ijsing self-assembled monolayers to understand the ... · the surface properties of both sams are...

Annu. Rev. Biophys. Biomol. Struc't. 1996. 25:55-78Copyright @ 1996 bt, Annual Review,s Int'. All rig,hts resen'ed

IJSING SELF-ASSEMBLEDMONOLAYERS TOUNDERSTAND THEINTERACTIONS OF MAN-MADE SURFACES WITHPROTEINS AND CELLS

Milqn Mrksich and George M. Whitesides

Department of Chemistry, Harvard University, Cambridge,Massachusetts 02138

KEY woRDS: biocompatibility, biomaterials, biosurfaces

AssrRncr

Self-assembled monolayers (SAMs) formed on the adsorption of long-chain alkanethiols to the surface of gold or alkylsilanes to hydroxylatedsurfaces are well-ordered organic surfaces that permit control over theproperties of the interface at the molecular scale. The abililty to presentmolecules, peptides, and proteins at the interface make SAMs especiallyuseful for fundamental studies of protein adsorption and cell adhesion.Microcontact printing is a simple technique that can pattern the forma-tion of SAMs in the plane of the monolayer with dimensions on themicron scale. The convenience and broad application offered by SAMsand microcontact printing make this combination of techniques usefulfor studying a variety of fundamental phenomena in biointerfacial sci-ence.

CONTENTSPERSPECTIVESBACKGROUND . . . . . . . . .

Man-Made sr*ii ' i ' i 'f i i| ti ' i 't:i ' i Eiotogi,oi M;di".......:.................Adsorption of Protein

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55

56 MRKSICH AND wHITESIDES

Self-Assembled Monolayers of Alkanethiolates on Gold..Self-Assembled Monolayers 6f ekvlsiloxonesTh? Physical-Organic e hemistry o7 Sey-, l ,ssembled Mono\ayer,s.. . . . . . . . . . . . . . . . . . . .

INTERACTIONS OF PROTEINS WITH SELF-ASSEMBLEDMONOLAYERS ... . . . . . . .

Adsorption of P r ote ins to S e lf-As s emble d M onolay err ................Surfaces That Resist the Adsorption of ProteinsI mmobil izat ion of P rote i ns to S e l f- As iembl e d M ono I ay ers.. . . . . . . . . . . . . . .Biospecific' Adsorption of Proteiis to Self-Assembled"Monolayer.r..................AttaV hnt"e nt of C e'l I s rc Se lf- As s e mb I e d lti onolay e rs ................

CONTROL OVER SPATIAL ADSORPTION OF PROTEINP atterning S e lf-As s e mble d M onolay e r s .........Patterning Adsorption of Protein on Self-Assembled Monolay-er,s....................P att ernin' ! Attacltme nt C e I I s o n S e l f- As iembl ed M onolay e r s . . . . . . . . . . . . .Attachment of Cells on Contoured Surfaces

APPLICATIONS OF SURFACES BASED ON SELF-ASSEMBLED

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MONOLAYERS IN BIOCHEMISTRY 12Drug Design and Screening 72BiosensorsElectrochemical Methods 7 3

PROSPECTS FOR SELF-ASSEMBLED MONOLAYERS IN BIOLOGY 1.7Self-Assembled Monoloyers as Model Surfaces.... 1,1Self-Assembled Monolayers in Cell Biologl' 71

PERSPECTIVES AND OVERVIEW

Man-made surfaces in contact with biological environments are impor-tant in biology, biotechnology, and medicine. These surfaces occur intools and reagents for studies in molecular and cell biology; in sub-strates for enzyme-linked immunosorbent assay (ELISA), cell culture,and tissue engineering; in materials for contact lenses, dentalprostheses, and devices for drug delivery; in coatings for catheters.indwelling sensors, and implant devices; and in materials for chroma-tography and storage of proteins (50, 65). The first event that usually'occurs on contact of the synthetic material with a medium that containsdissolved protein is the adsorption of protein to the surface: other re-sponses, such as the attachment of cells, are secondary and dependon the nature of the adsorbed layer of protein. Because of its centralimportance, the adsorption of protein to man-made surfaces has beenstudied extensively. Although much has been leamed, there are still nomechanistic models that rationalize (or predict) the interaction of aprotein with a surface in molecular detail. A broad goal of research inthis area is to understand the interactions of proteins with surfacesat the level of detail that is now common for characterization of theinteractions of proteins with water, ligands, and other proteins in solu-tion.

Model systems desisned to elucidate these mechanisms must have

INTERACTIONS OF SAMS WITH PROTEINS 57

three components: (a) a protein with known high-resolution structure

and properties (e.g. stability, conformational dynamics, and tendency

to uggt.gate); (b) a structurally well-defined surface with properties

that-Jan be tailored and controlled simply and allows the complex

functionality relevant to biochemistry to be introduced at the surface;

and (c) one or several analytical techniques that can measure adsorption

of protein in situ and in real time. Numerous proteins suitable for these

types of studies are now available. Analytical methodologies appropri-

ut. for these studies (e.g. surface plasmon resonance spectroscopy and

fluorescence spectroscopy) are becoming available. However, X-ray

and electron dffiaction studies of adsorbed crystalline monolayers are

still the only techniques that can provide information at the molecular

scale, and these techniques are applicable only in special cases. The

absence of methods to ptepa." well-defined surfaces has been a prob-

lem; for those surfacei that are well defined, such as metals, metal

oxides, and crystals, surface properties cannot be controlled precisely.

