Sensors & Transducers - Infoscience - EPFL

Sensors & Transducers Volume 125, Issue 2, February 2011

www.sensorsportal.com

ISSN 1726-5479

Editors-in-Chief: professor Sergey Y. Yurish, tel.: +34 696067716, fax: +34 93 4011989, e-mail: [email protected] Editors for Western Europe Meijer, Gerard C.M., Delft University of Technology, The Netherlands Ferrari, Vittorio, Universitá di Brescia, Italy Editor South America Costa-Felix, Rodrigo, Inmetro, Brazil

Editors for North America Datskos, Panos G., Oak Ridge National Laboratory, USA Fabien, J. Josse, Marquette University, USA Katz, Evgeny, Clarkson University, USA Editor for Asia Ohyama, Shinji, Tokyo Institute of Technology, Japan

Editor for Eastern Europe Sachenko, Anatoly, Ternopil State Economic University, Ukraine

Editor for Asia-Pacific Mukhopadhyay, Subhas, Massey University, New Zealand

Editorial Advisory Board Abdul Rahim, Ruzairi, Universiti Teknologi, Malaysia Ahmad, Mohd Noor, Nothern University of Engineering, Malaysia Annamalai, Karthigeyan, National Institute of Advanced Industrial Science and Technology, Japan Arcega, Francisco, University of Zaragoza, Spain Arguel, Philippe, CNRS, France Ahn, Jae-Pyoung, Korea Institute of Science and Technology, Korea Arndt, Michael, Robert Bosch GmbH, Germany Ascoli, Giorgio, George Mason University, USA Atalay, Selcuk, Inonu University, Turkey Atghiaee, Ahmad, University of Tehran, Iran Augutis, Vygantas, Kaunas University of Technology, Lithuania Avachit, Patil Lalchand, North Maharashtra University, India Ayesh, Aladdin, De Montfort University, UK Azamimi, Azian binti Abdullah, Universiti Malaysia Perlis, Malaysia Bahreyni, Behraad, University of Manitoba, Canada Baliga, Shankar, B., General Monitors Transnational, USA Baoxian, Ye, Zhengzhou University, China Barford, Lee, Agilent Laboratories, USA Barlingay, Ravindra, RF Arrays Systems, India Basu, Sukumar, Jadavpur University, India Beck, Stephen, University of Sheffield, UK Ben Bouzid, Sihem, Institut National de Recherche Scientifique, Tunisia Benachaiba, Chellali, Universitaire de Bechar, Algeria Binnie, T. David, Napier University, UK Bischoff, Gerlinde, Inst. Analytical Chemistry, Germany Bodas, Dhananjay, IMTEK, Germany Borges Carval, Nuno, Universidade de Aveiro, Portugal Bousbia-Salah, Mounir, University of Annaba, Algeria Bouvet, Marcel, CNRS – UPMC, France Brudzewski, Kazimierz, Warsaw University of Technology, Poland Cai, Chenxin, Nanjing Normal University, China Cai, Qingyun, Hunan University, China Campanella, Luigi, University La Sapienza, Italy Carvalho, Vitor, Minho University, Portugal Cecelja, Franjo, Brunel University, London, UK Cerda Belmonte, Judith, Imperial College London, UK Chakrabarty, Chandan Kumar, Universiti Tenaga Nasional, Malaysia Chakravorty, Dipankar, Association for the Cultivation of Science, India Changhai, Ru, Harbin Engineering University, China Chaudhari, Gajanan, Shri Shivaji Science College, India Chavali, Murthy, N.I. Center for Higher Education, (N.I. University), India Chen, Jiming, Zhejiang University, China Chen, Rongshun, National Tsing Hua University, Taiwan Cheng, Kuo-Sheng, National Cheng Kung University, Taiwan Chiang, Jeffrey (Cheng-Ta), Industrial Technol. Research Institute, Taiwan Chiriac, Horia, National Institute of Research and Development, Romania Chowdhuri, Arijit, University of Delhi, India Chung, Wen-Yaw, Chung Yuan Christian University, Taiwan Corres, Jesus, Universidad Publica de Navarra, Spain Cortes, Camilo A., Universidad Nacional de Colombia, Colombia Courtois, Christian, Universite de Valenciennes, France Cusano, Andrea, University of Sannio, Italy D'Amico, Arnaldo, Università di Tor Vergata, Italy De Stefano, Luca, Institute for Microelectronics and Microsystem, Italy Deshmukh, Kiran, Shri Shivaji Mahavidyalaya, Barshi, India Dickert, Franz L., Vienna University, Austria Dieguez, Angel, University of Barcelona, Spain Dimitropoulos, Panos, University of Thessaly, Greece Ding, Jianning, Jiangsu Polytechnic University, China Djordjevich, Alexandar, City University of Hong Kong, Hong Kong Donato, Nicola, University of Messina, Italy

