Structured thin films as functional components within biosensors

Biosensors and Bioelectronics 21 (2005) 1–20

Review

Structured thin films as functional components within biosensors

Frank Davis∗, Seamus P.J. HigsonInstitute of Bioscience and Technology, Cranfield University at Silsoe, Silsoe, Bedfordshire MK45 4DT, UK

Received 2 July 2004; received in revised form 4 October 2004; accepted 5 October 2004Available online 18 November 2004

Abstract

This review provides an introduction to the field of thin films formed by Langmuir–Blodgett or self-assembly techniques and discussesapplications in the field of biosensors.

The review commences with an overview of thin films and methods of construction. Methods covered will include Langmuir–Blodgett filmformation, formation of self-assembled monolayers such as gold–thiol monolayers and the formation of multilayers by the self-assembly ofpolyelectrolytes. The structure and forces governing the formation of the materials will also be discussed.

The next section focussed on methods for interrogating these films to determine their selectivity and activity. Interrogation methods to bec nce and othert

t on films y, activitya

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overed will include electrochemical measurements, optical measurements, quartz crystal microbalance, surface plasmon resonaechniques.

The final section is dedicated to the functionality of these films, incorporation of biomolecules within these films and their effectructure. Species for incorporation will include antibodies, enzymes, proteins and DNA. Discussions on the location, availabilitnd stability of the included species are included.The review finishes with a short consideration of future research possibilities and applications of these films.2004 Elsevier B.V. All rights reserved.

eywords: Langmuir–Blodgett; Thin films; Biosensors

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Methods of thin films formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.1. Langmuir–Blodgett (LB) films. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2. Polyelectrolyte multilayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3. Self-assembled monolayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.1. Alcohols, acids and siloxanes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.2. Gold–thiol monolayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Interrogation techniques for the study of thin films. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1. Monolayers at air–water interface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2. Spectroscopy and microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.3. Mass-sensitive techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.4. Surface plasmon resonance (SPR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.5. Electrochemical techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +44 1525 863455; fax: +44 1525 863533.E-mail address:[email protected] (F. Davis).

956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
oi:10.1016/j.bios.2004.10.001

2 F. Davis, S.P.J. Higson / Biosensors and Bioelectronics 21 (2005) 1–20

4. Biologically active films. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.1. Monolayers of biological molecules on water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.2. LB films, incorporation within biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.3. Self-assembled polymeric multilayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.4. Self-assembled monolayers in biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1. Introduction

The field of biosensors is a large and varied one. Biosensortechnology has developed into an ever-expanding and multi-disciplinary field since the Clark enzyme electrode was firstreported (Clark and Lyons, 1962). Biosensors generically of-fer simplified reagentless analyses for a range of biomedicaland industrial applications and for this reason this area hascontinued to develop into an ever-expanding and multidisci-plinary field during the last couple of decades. For any sensor,speed of response and reversibility are often paramount. Inany solid-state sensor, analyte molecules have to diffuse intoand react with the acting sensing component and any prod-ucts of the reaction must diffuse out. It therefore follows thatthe thinner the sensing layer is, the less time this will takeand thereby, speed and reversibility of sensor response, maywell be improved. Also the molecular dimensions of ultra-thin films make the use of even highly expensive moleculeseconomically possible, for example a typical self-assembledmonolayer has a mass of approximately 2× 10−7 g/cm2.

A wide range of materials have been electrodepositedonto electrode surfaces such as polyaniline, polyphenol, poly-thiophene, as reviewed (Bartlett and Cooper, 1993; Scoutenet al., 1995; Wink et al., 1997) and used to immobilisebiomolecules. These films tend to be relatively thick com-

withthe

isa-kingt im-ideuches,.s intols,0;98;avethinrro-pticaryst

microbalance measurements. Incorporation of biologicallyactive materials into these thin films leads to construction ofbiosensors.

2. Methods of thin films formation

2.1. Langmuir–Blodgett (LB) films

It has long been known that organic materials can spreadupon a water surface to form a thin film, hence the originof the saying “to pour oil on troubled waters”. Initial experi-ments carried out using vegetable oil and a convenient pond(Franklin et al., 1774) gave rough measurements of the thick-ness of such films, later work (Pockels and Rayleigh, 1891)suggested that these layers were but one molecule thick. Mostof the molecules which form these layers are amphiphilic, i.e.one end of the molecule is hydrophilic and usually attachedto a long hydrocarbon chain. Gaines in his 1966 book widelyreviewed many of these insoluble monolayers.

Much early work on the structure of these monolayers wasperformed byLangmuir (1917)who later in collaborationwith Katherine Blodgett first described the transfer of thesemonolayers onto a solid substrate. Blodgett later publisheddetails of extensive research into deposition of fatty acids

yer

lesent

Thef theface,packidd alld-

sur-ayerre.ughse, ahen

pared with the systems with which we are concernedhere and lie somewhat outside the scope of this article. Orelatively thick film systems can be used for the immobiltion of biomolecules, such as casting films and crosslinwith glutaraldehyde, often used for biosensors, covalenmobilisation with linkers such as dicyclohexyl carbodiim(DCC) or by coating slides with cast films of materials sas polylysine which show a high affinity for biomoleculused extensively for the production of DNA microarrays

Much research has been performed over many yearthe fabrication of thin films of a wide variety of materiaoften just one molecule thick (Gaines, 1966; Roberts, 199Ulman, 1991; Tredgold, 1994; Petty, 1995; Ulman, 19Birdi, 1999). These methods, as will be detailed later, hbeen used to immobilise biologically active species intofilms. A wide variety of techniques can be used to integate these thin films such as electrochemical methods, ospectroscopy, surface plasmon resonance and quartz c

r

lal

(Blodgett, 1935) and postulated they had a well-ordered lastructure.

In essence, Blodgett dissolved amphiphilic molecusuch as stearic acid in a water-immiscible organic solvwhich was carefully cast onto a clean water surface.solvent evaporates leaving behind a disordered layer oamphiphiles. Barriers are then moved across the surcompressing the layer and causing the molecules tomore closely (Fig. 1). Finally a quasi-two-dimensional solis obtained where the molecules are closely packed analigned in the same direction with their hydrophilic heagroups on the water surface (Langmuir, 1917; Gaines, 1966).This monolayer can often be compressed to quite a highface pressure until eventually over-compression of the lbeyond this point causes collapse to a multilayer structu

If a clean substrate (silicon, glass, etc.) is passed throa suitable compressed monolayer into the water subphamonolayer of the amphiphile attaches to the substrate, w

F. Davis, S.P.J. Higson / Biosensors and Bioelectronics 21 (2005) 1–20 3

Fig. 1. Formation of an ordered monolayer at the air–water interface.

withdrawn a second layer can attach as shown (Fig. 2). Ifthe surface pressure is maintained (usually by moving in thebarriers automatically via a feedback circuit) this process canbe repeated to build-up multilayers of any required thickness.

2.2. Polyelectrolyte multilayers

A more recently developed technique (Decher et al., 1992)for assembly of thin films takes advantage of the strong attrac-tion between oppositely charged polyelectrolytes. This pro-cess is a very simple one and shown schematically inFig. 3. Acharged solid substrate is placed in a dilute solution of an op-positely charged polymer. Strong multiple charge interactionscause the formation of a thin layer of polyelectrolyte, suchas polystyrene sulphonate, on the substrate, thereby generating a surface with the negative charge of the polyelectrolyte.This sample is then rinsed and placed in a second oppositely

charged polyelectrolyte, such as polyallylamine hydrochlo-ride, causing adsorption of a second layer and again reversingthe charge on the surface.

This process can be repeated to build-up multilayers of anydesired thickness. What makes this process so versatile is thata wide variety of charged materials such as polyelectrolytes,biomolecules, colloidal particles, etc. can be used in the as-sembly of these layers. The formation and structure of theselayers has been extensively reviewed (Decher, 1997; Decheret al., 1998; Hammond, 1999; Schonhoff, 2003). They are nothighly ordered crystalline-type films like LB films but tend tobe more amorphous in nature, with the polyelectrolytes tend-ing to interpenetrate somewhat (Decher, 1997). These filmsare highly stable, due to the multiple charge neutralisationinteractions and also any defects formed in the assembly ofa monolayer tend not to propagate through the structure butare “covered over” (Decher et al., 1992). The film structurecan often be greatly affected by pH and ionic strength, allow-ing for fine-tuning of the multilayer thickness and properties.They are not limited to flat surfaces but can be assembledonto charged particles (Sukhorukov et al., 1998) and a suit-able central core can be removed (Donath et al., 1998) to givestable, hollow polymeric shells (Fig. 4).

Biomolecules are often electrically charged and as thismethod works well from dilute aqueous or buffer solutions,it is a good method for easily assembling thin films withb e ise verysa ha theb ingm turei /at

2

ivelyr 998.

2the

1 n al-c rfacesrc nto Pts s,b ly re-m ptionot ly tot

lay-e1 and
Fig. 2. Deposition of a Langmuir–Blodgett multilayer.
-

iological activity. Also another variation of this techniquspecially suitable for biological components. There is apecific strong interaction between the biotin unit (Fig. 5)nd the protein avidin (Wilchek and Bayer, 1988). Since eacvidin molecule can bind four biotin units, it can be used inuild-up of multilayers, especially if molecules containultiple biotin sites are utilised, for example, in the struc

n Fig. 6, which is a schematic of a multilayer of avidinetra-biotin unit.

