Abstract In this work, we investigate the formation of redox protein Azurin (Az) monolayers on functionalized oxygen exposing surfaces. These metallo-proteins mediate electron transfer in the denitrifying chain of Pseudomonas bacteria and exhibit self-assembly properties, therefore they are good candidates for bio-electronic applications. Azurin monolayers are self-assembled onto silane functionalized surfaces and characterized by atomic force microscopy (AFM). We show also that a biomolecular field effect transistor (FET) in the solid state can be implemented by interconnecting an Azurin monolayer immobilized on SiO 2 with two gold nanoelectrodes. Transport experiments, carried out at room temperature and ambient pressure, show FET behavior with conduction modulated by the gate potential

A. Biasco

Alessandro Paolo Bramanti

Giuseppe Maruccio

P. Calogiuri

Paolo Visconti

Roberto Cingolani

Rosaria Rinaldi

English

In this work, we investigate the formation of redox protein Azurin (Az) monolayers on functionalized oxygen exposing surfaces. These metallo-proteins mediate electron transfer in the denitrifying chain of Pseudomonas bacteria and exhibit self-assembly properties, therefore they are good candidates for bio-electronic applications. Azurin monolayers are self-assembled onto silane functionalized surfaces and characterized by atomic force microscopy (AFM). We show also that a biomolecular field effect transistor (FET) in the solid state can be implemented by interconnecting an Azurin monolayer immobilized on SiO2 with two gold nanoelectrodes. Transport experiments, carried out
at room temperature and ambient pressure, show FET behavior with conduction modulated by the gate potential

BIASCO, Adriana Lucia Angela

MARUCCIO, Giuseppe

VISCONTI, Paolo

CINGOLANI, Roberto

RINALDI, Rosaria

A. Bramanti

Archivio Istituzionale della Ricerca- Università del Salento

Self-chemisorption of azurin on functionalized oxide surfaces for the implementation of biomolecular devices

