c) in chilledPBS for 15 min The 1047297xed cells were permeabilized with 200 ml 05 Triton X-100(SigmaeAldrich) in PBS at room temperature for 10 min and were washed 3times with PBS followed by blocking non-speci1047297c binding using 3 non-fat drymilk in PBS at 4 C for 1 h and washing the cells once with PBS10 ml methanolicstock solution of rhodaminephalloidin (Biotium Inc) was diluted with 200 mlPBS with 1 Bovine Serum Albumin (BSA Fisher Scienti1047297c) for each assay The

assay was incubated with this solution for 20 min at room temperature andwashed 2 or 3 times with PBS For nucleus staining ProLong gold antifadereagent with DAPI (Invitrogen) was added into the cells Immuno1047298uorescentimages were obtained on an inverted 1047298uorescent microscope (Axiovert 200 CarlZeiss MicroImaging Inc)

210 Atomic force microscopy (AFM) indentation

The elastic modulus of the detached cytoskeleton of cells was measured with anAutoprobe CP atomic force microscope system (Park Science Instruments) Allmeasurements were made at a low-indentation-speed of 10 nms to suppressa viscous damping effect in quantifying the elastic modulus of cells The elasticmodulus was determined by measuring the de1047298ection of an AFM tip(HYDRA2R e100N Nanoscience Instruments Inc) which indents the detached cellThe AFM tip with a nominal spring constant of 0011 Nm was calibrated so that itsreal spring constant was determined as 0016 0005 Nm which was used in the

AFM indentation

3 Results and discussion

31 Surface modi 1047297cations

Two kinds of surface modi1047297cations made on the assay wereexamined by contact angle measurement and XPS survey Thecontact angle measured from a PEG-modi1047297ed Pyrex glass substratewas 615 38 (mean standard deviation averaged from 10measurements) whereas that measured from an untreated Pyrexglass substrate was 257 15 (Fig 3A) This shows thePEG-modi1047297ed Pyrex glass substrate is changed to have stronghydrophobicity through PEG modi1047297cation and consequentlyprevents cell adhesion (and protein fouling) The effect of PEGmodi1047297cation on cell adhesion was also investigated with cellloading tests using NIH 3T3 1047297broblasts (Fig 3B) The imagesobtained after 24 h of cell loading show the Pyrex glass substrate ismodi1047297ed into cell-resistive as intended A thiol-functionalized RGDpeptide modi1047297cation on gold surfaces was characterized using thesame method The contact angles measured from bare gold thiol-modi1047297ed gold and thiol-functionalized RGD peptide-modi1047297edgold were 673 25 533 13 and 246 28 respectively(Fig 3C) This modi1047297cation was also characterized by an XPS survey

scan The XPS survey spectrum measured from an RGDthiolAuinterface (Fig 3D) shows the following results the peaks of Au 4 sAu 4 p Au 4d and Au 4 f indicate the presence of e-beam evaporatedgold (Au(111)) the peaks of S 2 p12and S 2 p32 (right inset) meansulfur from thiol compound is in existence on the RGDthiolAuinterface the peaks of C 1s O 1s O KLL and N 1s (left inset)demonstrate there are carbon oxygen and nitrogen from theamine functional group (eNH2) and carboxylic acid functionalgroup (eCOOH) of an RGD peptide For reference hydrogenwas notdetected due to XPS working principle This XPS survey spectrumdemonstrates the thiol-functionalized RGD peptide modi1047297cationon gold surfaces is well made as designed and provides a cell-binding site as close as possible to in vivo microenvironment