.3. Self-assembled monolayers

Much early work on these systems has been extenseviewed by Ulman in the books published in 1991 and 1

.3.1. Alcohols, acids and siloxanesSelf-assembly of organic monolayers was first noted in

950s when it was found that dilute solutions of long chaiohols would spontaneously adsorb onto clean glass suendering them hydrophobic (Bigelow et al., 1946). Longhain amines were also studied and shown to adsorb ourfaces (Shafrin and Zisman, 1949). However these layerased on simple physisorption were unstable and easioved. More robust layers were obtained by the adsorf fatty acids onto metal surfaces (Allara and Nuzzo, 1985),

he acids being deprotonated and binding quite stronghe surface.

The first types of strongly bound chemisorbed monors were those developed using siloxane chemistry (Sagiv,980) where a silicon surface is etched so as to clean


Fig. 3. Deposition of a self-assembled polyelectrolyte multilayer.

coat the surface with active silanol (SiOH) groups. Thesereacted with dilute solutions of long chain alkyl chlorosilanes(Fig. 5) to form siloxane attachments to the surface. Reactionwith adventitious water caused further hydrolysis and con-densation of unreacted SiCl bonds, leading to formation ofa stable, polysiloxane structure and a robust monolayer.

Unfortunately silyl chlorides are very reactive, precludingthe use of aqueous solutions and also meaning that only rela-tively inert substituents such as alkyl chains can be used. Whatis required is a simple functionality that binds very stronglyto a otherwise chemically inert surface, yet is relatively un-reactive chemically so other active groups (–OH, –COOH,–NH2, etc.) can be incorporated within the molecules.

2.3.2. Gold–thiol monolayersSulphur compounds are known for their reactivity towards

noble metals, for example, hydrogen sulphide is one of theculprits for the tarnishing of silver. Although a variety of sub-strates are possible candidates for deposition of sulphur moi-

F sem-b

eties, gold is the most commonly used for several reasons.The gold–sulphur interaction is based on binding between“soft” gold and sulphur atoms whereas many of the func-tional groups present in biological species, acids, amines,etc. are relatively “hard” and do not interact strongly withthe gold surface. This means the use of di- or polyfunc-tional molecules is possible without any interference to thegold–sulphur binding interaction. Also gold is relatively easyto clean, does not oxidise under standard lab conditions andany weakly physically adsorbed impurities are displaced bythe sulphur species. The best binding occurs between goldand thiol groups (Ulman, 1991, 1998) but other species suchas disulphides, thiones, thioesters, etc. have been used.

Early studies looked at simple alkyl disulphides(Troughton et al., 1988) and thiols (Nuzzo et al., 1997; Bainet al., 1989) on gold. These form very well-assembled mono-layers which are highly resistant to washing due to the strong

F ono-l

ig. 4. Formation of hollow polymer shells by the colloid templated asly of polyelectrolytes.

ig. 5. Deposition of a self-assembled polysiloxane or gold–thiol mayer.


Fig. 6. (a) Structure of the biotin headgroup. (b) Schematic of the structureof an avidin–biotin-type multilayer.

chemisorption of the sulphur atoms. Initially, it is thought,there is a rapid reaction between a –SH group (for a thiol)and a gold atom with formation of an SAu bond. After theinitial fast adsorption of the thiols, the alkyl side chains thenassemble together to maximise the Van der Waal’s interac-tions between them. Molecules such as hexadecanethiol leadto well-packed quasi-crystalline monolayers whereas shorterthiols such as hexanethiol give liquid-like monolayers.

The stability and thinness of these layers, plus the versa-tility of a gold substrate which makes them easily investi-gated by QCM, FTIR, AFM, SPR and electrochemical meth-ods rapidly led to these systems being investigated as pos-sible sensing films. Many chemical-sensing moieties suchas ferrocene (Chidsey et al., 1990), terpyridine (Maskus andAbruna, 1996) and calix-4-resorcinarene (Davis and Stirling,1996; Huisman et al., 1996; Collyer et al., 2003) can be easilyattached to gold surfaces and used to detect various species

Another factor which could be important in the develop-ment of these monolayers in a sensing field is their ability tobe easily printed onto gold using a soft polymeric stamp withresolution down to 30 nm (Xiu and Whitesides, 1998). Thismicrocontact printing gives us a method to directly manu-facture “circuits” from these monolayers. Besides being ofinterest themselves they can also be used to assemble patterns of proteins, enzymes, viruses and cells. Several reviewsare available on this subject (Kane et al., 1999; Gaspar et al.,2

3

iono ouldc f thet er inm 8

3.1. Monolayers at air–water interface

When a monolayer at the air–water interface is com-pressed, the surface tension of the water is reduced. This“surface pressure” can be measured by various means, suchas a piece of filter paper dipped into the subphase and attachedto a balance. Since the concentration and volume of solutionspread and the area of the monolayer are known, we can plotsurface area per unit against surface pressure. This plot isknown as the isotherm; a steep portion of the curve obtainedis indicative of the formation of a well-packed structure.

When deposition of a monolayer onto a solid surface oc-curs, a feedback circuit may be used to keep the surfacepressure content. Since the area of the substrate is known,we can obtain a deposition ratio, i.e. area of monolayer de-posited/area substrate. A ratio of 1 is indicative of good trans-fer of the monolayer.

3.2. Spectroscopy and microscopy

Many of the spectroscopic techniques used to characterise“bulk” samples can also be used to characterise deposited thinfilms. UV–vis spectroscopy can be performed on samples de-posited on suitable transparent or reflective substrates and cangive quantitative measurements of the amount of materialpresent. FTIR spectroscopy can also be used and can givei lacew meb ld-c spec-t givet epthp icall

ichg n thefi urefi n,1

i-c EM)c ma-t n ora ventst

3

artzc blef ngest tion.B f thefi ime.Ra

002; Gooding et al., 2003).

. Interrogation techniques for the study of thin films

It is in no way intended here to provide a full descriptf the methods of characterising thin films, since that womprise an entire book, not just a review article. Many oechniques described briefly below are reviewed moreovore detail elsewhere (Tredgold, 1994; Ulman, 1991, 199).

.

-

nformation about any reactions that may have taken pithin the film. The problems of sensitivity can be overcoy depositing the thin film on an ATR crystal or on a gooated substrate and using reflection/adsorption FTIRroscopy. X-ray photoelectron spectroscopy (XPS) canhe elemental composition of the film and also some drofiling-related information, giving an idea of the phys

ocation of various moieties within the film.Low-angle X-ray diffraction can give Bragg peaks wh

ive a measurement of order and repeat spacing withilm (Tredgold, 1994). Ellipsometry can be used to measlm thickness and refractive index (Tompkins and McGaha999).

Atomic force microscopy (AFM), scanning tunnelling mroscopy (STM) and scanning electron microscopy (San be used to visualise the thin film and can give inforion about the regularity of the film, any phase separatioggregation and may be able to visualise any binding e

hat have occurred (Ulman, 1998).

.3. Mass-sensitive techniques

The mass of a thin film can be monitored using a qurystal microbalance (QCM). This if often especially suitaor sensing applications since it can monitor mass chaaking place whilst the sample is immersed in a soluinding or desorption of species will cause the mass olm to be changed, and this can be monitored in real teviews on this subject includeMarx (2003)andO’Sullivannd Guilbault (1999).


3.4. Surface plasmon resonance (SPR)

If a gold or silver film attached to a quartz prism is irra-diated from the back with a laser, at a critical angle, energyfrom the laser will be lost in generating plasmons at the metalsurface. This process is affected by the presence of materialsat the metal surface and therefore provides a useful methodfor characterising binding and recognition events occurringwithin a thin film on the metal surface. This is the basis of sur-face plasmon resonance (SPR) spectroscopy and like QCM, itis capable of monitoring events occurring in a film in contactwith a solution in real time. This technique has been exten-sively reviewed (Ulman, 1998; Homola et al., 1999; Bairdand Myszka, 2001).

3.5. Electrochemical techniques

Finally many of these thin films have been deposited ontoconductive surfaces such as gold or carbon. This means wehave access to a whole range of electrochemical techniquessuch as cyclic voltammetry, amperometry and AC impedance(Pletcher, 2001). These are of great use within sensor appli-cations because of the relative ease and inexpensiveness ofthese methods. Should any binding event occur, it may re-lease or require electrons, cause generation or consumptionof an electrochemically active molecule such as hydrogenp pac-i cur,t t anyc lec-t

4

4

ateri icals lipids( iono ix-t arlys n ex-t oft

e be-h f theh ulesa s oft n hyd le.P r in-t ilicg doesn esters

(Kawaguchi et al., 1985) or amylose esters (Bardosova et al.,1994) can be used to form stable monolayers and depositedto form LB films. Often spreading species such as proteinsat the air–water interface can effect the conformation of themolecule such as causing unfolding. For example, insulin,BSA and ovalbumin unfold completely at the air–water in-terface whereas myoglobin and cytochrome C are only partlyunfolded (Birdi, 1999). This again is thought to be a functionof the ratio of polar to non-polar amino acids residues. Highlypolar proteins such as xanthine oxidase (isolated from milk)do not form stable monolayers (Birdi, 1999). Antibody pro-teins such as IgG have been spread at the air–water interfaceand their behaviour studied (Birdi, 1999). Other systems suchas mixed monolayers of proteins with lipids and membraneproteins have also been reviewed (Birdi, 1999).