Donato, Patricio, Universidad de Mar del Plata, Argentina Dong, Feng, Tianjin University, China Drljaca, Predrag, Instersema Sensoric SA, Switzerland Dubey, Venketesh, Bournemouth University, UK Enderle, Stefan, Univ.of Ulm and KTB Mechatronics GmbH, Germany Erdem, Gursan K. Arzum, Ege University, Turkey Erkmen, Aydan M., Middle East Technical University, Turkey Estelle, Patrice, Insa Rennes, France Estrada, Horacio, University of North Carolina, USA Faiz, Adil, INSA Lyon, France Fericean, Sorin, Balluff GmbH, Germany Fernandes, Joana M., University of Porto, Portugal Francioso, Luca, CNR-IMM Institute for Microelectronics and Microsystems, Italy Francis, Laurent, University Catholique de Louvain, Belgium Fu, Weiling, South-Western Hospital, Chongqing, China Gaura, Elena, Coventry University, UK Geng, Yanfeng, China University of Petroleum, China Gole, James, Georgia Institute of Technology, USA Gong, Hao, National University of Singapore, Singapore Gonzalez de la Rosa, Juan Jose, University of Cadiz, Spain Granel, Annette, Goteborg University, Sweden Graff, Mason, The University of Texas at Arlington, USA Guan, Shan, Eastman Kodak, USA Guillet, Bruno, University of Caen, France Guo, Zhen, New Jersey Institute of Technology, USA Gupta, Narendra Kumar, Napier University, UK Hadjiloucas, Sillas, The University of Reading, UK Haider, Mohammad R., Sonoma State University, USA Hashsham, Syed, Michigan State University, USA Hasni, Abdelhafid, Bechar University, Algeria Hernandez, Alvaro, University of Alcala, Spain Hernandez, Wilmar, Universidad Politecnica de Madrid, Spain Homentcovschi, Dorel, SUNY Binghamton, USA Horstman, Tom, U.S. Automation Group, LLC, USA Hsiai, Tzung (John), University of Southern California, USA Huang, Jeng-Sheng, Chung Yuan Christian University, Taiwan Huang, Star, National Tsing Hua University, Taiwan Huang, Wei, PSG Design Center, USA Hui, David, University of New Orleans, USA Jaffrezic-Renault, Nicole, Ecole Centrale de Lyon, France Jaime Calvo-Galleg, Jaime, Universidad de Salamanca, Spain James, Daniel, Griffith University, Australia Janting, Jakob, DELTA Danish Electronics, Denmark Jiang, Liudi, University of Southampton, UK Jiang, Wei, University of Virginia, USA Jiao, Zheng, Shanghai University, China John, Joachim, IMEC, Belgium Kalach, Andrew, Voronezh Institute of Ministry of Interior, Russia Kang, Moonho, Sunmoon University, Korea South Kaniusas, Eugenijus, Vienna University of Technology, Austria Katake, Anup, Texas A&M University, USA Kausel, Wilfried, University of Music, Vienna, Austria Kavasoglu, Nese, Mugla University, Turkey Ke, Cathy, Tyndall National Institute, Ireland Khelfaoui, Rachid, Université de Bechar, Algeria Khan, Asif, Aligarh Muslim University, Aligarh, India Kim, Min Young, Kyungpook National University, Korea South Ko, Sang Choon, Electronics. and Telecom. Research Inst., Korea South Kockar, Hakan, Balikesir University, Turkey Kong, Ing, RMIT University, Australia Kotulska, Malgorzata, Wroclaw University of Technology, Poland Kratz, Henrik, Uppsala University, Sweden

Kumar, Arun, University of South Florida, USA Kumar, Subodh, National Physical Laboratory, India Kung, Chih-Hsien, Chang-Jung Christian University, Taiwan Lacnjevac, Caslav, University of Belgrade, Serbia Lay-Ekuakille, Aime, University of Lecce, Italy Lee, Jang Myung, Pusan National University, Korea South Lee, Jun Su, Amkor Technology, Inc. South Korea Lei, Hua, National Starch and Chemical Company, USA Li, Genxi, Nanjing University, China Li, Hui, Shanghai Jiaotong University, China Li, Xian-Fang, Central South University, China Liang, Yuanchang, University of Washington, USA Liawruangrath, Saisunee, Chiang Mai University, Thailand Liew, Kim Meow, City University of Hong Kong, Hong Kong Lin, Hermann, National Kaohsiung University, Taiwan Lin, Paul, Cleveland State University, USA Linderholm, Pontus, EPFL - Microsystems Laboratory, Switzerland Liu, Aihua, University of Oklahoma, USA Liu Changgeng, Louisiana State University, USA Liu, Cheng-Hsien, National Tsing Hua University, Taiwan Liu, Songqin, Southeast University, China Lodeiro, Carlos, University of Vigo, Spain Lorenzo, Maria Encarnacio, Universidad Autonoma de Madrid, Spain Lukaszewicz, Jerzy Pawel, Nicholas Copernicus University, Poland Ma, Zhanfang, Northeast Normal University, China Majstorovic, Vidosav, University of Belgrade, Serbia Marquez, Alfredo, Centro de Investigacion en Materiales Avanzados, Mexico Matay, Ladislav, Slovak Academy of Sciences, Slovakia Mathur, Prafull, National Physical Laboratory, India Maurya, D.K., Institute of Materials Research and Engineering, Singapore Mekid, Samir, University of Manchester, UK Melnyk, Ivan, Photon Control Inc., Canada Mendes, Paulo, University of Minho, Portugal Mennell, Julie, Northumbria University, UK Mi, Bin, Boston Scientific Corporation, USA Minas, Graca, University of Minho, Portugal Moghavvemi, Mahmoud, University of Malaya, Malaysia Mohammadi, Mohammad-Reza, University of Cambridge, UK Molina Flores, Esteban, Benemérita Universidad Autónoma de Puebla, Mexico Moradi, Majid, University of Kerman, Iran Morello, Rosario, University "Mediterranea" of Reggio Calabria, Italy Mounir, Ben Ali, University of Sousse, Tunisia Mulla, Imtiaz Sirajuddin, National Chemical Laboratory, Pune, India Nabok, Aleksey, Sheffield Hallam University, UK Neelamegam, Periasamy, Sastra Deemed University, India Neshkova, Milka, Bulgarian Academy of Sciences, Bulgaria Oberhammer, Joachim, Royal Institute of Technology, Sweden Ould Lahoucine, Cherif, University of Guelma, Algeria Pamidighanta, Sayanu, Bharat Electronics Limited (BEL), India Pan, Jisheng, Institute of Materials Research & Engineering, Singapore Park, Joon-Shik, Korea Electronics Technology Institute, Korea South Penza, Michele, ENEA C.R., Italy Pereira, Jose Miguel, Instituto Politecnico de Setebal, Portugal Petsev, Dimiter, University of New Mexico, USA Pogacnik, Lea, University of Ljubljana, Slovenia Post, Michael, National Research Council, Canada Prance, Robert, University of Sussex, UK Prasad, Ambika, Gulbarga University, India Prateepasen, Asa, Kingmoungut's University of Technology, Thailand Pullini, Daniele, Centro Ricerche FIAT, Italy Pumera, Martin, National Institute for Materials Science, Japan Radhakrishnan, S. National Chemical Laboratory, Pune, India Rajanna, K., Indian Institute of Science, India Ramadan, Qasem, Institute of Microelectronics, Singapore Rao, Basuthkar, Tata Inst. of Fundamental Research, India Raoof, Kosai, Joseph Fourier University of Grenoble, France Reig, Candid, University of Valencia, Spain Restivo, Maria Teresa, University of Porto, Portugal Robert, Michel, University Henri Poincare, France Rezazadeh, Ghader, Urmia University, Iran Royo, Santiago, Universitat Politecnica de Catalunya, Spain Rodriguez, Angel, Universidad Politecnica de Cataluna, Spain Rothberg, Steve, Loughborough University, UK Sadana, Ajit, University of Mississippi, USA Sadeghian Marnani, Hamed, TU Delft, The Netherlands Sandacci, Serghei, Sensor Technology Ltd., UK Schneider, John K., Ultra-Scan Corporation, USA Sengupta, Deepak, Advance Bio-Photonics, India Shah, Kriyang, La Trobe University, Australia Sapozhnikova, Ksenia, D.I.Mendeleyev Institute for Metrology, Russia