32 Reductive desorption of gold-thiol SAM

The rapid desorption of a gold-thiol SAM with negative potentialwasinvestigated bymeasuringa cyclic voltammetryin DPBSsolution(pH74)usingathree-electrodesystemwherethegoldsurface(oftheassay) a platinum electrode and an AgAgCl electrode worked asworkingcounter andreference electrodes respectively(Fig3E)Thecyclic voltammetry was measured from the working electrode asa function of the applied potential with respect to the counter elec-trode (Fig 3F) At a section ldquoardquo (0 V to 09 V) the current wasnegligible This means the gold-thiol SAM impedes electron transferacross an electrolyteeelectrode interface due to no reductivedesorption of the SAM The reductive desorption of the SAM startedand 1047297nished at point ldquobrdquo (09 V) and point ldquodrdquo (155 V) respec-

tively This electrochemical reaction was maximized at 14 V Thismeasurement indicates the optimum potential to complete thiselectrochemical reaction is 09 V to 155V around 14 V Therelease of an RGD peptidefrom a gold surface by applying negativepotential to a gold-thiol SAM is also veri1047297ed

33 Cell adhesion of anchorage-dependent cells

The cell adhesion of anchorage-dependent cells was character-ized using our method NIH 3T3 1047297broblasts were detached fromgold surfaces modi1047297ed with a thiol-functionalized RGD peptide Todemonstrate the spatiotemporal manipulation of cell adhesion onthe gold surfaces we used an assay composed of two-by-one goldsurfaces where the gold surface on the left side was activated with

activation potential of 15 V but that on the right side was




inactivated When cells were loaded the loaded cells adhered onlyto the gold surface on the right side as shown in optical andimmuno1047298uorescent images (Fig 4A) This shows anchorage-dependent cells make integrin-mediated cell adhesion which hasmuch higher af 1047297nity for RGD peptide than for bare gold Thedependence of cell adhesion at a single cell level on the size andgeometric shape of a gold surface was also studied with forty typesof the assay Each array was designed to have twenty 1047297ve identicalgold surfaces whose size was 9 mm2 25 mm2 64 mm2 100 mm2225 mm2 400 mm2 625 mm2 or 900 mm2 and shape was a n-sidedregular polygon (n frac14 3 456NethcircleTHORN Fig 4B) NIH 3T3 1047297bro-blasts were loaded into the surface-modi1047297ed assays at a cellsuspension concentration of 1 106 cellsml As an index forquantifying cell adhesion at a single cell level a cell adhesion ratio(CA-ratio) de1047297ned as the ratio of the number of gold surfaces withcell adhesion to the total number of gold surfaces was measured asa function of the size and geometric shape of a gold surface(Fig 4C) The measured CA-ratio provides the following biologicalfacts First the CA-ratio in a single cell level is proportional to thesize of a gold surface and the minimum size of a gold surface forsingle cell adhesion is the diameter of a cell in a 1047298oating state(10 mm for NIH 3T3 1047297broblast) This indicates an anchorage-

dependent cell can make its FAs through cell-to-substrate interac-tionwhen it have a cell-binding site which is larger than (or at leastcomparable to) the size of a single cell Secondly a cell wants tomake its adhesion on the circumferential zone of a gold surfacerather than the central zone The CA-ratio is therefore proportionalto the circumferential length of a gold surface when each gold

surface has the same area This is likely because the circumferentialzone has its microbump dueto the side faces of the deposited CrAulayer so that provides additional cell-binding sites The micro-sizepattern is known to enhance cell adhesion [42] These 1047297ndings letus know how to design a gold surface for single cell adhesion Thesizeof a goldsurfaceshould belarger thanthe sizeof a single cell ina 1047298oated condition The geometric shape of a gold surface when itsarea is limited needs to be an equilateral triangle or square This isbecause the circumferential length S of an-sided regular polygonwith a side length of l and a surface area of A is inversely propor-tional to the number of sides n of the regular poly-gonS frac14 nl frac14 2

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffin A tanethp=nTHORN

p (Fig 4C inset)