Synthetic polypeptides have also been studied as modelsof protein structure. Polybenzyl glutamate (PBLG) has beenwidely studied at the air–water interface and shown to formboth stable monolayer and bilayer structures, depending onthe surface pressure (Malcolm, 1985). The PBLG monolayerwas shown to form a highly orientated structure with the longaxis of the helixes perpendicular to the compression direction,however, at higher pressures the resultant bilayer was shownto have the opposite orientation (Malcolm, 1985; Tredgoldand Jones, 1989). Deposited films of PBLG were shown byFTIR to retain this orientation (Jones and Tredgold, 1988).D enti

4

y-e sfert witht e-p llowm st pa-p dso ideh icalr wed(

uchw n glu-c atrixo yde.P re-s sec f thes in thisw

ublew LBm theyc ppo-s sur-

eroxide or oxygen, or even just affect the resistive or cative properties of the thin film. If any of these events ochey are detectable electrochemically. It is probable thaommercial sensor that includes these thin films will be erochemical in nature.

. Biologically active films

.1. Monolayers of biological molecules on water

Much of the early work on monolayers at the air–wnterface was performed using relatively simple chemtructures, e.g. fatty acids and amines, steroids, phosphoGaines, 1966). However, attention was turned to formatf thin films of biological molecules, either pure or as a m

ure with the more classical amphiphiles. Much of the etudies on monolayers of biological molecules have beeensively reviewed (Birdi, 1999) and lie outside the scopehis work. However, a brief summary can be given.

Whereas for simple molecules such as fatty acids, thaviour of the monolayer is dependent on the nature oydrophilic headgroup and the alkyl chain, for biomolecdifferent situation occurs. The monolayer propertie

hese species are dependent on the balance betweerophilic and lipophilic groups within the macromolecuroteins tend to form stable monolayers at the air–wate

erface because of their mixture of hydrophilic and lipophroups, whereas pectin (a hydrophilic polysaccharide)ot. Other polysaccharides however such as cellulose

-

ifferent forms (alpha and beta) of PBLG also give differsotherms (Birdi, 1999).

.2. LB films, incorporation within biosensors

After the initial work on bioactive molecules in monolars, it was inevitable that workers would attempt to tran

hese films onto solid substrates since a major problemhe use of LB films is their extreme fragility, requiring dosition onto a suitable substrate for support and to aeasurements to be made on the film. One of the earlieers (Arya et al., 1985) reports deposition of phospholipir cholesterol onto an ionically conductive polyacrylamydrogel, giving a structure capable of an electrochemesponse. Other early work in this field has been revieReichart et al., 1987).

The sensing of glucose is of paramount interest and mork has been done on developing biosensors based oose oxidase which can be adsorbed into a polymeric mr cast as a thick film and crosslinked with glutaraldehroblems with these sensors include stability and slowponse times.Table 1shows the behaviour of some of theonventional sensors and compares them with some oensors manufactured using the techniques describedork.Often species of biological interest are water-sol

hich means they cannot be directly deposited by theethod, however, should they be charged molecules,

an be dissolved in the subphase. If a layer of an oitely charged amphiphile is then spread on the water


Table 1Comparison of glucose sensors made by various strategies with a typical “commercial” glucose sensor

Immobilisation method Sample thickness Sensitivity(mM)

Response time Stability Reference

Typical commercial sensor N/A 1–30 15–40 s Single use; 7–18-monthshelf life

Newman et al. (2004)

Physisorption onto PVC membranes 50–110�m membranes 0.3–2.2 1 min 2 months Hirose et al. (1987)Crosslinked onto nylon mesh Not reported 0.01–3.0 45 s 4 months Moody et al. (1986)Glutaraldehyde crosslinking on Pt Not reported 0.2–2.5 25 s 10 days Badea et al. (2003)Polyaniline electrodeposition 1–12 nm of polyaniline 2–20 2–3 min Not reported Cooper and Hall (1992)Electrodeposition and glutaraldehyde Not reported 0.1–4 25 s 60 days Badea et al. (2003)LB deposition with C18H37N+Me3 1 layer 0.3–6 Not reported Not reported Sriyudthsak et al. (1988)LB deposition with lipids 2 layers 0.05–1 5 s 3 months Okahata et al. (1988)LB deposition with cationic polymer 1–4 layers 1–20 15 s 2–3 months Eremenko et al. (1995)LB deposition with lipids + UV 1–10 layers 0.4–4 10 s Not reported Zaitsev (1995)LB deposition with polythiophene 1 layer 0.5–2 2 min 40 days Singhal et al. (2002a, 2002b)Alternation with ferrocene polymer 1–5 layers 0.01–10 Not reported 21 days Hou et al. (1997)Alternation with polyacrylic acid 67 nm Not reported Not reported >68 days Franchina et al. (1999)Alternation with Co polymer 1–3 bilayers 1–10 Not reported >21 days in buffer Simonian et al. (2002)Coadsorption when encapsulated 10�m 1–10 <5 s Not reported Trau et al. (2003)Coadsorption with polyethylenimine 1 layer 0.05–2.5 Not reported 4 month or 245 h Dimakis et al. (2002)Self-assembly of thiolated GOD/Au 1 layer 1–50 <20 s 30 days McRipley et al. (1996)Binding GOD/thiol acid monolayer 1 layer 1–80 Not reported Not reported Gooding et al. (1998)

face and formed into an LB film, the biomolecule will thenbe incorporated into the LB film. Penicillinase could be co-deposited (Anzai et al., 1987, 1988) with stearic acid onto anISFET to give a penicillin sensor. Glucose oxidase (GOD)was also studied (Sriyudthsak et al., 1988; Tsuzuki et al.,1988) by using different lipids to adsorb the enzyme from so-lution and showing that a glucose sensor could best be madeby co-depositing GOD with octadecyltrimethylammoniumchloride.Okahata et al. (1988)mixed GOD with a cationiclipid and deposited a bilayer on a platinum electrode andshowed it to respond to glucose with a response time of 5 s,much faster than other techniques (Table 1). FTIR studies(Zhu et al., 1989) were performed on fatty acid/GOD andphospholipids/GOD monolayers deposited onto ATR crys-tals, the fatty acid was shown to incorporate more of theenzyme. Chymotrypsin and urease were also studied (Anzaiet al., 1989).

Glucose oxidase was dissolved in the subphase and incor-porated into behenic acid bilayers (Fiol et al., 1992). AFMimages of these systems showed a structure containing par-allel ridges with a periodicity of 6.5 nm, compatible with amorphology where the glucose oxidase molecules (thought tobe approximately 6 nm× 13 nm in size) are aligned in a close-packed structure. Later AFM images refine this and show theaggregation of the enzymes which are coated with a behenicacid monolayer (Sommer et al., 1997). Recent work exten-s enica no-lT ob-t anis-i avee

Spreading of bioactive molecules and transfer to solid sub-strates can often lower or destroy their bioactivity, as can stor-age. The stability and activity of glucose oxidase bound tolecithin:cholesterol monolayers and deposited (three layers)onto glass was found to be greatly increased by incorporationof submicron hydrophobic silica particles within the mono-layer, probably due to inhibition of leaching of enzyme fromthe film (Tang et al., 1992).

Glucose oxidase and monoamine oxidase were incorpo-rated from the subphase into monolayers of a variety ofcationic amphiphilic polyelectrolytes based on polyethylen-imine or polyvinyl pyridine (Eremenko et al., 1995) andtransferred onto polypropylene membranes. These were in-corporated into a Clark oxygen electrode and the resultingbiosensors gave linear responses to glucose (1–20 mM) andtyramine (8–100�M). These electrodes gave reproducibleresults for up to 100 measurements or after 2 months stor-age in air, whereas many commercial electrodes are singleuse only (Table 1). Similar work with glucose oxidase boundto monolayers of a cationic lipid containing a methacrylategroup, deposited onto Pt (Zaitsev, 1995) gave a biosensor(Table 1), the stability and reproducibility of which could beimproved by UV irradiation which causes polymerisation ofthe lipid. A cellulose polymer can also be used as a matrixfor the physisorption of glucose oxidase whilst retaining itsactivity (Guiomar et al., 1997).

osi-t poly-m ple,p andu r re-sp xed

ively studied the behaviour of the glucose oxidase/behcid monolayer system, the formation of the mixed mo

ayer and its deposition onto silicon (Chovelon et al., 2002).he best quality of films as shown by IR and AFM were

ained using high transfer pressures and importantly silng the Si/SiO2 surface, showing the substrate properties hffects on film structure.

