Ray, S; Steven, R T*; Green, F M; Hook, F*; Taskinen, B*; Hytonen, V P*; Shard, A G (2015) Neutralised chimeric avidin binding at a reference biosensor surface. Langmuir, 31 (6). pp. 1921-1930.

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Abstract

We describe the development of a reference biosensor surface, based upon a binary mixture of oligo-ethylene glycol thiols, one of which has biotin at the terminus, adsorbed onto gold as self-assembled monolayers (SAMs). These surfaces were analysed in detail by X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) to establish the relationship between the thiol solution composition and the surface composition and structure. XPS demonstrates that the biotin-containing thiol is well ordered and attaches exclusively through the thiol end with a packing density 1.5 times larger than the smaller oligo-ethylene glycol thiol which does not contain a biotin terminus. We report the use of argon cluster primary ions for the SIMS analysis of PEG-thiols, establishing that the different thiols are intimately mixed on the scale length of a primary ion impact (~10 nm) and that SIMS may be used to measure surface composition of thiol SAMs on gold with a detection limit approaching one molecule in a thousand. Surfaces with different biotin coverages ranging from 100% to 0.25% of maximum coverage were prepared.
The adsorption of neutralized chimeric avidin to these surfaces was measured simultaneously using in-situ ellipsometry and QCM-D. The total amount of avidin adsorbed was also determined by XPS and found to be in excellent agreement with the ellipsometry results. The results demonstrate that the reference biosensor surface produces repeatable, robust and reliable results. Comparison of the results from ellipsometry and QCM-D demonstrate the non-linearity between QCM-D areic mass and ellipsometric areic mass measurements. These are consistent with previous works describing the effect of mechanically coupled water on QCM-D frequency changes. The ellipsometric results display pronounced deviation from a simple Langmuir rate law. This is rationalized in the simplest possible manner using a model which incorporates both a random (Langmuir) adsorption process and a cooperative adsorption process in which adsorbed proteins promote the attachment of unbound protein at neighbouring sites. The model provides an excellent fit to the data.
The QCM-D dissipation data display unusual behavior. For biotin coverages greater than 1% of maximum, an initial rise in dissipation in the early stages of adsorption reaches a maximum after avidin is ~20% of the maximum adsorbed amount and then declines back to the original level. At 1% biotin coverage and below, this maximum is delayed and the dissipation signal continues to rise until, at 0.25% biotin coverage, there is no maximum and the dissipation signal is four times greater than the maximum observed for 100% biotin coverage. We show that the onset of the cooperative adsorption mechanism established from the ellipsometric data coincides with the maximum in the dissipation signal and that, at low biotin coverage, the lack of nearest-neighbour binding sites for avidin inhibits both the cooperative mechanism and the suppression of the dissipation signal. These results demonstrate that neutralized chimeric avidin, at least, attaches to these reference surfaces through two different mechanisms: A slow, random adsorption following Langmuir kinetics, which results in a compliant adsorbed layer and; a fast, cooperative mechanism catalyzed by preadsorbed neutralized chimeric avidin, which results in a rigid adsorbed layer.

Item Type:	Article
Subjects:	Nanoscience Nanoscience > Surface and Nanoanalysis
Identification number/DOI:	10.1021/la503213f
Last Modified:	02 Feb 2018 13:13
URI:	http://eprintspublications.npl.co.uk/id/eprint/6582

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