P450-Mediated Electrochemical Sensing of Drugs in Human Plasma for Personalized Therapy A. Cavallini, S. Carrara, G. De Micheli Integrated systems Laboratory EPFL Lausanne, Switzerland andrea.cavallini@epfl.ch V. Erokhin CRS SOFT CNR-INFM and Department of Physics - University of Parma, Italy Victor.Erokhin@fis.unipr.it Abstract— Nowadays, the concept of personalized therapy gains momentum. Pharmacogenomics, which represents a first answer to these needs, has the drawback of neglecting some variations of therapy response due to non-genetic factors. The aim of this paper is to investigate the feasibility of a non-genetic approach to personalized therapy, via the point-of-care drug monitoring in biological fluids with electrochemical biosensors. The proposed biosensor is based on the use of P450 enzymes as probe molecules, thanks to their key role in human metabolism. Multiwalled carbon nanotubes are used to enhance biosensor sensitivity. Results show how the proposed system is capable to detect drug amounts within the corresponding pharmacological ranges in human serum. Keywords – P450, personalized therapy, electrochemistry, human serum. I. INTRODUCTION At present, the most common clinical practice is the so called trial and error medicine [1]: according to the symptoms, a drug therapy is prescribed, and if it is not effective, or has significant side effects, it is modified. This approach, which is time consuming and expensive for both the patient and the health care system, is beneficial only to 20-50% of individuals while 7% experience severe adverse drug reactions [2]. Breakthrough targeted therapies could save many lives and a great deal of money. Pharmacogenomics, which considers the unique patient’s genetic polymorphism, represents a first step towards personalized medicine. However, lifestyle and drug interactions, which cannot be predicted with genetics, also play an important role in influencing the drug response. A system based on the direct drug monitoring in the patient’s blood represents a more accurate alternative to the pharmacogenomics. P450 proteins represent good candidates for the realization of biosensors for drug detection, since they account for the 75% of body metabolism [3], and often mediate the rate-limiting steps in the biotransformation of xenobiotics [4]. Biosensors based on P450s have already been proposed [4- 6]; however, they were always tested in artificial buffers. In this work we propose a biosensor based on carbon nanotubes (CNT) and two different cytochrome P450 isoforms, 3A4 and 2C9. These protein isoforms can detect drugs in undiluted human serum. The integration of carbon nanotubes greatly improves the sensor performance [7-9], while CYP 3A4 and 2C9 are chosen because they represent the two most important isoforms involved in drug metabolism [4, 10]. This sensor is an alternative as compared to traditional drug essay techniques, such as spectrophotometry and chromatography, which usually require sample dilution. II. MATERIAL AND METHODS A. Reagents Carbon paste screen-printed electrodes (model DRP-110 and DRP-110 CNT) were purchased from Dropsens. Cytochrome P450 3A4 and 2C9 microsomes were purchased from Sigma-Aldrich and used without further purification. Cyclophosphamide (CP) was purchased from Sigma and diluted in PBS 100mM pH 7.4 to the working concentrations. Naproxen (NAP) was purchased from sigma and diluted in ethanol to the working concentrations. Multi walled carbon nanotubes (MWCNT - diameter 10 nm, length 1-2 μm, COOH content 5 %) were bought in powder (95% purity) from DropSens (Spain), diluted in chloroform to the concentration of 1 mg/ml [4] and then sonicated for 20 minutes in order to break macro-aggregates. Human serum was purchased from Lonza and used without any dilution. B. Electrodes preparation The electrodes were made of a graphite working electrode (area, 12.56 mm 2 ), a graphite counter electrode and an Ag/AgCl reference electrode. The working electrode area was 12.56 mm 2 while the total area of the cell was 22 mm 2 . CNT nano-structuring was obtained by gradually dropping 30 μl of CNT solution onto the working electrode and waiting until complete evaporation of the chloroform. Electrode functionalization was obtained by drop cast of P450 solutions onto the working electrode and incubation at 4ºC overnight. The excess of cytochrome was then removed by washing with milliQ water. C. SEM measurement Morphological analysis of the structured and functionalized electrodes was carried out with Scanning Electron Microscopy (SEM). A Zeiss SUPRA 40 SEM instrument was used to acquire images for bare electrodes, electrodes structured with carbon nanotubes, and electrodes functionalized with the cytochrome P450. Images were acquired in 5-20 kV range. The research was financially supported by the EPFL - Integrated Center SI, and by the SNF Sinergia Project CRSII2_127547 / 1