Development of Highly Sensitive,Label-Free DNA Sensors Based on Conducting Polymers

Abstract

DNA-sensors show a great promise as tools for various applications, such as clinical diagnosis, forensic analysis, environmental monitoring and biological research. Current researchers in this field are attempting to design and synthesize well-structured transducers to realize high-performance DNA sensing devices. Electronically conducting polymers (ECPs) are attractive transducing materials for DNA sensing because they exhibit the electronic, magnetic and optical properties of metals and semiconductors while retaining the mechanical properties and processing advantages of polymers. These features, along with chemical sensitivity, room temperature operation, and tunable charge transport properties, have launched conducting polymers as a major class of chemical transducers, creating powerful thin / thick film sensors. Since their first application to biosensing by Foulds and Lowe (1986) and Umana and Waller (1986) conducting polymers have been widely used in the field of biosensing, both as biomolecule immobilisation platform and as transducers of the biomolecular recognition. Considerable efforts have been made in recent years to develop nanostructured conducting polymer for DNA detection, with the aim of making portable and affordable devices. New approaches to miniaturize the sensing element have been sought that would satisfy these demands, at the same time improving the simplicity, selectivity and sensitivity of DNA detection. In this thesis, specific gene sensing approaches are discussed that focus on the effect of miniaturization of conducting polymer (CP) sensing elements based on a functionalized polypyrrole (PPy) and functionalized poly(3,4,-ethylenedioxythiophene (PEDOT) on microelectrodes and microfabricated electrodes, respectively. A first type of DNA sensing transducer was constructed using a copolymer of pyrrole (Py) and a carboxylic acid functionalized 3-pyrrolylacrylic acid (PAA) [poly(Py-co-PAA)] on a glassy carbon microelectrode (11 μm in diameter). The interfacial electron charge transfer resistances on the poly(Py-co-PAA)-ODN films were studied before and after hybridization with complementary ODNs using electrochemical impedance spectroscopy (EIS). Such DNA sensors showed sensitivity down to 1 nM concentration of target ODN specific to salmonella virus gene. In another case, we electrochemically fabricated poly(3,4,-ethylenedioxythiophene) (PEDOT) nanowires across a gap between two wedge-shaped gold electrodes just by applying AC field without using any template. We attempted to grow these PEDOT nanowires with different dopants like poly(styrene sulfonic acid) (PSS), 1-butyl-3-methylimidazolium hexafluorophosphate (BMIPF6), sodium chloride (NaCl), lithium perchlorate (LiClO4) and sodium hexafluorophosphate (NaPF6) with different solvents like water, propylene carbonate and acetonitrile. The factors affecting the electrochemical growth of these nanowires like the solvents, the electrolyte conductivity, dopant concentration, monomer concentration, the excitation frequency and amplitude and the electrochemical cell geometry were studied in detail and the knowledge was applied to construct a DNA sensing transducer based on a copolymer single nanowire of 2,3-dihydrothieno[3,4-b]-1,4-dioxin (EDOT) and carboxylic functionalized 2-((2,3-dihydrothieno[3,4-b]-1,4-dioxin-2-yl)methoxy) acetic acid (C2-EDOTCOOH) [poly(EDOT-co-EDOT-COOH)] across the gap of the gold electrodes. To the constructed single poly(EDOT-co-EDOT-COOH) nanowire, probe ODN was covalently attached through EDC/NHS chemistry. The changes in resistance of the wires upon hybridizing the probe modified surface with target ODN were measured both in-situ and exsitu of buffer solutions. The Ex-situ study was performed by using a two terminal device setup and in-situ measurements were performed by utilizing double-barrel-pipette Scanning Ion Conductance Microscopy (SICM) in the presence of phosphate buffer solution. The resultant sensing elements showed selectivity and sensitivity down to 10 fM (20 zepto mole/20 μL) of target- ODN specific to Human breast cancer gene.