Saxena, Vibha, Bhbha Atomic Research Centre, Mumbai, India Seif, Selemani, Alabama A & M University, USA Seifter, Achim, Los Alamos National Laboratory, USA Silva Girao, Pedro, Technical University of Lisbon, Portugal Singh, V. R., National Physical Laboratory, India Slomovitz, Daniel, UTE, Uruguay Smith, Martin, Open University, UK Soleymanpour, Ahmad, Damghan Basic Science University, Iran Somani, Prakash R., Centre for Materials for Electronics Technol., India Srinivas, Talabattula, Indian Institute of Science, Bangalore, India Srivastava, Arvind K., NanoSonix Inc., USA Stefan-van Staden, Raluca-Ioana, University of Pretoria, South Africa Sumriddetchka, Sarun, National Electronics and Computer Technology Center, Thailand Sun, Chengliang, Polytechnic University, Hong-Kong Sun, Dongming, Jilin University, China Sun, Junhua, Beijing University of Aeronautics and Astronautics, China Sun, Zhiqiang, Central South University, China Suri, C. Raman, Institute of Microbial Technology, India Sysoev, Victor, Saratov State Technical University, Russia Szewczyk, Roman, Industrial Research Inst. for Automation and Measurement, Poland Tan, Ooi Kiang, Nanyang Technological University, Singapore, Tang, Dianping, Southwest University, China Tang, Jaw-Luen, National Chung Cheng University, Taiwan Teker, Kasif, Frostburg State University, USA Thirunavukkarasu, I., Manipal University Karnataka, India Thumbavanam Pad, Kartik, Carnegie Mellon University, USA Tian, Gui Yun, University of Newcastle, UK Tsiantos, Vassilios, Technological Educational Institute of Kaval, Greece Tsigara, Anna, National Hellenic Research Foundation, Greece Twomey, Karen, University College Cork, Ireland Valente, Antonio, University, Vila Real, - U.T.A.D., Portugal Vanga, Raghav Rao, Summit Technology Services, Inc., USA Vaseashta, Ashok, Marshall University, USA Vazquez, Carmen, Carlos III University in Madrid, Spain Vieira, Manuela, Instituto Superior de Engenharia de Lisboa, Portugal Vigna, Benedetto, STMicroelectronics, Italy Vrba, Radimir, Brno University of Technology, Czech Republic Wandelt, Barbara, Technical University of Lodz, Poland Wang, Jiangping, Xi'an Shiyou University, China Wang, Kedong, Beihang University, China Wang, Liang, Pacific Northwest National Laboratory, USA Wang, Mi, University of Leeds, UK Wang, Shinn-Fwu, Ching Yun University, Taiwan Wang, Wei-Chih, University of Washington, USA Wang, Wensheng, University of Pennsylvania, USA Watson, Steven, Center for NanoSpace Technologies Inc., USA Weiping, Yan, Dalian University of Technology, China Wells, Stephen, Southern Company Services, USA Wolkenberg, Andrzej, Institute of Electron Technology, Poland Woods, R. Clive, Louisiana State University, USA Wu, DerHo, National Pingtung Univ. of Science and Technology, Taiwan Wu, Zhaoyang, Hunan University, China Xiu Tao, Ge, Chuzhou University, China Xu, Lisheng, The Chinese University of Hong Kong, Hong Kong Xu, Sen, Drexel University, USA Xu, Tao, University of California, Irvine, USA Yang, Dongfang, National Research Council, Canada Yang, Shuang-Hua, Loughborough University, UK Yang, Wuqiang, The University of Manchester, UK Yang, Xiaoling, University of Georgia, Athens, GA, USA Yaping Dan, Harvard University, USA Ymeti, Aurel, University of Twente, Netherland Yong Zhao, Northeastern University, China Yu, Haihu, Wuhan University of Technology, China Yuan, Yong, Massey University, New Zealand Yufera Garcia, Alberto, Seville University, Spain Zakaria, Zulkarnay, University Malaysia Perlis, Malaysia Zagnoni, Michele, University of Southampton, UK Zamani, Cyrus, Universitat de Barcelona, Spain Zeni, Luigi, Second University of Naples, Italy Zhang, Minglong, Shanghai University, China Zhang, Qintao, University of California at Berkeley, USA Zhang, Weiping, Shanghai Jiao Tong University, China Zhang, Wenming, Shanghai Jiao Tong University, China Zhang, Xueji, World Precision Instruments, Inc., USA Zhong, Haoxiang, Henan Normal University, China Zhu, Qing, Fujifilm Dimatix, Inc., USA Zorzano, Luis, Universidad de La Rioja, Spain Zourob, Mohammed, University of Cambridge, UK

Sensors & Transducers Journal (ISSN 1726-5479) is a peer review international journal published monthly online by International Frequency Sensor Association (IFSA). Available in electronic and on CD. Copyright © 2011 by International Frequency Sensor Association. All rights reserved.

Sensors & Transducers Journal

Contents Volume 125 Issue 2 February 2011

www.sensorsportal.com

ISSN 1726-5479

Research Articles Microcantilever Sensors in Biological and Chemical Detections Qing Zhu .............................................................................................................................................

1

Design of a Low Voltage 0.18 um CMOS Surface Acoustic Wave Gas Sensor M. Moghavvemi and A. Attaran ..........................................................................................................

22

Glucose Monitoring System Based on Osmotic Pressure Measurements Alexandra Leal, António Valente, Ana Ferreira, Salviano Soares, Vitor Ribeiro, Olga Krushinitskaya, and Erik A. Johannessen ..........................................................................................

