Dual polarisation interferometry characterisation of DNA immobilisation and hybridisation detection on a silanised support.

Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterise the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19 mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was approximately 35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was approximately 85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.

Characterization and optimization of an optical DNA hybridization sensor for the detection of multi-drug resistant tuberculosis.

The development of an optical DNA hybridization biosensor based on ruthenium electrochemiluminescence (ECL) was designed to detect DNA hybridization, using the detection of specific DNA sequences, which are indicative for antibiotic resistant strains of Mycobacterium tuberculosis as a model. The development of the sensor involved the characterization and optimization of the individual elements of the biosensor. Using this approach it was possible to ensure that the complete biosensor system was optimized to its maximum sensitivity for the electrochemiluminescent optical signal, which is produced during the DNA hybridization event. The transduction element used in this work is a solid-state silicon PIN photodiode. The miniaturization of the optical element to 5 mm(2) and smaller has made it possible to integrate the optical element internally as part of the biosensor scheme. Ruthenium ECL is a chemiluminescent reaction that is initiated by electrical stimulation. Photolithographic techniques were used to deposit and pattern gold electrodes directly onto the surface of the PIN photodiodes for the initiation of the ECL reaction. The gold electrodes also act as a biological support layer for DNA immobilization via thiol -gold linkage chemistry. The characterization and optimization of the individual components of this biosensor have allowed us to fabricate a miniaturized and integrated system that is compatible with a flow-through system.

Development of a capacitive immunosensor: a comparison of monoclonal and polyclonal capture antibodies as the primary layer.

There is widespread interest in capacitance immunosensor systems which directly detect antigen binding to immobilized antibody. Our system comprises an active biolayer of antibodies bound to a silicon--silicon dioxide--silicon nitride (Si-SiO2-Si3N4) surface. As with other groups, our system initially gave poorly reproducible responses on addition of antigen. We mechanically degraded the Si-SiO2-Si3N4 surface, and the responses on addition of transferrin were monitored. The mechanical degradation allowed the affinity reaction to be 'seen' capacitively. Once the system was established, a comparison of capture antibodies was performed to establish the most effective biolayer. Three affinity reactions were examined: (a) 1D2A4, monoclonal antibody (mAb) to human transferrin, as the capture layer; (b) polyclonal goat anti-human transferrin antibody (PcAb) as the capture layer; and (c) 1D2A4 with transferrin (Tf) prebound as the capture layer. There was no response to addition of transferrin where 1D2A4 was the capture layer. Addition of transferrin when the polyclonal antibody was used as the primary layer resulted in a drop in measured capacitance. Addition of goat anti-human transferrin antibody to a device with 1D2A4 plus transferrin as the capture layer also resulted in a measured capacitance decrease. There is a difference in dielectric/blocking effectiveness between the monoclonal and polyclonal antibodies.