Steady-State Fluorescence Anisotropy Studies of Molecularly Imprinted Polymer Sensors

Molecularly imprinted polymers (MIPs) are used as recognition elements in biochemical sensors. In a fluorescence-based MIP sensor system, it can be difficult to distinguish the analyte fluorescence from the fluorescence of the polymer itself. We studied steady-state fluorescence anisotropy of anthracene imprinted in a polymer (polyurethane) matrix. Vertically polarized excitation light was incident on MIP films coated on silicon wafers; vertically and horizontally polarized emission was measured. We compared the fluorescence anisotropy of MIPs with imprinted molecules, MIPs with the imprinted molecules extracted, MIPs with rebound molecules, and nonimprinted control polymers. It is shown that differences in fluorescence anisotropy between the polymers and imprinted fluorescent molecules may provide a means to discriminate the fluorescence of analyte from that of the background polymer. INTRODUCTION Molecularly imprinted polymers (MIPs) used as the recognition elements in biochemical sensors are of great interest [1–3]. The advantages of MIPs include their stability in a wide range of environments, their facility in sensor micro-fabrication, and their ability to detect analytes that are difficult or impossible to sense by immunoassay. MIPs commonly rely on fluorescence of a bound analyte to monitor the presence of an analyte. Unfortunately, the MIP polymers themselves also have fluorescence properties. Background fluorescence from the polymer will contaminate the fluorescence signal from the analytes, thereby dramatically decreasing the sensitivity of this type of MIP sensor [4]. Fluorescence anisotropy has been used to investigate polymer processing and characterization [5–7], fluorescent molecules in various polymer environments [8, 9], and fluorescent MIPs [10]. Anisotropy methods are based on the principle of photoselective excitation of fluorophores by polarized light. Fluorescence anisotropy from the analyte bound to a rigid system reveals information about the analyte’s local environment. In this study, we investigated the steady-state fluorescence anisotropy properties of imprinted polymers, and the analytes after they were bound to MIP recognition sites. The differences in anisotropy between the polymers themselves and the bound analytes were large enough to allow discrimination of the fluorescent analytes from the polymers. EXPERIMENTAL DETAILS Materials The MIP system tested was polyurethane imprinted with anthracene [11]. Imprinted MIPs were made from mixture of anthracene, 1.25 M solutions of monomers (0.375 mmol bisphenol A