30

Chemical Vapor Identification by Plasma Treated Thick Film Tin Oxide Gas Sensor Array and Pattern Recognition J. K. Srivastava, Preeti Pandey, Sunil K. Jha, V. N. Mishra, R. Dwivedi ...........................................

42

A Preliminary Test for Skin Gas Assessment Using a Porphyrin Based Evanescent Wave Optical Fiber Sensor Roman Selyanchyn, Sergiy Korposh, Wataru Yasukochi and Seung-Woo Lee. ...............................

54

Optical Characterization and Humidity Sensing Properties of Praseodymium Oxide B. C. Yadav, Monika Singh and C. D. Dwivedi...................................................................................

68

Nanocrystalline SnO2-Pt Thick Film Gas Sensor for Air Pollution Applications M. H. Shahrokh Abadi, M. N. Hamidon, Abdul Halim Shaari, Norhafizah Abdullah, Rahman Wagiran and Norhisam Misron. ...........................................................................................

76

Characterization of WO3-SnO2 Nanocomposites and Application in Humidity Sensing N. K. Pandey, Akash Roy, Alok Kumar. .............................................................................................

89

Detections of Water Content Changes in a Nitrocellulose Membrane Based on Polarized Reflection Spectroscopy Hariyadi Soetedjo ...............................................................................................................................

100

Fabrication of Polyaniline/ TiO2 Nanocomposite Ammonia Vapor Sensor S. G. Pawar, S. L. Patil, M. A. Chougule, B. T. Raut, S. A.Pawar and V. B. Patil .............................

107

Impact of Mineral Composition on the Distribution of Natural Radionuclides in RigosolAnthrosol Z P. Tomić, A. R. Djordjević, M. B. Rajković, I. Vukašinović, N. S. Nikolić, V. Pavlović and Č. M. Lačnjevac...........................................................................................................................

115

Design of Photoreactor and Study of Modeling Parameters for Removal of Pesticides in Water: a Case Study of Malathion Amit K. Sharma, R. K. Tiwari and M. S. Gaur ....................................................................................

131

Studies on Gas Sensing Performance of Cr-doped Indium Oxide Thick Film Sensors D. N. Chavan, G. E. Patil, D. D. Kajale, V. B. Gaikwad, G. H. Jain ...................................................

142

Preparation and Studies on Gas Sensing Performance of Pure and Modified Sn-TiO2 Thick Film Resistor P. D. Hire, V. B. Gaikwad, N. U. Patil, R. L. Patil, R. M. Chaudhri, S. D. Shinde G. H. Jain ............. Electrocoductivity Studies of Grafted Polymer Thin Film

156 168

Muhammed Mizher Radhi .................................................................................................................. Ester Sensing with Poly (Aniline-co-m-aminobenzoic Acid) Deposited on Poly (Vinyl Alcohol) S. Adhikari, J. Singh, R. Banerjee and P. Banerji ..............................................................................

177

Fiber Bragg Grating Sensor for Detection of Nitrate Concentration in Water A. S. Lalasangi, J. F. Akki, K.G. Manohar, T. Srinivas, P. Radhakrishnan, Sanjay Kher, N. S. Mehla and U. S. Raikar .............................................................................................................

187

Study on Gas Sensing Performance of In2O3 Thick Film Resistors Prepared by Screen Printing Technique S. C. Kulkarni, R. Y. Borse .................................................................................................................

194

Periodically Tapered LPFG for Ethanol Concentration Detection in Ethanol-Gasoline Blend J. Linesh, T. M. Libish, M. C. Bobby, P. Radhakrishnan and V. P. N. Nampoori...............................

205

Chemically Deposited n-CuInSe2 / Polyiodide Based PEC Solar Cells R. H. Bari and L. A. Patil.....................................................................................................................

213

Sensitivity and Selectivity Studies on Polyaniline / Molybdenum Trioxide Composites to Liquid Petroleum Gas Aashis S. Roy, Machappa T, M. V. N. Ambika Prasad and Koppalkar R. Anilkumar ........................

220

Long-term Biosensors for Metabolite Monitoring by using Carbon Nanotubes Cristina Boero, Sandro Carrara, Giovanni De Micheli........................................................................

229

Modeling of a Bio Sensor Based on Detection of Antigens Concentration Using an Electrically Actuated Micro Cantilever Hadi Madinei, Ali-Asghar Keyvani-Janbahan, Mehdi Atashparva, Rasool Shabani, Ghader Rezazadeh ............................................................................................................................

238

A SAW Delay Line Sensor Combined with Micro-hotplate for Bio-chemical Applications Babak Vosoughi Lahijani, Habib Badri Ghavifekr...............................................................................

247

Bioelectrical Impedance Analysis Device: Measurement of Bioelectrical Tissue Conductivity in Dengue Patients Herlina Abdul Rahim, Mohd Nasir Taib, Fatimah Ibrahim and Ruzairi Abdul Rahim.........................

256

Authors are encouraged to submit article in MS Word (doc) and Acrobat (pdf) formats by e-mail: [email protected] Please visit journal’s webpage with preparation instructions: http://www.sensorsportal.com/HTML/DIGEST/Submition.htm International Frequency Sensor Association (IFSA).

Sensors & Transducers Journal, Vol. 125, Issue 2, February 2011, pp. 229-237

Sensors & Transducers ISSN 1726-5479 © 2011 by IFSA http://www.sensorsportal.com

Long-term Biosensors for Metabolite Monitoring by using Carbon Nanotubes *

Cristina BOERO, Sandro CARRARA, Giovanni DE MICHELI Laboratory of Integrated Systems, EPFL, 1015 Lausanne, Switzerland * Tel.: (+41)-21-693-6878 E-mail: [email protected]

Received: 20 January 2011 /Accepted: 15 February 2011 /Published: 28 February 2011

Abstract: The key-point for the development of an amperometric sensor is the immobilization of the enzyme. In the present work we use biosensors based on glucose oxidase (GOD) onto electrodes nanostructured with carbon nanotubes (CNT), to be employed in cell culture monitoring. The goal is to determine the best immobilization strategy from the point-of-view of sensor lifetime. We compared three types of immobilization: the spontaneous adsorption of the enzyme on nanotubes, the entrapment in a Nafion matrix (optimizing also the quantity), and the cross-linking with glutaraldehyde. The crosslinking gives the best sensitivity, 17.38 µA mM-1 cm-2, and the lowest detection limit, 25 µM. On the other hand, Nafion matrix allows to extend the linear range up to 7.5 mM. Finally, electrodes are tested over 35 days to analyze the lifetime. GOD cross-linking results to have 100% of retained activity after 35 days, while the adsorption and the entrapment retain only the 20 % of the original response. Copyright © 2011 IFSA. Keywords: Electrochemical biosensors, Glucose, Carbon nanotubes, Immobilization, Nafion, Glutaraldehyde.

