Materials Science and Engineering C 22 (2002) 449 – 452 www.elsevier.com/locate/msec
Development of a novel enzyme/semiconductor nanoparticles system for biosensor application M.L. Curri a,*, A. Agostiano a,b, G. Leo c, A. Mallardi a, P. Cosma b, M. Della Monica a,b a
Centro Studi Chimico-Fisici sulla Interazione Luce Materia (CS-CFILM)-CNR, Via Orabona 4, 70126 Bari, Italy b Dipartimento di Chimica-Universita` di Bari-Via Orabona 4, 70126 Bari, Italy c Istituto per lo studio di nuovi Materiali per l’Elettronica (IME)-CNR, Via Arnesano, 73100 Lecce, Italy
Abstract Nanosized semiconductor crystals can increase efficiency of photochemical reactions and can be effectively coupled to biomolecular units, such as enzyme, to generate novel photoelectrochemical systems. In this work, nanocrystalline CdS has been synthesized by using a microemulsive system and immobilised by self-assembling on a gold electrode in order to prepare, combined with formaldehyde dehydrogenase (FDH) enzyme, a biological-inorganic hybrid able to perform catalytic oxidation of formaldehyde. The preliminary results indicate that quantum-sized CdS layer on gold, in close contact with the enzyme, is an effective photoactive material to replace the NAD+/ NADH role as charge transfer in the enzymatic reaction. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Nanocrystalline semiconductor; Self-assembly; Enzyme; Photoelectrochemistry; Sensor
1. Introduction The use of semiconductor colloids as photocatalysts for a variety of chemical reactions, due to their peculiar optoelectronic and photocatalytic properties, is well stated in the recent literature [1,2]. When the semiconductor nanocrystal diameter is comparable to or less than the bulk exciton diameter, size-dependent electronic properties and ionization potentials result. Therefore, through particle size control, photocatalytic electron and hole redox potentials of size-quantized semiconductor nanocrystals can be tuned to achieve increased redox power for selective chemical reactions [3]. In addition, the use of nanosized semiconductor crystals represents a possible route to increase the efficiency of most photocatalytic processes because of their very high surface area to volume ratios. On the other side, nanoparticle architecture on electrode magnetises strong research efforts directed to the development of photoelectrochemical devices able to establish active interfaces for photocurrent generation. In this field, semiconductor particles coupled to biomolecular units represent an original route for the generation of novel photoelectrochemical systems: redox * Corresponding author. Email address:
[email protected] (M.L. Curri).
enzyme can indeed be considered as bioactive matrices of relevant importance for tailoring bioelectronic devices and biosensors [4,5]. Enzymes can be used as catalysts in a number of redox processes photosensitized by inorganic semiconductors, such as hydrogen formation, CO2 photofixation, photosynthesis of organic and amino acids, photochemical reduction of CO2, etc. [6,7]. In such systems, electrons and holes, generated respectively in the conduction and valence bands of the semiconductor, can be used by the enzyme for the reduction and oxidation of the substrate (Fig. 1). Usually, transfer of charge carriers from the semiconductor surface to the active center of the enzyme is performed by mediators able to carry out reversible redox processes, although the coupling of semiconductor and enzyme may proceed also without an electron carrier [5]. The aim of this work is the design of a biological-inorganic hybrid able to perform a catalytic oxidation of formaldehyde by using formaldehyde dehydrogenase (FDH) enzyme and quantum-sized CdS nanocrystals. The semiconductor particles can be used as charge carrier mediators instead of the nucleotidic co-factor, which plays the same role in the biological apparatus. The selectivity and peculiar properties of inorganic nanocrystalline moiety should allow to overcome the problems mainly due to the poor electrochemical performances of the NAD+/
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 1 9 1 - 1
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Fig. 1. Schematic illustration of photoinduced oxidation of formaldehyde to formic acid using FDH coupled to CdS as charge carrier mediator instead of NAD+ co-factor.