34 Cell detachment of anchorage-dependent cells at a cellular level

The cell detachment of anchorage-dependent cells was exploredat a cellular level An entire cell (NIH 3T3 1047297broblast) was detachedfrom gold surfaces modi1047297ed with a thiol-functionalized RGDpeptide thus characterizing the effect of cell con1047298uency initial cellshape (projected area) and activation potential magnitude on celldetachment behavior The cell detachment experiments were per-formed for 1047297ve cell con1047298uency conditions of 0 (single or two cellsFig 5A) 25 (Fig 5B) 50 75 and 100 (fully con1047298uent cellsFig 5C) A cell detachment or de-adhesion time (CD-time) sde1047297ned as the time required for detaching 95 of cells from a goldsurface was measured as an index for evaluating cell detachmentThe CD-times measured from 1047297ve cell con1047298uency conditions withactivation potential of 15 V were 452 68 s 367 87 s

Fig 4 Characterization of the cell adhesion of anchorage-dependent cells on gold surfaces modi 1047297ed with a thiol-functionalized RGD peptide (A) Optical and immuno1047298uorescent

images of the spatiotemporal manipulation of cell adhesion A two-by-one assay where a left gold surface is activated but a right one is inactivated is used here Cells are stained for

actin with rhodaminephalloidin (red) and for cell nucleus with DAPI (blue) (B) Single cell adhesion to a variety of gold surfaces with different size and shape No cell adhesion is

made on 25 mm2-sized equilateral triangle gold surfaces (1047297rst from left) and 64 mm2-sized square gold surfaces (second) with a cell suspension concentration of 1 106 cellsml Cell

adhesions are made on 25 of 100 mm2-sized regular hexagonal gold surfaces (third) 25 of 225 mm2-sized equilateral triangle gold surfaces (fourth) 50 of 400 mm2-sized square

gold surfaces (1047297fth) and 75 of 625 mm2-sized circle gold surfaces (sixth) (C) CA-ratio as a function of the size and geometric shape of gold surfaces Insect shows the circum-

ferential length of polygons as a function of number of sides when the polygons have the same surface area Scale bar of (A) is 100 mm and those of (B) are 50 mm (For interpretation

of the references to colour in this 1047297





311 42 s 248 55 s and 211 35 s respectively (Fig 5D) Alldata were averaged from at least 10 measurements The measuredCD-time was inversely proportional to cell con1047298uency This indi-cates cell-to-cell interaction through which cells are connected toeach other at a fully con1047298uent condition has a correlation to celldetachment To detach a cell from a substrate we need to break its

cell-to-substrate interaction as well as cell-to-cell interaction toneighboring cells The detachment of one cell therefore allowsneighboring cells to be detached fast by providing a vertical forcethrough cell-to-cell interaction The relation between CD-time andinitial cell shape (projected area) Ap was also explored at a singlecell condition (Fig 5E) The projected area of a cell was calculatedfrom the optical images of adhered single cells before cell detach-ment The images were analyzed using an image processingprogram ImageJ (National Institutes of Health USA) The measuredCD-time had an inverse relation to the projected area of a cellrepresented as sethsecTHORN frac14 182 thorn 96999= Apethmm2THORN The projectedarea of a cell means the degree of tension stress within the cellcytoskeletal stress That is a cell with a large projected area hashigher cytoskeletal stress than a cell with a narrow projected area

Thus the cell with a relatively large projected area is detached fast

due to its high cytoskeletal stress indicating in-plane cytoskeletalstress is also closely related to out-of-plane cell behavior celldetachment The dependence of cell detachment on activationpotential magnitude was also examined A CD-ratio was measuredbycounting the ratio of the number of detached cellsto the numberof all cells as activation potential was changed from 13Vto 18 V

(Fig 5E) The CD-ratio was on the decrease as the activationpotential was on the increase This is because the reductivedesorption of gold-thiol SAM gets faster as activation potentialincrease (Fig 3F) The measured CD-ratio was monotonicallyincreasing with two in1047298ection points s-shape curve This clearlydemonstrates there is a large deviation in the integrin binding toECM and other cells which is related to cell-to-substrate andcell-to-cell interactions respectively

