ISSN:1369 7021 © Elsevier Ltd 2010APRIL 2010 | VOLUME 13 | NUMBER 422

Chemical patterning in biointerface science

As society strives towards an improved quality of life, we care

for an ever increasing elderly population and attempt to combat

the increase in life-style associated health conditions, such as

diseases of the cardiovascular system. This drives researchers to

develop innovative biomaterials with new and improved surfaces.

In most cases the materials used to treat and diagnose disease

are in contact with biological fluids that contain proteins, which

spontaneously and irreversibly adsorb to surfaces. It is this surface

that cells contacting the material ‘see’, often triggering material

failure1-6. Cells respond to the biochemical signals contained

within the peptide motifs of the adsorbed proteins through extra

cellular matrix receptors such as integrins7. The consequences of

uncontrolled protein adsorption on man made surfaces can be

very severe indeed, resulting in not only device failure but also

additional health risks to patients8. Examples include:

1) attachment and colonisation of pathogenic bacteria to venous

catheters via adsorbed protein layers that can lead to high

patient mortality9;

2) thrombosis on the surfaces of cardiovascular devices such as

artificial hearts, catheters and prosthetic valves from platelet

adhesion and activation10;

3) fouling of hemodialysis membranes11; and

4) inflammatory responses that lead to restenosis following the

insertion of stents12.

On the other hand, the fields of tissue engineering13, biosensors

and diagnostic arrays14,15 and drug delivery systems16, which

Patterning of surfaces with different chemistries provides novel insights into how proteins, cells and tissues interact with materials. New materials, and the properties that their surfaces impart, are highly desirable for the next generation of implants, regenerative medicine and tissue engineering devices, and biosensors and drug delivery devices for disease diagnosis and treatment. Patterning is thus seen as a key technology driver for these materials. We provide an overview of state-of-the-art fabrication tools for creating chemical patterns over length scales ranging from millimeters to micrometers to nanometers. The importance of highly sensitive surface analytical tools in the development of new chemically patterned surfaces is highlighted.

Ryosuke Ogaki1, Morgan Alexander2, and Peter Kingshott1*1 The Polymer NanoInterfaces Group, Interdisciplinary Nanoscience Centre (iNANO), Faculty of Science, Aarhus University, 8000 Aarhus C,

Denmark.2 Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, Nottingham, NG7 2RD, UK.

*E-mail: peter.kingshott@inano.dk

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aim to regenerate, diagnose and treat diseased tissues, rely on

proteins and biomolecules being presented either to attach cells in

the aqueous environment in the correct conformation and spatial

configuration in 1, 2 and 3D to optimize bioactivity and minimize

adverse reactions.

The realization that interfacial phenomena were important

to biomaterials came after World War II when the concept of

‘biocompatibility’ emerged17. This lead to acceleration in the field of

surface and interface research targeted at biology and medicine. The

progress made has encouraged scientists and engineers to pursue

these goals further with the development of chemically patterned

surfaces with nanometer-scale precision, with a vision of ultimately

fabricating and controlling the biological system as desired through

the interface with the man made materials. The birth of a new

field, termed ‘nanobiotechnology’ arose, which utilizes biological

systems to fabricate functional nanostructured and mesoscopic

architectures comprised of organic and inorganic materials18. One of

the fundamental biological phenomena that has driven and continues

to underpin this field is the fact that cells respond to topographical and

chemical cues from their environment by interacting with extracellular

matrix (ECM) and other cells in vivo, and mechanical and chemical

properties of material-surface interface in vitro. The subsequent cell

signaling events ultimately influences cell function, shape, migration,

adhesion, survival, proliferation and differentiation19-24.

To understand the complex relationship between the surface

chemistry and biological systems, there has been a focus on combining

topographical and chemical modification to create multi-functional

surfaces. Highly defined topographical and chemical features from the

mm to nm range are intended to span the length scales of tissues to

cells to proteins and other biomolecules – these synthetic ‘models’ of

biology will allow us to rationally study and comprehend the complex

interfacial behavior possessed by the biological system in 2- and 3D.

The ability to mimic biological surfaces permits us to unravel the

essential controlling factors in biology by studying the interaction

between the mimicked surface and biological components.

Early developments of surface fabrication were inspired by the

‘top down’ approaches, traditionally used in microelectronics such as

photolithography and electron beam (e-beam) lithography25-27 for

devices such as microprocessors28, MEMS29 and NEMS30. Typically

it is possible to obtain a surface topographical feature of down to

~10 nm with e-beam lithography. Techniques such as Transmission

Electron Beam Ablation Lithography (TEBAL)31 have been recently

developed in an attempt to conquer the challenge of reaching the

sub-10 nm resolution limit. TEBAL is carried out by controllably

ablating evaporated metal films, pre-patterned with e-beam

lithography on silicon nitride membrane substrates, to produce a

variety of intricate nano-features such as gaps, rings, channels and

wires. Many technological methods for surface fabrication have been

inspired by the techniques from the printing industry; for instance,

ink-jet technology has a number of potential life science applications

such as genomics, combinatorial chemistry, drug discovery and tissue

engineering32-34. Current jet printer technology has reached the limit

of printing high resolution features on a surface down to ~ 1 μm in a

parallel manner35.