Self-assembled monolayers (SAMs)-particularly those formed by

the adsorption of long-chain alkanethiols on gold- are arecently devel-

oped class of organic surfaces that are well suited for studying interac-

tirons of surfacei with proteins and cells. The ability to control the

composition and properties of SAMs precisely through synthesis, com-

bined with the simple methods that can pattem their functional groups

in the plane of the monolayer, makes this class of surfaces the best now

available for fundamental mechanistic studies of protein adsorption

and cell adhesion. Here we review the use of two classes of SAMs,

alkanethiolates on gold and alkylsiloxanes on hydroxylated surfaces,

in the study of processes that occur at the interface between a man-

made surface und u biological medium. We do not discuss much of the

excellent work in which other classes of surfaces, such as Langmuir-

Blodgett films, lipid bilayers, and polymers, have been used (for re-

views. see 44. 60. 61, 75).

BACKGROUND

Man-Made Surfaces That Contact Biological Media

Many early studies of the interactions of artificial surfaces with biologi-

cal media were motivated by problems associated with the formation

of thrombus on foreign surfaces that contact blood-a process that is

now relatively well understood and involves the adsorption of proteins

intimately (13).For the past 3 decades, researchers have sought to

understand the interactions of man-made materials with proteins (and

58 MRKSICH AND WHITESIDES

processes dependent on protein adsorption), with applications in bio-compatible materials as a central motivation. Although many materialshave been identified that are compatible with tissue to varying degrees[e.g. titanium (implants), polymethylmethacrylate (contact lenses), ce-ramics (dental prosthesis), polyurethanes (artificial heart), pyrolyticcarbon (heart valves)l there has been much less progress in the elucida-tion of the mechanisms by which these materials function.

Adsorption of ProteinA survey of the extensive literature on the adsorption of proteins toman-made surfaces is not practical in this review (for reviews, see 44.6I, 63, 75). The adsorption processes are complicated. Even in thesimplest case, where a single, well-defined protein adsorbs to a uniform,well-defined surface, a substantial range of processes is usually in-volved (Figure 1). After an initial adsorption of a protein to a surface,the protein can (Figure 1a) dissociate from the surface and return tosolution; (d) change orientation; (d) change conformation but retainbiological activity; O denature and lose activity; or (g) exchange w'ithother proteins in solution. These processes are complicated further bya range of conformationally altered and/or denatured states accessibleto the adsorbed protein and by the many different microenvironmentsat the surface created by heterogeneities in the surface and the presenceand conformations of other proteins. Lateral protein-protein interac-tions may dominate the protein-surface interactions. Because many ofthese processes are essentially irreversible, models must emphasize thekinetic aspects of protein adsorption (59).

oRIENTATIoN AND coNFoRMATroN oF ADSoRBED pRorEIN Most studies

have generated empirical models by analyzing the amount and rates of

Solution.)

i l "[,

ol,l,

.f'

Figure 1 The complexities associated with studies of protein adsorption. Several equi-libria must be considered on adsorption of a protein to a surface (a); lateral mobilityof the adsorbed protein (b); dissociation of a protein adjacent to another protein (r ).reversible denaturation and changes in conformation of the protein (d); dissociation ofthe altered protein (e); denaturation of the protein that results in irreversible adsorption(fl; and exchange of the protein with a protein from solution (g). This scheme is notcomplete but is complicated further by the many different conformations and envrron-ments available to an adsorbed protein.

,1il l eil,


protein adsorption; few have investigated adsorption at the molecular

level. The information required for a detailed molecular description of

these processes, including infot ation concerning the orientation and

conformation of adsorbedproteins as a function of time and conditions,

has been difficult to obtain. Few analytical methods exist-and none

with the power to reveal structure comparable to X-ray crystallography

or multidimensional NMR spectroscopy-that can characterize the

conformation and orientation of proteins adsorbed to surfaces. Infrared

spectroscopy has been used to assess the degree of denaturation of

fibronectin adsorbed to SAMs that present different functional groups

(7). Lee & Belfort (37) conelated the activity of adsorbed RNase A to

a model that involved conversion between two orientations of the pro-

tein at the surface. Darst et al (14) probed the orientation and conforma-

tion of myoglobin adsorbed to a polydimethylsiloxane surface by using

a panel of fiu. monoclonal antibodies that had known epitopes on the

protein. This latter method and related footprinting methods that use

proteases (87) or selective chemical reagents for modification of resi-

bu.r of proteins (69) are perhaps the most general methods for the

direct chiracterization of unlabeled proteins adsorbed to surfaces. Korn-

berg and coworkers (13) have used electron diffraction to determine

theltructure of two-dimensional crystals of the protein streptavidin

adsorbed to a biotinylated lipid layer. Rennie and coworkers (18) have

used neutron reflection to determine the structure of B-casein adsorbed

to hydrophobic alkylsiloxane monolayers. An emerging technique

baseb on X-ray standing waves also provides direct structural informa-

tion with near-atomic resolution for cases in which the layer of protein

is ordered (6).