35 Cell detachment of anchorage-dependent cells at a subcellular

level

The subcellular detachment behavior of anchorage-dependentcells was also explored by our assay which releases one part of

a cell from the gold lines The assay composed of gold lines with

Fig 5 Characterization of cell detachment at a cellular level (A) Optical sequential images showing the spatiotemporal manipulation of the cell detachment of two cells (0 cell

con1047298uency) when activation potential is 12 V The measured CD-time is 452 68 s (B) Cell detachment of 25 con1047298uent cells whose average CD-time is 367 87 s (C) Cell

detachment of 100 con1047298uent cells whose average CD-time is 211 35 s (D) CD-time as a function of cell con1047298uency with a negative potential of 15 V where 0 cell con1047298uency

means single or two cells The measured CD-time is inversely proportional to cell con 1047298uency (E) CD-time s (sec) as a function of the projected area of a cell Ap (mm2) measured

from single cells with activation potential of 15 V The measured CD-time is inversely proportional to the projected area of a cell s frac14 182 thorn 96999= Ap (F) CD-ratio as a function

of activation time and potential measured from 100 con1047298uent cells




a width of 10 mm and a gap of 3 mm (Fig 2G) was used for thischaracterization When one part of a NIH 3T3 1047297broblast wasdetached with a single activation of 15 V the retracted itsdetached cytoskeleton within 16 s (Fig 6A) This fast retractioncompared to cell detachment at a cellular level is because thesingle cell has a higher strain (or stress) than cells in a con1047298uentcondition The single cell which has no constraint or interactionprovided by other cells stretches itself as wide as possible so that itis always under relatively high strain (or stress) In the nextsubcellular detachment one part of the single cell was sequentiallydetached with a series of activations where the 1047297rst activation(activation 1) was followed by the second one (activation 2) after16 s of the 1047297rst activation (Fig 6B) This subcellular detachmentwith sequential activations reveals repetitive activations to a cellwithin dozens of seconds do not damage the cell rsquos viability theamount of subcellular detachment is adjustable by the sequentialactivation of gold surfaces which are located below the cell of interest cell motility would be guided by spatiotemporal subcel-lular detachments on a large-scale assay

36 Changes in viscoelastic properties during subcellular

detachment

The changes in the viscoelastic properties of a NIH3T3 1047297broblastduring its subcellular detachment were also quanti1047297ed by detach-ing one part of the cell using this platform First of all the detachedcell was assumed as isotropic and viscoelastic The retractionmotion of the detached cytoskeleton of a cell was described witha standard linear viscoelastic solid model composed of two springsk1 and k2 and one dashpot c (Fig 6C top) From this modela normalized-strain ε at retraction step (STEP II Fig 6C (bottom))was mathematically expressed as [43]

ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e

k2c etht t 2THORN (3)

where ε0 isthe initialstrainof thecell atits protrusionstep (STEP I0 lt

t t 1) The changes in the viscoelastic propertiesof thedetached (andthen retracting) cytoskeleton were measured by combining (3) with

Fig 6 Subcellular detachment manipulation using the gold lines modi1047297ed with a thiol-functionalized RGD peptide and its applications to cellular dynamics characterizations (A)

Subcellular detachment using a single activation One part of a cell is detached and in turn the detached cytoskeletonstarts to retract (B) Subcellular detachment using a series of

activations One part of the cell is sequentially detached from the gold lines (C) Continuum model to describe the retraction of a detached cytoskeletonwhere the cell is assumed as

a homogeneous standard linear viscoelastic solid (top) Strain pro1047297le of an anchorage-dependent cell during cell adhesion and detachment (bottom) When a cell adheres to

a substrate etht frac14 0THORN the cell extends its protrusion and adheres again eth0 lt t t 1THORN the nucleus of the cell translocates etht 1 lt t t 2THORN one part of the cell is detached and retracts etht 2 lt t

t 3THORN (D) Normalized-strain as a function of time obtained from single cells which are detached at a subcellular level Arrows of (A) and (B) indicate the retraction direction of

detached cytoskeleton Scale bars of (A) and (B) are 100 mm (For interpretation of the references to colour in this 1047297gure legend the reader is referred to the web version of this

article)




