An HRP-based amperometric biosensor fabricated by thermal inkjet printing

Sensors and Actuators B 126 (2007) 252–257

An HRP-based amperometric biosensor fabricated by 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, Italy 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

b

Available online 15 February 2007

Abstract Direct inkjet printing of a complete and working amperometric biosensor for the detection of hydrogen peroxide, based on horseradish peroxidase (HRP), has been demonstrated. The device has been realized with a commercial printer. A thin layer of PEDOT:PSS, which was in turn covered with HRP, was inkjet printed on top of an ITO-coated glass slide. The active components of the device retained their properties after the thermal inkjet 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 very good sensitivity, in line with the best devices realized with other techniques, and a remarkable operational stability. This result paves the way for an extensive application of “biopolytronics”, i.e. the utilization of conductive/semiconductive polymers and biologically active molecules to design bioelectronic devices using a common PC, and exploiting normal commercial printers to print them out. © 2007 Elsevier B.V. All rights reserved. Keywords: Thermal inkjet printing; Biological ink; Electronic ink; Bioelectronics; Biopolytronics; Biosensors

1. Introduction Inkjet printing has many new practical applications, such as for example the production of printed electronic circuits. This latter application is of particular interest, due to the evident practical advantages of this approach. It permits infact to achieve shorter process times, higher rates of active material utilization, and a great versatility [1,2]. Among the various technologies, thermal, piezoelectric and electrostatic printing are the most diffused ones [3,4]. In the first technology, heat-generated vapour bubbles are exploited to eject ink droplets out of a chamber, in the second one the driving force for the ink ejection is provided by a piezo-electric actuator, while in the third one the ink is ejected following the application of a strong local electric field. Another advantage of inkjet printing is the easiness of management of the digital image; in addition, the absence of contact

∗ Corresponding author at: Department of Industrial and Materials Chemistry, Faculty of Industrial Chemistry, University of Bologna, V. Risorgimento 4, I40136 Bologna, Italy. E-mail address: [email protected] (L. Setti).

0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.12.015

between the printhead and the substrate makes this technique particularly useful for contact-sensitive surfaces. Today, the inkjet printing has already been used to fabricate all-polymer transistors [5,6], OLEDs [7], biosensors [8,9], arrays of bacteria colonies [10], biochips [11], to perform DNA synthesis [12], for the microdeposition of active proteins [13], and for freeform fabrication techniques aimed at the creation of acellular polymeric scaffolds [14]. On the basis of the cited technical possibilities, our research group has shown recently that it is possible to realize printed bioelectronic devices by means of thermal inkjet technology [9], printing enzymes such as GOD and ␤-galactosidase [15] and conjugated polymers like poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS) [16] without appreciable degradations of the specific functions of the organic molecule. This approach to the realization of bioelectronic devices has been named “biopolytronics”, linking polymer electronics and biological molecules by means of digitally controlled direct printing of dot matrices on a substrate, using inkjet technology with electronic and biological inks. These devices are in fact characterized by the electronic transport through the different active printed