Surfaces can be fabricated by ‘bottom up’ approaches, for example,

molecular self-assembly such as alkane thiols on gold is a commonly

used method to form well ordered surfaces. A range of surface

functionalities can be presented on the surface by introducing different

chemical terminal groups either as a single component or multiple

components36-40. Certain types of polymers such as block copolymers,

and particulate systems, can self assemble into a variety of nanoscopic

structures with topographical scales of 5 to 50 nm41-43. Examples of

how dimensions influence cellular behavior come from the Spatz group.

They have identified critical surface spacing ranges for the cell-adhesion

peptide sequence RGD, which influence the structure of integrin

receptors on cell surfaces. A spacing on 58 nm vs 108 nm showed

large variations in integrin mediated cell behaviour. On 58 nm RGD

patterns cell spreading was delayed and motility was erratic, whereas

on 108 nm patterns cell adhesion sites exhibited rapid turnover

indicating a critical RGD density was necessary for develop stable

integrins, and efficient cell spreading and focal adhesion formation44.

Furthermore, disordered patterns of > 70 nm cyclic RGD were found to

be necessary to ‘turn-on’ cell attachment compared to ordered 70 nm

patterns that ‘turned off’ attachment45. These nano dimensions have

been related to the size of the integrin structural elements indicating

that cell adhesion is sensitive to dimensions much smaller than the

size of an individual cell. Another emerging ‘bottom up’ fabrication

method is the implementation of micro and nanometer sized colloidal

particles known as ‘colloidal lithography’. Recent advances in colloidal

synthetic methods have enabled highly monodisperse particles to

be produced with good phase stability. Two dimensional structures

with lateral feature sizes in the range of micrometers to nanometers

can be readily assembled simply by selecting different sized particles.

By using colloidal patterns as masks, diverse topographical features

and geometrical control can be obtained; either directly by reactive

ion etching (RIE) on single and multi-layered colloidal surface46,47, or

by depositing various organic and inorganic materials on a substrate

surface through the interstitial spaces formed between the particles

via evaporation or sputtering followed by the subsequent removal

of particles48,49. A novel method ‘shadow nanosphere lithography’ is

capable of creating a range of topographical morphologies such as cups,

rods and wires in the nanometer size range on the surface by varying

the position and angle of the substrate with respect to the evaporation

source50,51. Various ‘bottom up’ and ‘top down’ fabrication methods

have been also employed in combination for surface patterning.

Non-photolithographic techniques including various soft lithography

methods52-56, dip pen nanolithography (DPN)57 and nanoimprint

lithography58 have been developed over the last two decades.

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Spatially controlling the surface chemistry for selective adhesion of

proteins and cells, while resisting non-specific interactions in passive

surface regions, is essential. Surface scientists have been attempting

to create ‘non-fouling’ surfaces, as unwanted protein adhesion to

implanted devices can encourage bacterial colonization which can

pose serious medical complications to the patients. A significant level

of research has been carried out in order to identify and define the

physical and chemical criteria required by the surface for preventing

bioadhesion.

Since the surface constitutes so little material, and surfaces are

very prone to contamination, by virtue of their location, surface

modification must always be accompanied by surface analysis. The

advent of sophisticated, highly surface sensitive analytical instruments

has certainly aided the technological advancement in lithographical

processes and their applications in biology and medicine.

In this article, we first provide examples of several modern surface

analytical techniques commonly used to study biologically relevant

materials. We then highlight some of the current research, process

and associated limitations for developing non-fouling surfaces and

biological patterning. Current applications and potential uses of such

patterns in the areas of drug discovery, combinatorial chemistry,

regenerative medicine and tissue engineering are also succinctly

discussed. Finally a summary and outlook is given in the areas of

fabrication and surface characterization.

Surface characterization and analysisEmphasis on the technical capability of surface analytical instruments

