Recombinant Cytochrome P450 Immobilization for Biosensor

Nov 25, 2004 - Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine s...
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Recombinant Cytochrome P450 Immobilization for Biosensor Applications Cristina Paternolli,† Mirco Antonini,† Paola Ghisellini,†,‡ and Claudio Nicolini*,†,‡ Nanoworld Institute and Biophysics Division, University of Genova, Corso Europa, 30, 16132 Genova, Italy, and Fondazione Elba, Via delle Testuggini, Roma, Italy Received July 29, 2004. In Final Form: October 5, 2004 As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.

1. Introduction All known cytochromes are heme proteins, which are involved in the electron-transfer process. Cytochrome P450 is known to catalyze a great deal of reactions such as hydroxylation, O-dealkylation, and N-oxidation.1-8 The presence of various important substrates for cytochrome P450s9 shows the possibility of utilizing the interaction between these enzymes and some specific substances for biodevice applications. In this study, recombinant cytochrome P450s were employed because, following earlier work on the P450SCC nanostructuring,10 only recombinant proteins have the purity required for the general procedure to organic sensor applications being sought here. In fact, they represent the homologous population of molecules with a controlled sequence which can be ad hoc modified by site specific mutagenesis, and they can undergo easy mass production and, furthermore, have a high level of purity.11 In particular, the reactions of the three cytochrome P450s that we focused our attention on are summarized in Table 1. In this field, the immobilization of the enzymes onto a solid support is crucial. In previous studies12 it was found that the Langmuir-Blodgett (LB) * Corresponding author. Tel.: +3901035338217. +3901035338215. E-mail: [email protected]. † University of Genova. ‡ Fondazione Elba.

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(1) Gonzalez, F. J. Trends Pharmacol. Sci. 1992, 13, 346-352. (2) Wrighton, S. A.; Stevens, J. C. Crit. Rev. Toxicol. 1992, 22, 1-21. (3) Lewis, D. F. V. Cytochromes P450: Structure, Function and Mechanism; Taylor & Francis: London, 1996. (4) Lewis, D. F. V. Guide to Cytochromes P450 Structure and Function; Taylor & Francis: London, 2001. (5) Guengerich, F. P. Cytochrome P450. In Enzyme systems that metabolize drugs and other xenobiotics; Ioannides, C., Ed.; Wiley: New York, 2002; Chapter 2, 33. (6) Ioannides, C. Cytochromes P450 - metabolic and toxicological aspects; CRC Press, Inc.: Boca Raton, FL, 1996. (7) Rendic, S.; DiCarlo, F. J. Drug Metab. Rev. 1997, 29, 413-580. (8) Evans, W. E.; Relling, M. V. Science 1999, 286 (5439), 487-491. (9) Prior, T. I.; Chue, P. S.; Tibbo, P.; Baker, G. B. Eur. Neuropsychopharmacol. 1999, 9, 301-309. (10) Nicolini, C.; Erokhin, V.; Ghisellini, P.; Paternolli, C.; Ram, M. K.; Sivozhelezov, V. Langmuir 2001, 17, 3719-3726. (11) Ortiz de Mantellano, P. R. In Cytochrome P-450: Structure Mechanism and Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York and London, 1986. (12) Nicolini, C. Trends Biotechnol. 1997, 15, 395-401.

