An Electrochemical Microfluidic Platform for Human P450 Drug

Nov 24, 2010 - Albertina 13, Torino, Italy, NanoBioDesign Ltd, Woodstock House, Winch Road, Kent Science Park, Sittingbourne,. Kent, ME9 8EF, United ...
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Anal. Chem. 2010, 82, 10222–10227

An Electrochemical Microfluidic Platform for Human P450 Drug Metabolism Profiling Andrea Fantuzzi,† Ennio Capria,† Lok Hang Mak,† Vikash R Dodhia,† Sheila J. Sadeghi,‡ Stephen Collins,§ Graham Somers,| Ejaz Huq,⊥ and Gianfranco Gilardi*,†,‡ Division of Molecular Biosciences, Imperial College London, Biochemistry Building, South Kensington, London, SW7 2AY, United Kingdom, Department of Human and Animal Biology, University of Torino, Via Accademia Albertina 13, Torino, Italy, NanoBioDesign Ltd, Woodstock House, Winch Road, Kent Science Park, Sittingbourne, Kent, ME9 8EF, United Kingdom, GlaxoSmithKline, PO Box 97, Stevenage SG1 2NY, United Kingdom, and Micro and Nanotechnology Centre, Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire, OX11 0QX, United Kingdom This paper is the first report of a P450-electrode in a microfluidic format. A 30 µL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of KM for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The KM values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron > nifedipine > quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested. Microfluidic technology has evolved from a fascinating concept to a rapidly growing field. The possibility of controlling and detecting chemical reactions in volumes many orders of magnitude smaller than in benchtop chemistry, combined with the ability to manipulate fluids in channels of dimensions of tens of micrometers, is an attractive perspective for the pharmaceutical industry. The need for high throughput approaches and for the use of small volumes in combinatorial screening has directed the research toward microfluidics in order to create new devices for proteomics and drug discovery.1-4 * Corresponding author. Phone: (+) 44 207 5945320. Fax: (+) 44 207 5945330. E-mail: [email protected]. † Imperial College London. ‡ University of Torino. § NanoBioDesign Ltd. | GlaxoSmithKline. ⊥ Rutherford Appleton Laboratory. (1) deMello, A. J. Nature 2006, 442, 394–402. (2) Whitesides, G. M. Nature 2006, 442, 368–373. (3) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M. R.; Weigl, B. H. Nature 2006, 442, 412–418.

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Few electrochemical techniques have been adopted in microfluidic devices aiming at different applications, ranging from single cell measurements to environmental pollutants and pathogen detection. Detection of analytes, either proteins or small organic molecules, has been tackled with both label and label-free methods integrated in microfluidic devices.5,8-11 Nevertheless, very rarely do these devices make use of immobilized redox enzymes such as label-free third generation sensors, making this approach still an interesting challenge. The great interest in cytochromes P450 very much reflects its importance in the elimination of drugs and other chemicals from the body and its role in chemical toxicity associated with drug-drug interaction. In particular, within the ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) framework the metabolic profiling of new chemical entities with respect to turnover by cytochromes P450 is fundamental in the drug discovery process12 and a prerogative for FDA approval. For this reason, the industry has been taking advantage of the progress made in the development of microfluidic devices with their ability to miniaturize assays and increase experimental throughput13 by using whole cell assays in microfluidic devices with fluorescent detection of the products14 and with purified enzymes immobilized onto agarose beads to create a PDMS microreactor.15 (4) Bange, A.; Halsall, H. B.; Heineman, W. R. Biosens. Bioelectron. 2005, 20, 2488–2503. (5) Sadik, O. A.; Aluoch, A. O.; Zhou, A. Biosens. Bioelectron. 2009, 24, 2749– 2765. (6) Sassa, F.; Morimoto, K.; Satoh, W.; Suzuki, H. Electrophoresis 2008, 29, 1787–1800. (7) Palchetti, I.; Mascini, M. Anal. Bioanal. Chem. 2008, 391, 455–471. (8) Wei, D.; Bailey, M. J. A.; Andrew, P.; Ryhanen, T. Lab Chip 2009, 9, 2123– 2131. (9) Pumera, M.; Merkoci, A.; Alegret, S. Trends Anal. Chem. 2005, 25, 219– 235. (10) Herva´s, M.; Lo´pez, M. A.; Escarpa, A. Analyst 2009, 134, 2405–2411. (11) Kovachev, N.; Canals, A.; Escarpa, A. Anal. Chem. 2010, 82, 2925–2931. (12) Ortiz de Montellano, P. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.; Kluwer Academics/Plenum Publishers: New York, 2005. (13) Kang, L.; Geun Chung, B.; Langer, R.; Knademhosseini, A. Drug Discovery Today 2008, 13, 1–13. (14) Hwan Sung, J.; Choi, J.; Kim, D.; Shuler, M. L. Biotechnol. Bioeng. 2009, 104, 516–525. (15) Srinivasan, A.; Bach, H.; Sherman, D. H.; Dordick, J. S. Biotechnol. Bioeng. 2004, 88, 528–535. 10.1021/ac102480k  2010 American Chemical Society Published on Web 11/24/2010

