Immobilization of Bio Macro Molecules on Self Assembled Mono Layers

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This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 25672592 2567

Cite this: Chem. Soc. Rev., 2011, 40, 25672592

Immobilization of bio-macromolecules on self-assembled monolayers:

methods and sensor applications

Debasis Samanta and Amitabha Sarkar*

Received 27th July 2010

DOI: 10.1039/c0cs00056f

Attachment of biomolecules on gold, silicon or glass surfaces has direct implications for the

development of novel biosensors in the context of nanoscale detection of pathogens and other

metabolites related to issues of human health. In this critical review, we have highlighted the

current developments in various techniques of immobilization of biomolecules, specifically

biological macromolecules on surfaces through the modification of a functional self-assembled

monolayer. The utility of such immobilized biomolecules in the area of biosensing in nanoscalehas been surveyed. Merits and demerits of some of the methods with reference to sensitivity of

detection and practical use have been discussed (221 references).

1. Introduction

Biosensors offer a simple, reagentless analytical method to

sense important biomolecules and can be used for detection of

pathogens, and other metabolites (often down to nanoscale)

related to issues of human health. Attachment of biomolecules

on different surfaces is an integral part of research for the

development of biosensors in the form of chips.

Biomolecules can be immobilized on a surface by

simple physical adsorption methods. For example, they can

be entrapped into a porous polymeric material such as

polypropylene membrane modified with polyaniline, aided

by electrostatic and hydrophobic interactions.1 However, such

an attachment is weak and pH dependant: they may be

removed by the buffer used for performing assays. Biomolecules

can also be entrapped into electrodeposited molecular layers

of polyphenol, polythiophene or polyaniline2,3 In this case,

molecules have to diffuse in and out through the layer. For

diffusion to be fast enough to speed up response, thinner layers

are preferred to improve the efficiency of sensing. Langmuirand Blodgett developed an advanced technique to build up a

layer of one molecular thickness or multi-layers of desired

molecular thickness (LB film)4,5 by attaching amphiphilic

molecules onto glass or silicon surfaces. Several groups

used the LB film technique to immobilize biomolecules on

Department of Organic Chemistry, Indian Association for theCultivation of Science, Jadavpur, Kolkata 700 032, India.E-mail: [email protected]; Fax: (91)3324732805;Tel: (91)3324734971

Debasis Samanta

Dr Debasis Samanta received

his BSc and MSc in Chemistry

from University of Calcutta.

He was awarded his PhD degree

in chemistry by Jadavpur

University, Kolkata, for hiswork on functional self-

assembled monolayers. In

2005, he joined as a research

associate in North Dakota

State University, USA, to

work on asymmetric synthesis.

Later, he moved to the

Department of Polymer Science

and Engineering at the

University of Massachusetts,

Amherst, USA, as a post-doctoral associate to work on the

synthesis of biocompatible polymers. Currently he is a senior

research associate at IACS, Calcutta.

Amitabha Sarkar

Dr Amitabha Sarkar is

currently a senior professor in

the Department of Organic

Chemistry at the Indian

Association for the Cultivation

of Science (IACS), Kolkata.He received his BSc and MSc

degrees in chemistry from IIT,

Kharagpur, followed by his PhD

from IISc, Bangalore, India.

After post-doctoral training

in Case Western Reserve

University and Washington

State University, USA, he

joined the National Chemical

Laboratory, Pune, India, in

1988 as a scientist and project leader. In 2002, he moved to IACS.

His research interests include organometallic chemistry, self-

assembled monolayers and stereoselective synthesis.

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2568 Chem. Soc. Rev., 2011, 40, 25672592 This journal is c The Royal Society of Chemistry 2011

surfaces.6,7

However, the LB film is fragile in nature and

biomolecules such as BSA, insulin, ovalbumin, myoglobin or

cytochrome C are often denatured when such a film is spread

in an airwater interface. In short, physisorption commonly

tends to afford arrays of immobilized biomolecules that are

less defined, less precise and more fragile.

For better reproducibility, durability and precision,

biomolecules have been attached to surfaces through an

organized monomolecular layer with defined orientation. Suitably

functionalized long-chain aliphatic molecules spontaneously

assemble on metal or glass surfaces to form stable covalent

bonds between its terminal function and the activated metal,

metal oxide or silicate. A densely packed, organized molecular

layer thus formed, which extends the hydrocarbon chains

approximately orthogonal to the surface (Fig. 1). Such

spontaneous molecular organization is referred to as a self-

assembled monolayer or SAM, whose thickness depends on

the length and orientation of the hydrocarbon chain. Early

examples include monolayers of long chain alcohols on glass,8

long chain amines on platinum,9 alkyl trichlorosilane on

silicon,10

and long chain thiols, thioesters, thiones and alkyl

disulfides11

on gold surface.

There are two factors that make SAMs viable for

commercial biosensor development: (1) a self-assembled

monolayer on a planar substrate can be prepared in the

laboratory easily by dipping the substrate in a dilute solution

of an organic molecule for a specific period of time followed by

thorough washing with the same solvent and drying under

nitrogen flow; (2) the formation of a self-assembled monolayer

needs only a very small amount (approximately 2 107 g cm2)

of chemicals.12 Thus it is economically viable to use even

expensive compounds for the development of SAM-based

biosensors for commercial use. One key issue in developing

such biosensors is the control of accessibility and molecular

orientation of the attached biomolecules. Since the SAM offers

a good control at molecular level, it is an excellent platform to

develop biosensors. In 2002, Vijayamohanan and others

reviewed the application of self-assembled monolayers for

biosensor development.13 We propose to revisit the

chemistry between biomolecules and the exposed functionalities

of the SAM that leads to covalent or noncovalent immobilization

of biomolecules on the surface and their application as

sensors. The discussion is restricted to silicon, silica, gold

and glass surfaces only. Direct attachment of biomolecules

to surfaces has also been achieved via their functional groups

or functional tethers, but those are beyond the scope of this

review.

Self-assembled monolayers can be formed on different

surfaces depending on the functional groups of the molecule

and properties of the surfaces. However, the monolayer

formation strategy should be based on the physicochemical

properties of both the surface and biomolecules. The active

biomolecules should retain their conformational integrity on

the surface for a long period of time. For this reason, well

defined and robust SAMs of siloxane on silicon and thiols on

gold surfaces are most extensively studied. For assembling a

monolayer on silicon, a trichlorosilane is commonly used. It is

highly reactive, incompatible with aqueous solution and

polar o-functionalities such as OH or NH2 on the linear

hydrocarbon. Relatively inert substituents such as hydro-

carbons (alkane, alkene or alkyne) or halogens can be used.

On the other hand, for the formation of SAMs on a gold

surface, the terminal thiol can accommodate functional groups

such as OH, COOH, NH2 within the molecule. In addition,

weakly adsorbed impurities are displaced from the gold

surface by the sulfur containing chain. One important factor

to maximize the activity of the biomolecule in the biosensor is

the order and crystallinity of the SAM. It was observed that

order and crystallinity is better when a monolayer is formed

with long chain alkane thiols (number of methylene groups

410).14 In 1996, Ulman reviewed various aspects of SAMs

formed by amines, carboxylic acids, chlorosilanes or organo-

sulfur compounds on different metal and metal oxide surfaces.15

The exposed functional terminus of a SAM is utilized for the

attachment of a biomolecule via covalent bond formation or

non-covalent interaction. Non-covalent attachment usually

requires a milder protocol, but there is always a possibility

that the concentration of active substance on the matrix would

gradually diminish on repeated use and washing. On the other

hand, covalently attached molecules survive such repetitive

operations over a longer period of time.

To maximize covalent and non-covalent attachment of

biomolecules to SAMs, accessibility of the end group of a

monolayer should be high. A spacer unit such as triethylene

glycol or hexaethylene glycol is often incorporated between the

exterior termini of the alkyl chain and the key functional

group in order to enhance its accessibility in the bulk medium

(Fig. 2).

The density of biomolecules on the surface can be controlled

by assembling a mixed monolayer by co-adsorption of two

different surfactants of similar dimension but different

terminal functional groups on the exteriorone reactive and

the other inert. In this case, reduced steric hindrance improves

the efficacy of bond-formation as well as sensing or recognition.

The ratio of two dissimilar molecular chains on the monolayer

is usually proportional to the ratio of the two molecules

Fig. 1 Formation of SAMs on gold and silicon surfaces.

Fig. 2 Example of a mixed monolayer with a spacer unit.