technique is a very suitable method for producing stable sensitive cytochrome matrixes. In fact, this type of immobilization preserves the functionality of the molecules in the working conditions of the biosensors (room temperature, dry conditions, etc.).13 In this study, therefore, different cytochrome P450s were immobilized by the LB technique, and the thin films were characterized. To choose cytochrome substrates, their relevance in the clinical field and in environmental monitoring was taken into consideration. The substrates utilized were clozapine for cytochrome P4501A2, styrene for cytochrome P4502B4, and cholesterol for cytochrome P450SCC. In fact, clozapine is an antipsychotic drug recently employed for schizophrenia therapy; styrene is a compound that enters the environment during the manufacturing of styrene-based products; and cholesterol is an organic substrate of clinical relevance that is present in the human blood. We originally immobilized cholesterol oxidase by the layer-by-layer technique14 only because we were unable at that time to obtain large multilayers of active LB or Langmuir-Schaeffer (LS) films with the cholesterol oxidase. The limitations of the layer-by-layer technique were later pointed out by Paternolli et al.,15 mainly because it requires a larger amount of material and appears less stable with respect to both LB or LS technology and the gel-matrix system. Similarly we have shown the limitations of the self-assembly approach in this context.15,16 The crucial importance of utilizing LB techniques to immobilize protein molecules is indeed largely documented in the literature (for a review, see Nicolini,12 and for the implication for biosensing, see Nicolini et al.10). In this manuscript we have then carried out a systematic experimentation utilizing the different recombinant cytochromes, transduction apparatus, and immobilization techniques introduced over the years to determine which is the best approach to construct effective organic sensors based on P450s. (13) Nicolini, C. Thin Solid Films 1996, 284-285, 1-5. (14) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16, 849-856. (15) Paternolli, C.; Ghisellini, P.; Nicolini, C. Mater. Sci. Eng., C 2002, 22, 155-159. (16) Antonini, M.; Ghisellini, P.; Pastorino, L.; Paternolli, C.; Nicolini, C. IEE Proc.: Nanobiotechnol. 2003, 150 (1), 31-34.

10.1021/la048081q CCC: $27.50 © 2004 American Chemical Society Published on Web 11/25/2004

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Table 1. Substrates and Primary Products of the Cytochrome P450s

a Cytochrome P4501A2 metabolizes the clozapine to produce N-desmethylclozapine and, secondarily, N-oxideclozapine.17 P450SCC converts the cholesterol into pregnenolone and isocapraldehyde.

2. Materials and Methods 2.1. Materials. Reagents for bacterial growth were purchased from Fluka (Buchs, Switzerland). Emulgen 913 was kindly provided by Kao Chemical (Tokyo, Japan). Protein molecular weight standards were obtained from Promega (Madison, WI), the hydroxyapatite column was from BioRad (Milan, Italy), and the glutathione sepharose 4B column was from Pharmacia (U.S.A.). The cholesterol (5-cholesten-3-β-ol), styrene (99% pure grade), clozapine (8-chloro-11-(4′-methyl)piperazine-5-dibenzo[b,e]-1,4diazepine), and other chemicals were purchased from Sigma Aldrich (Milan, Italy). 2.2. Recombinant Enzyme Expression and Purification. The cDNA encoding the mature form of cytochrome P4502B4 was then subcloned into the pGEX-KN expression vector and highly expressed in Escherichia coli by isopropyl-β-D-thiogalactoside induction. The expressed protein, fused to glutathione S-transferase (GST), was purified by glutathione sepharose 4B affinity chromatography.17 The purified cytochrome P4502B4 recombinant was in a 10 mM K-phosphate buffer (pH 7.4) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween 21, and 20% glycerol. Cytochrome P450SCC native recombinant (product of CYPA11A gene) was cloned in the E. coli system expression: the cDNA gene of P450SCC mature form was subcloned in the pTrc99A vector to obtain bacterial expression. The cDNA gene of the mature protein was obtained by deleting the N-terminal mitochondrial targeting sequence coding the first 39 amino acid residues.18,19 JM109 cells, transformed by using pTrc99A-P450SCC expression plasmid, were grown and induced as described by ref 18 with the following modifications: δ-aminolevulinic acid (1 mM), a precursor of heme biosynthesis, was added at the same time as isopropyl-1-thio-β-D-galactopyranoside (1 mM), and the cells were grown for 72 h at 28 °C by shaking at 150 rpm. (17) Pernecky, S. J.; Coon, M. J. Methods Enzymol. 1996, 272, 2534. (18) Wada, A.; Mathew, P. A.; Barnes, H. J.; Sanders, D.; Estabrook, R. W.; Waterman, M. R. Arch. Biochem. Biophys. 1991, 290, 376-380. (19) Amann, E.; Ochs, B.; Abel, K. J. Gene 1988, 69, 301-315.

