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ARTICLE IN PRESS+ModelNB-9764; No. of Pages 6

Sensors and Actuators B xxx (2007) xxx–xxx

An HRP-based amperometric biosensor fabricatedby thermal inkjet printing

L. Setti a,∗, A. Fraleoni-Morgera a, I. Mencarelli a, A. Filippini a, B. Ballarin b, M. Di Biase c

a Department of Industrial and Materials Chemistry, Faculty of Industrial Chemistry, University of Bologna, Italyb Department of General and Inorganic Chemistry, Faculty of Industrial Chemistry, University of Bologna, Italy

c School of Pharmacy and Pharmaceutical Sciences, University of Manchester, United Kingdom

Received 3 July 2006; received in revised form 28 November 2006; accepted 1 December 2006

bstract

Direct inkjet printing of a complete and working amperometric biosensor for the detection of hydrogen peroxide, based on horseradish peroxidaseHRP), has been demonstrated. The device has been realized with a commercial printer. A thin layer of PEDOT:PSS, which was in turn coveredith HRP, was inkjet printed on top of an ITO-coated glass slide. The active components of the device retained their properties after the thermal

nkjet printing. The whole device has been encapsulated by means of a selectively permeable cellulose acetate membrane.

The successful electron transfer between the PEDOT:PSS covered electrode and the enzyme has been demonstrated, and the biosensor evidenced

ery good sensitivity, in line with the best devices realized with other techniques, and a remarkable operational stability. This result paves the wayor an extensive application of “biopolytronics”, i.e. the utilization of conductive/semiconductive polymers and biologically active molecules toesign bioelectronic devices using a common PC, and exploiting normal commercial printers to print them out.

2007 Elsevier B.V. All rights reserved.

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eywords: Thermal inkjet printing; Biological ink; Electronic ink; Bioelectron

. Introduction

Inkjet printing has many new practical applications, such asor example the production of printed electronic circuits. Thisatter application is of particular interest, due to the evident prac-ical advantages of this approach. It permits infact to achievehorter process times, higher rates of active material utilization,nd a great versatility [1,2]. Among the various technologies,hermal, piezoelectric and electrostatic printing are the most dif-used ones [3,4]. In the first technology, heat-generated vapourubbles are exploited to eject ink droplets out of a chamber, inhe second one the driving force for the ink ejection is providedy a piezo-electric actuator, while in the third one the ink is

Please cite this article in press as: L. Setti et al., An HRP-based amperomB: Chem. (2007), doi:10.1016/j.snb.2006.12.015

jected following the application of a strong local electric field.Another advantage of inkjet printing is the easiness of man-

gement of the digital image; in addition, the absence of contact

∗ Corresponding author at: Department of Industrial and Materials Chemistry,aculty of Industrial Chemistry, University of Bologna, V. Risorgimento 4, I-0136 Bologna, Italy.

E-mail address: setti@ms.fci.unibo.it (L. Setti).

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925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2006.12.015

iopolytronics; Biosensors

etween the printhead and the substrate makes this techniquearticularly useful for contact-sensitive surfaces. Today, thenkjet printing has already been used to fabricate all-polymerransistors [5,6], OLEDs [7], biosensors [8,9], arrays of bacteriaolonies [10], biochips [11], to perform DNA synthesis [12],or the microdeposition of active proteins [13], and for free-orm fabrication techniques aimed at the creation of acellularolymeric scaffolds [14].

On the basis of the cited technical possibilities, ouresearch group has shown recently that it is possible toealize printed bioelectronic devices by means of ther-al inkjet technology [9], printing enzymes such as GOD

nd �-galactosidase [15] and conjugated polymers likeoly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid)PEDOT/PSS) [16] without appreciable degradations of thepecific functions of the organic molecule. This approacho the realization of bioelectronic devices has been namedbiopolytronics”, linking polymer electronics and biological

etric biosensor fabricated by thermal inkjet printing, Sens. Actuators

olecules by means of digitally controlled direct printing of dotatrices on a substrate, using inkjet technology with electronic

nd biological inks. These devices are in fact characterizedy the electronic transport through the different active printed

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ayers/dots; in particular, the electronic transport from anctive enzyme (Glucose Oxidase) to a conductive polymerPEDOT:PSS) has been demonstrated [9].