Conducting polymers, usually formed by electrodepion, have been of great interest in biosensors. Suitableers can be incorporated into LB films, and, for examolyaniline/glucose oxidase LB films can be depositedsed as electrochemical sensors for glucose with lineaponse from 5 to 30 mM (Ramanathan et al., 1995). Otherolymers include poly-3-dodecyl thiophene which mi


with stearic acid is a suitable matrix for deposition of glu-cose oxidase (Singhal et al., 2002a, 2002b), retains its elec-troactivity and detects glucose (Table 1). Similar work hasbeen performed using urease incorporated into polyvinyl car-bazole/stearic acid layers, which gives sensors that can detecturea down to 5 mM and have a shelf life of 5 weeks (Singhalet al., 2002a, 2002b).

Interferents such as ascorbate can cause a problem withelectrochemical sensing of glucose. One established ap-proach involves the inclusion of permselective barriers for theexclusion of interferents. An LB monolayer of a crosslinkedpolysiloxane (Kato et al., 2002), deposited on an electrodecan however be used as a permselective barrier in a glu-cose sensor. Neutral hydrogen peroxide produced by glucoseoxidase can diffuse across the membrane and be measuredwhereas charged interferents (ascorbate, paracetamol, etc.)are excluded.

In another example, tyrosine hydroxylase (TH) wasfound to form a stable monolayer on water (Wang et al.,1993) and could be transferred onto gold electrodes. Whenthese electrodes were placed into solutions of phenotiazinedrugs, for which TH is a receptor, they were selectivelybound by the TH layer. This lead to a preconcentration ofthe drugs and allowed their electrochemical detection atmuch lower levels than could be obtained for unmodifiedelectrodes.

n bes as aL nt h asa lutiona fullyd ss suf Ma wereu ld beuH vity,p

tod as asT sedt rasea e sur-f orkw thenu xin.A as ah hilstr -t ipidm nl wedt layera

Direct spreading of liposomes from aqueous solution isalso possible. A phospholipid/antibody conjugate could bespread on the water surface and transferred to a glass plate(Hirata et al., 2001). The film specifically bound its antigenwith aggregation of the conjugate as shown by AFM andfluorescence microscopy. Vesicles containing IgG and a gly-colipid could be spread and deposited in a similar manner(Godoy et al., 2003) and shown to bind acetylcholinesterasewith retention of enzyme activity.

A novel biosensor was developed using the LB deposi-tion of protein A complexed with an anti-ferritin antibodyonto pyrolytic graphite followed by crosslinking with glu-taraldehyde (Aizawa et al., 1995). AFM showed formationof an ordered structure and the resultant film was shown tobind ferritin. A similar method (Choi et al., 2001) was usedto bind antibodies for high- and low-density lipoprotein toa protein A film and to use it as the selective component ina fibre-optic fluorescence sensor. The detection ranges weresufficient to diagnose the risk of coronary heart disease.

The controllability of LB deposition enabled the formationof a step structure using a polyphthalocyanine deposited ontogold; this could be used as a base layer for protein depositionand was studied using near-infrared SPR (Brink et al., 1995).

An interesting effect was found when DNA is incorpo-rated into an LB film (Sukhorukov et al., 1996). When amineLB films are deposited from a subphase containing dissolvedD Bfi oc-t ded,w sedi n tob ilars re ofo tarys tiond NAs u-c pidsa andw itht -t ndo

erea edo tedsT thenb

eenw nb tiont Am 5%o film( ra

Glutathione-S-transferase is another enzyme which capread at the interface and compressed and depositedB film (Antolini et al., 1995). The resultant film was show

o maintain its biological activity towards pesticides suctrazine, even after being heated to 423 K, whereas in soll activity is lost at 353 K. The same group also successeposited enzymes such as urease onto silanised gla

aces (Paddeu et al., 1995) and studied the films using QCnd potentiometric measurements. Similar techniquessed to deposit antibodies onto QCM crystals and coused to detect antigens down to 10−9 M (Nicolini et al., 1995).eating to 423 K was found to actually increase sensitiossibly due to film re-arrangement.

Another method of incorporation of biomolecules iseposit an LB film of a phospholipid and then use thatubstrate for deposition of liposomes (Ohlsson et al., 1995).hey deposited phospholipids onto silicon or gold, then fu

hem with proteoliposomes containing acetylcholinestend showed the protein molecules had adsorbed onto th

ace by AFM and retained enzymatic activity. Other within this paper bound a ganglioside to the films andsed QCM and SPR to monitor the binding of cholera tophospholipid monolayer on water could also be used

ost for the liposomes and transferred onto a substrate wetaining activity (Pizzolato et al., 2000). Liposomes conainingp-glycoprotein could be deposited onto phospholonolayers (Diociauiti et al., 2002), the proteins were the

abelled with gold nanoparticles and imaged. Results shohe proteins had been successfully incorporated into thend that they retained specific reactivity.

n

r-

NA, the DNA is unsurprisingly incorporated into the Llm. However IR, UV and X-ray studies show that whenadecylamine is used, the DNA in the film is single-stranhereas if dimethyldioctadecyl ammonium bromide is u

t retains its double helix. The resultant films were showind specific reagents for DNA. Further work on a simystem deposited on QCM crystals showed that exposuctadecylamine/ssDNA films to solutions of complemeningle-stranded DNA lead to specific binding from soluue to hybridisation, giving us a sensor for specific Dequences (Nicolini et al., 1997). Single-stranded oligonleotides immobilised beneath monolayers of cationic lit the air–water interface have been shown to hybridisehen deposited as LB films, the DNA molecules align w

he dipping direction (Sastry et al., 2000). Table 2gives deails of the DNA containing thin films obtained by this ather techniques.

DNA was also included in a multilayer structure whbiotin containing phospholipid LB layer was first form

n gold, avidin adsorbed from solution and then biotinylaingle-stranded DNA adsorbed onto that (Xiao et al., 1998).he selective adsorption of complementary DNA coulde monitored by SPR.

Amphiphiles containing the diacetylene group have bidely studied (Ulman, 1991; Tredgold, 1994). These cae deposited and can be polymerised with UV irradia

o give LB films of highly coloured polydiacetylenes.ixed LB film containing diacetylene fatty acids andf a monoganglioside was polymerised to give a blueCheng and Stevens, 1997) which upon exposure to chole


Table 2Comparison of DNA monolayers constructed by various methods

Immobilisation method Film thickness (nm) Density (strands cm−1) Detects counter(conc)

Time exposed Reference

LB deposition with fatty amine Film 5.8 nm, DNA0.9 nm

Parallel to surface 25 mg/ml 16 h (60◦C) Nicolini et al. (1997)

Polyethyleneimine + DNA on carbon Not reported Parallel to surface 0.01 mg/ml 30 min–3 hDavis et al. (in press)Biotinylation and avidin/gold ∼5 nm Not reported 50�g/m 5 min Zhou et al. (2001)Assembly thiolated DNA Not reported 6× 1011 0.04 mg/ml 60 min Okahata et al. (1992)Assembly thiolated DNA 0.3 nm Parallel to surface 0.5�g/ml 40 min Caruso et al. (1997)Electrostatic (DNA/cationic thiol) 0.3 nm Parallel to surface No – Caruso et al. (1997)Thiolated DNA 3.3 nm 6.2× 1012 No – Herne and Tarlov (1997)Mixed layer thiol/thiolated DNA Not reported Up to 5× 1012 8 mg/ml 90 min Herne and Tarlov (1997)Thiolated DNA 17 nm (buffer), 2 nm

waterUp to 5.2× 1012 4 mg/ml 30 min Georgiadis et al. (2000)

Assembly thiolated DNA and marker Not reported 1–2× 1013 10 zmol 30 min Takenaka et al. (2000)Mixed layer thiol/thiolated DNA ∼2 nm Up to 4× 1012 8 mg/ml 60 min (35◦C) Steel et al. (1998)Assemble DNA on polysiloxane/thiol Thiol 1.3 nm, DNA

0.8 nm1× 1013 5 mg/ml 12 h Johnson and Levicky (2003)

DNA-containing polymers on Au Not reported 2–3× 1012 2�g/ml 60 min Taira and Yokoyama (2004)Biotinylated DNA on avidin/silica 2–3.6 nm Not reported 30 ng/ml 30 min Hook et al. (2001)

toxin (40 ppm) displayed a colorimetric transition to a redfilm, i.e. an optical biosensor. Similar materials were usedto detect influenza toxin (10–60 ppm), again by a colori-metric method (Song et al., 2002). Use of transmembrane-type lipids improved the colour response and sensitivity tocholera toxin (10 ppm). An interesting amphiphile based ona fullerene–glycodendron conjugate has potential to behavesimilarly (Cardullo et al., 1998).

The amphiphile chosen can often have a large effect onthe final film composition. When IgG is spread as mixtureswith phospholipids (Wang et al., 1995), care must be takenwith the surface pressure. If this is too high then the an-tibody is squeezed out from the monolayer as shown bypressure–area curves, ellipsometry and fluorescence imag-ing. Mixed LB monolayers of methyl docosanoate and doco-syl mesylate were fabricated and then exposed to solutions ofmyoglobin (Deckert et al., 1999). The myoglobin selectivelyattached itself to the docosyl mesylate, to which it covalentlybinds, displacing the mesylate group. Therefore the concen-tration and therefore the spacing of the biomolecules can becontrolled by the monolayer composition. The compositionof a mixture of two phospholipids in an LB monolayer hasbeen shown to affect the adsorption and domain structure ofantibody monolayers deposited on top of the phospholipids(Ihaleinan and Peltonen, 2003).