L. Setti et al. / Sensors and Actuators B 126 (2007) 252–257

layers/dots; in particular, the electronic transport from an active enzyme (Glucose Oxidase) to a conductive polymer (PEDOT:PSS) has been demonstrated [9]. In order to investigate the opposite electronic transport direction possibility, i.e. from the electronically conductive polymer to the enzyme, we present in this work a biosensor fabricated through the sequential deposition of an electronic ink containing the conductive polymer blend PEDOT/PSS and a biological ink containing the enzyme HRP, both deposited by thermal inkjet technology, onto an ITO-coated glass. HRP is infact characterized by using electrons for catalyzing the reduction of hydrogen peroxide, a step which requires an efficient electron transfer from the electrode to allow the enzyme to fully develop its activity. In order to preserve PEDOT/PSS and peroxidase from dissolving in water, the printed biosensor was protected with a cellulose acetate membrane, applied by dipcoating. 2. Experimental 2.1. Chemicals and materials Poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) 1.3 wt.% dispersion in water, polyoxyethylene-(20)-sorbitan monooleate (Tween 80), ethylenediaminetetraacetic acid, tetrasodium salt hydrate (EDTA) 98%, tetrahydrofuran 99%, potassium nitrate 99%, ferrocenemethanol 97%, peroxidase (HRP: EC 1.11.1.7. from horseradish, 133 U/mg), acetone (99.5%), 3-metil,2-benzothiazolinone hydrazone (MBTH) were purchased from Sigma–Aldrich. Cellulose acetate (Mn 29,000) and guaiacol were purchased from Fluka and Merck, respectively. Phosphate buffer 0.1 M (pH 6.5) and 0.02 M (pH 7.5) was prepared according to normal laboratory procedures. Brilliant blue FCF (E133) were purchased from Fiorio (Italy) and ITO-coated glass plates (12 /m2 ) were purchased from Technopartner (Italy). 2.2. Instruments Spectrophotometric measurements were performed with a UV–vis scanning spectrophotometer (Uvikon 923, Bio-teck Instruments srl, Milano). The electrochemical measurements were performed with an Autolab PGSTAT20 (Ecochemie, Utrecht, The Netherlands) potentiostat/galvanostat interfaced with a personal computer. The thickness of the films was evaluated by atomic force microscopy (AFM, Scanning Probe Microscope Vista 100, Burleigh Instruments Inc.), using a long˚ tip, operating in contact mode with a 10 nN force range 100 A constant. 2.3. Methods 2.3.1. Biological ink preparation The biological ink was obtained dissolving 1.7 mg/mL of HRP in a 0.1 M phosphate buffer, pH 6.5, which contained EDTA 1.5 mM as antimicrobial agent and 10% (w/v) of glycerol as stabilizer.

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2.3.2. Electronic ink preparation The electronic ink was prepared diluting 20 mL of the 1.3 wt.% PEDOT/PSS dispersion in distilled water, to a final volume of 50 mL, and filtering the obtained dispersion with a 0.25 ␮m filter (cellulose acetate). In order to obtain the necessary surface tension for the printing device, the dispersion was added of 0.426 g of Tween 80 (6.50 mM solution). 2.3.3. Description of the printing system A commercial inkjet printer Canon i905D with a thermal printhead was used for printing the electronic and biological inks. The configuration of the system permitted to realize matrices on solid supports, with a resolution up to 4800 × 1200 d.p.i., in which each dot was formed by an ejected ink drop of about 2 pL. The cartridge feeding the printhead was filled with the biological or the electronic ink, and layers/patterns printing was performed using a commercial software (CD label print). The ink deposition was realized setting the printing quality to “standard”. 2.3.4. Enzyme activity assay The HRP activity was tested with an assay based on previous work [17] relying on oxidative coupling between MBTH and guaiacol. To 3 mL of phosphate buffer 0.02 M, under stirring, 0.1 mL of HRP 1.7 mg/mL (192 U/mL) and 0.5 mL of guaiacol 0.1 M were added, followed by 0.5 mL of MBTH solution (7.4 mM) and 0.02 mL of hydrogen peroxide (500 mM). After the addition, the reaction was allowed to proceed for three minutes, under stirring, at 25 ◦ C, then stopped adding to the mixture 0.5 mL of a 2N solution of H2 SO4 and 1 mL of acetone. The so-formed red complex was analyzed by UV–vis spectrophotometry at 505 nm. 2.3.5. Determination of the deposited enzyme The amount of printed enzyme was determined realizing preliminary tests adding to the biological ink (containing 1.7 mg of HRP) 1.2 mg/mL of brilliant blue FCF (E133). The printing support was a hydrophobic polyester sheet on which the biological ink was not adsorbed. The printer was then connected to a standard PC and a filled rectangle (with an area of 8 cm2 ), defined by a word-processing software at a standard resolution, was printed on the substrate. The deposited ink was recovered washing off with 10 mL of water and the amount of printed E133 was determined spectrophotometrically at 628 nm, having in this way and indirect assessment of the amount of printed enzyme. 2.3.6. Electrochemical measurements A three electrode cell geometry was used in chronoamperometric experiments. The counter electrode was a Pt wire, the reference electrode was a SCE, while an ITO-glass inkjet printed device was used as the working electrode. The response of the inkjet printed electrode was measured by chronoamperometry, dipping the electrode in 25 mL of a stirred buffer solution (0.1 M phosphate buffer + 0.1 M KNO3 , pH 6.5) in presence of 14 mg/L ferrocenemethanol (FcMeOH), as mediator, at an applied potential of −0.10 V. After a stable current background was reached (30–60 s), aliquots of 250 ␮L of a 25 mM hydrogen peroxide