used in biological surface science has moved from spectroscopic based

analysis of mainly large area samples to include routine spatially

resolved chemical state analysis within the last three decades. This

is driven by the demand for methods to prepare spatially well-

defined surfaces59. Commonly a suite of complementary techniques

is applied to derive a full characterization of complex surfaces. X-ray

photoelectron spectroscopy (XPS)60-62 and time-of-flight secondary

ion mass spectrometry (ToF-SIMS)61,63,64 are routinely used to

elucidate quantitative and qualitative surface chemical information

respectively. Both are sensitive to the uppermost surface and are

routinely capable of achieving spatial resolution in the range of 5 μm

– 50 nm for chemical imaging. XPS provides quantitative elemental

and chemical functional information from the first 10 nm of the

surface for all elements but hydrogen and helium but is limited in

lateral resolution to a few microns on ideal samples. In contrast,

SIMS has excellent spatial resolution and sensitivity but is not readily

quantified. It has been extensively utilized to chemically image a range

of biologically relevant systems at high lateral resolution, including

lipids65,66 with lateral resolution as low as 100 nm in model systems

and biological cells67-69 and tissues with lateral resolution of 200 nm

– 1 μm70-72. Fig. 1 provides an example of how ToF-SIMS is currently

being exploited and developed to study the lateral distribution of

different chemistries in biological tissue samples to differentiate tissue

regions through their chemistry and thereby study disease. Such

an ability to differentiate tissue regions is envisaged as vital for the

guided design of artificial materials for tissue engineered constructs,

for example.

Atomic force microscopy (AFM)73 is a topographical imaging

technique that belongs to the family of scanning probe microscopy

(SPM)74 with the capability of obtaining highly resolved images,

with routine spatial resolutions down to 2-3 nm75. Although AFM

is primarily used for obtaining topographical information, chemical

specific (i.e. between chemical functional groups)76,77 and bio-specific

Fig. 1 ToF-SIMS imaging of mouse embryo. (a) Optical image of a 16-day-old H&E stained mouse embryo section and positive total ion ToF-SIMS image of brain (b), liver (c), rib (d) and heart (e). The secondary ion images contain structural and chemical information capable of identifying different tissue sections. (Reprinted with permission from72).

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(e.g. antibody-antigen78,79, ligand-receptor80,81, DNA-DNA82, cell-

cell83) interactions between the tip and the substrate surface can be

studied by functionalizing the probe tip84. Tips can also be modified

with molecules such as carbon nanotubes to decrease the probe size in

order to increase image resolution with added functionality85. Recent

efforts in the development of AFM have lead to atomically resolved

image in non contact (NC) AFM mode with tips functionalized with

CO molecule in vacuum86.

Immobilization and measurement of interactions between proteins

and surfaces, and between cells and surfaces, can be monitored without

labeling and in real-time by using sensing instruments such as quartz

crystal microbalance (QCM)87,88 and surface plasmon resonance

(SPR)89,90. SPR is also capable of imaging surface interactions by

monitoring the refractive index with spatial resolution of 2 μm91,92.

The techniques provide complementary information with recent

examples of successful application including the determination of DNA

hybridization efficiencies in streptavidin-biotinylated DNA complexes93,

estrogen receptor-DNA complexes94, the detection of different Ebola

virus glycoprotein species using monoclonal and polyclonal antibodies95

and monitoring the interactions between various photo-immobilized

biomolecules and specifically bound antibodies96. There are numerous

other important surface analytical techniques that have made a

significant contribution to the life sciences, summarised in Fig. 297.

In addition to the techniques and references listed above, interested

readers should refer to the following references for further reading in

surface analysis techniques97-100.

Fig. 2 Flowchart of the various analytical methods available to the biomedical researcher and their respective analytical features (Adapted from97).

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Recent developments in material surface patterning ‘Non-fouling’ surfacesMaterial surfaces that are resistant to bioadhesion have become an

essential platform for creating two dimensional selective patterning of

the biological components. The likelihood that a protein will adsorb to

a particular surface depends on numerous inter-relating physical and

chemical properties of the protein and the surface. So far, numerous

efforts have been made to unravel the relationships between surface

chemistry and protein adsorption. For instance, Whitesides et al.

employed a range of SAMs to produce model chemically functionalized

surfaces and studied the relationships between the surface chemistry

and protein adhesion. They found that surfaces that were hydrophilic,

and acted as hydrogen bond acceptors and were electrically neutral

properties resisted protein adsorption101,102. However, an exception

was observed by Mrksich’s group where the mannitol terminated SAMs

which contains a large number of hydrogen donors also resisted protein

adsorption103. Denis et al.104 has employed a combination of SAMs and

colloidal lithography to study the effect of both surface roughness and

chemistry on the adhesion of collagen. More recently, Roach et al.105

studied and noted the conformational changes and adsorption steps

of bovine serum albumin (BSA) and fibrinogen on hydrophilic and

hydrophobic SAMs by QCM and grazing angle infrared spectroscopy

(GA-FTIR).

For applications in biology and medicine, poly(ethylene glycol)

(PEG) and its derivatives continue to be the most promising materials

for creating ‘non-fouling’ surface due to their biocompatibility106.

Theoretical models have been put forward to understand the

effectiveness of PEG against protein and cell adhesion107-109.