b

Cytochrome

Expressed cytochrome P450SCC was purified in three different chromatographic steps: DEAE cellulose, hydroxyapatite, and Adx-sepharose 4B columns. The sample was solubilized in 10 mM K-phosphate buffer (pH 7.4) containing 0.1 mM EDTA, 0.2% sodium cholate, and 20% glycerol. Cytochrome P4501A2 cDNA clone was inserted into the expression vector and used to transform E. coli. Extraction and purification of cytochrome P4501A2 recombinant was performed according to ref 20. The purified cytochrome P4501A2 was solubilized in 100 mM K-phosphate buffer (pH 7.25) containing 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol. 2.3. Spectral Determination and Other Analytical Methods. The protein concentration was determined using the BCA assay (Pierce) or by the Bradford method, using bovine serum albumin as a standard. The 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis of protein samples was performed as described by ref 21. Spectra were recorded using a JASCO 7800 spectrophotometer (Japan) at room temperature. The purified cytochromes (P4501A2, P4502B4, P450SCC) were found to be almost completely in the low-spin iron configuration.22 The heme content for all isoforms was measured according to ref 23 using an extinction coefficient of 91 mM-1 cm-1 for the absorbance difference between 450 and 490 nm. The concentration of the recombinant enzymes is 1.2 mg/mL, and the pure grade of each sample was determined taking into consideration the absorbance values at A280 and the absorbance maximum at A417 (Soret peak). The absorbance A417/A280 ratios for cytochromes P4501A2, P4502B4, and P450SCC were 0.5, 0.8, and 0.9, respectively. 2.4. Immobilization and Characterization Techniques. LB films of cytochrome P450s were prepared using a Langmuir trough.12,24 Surface pressures of 15, 20, and 25 mN/m were used (20) Fisher, C. W.; Caudle, D. L.; Wixtrom, C. M.; Quattrochi, L. C.; Tuckey, R. H.; Waterman, M. R.; Estabrook, R. W. FASEB J. 1992, 6, 759-764. (21) Laemmli, U. K. Nature 1970, 227, 680-685. (22) Guryev, O.; Erokhin, V.; Usanov, S.; Nicolini, C. Biochem. Mol. Biol. Int. 1996, 39, 205-214. (23) Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2370-2378. (24) Nicolini, C.; Erokhin, V.; Antolini, F.; Catasti, P.; Facci, P. Biochim. Biophys. Acta 1993, 1158, 273-278.

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to transfer the monolayers from the air-water interface to solid supports, and 10 mM K-phosphate buffer, pH 7.4, was used as the subphase and as the working buffer. Cytochrome P450 solution was spread over the subphase. LB films were transferred onto slides by a horizontal lift.13,21 The samples were dried in a nitrogen flux following film deposition. The substrates used for immobilization were quartz slides for optical analysis, quartz resonators for gravimetric measurements, and indium tin oxide coated glass (ITO-glass) plates for electrochemical measurements. To immobilize cytochrome P4501A2, a mixture (10:20:70; v/v/ v) of cytochrome, glycerol (20% in working buffer), and agarose (1% in the same working buffer) was prepared. This mixture was prepared to anchor the enzyme and to preserve its activity for a long time25,26 and was then spread upon the screen-printed electrode (spe). The spectrophotometric analysis of cytochrome P450 layers deposited on quartz substrates was performed with a double beam spectrophotometer JASCO 7800 (Japan). The spectra of the films were recorded between 300 and 450 nm under dry conditions. All the experiments were carried out at room temperature. To study the interaction between styrene and cytochrome P4502B4, the spectra were recorded between 250 and 500 nm. The LB film of 30 layers was used under dry conditions. The acquisition data were recorded at different exposure times to the styrene atmosphere. The spin state equilibrium indexes were calculated using the formula22