MEASURING eROTEIN ADSoRPTISN The most useful of the many analyti-

cal techniques that have been used to measure protein adsorption are

those that are compatible with a variety of surfaces and that provide

measurements in silu and in real time (59). Methods that measure the

dielectric properties of an interface [surface plasmon resonance (SPR)

spectroscopy (41), waveguide interferometry (62), and ellipsometry

(45)l and ihbse that measure changes in the resonance frequency of a

piezoelectric material [quartz crystal microbalance (]1), surface acous-

ii. *uu. (17), and acoustic plate mode (12) devicesl are particularly

well suited. The primary drawback of these methods is that they mea-

sure bulk properties of the interface and provide little or no detail about

atomic-level interactions. Surface plasmon resonance spectroscopy is

particularly well suited for use in conjunction with SAMs, because both

iechniquer ur. thin films of gold as substrates: A commercial SPR


(* (* (' (* (^( ( ( ( (

( ( ( ( (( ( ( ( (

[ [ [ [ [

/ / / / / /nu/ / / / / / / / / / / s io r / / / / / /Fi,qure 2 Models for SAMs of alkanethiolates on gold and alkylsiloxanes on hydroxy-lated surfaces. (A) The thiol groups coordinate to the hollow threefold sites of the gold(l1l) surface. and the alkyl chains pack in a quasi-crystal l ine array. (B) The contorma-tions of alkylsilanes and the details of their bonding to surface hydroxyl groupsare less clear; a mixture of possible conformations and geometries is probably involved.The surface properties of both SAMs are controlled by controlling the terminal functiongroup X.

instrument has demonstrated many of the characteristics required forefficient studies of adsorption of proteins to functionalized SAMs ongold (53) .

Self-Assembled Monolayers of Alkanethiolates on Gold

Self-assembled monolayers of alkanethiolates on gold form on the ad-sorption of a long-chain alkanethiol [X(CH2),,5H, n : I I l8) fromsolution (or vapor) to a gold surface (Equation 1). The structure ofthese SAMs is now well established (16, l9):

RSH + Au(O), - RS-Au(I) Au(g),+ t/rHt( '?).

The sulfur atoms coordinate to the gold atoms of the surface. and thetrans-extended alkyl chains are tilted approximately 30 degrees fromthe normal to the surface (Figure 2).The properties of the interfacedepend on the terminal functional group X of the precursor alkanethiol;even structurally complex groups can be introduced onto the surfacethrough straightforward synthesis. The surface chemistry of SAMs canbe controlled further by forming so-called mixed SAMs from solutionsof two or more alkanethiols. Self-assembled monolayers are stable inair or in contact with water or ethanol for periods of several months:they desorb at temperatures greater than 70"C or when inadiated withUV light in the presence of oxygen. They have been used in cell culturefor periods of days. Self-assembled monolayers that are supported ongold 5-10 nm in thickness (on glass slides) are transparent, and those

BYX X " X

I s\ t) \ (, ii I-ois l&sl. o. d .?*o?' - on

A

INTERACTIONS OF SAMS WITH PROTEINS 6I

supported on gold of thickness greater than 100 nm are opaque and

reflLctive (15)a even the thin films of gold are electrically conductive.

Self-assembled Monolayers of Allqlsiloxanes

Alkylsiloxanes are obtained by reaction of a hydroxylated surfagg (usu-

ally the native oxide of silicon or glass) with a_solution of alkyltrichloro-

silane (or alkyltriethoxysilane) (48, 56, 12). The reactive siloxane

groups condense with water and with hydroxyl groups of the. surface

ind neighboring siloxanes to form a cross-linked network, the bonding

urrung.i,ent is-not well defined but depends on the conditions used to

formln. S4HA (Figure 2). These SAMs are significantly more stable

thermalty than alkinethiolates on gold and do not require evaporation

of a layei of metal for preparation of substrates. The siloxane monolay-

.r, ur. limited, however, in the range of functional groups that can be

displayed at the surface by the reactivity of the alkyltrichlorosilane

groupi of the precursors and by the technical difficulty of introducing

fun.iionul groups once the monolayer has formed'

The Physical-Oyganic Chemistry of Self-AssembledMonolayersAn important goal in interfacial science is to understand the relationship

between the microscopic structure of a surface and its macroscopic

properties: this relationship is particularly relevant in studies of protein

aasbrption where hydrophobic forces are dominant (71). Self-assem-

bled monolayers on-gold-and, to a lesser extent, alkylsiloxanec-of-

fer the level of structural control required for detailed studies of adsorp-

tion processes. Studies of the influence of a terminal functional group

X of a SAM on the wettability of the surface reveal that the hydropho-

bicity of the surfaces can be controlled precisely (2). Self-assembled

-onbluy.rs that present polar functional groups (e.g. carboxylic acid

and hydroxyl) are wetted by water. Those that present nonpolar. organic

g.oupt (e.g. trifluoromethyl and methyl) are autophobic and emerge

dry fto* water. Monolayers that present fluorinated groups are more

water repellent than Teflon.