As the reaction catalyzed by cytochromes P450 involves the use of two reducing equivalents from NADPH, the requirement of electrons is the basis for the immobilization of these enzymes on amperometric devices for the study of their interaction with new potential drugs.16,17 Several reports can be found in the most recent literature on the electrochemistry of cytochromes P450 and in some cases their potential exploitation for the characterization of new chemical entities.16-27 Our laboratory has previously reported the successful covalent immobilization of these enzymes on gold electrodes21,28,29 by using self-assembled monolayers to achieve controlled orientation.30 This paper reports for the first time the combination of a P450-based electrode with a microfluidic platform for enzymatic profiling of new potential drugs. The added advantages of using a microfluidic system integrated with a P450 amperometric sensor are immediately evident: the decrease in the volume of precious materials (such as newly synthesized chemical entities), the increase in the throughput, and the possibility of making either the electrodes or the whole chip disposable. Here we present the results obtained using the most important drug metabolizing enzyme, the human liver cytochrome P450 3A4, fused to the electron transfer module flavodoxin (FLD) in the P450 3A4/FLD construct, that we previously showed to have improved electrochemical response.28 Although FLD is not the physiological cytochrome P450 reductase present in the human liver, in our previous work we have shown that P450 3A4/FLD behaves catalytically like the wild type protein, possessing the same catalytic parameters measured for the natural enzyme. Here we report the Michaelis-Menten constants (KM) electrochemically measured using the microfluidic cell with four known substrates of P450 3A4, quinidine, nifedipine, alosetron, and ondansetron. MATERIALS AND METHODS Materials. All chemicals were purchased from Sigma-Aldrich (Poole, UK). Alosetron and Ondansetron were kindly provided by GSK (Stevenage, UK). (16) Dodhia, V. R.; Gilardi, G. Cytochromes P450: Tailoring a class of enzymes for biosensing. In Engineering the bioelectronic interface; Davis, J., Ed.; RSC Publishing: London, UK, 2009; pp 154-193. (17) Sadeghi, S. J.; Fantuzzi, A.; Gilardi; G. Biochim. Biophys. Acta 2010, DOI: 10.1016/j.bbapap.2010.07.010, in press. (18) Bistolas, N.; Wollenberger, U.; Jung, C.; Scheller, F. W. Biosens. Bioelectron. 2005, 20, 24082423. (19) Fleming, B. D.; Johnson, D. L.; Bond, A. M.; Martin, L. L. Expert Opin. Drug Metab. Toxicol. 2006, 2, 581–589. (20) Joseph, S.; Rusling, J. F.; Lvov, Y. M.; Friedberg, T.; Fuhr, U. Biochem. Pharmacol. 2003, 65, 1817–1826. (21) Fantuzzi, A.; Fairhead, M.; Gilardi, G. J. Am. Chem. Soc. 2004, 126, 5040– 5041. (22) Shumyantseva, V. V.; Ivanov, Y. D.; Bistolas, N.; Scheller, F. W.; Archakov, A. I.; Wollenberger, U. Anal. Chem. 2004, 76, 6046–6052. (23) Johnson, D. L.; Lewis, B. C.; Elliot, D. J.; Miners, J. O.; Martin, L. L. Biochem. Pharmacol. 2005, 69, 1533–1541. (24) Peng, L.; Yang, X.; Zhang, Q.; Liu, S. Electroanalysis 2008, 20, 803–807. (25) Liu, S.; Peng, L.; Yang, X.; Wu, Y.; He, L. Anal. Biochem. 2008, 375, 209– 216. (26) Yang, M. L.; Kabulski, J. L.; Wollenberg, L.; Chen, X. Q.; Subramanian, M.; Tracy, T. S.; Lederman, D.; Gannett, P. M.; Wu, N. Q. Drug Metab. Dispos. 2009, 37, 892–899. (27) Mie, Y.; Suzuki, M.; Komatsu, Y. J. Am. Chem. Soc. 2009, 131, 6646–6647. (28) Dodhia, V. R.; Sassone, C.; Fantuzzi, A.; Di Nardo, G.; Sadeghi, S. J.; Gilardi, G. Electrochem. Commun. 2008, 10, 1744–1747. (29) Ferrero, V. E. V.; Andolfi, L.; Di Nardo, G.; Sadeghi, S. J.; Fantuzzi, A.; Cannistraro, S.; Gilardi, G. Anal. Chem. 2008, 80, 8438–8446.