I ) (A393 - A470) × (A416 - A470)-1 Both solution and LB film data can be found by subtracting the respective time zero values, which point to a transition from low to high spin going from solution to film as previously shown.22 Gravimetric measurementswere carried out by means of a homemade gauge with a sensitivity of 0.57 ( 0.18 ng/Hz using quartz oscillators with a frequency of 10 MHz. Calibration of the quartz balance was performed according to Lvov et al.27 The protein film was deposited on both sides of the resonator and afterward dried by nitrogen flux; the frequency shift was registered after the covering.28-30 Circular dichroism (CD) spectra were carried out using a Jasco 710 spectropolarimeter (Japan). The spectra of proteins immobilized onto quartz slides were recorded at various temperatures. The data were collected in the range of 25-300 °C. CD spectra measurements for samples heated to 100 °C were carried out at the heating temperature using a Peltier cell (model PCT343, Jasco, Japan Spectroscopic Co., Ltd., Tokyo, Japan), while at higher temperatures the samples were heated in a muffle furnace (model ISM320, ISCO Inc., Lincoln, U.S.A.). All samples were maintained at the temperature for 10 min. The data were collected in the far-ultraviolet region (180-250 nm) with a wavelength step of 2 nm. Each spectrum was the result of an accumulation of three scans, and it was recorded at a rate of 50 nm/min with a time constant of 4 s. The electrochemical measurements were made by a potentiostat/galvanostat (EG & G PARC, model 263A) which was supplied with its own software (M270). The working cell was homemade. A standard three-electrode configuration was used, where LB films of cytochrome P450SCC were deposited on an ITO-coated glass plate which acted as a working electrode, having platinum as a counter electrode and Ag/AgCl as a reference electrode. The working electrode was cycled between initial and switch potentials of +500 and -500 mV, respectively, after holding the (25) Archakov, A. I.; Bachmanova, G. I. Cytochrome P450 and active oxygen; Taylor & Francis: London, New York, Philadelphia, 1990; pp 1-105. (26) Hara, M. Mater. Sci. Eng., C 2000, 12, 103-110. (27) Lvov, Y. M.; Erokhin, V.; Zaitsev, S. Biol. Membr. 1990, 7 (9), 917-937 (Russian). (28) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1993, 230, 86-89. (29) Antolini, F.; Paddeu, S.; Nicolini, C. Langmuir 1995, 11 (7), 2719-2725. (30) Paddeu, S.; Fanigliulo, A.; Lanzi, M.; Dubrovsky, T.; Nicolini, C. Sens. Actuators, B 1995, 25 (1-3), 876-882.

Paternolli et al.

Figure 1. Surface potential (grey line) and π-A isotherm (black line) of a monolayer of cytochrome P4501A2 at the air-water surface. The subphase was a 10 mM K-phosphate buffer, pH 7.4. electrochemical system at the initial potential for 10 s. The scan rate used was 20 mV/s because the cathodic peak was most evident at this speed and, at the same time, the low background current was minimized. All the measurements were repeated three times to verify the reproducibility. The current response of the cytochrome P4501A2 immobilized in the gel-matrix system, as a function of clozapine concentration, was measured by chronoamperometry. This analysis was performed using the spe: a three-electrode standard configuration was used, where platinum wire acted as the counter electrode and a spe served as both the reference and the working one. The screen-printed rhodium-graphite electrodes were produced using a modified protocol according to refs 31 and 32. The area of the working electrode was 2 × 10 mm2. The amperometric responses were obtained by adding aliquots of clozapine (40 µM in methanol) in a working stirred system of 10 mM K-phosphate buffer, pH 7.4 (2 mL). Equilibration time was 5 min for each measurement. The cytochrome was electrochemically reduced at the potential of -600 mV versus Ag/AgCl. Scanning electron microscopy (SEM) characterization was performed to see the actual film on the substrate. The samples were studied by using a Philips XL20 scanning electron microscope. The substrates were mounted on stubs with adhesive film and then coated with carbon by vacuum evaporation.