In order to investigate the opposite electronic transport direc-ion possibility, i.e. from the electronically conductive polymero the enzyme, we present in this work a biosensor fabricatedhrough the sequential deposition of an electronic ink contain-ng the conductive polymer blend PEDOT/PSS and a biologicalnk containing the enzyme HRP, both deposited by thermalnkjet technology, onto an ITO-coated glass. HRP is infactharacterized by using electrons for catalyzing the reductionf hydrogen peroxide, a step which requires an efficient elec-ron transfer from the electrode to allow the enzyme to fullyevelop its activity. In order to preserve PEDOT/PSS and per-xidase from dissolving in water, the printed biosensor wasrotected with a cellulose acetate membrane, applied by dip-oating.

. Experimental

.1. Chemicals and materials

Poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid).3 wt.% dispersion in water, polyoxyethylene-(20)-sorbitanonooleate (Tween 80), ethylenediaminetetraacetic acid, tetra-

odium salt hydrate (EDTA) 98%, tetrahydrofuran 99%,otassium nitrate 99%, ferrocenemethanol 97%, peroxidaseHRP: EC 1.11.1.7. from horseradish, 133 U/mg), acetone99.5%), 3-metil,2-benzothiazolinone hydrazone (MBTH) wereurchased from Sigma–Aldrich. Cellulose acetate (Mn 29,000)nd guaiacol were purchased from Fluka and Merck, respec-ively. Phosphate buffer 0.1 M (pH 6.5) and 0.02 M (pH 7.5)as prepared according to normal laboratory procedures. Bril-

iant blue FCF (E133) were purchased from Fiorio (Italy)nd ITO-coated glass plates (12 �/m2) were purchased fromechnopartner (Italy).

.2. Instruments

Spectrophotometric measurements were performed with aV–vis scanning spectrophotometer (Uvikon 923, Bio-teck

nstruments srl, Milano). The electrochemical measurementsere performed with an Autolab PGSTAT20 (Ecochemie,trecht, The Netherlands) potentiostat/galvanostat interfacedith a personal computer. The thickness of the films was

valuated by atomic force microscopy (AFM, Scanning Probeicroscope Vista 100, Burleigh Instruments Inc.), using a long-

ange 100 A tip, operating in contact mode with a 10 nN forceonstant.

.3. Methods

.3.1. Biological ink preparation

Please cite this article in press as: L. Setti et al., An HRP-based amperomB: Chem. (2007), doi:10.1016/j.snb.2006.12.015

The biological ink was obtained dissolving 1.7 mg/mL ofRP in a 0.1 M phosphate buffer, pH 6.5, which contained EDTA.5 mM as antimicrobial agent and 10% (w/v) of glycerol astabilizer.

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PRESStors B xxx (2007) xxx–xxx

.3.2. Electronic ink preparationThe electronic ink was prepared diluting 20 mL of the

.3 wt.% PEDOT/PSS dispersion in distilled water, to a finalolume of 50 mL, and filtering the obtained dispersion with a.25 �m filter (cellulose acetate). In order to obtain the neces-ary surface tension for the printing device, the dispersion wasdded of 0.426 g of Tween 80 (6.50 mM solution).

.3.3. Description of the printing systemA commercial inkjet printer Canon i905D with a thermal

rinthead was used for printing the electronic and biologicalnks. The configuration of the system permitted to realize matri-es on solid supports, with a resolution up to 4800 × 1200 d.p.i.,n which each dot was formed by an ejected ink drop of aboutpL. The cartridge feeding the printhead was filled with theiological or the electronic ink, and layers/patterns printing waserformed using a commercial software (CD label print). The inkeposition was realized setting the printing quality to “standard”.

.3.4. Enzyme activity assayThe HRP activity was tested with an assay based on previous

ork [17] relying on oxidative coupling between MBTH anduaiacol. To 3 mL of phosphate buffer 0.02 M, under stirring,.1 mL of HRP 1.7 mg/mL (192 U/mL) and 0.5 mL of guaia-ol 0.1 M were added, followed by 0.5 mL of MBTH solution7.4 mM) and 0.02 mL of hydrogen peroxide (500 mM). Afterhe addition, the reaction was allowed to proceed for three min-tes, under stirring, at 25 ◦C, then stopped adding to the mixture.5 mL of a 2N solution of H2SO4 and 1 mL of acetone. Theo-formed red complex was analyzed by UV–vis spectropho-ometry at 505 nm.