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solution were added, and the current response was measured after 60 s from the addition. 2.3.7. Biosensor fabrication ITO-coated glass plates were washed with chloroform, water and finally acetone in order to eliminate traces of dirt from the surface. To realize the biosensor, two different inkjet printed layers were deposed. In the first one, on a surface of 2.4 cm2 of an ITO-coated glass slide (7 mm × 50 mm), a thin film of PEDOT/PSS 230 nm, as determined using AFM, was deposed by thermal inkjet printing, repeating the deposition 10 times. On the so-obtained polymeric film, a biological ink containing 1.7 mg/mL HRP was printed with a single printhead pass. The biosensors were finally covered by dip-coating with a cellulose acetate membrane. To fabricate the membrane, a solution obtained adding 3% (w/v) of cellulose acetate (Mr 29,000), under stirring, to a solution of THF/acetone (60/40, v/v) was prepared, and the biosensors were dipped into the solution, with an extraction speed of 2.43 mm/s. The so-obtained membrane thickness, 199 nm, was determined by AFM. The layout of the final devices is shown in Fig. 1. 3. Results and discussion 3.1. HRP-based biological ink The inclusion of the enzyme in a formulation for realizing the biological ink did not alter appreciably its activity, as demonstrated comparing the latter parameter for HRP in pure water and in the biological ink, where almost the same activity values were found (1279 and 1293 U/mg, respectively). For the determination of the volume of the biological ink (and hence of the enzymatic activity) ejected by the printer, a dye (E133) was added to the ink and used as a colorimetric probe to evaluate the ink volume actually ejected from the printhead, following a method described elsewhere [15]. From this measurement it was found that the printhead ejects 0.712 ␮L/cm2 in the software-selected printing mode used for the realization of the biosensor (see Section 2 for details). Since the volume of each drop exiting from a single nozzle is 2 pL, the printhead ejects 2.3 × 106 dots/in.2 , with a final resolution of 1500 d.p.i. The enzyme activity values in the biological ink before and after the printing were, respectively, 210.3 and 238.0 U/mL, hence no appreciable denaturation effect of the HRP, due to the printhead heater, was evidenced, in agreement with the results obtained in previous works on GOD enzyme [9]. On this basis, the