Expression and Purification of 3A4-FLD. The N-terminally modified human CYP3A4 was fused at the genetic level to flavodoxin from Desulfovibrio vulgaris (FLD) to create the CYP3A4/FLD as described in Dodhia et al. 2008.28 Expression and purification of the protein was carried out as described previously.31 Protein concentration and activity were measured at 450 nm using the method of Omura and Sato32 using the difference spectra obtained subtracting the spectrum of the reduced protein from its carbon monoxide bound and reduced form. An ε450 of 91 mM-1 cm-1 was used. Electrode Preparation. Gold electrodes were provided by Rutherford Appleton Laboratories (RAL, UK). A layer of approximately 200 nm of silicon oxide was thermally grown on a clean silicon wafer. Subsequently, a layer of 50 nm of chromium was thermally deposited to improve the adhesion of the gold to the silicon oxide. Gold was deposited by evaporation onto the chromium layer, maintaining the chamber at a constant temperature of 300 °C. The thickness of the gold layer was monitored and kept at approximately 250 nm. A layer of photoresist was spin coated onto the gold, and the electrode features were imprinted with an optical mask. The excess material was removed by chemical etching and the wafer covered with a layer of silicon oxide. Upon scoring the surface of the wafer in order to cut out individual electrodes, the silicon dioxide was removed and the electrode prepared for use. All the operations for the electrode production were carried out in clean room environment. Prior to modification, the gold electrodes were cleaned with ethanol and acetone and then by oxygen plasma using an EMITECH K1050X (UK) instrument for 30 min at 90 W and 25 gauge gas flow. Screen printed counter and reference electrodes were purchased from Conductive Technologies Inc. (York, PA). All inks used were from DuPont (DuPont Research Triangle Park, NC). Electrode Modification. Following the plasma cleaning treatment, the electrodes were left immersed in ethanol for 20 min in order to remove the gold oxide generated by the plasma. The electrode surface was then rinsed with ethanol and immersed in a 1 mM ethanolic solution of 6-hexanethiol and the 7-mercaptoheptanoic acid (1:1). At the end of the incubation, the electrode was rinsed with ethanol, Milli-Q water, and 0.1 M MES buffer (2-(N-morpholino)ethanesulfonic acid). The electrodes were then immersed for 15 min in a solution containing 10 mM 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and 25 mM Nhydroxysuccinimide (NHS) dissolved in 0.1 M MES buffer pH 6.4, followed by a washing step in Milli-Q water. A 30 µL droplet of a 5 mM protein solution was then deposited on to the electrodes and incubated at 4 °C for 1 h in a high humidity compartment to limit evaporation of the droplet. FT-IR Characterization. The FT-IR measurements were carried out in a Bruker Tensor 27 FT-IR spectrometer. Spectra were collected using p-polarized light incident at grazing angle (75° from the surface normal) by using a grazing angle accessory (Harrick, Pleasantville, NY). The resolution was 4 cm-1 with 400 scans collected to improve the signal-to-noise ratio. All spectra (30) Mak, L. H.; Sadeghi, S. J.; Fantuzzi, A.; Gilardi, G. Anal. Chem. 2010, 82, 5357–5362. (31) Dodhia, V. R.; Fantuzzi, A.; Gilardi, G. J. Biol. Inorg. Chem. 2006, 11, 903– 916. (32) Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2370–2385.