INTERACTIONS OF PROTEINS WITHSELF-AS SEMBLED MONOLAYERS

Adsorption of Proteins to Self-Assembled Monolavers

The adsorption of several model proteins to SAMs that present different

functionaf gtoupt (e.g. alkyl, perfluoroalkyl, amide, ester, alcohol' ni-


trile, carboxylic acid, phosphonic, boric acids, amines, and heterocy-cles) correlates approximately with the hydrophobicity of the surfaces(43, 57); the degree of denaturation, as inferred by the density of alayer of adsorbed protein, increases with the hydrophobicity of thesurface and decreases with the concentration of protein in the contactingsolution. Adsorption on hydrophobic surfaces often is irreversible ki-netically, but the protein adlayer can be removed with detergents orreplaced by other proteins in solution. Although SAMs that presentionic groups have been used extensively to control protein adsorption,less is known about the relationships between the properties of chargedsurfaces and the structure and properties of the layer of protein. Vroman(73) has used ellipsometry and immunologic identification of adsorbedproteins to charactenze the exchange of plasma proteins at hydrophilicglass surfaces.

Surfaces That Resist the Adsorption of ProteinsMuch effort has been directed toward the identification of biologically"inert" materials, i.e. materials that resist the adsorption of protein.The most successful method to confer this resistance to the adsorptionof protein has been to coat the surface with poly(ethylene glycol) (PEG)(20,22); a variety of methods, including adsorption, covalent immobili-zatron, and radiation cross-linking, have been used to modify surfaceswith PEG (22).Polymers that comprise carbohydrate units also passi-vate surfaces, but these materials are less stable and less effective thanPEG (41,16). A widely used strategy is to preadsorb a protein-usuallybovine serum albumin-that resists adsorption of other proteins. Thisstrategy suffers from problems associated with denaturation of theblocking protein over time (3) or exchange of this protein with othersin solution. A further limitation of this strategy is the inability to presentother groups (e.g. ligands, antibodies) at the surface in controlled envi-ronments.

Self-assembled monolayers that are prepared from alkanethiolsterminated in short oligomers of the ethylene glycol group[HS(CH2)1r(OCH2CH2),,OH n : 2 - ]l resist the adsorption of sev-eral model proteins, as measured by both ex situ ellipsometry and insitu SPR spectroscopy (53, 58). Even SAMs that contain as much as50Vo methyl-terminated alkanethiolates, if mixed with oligo(ethyleneglycol)-terminated alkanethiolates, resist the adsorption of protein.Self-assembled monolayers that present oligo(ethylene glycol) groupsare useful as controls in studies of the adsorption of proteins to surfaces.The ability to prepare SAMs that present derivatives of these and other


groups will be useful for investigating the mechanisms by which these

surfaces resist adsorption.De Gennes, Andrade, and coworkers (29,30) have proposed that

surfaces modified with long PEG chains resist the adsorption of protein

by "steric stabilization." In aqueous solution, the PEG chains are sol-

vated and disordered. Adsorption of protein to the surface causes the

glycol chains to compress, with concomitant desolvation. Both the ener-

getic penalty of transferring water to the bulk and the entropic penalty

incurred on compression of the layer serve to resist protein adsorption.It is not clear that this analysis applies to thin, dense films of oligo(ethy-lene glycol) groups, as De Gennes and Andrade predicted that surfaces

comprising densely packed, nearly crystalline chains of PEG might not

resiJt the adsorption of protein. It is remarkable that SAMs presenting

densely packed tri(ethylene glycol) groups resist the adsorption of pro-

tein. These layers are almost certainly different in comformational flexi-

bility and solvation than are long, dilute PEG chains. We presume there

is sufficient free volume in the glycol layer of the SAMs to allow

solvation by water. An understanding of the properties of these SAMs

may permit the design of new classes of inert surfaces.

Immobilization of Proteins to Self-Assentbled Mcnolayers

The immobilization of proteins to substrates is important in many areas,

ranging from ELISA and cell culture to biosensors; consequently, many

strategies have been developed to confine proteins to surfaces (51,

63). Methods that rely on noncovalent association of proteins with

surfaces-with the use of both hydrophobic and electrostatic interac-

tions-are the most common and experimentally simplest, but they are

also the least well controlled.Methods that rely on covalent coupling of proteins to surfaces are

inherently more controlled and give layers of protein that cannot disso-

ciate from the surface or exchange with other proteins in solution. A

variety of surface chemistries have been used: the most successful have

been based on the formation of amide and disulfide bonds (26, 14,18).

The selectivity and rapid reaction of thiols with a-haloacetyl groups

constitutes a particularly attractive protocol (38). The use of well-de-

flned surfaces and proteins that have only a small number of reactivegroups permits a high degree of control over the attached protein. For

example, genetic engineering was used to construct a mutant of cyto-

chrome c that had only a single cysteine group; immobilizatron of thisprotein to a SAM terminated in thiol groups gave a uniformly orientedlayer of protein (26).