NADH couple, which exhibits kinetically unfavored processes of oxidation and reduction. In fact, the vast majority of NAD+/NADH-dependent redox enzymes reveal crucial difficulties because of their poor stability and electrical contacting with electrode, which prevent their broad use in biosensor applications. The final goal of this research is the development of new molecular recognition biosensors selective to formaldehyde, and able to detect and quantify this compound of large interest in food chemistry, classified as mutagen and possible human carcinogen. In order to prepare the hybrid semiconductor/enzyme coupled system, the synthesis of semiconductor nanocrystals with a good control of size and size distribution has been set up by using microemulsion systems [8,9]. The CdS nanocrystals have been successively immobilised on gold substrate by self-assembly [10 – 12]. The obtained self-assembled monolayers (SAM) have been characterised by scanning tunnel microscopy (STM) and UV – vis spectroscopy. To optimize the experimental conditions for effective coupling between the semiconductor and the enzyme, the characterisation of the interaction between enzyme and semiconductor nanocrystals and the kinetic study on the enzyme activity have been performed. Photoelectrochemistry of the CdS nanoparticles, linked onto gold substrate, in presence of FDH and formaldehyde has provided preliminary results on the effectiveness of the quantum-CdS (Q-CdS) particles upon photoexcitation of acting as charge carrier in substitution of the of NAD+/NADH couple.
2. Experimental section CdS nanoparticles have been prepared by using an improved synthetic route which exploits a quaternary water-in-
oil microemulsion to obtain CdS nanocrystals of high crystalline quality, small dimensions and a high degree of monodispersity. CdS nanoparticles have been prepared in the quaternary microemulsion, formed by Cetyl-trimethyl ammonium bromide (CTAB), pentanol, hexane and water, and their size has been modulated by properly changing water to surfactant (W0) and co-surfactant (pentanol) to surfactant ( P0) ratio, respectively [9]. The nanocrystals have been immobilised on gold by selfassembling technique, by using 1,6-hexanedithiol molecules as binding layer. The dithiol self-assembled monolayers on gold, obtained by immersing the gold substrates in an ethanol solution of the dithiol, have been exposed to solution of CdS nanoclusters in reverse micelles [12]. Spectra of the CdS nanocrystals and self-assembled nanocrystal layers have been recorded by using UV –vis spectrophotometer equipped with a Diffuse Reflectance Accessory (Varian). STM images of CdS quantum dots immobilised on gold have been recorded in air at sample voltage of 0.5 V and a reference current of 1 nA by using electrochemically thinned W tips with Explore Topometrix Microscope. The activity of FDH, from Pseudomonas putida (SIGMA), was measured in aqueous buffer and in reverse micelles at 25 jC. Aqueous buffer and the final water pool of reverse micelles consisted of MOPS buffer, pH = 7.5. The enzyme activity was followed by monitoring at 340 nm the NADH formation, by using UV – vis spectrophotometer Cary 3 (Varian). Kinetic parameters were determined by initial velocity analysis and were calculated from double reciprocal plots. This involved varying the formaldehyde concentration at fixed levels of NAD+. The substrate concentrations are expressed as overall. Electrochemical measurements, chronoamperometry, were performed by using a potentiostat PGSTAT10 Autolab at 20 F 2 jC in a properly designed electrochemical cell consisting of the working modified electrode, a saturated Ag/AgCl reference electrode and a Pt auxiliary electrode. N2 bubbling was used to remove oxygen from solution in the electrochemical cell.
3. Results and discussion A key point for preparation of nanostructured materials is the careful control of nanocrystallite size and size distribution and the optimization of the immobilisation procedures to combine chemical and physical properties of nanocrystalline semiconductor on mesoscopical scale. Self-assembling techniques, among various immobilisation approaches, seem to be particularly effective for the construction of highly ordered semiconductor nanocrystal monolayers. This method provides a stable bonding between particles and substrates and a strong adherence of the nanoparticles onto electrode, which is crucial for studying electron and energy transport within the nano-
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crystal monolayers and from the nanocrystals to the substrates. In this perspective, the use of the quaternary ‘‘water-inoil’’ microemulsion represents an effective pathway to obtain both nanosized semiconductor particles and stable nanocluster layers, as shown by data reported in Fig. 2A. The STM image clearly shows how nanoparticles are not fused together in the immobilised assembly, but maintain their original size and individuality. After repeated scans of the same region, the nanocrystals continue to be firmly attached to the gold surface. A further confirmation of the important role played by co-surfactant in SAM films is provided by UV – vis spectra of sample (Fig. 2B). Spectra show that, apart from the lower absorbance due to the reduced density of Q-sized CdS self-assembled layer compared to the solution, the immobilisation procedure does not affect absorption onset, peak position and width of the band. The UV – vis result is in accordance with the STM images and shows that the self-assembled layers of noncoalesced CdS nanocrystals obtained in the quaternary water-in-oil microemulsion form almost uniformly packed structure, durable and stable, having the energy band gap of nanoparticles in solution. In addition, the concordance between particle radius calculated from UV –vis spectra for CdS in solution and measured from STM image for the self-assembled layer indicates the absence of ripening during immobilisation procedure.
