Electrochemical studies of cystine modified self-assembled monolayer for Escherichia coli detection Chandra Mouli Pandey a , Gajjala Sumana a , K.N. Sood b , B.D. Malhotra a, ⁎ a Department of Science & Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, Materials Physics & Engineering Division, National Physical Laboratory (Council of Scientific & Industrial Research), Dr. K. S. Krishnan Marg, New Delhi 110012, India b Electron Microscope Section, Division of Material Characterization, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India abstract article info Available online 29 September 2010 Keywords: Cystine Escherichia coli Methylene blue Self-assembled monolayer Electrochemical DNA biosensor An electrochemical DNA biosensor based on cystine modified self-assembled monolayer (cys-SAM) onto gold electrode (AuE) has been fabricated for Escherichia coli (E. coli) detection. This biosensing electrode has been characterized using scanning electron microscopy (SEM), FT-IR spectroscopy, cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Under the optimum conditions, this DNA biosensor can be used to detect complementary target DNA concentration in the range of 1×10 -6 M to 1×10 -20 M within 60 s of hybridization time at 25 °C and has been found to be stable for about four months when stored at 4 °C. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Micro-organism detection in food and water is considered very important and the presence of Escherichia coli (E. coli) is used as a potential marker for diagnoses of water borne diseases affecting humans. Some strains of E. coli cause toxic effect resulting in diarrhoea, urinary tract infection, inflammation and peritonitis [1]. The conventional methods for E. coli detection include multiple-tube fermentation, membrane filter and plate counts that are accurate and sensitive. However, these techniques have certain limitations such as operational complications, lack of specificity and are time consuming (24–48 h). Therefore, rapid and sensitive technique for E. coli detection is urgently needed [2]. Electrochemical DNA biosensors based on the conversion of molecular recognition reactions into useful response signals, offer considerable promise for DNA analysis. Many approaches have been followed for the fabrication of electrochemical nucleic acid biosensors. In this context, self-assembled monolayers (SAMs) can be used for immobilization of desired biological molecules on a wide variety of substrates [3,4]. SAMs of organosulfur compounds such as alkane thiols and disulfides on Au surface have received much attention due to their potential applications in the field of electro-analytical chemistry [5–8]. SAMs are considered attractive for many applications including biotechnology, sensors, molecular recognition, etc. [9–13]. The orientation of SAM plays an important role in the design of artificial biomolecular recognition devices by providing flexibility and the possibility of controlling the biomolecular surface. Therefore, in the present work, studies related to the fabrication of cystine, (SCH 2 CHNH 2 COOH) 2 modified SAM/Au based E. coli biosensor are reported. 2. Experimental details 2.1. Materials All the chemicals and oligonucleotide sequences have been procured from Sigma-Aldrich (USA). Buffers have been prepared in Millipore water (Milli-Q, 18.2 MΩ). Cystine (cys) was prepared as 100% ethanolic solution. E. coli specific probe (17 bases) is identified from the16s rRNA coding region of the E. coli genome. The probe DNA sequences and complementary target sequence are as follows: Oligonucleotide probe sequence (ssDNA) Amine (CH 2 ) 3 —5′-GGT CCG CTT GCT CTC GC-3′ Amine (CH 2 ) 6 —5′-GGT CCG CTT GCT CTC GC-3′ Amine (CH 2 ) 12 —5′-GGT CCG CTT GCT CTC GC-3′ Complementary oligonucleotide sequence 5′-GCG AGA GCA AGC GGA CC-3′ 2.2. Preparation and modification of the electrode Pre-cleaned gold electrodes (AuE) are modified by immersion in cystine ethanolic solutions of 1 mM concentration for fixed time intervals followed by sonication in water–ethanol (1:1) solution for about 10 min and is finally rinsed with deionized water and dried. The cys-SAM electrode is modified with N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (EDC, 0.2 M) and N-hydroxysuccini- mide (NHS, 0.05 M) acting as a coupling agent [14–17], which activates Thin Solid Films 519 (2010) 1178–1183 ⁎ Corresponding author. Tel.: + 91 11 45609152; fax: + 91 11 45609312. E-mail address: bansi.malhotra@gmail.com (B.D. Malhotra). 0040-6090/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2010.08.065 Contents lists available at ScienceDirect Thin Solid Films journal homepage: www.elsevier.com/locate/tsf