3. Results and Discussion 3.1. Thin Films Characterization. 3.1.1. Pressurearea Isotherm and Surface Potential. Preliminary characterization was performed with enzyme at the air-water interface. Figure 1 shows the π-A isotherm of cytochrome P4501A2 on the 10 mM K-phosphate, pH 7.4, buffer subphase: similar behavior was found for cytochrome P450SCC and P4502B4 (data not shown). The X axis is expressed in barrier coordinate units, because the axis in the area per molecule units cannot be calibrated as a result of the impossibility of calculating the actual surface concentration of the protein. This is a widespread problem for protein monolayers, and it results from some partial solubility of proteins in the volume of the subphase.28 Initially, the surface pressure remains at zero, or more precisely at about 0.1 mN/m. Then a second phase begins with an abrupt increase caused by the linear reduction in area. At this point, molecules begin to orientate on the water surface, coming to the equilibrium state selfassembly, not yet closely packed. This phase can be (31) Kulys, J.; D’Costa, E. J. Biosens. Bioelectron. 1991, 6, 109-115. (32) Bachmann, T. T.; Schmid, R. D. Anal. Chim. Acta 1999, 401, 95-103.

Recombinant Cytochrome P450 Immobilization

Figure 2. Absorbance difference of cytochrome P4501A2, P450SCC, and P4502B4 LB films as a function of the number of layers. In the inset the absorbance spectrum of cytochrome P4501A2 in solution is shown.

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Figure 4. Dependence of the CD spectrum of the cytochrome P4502B4 LB films (30 layers) upon temperature.

substrate interaction by the electrochemical method. Starting from these considerations we have studied the gel-matrix entrapped method to exploit this low purity grade protein. 3.1.3. Quartz Crystal Nanobalance. The quartz crystal nanobalance is a well-established method for measuring small variations in mass, on the basis of the relationship between changes in the mass of the material attached to the crystal and the oscillation frequency of the crystal. It is possible to correlate this relationship by utilizing the Sauerbrey equation:34,35

∆x ) -K∆f

Figure 3. Dependence of the surface density of cytochrome P4501A2, P450SCC, and P4502B4 LB films upon the number of transferred layers.

considered a two-dimensional liquid state. The steeper gradient of isotherm represents a phase-change to an ordered arrangement of molecules within a quasi-solid layer which is highly incompressible. Further reduction of area will cause the monolayer to collapse or buckle. This behavior, which is characteristic of all proteins,33 can be seen in Figure 1. The surface potential is also reported so as to emphasize that the potential increases, in absolute value, linearly as the surface pressure increases. 3.1.2. UV-Vis Measurements. Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels, and UV-vis spectroscopy is applied to detect this change within molecules of cytochrome P450s in solution and deposited onto quartz slides. We collected absorbance spectra as a function of the number of layers in the LB film structure. The optimal number of layers in the LB film takes into consideration both the amount of the biological material and the diffusion process inside the layers. The absorbance values presented in Figure 2 were calculated by the difference between the absorbance at 470 nm and that at 416 nm, according to ref 22 , to verify the linear dependence of the absorbance peak upon the layers. Figure 2 also shows the best linear interpolation that was obtained using the data of all three cytochrome isoforms. It can be seen that the correlation for cytochrome P4501A2 is not linear. This is likely due to the low purity grade of the P4501A2 sample, which causes a shift in the absorbance spectrum, as reported in detail in Figure 2. This carries out several difficulties in the P4501A2 LB formation and in the following determination of enzyme(33) Nicolini, C.; Erokhin, V.; Paddeu, S.; Paternolli, C.; Ram, M. K. Biosens. Bioelectron. 1999, 14, 427-433.