1. Introduction Electrochemical enzyme-based biosensors have been largely used over the past few decades, because of their many advantages. First of all, they have a high specificity for the identification of a particular target molecule, due to enzyme selectivity. Then, they allow the direct transduction of a chemical reaction in an electrical measurement, which is easily detectable. These sensors have a high versatility, since the immobilization strategy used for one type of protein can be also employed for others of the 229


same family (i.e., oxidases). And finally, they can be easily miniaturized and used in disposable or even fully implantable devices. Among the wide variety of amperometric sensors developed in the last 20 years, those for metabolite detection have had the best success. Glucose detection has attracted lot of researches [1], due to the importance that these instruments have in the life of diabetic patients. Other kinds of metabolites have also captured the interest, like lactate [2], related to anaerobic conditions due to hypoxia, cholesterol [3], important for the diagnosis and prevention of a number of clinical disorders, and glutamate [4], the major neurotransmitter in the brain. Recently, an increased demand has arisen from the field of cell analysis. The monitoring of specific analytes involved in cell growth, cell proliferation and cell differentiation can contribute to a better understanding of the factors that influence metabolic processes, with a particular interest on stem cell mechanisms. In order to employ such biosensors to cell cultures, and in particular to stem cells, long-term stability plays a fundamental role, since they require a monitoring over several days. For example, mammalian cells can last for 5-10 days without contaminations [5]. Another interesting cell line is the one derived from the fusion of septal neurons with neuroblastoma cells. This cell line can mimic stem cell behavior, since it can be grown either in proliferation state, or it is possible to induce differentiation with some agents, like retinoic acid. These cells are able to survive up to 4 weeks [6]. Electrodes have been structured with different strategies, employing polymeric matrices, sol-gel, crosslinker, and mediators [7]. More recently, nanomaterials has been considered as alternative materials to wire the enzyme and the electrode surface, enhancing the electron transfer and the sensitivity of the sensors. Among the different nanomaterials, carbon nanotubes have revealed great electrical [8] and electrochemical [9] properties, suitable to be applied on biosensors. Many works have been published with the direct adsorption of the enzyme onto CNT surface [10]. Other researches have used polymer membranes to entrap the enzyme onto the electrode. Conductive polymers seem to be the best solution to develop amperometric biosensors, helping the exchange of charges. Nafion is a perfluorinated sulfonated cation exchanger, which has been widely used in electrochemical biosensors, due to its chemical inertness and its thermal stability, but especially for its anti-fouling properties [11]. Another immobilization strategy involves glutaraldehyde, an organic compound used as fixative, for its property to be a protein cross-linker. All the aforementioned strategies has been combined together in order to develop more durable electrodes, with a long-term stability. To date, all commercially available glucometer have disposable electrode for glucose measurement. From the point-of-view of handling blood, the use of disposable sensors is an advantage [1], avoiding contamination problems and allowing safe measurements. On the other hand, long-term stability is an important issue for cell culture monitoring, as already mentioned. The sensor should be stable over the culture time, in order to accurately follow metabolite concentrations in different cell states. For this purpose, the stability of a sensor has to last at least 20 days. The goal of the present work is a comparison between three different immobilization strategies of the enzyme for the development of a glucose biosensor with nanostructuration by using Multi-Walled Carbon Nanotubes (MWCNT). Glucose oxidase is let adsorbed onto the electrode surface, or entrapped in a Nafion matrix drop cast onto the electrode, or cross-linked with glutaraldehyde. Linear range, detection limit, sensitivity and lifetime are considered in order to evaluate the best immobilization strategy.

230


2. Experimental Section 2.1. Chemicals Carbon paste screen-printed electrodes (SPE - model DRP-110) and multi-walled carbon nanotubes were purchased from Dropsens (Spain). The electrodes consists of a graphite working electrode, which presents an active area equal to about 13 mm2, a counter electrode, also made of graphite, and a reference electrode, made of Ag/AgCl. The total area of the cell is 22 mm2. Multi-walled carbon nanotubes (diameter 10 nm, length 1-2 µm) where purchased in powder (90 % purity), and subsequently diluted in chloroform to the concentration of 1 mg ml-1 [12]. Samples were then sonicated in order to obtain a homogeneous solution. Glucose oxidase from Aspergillus Niger (GOD, EC 1.1.3.4, 129.9 units/mg solid), Nafion solution (5 wt% solution in a mixture of lower aliphatic alcohols and water), glutaraldehyde solution (25%), and D-(+)-glucose were purchased from Sigma-Aldrich (Switzerland). All the proteins were dissolved in Phosphate Buffer Saline (PBS) 0.01 M at pH 7.4, while glucose was dissolved in Milli-Q. 2.2. Preparation of Electrodes Three types of electrodes were prepared in order to evaluate different strategies of protein immobilization. All the three were nanostructured by using MWCNT. For each electrode, 40 µl of the MWCNT-chloroform solution were deposited by drop casting (5 µl each time) onto the working electrode, and the electrodes were allowed to dry. Then, for the protein autonomously adsorbed onto the surface (CNT/GOD), 20 µl of glucose oxidase (15 mg ml-1) were cast onto the working electrode and stored overnight at +4 ºC, in order to allow the adsorption of the protein onto the electrode surface. Then, the drop was rinsed out with Milli-Q. For the electrode with Nafion (CNT/GOD/Nafion), the procedure was similar to the previous one. Nafion was diluted at 0.5 wt% with a solution of 50 % of ethanol and 50 % of Milli-Q, according to what was reported in [13]. A drop of 2 µl of diluted Nafion was deposited onto the electrode and allowed to dry, forming a matrix of polymer which should protect the electrode from fouling and better immobilize the protein. For the immobilization with the cross-linker (CNT/GOD/Gluth), glutaraldehyde was diluted to 2.5 % with Milli-Q and the protein was dispersed in that solution (always with a concentration of 15 mg ml-1) and a drop of 20 µl was deposited to cover the working electrode and stored overnight at +4 ºC. Then, the drop was rinsed out with Milli-Q. All the three electrodes were conditioned for 10 minutes at constant potential (+550 mV) before the first use and they were stored at +4 ºC, covered with PBS, when not used. 2.3 Apparatus The electrochemical response of electrodes was investigated by chronoamperometries under aerobic conditions. Electrochemical measurements were acquired by using Versastat 3 potentiostat (Princeton Applied Technologies). For all the measurements, the electrode was dipped into a volume of 25 ml of PBS under stirring conditions. A volume of 25 µl per step of glucose was successively added into the solution with a time-step of 2 minutes. The applied potential was +550 mV vs Ag/AgCl.