printed HRP activity resulted to be 0.14 U/cm2 , corresponding to 1.95 × 10−5 U/dot. The found remarkable enzymatic stability is probably ascribable to a decreasing gradient of temperature occurring from the surface of the heater to the bulk of the ink solution during the thermal “shot”, allowing the enzyme to feel temperatures lower than those of the nozzle heater surface. Moreover, also the glycerol used in the biological ink as a wetting agent, to avoid the clogging effect on the external nozzle surface, could play a role in increasing the enzyme stability to the thermal shock, thanks to interactions between the polyol and the protein [18]. 3.2. PEDOT/PSS-based electronic ink The deposition of the electronic ink was realized according to already described procedures [16], performing 10 successive depositions of the printhead on the surface to be covered, at a printing standard resolution, obtaining a 230 nm-thick film, in line with already reported results [9]. 3.3. Electrochemical characterization of the HRP/PEDOT:PSS/ITO system The basic working mechanism of HRP in presence of oxygen peroxide consists in getting electrons from the external environment to reduce the oxygenated water to water. In order to maximize the electronic response of the device, a molecular mediator, i.e. ferrocenemethanol (FcMeOH), was used as a “shuttle” to increase the electron transfer rate between the enzyme and the electrochemical system [19]. To determine the most effective potential to analyze the device response, we first investigated the behaviour of the ferrocenic mediator by means of cyclic voltammetry; in particular, it was necessary to optimize the potential at which the electronic transfer between ITO and FcMeOH was maximum. The HRP-catalyzed oxidation of ferrocenes by H2 O2 , which was first reported by Epton et al. [20], follows Eq. (1). HRP

FcMeOH + H2 O2 + 2H+ −→2[FcMeOH]+ + 2H2 O

(1)

then the oxidized mediator is rapidly electrochemically reduced at the electrode surface at the appropriate applied potential: 2[FcMeOH]+ + 2e− → 2FcMeOH

(2)

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

Fig. 1. Layout of the inkjet printed biosensor.

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

in 0.02 M phosphate buffer, pH 7.5 at 40 ◦ C (see Fig. 2), shows a cathodic peak at +0.073 V (versus SCE). The behaviour of HRP in 0.02 M phosphate buffer solution, pH 7.5 (T = 40 ◦ C), with or without the presence of ferrocenemethanol (14 mg/mL), was investigated by chronoamperometric measurements on ITO glass electrodes at different applied potentials (i.e. +0.10, 0.00, −0.10 V) (see Fig. 3A–C, respectively). The results of the measurements brought to evidence that the optimal potential of the working electrode is −0.1 V (Fig. 3), and that the presence of ferrocenemethanol increases the overall response of the system, favouring the electron transfer mechanism at the interface, as reported in literature for these devices [21]. 3.4. Prototype of an amperometric HRP inkjet printed electrode Prototypes of HRP-based biosensor were realized printing an aqueous solution of the enzyme on top of a printed layer of PEDOT:PSS on ITO/glass. Given the hydrophilic nature of PEDOT:PSS, a partial dissolving of the polymer into the asdeposed enzyme solution occurs, during the enzyme deposition on the PEDOT:PSS layer, realizing a first step of the enzyme immobilization by instantaneous surface mixing between polymer and enzyme. The so-realized device was amperometrically tested dipping it into degassed aqueous solutions with phosphate buffers, stirred at a constant rate, by adding successive amounts of hydrogen peroxide, with fixed electrode potential (i.e. −0.10 V versus SCE). However, in these conditions, a partial dissolving of PEDOT:PSS into the solution occurred, making the device not suitable for measurements, nor even for practical use. Due to that, a water-resistant, selectively permeable membrane was used to encapsulate the whole device, by means of dip-coating the inkjet printed area in a solution of cellulose acetate (CA) in acetone:THF 60:40 [22]. The composition of the solution was calibrated for obtaining a semi permeable layer able to permit an efficient diffusion of ferrocenemethanol into the device and at the same time to retain effectively HRP inside the device, in addition to necessary action of protecting the PEDOT:PSS layer from water. From the assessment of the membrane permeability to HRP, carried on as described elsewhere [9], it turned out that

Fig. 3. Influence of the potential applied to an ITO electrode on the calibration curves for H2 O2 obtained by chronoamperometric measurements, with or without FcMeOH, at the applied potential vs. SCE of +0.10 V (A), 0.00 V (B) and −0.10 V (C); dQ is the amount of charge exchanged between the enzyme and the electrode.

for membranes prepared starting from CA solutions at 4–5% the enzyme permeation was negligible (Fig. 4). The behaviour of the mediator with respect to the membrane was investigated by means of cyclic voltammetry

Fig. 4. Amounts of HRP permeated through CA membranes obtained by dipcoating from solutions having different cellulose acetate concentrations.