.3.5. Determination of the deposited enzymeThe amount of printed enzyme was determined realizing pre-

iminary tests adding to the biological ink (containing 1.7 mg ofRP) 1.2 mg/mL of brilliant blue FCF (E133). The printing sup-ort was a hydrophobic polyester sheet on which the biologicalnk was not adsorbed. The printer was then connected to a stan-ard PC and a filled rectangle (with an area of 8 cm2), defined byword-processing software at a standard resolution, was printedn the substrate. The deposited ink was recovered washing offith 10 mL of water and the amount of printed E133 was deter-ined spectrophotometrically at 628 nm, having in this way and

ndirect assessment of the amount of printed enzyme.

.3.6. Electrochemical measurementsA three electrode cell geometry was used in chronoamper-

metric experiments. The counter electrode was a Pt wire, theeference electrode was a SCE, while an ITO-glass inkjet printedevice was used as the working electrode. The response of thenkjet printed electrode was measured by chronoamperometry,ipping the electrode in 25 mL of a stirred buffer solution (0.1 M

etric biosensor fabricated by thermal inkjet printing, Sens. Actuators

hosphate buffer + 0.1 M KNO3, pH 6.5) in presence of 14 mg/Lerrocenemethanol (FcMeOH), as mediator, at an applied poten-ial of −0.10 V. After a stable current background was reached30–60 s), aliquots of 250 �L of a 25 mM hydrogen peroxide

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olution were added, and the current response was measuredfter 60 s from the addition.

.3.7. Biosensor fabricationITO-coated glass plates were washed with chloroform, water

nd finally acetone in order to eliminate traces of dirt from theurface. To realize the biosensor, two different inkjet printedayers were deposed. In the first one, on a surface of 2.4 cm2

f an ITO-coated glass slide (7 mm × 50 mm), a thin film ofEDOT/PSS 230 nm, as determined using AFM, was deposedy thermal inkjet printing, repeating the deposition 10 times.n the so-obtained polymeric film, a biological ink contain-

ng 1.7 mg/mL HRP was printed with a single printhead pass.he biosensors were finally covered by dip-coating with a cel-

ulose acetate membrane. To fabricate the membrane, a solutionbtained adding 3% (w/v) of cellulose acetate (Mr 29,000), undertirring, to a solution of THF/acetone (60/40, v/v) was prepared,nd the biosensors were dipped into the solution, with an extrac-ion speed of 2.43 mm/s. The so-obtained membrane thickness,99 nm, was determined by AFM.

The layout of the final devices is shown in Fig. 1.

. Results and discussion

.1. HRP-based biological ink

The inclusion of the enzyme in a formulation for realiz-ng the biological ink did not alter appreciably its activity, asemonstrated comparing the latter parameter for HRP in pureater and in the biological ink, where almost the same activ-

ty values were found (1279 and 1293 U/mg, respectively). Forhe determination of the volume of the biological ink (andence of the enzymatic activity) ejected by the printer, a dyeE133) was added to the ink and used as a colorimetric probeo evaluate the ink volume actually ejected from the printhead,ollowing a method described elsewhere [15]. From this mea-urement it was found that the printhead ejects 0.712 �L/cm2

n the software-selected printing mode used for the realizationf the biosensor (see Section 2 for details). Since the volumef each drop exiting from a single nozzle is 2 pL, the printheadjects 2.3 × 106 dots/in.2, with a final resolution of 1500 d.p.i.he enzyme activity values in the biological ink before and after

Please cite this article in press as: L. Setti et al., An HRP-based amperomB: Chem. (2007), doi:10.1016/j.snb.2006.12.015

he printing were, respectively, 210.3 and 238.0 U/mL, hence noppreciable denaturation effect of the HRP, due to the printheadeater, was evidenced, in agreement with the results obtainedn previous works on GOD enzyme [9]. On this basis, the

2

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Fig. 1. Layout of the inkje

PRESStors B xxx (2007) xxx–xxx 3

rinted HRP activity resulted to be 0.14 U/cm2, correspondingo 1.95 × 10−5 U/dot.