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Figure 1. (A) Exploded view of the microfluidic cell made of micromachined poly(methyl methacrylate) and (B) of the evaporated gold on silicon working electrode (1) and screen printed carbon and silver/silver chloride inks on poly(ethylene terephthalate) counter/reference (2) electrodes.

are reported as absorbance. Microfluidic Platform. The microfluidic cell computer-aided design (CAD) was sent to AC Precision Engineering Ltd. (UK) for fabrication. O-rings were purchased from Precision Associates Inc. (Minneapolis, MN), while nanoports and PEEK tubing were purchased from Upchurch Scientific (Oak Harbor, WA). Silicone elastomer and curing agent were purchased from Dow Corning (Barry, UK) to build the poly(dimethyl sulfoxide) (PDMS) fluidic chamber. Mold design and construction were carried out at Rutherford Appleton Laboratories (RAL, UK). The mixture of the silicone elastomer with the curing agent was gently poured into the mold making sure no bubbles were formed and left to cure at 150 °C for 10 min. Syringe pump and injection system was provided by a CTC Analytics autosampler equipped with a 50 µL syringe. Washing and injection steps are fully controlled by the software and synchronized with the electrochemical measurements. Electrochemical Measurements. Electrochemical measurements were carried out at 25 °C in 50 mM potassium phosphate pH 7.4 containing 500 mM potassium chloride (working buffer). Drugs were dissolved in the appropriate solvent: methanol for quinidine, and DMSO for alosetron, ondansetron, and nifedipine. Stock solutions of 2 mM of the drugs were used to generate dilutions into the aforementioned buffer and placed in the autosampler tray. Electrochemical measurements were performed using a Uniscan PG580 potentiostat (Uniscan, UK). All potentials are reported versus the saturated calomel electrode (SCE). Data fittings were carried out using Sigmaplot (Systat Inc., Chicago, IL). The experiments were run in stopped flow mode with cycles of injection, incubation, and measurement. Cyclic voltammograms (CV) in the potential range +0.1 mV to -0.65 mV using a scan rate of 50 mV/s were recorded upon each 50 µL injection of the drug aliquots. A 1 min delay was allowed prior to CV measurement to permit the binding equilibrium to be reached. 10224

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RESULTS AND DISCUSSION Design and Construction of the Microfluidic Cell. Several methods and materials have been adopted to confine cytochromes P450 on an electrode surface with the aim of detecting a current as a function of increasing concentration of analytes.16-27 In all the successful applications, a three-electrode configuration consisting of a working, counter, and reference electrodes has been used. This has been ascribed to the necessity of maintaining a precise control over the applied potential in order to limit the adverse effect of nonspecific reactions, mainly associated with oxygen at the electrode surface.17 Bearing in mind these prerequisites, two microfluidic cell configurations were designed and constructed to be able to accommodate the three electrodes in the microfluidic chamber. The two initial configurations differed from one another by having the electrodes either on the same plane or on two parallel planes. The advantage of proximity of the electrodes in the coplanar configuration was found to be offset by the difficulties associated with the construction as well as the fouling of the working electrode during preparation (see Supporting Information). Therefore, the configuration with the electrodes on parallel planes, with the working electrode at the bottom and the reference and counter together at the top, facing each other defining a 25 µL microfluidic chamber, was the final preferred design (Figure 1A). The microfluidic cell was constructed as three parts in micromachined poly(methyl methacrylate) (PMMA) (Figure 1A). The sandwich structure consists of two plates with grooves positioning the electrodes on the microfluidic chamber and providing the tightening points for the clamping system. The central section was initially built in poly(dimethyl sulfoxide) (PDMS) because of its biocompatibility and ease of use in the early stages of the development of new prototype.33 Nevertheless, (33) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974–4984.