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

of ferrocenemethanol in a three electrode system with an ITO-covered glass slide as working electrode. The ITO was encapsulated with different cellulose acetate membranes (obtained from CA solutions of 2–6%, W/V). The CV measurements were performed in 0.1 M phosphate buffer solution, pH 6.5, with 0.1 M KNO3 and 14 mg/L ferrocenemethanol, and the cyclic voltammetry was carried out in a potential range between −0.10/+1.00 V, with a scan rate of 50 mV/s. The variations of anodic (ipa ) and cathodic (ipc ) peak current intensities of ferrocenemethanol obtained by cyclic voltammetry are reported in Fig. 5. When no membrane was used, the peak intensities were 3 × 10−6 and −3 × 10−6 A, for anodic and cathodic signal, respectively. The peak current intensity (ip ) is proportional to the concentration of the electroactive species and can be calculated with the Randles-Sevcik equation: ip = 0.4463(nF )3/2 (RT )−1/2 AD1/2 C0 ν1/2 where ip is the peak current value, A the surface area of the working electrode, D the diffusion coefficient of the electroactive species such as ferrocene methanol, C0 the bulk concentration of the electroactive species and ν is the scan rate [23]. When all the other parameters are kept constant, the ip values are strictly dependent on the diffusion capacity of the mediator through the CA membrane. As visible from Fig. 5, the mediator permeation reaches negligibility for membranes fabricated from CA solutions at 4–5% (w/v), while acceptable permeabilities were obtained for lower values of the CA concentration. On the basis of these data, it was evaluated that membranes fabricated from solutions at 3% of CA concentration are the optimal solution for permitting a good retention of the enzyme and a satisfactory permeation of ferrocenemethanol. Finally, the enzymatic activity before and after encapsulation of the device was measured as described elsewhere [9], evidencing that 97% of activity was retained by the enzyme after the membrane deposition. This datum confirmed that the membrane deposition step did not affect the HRP functionality, with the negligible enzyme inactivation ascribable to the organic solvents used for the formulation of the dip-coating solution. 3.5. Electrochemical response of the printed inkjet device The final HRP-inkjet printed biosensor was tested by means of chronoamperometry, as described in Section 2. The response of the device resulted remarkably linear up to 1 mM in hydrogen peroxide (Fig. 6), with a maximum sensitivity value of

Fig. 6. Calibration curve of the inkjet printed biosensor for H2 O2 at an applied potential of −0.10 V vs. SCE.

0.544 ␮A mM−1 cm−2 , in line with data reported in literature [24,25] for other HRP-based sensors. In addition, the device showed good mechanical resistance with respect to other inkjetfabricated devices based on GOD, since even after more than one hour of testing no visible degradation (i.e. membrane layer exfoliation, device malfunction, etc.) was found. 4. Conclusions In this work, we have shown that thermal inkjet printing could be a viable technology to realize a printed bioelectronic circuit, deposing an active protein such as a peroxidase (HRP) and a conductive polymer mixture such as PEDOT:PSS on electronically active substrates, through the formulation of specific biological and electronic water-based inks. In particular, our findings have evidenced that it is possible to realize a bioelectronic device characterized by an electron transfer from the conductive polymer to the enzyme, continuing our previous studies conducted on a glucose oxidase-based biosensor. As an outlook, such an approach opens the way for the development of multifunctional bioelectronic microdevices. Infact, the realization of printable and active biological and electronic inks is the first necessary step for the development of “biopolytronics”, i.e. the computer aided design of microsized, multifunctional and complex biosensors, followed by their immediate practical implementation on any surface via a suitable inkjet multi-ink printer. Acknowledgement The author is grateful to Dr. D. Frascaro for helpful discussion and for AFM measurements. References [1] Z. Bao, J.A. Rogers, H.E. Katz, Printable organic and polymeric semiconducting materials and devices, J. Mater. Chem. 9 (1999) 1895–1904. [2] B. Chen, T. Cui, Y. Liu, K. Varahramyan, All-polymer RC filter circuits fabricated with inkjet printing technology, Solid State Electron. 47 (2003) 841–847. [3] C. Ping-Hei, C. Wen-Cheng, S.H. Chang, Bubble growth and ink ejection process of a thermal ink jet printhead, Int. J. Mech. Sci. 39 (1997) 683–695. [4] H.P. Le, Progress and trends in ink-jet printing, J. Imaging Sci. Technol. 42 (1998) 49–62.