The found remarkable enzymatic stability is probably ascrib-ble to a decreasing gradient of temperature occurring from theurface of the heater to the bulk of the ink solution during thehermal “shot”, allowing the enzyme to feel temperatures lowerhan those of the nozzle heater surface. Moreover, also the glyc-rol used in the biological ink as a wetting agent, to avoid thelogging effect on the external nozzle surface, could play a rolen increasing the enzyme stability to the thermal shock, thankso interactions between the polyol and the protein [18].

.2. PEDOT/PSS-based electronic ink

The deposition of the electronic ink was realized accordingo already described procedures [16], performing 10 successiveepositions of the printhead on the surface to be covered, at arinting standard resolution, obtaining a 230 nm-thick film, inine with already reported results [9].

.3. Electrochemical characterization of theRP/PEDOT:PSS/ITO system

The basic working mechanism of HRP in presence of oxy-en peroxide consists in getting electrons from the externalnvironment to reduce the oxygenated water to water. In ordero maximize the electronic response of the device, a molecu-ar mediator, i.e. ferrocenemethanol (FcMeOH), was used as

“shuttle” to increase the electron transfer rate between thenzyme and the electrochemical system [19]. To determine theost effective potential to analyze the device response, we first

nvestigated the behaviour of the ferrocenic mediator by meansf cyclic voltammetry; in particular, it was necessary to optimizehe potential at which the electronic transfer between ITO andcMeOH was maximum. The HRP-catalyzed oxidation of fer-ocenes by H2O2, which was first reported by Epton et al. [20],ollows Eq. (1).

cMeOH + H2O2 + 2H+ HRP−→2[FcMeOH]+ + 2H2O (1)

hen the oxidized mediator is rapidly electrochemically reducedt the electrode surface at the appropriate applied potential:

+ −

etric biosensor fabricated by thermal inkjet printing, Sens. Actuators

[FcMeOH] + 2e → 2FcMeOH (2)

This mechanism is effective only when the electrode potentials optimized at the value relative to the reduction potential ofcMeOH. The cyclic voltammogram of the mediator, obtained

t printed biosensor.

ARTICLE IN PRESS+ModelSNB-9764; No. of Pages 6

4 L. Setti et al. / Sensors and Actuators B xxx (2007) xxx–xxx

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fenzyme permeation was negligible (Fig. 4).

The behaviour of the mediator with respect to the mem-brane was investigated by means of cyclic voltammetry

ig. 2. Cyclic voltammogram of ferrocenemethanol in the potential rangeetween −0.30 V and +0.70 V, with a scan rate of 50 mV/sec.

n 0.02 M phosphate buffer, pH 7.5 at 40 ◦C (see Fig. 2), showscathodic peak at +0.073 V (versus SCE).

The behaviour of HRP in 0.02 M phosphate buffer solu-ion, pH 7.5 (T = 40 ◦C), with or without the presence oferrocenemethanol (14 mg/mL), was investigated by chronoam-erometric measurements on ITO glass electrodes at differentpplied potentials (i.e. +0.10, 0.00, −0.10 V) (see Fig. 3A–C,espectively).

The results of the measurements brought to evidence that theptimal potential of the working electrode is −0.1 V (Fig. 3),nd that the presence of ferrocenemethanol increases the overallesponse of the system, favouring the electron transfer mecha-ism at the interface, as reported in literature for these devices21].

.4. Prototype of an amperometric HRP inkjet printedlectrode

Prototypes of HRP-based biosensor were realized printingn aqueous solution of the enzyme on top of a printed layerf PEDOT:PSS on ITO/glass. Given the hydrophilic nature ofEDOT:PSS, a partial dissolving of the polymer into the as-eposed enzyme solution occurs, during the enzyme depositionn the PEDOT:PSS layer, realizing a first step of the enzymemmobilization by instantaneous surface mixing between poly-

er and enzyme.The so-realized device was amperometrically tested dipping

t into degassed aqueous solutions with phosphate buffers, stirredt a constant rate, by adding successive amounts of hydrogeneroxide, with fixed electrode potential (i.e. −0.10 V versusCE). However, in these conditions, a partial dissolving ofEDOT:PSS into the solution occurred, making the device notuitable for measurements, nor even for practical use. Due tohat, a water-resistant, selectively permeable membrane wassed to encapsulate the whole device, by means of dip-coatinghe inkjet printed area in a solution of cellulose acetate (CA) incetone:THF 60:40 [22]. The composition of the solution wasalibrated for obtaining a semi permeable layer able to permitn efficient diffusion of ferrocenemethanol into the device and