LB films can also be used as transducers in various sensors.F dro-g s, theo sur-f ,1 rs ina

hasea of anI n-z fon

being able to be detected at levels down to 10−7 M. A biosen-sor for Salmonella(Olsen et al., 2003) could be made by de-positing a polyclonal antibody from the air–water interfaceonto a QCM crystal. Exposure to suspensions ofSalmonellacaused binding of the bacteria to the surface, a linear responsewas obtained from 103 to 109 cells/ml with at least 1000-foldselectivity overE. coli.

LB films have the potential to be deposited acrosssmall apertures to form structures similar to black lipidmembranes. Phospholipid bilayers containing�-hemolypsinfrom staphylococcus were successfully fabricated over small(25–250�m) apertures in silicon wafers (Peterman et al.,2002).

A new “protective plate” method (Pastorino et al., 2002)has been developed to allow continuous deposition ofbiomolecules onto polymeric tape without exposure of thespread monolayer to oxygen. Films containing penicillin Gacylase have been successfully deposited onto polymerictapes whilst retaining biological activity. This process haspotential to deposit 20–25 m of film in an 8 h day.

Thermal evaporation of amphiphiles has been shown togive structures similar to LB films (Jones et al., 1991). Evap-orated fatty acid and amine films were used to encapsulateproteins such as pepsin (Gole and Sastry, 2001; Gole et al.,2001, 2003).

4

lec-t uldb thes odesa ple, ap .,1 ed ont rrent

ilms of metal containing fatty acids were exposed to hyen sulphide gas to generate crystals of semiconductorrganic fraction then being removed by solvents. These

aces would be of use in field effect transistors (Facci et al.997). Phthalocyanine LB films were used as transducehydrogen peroxide sensor (Sergeyava et al., 1999).Butyrylcholinesterase can be dissolved in the subp

nd co-deposited with octadecylamine onto the surfaceSFET (Wan et al., 2000). The effects of pesticides on the eyme activity were monitored, with, for example, trichlor

.3. Self-assembled polymeric multilayers

After the work by Decher and other workers on polyerolyte multilayers, it was inevitable that biomolecules woe incorporated into these films. Early work concernedimple addition of polyelectrolytes to carbon paste electrnd using them to stabilise bound enzymes. For examolyvinyl pyridine-based cationic material (Parellada et al997) was added to carbon and glucose oxidase adsorb

he resultant paste electrode with a 25-fold increase in cu


density over non-polyelectrolyte containing electrodes andimprovements in linear range and half-life.

Glucose oxidase could also be adsorbed into thin layers(Table 1) of polyacrylic acid covalently bound to solid sur-faces (Franchina et al., 1999) and found to retain its activity.A ferrocene containing polyvinyl pyridine derivative couldbe assembled with glucose oxidase, the polymer acting asboth co-assembler and electron mediator (Hou et al., 1997).The resultant film (Table 1) deposited on a gold electrode ledto formation of an amperometric enzyme electrode with anextended range of 0.01–10 mM glucose with low interferencefrom uric acid. Alternating layers of Os-substituted polyvinylpyridine derivatives could be assembled with horseradish per-oxidase (Li et al., 2000) and used to detect hydrogen perox-ide electrochemically, with a linear response from 0.003 to3.7 mM and storage lifetimes in excess of 3 weeks. A hydro-gen peroxide sensor containing polyallylamine/horseradishperoxide multilayer with a detection limit of 2× 10−6 M hasalso been recently reported (Yang et al., 2004).

Other workers deposited alternate layers of polyally-lamine/polystyrene sulphonate with anti-immunoglobin onsolid substrates and characterised them with SEM andAFM. When the protein layers were separated with justone layer of polystyrene sulphonate, an aggregated disor-dered structure was observed but when five layers were used(PSS/PA/PSS/PA/PSS), a regular uniform multilayer wasf

ithp ase( s-t and< nts.S olo witht tivityt

intop oseo -c earr ate-r iron-m ue toe tivem 1c ma n ton

elec-t de-t1 then senceo inasec bec t

al., 2003) and reducedl-arginine to urea and then ammonia.When assembled on the tip of a commercial ammonia elec-trode,l-arginine could be detected at physiologically signif-icant concentrations.

An optical sensor for ammonia could be constructedby self-assembly of polyallylamine with a tetrasulphonatedmacrocycle which responded to ammonia by a colour change(Nabok et al., 1999). Incorporation of urease within the mul-tilayer led to the reduction of urea to ammonia and thereforean optical sensor for urea. Chitosan can be alternated withpolystyrene sulphonate to give layers which when incorpo-rated within an array and interrogated by impedance spec-troscopy are capable of distinguishing the four basic tastes(dos Santos et al., 2003). This array proved suitable as anelectronic tongue to distinguish different types of red wine.

The enzyme does not necessarily need to be in actualsolution for self-assembly to occur. Microcrystals of cata-lase encapsulated in a polyelectrolyte multilayer (polyal-lylamine/polystyrene sulphonate) (Yu and Caruso, 2003)could be deposited with layers of the oppositely chargedpolystyrene sulphonate. The advantage of encapsulation isthat it prevents enzyme leakage. A film containing a layerof encapsulated catalase deposited on gold was incorporatedwithin a hydrogen peroxide biosensor and gave five-foldhigher responses (between 3× 10−6 and 1× 10−2 M H2O2)with greater stability than the same sensor with a layer of non-e ncap-sa ctro-c ter re-s poly-m

bi-o wasd NAt 1w ter2 werelf tan-d e and5 n.

lec-t m tob ositedp s ass ta n toa ucoseo d andi de-p altosew

otini ny-l

ormed (Caruso et al., 1998).Cholesterol oxidase could be co-deposited w

olystyrene sulphonate onto a layer of microperoxidGobi and Mizutani, 2001) to give electrochemical choleerol sensors with linear response from 0.2 to 3 mM20 s response time with minimal effects from interfereimilar films (Ram et al., 2001) were made with cholesterxidase and esterase layered with polyethyleneimine,

he oxidase-containing electrodes showing similar sensio those above.

Redox-active materials based on Co incorporatedolyvinyl pyridine could be deposited with glucose, lactr pyruvate oxidases (Simonian et al., 2002). SPR of the gluose oxidase film showed sensitivity to glucose with a linesponse from 1 to 10 mM and also showed no loss of mial from surface even after 3 weeks in an aqueous envent, indicating that loss of activity of these systems is dnzyme inactivation rather than simple “leaching” of acaterial. Polyphenol oxidase (Coche-Guerente et al., 200)

ould be immobilised with polydiallyldimethyl ammoniund allowed electrochemical detection of catechol dowM concentrations.

Uricase and polyallylamine were deposited onto a Ptrode to form a uric acid sensor which can detect, via theection of hydrogen peroxide, the analyte between 10−6 and0−3 M (Hoshi et al., 2003). The response increased withumber of uricase layers and was unaffected by the pref ascorbate. Bienzyme films containing urease and argo-assembled with polydiallyldimethyl ammonium couldonstructed, their assembly monitored by SPR (Disawal e

ncapsulated catalase. Glucose oxidase was similarly eulated and incorporated (Trau and Renneberg, 2003) intothin film on a carbon electrode and shown to give ele

hemical responses to 1–10 mM glucose and a much fasponse time than a biosensor prepared by conventionaler entrapment.It is possible to co-deposit the polyelectrolyte and the

logically active species in one step. Pyruvate oxidaseeposited onto carbon with amine-modified dextran or D

o give a phosphate sensor (Gavalas and Chaniotakis, 200)ith detection limits of 0.1–10 mM and 49% activity af4 weeks storage. Polyethylenimine and aminodextran

ater used with glucose oxidase (Dimakis et al., 2002) andound give a sensor with excellent stability compared to sard techniques, no loss of activity after 4 months storag0% retention of activity after 245 h continuous operatio

Most work has entailed deposition of these films onto erodes, QCM crystals or SPR optical chips to enable thee used as sensors, however, other workers have depolyethylenimine on a variety of commercial membraneupplied or after grafting with polyacrylic acid (Nguyen el., 2003) and shown them to be suitable substrates thessemble layers of biomolecules such as heparin or glxidase. Sensors for glucose oxidase could be obtaine

f a mixture of glucose oxidase and glucoamylase wasosited, a sensor for higher polysaccharides such as mas obtained.Other early experiments utilised the strong avidin–bi

nteraction to form alternating layers of avidin and biotiated polyamines (Anzai and Nishimura, 1997). This work


was used as the basis for formation of multilayers containingcholine esterase and choline oxidase on Pt (Chen et al., 1997).A two-stage reaction from acetylcholine to choline (catal-ysed by esterase) to betaine (catalysed by oxidase) could beobtained for a membrane with two layers esterase and 10layers oxidase. The sensor could detect acetylcholine downto 10−6 M using QCM. Similar work deposited biotinylatedglucose oxidase and lactate oxidase as alternating layers withavidin with retention of catalytic activity (Anzai et al., 1998).Build-up of these types of films could be monitored by SPR(Cui et al., 2003) and films containing anti-IgG were shownto bind their antigens. The SPR response of the film to antigenwas shown to increase linearly with the number of antibodylayers up to three layers. Use of sensors containing four orfive layers gave responses similar to those containing threelayers. This shows that the antigens can penetrate no fartherthan three bilayers into the film.