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used.16 Murray and others reported that place exchange

reaction can be performed on monolayer protected nano-

particles by a suitable long chain functional molecule.17 Later,

the same group showed18 that when arylthiols were used, the

polarity of the substituent affects the exchange dynamics of

ligands in a place exchange reaction on the nanoparticle. The

author interpreted the phenomenon as more polar AuS

bonds at the defect sites favouring bonding with more electron

deficient sulfur moieties.

The o-functionality of SAMs is often required to be

chemically modified for the desired chemistry necessary for

attachment of target biomolecules or sensory molecules.

Such chemical modification should not destroy the organized

structure of the SAM.

1.1 Chemical reaction on the head-group of SAMs

The following chemical reactions are frequently performed for

the modification of SAMs on different surfaces.

1.1.1 Nucleophilic substitution reaction. In nucleophilic

substitution reaction, an electron rich nucleophile such ashydroxide, cyanide, azide, water, ammonia etc. replaces a

leaving group attached to a partially positive charged atom.

In 1990, Sukenik and others reported that bromine terminated

glass surfaces can be reacted with various nucleophiles such as

azide (N3), thiocyanate (SCN), sulfide (S2) to introduce

different end functionality on the monolayer.19 This was

achieved by dipping the monolayer coated substrate (glass

slides or ATR crystals) in the reagent for a short period of time

and withdrawing it with Teflon-coated tweezers. These

functional groups were converted to other functional groups

(thiocyanate to thiol, azide to amine, disulfide to thiol, etc.) by

subsequent oxidation or reduction reaction (Fig. 3). The

authors verified that the monolayers are unaffected by suchreaction conditions by carrying out control experiments with

monolayers terminated by a hydrocarbon end group. Study

of the reaction by IR spectroscopy, XPS spectroscopy and

contact angle measurements indicated a quantitative conversion

of bromo-terminated monolayers to other useful functional

monolayers.

However, Fryxell et al. claimed that on a mixed monolayer

on silicon wafers featuring bromo and methyl termini,

nucleophilic displacement of bromide by thiocyanate or cysteine

thiolate was slow and never went to completion.20 Only the

azide ion completely replaced the bromide. The authors argued

SN2 reactions require backside attack of the nucleophile on the

terminal bromomethylene. This low angle approach trajectory

is severely hindered sterically by the hydrocarbon matrix of the

SAM substrate. This observation is consistent with an earlier

report that described displacement of terminal benzyl chloride

with iodides. It concluded that less dense films allow for

essentially complete conversion of the iodinated product.21

This is also supported by the facile displacement reactions on

monolayers formed with (OMe)3Si(CH2)3NH2 on a silicon

surface.22 Yet, nucleophilic displacement reaction of bromide

of a bromo-terminated SAM by phthalimide is reported to

proceed efficiently under mild conditions. The phthalimide

was subsequently converted to amino function groups. One

needs to bear in mind that, however, a three-carbon high

monolayer is not tightly packed, hence stereochemical

consequences are not comparable with a dense monolayer.

1.1.2 Click chemistry. Copper-catalyzed 1,3-dipolar

cycloaddition of alkyne and azide is commonly known as a

click reaction because it is modular, wide in scope, gives

very high yields of one regioisomer and generates only

inoffensive byproducts. To perform the click reaction on a

surface, one needs to prepare either an azide or an alkyne

terminated surface. Heise and others described the preparation

of an azide terminated SAM.23 Alkyne terminated SAMs were

also prepared by several groups.2427

Hoffman and others reported 1,3-dipolar cycloaddition

reaction of different alkyne substrates on azide functionalized

silicon substrates (Fig. 4).28

Choi and others studied the click reaction on a self-

assembled monolayer on gold. They showed that the reaction

requires only mild reaction conditions.29 The progress of the

reaction was studied by Fourier transform infrared spectro-

scopy (FTIR), X-ray photoelectron spectroscopy (XPES),

ellipsometry, and contact angle measurement.

1.1.3 DielsAlder reaction. Mrksich and others studied the

DielsAlder reaction of cyclopentadiene with a benzoquinone

terminated monolayer.30 To study the steric effect on the rate

of reaction, the authors varied the accessibility of the

quinone by preparing mixed monolayers from hydroquinone-

terminated alkanethiols of variable lengths [HS(CH2)nHQ,

n = 614] and a hydroxyl-terminated alkanethiol [HS(CH2)11OH]

Fig. 3 Nucleophilic substitution reaction and oxidation or reduction

reaction on SAMs on a silicon surface.

Fig. 4 Click cycloaddition reaction on a silicon substrate containing

azide functionalities.

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of constant length. For monolayers that positioned the quinone

groups below the surrounding hydroxyl groups, the rate constants

of DielsAlder reaction decreased by approximately 8-fold. The

enthalpy of activation in this case was 4 kcal mol1

greater than

that with quinones which were more accessible at the interface

(Fig. 5).

Workentin and others prepared a maleimide modified

surface on gold by using retro-DielsAlder reaction

(Fig. 6).31

1.1.4 Palladium-catalyzed CC coupling reaction on

surfaces. Zhang and others showed for the first time that

palladium catalyzed Heck coupling reaction can be performed

on a G0 dendron SAM on a gold surface with bromophenyl

end group for the introduction of different functional groups.

During the Heck coupling process with 4-fluorostyrene, about

76% of bromine atoms were consumed for G0 dendrons

assembled on the gold plate, while for the G1 dendron film

about 68% bromine atoms were consumed32 (Fig. 7). The

variation of conversions may be attributed to different

orientation of bromophenyl groups and/or the presence of

adjacent thiol groups that may deactivate the catalyst in the

thin film assembly.

Cai and others studied the Heck reaction on G0, G1, G2

dendron SAMs with bromophenyl end group on SiO2/Si

surfaces and found that the overall yield decreases in the

following order: BrG0 4 BrG1 4 BrG2.33 Later, the

same group performed a Suzuki coupling reaction on an aryl

bromide terminated SAM on a silicon surface with an aryl-

boronic acid, and achieved excellent conversion (Fig. 8).34

Zhang and others reported Sonogashira reaction on a SAM

on a silicon (111) surface35 with moderate success.

1.1.5 Metathesis reaction. Alkene metathesis reactions36

(metal-catalyzed redistribution of alkene fragments by scission

of carboncarbon double bonds in alkenes) have been widely

used for the synthesis of important organic compounds and

polymers. In 2003, Sarkar and others reported for the first

time that cross metathesis (CM) reaction can be used to

graft a variety of groups such as Fischer carbene complex,

N-hydroxysuccinimide or ferrocene derivatives, by ruthenium-

catalyzed reaction on monolayer-protected gold clusters as a

mild and convenient strategy.37 Immobilized Fischer carbene

complex was further reacted with an amine, thus revealing the

possibility of immobilization of biomolecules on nanoparticles

through an amino function.

Almost simultaneously, Choi and others reported cross-

metathesis reaction on a vinyl-terminated SAM of undec-10-

ene-1-thiol on gold by Grubbs generation II catalyst (Fig. 9). 38

Fourier transform infrared spectroscopy, X-ray photoelectron

spectroscopy, and contact-angle measurement were performed

to study the reaction. The strategy was useful for the

introduction of various functional groups on SAMs on gold.

Brooksby and others electrochemically monitored the

cross-metathesis reaction on a vinyl-terminated SAM on gold

with a olefin-terminated ferrocenyl (Fc) derivative. This study

shows that surface concentration of alkene groups and

reaction conditions are important parameters for maximizing

CM yields on surfaces.39 Bowden and others reported the

cross-metathesis reaction on Si (111) surface to introduce

different groups such as carboxylic acid group, aldehyde,

alcohol or allyl bromide.40

1.1.6 Formation of amide and ester linkages. Ester and

amide linkages are widely used groups for surface modifications.

In this case, a monolayer with a carboxyl group at the end is

suitably activated by conversion to the anhydride,41 acyl

fluoride,42 or the active ester.43 This activated acid derivatives

are then reacted with alcohols or amines to form esters or

amides, respectively (Fig. 10). For example, Whitesides et al.

assembled a monolayer of mercaptohexadecanoic acid on gold

followed by treatment with trifluoroacetic anhydride to form

the mixed anhydride. a-Amino derivatives ofo-hydroxy and

o-methoxy-oligo(ethylene glycol)s was then reacted with this

carboxylic anhydride terminated SAM.44

The above reaction is particularly useful for anchoring

biological molecules to a SAM. Since many biomolecules such

as proteins contain amine functional groups on the lysine

residues, amide formation can occur readily. The hydroxyl

group of serine and phenolic OH group of tyrosine of protein

can similarly react with a mixed anhydride on the surface.

Alternatively, monolayers with terminal amine functional

groups can react with activated carboxyl functional group of

biomolecule (Fig. 11).

