pH Sensing with Temperature Compensation in a Molecular Biosensor for Drugs Detection Daniela De Venuto 1 , Sandro Carrara 2 , Andrea Cavallini 2 , Giovanni De Micheli 2 1 Dipartimento di Elettrotecnica ed Elettronica, Politecnico di Bari (IT) 2 EPFL - École Polytechnique Fédérale de Lausanne (CH) Abstract—This paper describes an electrochemical biosensor for molecules for personalized medicine including pH and temperature shift monitoring system. Electrochemical sensors based on the cytochromes P450 detect the large majority of drugs commonly used in pharmacological treatments. The same cytochrome detects different drugs at different electrochemical interface potentials. Therefore, the potential encodes the drug type meanwhile current encodes drug concentration. However, potential and current depend on pH variations that might occur in the patient sample. This paper presents evidence of these variations and proposes a novel design for multiplexing the bio- sensing with a new pH and temperature control system. I. INTRODUCTION The cytochromes P450 are the major enzymes involved in drug detoxification, and account for 75% of the total metabolism [1]. This feature makes them ideal candidates for the realization of biosensors for drug monitoring in personalized therapy, because cytochromes are suitable to metabolize a wide range of substrates [2]. Recently, several works have shown the possibility to detect endogenous compounds [3], single drugs [4] or drug mixtures [5] using different P450 isoforms. P450-mediated detection is mainly based on the recognition of current peaks which originate from cyclic voltammetry. In presence of a substrate, the P450 peak is subjected to a potential shift which varies according to the used drug [6]. This feature, called drug electrochemical signature, makes it possible to distinguish different compounds in the presence of a solution with multiple P450 substrates [5]. According to the electrochemical theory [7, 8], pH and temperature variations change the peak position and if not properly considered, can twist the results obtained with the peak analysis leading to the detection of false-positives in case of a solution with unknown substrates. Biological systems are very sensitive to pH and temperature levels: outside the acceptable ranges, proteins are denatured, enzymes lose their ability to function and cells apoptosis may occur. So, pH and temperature dependence of biological molecules became extremely important when working with biosensors, since a variation of these parameters can influence the output response [9, 10]. In this work we are showing a prototype of biosensor for drug detection based on P450 3A4, which supports pH and temperature measurement. The proposed system and the related circuits can represent a valid candidate for the realization of devices for multiple drugs monitoring in personalized therapy: P450 3A4 is involved in the metabolism of 36% of all known drugs [11], opening the possibility to use the sensor for a wide range of compounds. pH and temperature monitoring ensure strict and precise control of the electrochemical detection when multiple drugs are present in the sample and extend the applicability of the sensor to different biological fluids with different pH and temperature levels, like blood, urine and interstitial liquid. II. MOLECULAR BIOSENSING Reagents Carbon paste screen-printed electrodes (model DRP-110) were purchased from Dropsens. Cytochrome P450 3A4 microsomes were purchased from Sigma-Aldrich and used without further purification. Multi walled carbon nanotubes (MWCNT - diameter 10 nm, length 1-2 m, COOH content 5 %) were purchased 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. P450 biosensor The biosensor was assembled onto a screen printed electrode composed of a graphite working electrode, a graphite counter electrode and an Ag/AgCl reference electrode. The working electrode area was 12.56mm 2 while the total area of the cell was 22mm 2 . CNT nano-structuring was obtained gradually dropping 30l 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. 