because of issues with the leakage (see Supporting Information) of the injected liquid, it was substituted with micromachined PMMA, and the liquid containment was guaranteed by two O-rings with a section of 0.01 in. (from Precision Associates Inc.). The inlet and outlet channels were connected via two nanoports and PEEK tubing to the injection port and waste, respectively. The electrode materials are crucial for the performance of the amperometric sensor and were therefore chosen with care. The working electrode was constructed by evaporating gold onto silicon as described in Material and Methods (Figure 1B). The choice of gold stems from the successful immobilization of several human P450, and P450 3A4/FLD in particular, on SAM-modified gold surfaces,28 while the evaporation on silicon allowed controlling the roughness and cleanliness of the metal surface. The reference and counter electrodes were built by screen printing methodologies using a poly(ethylene terephthalate) (PET) support. Inks of carbon and silver/silver chloride were used for the counter and reference electrodes, respectively (Figure 1B). The stability of the reference electrode was tested, and it was shown to be very good over more than 48 h of continuous measurements in a buffer containing 500 mM KCl. Liquid delivery was carried out using a CTC Analytics autosampler where the injector works as syringe pump. This has the added advantage, with respect to the use of on-chip micropumps, of simplifying the cell design making the electrodes and potentially the whole cell disposable. Furthermore, autosamplers are equipment often available in research and development laboratories, making the integration of the microfluidic platform in the existing robotic systems quite straightforward and helping to solve the well-known issue of world-to-chip interfacing.34 Characterization of the Microfluidic Cell. The microfluidic chamber is a cylinder with an approximate geometrical volume of 25 µL with an inlet an outlet channel coaxially positioned in the walls perpendicular to those defined by the electrodes. Since the precise volume of the chamber depends on the compression level of the O-rings, a series of experiments were carried out with injections of a solution of a well-known and highly electrochemically active probe such as potassium ferricyanide (K3[Fe(CN)6]), using nonderivatized electrodes. A 10 mM solution of potassium ferricyanide in 0.1 M KCl gives a fast and reversible electron transfer on a clean gold surface and is therefore ideal to assess the fluid delivery to the cell. Sequential additions of 10 µL of the potassium ferricyanide were performed, and the cyclic voltammograms (CV) were recorded from +0.65 to -0.2 V. As expected, the CV showed the characteristics of a reversible diffusion controlled process, and the peak current reached a maximal plateau value when 30 µL was injected (Figure 2). From this value, consistent with the geometrical dimensions of the cell, we defined that additions of 50 µL during the titrations, run in stopped flow mode, would largely guarantee a homogeneous distribution of the analyte into the microfluidic electrochemical cell with the concentration set in the standard solution injected. FT-IR Characterization of the P450 3A4/FLD Immobilized on Gold Electrodes. The formation of the self-assembled monolayer (SAM) and of the protein layer on the electrode surface was characterized via grazing angle FT-IR. (34) Crevillen, A. G.; Hervas, M.; Lopez, M. A.; Gonzales, M. C.; Escarpa, A. Talanta 2007, 74, 342–357.

Figure 2. Determination of the optimal injection volume for the microfluidic cell. The current difference (∆Current) was calculated by subtraction of the current measured when only buffer was injected into the microfluidic cell from the one measured when potassium ferricyanide was present. Steps of 10 µL injections of 10 mM K3[Fe(CN)6] in 0.1 M KCl were performed.

Figure 3. FT-IR/GATR spectra of 1 mM hexanethiol and 1 mM carboxyheptanethiol on evaporated gold electrodes before (A) and after (B) immobilization of P450 3A4/FLD. The arrows at 1649 and 1531 cm-1 highlight the amide frequencies of the immobilized enzyme.

The spectrum of a sample modified with only the mixed SAM showed the same features identified in the transmittance spectra of the isotropic samples 6-hexanethiol and the 7-mercaptoheptanoic (Figure 3). The C-H stretches, in asymmetric and symmetric modes, are present at 2910 ± 4 cm-1 and 2842 ± 3 cm-1, respectively. The presence of the carboxylic group was confirmed by the occurrence of the CdO stretch at 1728 ± 1 cm-1 and by the asymmetric and symmetric COO- stretches at 1651 ± 3 cm-1 and 1536 ± 5 cm-1, respectively. Moreover, the presence of a broad peak centered at 1449 ± 5 cm-1 can be associated to a combination of a methylene scissoring and of a C-OH in plane bending, while a peak at 1232 ± 10 cm-1 is associated with the C-O stretch. When the SAM was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) followed by covalent linkage of the P450 3A4/FLD, new absorption bands became visible at 1649 ± 1 cm-1 and 1531 ± 4 cm-1. These bands are associated with the absorption of the amide group of the protein and confirm the presence of the enzyme on the surface of the electrode.30 Analytical Chemistry, Vol. 82, No. 24, December 15, 2010

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Figure 4. Cyclic voltammetry of a solution of 10 mM K3[Fe(CN)6] in 0.1 M KCl on bare gold (thin line) and on 1 mM hexanethiol and 1 mM carboxyheptanethiol modified gold (thick line). Scan rate was 50 mV/s.

