Review Article

Biotechnological applications of supersonic cluster beam-depositednanostructured thin films: Bottom-up engineering to optimizecell–protein–surface interactions

Ajay Vikram Singh1,2

1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180-35902Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180

Received 1 November 2012; revised 3 January 2013; accepted 4 January 2013

Published online 21 March 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34601

Abstract: Technological innovations in biomaterial sciences

harness nanoparticle (NP) production, manipulation, and dep-

osition with supreme precision, enabling the development of

industrial processes. This review first discusses the basic

components of this approach, introducing cluster sources,

experimental apparatus, and growth mechanisms for NP

formation. The second part of this review provides an

overview of how the nanoscale bottom-up engineering can

control protein adsorption, which in turn determines the fate

of nanostructured coating for prokaryotic and mammalian

(primary and stem) cell interactions. In addition, we briefly

address the implications of the cluster beam deposition tech-

nique for nanostructuration of biocompatible microdevices

and its potential as a facile coating method to promote

protein–surface interactions for microarray applications in

biotechnology. VC 2013 Wiley Periodicals, Inc. J Biomed Mater Res

Part A: 101A: 2994–3008, 2013.

Key Words: nanocluster, AFM, microarray, cell–surface inter-

actions, high throughput

How to cite this article: Singh AV. 2013. Biotechnological applications of supersonic cluster beam-deposited nanostructured thinfilms: Bottom-up engineering to optimize cell–protein–surface interactions. J Biomed Mater Res Part A 2013:101A:2994–3008.

INTRODUCTION

Nanoparticle (NP) applications have a long-stranding his-tory, with aerosol productions and robust steel coatingsdating back 2300 and 1800 years, respectively.1,2 In theindustrial realm, NPs have been used well before the adventof nanotechnology as both fillers (carbon in rubber tires)3

and additives (TiO2 in dye and paints).4,5 In the last fewyears, technological innovations and advances have ledresearchers to understand the complex interplay ofnanobiointeractions which stimulated the development ofmedicinal devices and drug-delivery systems based on NPs.6

However, further progress depends on fabricating new pro-tocols for superior NPs productions with controlled size/shape and biocompatible surface functionalities. In the longterm, nanoscience and technology (NST) either complementand strengthen existing technologies or substitute obsoletetechniques.7

Like other paradigms of science and technology, thecommercial success of NST depends on funding opportuni-ties and minimization of cost involved in scaling up at anindustrial level. The prerequisite of core manufacturing

areas containing NPs and nanomaterials in device platformneed capability of integrating different components in adefinite hierarchy defining structure and chemical composi-tions.7 This fact is closely associated with collaborativecommons of economic production technologies such assemiconductor industry, which improve in performance perunit cost.8 This could be achieved by synthesis of controlleddimensions with desired structure and chemical status ofNPs and meticulously position transfer on suitable matrices(bottom-up engineering).9 The care must be taken to main-tain a structure–function relationship of NPs in definedmatrices or areas. Lately, a technological breakthrough isachieved by manipulating and replicating micronanoscaleparticle in nonwetting templates (PRINT) to fabricate shape-and size-specific microparticles and NPs.10,11 Manipulationand controlled replications of PRINT technology put forwardthe ability to sort the objects in terms of a size or geometry,and positions. This in turn restores precise control overshape, size, surface stiffness, and modulus. Including PRINT,most of the controlled fabrication technologies are limitedto polymeric and/or biomolecular (protein/peptide)-specific

Correspondence to: A. V. Singh; e-mail: singha8@rpi.edu

Contract grant sponsors: Department of Biomedical Engineering (BME), Rensselaer Polytechnic Institute (RPI; AVS)

2994 VC 2013 WILEY PERIODICALS, INC.

NPs fabrication. However, inorganic metal and metal oxidesas biocompatible surface coating for implants and prostheticdevices in vivo, and lab-on-a-chip platforms for cell microar-ray/reverse transfection assays, yet to witness technologicalsuccess.12–14