Open Access Repository

www.elsevier.com/locate/msecMaterials Science and Engineering C 24 (2004) 563–567Self-chemisorption of azurin on functionalized oxide surfaces for theimplementation of biomolecular devicesA. Biascoa,b,*, G. Maruccioa, P. Viscontia, A. Bramantia, P. Calogiuria,R. Cingolania, R. RinaldiaaNational Nanotechnology Laboratory of INFM, University of Lecce, Via per Arnesano, 73100 Lecce, Italyb ISUFI, Innovative Materials and Technology School, University of Lecce, Via per Arnesano, 73100 Lecce, ItalyReceived 10 June 2003; accepted 16 February 2004Available online 22 April 2004AbstractIn this work, we investigate the formation of redox protein Azurin (Az) monolayers on functionalized oxygen exposing surfaces. Thesemetallo-proteins mediate electron transfer in the denitrifying chain of Pseudomonas bacteria and exhibit self-assembly properties, thereforethey are good candidates for bio-electronic applications. Azurin monolayers are self-assembled onto silane functionalized surfaces andcharacterized by atomic force microscopy (AFM). We show also that a biomolecular field effect transistor (FET) in the solid state can beimplemented by interconnecting an Azurin monolayer immobilized on SiO2 with two gold nanoelectrodes. Transport experiments, carried outat room temperature and ambient pressure, show FET behavior with conduction modulated by the gate potential.D 2004 Elsevier B.V. All rights reserved.Keywords: Adsorption; Scanning microscopy; Surface roughness; Transport properties1. Introduction tronic devices. First of all, they are composed of smallBiomolecular electronics is an interdisciplinary field thatconcerns the application of biological systems in electronicand optoelectronic devices. Application of biological sys-tems would provide a pathway toward the miniaturization ofelectronic devices down to the nanometer level and towardthe generation of a densely packed biomolecular nanochip[1]. In particular, metalloproteins and metalloenzymes arebiological polymers that catalyse the oxidation or reductionof substrate molecules via an active redox centre containinga metal ion. Hence, the way in which electrons movethrough protein strands has attracted wide attention [2].The past two decades have witnessed a growing interestin new methods of integrating solid state devices withbiomolecules that present unique electronic functions.Among biomolecules, proteins have properties that makethem good candidates for the assembly of nanoscale elec-0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2004.02.006* Corresponding author. National Nanotechnology Laboratory ofINFM, University of Lecce, Via per Arnesano, 73100 Lecce, Italy. Tel.:+39-832-321877; fax: +39-832-326351.E-mail address: adriana.biasco@unile.it (A. Biasco).covalently linked monomeric subunits of relatively fewkinds (amino acids) by means of which they can recognizeeach other, self-assembly, react specifically with surfaces orother molecules. Secondly, proteins can be further engi-neered to tailor their properties for nanodevices [3].Redox proteins and enzymes are attractive targets aselectron conductive materials for bioelectronics. Protein-mediated electron transfer, the essential process carried outby these molecules, has been increasingly investigated sincethey play a significant role not only in cellular processes butalso in biotechnological applications.In this work we face the problem of exploiting redoxproteins as molecular wires that promote a strong couplingbetween the redox centres of two proteins or a protein andan electrode. From a practical point of view, azurin (Az)proteins have been adsorbed on the insulating gap betweentwo metal electrodes through an organic linker in order tocreate a protein array (or a protein monolayer) throughwhich electrons can flow. The aim is to fabricate a fieldeffect transistor, working in air and at room temperature,which has redox proteins as the active layer and allows tomodulate the current between the source and drain elec-trodes by means of the gate voltage.Fig. 2. Azurin protein solution is deposited onto the gap.A. Biasco et al. / Materials Science and Engineering C 24 (2004) 563–5675642. Design and fabricationRecently, different molecular electronic devices have beenfabricated that make use of individual molecules to performelectronic functions now performed by semiconductor devi-ces [4–6]. In a previous work we demonstrated a field effecttransistor based on amodified DNA base entrapped in the gapbetween source and drain electrodes [7].In this perspective, the use of electron-transfer proteinsappears to be particularly attractive due to their naturalcharge transfer activity in biological systems.A special kind of electron transfer (ET) reaction is thereaction between the reduced and the oxidized form of thesame protein, the so-called electron self-exchange (ESE)reaction.In this work we use the blue copper protein Pseudomonasaeruginosa azurin (14.6 kDa), one of the best characterizedredox metalloproteins [8]. It mediates electron transfer in thedenitrifying bacteria; specifically, it transports electrons fromcytocrome c551 to nitrite reductase in the electron transportchain of respiratory phosphorilation [9]. The central Cu atom,the redox active site, is coordinated to five aminoacid ligandsin a distorted trigonal bipyramid geometry [9]. The tertiarystructure of azurin is strongly asymmetric for the presence ofa S–S bridge opposite to copper, as clearly shown in Fig. 1.Azurin from P. aeruginosa fold into an eight-stranded Greekkey h-barrel motif with only a a-helix present. Given to thissecondary structure and also to the disulfide bond, it can beregarded to as a very stable protein. Due to their physical andchemical properties, Az proteins are good candidates for bio-electronic applications.Fig. 1. 