1.2 Analytical techniques to characterize functional SAMs

Several analytical techniques can be used to characterize a

functional monolayer on surfaces. Since the total amount of

organic material in the monolayer is very small, it is impossible

to get a complete molecular picture of SAMs using a single

analytical technique. In the following section, we briefly

qdiscuss some of the important characterization techniques.

Fig. 5 DielsAlder reaction on a SAM on a gold plate.

Fig. 6 Retro-DielsAlder reaction on a gold nanoparticle.

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One of the simplest and most effective methods to monitor

the structure and composition of monolayer is contact angle

measurement. The contact angle is the angle of contact of the

droplet of a probe liquid (water for water-contact angle

measurement) on a monolayer surface. The contact angle is

high when the end group of the monolayer is hydrophobic,

such as methyl or vinyl group, and the contact angle is low

when the end group is hydrophilic, such as hydroxyl or

carboxylate group. So, contact angle measurement is an

important tool to study chemical change of the end functional

group of a monolayer (hydrophilic to hydrophobic and

vice versa).

The quartz crystal microbalance is a highly sensitive device

that can detect very small changes in mass, usually in the

range of nanograms (e.g.: when a biomolecule binds to a self-

assembled monolayer).

Infrared (IR) spectroscopy is a powerful tool to unambiguously

identify several common functional groups present on SAMs.

To characterize SAMs on gold surface, incoming IR light is

reflected under a large angle of incidence (grazing angle

reflection configuration). SAMs on silicon surface are

characterized by transmission IR under Brewster angle. IR

spectroscopy is effectively used to monitor the fate of a

functional group after a chemical reaction.

UV-vis absorption spectroscopy and fluorescence spectro-

scopy provide information concerning the packing of a SAM.

Since the signal is concentration dependent, this spectroscopic

characterization technique is used to estimate the density of

the adsorbates on the SAM.

Ellipsometry and surface plasmon resonance (SPR) are

widely used to determine the thickness of the layer. In case

of ellipsometry, a plane polarized laser beam is allowed to be

reflected by the substrate resulting in a change of phase and

amplitude. By comparing the change of phase and amplitude

before and after formation of the SAM, the thickness of the

layer can be calculated. In case of surface plasmon resonance,

angle-dependent reflection of a p-polarized laser beam

on the monolayer is studied. At a certain angle of incident

light (plasmon angle) the reflectance is minimum due to the

excitation of surface plasmons by laser light. The plasmon

angle is dependent on the refractive index of the contacting

medium of the surface, so it can be used to study in situ the

Fig. 7 Heck reaction of (A) G0 dendron and (B) G1 dendron on gold

with 4-fluorostyrene.

Fig. 8 Suzuki coupling reaction on a silicon platform.

Fig. 9 Cross metathesis reaction on a SAM on a gold surface.

Fig. 10 Acid-amine coupling reaction on a SAM on a gold surface.

Fig. 11 Coupling reaction on an amine functionalized monolayer.

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growth of the SAM. It is also useful to study the real-

time proteinprotein interaction kinetics thus allowing for

multichannel biosensing.45,46

With XPS one can determine the elemental composition of

the self-assembled monolayer. The sample is irradiated with

monochromatic X-rays, which results in the emission of core

electrons of different nuclei. Based on the energy of the ejected

electrons the elemental composition and oxidation state of the

elements on the monolayer as well as outer surface of the metal

can be determined.

Atomic force microscopy (AFM) and scanning tunneling

microscopy (STM) are used to visualize the thin film in

molecular resolution. In this case, a tip is used which measures

the force between the tip and surface (AFM) or tip and surface

electron density (STM). Those characterization techniques are

also used to visualize any binding event that occurs on SAMs.

Scanning electron microscopy (SEM) and transmission

electron microscopy (TEM) are important viewing techniques

to study solid materials in nanometre scale. In SEM, an image

is generated with the help of secondary electrons to give the

viewer an impression of the surface of an object in three

dimensions. TEM produces a two-dimensional image by

projecting electrons through an ultrathin slice of specimen.

Those techniques can be used to identify defects on surface

regions and study molecular interactions.

Various electrochemical techniques such as cyclic voltammetry

(CV), amperometry, impedance measurements can be used to

study the electron transfer on the SAM. Since these methods

are inexpensive and operations are easy, it is suitable for

sensor applications. If any binding event occurs on the

SAM, it causes generation or consumption of an electro-

chemically active molecule such as hydrogen peroxide or

oxygen, or changes the resistive or capacitive properties of

the thin film. For any of the above events, the signal can be

detected electrochemically.

Recently Mrksich and others showed that matrix-

assisted laser desorption/ionization and time-of-flight mass

spectrometry (MALDI-TOF MS) can be used to characterize

the products and yields of reactions that occur with molecules

attached to monolayers.47 The Maldi-TOF MS method can

identify species by their masses so it can be applied for the

analysis of different chemical reactionsindependently or

sequentially. It is very sensitive technique which can identify

even an exchange of hydrogen by deuterium. In 2007, Mrksich

reported the study of 16 different chemical reactions

on a self-assembled monolayer using MALDI-TOF mass

spectrometry.48

2. Attachment of biomolecules to SAMs:

methodology

For decades, several methods have been developed to attach

biomolecules on SAMs on surfaces. All the methods can be

classified into two categories based on the mechanism of

attachment: (1) attachment through non-covalent interaction

(such as electrostatic, hydrophilic, hydrophobic or a combination

thereof) between biomolecules and the head groups of the

SAM, (2) attachment through covalent bond formation

between biomolecules and the head groups of the SAM.

Affinity interaction methods such as antigenantibody inter-

action or biotinavidin also falls under the first category.

They involve multipoint interaction which are non-covalent

in nature and provides a substantial binding energy.

Choice of suitable method is important for the design

of a biosensor since the activity of biomolecules may diminish

due to random orientation or deformation of its native

structure.49

2.1 Immobilization via non-covalent interactions

Immobilization of biomolecules on SAMs on surfaces can be

achieved through electrostatic, hydrophobic or polar inter-

actions.50,51 SAMs that contain positively charged amine or

negatively charged carboxy groups are most suitable for

biomolecule immobilization through electrostatic interactions.

For example Cag layan and others reported the attachment of

a robust protein BSA on a carboxyl terminated SAM using

electrostatic interaction between CO2 of the SAM head

group and pendent NH3+ on BSA (Fig. 12).52

Similarly, de Groot et al. studied electrostatic immobilization

of cytochrome c0

on a carboxylic acid terminated SAMon gold.53 They found that immobilization could only be

achieved for pH in the range 3.55.5 reflecting the fact that

the protein is only sufficiently positively charged below pH = 5.5.

Murgida and others showed that54

electrostatic immobilization

of cytochrome c on o-carboxyl alkanethiol on silver does not

result in change of the conformation of protein. A few years

ago, a surface was designed that exhibits a change in inter-

facial properties such as wettability in response to an applied

electrical potential by forming a SAM of terminal carboxylate

functional groups.55 Later, Mu and others designed an electro-

chemically switchable gold surface in a similar way to assemble

charged proteins avidin and streptavidin selectively.56 In

Fig. 12 Attachment of BSA on a carboxylate terminated SAM.

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this case, a surface was designed so that mercaptohexanoic

acid molecules of the self-assembled monolayer undergoes

conformational transitions between bent and straight

states when different potentials are applied. As a result, the

SAM can exhibit either hydrophilic or hydrophobic properties

as desired. Thus, the potential-controlled selective protein

assembly at pH 7.2 was achieved by the potential-driven

adsorption of two kinds of fluorescent-labelled proteins that

have different isoelectric points (IP) (Fig. 13).

Yamauchi et al. developed a method for electrostatic

layer-by-layer assembly of cationic lipid/plasmid DNA onto

a carboxylic acid terminated SAM on gold surface.57 The

advantage of the electrostatic immobilization method is that it

is a single step reagentless method of molecular self-assembly that

can be universally applicable to any protein or biomolecules.

Disadvantages include possible leaching of biomolecules from

the support and possible denaturation. There is also no control

over the packing density of the biomolecules.

The hydrogen bonding and/or hydrophobic packing

between the sugar moiety and the amino acid side chains

of protein is useful to noncovalently immobilize type II

cytochrome c3 on the SAM of thiol-derivatized neoglyco-

conjugate on a silver surface.58

Du and co-workers used the process of controllable adsorption

of protein on multiwalled carbon nanotubes for the immobil-

ization of acetyl cholinesterase on an alkanethiol self-assembled

monolayer.59

For the immobilization of protein, an oriented (i.e. site-

specific) attachment strategy is often undertaken to increase

the accessibility of the active site of the protein towards

different analyte molecules. Loss of activity due to random

orientation of protein to the SAM on the surface has been

reported.60 Cha et al. compared the catalytic activity of

sulfotransferase enzyme immobilized on silicon surfaces with

or without controlled orientation61 and found that oriented

immobilization is essential to retain the activity of the enzyme

and no activity was reported for randomly oriented biomolecules.