Significant effort has been expended in an attempt to understand

the effect of the various oligoEG preparation conditions; grafting

density, molecular weight, chain conformation, hydration, ionic

strength, surface charge and temperature on the effectiveness of PEG

in resisting protein and bacterial adhesion110-119. From the theoretical

and experimental work, the graft density, molecular weight and

hydration have been found to be the most prominent factors in the

effectiveness of PEG to resist protein adsorption118. However the

detailed understanding of the exact parameters required for resisting

protein adsorption and cell adhesion in practice still remains complex

and unsolved, particularly for preventing bacterial adhesion in vitro and

bioadhesion in vivo. A variety of methodologies have been developed

for preparing PEG-modified surfaces. Examples of PEG modified

Fig. 3 The effect of spatially controlled surface modification on protein adhesion and cell growth. (a) Confocal laser scanning microscopy highlighting the preferential binding of collagen I on acetylaldehyde plasma polymer film (AAPP, left) over acetylaldehyde-allylamine plasma polymer film with covalently immobilized PEG (AAPP-ALAPP-PEG, right). (b) The direction of outgrowth on different surface chemistries from a central piece of bovine corneal tissue over an 8 day period. Unrestricted outgrowth on AAPP substrate (1), no outgrowth on AAPP-ALAPP-PEG substrate (2) and controlled outgrowth on chemically patterned substrate with central 13 mm diameter of AAPP and AAPP-ALAPP-PEG on the peripheral (3). (c) Histological analysis of the tissue outgrowth progression over 21 days cultures on chemically patterned substrate. The direction of the tissue migration is depicted as dashed arrows. (1) Migration of epithelial layer (ep) from the corneal disk (cd) onto the supportive AAPP substrate. (2) The central area of the cell supportive (cs) region (AAPP). (3) Boundary point (bp) between cell supportive and the non-supportive (ncs) region (AAPP-ALAPP-PEG) where a disorganized pile up of the cells can be seen behind the boundary point, restricting further migration. (Reprinted with permission from134).

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surfaces include PEG-like coatings via plasma polymerization120-122,

PEG grafting via ‘cloud point’112, co-polymer systems containing

PEG (e.g. poly-l-lysine-grafted-PEG (PLL-g-PEG)123,124, polysulfone-

grafted-PEG (PSf-g-PEG)125, polystyrene-block-PEG (PS-b-PEG)126),

development of poly(oligoethylene glycol methyl methacrylate)

(POEGMA) via atom transfer radical polymerization (ATRP)127,

multi-component cross linked PEG128,129, incorporation of EG to

self assembled monolayer130-132 and poly electrolyte multilayers133.

In recent work, George et al.126 demonstrated that PS-b-PEG block

co-polymer system can self assemble into various controllable

domain morphologies while retaining protein and cell resistance.

Thissen et al.134 used combination of cloud point grafting and plasma

polymerization and demonstrated the controlled deposition of ECM

protein collagen I and the growth of bovine corneal epithelial (BCEp)

tissue in two dimensions (Fig. 3). Multi-component cross linked PEG

has been developed as a single commercial formulation that can be

covalently attached to any substrates without the need of primers129.

Ma et al.135 has demonstrated the effect of surface density of POEGMA

towards the resistivity of fibronectin by controlling the number of ATRP

initiators presented by the mixed SAMs. These novel methodologies

and materials possess diverse physical and chemical characteristics

that facilitate the fabrication of non-fouling backgrounds on which to

accurately pattern biomolecules on surfaces.

Current strategies in biological surface patterningSite specific immobilization of proteins and peptides with nanometer

resolution is of fundamental importance for subsequent recognition

Fig. 4 Self-selective immobilizations of different proteins onto respective surface chemistries from a single mixed solution. (a) Binary protein patterning is carried via a photolithographic method to create a patterned surface contrast of biotin and chloroalkane onto hydrogel substrate. Subsequently this surface is exposed to the mixed solution of streptavidin and HaloTag proteins. (b) Fluorescent images of affinity ligand patterned hydrogel surface after 1 hour exposure to the fluorescently labeled protein conjugates with different excitation wavelengths (streptavidin – excitation/emission 532/557-592 nm, HaloTag – excitation/emission 635/650-690 nm). Methoxy terminated surface chemistry was included as a control. The three binary surface chemical patterns are biotin/methoxy (BM), methoxy/chloroalkane (MC) and biotin/chloroalkane (BC). (Reprinted with permission from139).

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and adhesion of cells and organization of tissues. While a number of

methodologies have been developed and utilized for the patterning of

biological molecules, recent trends indicate that some techniques are

exploited more extensively, most likely due to limitations posed by

some processes. For example, we have observed significant decrease

in the implementation of photolithographic based techniques for

protein patterning. Although the major limitation associated with

spatial capability has recently been rectified by techniques such

as plasmonic nanolithography136 and scanning near field optical

microscopy (SNOM)137 to reach sub 100 nm resolution, high running

costs and need for a clean room is still a deterring factor. Despite

these limitations, a number of recent studies using photolithographical

techniques are worth mentioning. For instance, Carrico et al.138 has

utilized a recombinant photoreactive protein which exhibits certain

mechanical properties at a given wavelength and demonstrated

Rat-1 fibroblast patterning without the use of traditional chemical

modification that may compromise protein function. Dubey et al.

demonstrated the self-selective patterning of streptavidin and

HaloTag™ into adjacent regions of biotin and chloroalkane respectively

that were prepared onto a PEG based multicomponent hydrogel via

selective photolithographic patterning from the same solution. The

pattern was subsequently characterized by fluorescent microscopy and

ToF-SIMS139 (Fig. 4).