3D structure of P. aeruginosa azurin. Opposite to Cu atom there isthe unique disulfide bridge between Cys3 and Cys26. Alpha helices areevident on the right. Five aminoacids coordinate the Cu ion giving adistorted trigonal bipyramid geometry.The immobilization procedure is a particularly demand-ing aspect: it should assure an appropriate and permanentlocalization of biomolecules on a support and it mustpreserve its necessary function [10]. Different immobiliza-tion methods can be employed in order to attach the proteinto the substrate: among others (physical entrapment withinmembranes, Langmuir Blodgett, physical or chemical ad-sorption), covalent attachment on self assembled mono-layers (SAMs), consisting of molecules adsorbed fromsolution onto solid substrates [11], are the most promisingsince they provide control over the reproducibility andorientation of the anchored protein.Apart from being oriented and higly ordered, SAMs arechemically and thermodynamically stable even after cou-pling them with the immobilising molecule. They can alsobe prepared in laboratory using very simple method andequipment.By immobilizing proteins in thin monolayer films ofpolymers, it is possible to interface molecules with metallicelectrodes. Chemical functionalization of oxygen-exposingsurfaces by means of linker molecules creates bio-compat-ible interfaces that communicate with metallic circuits.Three-terminal nanodevices were fabricated using acombination of photolithography and electron-beam lithog-raphy (EBL), as previously described [12]. They consist oftwo Cr/Au (8 nm/35 nm) tip-shaped electrodes on a Si/SiO2substrate with a tip separation between 100 and 10 nm.The technique for protein immobilization is based uponthe self-assembly of 3-MPTS on SiO2 surfaces. A verydiluted solution (2%) of 3-MPTS has been used to obtainordered monolayers and to prevent from the multilayerformation. Sulfide groups are adsorbed from high purityethanol solution and at the solid–liquid interface a strongchemisorption of the head group (Si) gives rise to highlyordered architectures.The SAMs were constructed by using a two-stepprocedure. Substrates were incubated in 3-MPTS ethanolsolution. The promoted surfaces were exposed to commer-cial Az solutions and gently dried at room temperature.Adsorption of Az was performed from 10 4 M solutionA. Biasco et al. / Materials Science and Engineering C 24 (2004) 563–567 565with a pH 4.6 prepared dissolving Az in 50 mM ammo-nium acetate buffer. The choice of a 4.6 pH value is due tothe fact that it corresponds to the Az isoelectric point. Inthese conditions no charges are present on the biomoleculesurface and the adsorption process is not affected byelectrostatic attractive or repulsive forces. Fig. 2 showsthe deposition of the azurin solution on the final deviceand between the electrodes.3. Characterization and discussionIn the present work we took advantage of atomic forcemicroscopy (AFM) to provide qualitative information aboutadsorbed proteins with molecular resolution [13].The SAM preparation was performed on the cleaned anddried surfaces (pre-treated with a oxygen plasma treatment)in absolute ethanol solvent and in a clean and dust-proofenvironment.3-MPTS was used as a cross-linking agent because itensures orientation of the protein deposited film. In fact, theprotein adsorption on 3-MPTS SAM occurs by the uniquedisulfide bridge. Therefore, proteins are well ordered in thenanomolecular film and the molecular architecture is similarto that of immobilized protein molecules on gold [14].Fig. 3 displays the obtained tapping-mode AFM imageswith a scan size of 0.88 0.88 Am.This is an evidence for azurin adsorption since it revealsfeatures uniformly distributed across the surface, whichwere not present on SAMs substrates (images not shown).The presence of these topographic features was reflected inFig. 3. About 0.88 0.88 mm2 topography image of a protein adsorbed surface. Itcontact mode. The scan rate was 1 Hz and the set point 2.9 mV; (columns rowthe roughness. The protein adsorption, in fact, is responsiblefor a surface root mean square (rms) roughness increasingfrom 0.3 to 3.17 nm. The recognizable granular structuresare adsorbed proteins and the surface is totally covered byproteins. The images have not been processed with theexception of flattening.AFM techniques can add size and shape to imagedstructures due to tip convolution effect; consequently, thelateral size of nanoparticles dispersed onto a substrateresults overestimated. The real surface topography couldbe obtained only by using an infinitely sharp tip (zerocurvature radius) or, as in the specific case of adsorbedproteins, by performing AFM imaging in a liquid cellcontaining physiological buffer [15]. In our experiments,since the tip could not be reduced under a certain limit andsince a dried environment was requested, we used anapproximation to find out protein real dimensions. It isknown that Az has a dimension of 4.4 nm [14]. If weapproximate the tip to a sphere of diameter D and theprotein to a sphere of actual diameter d, then the apparentwidth