(687f) Ask a Simple question… Pitfalls in Analysis of Thread/Loop Polymer Structures On Solid Surfaces

A common technique for immobilization of antibodies, enzymes and other biomolecules is to include a polymer ?tether? between the surface and the target. The tether presumably increases the biomolecule's solvent accessibility and mobility, improving activity and stability by reducing steric hindrance and surface-induced denaturation. One practical approach to making these tethers is to immobilize a commercial telechelic (end-functionalized) polymer on an activated surface. The pendant reactive ends of ?threads? can be further activated to immobilize the target biomolecules. However, ?loops? may also form if the free end of the ?thread? reacts with adjacent surface sites, anchoring the polymer at both ends.

In a typical experiment from the literature, a metal oxide surface (e.g. silica) is first activated with an epoxy- or aldehyde-bearing organosilane. The resulting reactive surface is then exposed to an amine-functional telechelic polymer under a variety of conditions, and the immobilized brush is used ?as-is? for further conjugation. Since few authors characterize the structure of their brush layers in detail, we set out to investigate the reaction conditions that affect the ratio of threads and loops. Such tailored reactive brush layers could be useful in enzymatic microreactors or biosensors.

The question begs to be asked: If a homobifunctional end-functional polymer is immobilized on a reactive surface, does it form a ?thread? or a ?loop?? In this cautionary tale, we describe how a research project started from this apparently simple question, and became progressively more and more complicated. A variety of schemes from the bioconjugation and peptide chemistry literature were attempted, but no simple reproducible analytical method was forthcoming. We discuss experimental difficulties related to the analysis and characterization of brush layers, and some pitfalls caused by cross-reactivity and side-reactions of common reagents.

Materials and Methods: In this study, silica microspheres were functionalized to produce amine-reactive epoxide surface coatings. Various mono- and bi-functional amine-terminal compounds and polymers were covalently linked to the epoxide surface by nucleophilic attack or reductive amination in dry organic solvent. The distribution of primary and secondary amines on the modified microspheres was investigated by physical methods (IR, TGA, and XPS), and differential chemical labeling procedures. Typically, amine-reactive dyes (e.g. FITC, dabsyl chloride, etc.) were used to label surface-bound amines. The labeled silica was then dissolved in NaOH solutions, and spectrophotometry used to quantify dye in the solution. Other chemistries were also attempted, including protection or blocking of primary amines with anhydrides prior to labeling, and specific labeling of primary amines with reagents such as Traut's reagent in combination with pyridyl disulfides. None of these methods provided reproducible results, partially due to side-reactions and other effects that are often poorly documented in the bioconjugation literature.