A common problem that limits the use of immobilized proteins isdenaturation of the protein, with concomitant loss in activity. Methodsto increase the lifetimes of these proteins have involved coupling to"inert" materials (76). A commercial SPR instrument that quantitatesprotein-protein interactions uses a gel layer of carboxylated dextranto stabilize immobilized proteins (41). Self-assembled monolayers thatare terminated in oligo(ethylene glycol) groups may have broad useful-ness as inert supports, because a variety of reactive groups can beincorporated in SAMs in controlled environments.

Biospecific Adsorption of Proteins to Self-AssembledMonolayersThe design of surfaces to which analytes bind specifically is importantfor biosensors and other technologies, e.g. affinity chromatography,cell culture, coatings for implants, and artificial organs. These surfacesmust possess specificity for a particular protein and simultaneouslyresist the nonspecific adsorption of other proteins.

Immobili zatron schemes based on the biotin-streptav idin interactionhave been investigated widely. Spinke et al (68) studied the recognitionof streptavidin by SAMs that present biotin ligands. The effectivelyirreversible complexation in this system is useful for many applications,such as immobilization of proteins or nucleic acids, but is not relevantto the weak, reversible recognition that is more common in biology.In an early model system, Mosbach and coworkers (45) used ellipsome-try to characterize the reversible binding of lactate dehydrogenase toSAMs that present analogues of nicotinamide adenine dinucleotide(NAD). Several groups have studied the recognition of immobilizedantigen by antibodies, because of the availability of antibodies to avariety of antigens and the high specificity displayed by antibodies(35) .

Self-assembled monolayers that present oligo(ethylene glvcol)groups were used as supports to which ligands for proteins were at-tached (Figure 3). With the use of SPR spectroscopy. Sigal et al (64)measured the binding of a His-tagged T-cell receptor to a SAM present-ing a Ni(II) complex. Likewise, carbonic anhydrase bound to SAMsthat presented a benzenesulfonamide group (Figure 4) (52). In bothcases, the amount of protein that bound increased with the density ofligand on the SAM. Both SAMs also resisted the nonspecific adsorptionof other proteins. The SAM terminated in benzenesulfonamide groupscould be used to measure the concentration of carbonic anhydrase (CA)in a complex mixture that contains several other proteins (52). Theeffectiveness of the oligo(ethylene glycol) groups to resist nonspecific


Ligand

(EG)r

Anchor region-(cH)rr_ras

Figure 3 Design of SAMs for biospecific adsorption. Mixed SAMs that present ligands

and oligo(ethylene glycol) groups permit control over the density of adsorbed protein.

The glycol layer is effective at preventing nonspecific adsorption of protein.

Buffer CA Buffer

1000

Time (s )

Fi,qure.l Surface plasmon resonance spectroscopy was used to measure the rate and

quantity of binding of CA to a SAM terminated in EGr groups and benzenesulfonamide

groups (A). The change in resonance angle 6A of light reflected from the SAM/gold

is plotted against time; the time over which the solution of CA (5 pM) was allowed to

flou, through the cell is indicated at the top of the plot (B). (upper curve) Binding (and

dissocration) of CA to a SAM containing approximately 57o of the ligand-terminated

alkanethiolate. Carbonic anhydrase did not adsorb to a SAM that presented only ethylene

glycol groups (lov.er curt'e). A response caused by the change in index of refraction of

the CA-containing solution was observed on introduction of protein into the flow cell

(evident in lov,er curt,e). The difference between the measured response and this back-

ground signal represents binding of the CA to the SAM.

B

I1-500


adsorption, combined with the ability of SAMs to present a range ofgroups in controlled environments, makes this system well suited forother studies of biospecific adsorption and for applications dependenton specific adsorption.

Attachment of Cells to Self-Assembled MonolayersThe attachment and spreading of anchorage-dependent cells to surfacesare mediated by proteins of the extracellular matrix, e.g. fibronectin.laminin, and collagen. A common strategy for controlling the attach-ment of cells to a surface therefore relies on controlling the adsorptionof matrix proteins to the surface. Both hydrophobic (42,46), and ionic(34) SAMs have been used as substrates for cell culture. A significantproblem with these preparations is the lack of control over the adsorp-tion process. It generally has been assumed that the density of matrixprotein is the parameter that influences the behavior of attached cells.Studies of the differentiation response of fibroblasts and neuroblastomacells on siloxane SAMs terminated in different groups that had beencoated with fibronectin suggested that cell behavior depended on rheconformation of fibronectin and not on the density of protein (7. 40).The role of protein adsorption in most instances remains poorly under-stood.

Cell attachment to and spreading on fibronectin involve binding ofintegrin receptors of the cell to the tripeptide RGD of the matrix. Mas-sia & Hubbell (47) demonstrated that a siloxane SAM that presents theRGD peptide supported the attachment and spreading of fibroblast cells.These synthetic culture substrates have advantages over the traditionalmatrix-coated substrates of increased reproducibility in culture and util-ity for fundamental studies of cell-matrix interactions. Correspondingwork has focused on the development of substrates for serum-free ce llculture by immobilizing essential growth factors at the surface clt'thesubstrate (86).