two experimental results time-sequential images of the retractionmotion of the detached cytoskeleton (obtained from twenty cellsdetached at a subcellular level) and AFM indentation results on thedetached cytoskeleton From the time-sequential images of subcel-lular detachment the normalized-strain of the detached cytoskeletonwas described as ε

frac14 0799e0055t (Fig 6D) Based on the AFMindentationresults on 10samples [43] the totalelastic modulusof thedetached cytoskeleton ethk

total frac14 k

1 k

2=ethk

1thorn k

2THORNTHORN was determined as

1320 310 Pa These experimental results with (3) determined theviscoelastic properties of the detached cytoskeleton (k1 frac14 6567 Pak2 frac14 1652 Pa and c frac14 30037 Pa s Compared to the previous results(ktotal gt 4000 Pa and c lt 100 Pa s) obtained from the adhered (notdetached) cytoskeleton of 1047297broblasts [4445] the detached cytoskel-eton showeda three-timesdecreasein itselasticmodulusand a thirty-times increase in its damping coef 1047297cient This measurement suggeststhat a detached cytoskeleton becomes softer and consequently hasa remarkable increase in its damping coef 1047297cient aftera few seconds of subcellular detachment This phenomenon is likely owing to thegelesoltransitionof actin1047297lamentsat celldetachment which changesthe viscoelastic properties of the detached cytoskeleton The subcel-lular detachment depolymerizes the cross-linked network of actin1047297laments Thus the structural strength of the detached cytoskeleton

decreases but its viscous damping capacity increases This resultshows that the subcellular adhesiondetachment platform can befurther exploited forstudies of cellular rheology andforquanti1047297cationof viscoelastic properties of the cytoskeleton to supplement compu-tational modeling efforts [4647]

4 Conclusion

We have developed a method for the spatiotemporal manip-ulation of cell adhesion and detachment at cellular and evensubcellular levels thus quantitatively characterizing the adhesionand detachment behaviors of anchorage-dependent cells on goldsurfaces modi1047297ed with a thiol-functionalized RGD peptide Ourassay composed of an array of identical gold surfaces patterned on

a Pyrex glass substrate is surface-modi1047297ed with a thiol-functionalized RGD peptide This assay manipulate cell adhesionand detachment using the reductive desorption of a gold-thiolSAM with activation potential of 09 V to 18 V while main-taining cells of interest living and intact In the experiments usingNIH 3T3 1047297broblasts cell adhesion is proportional to the size of thegold surface and is made on the circumferential zone of the goldsurface rather than the central zone These 1047297ndings lead to severalpropositions for gold surface design the gold surface for singlecell adhesion must be larger than (or at least comparable to) thesize of a single cell in a 1047298oating state the geometric shape of thegold surface when its area is limited needs to be an equilateraltriangle or square Cell detachment behavior at a cellular levelcharacterized here yields the following results cell-to-cell

interaction is one of the main factors which determine thevelocity of cell detachment a fully stretched cell with a relativelylarge projected area is detached fast indicating the in-plane stresswithin a cell has a correlation with an out-of-plane cell behavior(cell detachment) In the characterization on subcellular detach-ment the detached (and then retracting) cytoskeleton experi-ences a three-times decrease in its elastic modulus and alsoa thirty-times increase in its damping coef 1047297cient within a fewseconds showing cell detachment has a dynamic nature Extrap-olation of this method to other anchorage-dependent cells mighthelp us to investigate critical cellular function and behaviorthereby leading to a better understanding of cellular dynamicsOngoing work is focusing on more in-depth control of cell motilityby developing a large-scale assay to shed light on the dynamics of

cell motility Combined with molecular dynamics models [48e

50]

the proposed device for programmable subcellular adhesiondetachment will offer a platform for studies of molecular biome-chanics of the cell especially as related to mechanotransduction atthe integrin-mediated focal adhesions [5152]

References

[1] Geiger B Bershadsky A Pankov R Yamada KM Transmembrane crosstalk

between the extracellular matrix and the cytoskeleton crosstalk Nat Rev MolCell Biol 20012(11)793e805