Micron scale protein and cell surface patterning has been

achieved two decades ago by Whitesides’ group using micro contact

printing (μCP)52,140,141 as an alternative method to overcome the

drawbacks associated with photolithographic based techniques and

the popularity of the method still remains today. In μCP, polymer

stamps with features are developed from a master fabricated from

a range of lithographical processes. The stamp is dipped in the ‘ink’

of protein solution and brought to contact with a substrate surface.

More recent development in the patterning process has focused

on utilizing a combination of fabrication techniques to produce

nanometer scale protein patterns on the surface in an extremely

parallel fashion. Nanometer scale contact printing (nano contact

printing, nCP) of single142 and multiple108 protein patterning has been

achieved by Delamache’s group (Fig. 5). A number of workers have

highlighted the contact transfer of hydrophobic oligomeric species

from the poly(dimethyl siloxane) (PDMS) stamp as a problem in

many applications143. Csucs et al. have published work illustrating the

utility of an alternative stamp material, polyolefin plastomer (POP),

with improved nanometer scale protein patterning144. To overcome

the issues associated with contact printing such as reproducibility

of the pattern over large areas and transfer contamination, Textor’s

group developed a technique called selective molecular assembly

patterning (SMAP)145. In SMAP, nanometer scale spatially resolved

contrast of hydrophilic (non-fouling) and hydrophobic (fouling) surface

can be created by immobilizing PLL-g-PEG and alkyl phosphates onto

silicon dioxide and titanium dioxide respectively. Nanometer scale

patterns can be created over large areas by combining processes such

as colloidal lithography, hot embossing and template synthesis145.

More recently the same group has demonstrated the improved SMAP

process by combining e-beam lithography into the process and the

incorporation of indium tin oxide into substrate, producing large scale

patterning of streptavidin146.

Another printing based technique is nanoimprint lithography (NIL)

and the first patterning of protein using this method was reported by

Hoff et al. in 200458. NIL typically employs a Si stamp topographically

patterned using conventional lithography which is pressed against

polymer coated substrate heated above the glass transition

temperature of the polymer. The stamp is removed after cooling,

leaving the imprint of the polymer. Residual polymer is removed

further by RIE and the patterned substrate is further modified to create

contrasts of protein adhering / resistant regions. In the recent work,

Fig. 5 μCP for protein surface patterning. (a) Micro-fabrication of a stamp is carried out by pouring liquid polydimethylsiloxane (PDMS) pre-polymer against silicon master mold and cured. (b) The PDMS stamp is released from the master and inked in protein solution. The subsequent rinsing and drying of the inked stamp produces a monolayer of protein and transferred to the target substrate by printing. Multiple proteins can be patterned either by sequential inking and printing (c), parallel inking of stamp followed by a single printing (d) or blocking of certain surface sites by adsorbing blocking agents such as bovine serum albumin (e). (Reprinted with permission from108).

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regeneration and reusability of N-nitrilotriacetic acid (NTA) patterned

substrate via NIL was successfully demonstrated by the repeatable

sequential immobilization and rinsing of DsRed protein and Green

fluorescent protein via histidine functionalization147.

A range of AFM based surface patterning methods have been

developed and employed extensively for the surface patterned

immobilization of proteins and cells. AFM based methods allow

immobilization in ambient conditions and enables patterning of

proteins with high spatial resolution and density. Nanoshaving148 and

the extended method of nanografting149 was developed in 1997 and

utilized most commonly on SAMs. The process involves the physical

scraping and removal of the SAM surface by the AFM tip (nanoshaving)

and the molecules with affinity towards the exposed surface are

subsequently grafted on the removed region (nanografting). To date,

SAM feature sizes as low as 2 x 4 nm2 has been demonstrated by

Liu et al.150. For surface protein patterning, EG containing SAMs can

be employed in the resist and immobilization of protein can be carried

out to avoid non specific adsorption of the surrounding unshaved

areas151. The recent development in the extended technique, reversal

nanografting, have overcome the issues associated with the original

nanografting process such as difficulty in maintaining the designed

geometry due to thermal drift, thiol in solution may exchange with

the chemisorbed thiol on substrate due to prolonged soaking, and

tip wear152. In general, reversal nanografting is almost the same

process as the original nanografting procedure, except that the protein

binding terminal containing thiols are bound to the substrate while

protein resistant thiols are in solution. Tan et al.152 demonstrated

the regulation in the coverage and immobilization of antibiotin IgG

by controlling the shaving size and spacing between the shaving lines

on reversal nanografted array of biotin with individual dot size of

25 – 300 nm2 (Fig. 6).

