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

Lookup NU author(s): Dr Helen Berney

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Abstract

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 ∼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 ∼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.


Publication metadata

Author(s): Lillis B, Manning M, Berney H, Hurley E, Mathewson A, Sheehan MM

Publication type: Article

Publication status: Published

Journal: Biosensors and Bioelectronics

Year: 2006

Volume: 21

Issue: 8

Pages: 1459-1467

ISSN (print): 0956-5663

ISSN (electronic): 1873-4235

Publisher: Elsevier

URL: http://dx.doi.org/10.1016/j.bios.2005.06.009

DOI: 10.1016/j.bios.2005.06.009

PubMed id: 16112566


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