where ∆x and ∆f represent the surface density and the frequency shift, respectively, and K is a constant determined by the physical parameters of the resonator that is used. The value of this constant was estimated at 0.034 ng Hz-1 mm-2 according to the procedure reported in the literature.28 This equation was changed to obtain a direct connection between the frequency shift and the surface density, but only assuming a thin, uniform, rigidly attached mass. The linear dependence of the frequency shift upon the number of deposited layers indicates the reproducibility and homogeneity of the deposition (Figure 3). The results obtained by UV-vis spectroscopy for P4501A2 are confirmed by nanogravimetric measurements. In fact, the surface density values of this cytochrome are greater than those of P450SCC and P4502B4. This is in accordance with the presence of impurities hypothesized in the UV-vis spectroscopy study. 3.1.4. CD. The phenomenon of CD is very sensitive to the structure of proteins in solution and in thin films.13,24,29 The temperature variations of the CD spectra of the LB films of the three P450s confirms that heating has practically no effect on the secondary structure of proteins in LB films up to 150 °C (Figure 4), unlike the normal behavior of corresponding proteins in solution that already become denatured at 60-70 °C. As it is well-known, similar CD spectra were observed for all three cytochromes being here used (data not shown). Such behavior is typical of all protein LB films as determined previously.12,13,24,36 Two factors are responsible for such improved heat stability, namely, molecular close packing and low inner water content, as shown by the atomic resolution study of mesophilic proteins.37 It seems that both factors work in (34) Sauerbrey, G. Z. Z. Phisik. 1959, 155, 206-222. (35) O’Sullivan, C. K.; Guibault, G. G. Biosens. Bioelectron. 1999, 14, 663-670. (36) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 243, 403-406. (37) Bartolucci, S.; Gagliardi, A.; Pedone, E.; De Pascale, D.; Cannio, R.; Camardella, L.; Carratore, V.; Rossi, M.; Nicastro, G.; De Chiara, C.; Nicolini, C. Biochem. J. 1997, 328, 277-285.

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Paternolli et al. Table 2. Physiological and Minimum Detectable Concentrations of Clozapine and Cholesterol in the Blood and of Styrene in the Atmosphere

detected metabolite clozapine styrene

physiologiocal concentration range

50 ng/mL 500 mg/mc (4 µg P4502B4 in 40 LB) b cholesterol 100-220 mg/dL NCEPEP, 1988 11.6 mg/dL (5 µg P450SCC in 40 LB)

Figure 5. SEM image of 10 layers of the cytochrome P4502B4 LB film.

50-600 ng/mL 0-85.2 mg/mc

references

minimum detectable concentration (P450 µg in the LB per electrode)

40-42 ACGIH, 1997a

a American Conference of Governmental Industrial Hygienists, Inc., ACGIH, 1997. b Report of the National Cholesterol Education Program Expert Panel (NCEPEP) on Detection, Evaluation and Treatment of High Blood Cholesterol, 1988. Adults. Arch. Intern. Med. 1998, 148, 36-58.

Figure 7. Cyclic voltammetry of cytochrome P450SCC LB film with the addition of (1) 100, (2) 400, and (3) 750 µM cholesterol. Figure 6. Graph of the spin state equilibrium index of cytochrome P4502B4 in solution and in the LB film (30 layers). The data were acquired at different exposure times to the styrene atmosphere.

synergy, and it is difficult to say which one plays the dominant role;38 however, molecular close packing appears to play a dominant role in the thin solid film. 3.1.5. SEM Characterization. The SEM image of the cytochrome P4502B4 LB film (10 layers deposited at 25 mN/m) is presented in Figure 5. The figure shows a rather uniform surface; the domains are probably caused by the closed package that takes out the subphase. 3.2. Biodevice Applications. 3.2.1. Spectroscopic Styrene Biosensor Based on P4502B4. Moreover, to develop a sensor for styrene, the characteristic Soret peak of the cytochrome P4502B4 free and complexed with its substrate was monitored in the LB film. In fact, it is known that the interaction between cytochrome and the substrate changes the protein spin state. The results are summarized in Figure 6. The indexes of the P4502B4 spin state equilibrium were calculated from the absorbance spectra acquired at different times of exposition to the styrene atmosphere. It was found that the exposure of cytochrome P4502B4 to a styrene saturated environment determines the progressive shift to higher spin values following the P4502B4 molecules binding to their substrates in both solution and film, as it is the case going from solution to the LB film without styrene. The styrene detectable quantity found (Table 2) is limited by the homemade measurement system, in which the control of the working volume is difficult. 3.2.2. Amperometric Sensors for Cholesterol Based on P450SCC in LB Films. Protein studies utilizing electrochemical techniques such as cyclic voltammetry have (38) Nicolini, C.; Erokhin, V.; Ram, M. K. Supramolecular layer engineering for industrial nanotechnology. In Nano Surface Chemistry; Rosoff, M., Ed.; Marcel Dekker: New York, 2001; pp 4, 141-212.