In 2007, Camarero reviewed some methods of site-specific

immobilization of proteins on solid supports.62

Recombinant proteins expressed with histidine or polyhistidine

tags are often used to perform site specific protein immobil-

ization.63 In this case, protein with poly(His) tag was placed

far away from the active site by genetic modification. It

was immobilized via a nickel-chelated complex, such as

Ni-nitriloacetic acid (Ni-NTA) already immobilized on the

surface (Fig. 14). The tetradentate ligand NTA forms a

hexagonal complex with Ni2+, Cu2+, Zn2+, Co2+ leaving

two binding positions available for binding to a His6 sequence.

The histidine or polyhistidine tag may be at the C- or

N-terminus or inserted in the exposed loop of the protein

and are widely incorporated into commercial expression vectors.

NTA can be assembled on the surface via the formation of a

SAM, or NTA can be attached covalently to a SAM already

formed on the surface. For example, Vogel and others

reported immobilization of NTA-maleimide on a silica surface

via thiolmaleimide reaction (Fig. 15).63 Corn and others

reported the formation of the SAM of an NTA-based

molecule on gold surface for the oriented attachment of

protein on gold surface.64

NHS-terminated SAMs can also be used to attach amino-

terminated NTA on gold surface.65 This NTA on gold surface

can be used for oriented immobilization of enzyme through

biotinavidin interaction (Fig. 16).

Patrie et al. immobilized histidine-tagged protein G (or A)

to a monolayer followed by IgG antibodies.66 In this strategy,

a maleimide terminated SAM was used to immobilize the aza

ligand. The monolayer was then treated with Ni2+ followed by

treatment with histidine-tagged protein G/A. IgG antibodies

with specificity for the intended analyte was then attached to

the protein (Fig. 17).

Nuzzo and others used self-assembled polymer on gold plate

containing nitrilotriacetic acid for the immobilization of

histidine-tagged protein.67

However, the binding between the histidine-tag and Ni-NTA

is relatively weak, and may cause unwanted dissociation

of protein. For this reason, Tempe and others synthesized

different multivalent metal-chelating thiols suitable for stable

Fig. 13 Assembly of charged protein on a switchable surface.

Fig. 14 Binding of a His6-tagged protein to an Ni-NTA functionalized

quartz surface.

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binding of histidine-tagged proteins on biocompatible SAMs,

e.g. bis-NTA-thiol (Fig. 18).68

Brellier et al. reported that although bis-NTA binds

with His6-tagged proteins via Ni2+ with improved binding

capabilities, however, a cooperative effect less than theoretical

maximum was observed in this case.69 It was suggested that

the susceptibility of divalent molecules to form discrete species

and oligomers is the reason for the absence of a strong

cooperative effect. It was also observed that when bis-NTA

ligands were preloaded with two equivalents of Ni2+, a

number of discrete species with defined stoichiometries were

generated instead of exclusive formation of bis-NTA-(Ni2+)2species.

The weak binding between histidine-tag and Ni-NTA is

advantageous in some cases in view of reversibility of

immobilization because the biosensor surfaces might be suitable

for repeated use. For example, Gondran and others showed that

immobilization can be reversed by the addition of EDTA that

removes the metal and thus removes the protein (Fig. 19).70

Limogase and others showed that protein laccase does not

lose its activity when oriented immobilization is performed

using NTA-Cuhistidine interaction (Fig. 20).71

In 2007, Mrksich and others reported attachment of the

photoactivable protein rhodopsin to a SAM on gold via

Ni-NTA complex formation.72 Subramaniam and others

reported a method of protein patterning using Ni(II) ion

templates.73 In this case, patterns of nickel(II) ions were prepared

first on nitrilotriacetic acid SAM-functionalized glass slides by

microcontact printing and dip-pen nanolithography followed by

dipping of the slides in His6-protein solution. This method

prevented the denaturation of fragile biomolecules as observed

for direct printing.

Iwata and others reported the oriented immobilization of

histidine-tagged epidermal growth factor (EGF) onto a mixed

SAM containing COOHthiol and triethylene glycolthiol.74 The

chelate NTA-Ni(II) is formed at the terminal of the carboxylic

acid, so that a His-tagged protein EGF can coordinate to the

Ni(II).

Thompson and others showed that silica surface modified with

nitrilotriacetic acid can be used to immobilize different his-tagged

proteins such as hexahistidine-tagged green fluorescent protein

Fig. 15 Attachment of NTA through a maleimide linker.

Fig. 16 NTA on a gold surface for the oriented immobilization of

protein.

Fig. 17 Immobilization of His-tagged protein G (or A) to a mono-

layer presenting Ni2+ chelates.

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(His6-GFP), biotin/streptavidin-AlexaFluor555 (His6-biotin/

SA-AF), and gramicidin A-containing vesicles (His6-gA).75

Although this attachment method of using the complexation

of histidine-tagged protein with first-row transition metal is

applicable to a wide variety of proteins and commercially available

supports, protein leaching is still possible (Kd B110 mM).

Moreover, the selectivity is poor compared to other biological

methods.

Strong non-covalent interactions such as avidinbiotin,

streptavidinbiotin or neutravidinbiotin interactions have

also been frequently used to immobilize biomolecules to

surfaces. Avidin, streptavidin and neutravidin are tetrameric

glycoproteins while biotin is a naturally occurring vitamin

found in all living cells. It contains a bicyclic ring and a

carboxyl group on the valeric acid side chain. Avidin can bind

up to four molecules of biotin (bicyclic ring part). The

biotinavidin interaction is one of the strongest non-covalent

interactions (Kd = 1015 M), the bond formation is rapid and

usually unaffected by pH, temperature, organic solvents; hence

several groups used it to attach biomolecules to surfaces.

However, Holmberg and others showed that incubation

in non-ionic aqueous solvent above 70 1C can break the

biotinavidin bonding.76

In biotin, the carboxyl group can be modified to generate

the biotinylation agent (Fig. 21). NHS ester of biotin,77

hydrazide78 or maleimide functionalized biotin, can be used

keeping the bicyclic ring intact for interaction with avidin.

These functionalized biotin molecules can be grafted on the

SAM on surface by reaction with amine, thiol or other suitable

functionality.

Biotin molecules can be grafted directly on a gold surface via

the formation of a SAM. Knoll and others reported the

synthesis of different sulfur-based biotin compounds for the

formation of a SAM of biotin on a gold surface (Fig. 22).

Interestingly, they observed that binding properties of

the biotin monolayer and streptavidin can be improved by

introducing the spacer segment (D, F, G, Fig. 22) and forming

a mixed SAM with hydroxythiol (E, Fig. 22).79

Knoll and others immobilized biotinylated enzyme

lactamase on a gold surface to monitor enzymatic activity

on surfaces. In this case, a cysteine was incorporated at a

specific site on b-lactamase via genetic modification. This

cystein part was used to biotinylate b-lactamase. The protein

was then attached to a biotin-terminated SAM via

biotinNeutrAvidinbiotin interaction (Fig. 23).80

Spinke and others generated a multilayer system of

biotinylated Fab-human chorionic gonadotrophin-monoclonal

antibody using sequential biotinavidinbiotin interaction

(Fig. 24).81 Although the binding is highly specific, the detection

limit of human chronic gonadotrophin using the surface

plasmon resonance technique is approximately 1 108 M,

which is higher than the detection limit required for a

commercial pregnancy test.

Fig. 19 Reversible immobilization of a histidine-tagged protein.

Fig. 20 Oriented immobilization of laccase on a gold electrode.

Fig. 21 Substituted biotin compounds.

Fig. 18 Bis-NTA-thiol for binding with a gold surface.

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Yoon et al. developed a methodology to reversibly attach

avidin to a dendrimer SAM via biotinavidin interaction

(Fig. 25).82

Protein patterning can be performed using biotinavidin

chemistry. For example, Pritchard et al.83 immobilized rabbit

IgG and rat IgG using biotin photochemistry. In this case,

avidin was covalently bound with a thiol monolayer on gold

slide followed by incubation in a solution of photoactivable

biotin (Fig. 26). Selected areas of the surface were exposed to

light, resulting in the formation of aryl nitrene that can react

with alkyl residues of proteins in the sample. Along the same

line, Kim et al. reported protein patterning using hydro-

quinone-caged biotin through electrochemical oxidation.84

This immobilization technique (using biotinavidin inter-

action) is advantageous because protein leaching can be avoided

due to very strong non-covalent binding (KdB1015 M). One

disadvantage is that the protein must be labelled with biotin

prior to immobilization.