3. Biosensor Characterization 3.1. Comparison for Different Immobilization Strategies Calibration lines are derived from chronoamperometries within the concentration range from 0 to 4 mM of glucose. The substrate is dissolved in Milli-Q at a concentration of 0.5 M. 231


Chronoamperometries are carried out by using 25 ml of PBS as support electrolyte in stirring conditions. Screen-printed electrodes are dipped into the solution and 25 µl of the substrate are added, in order to obtain step of 0.5 mM. The addition are performed every 120 s, allowing the system to reach the steady-state. The response time of the system after each addition is around 30 s (data not shown). The three electrodes are tested in the same condition and calibration curves are shown in Fig. 1.

Fig. 1. Linear range: (a) adsorbed GOD; (b) GOD entrapped in Nafion; and (c) GOD cross-linked with glutaraldehyde. Data are represented as means ± Standard Deviation (SD).

The sensitivity related to GOD simply adsorbed onto MWCNT is 10.24 µA mM-1 cm-2; the value related to GOD entrapped in Nafion is 8.18 µA mM-1 cm-2; and finally, the sensitivity for GOD crosslinked with glutaraldehyde is 17.38 µA mM-1 cm-2. Note from Fig. 1 that all the behaviors are almost linear within the concentration range. Fig. 2 illustrates the maximum linear range: the adsorption allows measurements from 100 µM to 6.5 mM, while the entrapment results in a linear range from 200 µM to 7.5 mM. The cross-linking shows the narrowest linear range, from 25 µM up to 5 mM, probably due to the fact that glutaraldehyde denaturates part of the enzymes, leaving a smaller amount able to react with the substrate. Regarding the case of Nafion, this fact is also confirmed by Rahman et al. [14]: they showed that electrodes modified with MWCNT and Nafion exhibit wider range compared to unmodified electrodes. On the other hand, the same electrodes show a higher inferior limit for the concentration window: the sensor seems to be insensitive for values lower than 200 µM, due to its anti-fouling properties. Nafion films were extensively used for their anti-interferent and antifouling properties in electrochemical sensing, since they work like an effective pre-selective barrier, eliminating anionic biological interference and enhancing the selectivity of the sensor. In particular, it has been used to avoid interferences due to ascorbic and uric acid [15]. Tsai et al. [11] showed that peak current decreases drastically if the glassy carbon electrode is coated with 0.5 wt% Nafion. It was because Nafion film becomes a barrier in diffusion on the surface of the electrode, as demonstrated by the detection limit. On the other hand, the presence of MWCNT improves significantly the sensitivity of the sensor, as demonstrated in our previous work [16]. The detection limit is also investigated to see what is the effect of different immobilization strategies, considering a signal-to-noise ratio of 3. Adsorbed GOD shows a limit of detection of 97 µM, entrapped enzyme results in a detection limit of 59 µM, and cross-linked oxidase has a detection limit of 16 µM. From a calibration point-of-view it seems that the cross-linking offers better advantages respect to the other techniques, even if the detection is not very linear for high concentration of glucose (refers to Fig. 2 curve c). 232


Fig. 2. Wider linear range: (a) adsorbed GOD; (b) GOD entrapped in Nafion; and (c) GOD cross-linked with glutaraldehyde. Data are represented as means ± SD.

3.2. Effect of Nafion Quantity on Electrocatalytic Properties of the Biosensor We investigated the quantity of Nafion cast onto the electrode, in order to determine its influence on the biosensor response. Electrodes are prepared with carbon nanotubes, GOD and three different amounts of Nafion 0.5 wt% (volumes of 1, 2, and 3 µl). Fig. 3 illustrates calibration line for the developed biosensors.

Fig. 3. Calibration line for GOD entrapped in different quantities of Nafion. Data are represented as means ± SD.

It is possible to observe that sensitivity decreases with Nafion volume cast onto the surface, as shown also by Tang et al. [15]. The values of sensitivity for 1 µl, 2 µl, and 3 µl are 8.42 µA mM-1cm-2, 7.02 µA mM-1cm-2 and 5.33 µA mM-1cm-2, respectively. Note that sensitivity decreases drastically for higher volume of Nafion: the higher the volume, the thicker the matrix. Although Nafion is a cation exchanger, it obstructs glucose molecules to reach the electrode surface, and then glucose oxidase. Moreover, although the calculated limit of detection (S/N = 3) is around 60 µM for the three cases, the 233


lower limit of the linear range is around 1 mM. It seems to be not so dependent from the cast volume, but it confirms the tendency of Nafion to behave like a barrier in diffusion on the surface of the electrode, which is more evident for lower concentrations.

4. Sensors Lifetime Measurements of glucose are carried out for 38 days, in order to verify the stability of the different strategies for enzyme immobilization. The three electrodes are tested for a range of concentration from 0.5 up to 4 mM every 2-3-4 days over 24 days. After a pause of 1 week, they are eventually tested to see the retained activity. Fig. 4, Fig. 5 and Fig. 6 illustrate the aging of the three immobilization strategies for 2.5 mM of glucose. For the case of GOD cross-linking with glutaraldehyde, the 100 % of the retained activity is considered the one related to the 3rd day, because we observed a more evident increase of enzyme activity after some days respect to the other cases. Moreover, the measurement is unstable during the first ten days after the immobilization. It could be due to the fact that the cross-linking process is still under development, and the enzyme needs some days to stabilize.