Conventional route for nanostructuredmaterial productionNanostructured materials covers a vast area of materials atnanoscale as thin film metal and metal oxide coating forenergy applications and enhance mechanical stability ofsurfaces. Biocompatible transition metal oxides (e.g., gold,silver, titania, and zirconia, iron is excluded as it is toxic tocells15 are among the most popular nanostructured materi-als for creating biocompatible surfaces and functional bioen-gineered materials for promoting tissue, organs and cellularmatrix interaction.16 A fascinating prospect of engineeringbiopolymer via docking nanoscale peptides, proteins, carbo-hydrates, and lipid moieties makes them particularly suita-ble for tissue engineering and scaffold design. Anotheremerging area in nanomedicine which involves in vivoinvasive and noninvasive theranostic (therapy þ diagnosis)application17 for soft tissue is polymer thin films and versa-tile hydrogel.18 The efficient application of nanomaterialsdepends upon its synthesis routes; however, biologicalroutes are preferred over chemical routes due to cellular,immune, genotoxicity, and carcinogenesis consideration.Newly emerging techniques such as electrophoretic deposi-tion and conventional deposition of biomaterials as nano-layers via self-assembled monolayer, Langmuir–Blodgettfilms, and layer-by-layer assemblies provide precisionin coating at air–water interface.19,20 NP production ofdiverse inorganic materials using colloidal phase, wet lab,and gas phase procedure are other popular conventionalroutes.9,21,22 Among diverse protocols for the NPs produc-tion, gas-phase approaches are very popular due to massproduction via facile scaling route.23–26 These approachesprovide a high level of control on particle physicochemicalproperties such as surface charge, energy, roughness, andporosity with wettability composition.27 In situ synthesis inaerosols and postproduction thermal treatment to improveperformance are other factors attracting this route of metaland oxide NP synthesis. More interesting, surface manipula-tions with a resolution in the 100 nm range and below hasbeen demonstrated using contemporary physical vapor dep-osition (PVD) techniques.28,29

In current review, we present and discuss features ofgas-phase nanocluster production and deposition relevantfor the fabrication of nanostructured systems. In particular,we will discuss the fundamental aspects related with super-sonic cluster beam depositions (SCBDs) for gas phase NPsproduction and possibility of patterning, and coupling toplanar technologies. In last part, we present an overview ofnanostructured thin films in biological applications.

SUPERSONIC CLUSTER BEAM DEPOSITION

Among different gas phase approaches to nanofabrication,the deposition of clusters from supersonic beams is gaining

increased attention extending the interest for this field frombasic to applied research.28 Aggregates ranging from a fewto thousands of atoms called cluster are produced and car-ried in a supersonic regime. Supersonic expansions haveseveral advantages for cluster manipulation over effusivebeams, which make this approach very powerful for thedeposition of nanostructured films. Due to the fact that clus-ter beam depositions favor the manipulation and positioningof NPs by the exploitation of NPs inertial properties, thereis great possibility to couple the SCBD with contemporarymicronanofabrication technologies.29,30

Many theoretical and experimental approaches havebeen developed to solve the problem of neutral NPs manip-ulation in the gas phase.31 The merging of solutions andmodels developed for aerosols, and for supersonic expan-sions has stimulated a novel and interdisciplinary route tosolve this problem.31 The solutions proposed and tested inthe last couple of decades have shown that the synthesis ofnanostructured materials with tailored structural and func-tional properties can be obtained by exploiting aerodynami-cally, focusing on seeded supersonic beams. In particular,the use of aerodynamic lenses32 allows an unprecedentedcontrol on NPs spatial and mass distribution, while keepingvery high fluxes and deposition rates.30 A further improve-ment in aerodynamic manipulation techniques for differentgas phase synthesis methods is opening new perspectivesfor the integration of gas phase NPs production into thelarge and well-consolidated arena of PVD technologies.33

Thanks to the use of collimated beams and stencil masks,patterned depositions with very high lateral resolution canbe obtained. For all these reasons, recently SCBD broughtforth great technological and theoretical interest consideringminimizing the cost/time involved in gas phase NPs produc-tion, manipulation and deposition of nanostructured materi-als using this technique.34,35

The SCBD apparatus houses mainly two chambers: anexpansion (source) chamber and a deposition chamber [seeFig. 1(A); for detail see Ref. 36]. The expansion chamber isequipped with a pulsed microplasma cluster source (PMCS),which represents a combination of different elements typi-cal of sputtering sources, and a laser vaporization clustersource37 as demonstrated in Figure 1(B,C). The clusterdeposition operation principle is based on the three mainevents:

1. The extraction of atoms from a solid sample2. The condensation of the atoms in clusters3. The escaping of the cluster from the source due to the

flow of an inert gas acting as the carrier of clusters.

PMCS and working principleThe group at CIMAINA (Milan, Italy) has developed thePMCS36,37 for the production of nanostructured thin films todeposit many samples in parallel minimizing both time andlabor. It consists of a ceramic cubic body [see Fig. 1(C)] inwhich a cylindrical cavity (of 1.8 cm3 volume) is drilled. Asolenoid-pulsed valve faces one side of the cavity, while theother side is closed by a removable nozzle. A channel

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crosses the cavity perpendicularly to its axis and hosts atarget (generally a rod) of the material to be vaporized.Before the valve opening, the cavity has the same pressureof the vacuum chamber that hosts the source (10�6 Torr),while the pressure of the inert gas (helium or argon typi-cally) at the back of the valve is 50 bars. Once the valveopens the large pressure difference causes an adiabaticexpansion which accelerates the gas atoms to overcome thesonic speed, thus, producing a supersonic cluster beam.38