Figure 8. Dependence of the cathodic peak current of the cytochrome P450SCC LB films (40 monolayers) as a function of the cholesterol concentration.

provided insight into the functional properties of redoxactive centers. Electrochemical measurements are thereby performed to study the binding between cholesterol and cytochrome P450SCC. The current values of the P450SCC LB film (40 monolayers) are plotted as a function of cholesterol concentration additions in Figures 7 and 8. Each addition consists of 50 µL of cholesterol solution (10 mM in TritonX100) until reaching a final concentration of 100-750 µM. The time of the process is estimated to be about 3 min. The data show that the electrochemical process itself gives cytochrome P450SCC electrons, allowing it to react with cholesterol. The kinetics of the absorption and reduction process might be the result of the ion-diffusion-controlled process. Moreover, the data plot suggests that the steadystate current is directly proportional to the cholesterol concentration until the beginning of the saturation trend. 3.2.3. Amperometric Sensor for Clozapine Based on P4501A2 in the Gel-Matrix. The electrochemical studies proved, however, that while the behavior of P4502B4 is comparable to that of P450SCC in LB films (data not

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sensitivity extrapolated by our experiments is sufficient for routine measurements. 4. Conclusions

Figure 9. Current response of cytochrome P4501A2 gel-matrix as a function of the clozapine concentration by rhodium-graphite spe. Working solution is 10 mM K-phosphate buffer, pH 7.4. The potential electrode was poised at -600 mV.

Figure 10. Cyclic voltammetry of cytochrome P4501A2 in solution with spe rodhium-graphite electrodes in a 10 mM K-phosphate buffer, pH 7.4, with additions of clozapine: (black line) cytochrome P4501A2, (dotted gray line) P4501A2 + 1.5 nM and (grey line) P4501A2 + 4.5 nM. Potentiostat scan rate used is 20 mV/s.

shown and Table 2) the cytochrome P4501A2 LB films cause some problems with the cyclic voltammetry, particularly in the study of the interaction between this enzyme and its substrate (clozapine), likely because of the low purity grade (as shown in Materials and Methods). In fact, it is well-known that molecular impurities on the electrodes may impede electron transfer and prevent enzyme-electrode electrical communication.39 For this reason, we immobilized P4501A2 in a gel-matrix as described in Materials and Methods, and we employed chronoamperometry to verify the possibility of producing an amperometric sensor to detect clozapine. Aliquots of clozapine (40 µM in methanol) were added to the working mixture to obtain an amperometric response curve. Figure 9 shows the resulting current as a consequence of constant potential. Figure 10 shows the cyclic voltammetry in the same experimental conditions. It can be observed that raising the clozapine concentration increased the response current, which appears stable at room temperature for over 30 days to 60% of its original value. Because the therapeutic range of clozapine in plasma is between 0.16 and 1.83 µM (50-600 ng/mL),40-42 we conclude that the (39) Joseph, S.; Rusling, J. F.; Lvov, J. M.; Friedberg, T.; Fuhr, U. Biochem. Pharmacol. 2003, 65, 1817-1826. (40) Haring, C.; Barnas, C.; Saria, A.; Humpel, C.; Fleischhacker, W. W. J. Clin. Psychopharmacol. 1989, 9, 71-72. (41) Buur-Rasmussen, B.; Brøsen, K. Eur. Neuropsychopharmacol. 1999, 9, 453-459. (42) Rahden-Staron, I.; Czeczot, H.; Szumilo, M. Mutat. Res. 2001, 498 (1), 57-66.