Bienzyme films could be obtained by co-depositing con-canavalin A with either horseradish peroxidase or glucoseoxidase onto carbon (Kobayashi and Anzai, 2001). Thesefilms depend not on electrostatic interactions but rather thosebetween lectins and sugar groups. Electrochemical responseto 1 mM glucose increased with number of GOD layers buta bilayer of HRP sufficed for maximum response.

One material that particularly lends itself to binding in aself-assembled polyelectrolyte multilayer is DNA due to itsl lymerc intoa bleh ithw iu,1 oft

18-m atica ingt ec din-c itht ow-i al”D

ifiedu gle-s ,i tod tion,t suchd am-p rringa

reeo s, al iny-l ft di-

fied with a gold nanoparticle could then be studied, the goldnanoparticle after adsorption is coated with silver which isthen removed electrochemically. The presence of the poly-electrolyte multilayer is a barrier to deposition of silver di-rectly on the gold electrode.

Multilayer films containing DNA could be constructedon top of a Ru-containing polyvinylpyridine layer adsorbedonto a pyrolytic graphite electrode (Mugweru and Rusling,2002). Square-wave voltammetry techniques were used tostudy the resultant films and it was found that the resultscould be affected by exposing the samples to styrene oxidewhich is a carcinogen and damages DNA. It was estimatedthat one damaged base in 1000 could be detected by thistechnique.

As a simple model of DNA, rigid helical polypep-tides, both cationic and anionic (polylysine, polyglutamicacid) have been incorporated into polyelectrolyte (polydi-allyldimethyl ammonium or polyvinyl sulphate) multilay-ers and then assembled onto nanogrooved silicon. The stiffchains tend to align in the direction of the grooves (Mulleret al., 2003). Better alignment is found for thicker films andshorter peptide chains.

4.4. Self-assembled monolayers in biosensors

A variety of SAMs have been used to immobiliseb work(h itablef reda ableh heya fort hata

ar-b vert-i uricc binde e( et-r iquesi tiono ,1V ofS omi-n

forg stemsf tivem im-m o-l leculev g. As ith

arge number of negative phosphate groups along the pohain. Initial work showed that DNA could be assembledmultilayer with polyamines with retention of the dou

elix structure to give a stable film which interacted water-soluble dyes (Sukhorukov et al., 1996; Lang and L999; Shabarchina et al., 2003). The process of assembly

hese films could be monitored by SPR (Pei et al., 2001).QCM crystals could be coated with a single-stranded

er DNA oligonucleotide by covalent bonding, electrostssembly with polyallylamine or biotinylation and bind

o avidin (Zhou et al., 2001). Exposure to solutions of thomplementary DNA strand showed that for the aviontaining film, rapid hybridisation (5 min) took place, whe control experiment with non-complementary DNA shng no binding. This test could distinguish between “normNA and that from a�-thalassemia sufferer.Screen-printed carbon electrodes could be first mod

sing a polyethylenimine layer upon which a layer of sintranded DNA could be electrostatically bound (Davis et al.n press). AC impedimetric techniques were then usedetect the presence of complementary ssDNA in solu

he presence of which caused a drop in impedance. Norop was noted for non-complementary ssDNA, for exle, the sensor could distinguish between genomic hend salmon DNA.

Polyallylamine/polystyrene sulphonate multilayers (thf both polymers) were deposited onto gold electrode

ayer of avidin then adsorbed and finally a layer of biotated DNA deposited (Lee et al., 2003). The hybridisation ohis probe DNA with sample DNA which had been mo

iomolecules and reviews have been published of earlyWink et al., 1997; Chaki and Vijayamohanan, 2002). SAMsave several advantages that make them especially su

or biosensors, namely their ability to form ultrathin, ordend stable monolayers, and the wide variety of availeadgroups leading to the ability to tailor the surface. Tlso often provide a membrane-like microenvironment

he biomolecules and only require minimal amounts of wre often expensive biomolecules.

Some of the simplest early SAMs involved modifying con electrodes, first with a oxygen plasma and then con

ng the resultant reactive groups chemically into cyanhloride groups which then could be used to covalentlynzymes such as glucose oxidase orl-amino acid oxidasIanello and Yacynych, 1981) and using these as amperomic sensors for the target species. Other chemical technnvolve the use of silanes, for example, in the immobilisaf aminosilanes on oxidised platinum surfaces (Katz et al.989). Much of this early work has been reviewed (Chaki andijayamohanan, 2002) and although of interest, the fieldAMs especially for biosensor applications has been dated by gold–thiol monolayers.

The strong, selective interaction of sulphur groupsold makes self-assembled monolayers very suitable sy

or immobilising biomolecules at a surface. The bioacolecule itself can contain sulphur atoms enabling it to beobilised directly on gold. Alternatively a gold–thiol mon

ayer can be deposited and then used to adsorb a biomoia adsorption, electrostatic attractions or covalent bindinimple example is the modification of a gold electrode w


mercaptocarboxylic acid. The resultant negatively chargedsurface allowed the accurate electrochemical detection ofcationic dopamine even in the presence of high concentra-tions of anionic ascorbate which was repelled by the mono-layer (Malem and Mandler, 1993).

If higher selectivities are required, then the incorporationof biologically active molecules in the monolayer is essential.Although the initial work on gold–thiol monolayers producedquasi-crystalline monolayers of long chain alkyl thiols, it isunlikely that more complex molecules should pack as well.Size and rigidity of species such as DNA and proteins couldhinder adsorption and although simple amines tend not bindto gold, the multiple amines present in many biomoleculescould easily bind the biomolecules to gold via multiple co-operative interactions. However, this may improve their suit-ability as sensors since a highly ordered crystalline mono-layer may have steric interactions which prevent the bindingof substrates or the hybridisation of complementary DNAstrands.

Glucose oxidase could be chemically modified to attachthiol groups which were then utilise to attach the enzymeto gold microelectrodes (McRipley and Linsenmeier, 1996)to give an amperometric glucose sensor with fast (<20 s)response to glucose in the range 0–50 mM. Alternatively agold–thiol monolayer can be deposited onto gold and usedas a point of attachment for biomolecules. Covalent bindingo -p etricg )w d bee me-d

tog acid( er( w-i ow-i e-i (e olde kede eens

is-i yersw fh d, ther ch asC

evel-o ase)a layer(w oulda -o te to

allow an increased sensitivity and lowered response times incomparison to horseradish peroxidase. Direct electron trans-fer was also observed for alcohol dehydrogenase adsorbed ona series of gold–thiol monolayers (Schuhmann et al., 2000).The electron transfer was more efficient than that for enzymeentrapped in polypyrrole, possibly due to preferential ori-entation of the enzymes heme units towards the negativelycharged monolayer. Nile Blue dye, often used as a medi-ator, could also be covalently bound to mercaptopropionicacid monolayers (Liu et al., 2002) and this layer coated withhorseradish peroxidase to give rise to a biosensor for hydro-gen peroxide.

A series of proteins were covalently bound to mixed mono-layers of carboxylic acid and PEG-terminated thiols (Lahiriet al., 1999) under a wide variety of binding conditions. Theuse of mixed monolayers of this type prevented unspecificadsorption of proteins and allowed the level of binding to becontrolled by varying the ratio of the two thiols.

Other work (Darder et al., 2000) involved the adsorptionof a difunctional disulphide containing an alkyl chain and anaromatic acid on gold. This monolayer proved very efficientat binding membrane-bound type enzymes such as fructosedehydrogenase as measured by simple adsorption onto QCMcrystals. Up to 3× 1012 molecules/cm2 of enzyme could bebound, using this technique to attach the enzyme to gold elec-trodes led to the construction of a voltammetric sensor forf to0

lde-h ers( m-b gaveo be-t

lay-e rode,i trate.T eenw ch-m eses golde tionstg nallyh lloid.T a de-ta ith at idalg f sim-i tedw elec-t (a ishp xideb

f glucose oxidase (Gooding et al., 1998) to mercaptoproionic acid monolayers was used to give an amperomlucose biosensor when either a mediator (p-benzoquinoneas utilised or alternatively the enzyme electrode coullectrochemically platinised to remove the need for theiator.

Cytochrome-c oxidase could be covalently attachedold via carbodiimide couple to a mercaptohexadecanoicCollinson et al., 1992) or mercaptopropionic acid monolayLi et al., 1996), with the close proximity of the enzyme allong direct electron transfer to the substrate whilst still allng catalysis of the oxidation and reduction of cytochromcn solution. Viologen-substituted thiols were also utilisedLit al., 1997) to immobilise horseradish peroxidase onto glectrodes, the viologens allowing formation of a well-pacnzyme monolayer and facilitating electron transfer betwubstrate and enzyme.