This highly collimated gas jet impacts the rod (the cathode),and after a few microseconds from the valve opening apulsed voltage is applied to the two electrodes (800 V) fora typical duration of few tens of ls. This voltage ionizes theinert gas atoms, producing plasma. Ions are then acceleratedagainst the cathode (by the negative potential) ablating thetitanium rod by sputtering. Since only the gas atoms in theMach disk of the supersonic pulse are ionized,32,39 theplasma is spatially confined. As a result, the cathode rod,which is uniformly rotated during the operation of thesource, is ablated in a well-defined region. The cluster’s con-densation begins immediately after the ablation of the tita-nium atoms in a region very close to the cathode surface.Due to the higher kinetic energy of the sputtered atoms;they expand very rapidly from the hot ablation region, thusshortening the primary aggregation process. As the gas

atoms ionized by the polarization of the electrodes repre-sent only a small fraction of the injected gas (the gas pulselasts near 300 ls, while the voltage pulse is only 60 lslong), and due to the adiabatic cooling of the gas atoms dur-ing the supersonic expansion, the mixture of gas and pri-mary clusters thermalize at a rapid pace.40 In this cold mix-ture, the secondary aggregation takes place. The blend ofcarrier gas and titanium/zirconium clusters can then gradu-ally exit the source cavity through the nozzle, and due tothe high pressure difference between the inside and the out-side of the source, a supersonic expansion of the beamoccurs [see Fig. 1(D–F)].41

In a supersonic jet, the collision rate between the atomsbecomes extremely slow, representing a transition from afluid flow dynamics to a free molecular flow dynamics. Allthe processes based on binary collisions are thereforefrozen, and thus the cluster structures are quenched. Fur-thermore, supersonic beams are much more collimated thaneffusive beams, and offer the opportunity of further decreas-ing their divergence by the nozzle. An aerodynamicallyfocusing assembly consist of five aerodynamic lenses(drilled thin plates) mounted at the end of cylindricalspacers in a sequence [Fig. 1(B)]. This configuration allowsthe clusters to concentrate on the axis of the beam. More-over; this system aerodynamically selects the size of the

FIGURE 1. Schematic of supersonic cluster beam apparatus (A); 3D view of PMCS components working (B) and its internal configuration. PMCS

working principle exhibiting microplasma formation (D), thermalization of nanoclusters (E), and expansion as a supersonic beam (F). [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

2996 SINGH BOTTOM-UP ENGINEERING TO OPTIMIZE CELL–PROTEIN–SURFACE INTERACTIONS

clusters depending on the lens geometry and on the carrier,gas used.32,40 Considering the dimension of the clustermass, there could be three outcomes:

1. If the cluster mass is too big, due to their higher inertiain comparison to the gas atoms, cluster will follow trajec-tories that cause their impact with the focus, preventingexiting from the source.

2. If the mass is too small, the clusters will substantially fol-low the gas streamlines and no focusing will happen.

3. For the proper mass range cluster with a diameter closeto a few tens of nanometers, the trajectories will be con-centrated along the beam axis. The mass range is deter-mined by the focus geometry and by the gas pressure inthe cavity.

As shown in Figure 1(D,E), at step D, ablation of a tita-nium rod takes place by an argon plasma jet, ignited by apulsed electric discharge. After the ablation, TiO2 (or anyother metal oxide) ions thermalize with argon and condenseto form clusters. The mixture of clusters and inert gas isthen extracted in the vacuum through a nozzle to form aseeded supersonic beam, which is collected on a physicalsubstrate located in the beam trajectory. The use of super-sonic beams of clusters allows an excellent control of thecluster mass distribution and kinetic energy with the possi-bility to obtain a highly collimated beam (due to aerody-namically focusing assembly) and very high depositionrates.31,42 The mass range is determined by the focalizer ge-ometry and by the gas pressure in the cavity. Exploitingsimple aerodynamic effects, one can control the divergenceof the supersonic beam and the mass distribution of theparticles.31,43 The size of the exiting nanoclusters dependson the lens geometry and on the carrier gas used. Simply,using stencil masks, patterned films with very high lateralresolutions can be obtained, and this makes SCBD compati-ble with the other planar microtechnologies.42 In a super-sonic jet, the collision rate between the atoms becomesextremely slow, representing a transition from fluid flowdynamics to free molecular flow dynamics. All the processesbased on binary collisions are therefore frozen, and thus thecluster structures are quenched. This allows a low-energyrandom stacking of NPs on the substrate: the particles donot suffer significant fragmentation at the impact, and there-fore, the resulting film keeps memory of the precursorclusters, hence this kind of material is also referred to as acluster assembly. Therefore, SCBD consists in a randomstacking of NPs that leads to materials characterized by alower density with respect to the one shown by filmsobtained from highly energetic particles.34

PMCS-SCBD as an innovative toolto produce ns-TiO2 filmsThe control of film nanotopography and coexisting capabil-ities to regulate physicochemical properties can openstimulating perspectives for applications in surface coatingin biomedicine.