Immobilization of biomolecules (particularly proteins) can

also be achieved by DNA-directed immobilization taking

advantage of specific WatsonCrick base pairing of two

complementary single-stranded nucleic acids.85 Protein

Fig. 22 Different sulfur-based biotin compounds for the formation of

SAMs on gold.

Fig. 23 Biotinylated b-lactamase on a biotin-terminated SAM.

Fig. 24 The construction of a multicomponent multilayer using

biotinavidin interaction.

Fig. 25 Avidin binding to a dendritic monolayer.

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immobilization by using this technique requires two steps: (1)

conjugation of protein with a single-stranded DNA of

a specific sequence; (2) hybridization with another single

stranded DNA of complementary sequence that is attached

to the surface (Fig. 27). Since the hybridization of comple-

mentary DNA strands is highly efficient, the method can be

utilized for generating reliable biosensor chips. Since the

hybridization of complementary DNA strands is highly spe-

cific, simultaneous grafting of different proteins at different

specific locations is possible.86 The surface can be reused after

alkaline denaturation of DNA double helix.

Prior attachment of an oligonucleotide to protein is a

primary requirement for this strategy. Several chemical reactionscan be performed to attach chemically modified protein and an

oligonucleotide.87

A homobifunctional or heterobifunctional linker can be used

to link DNA with protein. For example, bis(sulfosuccinimidyl)

linker can covalently link amino groups of protein and amino-

terminated nucleotide. Sulfo-succinimidyl 4-[N-maleimidomethyl]-

cyclohexane-1-carboxylate can be used to link an amino group

of oligonucleotide with thiolated protein. Other maleimide-

substituted compounds such as N-[g-maleimidobutyryloxy]-

succinimide ester or sulfo succinimidyl 4[p-maleimidophenyl]-

butyrate have also been used as cross linker.86,88

Non-covalent interaction such as biotinavidin reaction can

also be used to prepare DNAprotein conjugate89 andin turn, graft protein on the SAM. Jiang and others

reported the attachment of specific antibody to a SAM

through biotinavidin interaction and DNA hybridization

at the specific location (Fig. 28).90 The specificity of DNA

hybridization was utilized in this work and it was shown that

antibodies conjugated to a non-complementary ssDNA did

not bind.

Chevolot and co-workers demonstrated that DNA directed

immobilization of glycomimetics and subsequent immobilization

of fluorescently labelled lectin RCA 120 led to a stronger

fluorescence signal than for a covalently immobilized system.91

Recently, a versatile biolinker was prepared for efficient

antibody immobilization by using the site specific coupling

reaction of protein G to immobilized DNA.92 The immobilized

protein G ensures the controlled immobilization of antibody

to the intended area.

The hybrid bilayer membrane (HBM) approach is another

excellent method to immobilize biomolecules non-covalently.

Choi and others used this approach to attach an important

protein avidin to a SAM on the gold surface of quartz crystal

used in a quartz crystal microbalance (QCM) device.93 In this

approach, HBM consisting of egg phosphatidyl choline

and biotinylated lipid was first fused to a hydrophobic

SAM. Avidin was then immobilized through the biotin

molecule that was already present on the HBM (Fig. 29).

The strategy maintains biological activity of avidin more

effectively than most of the other methods. The strategy

presents several advantages over other immobilization

methods such as EDC/NHS coupling: (1) it does not need

the biomolecules to be exposed to damaging chemicals,

(2) the HBM method is composed of only two stepsfusion

of lipid layer with a hydrophobic SAM and association of

avidin with biotin (NHS/EDC coupling method requires three

steps: activation of surface carboxyl group to NHS ester

group, reaction of avidin with surface NHS-ester group,

blocking of residual NHS ester group), (3) the entire HBM

method can be executed in less than 1 h, which is considerably

shorter than the time required for EDC/NHS coupling

method.

Xu et al. combined nanogold and SAM technology94 for the

immobilization of antibodies. In this process, nanogold was

immobilized on a mixed SAM containing thiol and amine end

functional groups via goldthiol and goldNH3+ interactions

(Fig. 30). Subsequently, antibodies were immobilized on the

other bare side of the gold nanoparticle. In this case high

efficiency of immobilization was achieved by using alkanethiols

of different chain length and end group functionality

which caused reduced steric hindrance and increased specific

interface area.

Brunsveld and others95 developed a strategy to immobilize

ferrocene-conjugated protein onto a cucurbit[7]uril (CB7)

monolayer using strong ferroceneCB7 interactions (Fig. 31).

In this case CB7 was spontaneously absorbed on the gold plate

to form a monolayer. This methodology offers the advantage

of stable, reversible and site specific immobilization of protein.

Using a similar strategy, CB7 was modified with a disulfide

moiety which was assembled on a gold plate. Protein attached

with the ferrocene moiety was then directly interacted with the

CB7 part (Fig. 31).96

Fragoso et al. immobilized cytochrome c (Cyt c) on an

electrode surface through the formation of hostguest complexes

between adamantane (A) units located at the protein

surface (Cyt c-A) and chemisorbed thiolated b-cyclodextrin

(CDSH).97 Although the interaction is non-covalent, one

initial covalent attachment of protein with the adamantane unit

was necessary.

Fig. 26 Photoactivable derivative of biotin.

Fig. 27 DNA directed protein immobilization.

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Yamamoto et al. used an electrochemical method to

immobilize cytochrome c through interaction between a pyridine

terminal unit and the heme of the cytochrome (Fig. 32).98 Unlike

electrostatic binding with surface functionalities, the pyridine

unit associates directly with the redox-center thus creating a

better defined geometry between protein and electrode.

Bonanni and others showed that biorecognition ability

of azurin for cyt c 551 can be used for the immobilization of

cyt c.99

Recently, thiol functionalized DNA was immobilized on

a gold nanoparticle modified electrode via a mercapto-

diazoaminobenzene monolayer.100

2.2 Immobilization via covalent bond formation

Proteins can be anchored on suitably functionalized SAMs via

covalent bond formation by reaction with exposed functional

groups on side-chains of the protein. Covalently bound

protein is not removed by buffers during assays. Typically,

amine groups of lysine side chains of protein would react with

NHS-esters of carboxylic acids, epoxide groups or aldehydes;

the carboxylic acid group of aspartic acid or glutamic acid

would react with amino termini of a SAM; hydroxyl groups of

serine and threonine side chain would react with epoxy

functional groups of SAMs.

Amine groups of lysine residues of proteins are widely used

for anchoring proteins to SAMs on surfaces. For example,

amine groups of lysine residues of proteins react with

N-hydroxysuccinimide (NHS) ester of the SAM forming a

stable amide bond.43,101 In 1993, Herrwerth and co-workers

Fig. 28 Immobilization of protein to a SAM through biotinavidin

interaction and DNA hybridisation.

Fig. 29 Schematic presentation of the hybrid bilayer membrane

(HBM) approach to attach avidin (reproduced from the article as in

ref. 92).

Fig. 30 Antibody immobilization process using nanogold.

Fig. 31 Site selective immobilization of ferrocene-conjugated protein.

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reported the activation of carboxylic acid functional group

on a SAM via the formation of NHS ester that was used

for covalent coupling of an antibody (Fig. 33).102 This

method of immobilization is one of the most widely used

methods for covalent immobilization of proteins including

native proteins because it is a universally applicable single-step

immobilization method and does not require an external

coupling agent (reagent free process). However, it generates

the product in a modest yield. Moreover, NHS-esters are

susceptible to hydrolysis, so the NHS-functionalized SAM

must be carefully stored or freshly prepared prior to use.

Shannon and others103 immobilized antibodies on a mixed

monolayer of carboxylic acid and methyl end group on gold

using EDC. Dong and others immobilized cytochrome c,

cytochrome c oxidase, horseradish peroxidase onto a SAM

of 3-mercaptopropionic acid104 on gold using EDC coupling

method.103

Based on the coupling reaction of amine modified protein

and acid functionalized SAM, Tjin and others developed a

microfluidic immunoassay card based on polystyrene substrate

for the detection of horse IgG.105

Nakamura and others immobilized amine terminated

oligonucleotide on an acid terminated self-assembled monolayer

on gold using a suitable condensing agent.106

Kerman and others immobilized amine terminated DNA

and oligonucleotides onto a carboxylate terminated alkanethiol

SAM using NHS and EDC coupling method.107 Pendent

amino groups of the protein also undergo reductive alkylation

with aldehyde groups on the SAM. Smith and others used

aldehyde-terminated SAMs on gold surfaces to form imine

that can be reduced to secondary amine using NaBH3CN

(Fig. 34).108 The immobilized proteins were employed to

capture intact living cells through specific ligandcell surface

receptor interactions.

