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

B Lillis, M Manning, H Berney, E Hurley, A Mathewson, M M Sheehan
Biosensors & Bioelectronics 2006 February 15, 21 (8): 1459-67
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.

Full Text Links

Find Full Text Links for this Article


You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read

Save your favorite articles in one place with a free QxMD account.


Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"