W can be calculated from the formula [16]W ¼ 2ðDdÞ1=2:The nominal D value is 20 nm, therefore the apparentwidth measured by AFM (19–21 nm) corresponds tod = 4.5–5.5 nm, not very different from the literature value.On the contrary, the height measured is not affected by thesize of the AFM probe. The average height is 3.26 nm that islower then the expected one. This could be explained bytaking into consideration the compression over the proteinwas recorded using a Bio Scope AFM (Veeco Metrology Group) in the non-s = 512 512).A. Biasco et al. / Materials Science and Engineering C 24 (2004) 563–567566induced by tip force while scanning the surface and thedistortion effect after the adsorption and drying process.Current–voltage experiments were carried out by usinga semiconductor parameter analyzer (HP Agilent 4155B) inthe voltage range between  6 and 6 V, at room temper-ature and ambient pressure. The gate voltage (VG) waschanged in order to investigate its influence on the current(Ids) between the source (s) and drain (d) electrodes.Before the deposition of molecules, all devices were testedto verify that the channel was insulated (resistance as largeas 100 GV).Among all the investigated samples, only few behaved asa FET since a modulation of the transport between S and Dvoltage by means of the gate electrode was observed. Inmost cases, in fact, protein solution is physically excludedfrom the few nanometer gap between electrodes and thisprevents from obtaining an azurin active layer. A typicalcurrent–voltage (I–V) curves measured in air at roomtemperature is shown in Fig. 4. The figures represent theIds current vs. Vds voltage at different Vg voltages. Vds wasvaried between 0 and 6 in one case (a) and between 0 and 6 (b) in the other. In both cases a low-current plateau isdiscernible meaning that our system behaves like an insu-Fig. 4. (a) I–V characteristics of an azurin device. Vds>0, Vg>0.lator at low voltages. Then current rises steeply above athreshold voltage of the order of 3 V and saturates. At afixed value of Vds = 4.5 V it has been observed that Idsincreases with Vg, it reaches a maximum at approximatelyVg = 1.2 V and then decreases. This behaviour is not evidentin semiconductor devices and is certainly due to the pres-ence of the biological material. Moreover, it could beexploited for the fabrication of nanodevices with newfunctionalities, unknown to the semiconductor industry.Azurin monolayers chemisorbed on functionalized oxidesurfaces have been fabricated successfully and assessed byAFM. A blue-copper protein modified field effect transistoris presented. The characteristic of the developed device isdescribed. We suggest that transport through the proteincopper site is responsible for conduction whereas themodulation is correlated to the field effect. However, aftersome cycles of measurement, current level drops because ofdevice aging, due to both molecular active layer degradationand electrode burning. As revealed by SEM investigations(images not shown), a correlation between electrical re-sponse and morphological changes of the molecular activelayer is observed. Though a better control of the mainparameters affecting the reproducibility has to be achieved,(b) I–V characteristics of an azurin device. Vds < 0, Vg>0.A. Biasco et al. / Materials Science and Engineering C 24 (2004) 563–567 567the demonstration of such field effect device is an importantstep towards the development of molecular electronics.AcknowledgementsThis work is the result of recent research carried out bythe biomolecular electronics division of the NationalNanotechnology Laboratory (NNL)–Lecce. Special thanksgo to the team of the European Community projectSAMBA. Financial support by EC through SAMBA projectis gratefully acknowledged.References[1] A.B. Kharitonov, M. Zayats, A. Lichtenstein, E. Katz, I. Willner, En-zyme monolayer-functionalized field-effect transistors for biosensorapplications, Sens. Actuators, B, Chem. 70 (1–3) (2000) 222–231.[2] M.L. Kennedy, B.R. Gibney, Metalloproteins and redox protein de-sign, Curr. Opin. Struck. Biol. 11 (4) (2001) 485–490.[3] G. Gilardi, A. Fantuzzi, Manipulating redox systems: application tonanotechnology, Trends Biotechnol. 19 (11) (2001) 468–476.[4] C. Joachim, J.K. Gimzewski, A. Aviram, Electronics using hybrid-molecular and mono-molecular devices, Nature 408 (2000) 541–548.[5] J.M. Seminario, A.G. Zacarias, J.M. Tour, Theoretical study of amolecular resonant tunneling diode, J. Am. Chem. Soc. 122 (13)(2000) 3015–3020.[6] M.A. Reed, J. Chen, A.M. Rawlett, D.W. Price, J.M. Tour, Mo-lecular random access memory cell, Appl. Phys. Lett. 78 (23) (2001)3735–3737.[7] G. Maruccio, P. Visconti, V. Arima, S. D’Amico, A. Biasco, E.D’Amone, R. Cingolani, R. Rinaldi, S. Masiero, T. Giorni, G. Gottar-elli, Field effect transistor based on a modified DNA base, Nano Lett.3 (4) (2003) 479–483.[8] E.T. Adman, R.E. Stenkamp, L.C. Sieker, L.H. Jensen, A crystallo-graphic model for azurin at 3 Å resolution, J. Mol. Biol. (123) (1978)35–47.[9] M.A. Webb, C.M. Kwong, G.R. Loppnow, Excited-state charge-trans-fer dynamics of Azurin, a blue copper protein, from resonance ramanintensities, J. Phys. Chem., B 101 (25) (1997) 5062–5069.[10] N.K. Chaki, K. 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Self-chemisorption of azurin on functionalized oxide surfaces for the implementation of biomolecular devices

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