Fig. 4. Stability of the adsorbed GOD onto the electrode surface for the detection of 2.5 mM glucose. The applied potential was +550 mV. Data are represented as means ± SD.

Fig. 5. Stability of the entrapped GOD in a Nafion matrix for the detection of 2.5 mM glucose. The applied potential was +550 mV. Data are represented as means ± SD. 234


Fig. 6. Stability of the cross-linked GOD with glutaraldehyde for the detection of 2.5 mM glucose. The applied potential was +550 mV. Data are represented as means ± SD.

None of the three immobilization strategies highlights a specific trend over 25 days, but all the measurements oscillate around the 100 % of the retained activity. The difference is really evident after 30 days, where GOD cross-linked with glutaraldehyde shows the highest lifetime. In fact, the measurements are still around 100 % after 35 days, without any identifiable decay over the time. On the contrary, for the other two strategies of immobilization there is a clear decay after 24 days, with a consequent retained activity of around 16 % and 23 %, for adsorption and entrapment strategies, respectively. The entrapment in Nafion (over the first 24 days) and the cross-linking (after the first 10 days) are the methods with the highest reproducibility, with an error around 14 %. GOD adsorbed on the electrode surface shows an error around 23 %, denoting lower reproducibility. The obtained values related to the retained activity over the time are quite similar with what was previously found in literature. Regarding the adsorption of GOD onto MWCNT, Wang et al. [10] found that for the detection of 1 mM of glucose, the retained activity after 25 days was about 97 %. Their sensor was quite similar to the one of the present work, since they adsorbed GOD onto grown MWCNT, without any other nanocomposite. The best result for the case of GOD immobilized without any other compound was obtained by Crounch et al. [17], where there was no apparent reduced activity of the sensor after 550 days. The immobilization strategy was instead different, since they developed a water-based carbon ink containing glucose oxidase and they stored the sensor in desiccated conditions, when not used. For the case of GOD entrapped in a Nafion membrane, Tang et al. [15] showed that the current response was still retaining the 73.5 % of the initial value after 22 days, coherently with what was found in the present work. For the case of enzyme cross-linking, the work of Kang et al. [18] showed a retained activity of about 92 % after 35 days and 85% after 50 days, in the case of 1 mM of glucose. Table 1 reports the main features of biosensors developed with the three immobilization strategies. From the point-of-view of cell culture monitoring, the three presented techniques show an appropriate stability to be employed for this purpose. Although previous work presented good results in terms of lifetime for implantable sensors [19, 20], the research is not so focused on cell culture applications. For some biological studies, cell lines are not monitored for more than 40 days and there is a lack of research to develop suitable biosensors for this purpose. From the present research, we can conclude that glutaraldehyde cross-linking seems to be the most suitable to monitor septal neuron and stem cell lines, surviving for more than 40 days.

5. Conclusions In the present work, we have investigated three different immobilization strategies to develop enzymebased glucose biosensors using nanostructured electrodes for long-term cells monitoring. 235


Table 1. Resume of the main futures for the three immobilization techniques.

Adsorption Entrapment Cross-linking

Sensitivity (µA mM-1 cm-2) 10.24 8.18 17.38

Limit of detection (µM)

Linear range (mM)

97 59 16

0.1 – 6.5 0.2 – 7.5 0.025 - 5

Lifetime (days) 24 34 > 38

We have shown a comparison in terms of linear range, detection limit, sensitivity and long-term stability. For the case of GOD adsorbed onto the electrode surface, we found a linear range of 0.1 – 6.5 mM, a detection limit of 97 µM, and a sensitivity of 10.24 µA mM-1 cm-2. In the case of GOD entrapped in a Nafion matrix, we found a linear range of 0.2 – 7.5 mM, a detection limit of 59 µM, and a sensitivity of 8.18 µA mM-1 cm-2. For the GOD cross-linked with glutaraldehyde we observed a linear range of 0.025 – 5 mM, a detection limit of 16 µM, and a sensitivity of 17.38 µA mM-1 cm-2. We also investigated the dependence of the sensor response related to Nafion quantity drop cast onto the electrode surface. We found that the more the volume of deposited Nafion, the lower the value of sensitivity. The best amount of Nafion results in 1 µl of 0.5 wt% Nafion. Finally, we investigated the lifetime of the three developed biosensors. All the three electrodes have a good stability over 25 days, with higher reproducibility in the case of GOD entrapped in Nafion and cross-linked with glutaraldehyde (errors around 14 %). In addition, the best result in terms of long-term stability is obtained with cross-linking, able to maintain stable measurements for more than 40 days. Further experiments will focus on other kinds of oxidases, in order to confirm the best strategy for enzyme immobilization, and to develop sensor array for the long-term monitoring of multiple metabolites in cell cultures.

Acknowledgements The authors want to thank M. Mascini and his co-workers for useful discussions on protein immobilization. The research is financed by SNF Sino-Swiss cooperation project n. IZLCZ2 123967.

References [1]. J. D. Newman, A. P. F. Turner, Home blood glucose biosensors: a commercial perspective, Biosensors and Bioelectronics, Vol. 20, Issue 12, 2005, pp. 2435-2453. [2]. N. Nikolaus, B. Strehilitz, Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing, Microchimica Acta, Vol. 160, No. 1-2, 2008, pp. 15-55. [3]. V. Shumyantseva, G. Deluca, T. Bulko, S. Carrara, C. Nicolini, S. A. Usanov, A. I. Archakov, Cholesterol amperometric biosensor based on cytochrome P450scc, Biosensors and Bioelectronics, Vol. 19, Issue 9, 2004, pp. 971-976. [4]. S. Qin, M. van der Zeyden, W. H. Oldenziel, T. Cremers, B. Westeink, Microsensors for in vivo measurement of glutamate in brain tissue, Sensors, Vol. 8, Issue 11, 2008, pp. 6860-6884. [5]. K. B. Male, P. O. Gartu, A. A. Kamen, J. H. Luong, On-line monitoring of glucose in mammalian cell culture using a flow injection analysis (FIA) mediated biosensor, Biotechnology and Bioengineering, Vol. 55, Issue 3, 1997, pp. 497-504. [6]. H. J. Lee, D. N. Hammond, T. H. Large, B. H. Wainer, Immortalized young adult neurons from the septal region: generation and characterization, Developmental Brain Research, Vol. 52, Issue 1-2, 1990, pp. 219-228. [7]. J. Wang, Electrochemical glucose biosensors, Chemicals Reviews, Vol. 108, Issue 2, 2008, pp. 814-825. [8]. V. Bavastrello, S. Carrara, M. K. Ram, C. Nicolini, Optical and electrochemical properties of poly(o-toluidine) multiwalled carbon nanotubes composite langmuirschaefer films, Langmuir, Vol. 20, Issue 3, 2004, pp. 969-973. 236