Please cite this article in press as: L. Setti et al., An HRP-based amperomB: Chem. (2007), doi:10.1016/j.snb.2006.12.015

t the same time to retain effectively HRP inside the device, inddition to necessary action of protecting the PEDOT:PSS layerrom water. From the assessment of the membrane permeabilityo HRP, carried on as described elsewhere [9], it turned out that

Fc

ut FcMeOH, at the applied potential vs. SCE of +0.10 V (A), 0.00 V (B) and0.10 V (C); dQ is the amount of charge exchanged between the enzyme and

he electrode.

or membranes prepared starting from CA solutions at 4–5% the

etric biosensor fabricated by thermal inkjet printing, Sens. Actuators

ig. 4. Amounts of HRP permeated through CA membranes obtained by dip-oating from solutions having different cellulose acetate concentrations.

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ig. 5. Variation of the anodic (ipa) and catodic (ipc) peak current intensities ofcMeOH (obtained by CV) as a function of the membrane characteristics.

f ferrocenemethanol in a three electrode system with anTO-covered glass slide as working electrode. The ITO wasncapsulated with different cellulose acetate membranesobtained from CA solutions of 2–6%, W/V). The CV mea-urements were performed in 0.1 M phosphate buffer solution,H 6.5, with 0.1 M KNO3 and 14 mg/L ferrocenemethanol,nd the cyclic voltammetry was carried out in a potential rangeetween −0.10/+1.00 V, with a scan rate of 50 mV/s.

The variations of anodic (ipa) and cathodic (ipc) peak currentntensities of ferrocenemethanol obtained by cyclic voltamme-ry are reported in Fig. 5. When no membrane was used, theeak intensities were 3 × 10−6 and −3 × 10−6 A, for anodicnd cathodic signal, respectively. The peak current intensity (ip)s proportional to the concentration of the electroactive speciesnd can be calculated with the Randles-Sevcik equation:

p = 0.4463(nF )3/2(RT )−1/2AD1/2C0ν1/2

here ip is the peak current value, A the surface area of the work-ng electrode, D the diffusion coefficient of the electroactivepecies such as ferrocene methanol, C0 the bulk concentrationf the electroactive species and ν is the scan rate [23]. When allhe other parameters are kept constant, the ip values are strictlyependent on the diffusion capacity of the mediator throughhe CA membrane. As visible from Fig. 5, the mediator perme-tion reaches negligibility for membranes fabricated from CAolutions at 4–5% (w/v), while acceptable permeabilities werebtained for lower values of the CA concentration. On the basisf these data, it was evaluated that membranes fabricated fromolutions at 3% of CA concentration are the optimal solutionor permitting a good retention of the enzyme and a satisfactoryermeation of ferrocenemethanol. Finally, the enzymatic activ-ty before and after encapsulation of the device was measureds described elsewhere [9], evidencing that 97% of activity wasetained by the enzyme after the membrane deposition. Thisatum confirmed that the membrane deposition step did notffect the HRP functionality, with the negligible enzyme inacti-ation ascribable to the organic solvents used for the formulationf the dip-coating solution.

.5. Electrochemical response of the printed inkjet device

Please cite this article in press as: L. Setti et al., An HRP-based amperomB: Chem. (2007), doi:10.1016/j.snb.2006.12.015

The final HRP-inkjet printed biosensor was tested by meansf chronoamperometry, as described in Section 2. The responsef the device resulted remarkably linear up to 1 mM in hydro-en peroxide (Fig. 6), with a maximum sensitivity value of

ig. 6. Calibration curve of the inkjet printed biosensor for H2O2 at an appliedotential of −0.10 V vs. SCE.

.544 �A mM−1 cm−2, in line with data reported in literature24,25] for other HRP-based sensors. In addition, the devicehowed good mechanical resistance with respect to other inkjet-abricated devices based on GOD, since even after more thanne hour of testing no visible degradation (i.e. membrane layerxfoliation, device malfunction, etc.) was found.