Insulation of the Self-Assembled Monolayer. As the P450 catalytic reaction is driven by the application of a potential bias of -550 mV, some of the drugs tested, having electroactive properties, could be directly reduced at the gold surface. As this platform is intended to be used with purified samples of the drug to be tested in the drug discovery process, we identified this as the only potential source of interference. Nevertheless, in order to minimize this possible interference, the SAM was deemed to function not only as the anchoring point for the protein but also as a barrier limiting the diffusion of molecules to the gold surface. For this reason, a long chain alkenthiol was used to improve the insulation properties of the SAM, as the strong van der Waals interactions between the long aliphatic chains will make a compact and uniform layer. Figure 4 reports the CVs obtained at 50 mV/s scan rate for a solution of 10 mM potassium ferricyanide in 1 mM KCl on a bare gold electrode compared to the CV signal of the same solution when the long chain alkenthiol is used as a SAM. The dramatic disappearance of the electrochemical signal of the freely diffusible probe in the electrode modified with the SAM confirms the successful linkage of the spacer with the desired insulating properties. Performance of the P450 3A4/FLD Microfluidic Cell. We previously demonstrated that the immobilized CYP3A4/FLD

responds to the presence of substrate in a Michaelis-Menten fashion, with catalytic currents increasing as a function of the concentration of the substrate with a hyperbolic behavior.28 Because the calculated kinetic parameters were found to be in keeping with the values reported in the literature, we suggested that the P450-modified electrode can be used for the metabolic profiling of new chemical entities in the drug discovery process. Following the same approach, here we investigated the behavior of the P450-modified electrode in the microfluidic cell, measuring the catalytic current as a function of increasing concentrations of specific substrates. On the basis of the knowledge of the literature data on their metabolism either in vivo or in vitro by P450 3A4 and their commercial availability, quinidine, nifedipine, alosetron, and ondansetron were chosen for testing the performance of P450 in the microfluidic cell. The working buffer was initially flushed into the cell with repeated injections. Upon each injection a cyclic voltammogram (CV) was recorded between +0.1 mV and -0.65 mV. This initial equilibration procedure allows the electrochemical signal to reach stability prior to titration with the substrate. Such equilibration minimizes errors due to the drift of the signal. Once the electrode reached stability, fixed volumes of buffer with increasing concentrations of drug were injected into the microfluidic cell. The volume of the injection was set to 50 µL. Upon each addition, an incubation time of 1 min was allowed to permit the binding equilibrium to be reached prior to the electrochemical measurement. A CV was recorded after each step. In order to eliminate any contribution due to nonspecific redox reactions occurring during the titration, control CVs were measured in the absence of substrate, corresponding to the last CV collected during the equilibration scan. Titrations of the selected drugs were performed as detailed in Materials and Methods. The necessity of a series of potential scans prior to the titration was found to be crucial to increase the signalto-noise ratio by stabilizing the background signal. The normalized CVs (Figure 5A) show that, upon each addition of substrate, the cathodic current increases significantly as expected from the consumption of electrons in the P450 catalytic cycle. The values of the current at -550 mV (i.e., maximum of the reductive wave) were extrapolated from the normalized CVs and plotted in function

Figure 5. (A) Cyclic voltammetry of P450 3A4-FLD immobilized on 1 mM hexanethiol and 1 mM carboxyheptanethiol modified gold upon titration with increasing concentrations of quinidine. (B) Michaelis-Menten plot based on the current measured at -550 mV upon addition of aliquots with increasing concentrations of quinidine (filled squares) and, as a control, aliquots of buffer without substrate (filled circles). 10226

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Table 1. Michaelis-Menten Constants (KM) for P450 3A4/FLD with Quinidine, Nifedipine, Alosetron, and Ondansetron Determined by Using the Microfluidic Platforma drug