[9]. V. Bavastrello, E. Stura, S. Carrara, V. Erokhin, C. Nicolini, Poly(2,5-dimethylaniline)-mwnts nanocomposite: a new material for conductiometric acid vapours sensor, Sensors and Actuators B: Chemical, Vol. 98, Issue 2-3, 2004, pp. 247-253. [10].S. G. Wang, Q. Zhang, R. Wang, S. F. Yoon, A novel multi-walled carbon nanotube-based biosensor for glucose detection, Biochemical and Biophysical Research Communications, Vol. 311, Issue 3, 2003, pp. 572-576. [11].Y. Tsai, S. Li, J. Chen, Cast thin film biosensor design based on a Nafion backbone, a multiwalled carbon nanotube conduit, and a glucose oxidase function, Langmuir, Vol. 21, Issue 8, 2005, pp. 3653-3658. [12].S. Carrara, V. Shumyantseva, A. I. Archakov, B. Samorì, Screen-printed electrodes based on carbon nanotubes and cytochrome P450scc for highly sensitive cholesterol biosensors, Biosensors and Bioelectronics, Vol. 24, Issue 1, 2008, pp. 148-150. [13].S. Kröger, S. J. Setford, A. P. F. Turner, Assessment of glucose oxidase behaviour in alcoholic solutions using disposable electrodes, Analytica Chimica Acta, Vol. 368, Issue 3, 1998, pp. 219-231. [14].M. M. Rahman, A. Umar, K. Sawada, Development of amperometric glucose biosensor based on glucose oxidase co-immobilized with multi-walled carbon nanotubes at low potential, Sensors and Actuators B: Chemical, Vol. 137, Issue 1, 2009, pp. 327-333. [15].H. Tang, J. Chen, S. Yao, L. Nie, G. Deng, Y. Kuang, Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode, Analytical Biochemistry, Vol. 331, Issue 1, 2004, pp. 89-97. [16].C. Boero, S. Carrara, G. De Micheli, Sensitivity enhancement by carbon nanotubes: applications to stem cell cultures monitoring, in Proceedings of the Conference on PhD Research in Microelectronics and Electronics (PRIME’ 2009), Cork, Ireland, 12-17 July 2009, pp. 72-75. [17].E. Crounch, D. C. Cowell, S. Hoskins, R. W. Pittson, J. P. Hart, A novel, disposable, screen-printed amperometric biosensor for glucose in serum fabricated using a water-based carbon ink, Biosensors and Bioelectronics, Vol. 21, Issue 5, 2005, pp. 712-718. [18].X. Kang, Z. Mai, X. Zou, P. Cai, J. Mo, A novel glucose biosensor based on immobilization of glucose oxidase in chitosan on a glassy carbon electrode modified with gold-platinum alloy nanoparticles/multiwall carbon nanotubes, Analytical Biochemistry, Vol. 369, Issue 1, 2007, pp. 71-79. [19].D. A. Gough, L. S. Kumosa, T. L. Routh, J. T. Lin, J. Y. Lucisano, Function of an implanted tissue glucose sensor for more than 1 year in animals, Science Translational Medicine, Vol. 2, Issue 42, 2010, pp. 42ra53. [20].W. K. Ward, L. B. Jansen, E. Anderson, G. Reach, J. C. Klein, G. S. Wilson, A new amperometric glucose microsensor: in vitro and short-term in vivo evaluation, Biosensors and Bioelectronics, Vol. 17, Issue 3, 2002, pp. 181-189. ___________________ 2011 Copyright ©, International Frequency Sensor Association (IFSA). All rights reserved. (http://www.sensorsportal.com)

237

Sensors & Transducers Journal

Guide for Contributors Aims and Scope Sensors & Transducers Journal (ISSN 1726-5479) provides an advanced forum for the science and technology of physical, chemical sensors and biosensors. It publishes state-of-the-art reviews, regular research and application specific papers, short notes, letters to Editor and sensors related books reviews as well as academic, practical and commercial information of interest to its readership. Because of it is a peer reviewed international journal, papers rapidly published in Sensors & Transducers Journal will receive a very high publicity. The journal is published monthly as twelve issues per year by International Frequency Sensor Association (IFSA). In additional, some special sponsored and conference issues published annually. Sensors & Transducers Journal is indexed and abstracted very quickly by Chemical Abstracts, IndexCopernicus Journals Master List, Open J-Gate, Google Scholar, etc. Since 2011 the journal is covered and indexed (including a Scopus, Embase, Engineering Village and Reaxys) in Elsevier products.

Topics Covered Contributions are invited on all aspects of research, development and application of the science and technology of sensors, transducers and sensor instrumentations. Topics include, but are not restricted to:             

Physical, chemical and biosensors; Digital, frequency, period, duty-cycle, time interval, PWM, pulse number output sensors and transducers; Theory, principles, effects, design, standardization and modeling; Smart sensors and systems; Sensor instrumentation; Virtual instruments; Sensors interfaces, buses and networks; Signal processing; Frequency (period, duty-cycle)-to-digital converters, ADC; Technologies and materials; Nanosensors; Microsystems; Applications.

Submission of papers Articles should be written in English. Authors are invited to submit by e-mail [email protected] 8-14 pages article (including abstract, illustrations (color or grayscale), photos and references) in both: MS Word (doc) and Acrobat (pdf) formats. Detailed preparation instructions, paper example and template of manuscript are available from the journal’s webpage: http://www.sensorsportal.com/HTML/DIGEST/Submition.htm Authors must follow the instructions strictly when submitting their manuscripts.

Advertising Information Advertising orders and enquires may be sent to [email protected] Please download also our media kit: http://www.sensorsportal.com/DOWNLOADS/Media_Kit_2011.pdf