[2] Park TH Shuler ML Integration of cell culture and micro-fabricationtechnology BiotechnolProg 200319(2)243e53

[3] Ridley AJ Schwartz MA Burridge K Firtel RA Ginsberg MH Borisy G et al Cellmigration integrating signals from front to back Science 2003302(5651)1704e9

[4] Beningo KA Dembo M Kaverina I Small JV Wang YL Nascent focal adhesionsare responsible for the generation of strong propulsive forces in migrating1047297broblasts J Cell Biol 2001153(4)881e8

[5] Zamir E Geiger B Molecular complexity and dynamics of cell-matrix adhe-sions J Cell Sci 2001114(20)3583e90

[6] Galbraith CG Yamada KM Sheetz MP The relationship between force andfocal complex development J Cell Biol 2002159(4)695e705

[7] Wiesner S Legate KR Faumlssler R Integrin-actin interactions Cell Mol Life Sci200562(10)1081e99

[8] Bershadsky A Kozlov M Geiger B Adhesion-mediated mechanosensitivitya time to experiment and a time to theorize CurrOpin Cell Biol 200618(5)472e81

[9] Palecek SP Loftus JC Ginsberg MH Lauffenburger DA Horwitz AF Integrin-ligand binding properties govern cellmigration speed throughcell-substratumadhesiveness Nature 1997385(6616)537e40

[10] Clark P Connolly P Curtis AS Dow JA Wilkinson CD Topographical control of cell behaviorI Simple Step Cues Dev 198799(3)439e48

[11] Chehroudi B Gould TR Brunette DM Titanium-coated micromachinedgroovesof different dimensions affectepithelial and connective-tissue cells differentlyin vivo J Biomed Mater ResA 199024(9)1203e19

[12] Lussi JW Tang C Kuenzi P-A Staufer U Csucs G Voumlroumls J et al Selectivemolecular assembly patterning at the nanoscale a novel platform forproducing protein patterns by electron-beam lithography on SiO2indium tinoxide-coated glass substrates Nanotechnology 200516(9)1781e6

[13] Lee K-B Park SJ Mirkin CA Smith JC Mrksich M Protein nanoarrays gener-ated by dip-pen nanolithography Science 2002295(5560)1702e5

[14] Hoff JD Cheng L-J Meyhoumlfer E Guo LJ Hunt AJ Nanoscale protein patterningby imprint lithography Nano Lett 20044(5)853e7

[15] Chen CS Mrksich M Huang S Whitesides GM Ingber DE Geometric control of cell life and death Science 1997276(5317)1425e8

[16] Lee NY Lim JR Kim YS Selective patterning and immobilization of biomole-cules within precisely-de1047297ned micro-reservoirs BiosensBioelectron 200621(11)2188e93

[17] Folch A Jo BH Hurtado O Beebe DJ Toner M Microfabricated elastomericstencils for micropatterning cell cultures J Biomed Mater ResA 200052(2)346e53

[18] Roth EA Xu T Das M Gregory C Hickman JJ Boland T Inkjet printing forhigh-throughput cell patterning Biomaterials 200425(17)3707e15

[19] Birkbeck AL Flynn RA Ozkan M Song D Gross M Esener SCVCSEL arrays asmicromanipulators in chip-based biosystems Biomed Microdevices 20035(1)47e54

[20] Ozkan M Pisanic T Scheel J Barlow C Esener S Bhatia SN Electro-opticalplatform for the manipulation of live cells Langmuir 200319(5)1532e8

[21] Rosenthal A Voldman J Dielectrophoretic traps for single-particle patterningBiophys J 200588(3)2193e205

[22] Lahann J Mitragotri S Tran T-N Kaido H Sundaram J Choi IS et alA reversibly switching surface Science 2003299(5605)371e4

[23] Truskey GA Pirone JS The effect of 1047298uid shear stress upon cell adhesion to1047297bronectin-treated surfaces J Biomed Mater ResA 199024(10)1333e53