CONTROL OVER SPATIAL ADSORPTIONOF PROTEIN

P atterning S elf-As sembled M onolayers

MICRocoNrACr pRrNrrNG Microcontact printing (pCP) (36, 54. 8l )provides a new and convenient method for patterning SAMs of alka-nethiolates on gold with features of sizes ranging down to I p.m (Figure

Master

INTERACTIONS OF SAMS WITH PROTEINS 6l

Yl^

wl iV

w

*z t . "

l aY

1l;l;l;l;l;l;l;l PDMS ;l;l;l;l;l;l;l;i'l\Rrt, )',.tRr','i,)Ri,'

l nV"

;l;l;l;l;l;l;l;l' PDMS ;l;i1l;l;l1i;l'/\/\ir+'/\/\/-+:-\'/'2\/\i-'+'/'?

i '

l*l*l I // SAM (2-3 nm),/z Au(10-200nm),z/ nu \ru-zuu rul'

T i (1 -10 nm)- Si (0.5-2 mm)

Figure 5 Microcontact printing starts with a master template containing a pattem of

relief (a); this master can be fabricated by photolithography or by other methods. A

PDMS sramp cast from this master (b) is inked with a solution of alkanethiol in ethanol(c) and used to transfer the alkanethiol to surface of gold (d); a SAM is formed only at

those regions where the stamp contacts the surface (e). The bare regions of gold can

then be derivatized with a different SAM by rinsing with a solution of a second alkanethiol

(/). The initial pattemed SAM can also be used to protect the underlying gold from

dissolution in a corrosive etchant (g). Anisotropic etching of the exposed silicon gives

contoured surfaces (/i). The gold mask can be removed by washing w\th aqua regiai:

the resulting silicon substrates are useful as new masters from which stamps can be cast

or as substrates for a variety of applications.

5); features as small as 200 nm have been formed with the use of thisrechnique (85). Microcontact printing starts with an appropriate reliefstructure from which an elastomeric stamp is cast; this "master" tem-plate usually is generated photolithographically, but any substrate thathas an appropriate pattern of relief can be used. The polydimethylsilox-ane (PDMS) stamp is "inked" with a solution of alkanethiol in ethanol,dried, and manually brought into contact with a surface of gold. Thealkanethiol is transferred to the surface only at those regions where thestamp contacts the surface. This process produces a pattem of SAM

l "V0.2 - 100 pm

cHq(cH2)1ssH


that is defined by the pattern of the stamp. Conformal contact betweenthe elastomeric stamp and surface allow surfaces that are rough (at thescale of 100 nm) to be patterned over areas several square centimetersin size with edge resolution of the features better than 50 nm. Multiplestamps can be cast from a single master, and each stamp can be usedhundreds of times. Microcontact printing also was used to pattem silox-anes on the surfaces of SiO2 and glass (84) and to pattern SAMs onnonplanar and contoured surfaces (27). Because pCP relies on molecu-lar self-assembly and does not require stringent control over the labora-tory environment, it can produce pm-scale patterns conveniently andat low cost relative to methods that use photolithography.

PHoroLIrHocRApHy Photolithographic methods illuminate a surfacewith uv light through a mask (5, 17,34). In rhe common "lift-off"method (34), a silicon oxide substrate is coated with a thin layer ofphotoresist. The resist is exposed to UV light through a mask, and theexposed regions are subsequently removed in a developing bath: thisprocess creates a pattern of silicon dioxide that can be derivatized w'ithan alkylsiloxane SAM. The remaining regions of photoresist are rhenremoved, and a different SAM is formed on the complementarv regions.Other variants of photolithography create patterns by usin-e UV lightto damage, or modify, a SAM. wrighton and coworkers (19) preparedSAMs of alkanethiolates terminated in an aryl azide group; near-UVirradiation of the SAM through a lithographic mask and a thin film ofan amine resulted in the attachment of the amine in the exposed regions.Hickman et al (24) inadiated thiol-terminated siloxanes through a maskin the presence of oxygen to form sulfonate groups. These methods arcless well controlled and less general than such methods as plCP. w,hichpattern the adsorption of preformed components. Photolithographicmethods can produce patterns that have features down to I trrm conven-iently. Capital costs for the equipment and controlled environment facil-ities, however, make this technique expensive and inconvenient for thebiological researcher.

FABRICATION oF CONTOURED SURFACES Both pCP (32) and photol i-thography (10) have been used to pattern silicon substrates with a layerof resist that protects the substrate from dissolution in a chemical et-chant (Figure 4). Chemical etching of these patterned substrates pro-duces contoured features whose shapes depend on the orientation ofthe silicon and the time of etching; anisotropic etching of a silicon( 100) surface produces controlled V-shaped grooves. The properties ofthese etched substrates can be tailored either by forming an alkylsilox-


ane SAM or by evaporating a layer of gold and forming a SAM ofalkanethiolates. Alternatively, the topographic pattern can be trans-ferred to other substrates (e.g. prepolymers) with the use of a PDMSstamp cast from the etched master.