Recombinant cytochrome P450s consist of a large and highly diverse enzymes family that plays a pivotal role in the metabolism of a wide variety of xenobiotic compounds and drugs. P450 enzymes catalyzed the process of biotransformation by oxidizing, reducing, or hydrolyzing toxins, creating a biotransformed water-soluble substance with a minor toxicity. For these attractive properties we have performed several studies to obtain nanostructures for device applications. In particular, we have investigated the possible employment of three cytochrome isoforms (P450SCC, P4501A2, and P4502B4) immobilized onto solid supports. For each enzyme we have carried out structural and functional characterizations to optimize the immobilization process. As can be easily seen, in earlier works on cholesterol,10,14 styrene,15,16 and clozapine sensing we have used several immobilization techniques, including layer-by-layer, LB, gel-matrix, and self-assembly (solution spreading), and several forms of the given cytochromes, including the wild type, the native recombinant, and the GST-fused P4501A2, P4502B4, and P450SCC cytochromes, the latter being different from the mutant one utilized in this work. The conclusive new insights with respect to all our previously published papers are a comparative undertaking capable of identifying the optimal sensing results achieved with LB immobilization and recombinant P450 of high grade, which both prove superior to all other approaches being tested. This confirms and generalizes all P450-based sensors being tested, which has been shown recently by refs 10 and 15, namely, that the sensing can be optimized in terms of performance, stability, reusability, and efficiency whenever LB immobilization and proper mutant are utilized. The results underline that the LB technique can be used to immobilize molecules of cytochrome P450 onto solid supports in a stable and reproducible way and to employ heme-protein structures as sensitive elements in biodevice applications. Nevertheless, to detect clozapine by the electrochemical method we have found that, because we are dealing with a low purity grade cytochrome, it appears more advantageous to use cytochrome P4501A2 entrapped in the gel-matrix. The data demonstrated that utilizing both types of systems (LB film and gel-matrix) could properly detect the presence of styrene, cholesterol, and clozapine employing cytochrome P4502B4, P450SCC, and P4501A2, respectively. The detectable quantities found in this work are in agreement with the range of routine measurements such as those summarized in Table 2. The limit in the styrene detection is attributable to the homemade measurement system in which the control of the working volume is difficult. These results are encouraging for their possible future applications in the medical and ecological fields. In conclusion, because of the very attractive pleiotropic properties of the heme-enzymes, P450 cytochrome isoforms confirm their potential to be used as sensing elements for a wide range of organic substances. It does not escape our notice, however, that even if the LB/LS films appear to yield the best stable working conditions, the sensor technology may yield optimal results also with other immobilization techniques, such as solution casting, improving cholesterol sensing to the range 11.5-1.2 mg/ dL without gold nanoparticles43 and to the range 2.7-0.4 (43) Shumantseva, V.; De Luca, G.; Bulko, T.; Carrara, S.; Nicolini, C. Usanov, S. A.; Archakov, A. Biosens. Bioelectron. 2004, 19, 971-976.

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mg/dL with gold nanoparticles44 in the presence of 200 µg of P450SCC on the electrode.

technology to the Nanoworld Institute of the University of Genoa and to the Fondazione Elba.

Acknowledgment. This work was supported by a FIRB-MIUR grant on Organic Nanoscience and Nano-

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(44) Shumyantseva, V. V.; Carrara, S.; Bavastrello, V.; Bulko, T. V.; Skryabin, K. G.; Archakov, A. I.; Nicolini, C. Biosens. Bioelectron. 2004, in press. (45) Brøsen, K. Clin. Invest. 1993, 71, 1002-1009. (46) Brøsen, K.; Skjelbo, E.; Rasmussen, B. B.; Poulsen, H. E.; Loft, S. Biochem. Pharmacol. 1993, 45, 1211-1214.

(47) Pirmohamed, M.; Williams, D.; Madden, S.; Templeton, E.; Park, B. K. J. Pharmacol. Exp. Ther. 1995, 272, 984-990. (48) Miller, W. L. Endocr. Rev. 1988, 9, 295-318. (49) Vaz, A. D. N.; McGinnity, G. N.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555-3560. (50) Waterman, M. R.; Simpson, E. R. Mol. Cell. Endocrinol. 1985, 39, 81-89.