Microchim Acta (2008) 160: 447–454 DOI 10.1007/s00604-007-0793-0 Printed in The Netherlands Original Paper Development of a myoglobin impedimetric immunosensor based on mixed self-assembled monolayer onto gold Morsaline Billah, Henry C. W. Hays, Paul A. Millner Institute of Membrane and Systems Biology, Garstang Building, University of Leeds, Leeds, United Kingdom Received 21 December 2006; Accepted 20 April 2007; Published online 9 July 2007 # Springer-Verlag 2007 Abstract. Immunosensors rely on antibody-antigen binding with a range of possible detection method- ologies. In this study, electrochemical impedance spectroscopy was used to monitor the sensor surface assembly and recognition of the analyte (myoglobin). Myoglobin is rapidly released into the circulatory sys- tem after an acute myocardial infarction and rapidly rising levels make it the first biochemical marker of myocardial damage. The immunosensor fabrication steps comprised the steps of (a) formation of mixed self-assembled monolayers (mSAM) on gold electrodes using a mixture of biotinyl-phospholipid and mer- captohexadecanoic acid; (b) neutravidin functionali- sation and (c) attachment of biotinyl anti-myoglobin antibodies. A range of analyte concentration (10 12 – 10 6 M) was successfully detected in phosphate buf- fered saline and in serum concentration ranging from 10% (v=v) to 100% (v=v) serum. Quartz crystal micro- balance and atomic force microscopy studies were carried out to study each step of fabrication to eluci- date binding characteristics and surface topography. Keywords: Mixed self-assembled monolayer; myoglobin; immu- nosensor; acute myocardial infarction; impedance The requirement of rapid identification and accurate biochemical diagnostics for chest pain and acute myo- cardial infarction (AMI) has led to the development of point-of-care devices including biosensors [1–3]. Monitoring a specific analyte in a complex envi- ronment such as blood plasma utilises recognition ele- ments such as enzymes, antibodies, receptors to detect the target analyte from a sample with high specificity and sensitivity [4]. Receptor based biosensors such as immunosensors have gained increasing popularity due to their inherent recognition capabilities against specif- ic analytes [5]. The absence of labelling requirements and the speed of analysis mean that immunosensors have gained increased recognition against conven- tional immunoassay techniques such as enzyme- linked immunosorbent assay (ELISA). Physical and chemical changes from immunodetection can be iden- tified using electrochemical [6], mass [7], calorimetric [8], or optical [9] properties. Various immobilisation procedures have been used to couple antibodies and antibody fragments onto suitable transducers [10–14]. Over the past decade, self-assembled monolayers (SAMs) have been employed as a platform for im- mobilisation [15–21]. The alkanethiol SAMs are employed to tether the recognition layers towards the detection specific analytes through immunoreac- tions using impedance and=or capacitance measure- ments [22–25]. Furthermore, it has also been shown possible to incorporate proteins and phospholipids Correspondence: Morsaline Billah, Institute of Membrane and Systems Biology, Garstang Building, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, United Kingdom e-mail: bmbmbi@bmb.leeds.ac.uk