[24] van Kooten TG Schakenraad JM van der Mei HC Dekker A Kirkpatrick CJBusscher HJ Fluid shear induced endothelial cell detachment from glass-in1047298uence of adhesion time and shear stress Med EngPhys 199416(6)506e12

[25] Garciacutea AJ Huber F Boettiger D Force required to break a5b1 integrin-1047297bronectin bonds in intact adherent cells is sensitive to integrin activationstate J BiolChem 1998273(18)10988e93

[26] Cargill RSII Dee KC Malcolm S An assessment of the strength of NG108-15cell adhesion to chemically modi1047297ed surfaces Biomaterials 199920(23e24)2417e25

[27] Kuo SC Lauffenburger DA Relationship between receptorligand bindingaf 1047297nity and adhesion strength Biophys J 199365(5)2191e200

[28] Kuo SC Hammer DA Lauffenburger DA Simulation of detachment of speci1047297-callyboundparticlesfromsurfacesbyshear 1047298owBiophysJ 199773(1)517e31

[29] Goldstein AS DiMilla PA Effect of adsorbed 1047297bronectin concentration on celladhesion and deformation under shear on hydrophobic surfaces J BiomedMater ResA 200259(4)665e75

[30] McClay DR Wessel GM Marchase RBIntercellular recognition quantitation of

initial binding events ProcNatlAcadSci U S A 198178(8)4975e

9




[31] Lotz MM Burdsal CA Erickson HP McClay DR Cell adhesion to 1047297bronectinand tenascin quantitative measurements of initial binding and subsequentstrengthening response J Cell Biol 1989109(4)1795e805

[32] Burdsal CA Alliegro MC McClay DR Tissue-speci1047297c temporal changes in celladhesionto echinonectin in the sea urchinembryo DevBiol1991144(2)327e34

[33] Burdsal CA Lotz MM Miller J McClay DR A quantitative switch in integrinexpression accompanies differentiation of F9 cells treated with retinoic acidDevDyn 1994201(4)344e53

[34] Sung KL Sung LA Crimmins M Burakoff SJ Chien S Determination of junctionavidity of cytolytic T cell and target cell Science 1986234(4782)1405e8

[35] Evans E Ritchie K Merkel R Sensitive force technique to probe molecularadhesion and structural linkages at biological interfaces Biophys J 199568(6)2580e7

[36] Shao J-Y Hochmuth RM Micropipette suction for measuring piconewtonforces of adhesion and tether formation from neutrophil membranes Biophys J 199671(5)2892e901

[37] Richards RG ap Gwynn I Bundy KJ Rahn BA Microjet impingement followedby scanning electron microscopy as a qualitative technique to comparecellular adhesion to various biomaterials Cell BiolInt 199519(12)1015e24

[38] Jiang X Bruzewicz DA Wong AP Piel M Whitesides GM Directing cellmigration with asymmetric micropatterns ProcNatlAcadSci U S A 2005102(4)975e8

[39] Inaba R Khademhosseini A Suzuki H Fukuda J Electrochemical desorption of self-assembled monolayers for engineering cellular tissues Biomaterials200930(21)3573e9

[40] Guillaume-Gentil O Gabi M Zenobi-Wong M Voumlroumls J Electrochemicallyswitchable platformfor the micro-patterning and release of heterotypic cellsheets Biomed Microdevices 201113(1)221e30

[41] Karp G Cell and molecular biology concepts and experiments New York John Wiley amp Sons 2005

[42] Dalton BA Walboomers XF Dziegielewski M Evans MD Taylor S Jansen JAet al Modulation of epithelial tissue and cell migration by microgrooves J Biomed Mater Res A 200156(2)195e207

[43] Yoon S-H Lee C Mofrad MRK Viscoelastic characterization of the retractingcytoskeleton using subcellular detachment ApplPhysLett 201198(13)133701