A variety of different methods of covalently immobilng heavy-metal-sensitive proteins on gold–thiol monolaere compared (Bontidean et al., 1998) and the effect oeavy metals on the capacitance of the system measureesulting biosensor being capable of detecting metals sud, Cu, Hg and Zn in femtomolar concentrations.A reagentless sensor for hydrogen peroxide was d

ped by binding both an enzyme (horseradish peroxidnd a mediator (thionine) onto a self-assembled monoRuan et al., 1998) which could detect�M levels of peroxideith a response time of <4 s. Other plant peroxidases clso be deposited (Gaspar et al., 2000), with tobacco perxidase showing direct electron transfer to the substra

ructose in fruit juice with linear response behaviour up.7 mM and detection limit of 0.02 mM.

Laccase enzymes could be immobilised via glutarayde coupling onto amine-terminated thiol monolayGupte et al., 2002) and then used to detect catechol. A coination of fungal laccase and a cystamine monolayerptimal results, giving a linear amperometric response

ween concentrations of 0.001 and 0.4 mM.An interesting variant on the use of gold–thiol mono

rs has been rather than attaching the thiol to gold electnstead to use small colloidal gold particles as the subshe attachment of simple alkyl thiols to gold colloid has bell studied (Ulman, 1998) and has been extended to attaent of biological molecules. Of interest in some of th

ystems is that no mediator is required. For example, alectrode was modified with cystamine and further reac

o give a reactive thiol surface (Xiao et al., 1999). Colloidalold particles were then attached to this surface and fiorseradish peroxidase adsorbed onto to the bound cohis enabled the construction of a peroxide sensor with

ection time <5 s and limit 0.15�M (linear between 0.39�Mnd 0.33 mM). Other workers have coated gold slides w

hiol-containing sol–gel film and then adsorbed first colloold and then horseradish peroxidase to give sensors o

lar ability (Jia et al., 2002). Polymer nanospheres coaith thiol groups have also been assembled onto gold

rodes and then a layer of colloidal gold bound on topXund Han, 2004). Once again immobilisation of horseraderoxidase allowed the construction of a peroiosensor.


Immunosensors have also been constructed utilising thegold–thiol linkage to immobilise antibodies on gold surfaces.Early work studied the attachment of immunoglobin G anti-bodies to gold via (i) physisorption from solution, (ii) cova-lent linkage to a mercaptododecan-1-ol layer, (iii) thiolatingthe biomolecule chemically and using the resultant thiols tobind to gold and (iv) direct adsorption onto a protein A layer(Caruso et al., 1996). AFM studies showed the antibodiesaggregated and to be randomly distributed on the surface.Exposure to the antigen was monitored by QCM and showedthat protein A coupling layer facilitated antigen binding com-pared to the other methods. A synthetic thiol containing pep-tide containing a foot-and-mouth epitope could also be ad-sorbed on QCM crystals (Rickert et al., 1997) and shown tobind antibodies.

Proteins such as anti-IgG and BSA were immobilisedusing adsorption, binding to a gold–thiol-monolayer andavidin–biotin binding. The monolayer and avidin protocolswere found to give the best reproducibility and also werethe easiest to regenerate (Storri et al., 1998). Similar workwas performed using human cytomegalovirus (Susmel et al.,2000) antibodies in a variety of binding formats, the best be-ing binding to a thiosalicylic acid monolayer via a polylysinecoupling layer. An epitope of the virus could be selectivelydetected via QCM at 1�g/ml. ThiolatedSalmonellaantibod-ies could be directly bound to the gold surface of a QCM (Parka s oft ncys tingS

i oldd mea-s de-t hiolo anti-cT t thea .

phurg ono-l ariedfi .,1 NAos non-c ctiveb hesb ting.R dw eacho copya hada

te a-

tively the electrostatic binding of DNA to a gold surfacemodified with a cationic thiol. This paper first showed theimportance of DNA configuration at the surface, the electro-statically bound DNAs were shown to lie flat on the surface,to pack very inefficiently and would not hybridise with theircomplementary strands. Conversely the thiol-bound DNA,although it had a flat orientation would bind complemen-tary DNA from solution.Herne and Tarlov (1997)studiedthe effect of surface coverage on hybridisation and foundthat efficiency (up to 100%), stability and reversibility ofhybridisation were improved by formation of mixed mono-layers of thiol DNA with mercaptohexanol. These layers areprobably less ordered than a “pure DNA” monolayer but thismay actually help allow hybridisation to take place since lay-ers of solely thiolated DNA did not allow hybridisation tooccur. Ferrocenylnaphthalene diimide was used as an elec-trochemical marker (Takenaka et al., 2000) to detect hy-bridisation of thiol-ssDNA with its complementary strandat a gold electrode. This could be used to detect speciessuch as the yeast choline transport gene down to levels of10 zmol.

Mercaptohexan-1-ol/thiolated ssDNA mixed monolayerswere made with varying compositions and the hybridisationof complementary ssDNA studied electrochemically (Steelet al., 1998). The hybridisation efficiency as a function ofimmobilised DNA surface density showed good uptake upt th

ur-f tud-i ec ith as -t nb inals ox-a freet

e erem to-c ino-s ndt sur-f thens inso ag-i idi-s nu-c -o2 ichcp anda FMo

nd Kim, 1998). Exposure of the crystals to suspensionheSalmonellabacterium gave rise to measurable frequehifts, giving a rapid (<1 h), selective method of detecalmonellain the range 106–108 CFU/ml.Other systems that have been studied (Murata et al., 2001)

nclude binding estrogen receptors to thiol-modified gisk electrodes and by using the modified electrode toure the ferricyanide redox couple, estrogen could beected at nM concentrations. Also a variety of gold–tr sulphide monolayers were used to covalently bindhloramphenicol to QCM crystals (I.-S. Park et al., 2004).he resultant biosensors could rapidly (10 min) detecntigen with linear response in the range 0.01–100 mM

Since polynucleotides can be easily made with a sulroup located at one end of the strand, use of these m

ayers as potential DNA sensors comprises a large and veld. One of the earliest papers in this field (Okahata et al992) immobilised a thiol-substituted single-stranded Dnto a QCM crystal (about 6× 1011, molecules/cm2 corre-ponding to 8% coverage). Exposure of this sample toomplementary and complementary DNAs led to seleinding of DNA, dependent on the number of mismatcetween the strands, which could be reversed by heaabke-Clemmer et al. (1994)compared DNAs substituteith sulphur atoms at the terminus of the strand or atf the phosphate groups, with Auger electron spectrosnd ESCA showing that the multiple substituted strandmuch higher level of adsorption.QCM and SPR were used (Caruso et al., 1997) to detec

ither binding of thiol-substituted DNA to gold or altern

o about 4× 1012 molecules/cm2 but the efficiency fell off aigher surface densities.

Use of thiol monolayers as modifiers of the gold sace to allow the covalent binding of DNA has been sed (Smith et al., 2001) and by way of sulphydryl-maleimidhemistry, oligonucleotides can be attached to gold wurface density of 1.5× 1012 molecules/cm2. Higher densiies (1013 molecules/cm2) while still retaining hybridisatioehaviour can be obtained by binding a thiol with a termiloxane group to gold to form a thiol-substituted polysilne surface and then covalently linking the DNA to the

hiol groups (Johnson and Levicky, 2003).A more complex procedure has been adopted (Brockmann

t al., 1999) to create DNA arrays. Gold surfaces wodified with thiols which were then patterned by pho

hemically removing the thiols. Readsorption of a amubstituted thiol, followed by chemical grafting of DNA ahen polyethylene glycol modification of the unirradiatedace were used to form a DNA array. This array washown by SPR to selectively adsorb DNA-binding protento the DNA-modified portions of the substrate. SPR im

ng (Nelson et al., 2001) was also used to detect the hybration of DNA probes with either complementary oligoleotides or RNA fromE. coli down to levels of 2 nM withut the need for labelling. Similar methods (O’Brien et al.,000) were used to deposit dsDNA mixed monolayers whontained a recognition sequence for the enzymeEcoR1. Ex-osure to the enzyme led to digestion of the DNA strandchange in film thickness which could be detected by Ar loss of a fluorescent marker.


SPR was utilised to study the kinetics of thiol-DNA ad-sorption onto gold and hybridisation (Georgiadis et al., 2000).DNA was shown to adsorb to the surface both covalentlyand electrostatically, addition of mercaptohexanol displac-ing the electrostatically adsorbed DNA to give films withup to about 20% of the calculated maximum possible cov-erage of DNA. Further work (Heaton et al., 2001) demon-strated that control of the electric field could be utilised toenhance, retard or reverse surface hybridisation without des-orption of the thiol-DNA. Control of the electrical potentialcan be used to distinguish between matched and mismatchedhybrids.

Scanning probe microscopy was used (Sam et al., 2001)to study the morphology of 15-mer dsDNA thiol-linked ateither its 3- or 5-position to gold, the 3-substituted DNAgiving a more regular structure. An 18-mer single-strandedoligonucleotide modified in similar ways to have thiols ateither the 5- or both ends was immobilised on gold alongwith mercaptoundecanoic acid (Hianik et al., 2001). Addingcomplementary DNA caused the conductivity of the mono-layer to increase for the vertically orientated (5-bound) DNAcontaining film whereas for the disubstituted, horizontallyorientated DNA containing film a decrease in conductivitywas noted. Minimal effects were noted for addition of non-complementary DNA.

