Biosensors and Bioelectronics 25 (2009) 211–217 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor Wei Choon Alvin Koh a,b , Eun Sang Choe c , Dong Kun Lee c , Seung-Cheol Chang b , Yoon-Bo Shim a,b,∗ a Department of Chemistry, Pusan National University, Busan 609-735, Republic of Korea b Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, Republic of Korea c Department of Biological Sciences, Pusan National University, Busan 609-735, Republic of Korea article info Article history: Received 6 April 2009 Received in revised form 8 June 2009 Accepted 25 June 2009 Available online 3 July 2009 Keywords: All solid state ISE Immunosensor Neuronal nitric oxide synthase Potentiometry Urease conjugate abstract An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H + -selective ISE was fabricated with the carboxy- lated poly(vinyl chloride) (PVC-COOH) film containing H + ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to inter- action between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 g/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was -0.073 ± 0.002 mV/g ml -1 . The detection limit of nNOS was determined to be 0.2 g/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. 1. Introduction Nitric oxide (NO) is an important molecule due to its small size, high reactivity, and simple structure. This free radical species has been the subject of much research spanning many fields including physiology, medicine, environmental chemistry, and fundamental catalysis (Cheng and Richter-Addo, 2000). Evidence accumulated over the years has pointed to important roles of NO in a myriad of physiologic processes, including regulatory cardiovascular mech- anisms, neurotransmission, and immune host-defense (Guzik et al., 2003; Hibbs et al., 1990). NO is produced in vivo by the fam- ily of enzymes known collectively as nitric oxide synthases (NOSs). Although diverse research has been carried out to date, a number of issues still remain unclear regarding the exact mechanism or mech- anisms of NO synthesis by NOSs (Alderton et al., 2001; Hurshman and Marletta, 2002; Hurshman et al., 1999). Neuronal NOS (nNOS) is found predominantly in the brain. Activation of nNOS by phos- phate group removal is structurally distinct, and it is speculated ∗ Corresponding authors at: Chemistry Department and IBST, Pusan National Uni- versity, Busan 609-735, Republic of Korea. Tel.: +82 51 510 2244; fax: +82 51 514 2430. E-mail address: ybshim@pusan.ac.kr (Y.-B. Shim). that this process is uniquely tuned to support the nNOS function (Crane et al., 1998; Raman et al., 1998; Zhang et al., 2001). Careful investigation of the activation process of nNOS is therefore fun- damentally important and would contribute to our understanding of NOS catalysis (Raman et al., 1998; Wei et al., 2001). Thus, it is essential to develop a reliable detection method for nNOS. Generally, Western blot analysis (Smith and Titheradge, 1997) and fluorescent labeling (Wei et al., 2001) have been used for detection of nNOS. Other analytical methods, such as electron para- magnetic resonance (Zhang et al., 1991), fluorometry (Griffith and Stuehr, 1995), etc. have also been known to detect nNOS activity indirectly. These analytical techniques allow the specific determi- nation of various biologically active compounds using standard immunoanalytical methods, but they are complicated and time consuming. Thus, more simple and reliable methods such as elec- trochemical detection methods are developed in that they are less time-consuming, more sensitive, and fast. As of yet, there has been no extensive attempt to obtain more accurate and simple electro- chemical methods for the direct determination of nNOS. Of the electrochemical methods, potentiometry using ion-selective elec- trodes (ISEs) is used to detect gases or changes in pH. They are more advantageous due to simplicity, speed, sensitivity, being able to perform in vivo measurements, and have been widely used in many fields, including clinical diagnostics, industrial process 0956-5663/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.06.039