Recently, we have demonstrated that bacterial coloniza-tion on biomaterial surface can be successfully controlled

by tuning the surface nanotopography using SCBD tech-nique.44 Moreover, facile operation and ambient depositionconditions in SCBD make it technology of choice not onlyfor thin film coating [Fig. 2(A–H), left panel] but also inte-grate contemporary micronanofabrication approaches, suchas colloidal lithography [Fig. 2(I–Q), right panel].45 Albeitcomplex, objects characterized at different scales anddimensions are hierarchically organized in biological sys-tems.44 The study of nanostructured titanium oxide (ns-TiO2) thin films production using argon as carrier gas beganapproximately 10 years ago. Cluster-assembled ns-TiO2 filmshave a semiconducting behavior that was initially studiedfor application in catalysis and sensors.30,45–47 Recently, webegan to study and to use ns-TiO2 thin films as a substratefor biological applications because it is possible to use themwith optical instruments, like phase contrast microscopy orconfocal microscopy,48,49 since ns-TiO2 argon thin films areoptically transparent with an estimated refraction index of1.7.50 Also ns-TiO2 thin films have structure constituted bynanocrystals embedded in an amorphous material whichshow good adhesion on various substrates (e.g., glass, mica,and polymer). It is an important parameter for the evalua-tion of the successful performance of cluster-assembledcoatings for various technical and technological applica-tions.51,52 Through the use of a device manipulator and ras-tering, it is possible to produce films whose thickness andcharacteristics are uniform over an extended area, by slidingthe substrate in the plane perpendicular to the beam. Weobtain, on micro- to nanometer scale uniform layer ofns-TiO2 in which grain features are the result of growthdynamics, which follow a ballistic aggregation process, typi-cal of low mobility deposition regimes, provided by lowenergy deposition on cold substrates.30,53

Physical and chemical properties of the clusterassembled filmsAtomic force microscopic (AFM) images of a cluster-assembled TiO2 (left panel) films with nanoscale roughnessare shown in Figure 2(A–H). At the nanoscale, both thefilms expose a granular surface with a grain diameter rang-ing from few up to 20 nm mimicking to that of extracellularmatrix (ECM). This is the result of ballistic deposition ofsize-dispersed clusters, which impinge on the surface andstick together. The cluster growth mechanism leads to ahighly porous material, with a large specific area and asmall average surface slope despite the large surfaceroughness.54

The nanostructure is characterized by nanocrystallineregions embedded in an amorphous matrix. The nanograinsare randomly assembled to constitute a porous structuresuch as those typical of the ballistic aggregation regime, thedensity of the films being roughly half of the correspondingbulk phase (2.5–2.7 g/cm3 against 3.9–4.3 g/cm3 for bulkTiO2). As reported in previous works49 the surface of anas-deposited cluster assembled TiOx is characterized by aremarkable abundance of Ti-3d defect states in the bandgap related to surfacing oxygen vacancies and under coordi-nated titanium atoms.55 These states are known to favor the

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dissociative adsorption of water molecules and to play arole in the chemisorptions of a variety of organic mole-cules.56–58 Despite the mean clusters’ diameter, nanostruc-tured films are actually comparable with the thickness ofthe native oxide film grown on pure titanium,55,58 as the de-posited cluster assembled titania exhibit a surface muchmore under-stoichiometric (TiOx). However, post depositionannealing in a dry air reveals surface stoichiometric strictlyclose to MO2 for inorganic materials such as zirconium andtitanium (M: Ti/Zr).59,60

Biotechnological application of nanostructuredthin filmsBiocompatible substrate for primary, cancerous cell cul-ture, and stem cell expansion. Referring to biocompatibil-ity of inorganic and polymeric materials, over the years,many definitions have been offered, nevertheless, updated

manifesto and biocompatibility definition for the 21st cen-tury smart biomaterials, as stated by Prof. BD Ratner ‘‘Bio-compatibility refers to the ability of a material to locallytrigger and guide non-fibrotic wound healing, reconstructionand tissue integration’’.61 In true clinical in vivo set up, it fol-lows that biocompatibility is not just a property of the ma-terial, but it implies the interaction between the materialand the host relatively to a precise function.61,62 This inter-action is strictly connected to the protein-adsorption pro-cess, as cells or any other biomolecules never interactdirectly with the surface. Indeed, as soon as a biomaterialsurface comes into contact with blood, serum or any otherbiological fluid, it is immediately covered by proteins thatform an adsorbed layer. In other words, the interactionbetween a material and the biological environment is medi-ated by the protein adsorption process.58,63,64 More relevantliterature explaining the role of nanostructured surfaces in

FIGURE 2. Cluster beam deposition as a tool for controlled synthesis of nanostructured thin films deposition (left panel) and integrating it with

contemporary planar technologies (right panel). (A–D) Representative height maps in three-dimensional view of ns-TiO2 films produced by

