Biotin selective polymer nano-films

Abstract

Background: The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements.

Results: Biotin-selective thin (3-5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N,N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts.

Conclusions: Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems.

Figure 1

Structures of chemicals used. Biotin methyl ester (1), N,N'-methylenebisacrylamide (MBA, 2), 2-acrylamido-2-methylpropanesulfonic acid (AMPS, 3), benzophenone (4) and hexadecanethiol (HDT, 5).

Figure 2

Schematic illustration of molecularly imprinted polymer grafted SAM-Au/quartz nano-films. (i) Self-assembly of hexadecanethiol on Au, (ii) physisorption of benzophenone photoinitiator, (iii) cover surface with pre-polymerization mixture, (iv) cover with glass slide, (v) irradiate with UV light and (vi) template extraction.

Figure 3

Surface characterization of the polymer films. Topography of (a) Au/quartz resonator (b) MIP and (c) REF polymer films measured using AFM. (d) Scanning electron micrograph of the REF polymer film. (e) RAIR spectra of MIP and REF polymer films.

Figure 4

Sensitivity and kinetics of polymer-ligand recognition. (a) Resonant frequency change as a function of BtOMe concentration. Inset is the calibration plot for BtOMe on the MIP (n = 3, 6 injections per surface) and REF (n = 1, 6 injections) films. (b) Variation of observed k_app calculated from the associative part of the frequency vs time response curves for BtOMe binding to MIP and REF films .

Figure 5

Selectivity of polymer-ligand recognition. (a) Frequency response for dextran and biotinylated dextran (B-Dextran) binding to the MIP film. (b) Resonant frequency changes for the binding of different analytes to MIP and REF films. * p > 0.05, ** p < 0.01, n = 3-8, 3–6 injections per surface.

Figure 6

Stability of polymer films. Resonant frequency changes for the binding of BtOMe to the MIP film after prolonged storage in the dry state. Analyte concentrations used were 10 mM (red), 7.5 mM (green), 5 mM (black), 4 mM (yellow) and 2 mM (pink). Calibration was carried out on one MIP film at specified time intervals after fabrication (6 injections for one surface). Inset shows the sensor response for BtOMe (5 mM) injections under continuous flow (20 μL/min) after 0, 24, and 48 h.