The reaction of aldehyde and amine groups has been used

frequently for the immobilizing of proteins on different

surfaces because of its universal applicability.109113 However,

it forms a reversible covalent bond, unless converted to amine

with a reducing agent (Fig. 34).

This aminealdehyde reaction has also been used to

generate protein arrays.114,115 Smith and others used the

aminealdehyde reaction for the immobilization of amine-

modified oligonucleotides on aldehyde terminated alkane thiol

monolayers on gold.116

Pendant lysine groups of proteins also react with isocyanate-

functionalized SAM117

or epoxy modified SAM. Zhang and

others demonstrated that amine terminated DNA can be

immobilized on epoxy terminated SAM on a gold surface

(Fig. 35).118 However, the reaction of amine with epoxides

require the reaction medium to be at high ionic strength, which

can cause denaturation of protein.118,119

Smith and others showed that SAMs of tetrafluorophenyl

(TFP) ester end functional group can be used for the fabrication

of a DNA array on gold.120

Sarkar and others reported a method for anchoring proteins

to glass and silicon surfaces using a grafted Fischer carbene

complex. For this purpose, an alkyne tethered Fischer carbene

complex was grafted first on an azide terminated SAM on

glass or silicon using a click reaction.121 The amine functional

group of protein was reacted with grafted carbene complex for

the immobilization of BSA.

Reaction of carboxylic acid functional groups of bio-

molecules and amine functional group on the surface

can be used for the immobilization of biomolecules. The large

number of aspartic acid and glutamic acid side chains

of proteins can be used for immobilization of proteins

by coupling reaction with amines. In 2008, Baldrich and

others reported antibody immobilization using side chain

COOH after NHS coupling on an amine terminated SAM.

Fig. 32 Cytochrome c immobilization on a gold electrode via

pyridineheme interaction.

Fig. 33 Covalent immobilization of antibodies to a SAM on a gold

surface.

Fig. 34 Reaction of pendent amine group of protein with aldehyde

on a gold surface. The imine formed in the first step is then reduced to

a secondary amine using NaBH3CN.

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Although in this case antibody immobilization is random, it isstill effective for protein detection (Fig. 36).122

Carboxyl groups can also be activated with carbodiimide

(CDI) or other reagents. However, carbodiimides and NHS

esters are susceptible to hydrolysis, and competitive hydrolysis

gives poor yield. Moreover, CDI coupling method for protein

immobilization may cause cross-linking of proteins due to the

reaction of activated carboxylic ester with the amine groups of

lysine residues. Also, a high concentration of CDI may prove

detrimental to enzymatic activity.

Proteins can be immobilized on SAMs by using suitable

photoreagents such as diazirines, benzophenones, aryl azides

and nitrobenzils upon irradiation of light at wavelength

Z350 nm, for which biomolecules are usually inactive. Thephotoreagents usually generate suitable intermediates upon

irradiation that leads to covalent bond formation with the

biomolecules. There are certain advantages and disadvantages

of using the photoimmobilization technique. Advantages are

that the process is fast, requires mild reaction condition such

as ambient temperature and pH, compatible with most

biomolecules. The most significant disadvantage is that some

proteins may degrade in photoirradiation condition.

Upon photoirradiation, aryl azides generate a reactive

nitrene, diazirines generate reactive carbenes and benzophenones

generate reactive ketyl radicals (Fig. 37). Those intermediates

can be used to immobilize proteins to surfaces. For example,

heparin was photoimmobilized on a methyl terminated SAM

on a silicon plate.123 In this process, an octadecyltrichlorosilane

(OTS) SAM was coated on the silicon surface as the bridging

layer, and heparin was modified by attaching photosensitive

aryl azide groups. Upon UV illumination, the modified

heparin was then covalently immobilized onto the surface

(Fig. 38). This photo-immobilized heparin on silicon with

the OTS SAM as the bridging layer showed superb stability.

Michel and others performed photoimmobilization of IgG

on a benzophenone functionalized surface.124 Simon and

others reported the photoimmobilization of phenyl azide

modified protein on a methyl terminated SAM on silicon

to compare the biocompatibility of sapphire and silicon

surface.125

Although the above methods of covalent immobilization

using amine or carboxyl acid groups and or photoirradiation

reaction create a strong bond between the protein and the

SAM, the attachment is random and non-specific in nature.

However, to get better control in immobilization, immobilization

techniques such as thiolmaleimide reaction, click reaction,

DielsAlder reaction, and Staudinger ligation reaction can be

utilized.

Since thiol group containing amino acid cysteine is usually

present in low abundance in protein outside the active site, it

can be used for controlled immobilization of protein at the

specific site. Cysteine residue of proteins is usually reacted with

a-haloacetyl or maleimide terminated SAM forming a stable

thioether bond at physiological pH (6.57).126128 Since at this

condition most surface amine groups are predominantly

protonated, thiol groups can specifically react in the presence

of amines. If necessary, cysteine residue can also be introduced

at a specific position of the protein, preferably far from the

active site by the genetic modification or removal of all but one

surface cys by genetic engineering.129,130 Gaub and others used

thiolmaleimide chemistry for site-specific immobilization of

genetically engineered variants of Candida Antarctica lipase B,

on a glass surface by means of heterobifunctional poly(ethylene

glycol) (PEG) spacer.128

Fig. 35 Immobilization of DNA on epoxy terminated monolayer on

gold.

Fig. 36 Immobilization of antibody on an amine terminated SAM

containing ethylene glycol spacer. Fig. 37 Photoactivable groups for protein immobilization.

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Staii and others showed that AFM tip can be used to

immobilize a dicysteine-terminated protein (maltose binding

protein) at well defined locations directly on gold substrates

via nanografting. Nakamura and others used a protein tag

consisting of five tandem cysteine repeats for the covalent

attachment of proteins to the surface of a maleimide-modified

diamond such as a carbon coated silicon substrate.127 Wang

et al. immobilized thiol-tagged DNA through conjugate addition

on a maleimido-functionalized SAM. These immobilized

oligonucleotides were further hybridized with complementary

strands (Fig. 39).131

A biomolecule with glyoxyl functional group can be

immobilized on a surface containing a semicarbazide functional

group via the formation of a semicarbazone bond. Melnyk

et al. reported the generation of peptide arrays by the reaction

of glyoxylyl peptide onto a semicarbazide-functionalized glass

surface for specific antibody detection in small amount of

blood sample (Fig. 40).132

A similar strategy was used by Coffinier et al. for the oriented

peptide immobilization on a silicon surface.133 Smaihi and others

reported the preparation of semicarbazide-functionalized silicate

nanoparticles for site-specific ligation of COCHO-modified poly-

peptides (Fig. 41).134

Staudinger ligation135 has been used in a large number of

bioapplications such as protein labeling, chemical synthesis of

proteins136 and protein immobilization. Staudinger ligation is

the reaction between an azide group and a phosphine

containing ester or thioester that involves the formation of

an iminophosphorane intermediate. It is followed by nucleo-

philic attack of an iminophosphorane nitrogen atom on the

thioester leading to the formation of an aminophosphonium

salt that hydrolyzes to yield an amide bond (Fig. 42).

Soellner first utilized the Staudinger ligation reaction for the

immobilization of RNAase S (after modification, the azide

group was incorporated in a specific position of the protein)

on microarray slides (Fig. 43).137

Recently Raines and others used Staudinger ligation for the

site specific attachment of protein on a gold surface.138 For

this purpose, expressed protein ligation method was used to

incorporate an azido group at the C-terminus of a model

protein, bovine pancreatic ribonuclease. Staudinger ligation

took place between the azide group of protein and the

phosphothioester functionalized SAM on a gold surface. The

activity of the immobilized enzyme was retained and hence

could bind to the ribonuclease inhibitor protein.

Advantages of Staudinger ligation reaction are manifold as it

only requires mild reaction conditions and yields product

quantitatively without the formation of much side products.

Although naturally occurring proteins do not contain azide

functionalities, it can be incorporated into recombinant proteins

by Bertozzis procedure,139

which involves a methionine surrogate,

azidohomoalanine, that is activated by the methionyl-tRNA

synthetase of Escherichia coli and replaces methionine in

proteins expressed in methionine-depleted bacterial cultures.

Waldmann and others reported the site-selective covalent

immobilization of proteins by reaction of the azide-modified C

terminus of a protein generated by expressed protein ligation

(EPL) in vitro with a phosphane-functionalized glass surface140

(Fig. 44).