. Conclusions

In this work, we have shown that thermal inkjet printing coulde a viable technology to realize a printed bioelectronic circuit,eposing an active protein such as a peroxidase (HRP) and a con-uctive polymer mixture such as PEDOT:PSS on electronicallyctive substrates, through the formulation of specific biologicalnd electronic water-based inks. In particular, our findings havevidenced that it is possible to realize a bioelectronic deviceharacterized by an electron transfer from the conductive poly-er to the enzyme, continuing our previous studies conducted

n a glucose oxidase-based biosensor.As an outlook, such an approach opens the way for the devel-

pment of multifunctional bioelectronic microdevices. Infact,he realization of printable and active biological and elec-ronic inks is the first necessary step for the developmentf “biopolytronics”, i.e. the computer aided design of micro-ized, multifunctional and complex biosensors, followed by theirmmediate practical implementation on any surface via a suitablenkjet multi-ink printer.

cknowledgement

The author is grateful to Dr. D. Frascaro for helpful discussionnd for AFM measurements.

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rrait

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iographies

eonardo Setti was graduated in 1988 at the University of Bologna, wheree obtained a PhD in biocatalysis applied to industrial fermentations in 1993.rom 1998 on, he joined the Department of Industrial and Materials Chem-

stry of the University of Bologna as researcher. His main research interestsre in the field of sensing and biosensing, recovery of valuable chemicals fromndustrial wastes, energy generation. He is author of more than 50 scientificapers and is currently responsible for several research projects, funded by pri-ate companies and public institutions, coordinating a research group of about0 people.

lessandro Fraleoni Morgera obtained his degree in industrial chemistry in995 at the University of Bologna (Italy), where he gained also a master innterprise management in 1996. From 1996 to 2000, he worked for a privateompany in Italy. After that, always in Bologna he earned a PhD in industrialnd materials chemistry in 2002, on conjugated polymers, and from that timee works as a post-doc researcher at the Department of Industrial and Materialshemistry of the University of Bologna, in the research group of Dr. Setti.is main scientific interests are in the field of organic conjugated materials for

pplications in electronic, optoelectronics and bioelectronics.

van Mencarelli earned his degree in industrial chemistry in 2006 at the Depart-ent of Industrial and Materials Chemistry of the University of Bologna, withthesis on peroxidase-based biosensors fabricated by inkjet printing. After an

nvolvment in a one-year project focused on the development of a low envi-onmental impact ink for inkjet printing, he is now actively working on severalpplications (optoelectronic, biosensors and photovoltaics) of semiconductingolymers.

lessandro Filippini earned his degree in industrial chemistry in 2004 at theepartment of Industrial and Materials Chemistry of the University of Bologna

Italy). From 2004 to 2006, he worked as a contract researcher in the sameepartment on the themes of biosensing, inkjet printing of special inks, andntegrated valorization of agroindustrial wastes. From 2006 on, he works in aompany dedicated to vegetal extracts for applications in cosmetology, of whiche is co-founder.

arbara Ballarin took her degree in industrial chemistry in 1998 at the Depart-ent of Analytical Chemistry of the University of Venice (Italy), after which she

arned a a PhD focused on electrochemistry and electrocatalysis at the Depart-ent of Physical Chemistry of the University of Venice. After a one year postdoc

t the Colorado State University (USA) under the supervision of prof. C.R. Mar-in, she worked as a postdoc at the University of Padova (Italy) from 1992 to995. From 1995 on, she is researcher at the Department of Inorganic and Physi-al Chemistry of the University of Bologna, working on electrochemistry mainlypplied to sensors and biosensors.

anuela Di Biase obtained the master degree in industrial chemistry in 2005rom the University of Bologna, Italy (Faculty of Industrial Chemistry, Depart-ent of Industrial Chemistry and Materials), with a thesis on the manufacturing

f biosensors by thermal inkjet printing. From 2005 to 2006, she worked as

etric biosensor fabricated by thermal inkjet printing, Sens. Actuators

esearch assistant in the School of Materials, University of Manchester, UK. Cur-ently she has the same position in the School of Pharmacy and she is involved inPhD program in the School of Materials, University of Manchester, UK, work-

ng on a project focussed on the use of piezoelectric inkjet printing of hydrogelso build a cell-containing scaffold.