SCBD with increasing thickness (50, 100, 200, and 300 nm)44; (E–H) representative surface profiles exhibiting variations in root mean square

roughness (Rq), specific area (Aspec), correlation length, skewness, and kurtosis, as well as in pore width and depth distributions, as discussed

in the main text. (I–Q) Schematic presentation of the different steps of the NSL-SCBD-based micro- and nanopatterning.45 (J) Glass cover slip as

the starting substrate. (J) Drop-coated FITC-BSA (green) and spin-coated polystyrene microspheres (light blue); the microspheres form a closely

packed hexagonal lattice. (K) ns-TiO2 coating over microspheres obtained by SCBD (yellow). (L) ns-TiO2 micro/nanopatterns generated by soni-

cation in DI. (M) Plating of bacterial cells limits the bacterial interaction with nanotriangles. Right panel (N): AFM image of micro/nanopatterns

of ns-TiO2 þ BSA obtained with 3 lm microspheres (60 lm � 60 lm). (O) Magnified topographic image of (P) showing hexagonal distribution of

triangular nanopatterns (ns-TiO2; 15 lm � 15 lm; z scale: 50 nm). (Q) Magnified image of the ns-TiO2 triangle inside the encircled area of image

(G) showing granular morphology of ns-TiO2 (500 nm � 500 nm; z scale: 50 nm). (D) Overlay of reflectance and fluorescence images showing

both the reflective ns-TiO2 islands and the precoated FITC-BSA underneath, visible as the green fluorescent signal (scale bar: 10 lm). [Color fig-

ure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

2998 SINGH BOTTOM-UP ENGINEERING TO OPTIMIZE CELL–PROTEIN–SURFACE INTERACTIONS

proteins and biointerfacial interactions which are beyondthe scope of this review can be found elsewhere.65,66

A recent work characterized the biocompatibility of ns-TiOx films produced by the deposition of a supersonic beamof TiOx clusters via analyzing different interactions (van derWaals, electrostatic, and chemical bonding) between AFMtip and surface.53 The findings corroborate past andcurrent cellular studies showing the new material is highlybiocompatible, and it supports normal growth and adhesionof immortalized, tumor and primary cells, and stemcells without coating with ECM proteins (Fig. 3).41,67,68

Therefore, ns-TiOx is proposed as an optimal substrate fordifferent applications in cell-based assays (cell microarray),biosensors, or microfabricated devices for medical applica-tions69–72 (Table I). The size of the clusters composing thens-TiOx film as well the size of many ECM proteins is of theorder of a few nanometers. As an immediate implication, ona sufficiently small scale (i.e., few hundreds of nanometers)the surface morphology of cluster-assembled ns-TiOx canmimic the natural ECM environment. This topography pro-vides a synthetic cue for ECM protein adsorption from cellculture media, thus, generating biochemical cue with correctorientation and clustering of integrin proteins, promotingcell–ECM interaction.

Given that the topographic features of the substrate playan essential role, the morphology of the cluster assembledns-TiOx may favor the interaction of cells with its surface.48

Therefore, it is of extreme interest to quantitatively charac-terize the interaction of this new material with proteins, asthis is the basis for controlling cell–surface interactions.67

Considering our recent study on nanostructured zirconiumoxide, to the best of our knowledge, bottom-up approach ofzirconia film synthesis in form of nanoclusters, using PMCStechnique is truly novel with defined nanoscale physical andchemical properties.9 The nanoscale morphology and sur-face topography of zirconia shares much similarity with thetitania except surface chemistry. Titanium oxides films haveversatile chemistry as described in above sections, whereaszirconium oxide behaves like almost bioinert surfaces.Human osteosarcoma cell line (MG-63) and Pheochromocy-toma (PC12) cell lines seeded on the cubic ns-ZrOx filmsmade by the bottom-up approach demonstrated precisesensing of nanoscale roughness of different zirconia filmswith increasing focal adhesion complex formation (Fig. 4).9

Application of nanostructured film in designing cell andprotein microarray. Living-cell microarrays are powerfultools for functional genomics and drug discovery.70–72 Inthis perspective, new approaches have been proposed tophenotype screening through the creation of living-cellmicroarrays, which exploit the ‘‘reverse transfection’’ of com-plementary DNAs or small interfering RNAs or the ‘‘reverseinfection’’ by lentiviral vectors to allow the simultaneoushigh-throughput analysis of the function of many differentgenes on a glass slide.71,73,74 However, despite severalattempts to improve this technology, it is still a challenge toobtain microarrays of cells with competence to overexpress-ing or downregulating specific genes to address complex

phenotypes.75–77 The bottleneck of technology relies in effi-cient immobilization of molecule of interest on a putativesurface, exhibiting correct functional state to be expressedin living cells.78–80 Other than this, there is a need of experi-mental protocols to fabricate substrate topography andchemistry on an artificial substrates which ‘‘mimic’’ theextracellular environment surrounding cells in vivo, likely topresent more appropriate physiological cues to culturedcells.81,82 In this regard, researchers immobilized viral vec-tors on a ns-TiO2 film obtained by depositing a supersonicbeam of titania clusters on a glass substrate. The SCBD-based technique demonstrated the validation of the retrovi-ral cell array by overexpression of green fluorescent protein(GFP) reporter genes in primary and cancer cells, and byRNA interference of p53 in primary cells by analyzingeffects in cell growth (Fig. 5). It has been demonstrated thatns-TiO2 retroviral arrays are an enabling tool for the studyof gene function of families of genes for complex pheno-types and for the identification of novel drug target.83