Although the Staudinger ligation process is an excellent

technique for site-specific immobilize protein on a phosphine-

terminated SAM, an extra step of manipulation of expressed

proteins before immobilization limits the generality of the

method. Moreover, all components are not commercially

available and immobilized phosphines are susceptible to

oxidation; hence the surface should be stored carefully or

freshly prepared.

Since the click reaction141 requires only simple reaction

conditions and is an easy work up process it has been applied

to biological systems extensively. However, biomolecules need

to be modified to incorporate an azide functional group

or an alkyne functional group so that it can react with

alkyne functionalized surface or azide functionalized surface,

respectively.

Choi and others studied the click reaction29 on SAMs on

gold. The reaction was applied to alkyne-terminated SAMs for

the immobilization of an azide-functionalized nucleoside

moiety 30-azido-30-deoxythymidine without difficulty (Fig. 45).

Gauchet et al. immobilized protein on a glass surface in a

regioselective and chemoselective manner.142 Waldman and

Fig. 38 Photoimmobilization of heparin on a methyl terminated

SAM on a silicon surface.

Fig. 39 Immobilization of DNA on a maleimido terminated

monolayer on a glass surface.

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others used the click reaction for the immobilization of alkyne

modified biomolecules such as sugar, peptide, biotin, etc. on

sulfonyl azide slides with high chemoselectivity. Successful

immobilization of functional proteins via this approach was

demonstrated by immobilizing an alkyne-modified Ras-binding

domain of cRaf1 on a sulfonylazide slide.143

Devaraj et al. applied a Cu(I) tris(benzyltriazolylmethyl)-

amine catalyzed click reaction to attach an oligonucleotide

probe to an azide terminated self-assembled monolayer on a

gold surface (Fig. 46).144 Interestingly, the reaction proceeded

selectively in the presence of other nucleophilic and electro-

philic impurities and the density of the oligonucleotide

probe can be controlled by controlling the amount of azide

functionality.

Although the click reaction is an excellent process to

site-specifically immobilize proteins or other biomolecules on

suitably functionalized SAMs, use of Cu(I) that is cytotoxic,

limits its usability in sensitive biological systems.

Fig. 40 Semicarbazide-functionalized peptide immobilization on a

glass surface.

Fig. 41 Peptide immobilization on silicate nanoparticles.

Fig. 42 Staudinger ligation reaction.

Fig. 43 Attachment of S-protein by Staudinger ligation.

Fig. 44 Immobilization of the azide-functionalized N-Ras protein on

phosphane-functionalized glass slides.

Fig. 45 Click reaction on gold.

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DielsAlder reaction, a cycloaddition reaction between a

dienophile and a diene can be applied for biomolecule

immobilization since it is an efficient process that can also beperformed in a biocompatible solvent such as water.

Waldmann and others used DielsAlder reaction for the

immobilization of proteins.145 To achieve this, they first

prepared a maleimide-terminated glass surface by the reaction

on the amino terminated surface with the functionalized

maleimide (Fig. 47) through amide bond formation. Next, to

perform the immobilization step, both diene modified streptavidin

(positive control) and non-modified streptavidin (negative

control) were treated with the maleimide terminated surface,

followed by treatment with fluorescent-labeled biotin and

washing. There is a clear fluorescence signal that indicates

attachment of avidin via DielsAlder reaction. This signal is

absent for the negative control.Native thiol ligation is a chemoselective transesterification

reaction between a thioester and a cysteine residue (Fig. 48).

Following its development by Kent,146 this technique has been

widely used for the chemical synthesis and semisynthesis of

proteins147 as well as recently for the site selective immobiliza-

tion of proteins and peptides. For example, Lesaicherre et al.

used a native chemical ligation method for the attachment

of N-terminally cysteine-containing peptides on a thioester-

functionalized glass surface.148

Yao and others used native thiol ligation reaction to site

specifically immobilize biotinylated protein on an avidin-

functionalized glass slide (Fig. 49).149

Camarero and others used native thiol ligation for the

selective attachment of protein with C-terminal a-thioester

and a SAM containing N-terminal cys-residues for the

production of biochips. a-Thioester in protein was obtained

using standard recombinant technique by using an expression

vector containing engineered inteins. This method was used to

immobilize two fluorescent proteins (Fig. 50).62

Meijer and co-workers immobilized thioester-modified

peptides and proteins onto a microfluidic biosensor chip that

is functionalized with cysteine derivatives. The process allows

the control of ligand density in a programmed way with good

homogeneity. The modified surfaces are selectively responsive

towards complementary analytes, suitable for biosensor

development (Fig. 51).150

Camarero et al. reported a traceless capture ligand

approach for selective immobilization of proteins through

their C-termini onto a modified glass surface.151

In this

approach the C-intein fragment of the protein was covalently

immobilized on a glass surface through a PEGylated-peptide

linker, whereas the N-intein fragment was fused to the

C-terminus of the protein that was intended to be attached

to surface. When both intein fragments interacted, they

formed an active intein domain, which ligated the protein of

interest to the surface. At the same time, the split intein was

spliced out into solution (Fig. 52).

Waldmann et al. developed a method for fast, oriented and

covalent immobilization of proteins from lysates using the

thiolene reaction.152

In this process, genetically modified

protein was farnesylated at a specific position. A photochemical

thioether bond was formed between an olefin of the isoprenoid

and surface-exposed thiols (Fig. 53). The whole process takes

only 10 min and protein from expression lysates can be

immobilized without additional isolation, purification, or

chemical derivatization steps otherwise required for oriented

and covalent immobilization.

Mrksich and others described a method for the selective and

covalent immobilization of proteins to surfaces with control

over the density and orientation of the protein.153

The strategy

is based on binding of the serine esterase cutinase to a SAM

presenting a phosphonate function. Subsequent displacement

reaction covalently links the ligand to the enzyme active site.

Taki and others reported a chemoenzymatic method for site

specific immobilisation of proteins through N termini.154 They

combined L/F-transferase-mediated functionalization with

tRNA-aminoacylation using engineered ARS in the same test

tube and named the whole method NEXT-A (N-terminal

extension of protein by transferase and aminoacyl-tRNA

synthetase). Using this method, target protein was modified

by introducing an amino acid bearing a reactive group at the

N-terminus, followed by reaction on surface.

Recently, Mendes and others compared the effects of

different SAMs on the enzyme immobilization procedure.155

They found that for the immobilization of horseradish

peroxidase, amine terminated monolayer provided the best

results when glutaraldehyde was used as ligand. Corn and

others immobilized maleimide-functionalized DNA on the

Fig. 46 Click reaction to attach oligonucleotide probes on an azide

terminated SAM on a gold plate.

Fig. 47 DielsAlder reaction on a maleimide-terminated glass

surface.

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thiol functionalized SAM on gold.156 Alternatively, an

exchange reaction between the pyridyl disulfide surface and

sulfhydryl-containing oligonucleotide was also used for

anchoring DNA onto the surface.

Nakano and others157 immobilized DNA on a gold surface

using psoralen photochemistry. In this method, SAM of an

amine functionalized psoralen derivative was formed on a gold

surface followed by irradiation with UV light so that DNA

molecules are covalently immobilized on the monolayer

surface through the photoadduct formation of the psoralen

molecules with the DNA bases. Zhang and others prepared

a DNA-modified surface by attachment of succinimidyl

4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC)

crosslinker on amine terminated SAMs on silicon.158

3. Sensor applications

Depending on the mechanism of analyte recognition, biosensors

can be classified into three categories: electrochemical, optical

and piezoelectric sensors.

3.1 Electrochemical biosensors

An electrochemical sensor works by recognizing the electrical

signal that is produced by selective reaction of a biological

recognition element such as protein, nucleic acid, cell or tissue

Fig. 48 Native thiol ligation on a thioester-functionalized glass

surface.

Fig. 49 Native thiol ligation on an avidin-functionalized glass

surface.

Fig. 50 Native thiol ligation for protein immobilization.

Fig. 5 1 Protein immobilization by pulsed native chemical

immobilization.

Fig. 52 Site-specific immobilization of proteins onto a solid support

through split-intein mediated protein trans-splicing.

Fig. 53 Oriented immobilization of farnesylated protein.

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with a target analyte. Since the electrical signal is concen-

tration dependent, it is used to determine the concentration ofan analyte.

Fig. 54 depicts the design of an electrochemical biosensor. It

contains three parts: (1) a biological sensing element that

generates a selective response for a particular analyte or group

of analytes, (2) a transducer which is actually an electrode that

produces the signal, (3) a signal processor that collects,

amplifies and displays the signal.