Patterning Adsorption of Protein on Self-AssembledM onolayersPatterned SAMs on gold have been used extensively to control theadsorption of protein to surfaces (Figure 6). This method relies on theability of a SAM terminated in oligo(ethylene glycol) groups to resistthe adsorption of protein. Microcontact printing was used to pattern aSAM into regions terminated in methyl groups and oligo(ethylene gly-col) groups (42,54). Immersion of these SAMs in aqueous solutionsthat contain proteins resulted in the adsorption of a monolayer of proteinonly on the methyl-terminated regions; this pattern of protein could beimaged by scanning electron microscopy (43). Bhatia et al (4 patterneda siloxane film terminated in thiol groups by inadiation with UV lightthrough a mask. The fluorescent protein phycoerythrin was immobi-bzed to the thiol groups in regions that were protected from the UVlight with the mask; photo-induced oxidation of the thiol groups inregions of the surface that were irradiated presumably gave negativelycharged sulfonate groups, which resisted the adsorption of protein.

Patterned Attachment of Cells on Self-AssembledM onolayersThe same methods used to pattern the adsorption of proteins to surfaceshave been used to direct the attachment of cel ls to surfaces (5,23,,34,66,61).In an early example, Kleinfeld et al (34) used photolithographyto pattern siloxane SAMs into regions terminated in methyl and aminogroups. When plated in the presence of serum, neural cells attachedand spread selectively on the amino-terminated regions; in the absenceof serum, the cells attached to all regions. Others have also found thatamino-terminated siloxanes are excellent substrates for culture of neuralcells (241. We presume that proteins of the serum that do not supportcell attachment adsorbed to the hydrophobic areas. Regions terminatedin perfluoro groups have also been used to resist the attachment of cells( 3 , 6 1 , 7 0 ) .

Self-assembled monolayers patterned into regions terminated inmethyl and oligo(ethylene glycol) groups permit spatial control overthe attachment of cells. Microcontact printing was used to pattern SAMsinto adhesive lines ranging from l0 to 100 pm in width; after coatingthese substrates with fibronectin. endothelial cells were confined to


CHzlProtein

I

;;ru

1GmFigure 6 Scanning electron micrograph of fibrinogen adsorbed on a pattemed SAM.A patterned hexadecanethiolate SAM on gold was formed b1 7-rCP. and the remainderof the surface was derivatized by immersion in a solution containing a hexa(ethyleneglycol)-terminated alkanethiol IHS(CHr)11(OCH2CH:)oOH].The patterned substrate was

immersed in a solution of fibrinogen (1 mg/ml) in phosphate-buffered saline for 2 h,removed from solution, rinsed with water, and dned. Fibrinogen adsorbed only to themethyl-terminated regions of the SAM. as illustrated by the dark regions in the mi-crograph: Secondary electron emission from the underlying gold is attenuated by theprotein adlayer.

EG6OH

INTERACTIONS OF SAMS WITH PROTEINS l l

Figure Z Control over the attachment of bovine capillary endothelial cells to planarsubstrates that were patterned into regions terminated in methyl groups and tri(ethyleneglycol) groups using pCP. The substrates were coated with fibronectin before cell attach-ment; fibronectin adsorbed only to the regions of methyl-terminated SAM. (A) An opticalmicrograph showing attachment of endothelial cells to a nonpatterned region (left\ andto lines 3O pm in width. (B) A view at higher magnification of cells attached to thel ines.

grow on these lines (Figure 7). This technique was also used to patternSAMs into adhesive regions approximatelv 20 X 50 pm in size thatwere surrounded by EG6-terminated SAM (66). After the hydrophobicregions were coated with laminin, hepatocytes attached to the rectangu-lar islands and conformed to the shape of the underlying pattern. Thesize of the islands controlled DNA synthesis, cell growth, and proteinsecretion of the attached cells. The ability to pattern the attachment ofindividual cells may be useful for single cell manipulation, toxicologyand drug screening.

Attachment of Cells on Contoured SurfacesSeveral groups have used surfaces contoured into grooves and ridges,which were fabricated with the use of photo- or electron-beam lithogra-phy, to study their effects on the behavior and growth of attached cells(9, 10, 25,49). Chou et al (9) found that human fibroblasts adherentto surfaces contoured into V-shaped grooves had increased levels offibronectin synthesis and secretion. Surfaces with arrays of grooves ofvarying dimensions controlled the alignment and orientation of attacheclmammalian cells (10, 49). Surfaces with arrays of ridges directed themotility and induced differentiation of the fungus (Jromyc'es (25).