After the successful completion of the Human GenomeProject, the researchers involved in Human Proteome Orga-nization are looking forward in exploring a global HumanProteome Project, which is designed to map the entirehuman protein set (proteome).84 However, analysis of pro-tein expression and functions of human proteome data baseavailable has revealed there is tremendous complexity andvariability involved in the individual amino acids in proteinsequences.85,86 This inherent complexity of the human pro-teome has encouraged the development of sophisticatedmultiplexed technologies for more appropriate methods ofanalysis. As a consequence, the trend of detection technolo-gies has moved from low-throughput analytical methodsuch as enzyme-linked immunosorbent assay, Western blotto ‘‘high-content/high-throughput’’ approaches.87 In this con-text, the development of novel high throughput miniaturizeddevices, such as biochips and microarrays, can offer a validapproach to interrogate the high content proteome by multi-plexing the information at a reasonable cost.87,88 Due to thisfact, other than living cell microarray, protein microarraytechnologies are rapidly expanding to fulfill current needsof proteome discovery for disease management.87,89,90

As explained in the previous sections, ns-TiOx surface ischaracterized by morphology at the nanoscale that can betuned to modulate specific biomolecule–material interactions.58

It is important to note that topographical features withdimensions similar to the surface bound biomolecule (�10nm) can significantly affect their morphology and activityfor cell–surface interactions.56,91 Carbone et al.50 presenteda systematic characterization of ns-TiOx coatings as proteinbinding surfaces for antibody microarray using SCBD pro-duced nanostructured film. Comparing the performances ofthe coating with those of the most common commercialsubstrates in protein and antibody microarray assays, thismethod demonstrated equivalent efficiency. Through a ro-bust statistical evaluation of repeatability in terms of coeffi-cient of variation analysis, they demonstrate that ns-TiOxcan be used as a reliable substrate for biochips in analyticalprotein microarray application.48

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FIGURE 3. Immunofluorescence analysis of cytoskeleton and cellular adhesion markers on gelatin- and cluster-assembled nanostructured (ns)

TiO2-coated coverslips at short and long-term points. (A) MEFs, (B) Tig3-hTert, (C) U2OS, and (D) human primary melanocytes (small arrows

identify ‘‘long’’ and big arrows identify ‘‘short’’ focal adhesions). (E–G) In vitro endothelial differentiation of human circulating CD133Þ cells after

expansion on cluster-assembled ns-TiO2 layers and control condition, performed in coculture with a line of human umbilical vein endothelial

cells (HUVEC), used also as a positive control. 2 h after deposition, CD133Þ cells still can be distinguished from HUVEC cells used as feeder (A);

after 14 days of coculture, CD133Þ cells appear as endothelial structures and adherent mature endothelial cells CD31Þ (red) (B). Endothelial

marker expression has been verified through reverse transcriptase polymerase chain reaction analysis (C), both for CD133Þ cells previously

expanded on cluster-assembled ns-TiO2 layers (1, as-deposited; 2, annealed at 400�C) and in control conditions. (Reproduced with permission

from Refs. 51, 68.) [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

3000 SINGH BOTTOM-UP ENGINEERING TO OPTIMIZE CELL–PROTEIN–SURFACE INTERACTIONS

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116

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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | OCT 2013 VOL 101A, ISSUE 10 3001

Immunocell arrays technology based on antibody–nanostructured surface interaction. The knowledge of sig-naling pathways, which are triggered by physiological andpathological conditions or drug treatment, is essential for thecomprehension of the biological events that regulate cellularresponses.92,93 Recently novel platforms based on ‘‘Reverse-Phase Protein Arrays or Reverse Phase Protein Microarray(RPMA)’’ have proven to be useful in the study of differentpathways.94 However, it still lack the possibility to detectevents in the complexity of a cellular context.95,96 RPMA istechnically efficient in analyzing any fluid from cellularlysates or body fluids (serum, cerebrospinal fluid, urine, vit-reous, saliva, etc.) printed with micro- or nanospotter anddetected by secondary labeled antibody via chemilumines-cent, fluorescent or colorimetric assays. Unlike conventionalprotein array which are preferably designed with microscaleddot-blot platforms, RPMA utilizes either of the micro- ornanoscaled dot-blot platform which allows measurement ofprotein expression.97 The advantage of RPMA technologyover its contemporary counterparts is that it provides high

