Fabrication of electrochemical biosensors for detection of microorganisms

Pathogenic bacteria are responsible for around two million deaths and many reported hospitalization per year worldwide. Streptococcus pyogenes is a Gram +ve bacteria responsible for invasive and non-invasive infections. Traditional molecular and immunological detection systems are usually time consuming, require well equipped laboratory facilities and skilled personnel. Biosensors are ideal for the early detection of microorganisms because of their on-site, sensitive and rapid detection capability and electrochemical impedance offers a highly sensitive signal following target analyte interaction. In this study, immunosensors were fabricated on commercial screen printed gold electrodes to detect S. pyogenes. Electrochemical impedance spectroscopy (EIS) was carried out as the primary technique with other supporting methods including cyclic voltammetry (CV), on-sensor chemiluminescence characterization (termed ‘Midland blotting’) and fluorescence microscopy to successfully detect bacteria. With unstable impedance using conducting polymer and inconsistent signal using self-assembled monolayer (SAM), a non-conducting polymer, polytyramine was selected and optimized. Polyclonal antibodies against heat killed intact S. pyogenes was immobilized using two methods. First, biotinylated antibodies were immobilized via biotin-NeutrAvidin; second, reduced half antibody fragments were coupled using a bifunctional linker. The latter produced a higher signal due to the oriented half antibodies and close distance from the sensor surface. The surface density of antibodies was found to be critically important to get optimum signal. S. pyogenes cell surface proteins M and H, contributed towards the non-specific signal and this was reduced using protein A/ G as specific blocking agents. In addition, an attempt was made to polymerize novel phenolic copolymers in order to minimize non relevant protein interference from biological samples. The optimized full antibody based sensors detected S. pyogenes both in buffer to 104 cells/ml and in spiked 50% human saliva in PBS (v/v) with high specificity and selectivity.