Immobilized Streptavidin Gradients as Bioconjugation Platforms

Abstract

Surface density gradients of streptavidin (SAV) were created on solid surfaces and demonstrated functionality as a bioconjugation platform. The surface density of immobilized streptavidin steadily increased in one dimension from 0 to 235 ng cm(-2) over a distance of 10 mm. The density of coupled protein was controlled by its immobilization onto a polymer surface bearing a gradient of aldehyde group density, onto which SAV was covalently linked using spontaneous imine bond formation between surface aldehyde functional groups and primary amine groups on the protein. As a control, human serum albumin was immobilized in the same manner. The gradient density of aldehyde groups was created using a method of simultaneous plasma copolymerization of ethanol and propionaldehyde. Control over the surface density of aldehyde groups was achieved by manipulating the flow rates of these vapors while moving a mask across substrates during plasma discharge. Immobilized SAV was able to bind biotinylated probes, indicating that the protein retained its functionality after being immobilized. This plasma polymerization technique conveniently allows virtually any substrate to be equipped with tunable protein gradients and provides a widely applicable method for bioconjugation to study effects arising from controllable surface densities of proteins.

Properties and reactivity of polyoxazoline plasma polymer films

“…Although the amount of gold detected via XPS is small, it is in good agreement with previous reports. 44 This suggests that after immobilization to PPOx coatings, the streptavidin molecules are in their functional, bioactive conformation.…”

A Mechanistic Study of the Plasma Polymerization of Ethanol

“…Deposition at low RF power allowed higher retention of oxygen into the films which in turn almost completely eliminated protein fouling. The same authors used this knowledge to create advanced gradient surfaces of protein number density . One end of the surface was deposited from plasma of pure ethanol which provided low protein adsorption.…”

A novel flash detection algorithm for single molecule counting with TIRF microscopy

“…Surface chemistries for studying albumin adsorption were generated by RFGD plasma deposition of allylamine or ethanol groups onto glass coverslips (Menzel, Germany) as previously described [15]. Labelled albumin solutions were loaded into Flexiperm ® wells (Greiner, Germany) on coverslips ½ hour prior to imaging.…”