One important advantage for an electrochemical sensor

is it is not affected by the presence of chromophores or

fluorophores and measurement can be reliably made on

coloured or turbid samples.

One of the commercially most successful biosensors

developed so far is a glucose sensor for monitoring bloodglucose levels of diabetic patients. Following the seminal work

by Clark and Lyons,159 glucose oxidase (highly specific

enzyme for b-D-glucose) has been routinely immobilized160

to measure blood glucose level by using the following oxidation/

reduction reaction.

b-D-Glucose + GOx-FAD - GOx-FADH2

+ d-D-gluconolactone (1)

GOx-FADH2 + O2- GOx-FAD + H2O2 (2)

H2O2- 2e + O2 + 2H

+ (3)

During the oxidation by H2O2 at the working electrode, twoelectrons are transferred to the electrode (eqn (3)) that results

in the current response of the enzyme electrode which is

monitored and displayed on the detector. However, this

process requires the constant presence of oxygen as is evident

from eqn (2), hence limiting the practical usability of the

process since oxygen is not very soluble in water. So, a

mediator such as quinone, ferrocene or ferricyanide derivatives

is often used to eliminate the oxygen dependence. As evident

from eqn (4) and (5), the mediators act as electron shuttles.

They are reoxidized thus generating a current while coming in

contact with the working electrode.

GOx-FADH2 + 2MediatorOx- GOx-FAD+ 2MediatorRed + 2H

+ (4)

2MediatorRed- 2MediatorOx + 2e (5)

In the commercial biosensor, electrodes hold both the enzyme

and the mediator. It can detect 1.133.3 mM glucose with a

precision of38% and test time of about 30 s or less.161

Xanthine sensor is another important biosensor that is used

for monitoring the freshness of fish. After the death of a fish,

ATP starts decomposing to produce xanthine, and hence the

concentration of xanthine indicates the freshness of fish.

Several reports describe the development of amperometric

biosensors using immobilized xanthine oxidase that utilizes

the following equation.162

Xanthine + O2 + XO- Uric acid + H2O2 + XO (6)

In a similar way, lactate dehydrogenase, lactate oxidase or

lactate monooxidase has been used for the detection of lactate

concentration in blood. Since the concentration of lactate in

blood is an indication of oxygen deprivation resulting

from ischemia, trauma, and hemorrhage, it is an important

component of many medical monitoring devices.163

Mathebula and others developed an electrochemical sensor

for the recognition of anti-mycolic acid antibody that is

present in tuberculosis-positive human serum co-infected with

human immunodeficiency virus.164 In this sensor development,

mycolic acid was integrated into a SAM of N-(2-mercaptoethyl)-

octadecanamide on a gold electrode, so that the electrode can

discriminate between TB-positive and a TB-negative serum.

Abrantes and others165 developed an electrochemical

immunosensor based on physical and chemical immunoglobulin

adsorption onto a mixed self-assembled monolayer. They used

a non-ionic detergent, Tween 20 to block the physical adsorption

of biomolecules on hydrophobic CH3-terminated SAMs and

mixed SAMs with a few COOH terminal groups.

Fowler and others prepared a SAM of thiolated protein G

on a gold electrode in such a way so that it can afford

maximum binding with the specific antigen.166 For this

purpose, a SAM of protein G was exposed to a solution of

capture antibody (mAb1) so that these antibodies could attach

to the protein G layer through their non-antigenic regions,

leaving antigen binding sites available with minimal steric

hindrance for binding of target analyte. Akyilmaz and others

developed a bienzymatic biosensor based on co-immobilization

of alcohol oxidase and glucose oxidase on the same electrode

for selective determination of alcohol and glucose.167

Sadik and others developed a metal-enhanced electro-

chemical immunosensor for the quantitative detection of

Bacillus globigii using the immobilized antibacillus globigii

(BG) antibody onto a gold quartz crystal electrode via a

cystamine bond.168

Several groups used DNA sensors for the diagnosis of

genetic or infectious diseases. In this type of sensor, a short,

single stranded nucleotide is immobilized on the electrode

surface and an electrical signal is produced when the target

DNA binds to the complementary strand of probe DNA. To

generate the electrical signal, an electroactive indicator such as

ferrocenyl naphthalene diimide (FND) can be used that could

bind preferentially to the DNA duplex instead of a single

stranded DNA probe.169 Alternatively an electrochemical

signal can be generated from an enzyme label such as

horseradish peroxidase or alkaline phosphatase. This method

allows very high sensitivity in measurementup to 3000

copies of target DNA.170 Although the oxidation of nucleotide

bases such as guanidine can also be used for generating an

electrical signal, the signal can be amplified by using a redox

mediator such as [Ru(bpy)3]2+. Recently a company called

Osmetech developed an electrochemical DNA biosensor

named esensor for the detection of cystic fibrosis carriers. In

this chip design, a DNA capture probe is first immobilized on

Fig. 54 Design of an electrochemical biosensor.

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an electrode surface followed by its exposure to the sample of

target DNA.171 In this case, the capture probe is shorter than

the complementary target probe so that a signal probe

containing an electroactive label such as ferrocene can bind.

The ferrocene label can be detected by the generation of an

electrical signal.

Meyerhof and others,172 designed a novel electrochemical

detection system for detecting a physiologically important

protein marker, the human chorionic gonadotrophin (hcg).

In this case, anti hcg antibody was immobilized on a gold

electrode and alkaline phosphatase was used as a label.

4-Aminophenyl phosphate was used as substrate and the

product 4-aminophenol was detected electrochemically. The

detection limit in this case is 500 ng l1 of hcg in whole blood.

Maida and co-workers designed a sensor based on electro-

chemical impedence measurement for identifying a single-

base mismatch of DNA duplex at the distant end using

[Fe(CN)6]3/4 as redox marker ions.173 Lee and others

devised a method for detecting a single nucleotide mismatch

in unlabeled duplex DNA by electrochemical methods after

converting to M-DNA (a metallated duplex). The resistance to

charge transfer (RCT) was found to decrease for duplexes

with mismatches, a feature that can be used for diagnostic

purposes.174 Kraatz and others used gold electrode arrays for

the detection of eight single nucleotide mismatches, in unlabeled

and prehybridized DNA by electrochemical impedance

measurement.175 They used the differences in the electrical

properties of films of duplex DNA in the presence and absence

of Zn2+ at pH Z 8.6. The differences in the charge transfer

resistance (DRCT) between B-DNA (absence of Zn2+

at

pH Z 8.6) and M-DNA (presence of Zn2+

at pH Z 8.6)

allows unequivocal detection of all eight single-nucleotide

mismatches within a 20-mer DNA sequence.

Kraatz and others used hairpin-DNA probe for the

detection of a single nucleotide mismatch by electrochemical

impedence spectroscopy. Upon hybridization of the target

strand with the hairpin DNA probe, the loop structure is

opened and a duplex DNA is formed. Consequently, the film

thickness is increased which causes differences in the electrical

properties of the film that is manifested in the differences in

charge-transfer resistance (DRCT) between hairpin DNA

(before hybridization) and duplex DNA (after hybridization)

(Fig. 55). The detection limit for the concentration of target

strand can reach as low as 10 pm.176

Willner and others reported177 the use of tagged, negatively

charged liposomes to amplify the signal for DNA sensing. Yao

and others developed an aptamer-based electrochemical

sensor for the label-free and selective detection of leukemia

cells based on an aptamer-modified gold electrode. In this

process, the thiol-terminated aptamer sgc8c (a single-stranded

1540 base long DNA or RNA oligonucleotide sequences that

are used as biorecognition element) that is selective for

CCRF-CEM acute leukemia cells, was self-assembled on the

gold electrode surface first. The measurement of change in

electron transfer resistance (RCT) of [Fe(CN)6]3/4 on the

sensor surface allows the electrochemical recognition and

detection of cancer cells.178 Woollenberger and others

reported the use of glucose dehydrogenase on a gold electrode

for sensor development. The sensor showed good stability;

after 2 months, about 75% of its original activity was

retained.179 Collinson and others reported an amperometric

sensor by immobilizing cytochrome c on a carboxylic acid

terminated SAM.180 Kalaji and others181 developed an

amperometric biosensor for the detection of nitroaromatics

using a genetically engineered enzyme nitroreductase with a

detection limit of 100 parts per trillion, thus promising the

development of in situ sensor for the detection of explosives.

Fan and others reported an electrochemical DNA sensor

based on a nonfouling self-assembled monolayer structure

on a gold plate.182 In this case, a mixed SAM of thiolated

oligoethylene glycol and thiolated DNA was used for designing

the non-fouling surface.

Reynes and others developed an electro

Immobilization of Bio Macro Molecules on Self Assembled Mono Layers

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