Out of the AFM based surface patterning methods available to

date, DPN has been attracting most attention since the technique was

pioneered by Mirkin’s group in 199957. DPN is a scanning probe based

technique where AFM cantilever tip is ‘inked’ in solution containing

particular molecules of choice and these are transferred from the tip

Fig. 6 Nanografting for protein patterning. (a) Reversal nanografting is carried out by (1) imaging the target surface immobilized with self assembled monolayer containing protein binding termini (2 and 3) shaving of the surface is carried out by AFM tip and shaved areas are immobilized with protein resisting thiols in solution (4) the resulting surface is imaged. (b) 1500 x 1500 nm area of thiolated biotin nanoarrays fabricated by reversal nanografting. (c) Same area as (b) after anti biotin Ig G protein immobilization. (d) The combined cursor plot in (b) and (c) revealing the local height change before and after Ig G immobilization. (e) 300 x 300 nm AFM image of area 1 in (b) containing 1089 biotin nanofeatures with 5.2 x 5.2 nm (f) 300 x 300 nm AFM image of area 2 in (b) containing 288 biotin nanofeatures with 12.7 x 12.7 nm. (g) 300 x 300 nm AFM image of area 3 in (b) containing 144 biotin nanofeatures with 10.3 x 31.9 nm. (Reprinted with permission from152).

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to the substrate surface via capillary transport. Direct and indirect

DPN methods have been developed; the direct method utilizes inks of

protein solution for single-step immobilization of protein onto surface

whereas the indirect method is carried out in a two-step process where

organic molecules such as alkanethiol are deposited first and proteins

are subsequently immobilized onto the surface from solution. The first

indirect DPN for protein patterning was demonstrated by Lee et al.153,

who has successfully immobilized lysozyme and immunoglobulin G

(IgG) onto arrays of 16-mercaptohexadecanoic acid (MHA) surrounded

by the protein resistant area of 11-mercaptoundecyl-tri(ethylene

glycol). The same group has also demonstrated direct DNP writing of

IgG onto base treated and aldehyde modified SiO2 substrates using

PEG modified tip to facilitate transport of protein to the surface

with sub-100 nm resolution154. Hyun et al. utilized indirect DPN and

produced ~ 230 nm array of biotinylated BSA mediated by specific

molecular recognition with streptavidin-biotin complex via covalent

attachment onto MHA155. More recent work in this area has attempted

to overcome some drawbacks associated with the technique, such

as retaining the biological activity of protein, the limited size of the

pattern that can be written and the amount of protein deposited.

Mirkin’s group has recently rectified the issue associated with the

limited fabrication area by utilizing 26 cantilever tips and successfully

immobilized biologically active IgG proteins in array format covering

~ 1 cm156. They have also demonstrated the capability of the parallel

DPN by using arrays of up to 55 000 cantilever tips simultaneously157

(Fig. 7). Wu et al.158 have developed layer-by-layer tip modification

method to form hydrophilic porous structures on the surface. The

modification increases the amount of ink that the tip can hold while

retaining biological activity of protein. Bellido et al.159 recently

demonstrated the possibility of controlling the number of proteins

deposited onto surface by direct DPN. Nanoarrays of ferritin were

fabricated directly on TEM grid and the number of molecules deposited

was controlled by the initial protein concentration, array dot diameter

and the contact angle between the ink solution and the substrate

(Fig. 8).

Implementation of colloidal lithography for protein and cell

patterning is relatively new. In general, colloidal lithography templating

prior to protein and cell immobilization can be carried out in two ways;

the colloids can be deposited electrostatically onto various substrates

in a geometrically random manner with defined separation distance or

Fig. 7 Massively parallel DPN. (a) Gravity driven alignment method used for parallel DPN with each pen capable of contacting with the substrate under the weight of the whole pen array. The exact position of the pen array is controlled by the AFM scanner head attached to the pen array via epoxy resin. (b) Optical micrograph of part of the two dimensional array of cantilevers. (c) Dot matrix map of the front face of the US five-cent coin. (d) Optical micrograph of a representative region of the substrate with 55,000 duplicates produced by parallel DPN. Each circular feature represents the front face of the US five cent coin made from 1-octadecane thiol dot features as mapped in (c). Inset: AFM image of single circular feature. (Reprinted with permission from157).