Fig. 2. Sketch of self-assembled layer of Q-CdS 1,6-hexanedithiol functionalised Au substrate. (A) STM image of the semiconductor ˚ , z range 1.20 nm). (B) Absorption nanoparticle layer (scan size 530 530 A spectra of CdS nanoparticles in solution (solid line) and after selfassembling on 1,6-hexanedithiol functionalised substrate (dotted line) prepared by using the quaternary water-in-oil microemulsion.
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Table 1 Sample
Aqueous solution P0 = 8; W0 = 20 P0 = 8; W0 = 30 P0 = 10; W0 = 20 P0 = 10; W0 = 30 P0 = 14; W0 = 20
Km (AM) No Q-CdS
Q-CdS
1.80 2.44 4.81 3.09 3.65 3.09
– 4.91 7.61 2.9 4.44 4.02
After the optimization of the immobilisation procedures, the reactivity of the enzymatic system formaldehyde dehydrogenase (FDH) in water and in micellar solution used for the preparation of the CdS nanocrystals has been investigated, followed by a study of enzyme – semiconductor nanocrystal interaction in the micellar system. This step is fundamental in order to exclude inhibition processes of the FDH by CdS nanoparticles. The data relative to the measurements of FDH activity in the quaternary microemulsion CTAB/hexane/pentanol/water are reported in Table 1 as a function of both W0 and P0. In the same table, the value of the rate constant in the aqueous buffer is also reported. As the enzymatic activity followed as a function of pH in aqueous solution and in reverse micelles does not show any substantial difference between the two systems, the kinetic measurements were performed at pH = 7.5, value at which the enzyme is most active and stable [13] and the results are not influenced by the spontaneous hydrolysis of NADH. As well documented in the literature [14,15], the solubilization of enzyme in reverse micelles may differently affect their activity. In our case, no relevant change in the kinetic parameters determined in
Fig. 3. Chronoamperometry registered at 78 mV (vs. Ag/AgCl sat.) in a buffer solution containing FDH under illumination (ON) and in the dark (OFF). In (a) and in (b), added was the same amount of formaldehyde. Inset: time course of photocurrent registered at 78 mV (vs. Ag/AgCl sat.) with formaldehyde (B) and with formaldehyde and FDH (A) under illumination.
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water and in micelles and between the values recorded in micelles of different dimensions has been found. These results suggest that the enzyme is poorly modified in its behaviour after confinement into the reverse micelles. Similarly the enzymatic activity is nearly completely retained in the presence of CdS nanoparticles, while Cd ions used as nanocrystal precursors completely inhibit the formaldehyde dehydrogenation reaction (in accordance to Ref. [13]). Particular attention has therefore to be paid in the preparation of the CdS nanocrystals to avoid the presence of excess of cadmium ions that negatively influence the enzyme behaviour. The electrochemical behaviour of CdS nanoparticles, immobilised on gold electrodes through dithiols, as previously described, has been finally investigated in the presence of the FDH enzyme and its substrate formaldehyde. In Fig. 3, the chronoamperometric response of the electrode in different experimental conditions is shown. When only the enzyme is present in solution, a stable dark current is recorded whose value increases under illumination. The addition of a certain amount of HCHO (a) results in a noteworthy increase of the anodic photocurrent, which, however, decreases in time up to the value detected before formaldehyde injection. After restoring the dark conditions, the same procedure was repeated without sorting any observable increase of the current. The behaviour of the current time course can be explained by assuming a charge injection from the photoactivated CdS to the active center of the enzyme, which makes possible the catalytic oxidation of the formaldehyde. Further confirmation of the abovereported assumption is given by the data shown in the inset of Fig. 3, where the photocurrent recorded in the presence and in the absence of the enzyme in a solution containing the HCHO is reported. The overall data shown in the figure clearly indicate the necessity of the contemporary presence of the enzyme and of its substrate in determining the increase of the anodic photocurrent, and the catalytic role played by the CdS in the occurrence of the enzymatic reaction. Although with the low value of the photocurrents and their decrease in time, which indicate the occurrence of photocorrosion effects on electrode and the need for a better engineering of the photoelectrochemical device, the above-
reported preliminary results on quantum-sized CdS immobilised on gold electrodes, in close contact with enzyme and substrate, are encouraging. Experimental data clearly point out that the nanocrystalline semiconductor effectively acts as charge shuttle by involving electrons and holes in the enzymatic oxidation of formaldehyde to formic acid carried out by formaldehyde dehydrogenase.
Acknowledgements This work was partially supported by CNR Progetto Finalizzato Biotecnologie.
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