[44] Haga H Sasaki S Kawabata K Ito E Ushiki T Sambongi T Elasticity mappingof living 1047297broblasts by AFM and immuno1047298uorescence observation of cyto-

skeleton Ultramicroscopy 200082(1e4)253e8[45] Haga H Nagayama M Kawabata K Imaging mechanical properties of living

cells by scanning probe microscopy CurrNanosci 20073(1)97e103[46] Mofrad MRK Rheology of the cytoskeleton Annu Rev Fluid Mech 200941

433e53[47] Jamali Y Azimi M Mofrad MRK A sub-cellular viscoelastic model for cell

population mechanics PLoS One 20105(8)e12097[48] Lee SE Chunsrivirot S Kamm RD Mofrad MRK Molecular dynamics study of

talin-vinculin binding Biophys J 200895(4)2027e36[49] Golji J Mofrad MRK A molecular dynamics investigation of vinculin activa-

tion Biophys J 201099(4)1073e81[50] Golji J Lam J Mofrad MRK Vinculin activation is necessary for complete talin

binding Biophys J 2011100(2)332e40[51] Bao G Kamm RD Thomas W Hwang W Fletcher DA Grodzinsky AJ et al

Molecular biomechanics the molecular basis of how forces regulate cellularfunction Mol Cell Biomech 20103(2)91e105

[52] Mofrad MRK Kamm RD Cellular mechanotransduction diverse perspectivesfrom molecules to tissues New York Cambridge University Press 2010




contact printing [1516] elastomeric stencil [17] ink-jet printing[18] optical tweezer [19] electrophoresis [2021] and switchablesurface [22] the techniques for cell detachment manipulation were

hydrodynamic shear force assay [23e

29] centrifugal assay

[30e33] and micropipette aspiration [34e36] These techniquessuccessfully manipulated either cell adhesion or detachment Theywere however designed not to characterize cell behavior during

adhesion and detachment but to achieve cell adhesion or

Fig 1 An assay for spatiotemporally controlled manipulation of cell adhesion and detachment(A) A schematic of the assay composed of identical gold surfaces a SiO2 insulator

layer and a Pyrex glass substrate This assay is surface-modi1047297ed with thiol-functionalized RGD peptide (for the gold surfaces) and PEG (for the Pyrex glass substrate) to spatio-

temporally control cell adhesion and detachment (B) Spatiotemporal manipulation of cell adhesion Before cell adhesion manipulation RGD peptide is bound to all gold surfaces via

thiol and PEG is coated on the Pyrex glass substrate On cell adhesion manipulation the RGD peptide on a target gold surface (on the right side) is detached by activating the target

gold surface with activation potential followed by cell loading The loaded cells adhere only to an inactivated gold surface (on the left side) (C) Spatiotemporal manipulation of cell

detachment Before cell detachment manipulation cells are loaded and then adhere to all gold surfaces modi1047297ed with a thiol-functionalized RGD On cell detachment manipulation

the cells or one part of the cell is detached from a target gold surface (on the right side) with activation potential which yields the reductive desorption of a gold-thiol SAM (For

interpretation of the references to colour in this 1047297gure legend the reader is referred to the web version of this article)






























































characterization





gold-thiol SAM




28 Cell culture



6







AFM indentation


















p (Fig 4C inset)





















level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9
























































































characterization





gold-thiol SAM




28 Cell culture



6







AFM indentation


















p (Fig 4C inset)





















level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9










































































characterization





gold-thiol SAM




28 Cell culture



6







AFM indentation


















p (Fig 4C inset)





















level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9




































































characterization





gold-thiol SAM




28 Cell culture



6







AFM indentation


















p (Fig 4C inset)





















level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9































characterization





gold-thiol SAM




28 Cell culture



6







AFM indentation


















p (Fig 4C inset)





















level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9


































p (Fig 4C inset)





















level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9




































level














detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9
































detachment


ε frac14

εetht t 2THORN

ε0frac14

1

k2

k1 thorn k2

e











article)






total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9
































total frac14 k

1 k

2=ethk

1thorn k




4 Conclusion





50]


References

































9






















































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Cell Adhesion and Detachment on Gold Surfaces Modified With a Thiol Functioalized RGD Peptide

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