KM, microfluidics (µM)

KM, literature (µM) [reference]

quinidine nifedipine alosetron ondansetron

2.9 ± 1.3 29.1 ± 8.3 113.4 ± 51.8 114.1 ± 36.9

1.3 ± 0.3 [ref 35] 7.9 ± 2.2 [ref 36] 88.0 ± 109 [ref 37] 87 ± 107 [ref 37]

a The KM values are compared with literature data obtained from conventional incubations with HLM and baculosomes. Despite the large errors (particularly for the available published data) the ranking of the KM values follows the order [ondansetron ≈ alosetron > nifedipine > quinidine].

of the concentration. The data points were fitted to the MichaelisMenten equation, and the kinetic parameters were determined (Figure 5B). The calculated KM values for each substrate are shown in Table 1. These drugs are all well studied substrates of P450 3A4 and their KM values have been determined in vitro using different sources of enzyme, ranging from human liver microsomes to baculosome systems, and are reported in the literature. The source of the P450 3A4 is an intrinsic problem associated with the comparison of kinetic data deriving from purified proteins, microsomes, and hepatocytes. This often yields to a spread of results. Recombinant P450 3A4 was used for quinidine35 and nifedipine36 while a comparison of results obtained with microsomes and hepatocytes for ondansetron and alosetron showed different values in the measured KM.37 Table 1 shows that there is good agreement between the values obtained electrochemically in the microfluidic cell and the published ones, with only a small deviation observed for nifedipine that is know to be affected by poor solubility issues. The key point to note is that the ranking of the KM values from low and high affinity of the substrates is the same in the two sets of data, and it corresponds to [ondansetron ≈ alosetron > nifedipine > quinidine]. Furthermore, Figure 5B shows the plot of a control experiment performed by injecting equivalent aliquots of the solvent used to dissolve the drugs. In this case, the current did not show a catalytic behavior, and the values of current at -550 mV remained substantially unchanged during the titration. Unfortunately, the (35) Ngui, J. S.; Tang, W.; Stearns, R. A.; Shou, M.; Miller, R. R.; Zhang, Y.; Lin, J. H.; Baillie, T. A. Drug Metab. Dispos. 2000, 28, 1043–1050. (36) Niwa, T.; Shiraga, T.; Yamasaki, S.; Ishibashi, K.; Ohno, Y.; Kagayama, A. Xenobiotica 2003, 33, 717–729. (37) Somers, G. I.; Bayliss, M. K.; Houston, J. B. Xenobiotica 2007, 37, 832– 854.

uncoupled nature of human cytochromes P450 prevented the calculation of accurate kcat values. CONCLUSIONS The microfluidic integrated system presented in this paper maintains the analytical properties of the previously reported P450modified electrode with the added advantage of decreasing the required volume of drug to 30 µL, with the attractive possibility of further miniaturization. The results of the titrations with the specific substrates clearly show how the electrochemical determination of the KM in the microfluidic platform correctly ranks the affinity for the different drugs, opening the possibility for using such a device in the early stages of the drug discovery process. Furthermore, the use of an autosampler as the pumping system performs the double role of increasing the repeatability and controlling the flow with the added potential of ease of integration in the robotic high throughput system currently available in the pharmaceutical industry. In addition, the recombinant nature of the immobilized P450, combined with the standardization of the methodologies and the materials used in the construction of the microfluidic cell, decreases the scattering currently observed in the kinetic values determined for P450 substrates as well as making the sensor potentially disposable. The platform presented in this paper is not a biosensor intended to detect an analyte in a complex mixture but a device for the identification of specific metabolic parameters (KM) of pure samples of new potential drugs in controlled experimental conditions. Taking advantage of the integrating capability of the microfluidic chip with other devices, the amperometric detection could be integrated with the product identification by HPLC-MS, greatly increasing the information gathered on a potential new drug. Finally, this work opens the way for the immobilization of other cytochrome P450s and the construction of a microfluidic platform where multiple cells containing electrodes with various P450s are connected in series, allowing the complete metabolic profiling of new drugs. ACKNOWLEDGMENT This research was supported by the Micro & Nanotechnology program of the Technology and Strategy Board, project MNT142 and NanoBioDesign Ltd. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 19, 2010. Accepted November 4, 2010. AC102480K

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