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colloids can be self assembled into a two dimensional hexagonal close

packed structure. In geometrically random templating, Michel et al.160

has combined the SMAP approach and colloidal lithography to create

nanopillers of TiO2 which was made protein adherent with dodecyl

phosphate (DDP) while SiO2 was made protein resistant by PLL-g-PEG

grafting. Streptavidin was patterned on the nanopillers with 50 nm

feature size over a large area. Similar approach was demonstrated by

Agheli et al.161, where electrostatically assembled polystyrene particles

were first made into gold ‘nanodiscs’ via particle annealing and dry

etching with SiO2 background. Alkanethiol was deposited onto gold,

followed by the PLL-g-PEG layer on SiO2. Laminin, BSA and polyclonal/

monoclonal anti-mouse laminin were immobilized and quantitatively

characterized using AFM height histogram and QCM. Cai et al.162

created hexagonally arranged ~ 120 nm nanoarrays of lysozyme over

1cm2. 10-undecenyltrichlorosilane (UTS) was initially deposited onto

Si substrate followed by colloidal template and PEG silane. Colloids

were removed ultrasonically and the lysozyme was deposited on

the UTS exposed surface. Blatter et al.163 has also employed similar

methodology to create submicron protein array. PS particles were

first assembled onto TiO2/SiO2 substrate and combinations of O2/N2

and SF6/O2 etchings were carried out, followed by the lift-off of the

particles to create TiO2 and SiO2 contrast. Protein immobilization onto

TiO2 was carried out by pre-functionalizing the TiO2 and SiO2 with

DDP and PLL-g-PEG, respectively.

Use of programmable self assembly of DNA as a template

for protein patterning has been recently attracting attention.

Two dimensional DNA template for protein patterning was first

demonstrated by Yan et al. in 2003164. Periodic pattern of streptavidin

was achieved by the self assembly of DNA via the reprogrammed

sticky ends into 4 x 4 tiles of nanogrids containing biotinylated

oligonucleotides on each tile junctions separated by ~ 19 nm.

A variety of positioning of streptavidin was achieved further by

selectively modifying each tile with biotin and combining two DNA

tiles together165. Cohen et al.166 demonstrated the use of polyamide-

biotin conjugate to achieve multiple arrangements of streptavidin on a

single DNA tile with ~ 25 nm separations. In a similar manner, human

α-thrombin binding aptamer and platelet derived growth factor (PDGF)

binding aptamers have been incorporated into the DNA template

and the thrombin and PDGF were subsequently patterned167. DNA’s

ability to form an intricate two dimensional geometry with nanometer

precision programmable control makes this technique an attractive

candidate for protein patterning.

3D PatterningIn vivo, cells inhabit a 3D rather than a 2D world. Thus, in regenerative

medicine and the construction of cellular tissue models efforts are

made to achieve this environment using a variety of strategies

including coalesced particles, porous scaffold materials and hydrogels.

Patterning of surface chemistry over long length scales (up to 10s of

millimetres) has been shown to be of great utility in controlling cell

distribution in macroscopic tissue engineering scaffolds168. Gradients

of surface chemistry within porous 3D objects were first exploited to

control fibroblast adhesion in a porous poly(lactic acid) tissue scaffold

formed by super critical CO2 processing by Barry et al.168. Plasma

polymers were deposited to introduce nitrogen functionalities which

enhanced cell adhesion in the centre of a porous 3D object. The

graduated deposition thickness achieved from the periphery to the

centre of the thick disc placed in the plasma was exploited to form

a radial chemical gradient of a second cell resistant plasma polymer

(a hydrophobic hexane plasma polymer). This chemical gradient

achieved a decreasing fibroblast density at the periphery and increase

in the centre, as seen in Fig. 9, thus, counteracting the tendency of

cells to adhere to the periphery of the porous object. This method

achieved a uniform cell distribution in the scaffold, one of the pre-

requisites to tissue formation. Recently a 3D photo patterning method

achieving in hydrogels has been published which allows spatial control

Fig. 8 Controlling the number of protein by direct DPN. (a) Schematic illustration of ferritin pattern produced onto the surface of the TEM grid. (b) TEM images of a ferritin nanoarray (20 x 20 μm, 150 nm feature) generated by direct DPN on a TEM grid. The scale bars are 2 μm, 500 nm and 100 nm for different magnifications. (Reprinted with permission from159).

(b)

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with micrometer scale resolution of biological functionalities such as

RGD (Fig. 10). This exciting new method based on orthogonal click

chemistries shows great promise with the initial report showing control

of cell migration, proliferation and morphological changes169.

High throughput screening approaches to find new materialsPolymer spots of a few hundred microns in diameter formed by micro

array printing are being exploited as material discovery platforms

in high throughput (HT) screening analogous to HT drug discovery.

Anderson and Langer developed an approach to print 576 unique

materials in triplicate on a slide coated with a cell resistant hydrogel170.

They used UV photoinitiated acrylate monomers which provided a

large library of chemical moieties. A convenient combinatorial on-slide

production method was subsequently developed by Bradley et al.

to allow on-slide monomer mixing and thereby potentially further

increasing throughput171. Cell-surface interactions can be studied

on these large micro array libraries of materials (Fig. 11)172,173.