dimensional proteomic data in a high throughput, sensitiveand quantitative manner. An efficient modified version ofRPMA is immune cell array for multiplexed analysis of signal-ing pathways in drug response screening. An ‘‘immunocell-array’’ of cells on chip was developed using ns-TiOx as novelcoating substrate, where upon cell plating, culture, drugtreatment, and fixation, by spotting specific antibodies (Fig.6).98 On the chip, one can detect the localization and state ofhundreds of proteins involved in specific signaling pathways.By applying this technology to mammalian cells, the group atTethis s.r.l. analyzed signaling proteins involved in theresponse to DNA damage and identified a chromatin remod-eling pathway following bleomycin treatment.99 The technol-ogy developed manifests a new tool for the array-based mul-tiplexed analysis of signaling pathways in drug responsescreening.100 It also applies to the proteomics of profilingpatient cells, and ultimately for the high-throughput screen-ing of antibodies for immunofluorescence application.101,102

The conational microarray platforms utilizes surfacecoating to immobilize protein/peptide/antibody in chip

FIGURE 4. Zirconium oxide as novel coating for cell culture substrate. (A–F) Fluorescent immune labeling of adhesion complexes and stress

fiber organization of the Pheochromocytoma (PC12) cell line on nanostructured ZrOx films. PC12 cultured on ns-ZrOx (50–100–200 nm) sub-

strates formed well-organized stress fiber and numerous premature (large arrows) and mature adhesions (small arrows). (G–L) MG-63 cells.

Adhesions plaques were numerous and located predominantly at the cell periphery. (A, C, and E) cell clusters and an individual [squared cell

from cluster, (B, D, and F)] magnified (4�) view of PC12 cell. Similarly, (G, I, and K) MG-63 cell cluster and magnified (H, J, and L) view.9 [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

3002 SINGH BOTTOM-UP ENGINEERING TO OPTIMIZE CELL–PROTEIN–SURFACE INTERACTIONS

format with correct accessibility of the antibody active moi-eties or functional groups involved in complementary reac-tion. On the other hand, immobilizing whole cells on bio-compatible metal/metal oxide coated substrate expressingantibodies to capture antigen could be more productive inpreparing sensitive and specific microarrays for antigendetection. In a similar approach, nanostructured thin filmswere used for spotting bacteria that expose recombinantantibodies on their external surface directly on nanostruc-tured-TiO2 or epoxy slides [purification-independent micro-array (PIM)]. This is a simple and reliable alternative forpreparing sensitive and specific microarrays for antigendetection. Variable domains of single heavy-chain antibodiesagainst fibroblast growth factor receptor 1 (FGFR1) wereused to capture the antigen diluted in serum or bovine se-rum albumin (BSA) solution.103–106 The FGFR1 detectionwas performed by either direct antigen labeling or using asandwich system in which FGFR1 was first bound to itsantibody and successively identified using a labeled FGF. Inboth cases, the signal distribution within each spot was uni-

form and spot morphology regular. The signal-to-noise(S/N) ratio was extremely elevated, and the specificity ofthe system was proven statistically.104 The level of detectionof the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4 ng/mL and 10lg/mL. The microarrays prepared with bacteria exposingantibodies remain fully functional for at least 31 days afterspotting on the ns-TiO2 films. The current method is suita-ble for other antigens–antibody pairs and expects that itcould be easily adapted to further applications such as thedisplay of scFv and IgG antibodies or the autoantibodydetection using protein PIMs.106

Protein–surface interaction microarrays. Protein surfaceinteractions is the first of complex cascade of events regu-lates many phenomena at the nanobiointerface such asin vivo inflammatory responses, cell adhesion and differen-tiation on synthetic surfaces in vitro. Protein surface interac-tion microarray (PSIM) is a powerful method for studyingin details the interaction between proteins and

FIGURE 5. Retroviral microarrays for the overexpression of Enhanced Green Fluorescent Protein-EGFP-fused proteins; (A) 10� immunofluores-

cence panel of a 10 � 24 arrays of MCF10A cells infected by PINCO and PINCO GFP-NPM vectors in an alternate fashion. Colors as in panel (A)

blue, cell nuclei (DAPI staining); green, GFP fluorescence. (B) 20� magnification of fields from spots in a selected area of the array; GFP fluores-

cence only is shown. (C) 10� immunofluorescence panel of a 12 � 24 arrays of U2OS cells infected by the same vectors in an alternate fashion.