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Biographies Leonardo Setti was graduated in 1988 at the University of Bologna, where he obtained a PhD in biocatalysis applied to industrial fermentations in 1993. From 1998 on, he joined the Department of Industrial and Materials Chemistry of the University of Bologna as researcher. His main research interests are in the field of sensing and biosensing, recovery of valuable chemicals from industrial wastes, energy generation. He is author of more than 50 scientific papers and is currently responsible for several research projects, funded by private companies and public institutions, coordinating a research group of about 10 people. Alessandro Fraleoni Morgera obtained his degree in industrial chemistry in 1995 at the University of Bologna (Italy), where he gained also a master in enterprise management in 1996. From 1996 to 2000, he worked for a private company in Italy. After that, always in Bologna he earned a PhD in industrial and materials chemistry in 2002, on conjugated polymers, and from that time he works as a post-doc researcher at the Department of Industrial and Materials Chemistry of the University of Bologna, in the research group of Dr. Setti. His main scientific interests are in the field of organic conjugated materials for applications in electronic, optoelectronics and bioelectronics. Ivan Mencarelli earned his degree in industrial chemistry in 2006 at the Department of Industrial and Materials Chemistry of the University of Bologna, with a thesis on peroxidase-based biosensors fabricated by inkjet printing. After an involvment in a one-year project focused on the development of a low environmental impact ink for inkjet printing, he is now actively working on several applications (optoelectronic, biosensors and photovoltaics) of semiconducting polymers. Alessandro Filippini earned his degree in industrial chemistry in 2004 at the Department of Industrial and Materials Chemistry of the University of Bologna (Italy). From 2004 to 2006, he worked as a contract researcher in the same department on the themes of biosensing, inkjet printing of special inks, and integrated valorization of agroindustrial wastes. From 2006 on, he works in a company dedicated to vegetal extracts for applications in cosmetology, of which he is co-founder. Barbara Ballarin took her degree in industrial chemistry in 1998 at the Department of Analytical Chemistry of the University of Venice (Italy), after which she earned a a PhD focused on electrochemistry and electrocatalysis at the Department of Physical Chemistry of the University of Venice. After a one year postdoc at the Colorado State University (USA) under the supervision of prof. C.R. Martin, she worked as a postdoc at the University of Padova (Italy) from 1992 to 1995. From 1995 on, she is researcher at the Department of Inorganic and Physical Chemistry of the University of Bologna, working on electrochemistry mainly applied to sensors and biosensors. Manuela Di Biase obtained the master degree in industrial chemistry in 2005 from the University of Bologna, Italy (Faculty of Industrial Chemistry, Department of Industrial Chemistry and Materials), with a thesis on the manufacturing of biosensors by thermal inkjet printing. From 2005 to 2006, she worked as research assistant in the School of Materials, University of Manchester, UK. Currently she has the same position in the School of Pharmacy and she is involved in a PhD program in the School of Materials, University of Manchester, UK, working on a project focussed on the use of piezoelectric inkjet printing of hydrogels to build a cell-containing scaffold.