A simple technique based on pCP and micromolding (33) was

1@'" 50 pm


Me/ FN Me/ FN EG"

r lffivl v

50 umFigure I Control over the attachment of endothelial cells to contoured surfaces usingSAMs. The substrates are films of polyurethane (supported on glass slides) that werecoated with gold and modified with SAMs of alkanethiolates terminated in methyl groupsand tri(ethylene glycol) groups; the substrates were coated with fibronectin before cellattachment.(left) Cells attached to both the ridges and grooves of substrates that presentfibronectin at all regions. (right) Cells attached only'to the ridges when the grooves vuercmodified with a SAM presenting tri(ethylene glycol) groups

used to fabricate contoured surfaces of optically transparent filmsof polyurethane on glass coverslips. After evaporating a thin filmof gold onto these substrates, the ridges were derivatized with aSAM by stamping with a flat PDMS stamp, a different SAM wasformed in the grooves by immersing the substrate in a solution ofalkanethiol. By modifying the ridges with a SAM of hexadecanethio-late, and the grooves with a SAM terminated in oligo(ethylene glvcol)groups, endothelial cells were confined to attach and spread onlyon the ridges (Figure 8). By the reverse process, cells were confinedto attach and grow in the grooves.

APPLICATIONS OF SURFACES BASED ON SELF-ASSEMBLED MONOLAYERS IN BIOCHEMISTRY

Drug Design and ScreeningSeveral combinatorial strategies for drug design screen mixtures ofpotential ligands to identify those with a desired property-usually theability to bind a protein. Methods that use immobilized libraries have

INTERACTIONS OF SAMS WITH PROTEINS ]3

the primary advantage that the identity of each ligand is defined by itslocation in the matrix; amplification and sequencing of selected ligandsare not required. Fodor and colleagues (28) used photolithography andsolid-phase synthesis to pattem SAMs of siloxanes into arrays ofhundreds of different peptides. Confocal microscopy was used to assaythe in situ binding of a monoclonal antibody to each of these peptidesin a single experiment. This technology also was used to create an arraycontaining 256 octanucleotides that served as a hybridization probefor sequencing DNA. The demonstration of libraries that contain non-natural biopolymers (8), and the potential to screen for reactions atsurfaces (78), widens the scope of this technology. Scanning probemicroscopies may be useful for screening libraries, because these tech-niques can assay functionalized surfaces at the sub-micron scale rapidly(39, 80).

Biosensors

Self-assembled monolayers are finding increasing use to tailor the mo-lecular recognition properties of surfaces used in biosensing. The sur-face of a TiO2-SiO2 waveguide was modified with an alkylsiloxanemonolayer terminated in amino groups to which the antibody anti-HBsAg was conjugated; this difference interferometer measured thebinding of hepatitis B down to a concentration of 2 x 10-13 M inundiluted serum (621. Self-assembled monolayers have been used insimilar ways to control the properties of sensors on the basis of thequartz crystal microbalance (77), acoustic plate modes (3 1), and surfaceplasmon resonance (52, 53, 64). Nonspecific adsorption of protein isa common problem with these devices. A commercial technology usesgel layers of dextran to control unwanted adsorption (41), althoughmost applications use bovine serum albumin-coated surfaces. Recentwork with ligands immobilized on SAMs terminated in oligo(ethyleneglycol) groups provides a route to biospecific surfaces with a highcontrol over the properties of the surfaces (52, 64).

E I e ctroc hemical M ethods

Self-assembled monolayers can either mediate or inhibit the transferof electrons from the underlying gold to electrolytes in solution. Theproperties of SAMs terminated in electroactive groups (e.g. ferroceneor quinone groups) could be switched reversibly by adjusting the poten-tial at the underlying gold (l). Several groups have studied the transferof electrons from gold to electroactive proteins immobilized to SAMs( I I, 82). There is now commercial technology for analysis of biological


analytes on the basis of electrochemiluminescence from tris-bipyridineruthenium(Il) tags (21): This technology is well suited for SAMs.

PROSPECTS FOR SELF-ASSEMBLED MONOLAYERSIN BIOLOGY

Self-Assembled Monolayers as Model SurfacesSelf-assembled monolayers of alkanethiolates on gold provide the bestsystem available with which to understand interactions of proteins andcells with man-made surfaces. The ease with which complex and deli-cate groups of the sorts relevant to biochemistry can be presented incontrolled environments, combined with simple methods that can pat-tern the formation of SAMs in the plane of the monolayer, make thesesurfaces well suited for studies of fundamental aspects of biointerfacialscience. Other advantages with this system include the optical transpar-ency of SAMs when supported on thin films of gold, the electricalconductivity of the underlying gold, the compatibility of these sub-strates with a range of analytical methodologies, the stability of thesesubstrates during storage and in contact with biological media, and therange of surfaces, including curved and nonplanar substrates. that canbe used.

Self-Assembled Monolayers in Cell Biology

Designed substrates permit strategies for the noninvasive control overthe activity of attached cells. Langer and coworkers (83) cultured endo-thelial cells on optically transparent films of electrically conductingpolypynole. Cells attached and spread normally on fibronectin-coatedpolypynole in the oxidized state; on application of a reducing potential.however, the extension of cells and the synthesis of DNA were bothinhibited. Okano et al (55) grafted thermoresponsive gels of poly(l/-isopropylacrylamide on cell culture substrates. Hepatocytes attached tothe substrates normally at 37'C: when the culture was chilled to 4oC,the cells detached from the substrate. The features of SAMs describedin this review make them well-suited model surfaces for studies inbiology that require substrates with tailored properties.

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