STM results have also been obtained (Wackerbarth eta ived tidesw ecifica bea stud-ih asesw d-s akeri ithh thes

ong NAl theth omc r re-sb ions.A thep ie lda XPSa n ofs on-d or-p -m died( ur

atom position on binding and hybridisation, with the oligonu-cleotides with thiols substituents at the 3-position giving su-perior adsorption and hybridisation.

A series of single- and double-stranded oligonucleotidessubstituted at 3- and 5-positions and using ethane, hexaneor xylene spacers were synthesised and self-assembled ongold (Taft et al., 2003). All the molecules formed well-packed monolayers, however, use of the xylene spacerslowed adsorption compared to alkyl spacers and double-stranded species were found to be less efficient than single-stranded. Charge transport through the film was affected bythe DNA–electrode connection, with 5-substituted DNA giv-ing the best charge transfer.

Polyallylamine or polyacrylic acid was used as scaffoldto attach both a 30-mer ssDNA probe and a suitable sulphur-containing species (Taira and Yokoyama, 2004). These poly-mers can form self-assembled layers on gold, although prob-ably with a low degree of order, having the more amorphousstructure of typical polyelectrolyte layers (Decher, 1997)and as the DNA loading of the polymer can easily be al-tered so can the DNA loading on the surface. Both polymersgave better hybridisation selectivity than a comparable thiol-immobilised ssDNA, with the polyacrylic acid scaffold beingthe more selective of the two.

One of the fields of most interest is to use DNA sensors fordetermination of bacterial contamination. Biotinylated DNAw CMc randfb ndq r hu-m etectp rombc fs threet

ichw andsl d tovA hera n byA etectt .,1

w isedwa ouldt -t alsou At thiolo oft e’s,

l., 2004) which indicate that thiol-substituted 10-mers gensely packed domains of tilted ordered oligonucleohereas unsubstituted controls show much less spdsorption. The domain structure was also found toffected by the surface electrochemical potential. SPR

es on thiolated poly-dA, dC or dT (Wolf et al., 2004)ave shown that strong interaction of the nucleotide bith gold as found for poly-dA hinders long-time thiol aorption leading to lower overall coverage whereas wenteractions such as for poly-dT give rise to films wigher coverage’s and a more vertical orientation oftrands.

XPS was used to study binding of a thiolated ssDNAold and showed that at lower coverage’s most of the D

ies flat on the gold surface with a substantial fraction ofhymine residues chemisorbed (Petrovykh et al., 2003). Atigher concentrations, the DNA is in the form of a randoil anchored at the thiol end. Divalent salts in the buffeult in high absorbances (5× 1013 mol/cm2) that would onlye possible if there were strong intermolecular interactFM techniques also indicate that at the beginning ofrocess, adsorption is very unspecific (Mourougou-Candont al., 2003) followed by the reaction of thiol groups with gond film rearrangement to give a more ordered structure.nd AFM techniques were also used to study adsorptioimple thiol-substituted oligonucleotides and optimum citions for their adsorption and its effects on surface mhology determined (Casero et al., 2003). A range of 12ers substituted with thiols or thiophosponates were stu

Wirtz et al., 2004) to determine the effects of the sulph

as bound to a thiol-dextran-streptavidin-modified Qrystal and shown to hybridise with a complementary stound in pathogenic bacteria (Tombelli et al., 2000a). Com-ination of this technique with PCR allowed isolation auantification of bacterial presence in water, vegetable oan specimens. Similar techniques could be used to dolymorphisms in human DNA (apoE gene) extracted flood piezoelectrically (Tombelli et al., 2000b) or electro-hemically (Marrazza et al., 2001) leading to detection oingle base mismatches and distinguishing between theypes of polymorphism.

Gold surfaces could be used to immobilise ssDNA whas then exposed to target ssDNA complexed with str

abelled with ferrocene to give a ternary complex which leoltammetric detection of hybridisation (Ihara et al., 1997).similar procedure using biotin-labelled DNA and furt

vidin complexation could be used to detect hybridisatioC impedance techniques and could also be used to d

he presence of the Tay-Sachs mutant gene (Bardea et al999).

A 23-mer DNA strand derived fromBacillis anthracisas adsorbed onto a gold–thiol monolayer and hybridith a target ssDNA tagged with a ruthenium marker (Miaond Bard, 2003). Electrogenerated chemiluminescence c

hen be used to detect the target down to�M/ml levels. Elecrochemical detection using a ferricyanide probe wassed (J.W. Park et al., 2004) to determine the binding of DN

o complementary samples adsorbed by either a gold–r streptavidin–biotin type immobilisation on gold; use

he streptavidin–biotin system led to higher coverag


less non-specific interaction and better hybridisation res-ponses.

Immobilisation of DNA probes onto an MEMS device(Gau et al., 2001) was used to develop an amperometricdetector capable of detectingE. coli within 40 min. Use ofPNA-modified gold electrodes (Aoki and Umezawa, 2002)along with a electroactive ruthenium marker could detectcomplementary nucleotides with a limit of 50 nM, with highselectivity over non-complementary nucleotides. An elec-trochemically active DNA intercalator was used along withthiol-substituted oligonucleotides on gold electrodes (Leeand Hsing, 2002) to distinguish the PCR products from dif-ferent Chinese medicinal plant species.

The use of genetically modified species is a controver-sial topic and recently work has involved the developmentof a method for detection of specific DNA sequences oftenused in genetic modification (Manelli et al., 2003). Biotiny-lated or thiolated DNA probes complementary to the genesequences are attached to QCM crystals and then exposed totarget solutions. Both sensors give comparable results but thegold–thiol chemistry allows faster fabrication of sensors. Thesensors can be regenerated up to 20 times with 1 mM HCl,analysis takes 15 min and can detect the modified sequencesat �M concentrations with high specificity.

Deposition of a range of thiolated oligonucleotides ontogold microelectrodes (Albers et al., 2003; Nebling et al.,2 beu mi-c os-p sim-p ofa

tion( in-fi ateda es.P de-t Ms

ou-b s hasb lyly-s co-v wn tod ta t-t oro hel gleb A.

te forD dd rcesb meas isa-t

5. Conclusions

The use of thin films, especially polyelectrolyte self-assembly and gold–thiol monolayers provides a simplemethod for the functionalisation of electrode surfaces usingnanogram amounts of material. Different techniques can giveeither highly ordered or amorphous films, giving a high levelof control of the environment and often resemble the environ-ment found inside biomembranes, thereby helping to stabiliseenzymes and proteins.

Biosensors produced using these various types of filmscan display high sensitivities, be easily interrogated usingelectronic, optical or mass-sensitive techniques, can often beregenerated and display good stability. The potential for us-able devices based on these types of devices in the medicaldiagnostic and environmental monitoring applications is im-mense. The current market for biosensors is $5 billion p.a.with glucose sensing contributing 85% of that (Newman etal., 2004).

It is unlikely that sensors constructed using techniquessuch as those described within this review will displace thecurrent range of simple, relatively inexpensive disposable teststrips available for analytes such as glucose due to these de-vices already having reasonable speed of detection and stabil-ity (Newman et al., 2004). However, for systems requiring useof very small amounts of material (a typical self-assembledm hs ionso quest ech-n

andt Msc izesm e then

A

forf tionw

R

A , T.,sors.

A 003.us

A cularofsur-

004) gave rise to formation of arrays which could thensed to detect a range of viral DNA’s. Coupling of theroarray with PCR and labelling targets with alkaline phhatase led to detection of a variety of viral (e.g. herpeslex, cytomegalovirus and Epstein-Barr) DNA at levelsbout 2 nM.

A recent variation on mass detection of hybridisaGabl et al., 2004) has been reported, using 2 GHz thlm bulk acoustic wave resonators. These were Au-cond then modified with thiol-substituted oligonucleotidrotein adsorption or DNA hybridisation could then be

ected, with potentially higher sensitivities than for QCystems.

As an alternative to gold–thiol chemistry, grafting of dle bond substituted esters to H-terminated Si surfaceeen used to generate a reactive surface to which first poine is electrostatically adsorbed and then thiol–DNAalently bound. The resultant surfaces have been shoemonstrate excellent specificity and stability (Strother el., 2000). Hook et al. (2001)compared the kinetics of a

achment of DNA either directly to gold via a thiol groupnto SiO2 via the avidin–biotinylated DNA interaction, t

ayers on silica proving to be superior in distinguishing sinase mismatches and also distinguishing DNA from PN

Silver-coated mica has also been used as a substraNA adsorption (Cho et al., 2001). Layers of single- anouble-stranded DNA could be adsorbed and the foetween these surfaces and uncoated mica surfacesured, showing an increase in chain stiffness with hybridion.

-

onolayer requires 2× 10−7 g/cm2) such as antibodies, higensitivity or an array of sensors, the control and dimensf the ultrathin films described could enable these techni

o compete with current screen printing and lithographic tology.

The manufacture of smaller and smaller chips goes onechniques such as soft lithography combined with SAan be used to print chips and microfluidic devices with seasured in nanometers. These devices could well bext leap in biosensor technology.

cknowledgement

The authors would like to acknowledge the BBSRCunding F.D. as part of the Centre for Bioarray innovaithin the post-genomic consortium.

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Structured thin films as functional components within biosensors