Importantly, as with all materials the surface chemistry must be

well characterised in order to correctly interpret cell response to the

surface, which poses a challenge when dealing with so many samples.

A complementary range of surface analysis techniques, including ToF

SIMS, XPS and sessile drop water contact angle have been developed

to allow high throughput surface characterisation (HT-SC) to be carried

out within the same time frame as the cell culture experiments (a few

days)171. The challenge in HT-SC is automation of the data handling

methods to identify correlations in the large amount of data generated

from this type of combinatorial materials platform174. However, the

method is proving extremely useful at identifying polymers with

application in biotechnology and in understanding the cell response to

materials.

Summary and outlookChemical manipulation and patterning of material surfaces embrace

exciting opportunities in biology and medicine that aim to improve

the quality of human life. The repertoire of patterning methods and

techniques that has been pioneered over the last several decades,

and their advancement have been significantly helped by scientific

and instrumental progressions made in the field of surface analysis.

This linked progression has provided us with the ability to influence

and control a range of biological components from mm to nm scales.

Control over the length scale allows us to integrate newly patterned

Fig. 9 Controlling cell distribution in tissue engineering scaffolds via surface chemical gradients. Surface chemical contrast were created in the core and sheath of Poly-DL-lactic acid (PDLLA) scaffold by plasma polymer depositions of cell adhesive allyl amine (ppAAm) and cell repulsive hexane (ppHex). (a) Scaffolds cultured with 3T3 fibroblasts for 24 h (the scale bars are 1 mm and cells are color-coded in red): (1) PDLLA, (2) PDLLA/ppAAm, and (3) PDLLA/ppAAm/ppHex. The lower images show X-ray μCT images from approximately 2 mm slices through the centers of the scaffolds. (b) Cumulative cell area in the 0.01 mm slices through the centers of the scaffold within the core and the sheath denoted by the black dotted lines in (a). (Reprinted with permission from168).

(b)

(a) 1 2 3

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Fig. 10 Cytocompatible, 3D biochemical patterning within preformed click hydrogels. (a) The thiol–ene reaction mechanism provides a means to quantitatively couple sulphhydryls (–SH) with vinyl functionalities (–C=C) in the presence of light. (b) On swelling into the material, relevant thiol-containing biomolecules are covalently affixed to the hydrogel network at varying concentrations by altering the dosage of exposed light (intensity and exposure time). (c) A live/dead stain at 24 h after photolithographic patterning of 3T3s indicates a predominantly viable population (live cells are shown in green, whereas dead cells are shown in red) and that the patterning process is cytocompatible. (d) The thiol–ene reaction is confined to user-defined regions in space using photomasks to introduce three different fluorescently labelled peptide sequences within the gel, a process that can be repeated at desired times and spatial locations to introduce additional biochemical cues. (e) By controlling the focal point of the laser light in three dimensions using a confocal microscope, micrometre-scale spatial patterning resolution is achieved. Values in (b) are reported as mean ± s:d: (n = 5). The image in (c) represents a 200 μm confocal projection. The images in (d )and (e) represent confocal micrographs of fluorescently tagged peptides patterned within the networks. (Reprinted with permission from169).

Fig. 11 Human embryonic stem (hES) cells grown on polymer arrays. (a-c) Six million hES cell embryoid body day-6 cells were added on the polymer array in the presence of retinoic acid for 6 day and then stained for cytokeratin 7 (green) and vimentin (red). Polymer spots can be identified by blue fluorescence. (d) Nuclei were also stained (green) (not shown in other images to simplify presentation). (e) Typical cytokeratin 7 positive spot. (Reprinted with permission from170).

(b)(a) (c)

(d) (e)

(b)(a) (c)

(d)

(e)

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chemistries and functionalities onto a variety of medically relevant

device surfaces of various sizes and shapes for resisting protein

adsorption and mammalian and bacterial cell attachment which may

threaten health and well being. Despite these remarkable advances,

each of the chemical and biological patterning processes possess

drawbacks, and improvement of conventional techniques, as well as

the development of new methods can only be made into reality by

overcoming analytical challenges, particularly in 3D where one might,

for example, want to display multiple signals with nanoscale spatial

distribution. The drawbacks associated with the chemical and spatial

sensitivities may hinder meaningful characterization of the patterns.

In addition many of the current surface analytical techniques are

carried out in vacuum environment which is not the representative

of the native state of the biological components. Thus a combined

use of a range of surface analytical techniques is essential for gaining

the thorough characterization of the patterned surface needed to

understand its biological response. The synergy among the surface

engineers and surface scientists in the development of surface

analytical instruments and the patterning processes must continue,

in order to desirably harness the biological components in a range

of scales that are essentially invaluable in progressing materials to

improving human health.

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