(D) 20� magnification of fields from spots in a selected area of the U2OS array; GFP fluorescence only is shown. (E) Scatter plots of GFP inten-

sity versus DAPI intensity in populations infected by PINCO (blue) and PINCO GFP-NPM (red) in MCF10A cells. The percentage of infected cells

is reported. (F) Same as the panel (E) for U2OS cells (Printed with permission Ref. 84). [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]

REVIEW ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | OCT 2013 VOL 101A, ISSUE 10 3003

nanostructured surfaces considering relevance of the media-tion adsorbed protein in biomaterial–cell interac-tions.12,67,106,107 PSIM is based on protein array technology,and it allows to study in one single experiment hundreds ofdifferent protein surface interactions. PSIM protocol consistsin spotting small-volume droplets (30 nL) of fluorescent-labeled proteins on the surface under investigation[Fig. 7(A)]. After incubation, blocking, washing, and drying,the amount of adsorbed proteins is evaluated by readingthe fluorescent signal with a commercial microarray scan-ner108 [Fig. 7(B)]. Using PSIM it is possible to compare, onthe same biomaterial sample, the amount of adsorbed pro-teins for a panel of proteins under various conditions suchas protein concentration and pH.109 Since the experimentcan be performed in parallel on several biomaterial samples,PSIM allows characterizing the role of surface synthesis pa-rameters in protein immobilization. PSIM is applicable tomany nanostructured surfaces, and it is a very flexiblemethod, in each of the 300 drops that can be spotted on aglass slide (25 mm � 75 mm), it is possible to change pro-

tein concentration, pH, buffer, salt concentration or proteintype. New spotting technologies give the possibility to study1200 different protein surface interactions in a singleexperiment. The high number of spots can also be used tomake replicates, and to produce very good statistics foreach interaction.110,111

The recent application of a high-throughput characteriza-tion approach to study the effect of surface nanoscale mor-phology on protein adsorption shed more light on this phe-nomenon. PSIM has been applied to ns-TiOx for carrying outsystematic characterization of the adsorption of proteins onnanostructured surfaces.64,67 With PSIM, it was possible tostudy the adsorption of a panel of three proteins (BSA, fibri-nogen, and streptavidin) on a ns-TiOx library composed byfive families of samples each with different surface morphol-ogy110 [Fig. 7(C–E)]. Thanks to PSIM high-throughput power,it was possible to design an experiment in which 1200 pro-tein–surface interaction were studied in order to reproduceprotein adsorption isotherms for the different proteins [Fig.7(C–E)]. Results confirm that nanoscale morphology signifi-cantly enhances the adsorption of proteins. Varying surfaceroughness from 15 nm to 30 nm, amount of adsorbed pro-teins increases up to 600%, well beyond the correspondingincrease of specific area.111 The application of PSIM, in com-bination with other quantitative methods, demonstrated thatthe increase is related to the formation of protein clusters incorrespondence of surface nanometric pores. The nanometricpores inducing protein clusterization112 are characterized byan aspect ratio higher than a threshold value (depends onthe protein characteristic).67,113

CONCLUDING REMARKS

The SCBD technique provides a cutting-edge solution tocontrol surface texture without changing chemistry (biomo-lecular information) at the biomaterial–tissue interface in aspatially controlled manner, which is greatly compromisedin both contemporary gas and chemical vapor depositiontechnologies. In the present review, we demonstrated thatthe cluster beam depositions technique is a vital tool fornanostructured film production with controlled surface to-pography, without changing surface chemical compositions.The most important property of the clusters produced atnanoscale via SCBD is the ECM mimicking topography,which opens a plethora of applications in biotechnology: anefficient surface coating to the cell culture substrate formammalian as well as prokaryotic (bacterial) cell adhesion.Many cell culture-based biological assay need surface coat-ing by ECM proteins or poly-L-lysine for cell adhesion whichoften alters surface chemistry. This in turn might affect thegenomic and proteomic profile of the cells. In addition, bio-compatibility of ns-TiOx/ZrOx films makes it future technol-ogy for the coating implants surface to promote interactionsof the prosthesis with surrounding tissue/organ in vivo. Theprotein surface interactions make it a valuable tool fordesigning HTP protein microarrays for functional genomicsand drug screening; immunocell array and ECM-based cellarrays. Moreover, SCBD compatibility with the planar tech-nologies makes it the technique of choice for the

FIGURE 6. (A) Mouse IgG spot morphology and background on the

ns-TiOx slide. The excellent morphology is demonstrated by surface

plot (B) and plot profile (C) of a selected 3 � 3 arrays of mouse IgG

spots detected by anti-mouse Cy3 antibody. (D) ns-TiOx slide back-

ground in comparison with epoxy and NC slides. (E) Schematic depic-

tion of the immunocell-array technology. Cells are cultured directly on

slides and processed at the appropriate time for immunostaining, and

images are acquired with an automated fluorescence microscope and

analyzed by imaging software (adopted with permission from Refs.

50,101. [Color figure can be viewed in the online issue, which is avail-

able at wileyonlinelibrary.com.]

3004 SINGH BOTTOM-UP ENGINEERING TO OPTIMIZE CELL–PROTEIN–SURFACE INTERACTIONS

future Bio-MEMS and Bio-NEMS device productions(Table I).114–116 Further exploration should be focused onintegrating SCBD with silicon-based microtechnologies toreach the next level of technological innovations.

ACKNOWLEDGMENTS

We thank for the support from Ahmad Abu-Hakmeh in revisingthe manuscript for syntax, grammatical errors, and specific re-vision to biological part.

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