Perturbation of Fluorescence by Nonspecific Interactions between Anionic Poly(phenylenevinylene)s and Proteins:  Implications for Biosensors

The use of anionic water-soluble conjugated polymers (CPs) for sensing the presence of avidin by use of a biotin-modified fluorescence quencher was studied. The molecules involved in the study included poly[2-methoxy-5-(3‘-propyloxysulfonate)-1,4-phenylenevinylene] with either lithium (Li⁺-MPS-PPV) or sodium (Na⁺-MPS-PPV) countercations, the well-defined oligomer pentasodium 1,4-bis(4‘(2‘ ‘,4‘ ‘-bis(butoxysulfonate)-styryl)-styryl)2-butoxysulfonate-5-methoxybenzene (5R5^-), the quenchers N-methyl-4,4‘-pyridylpyridinium iodide (mMV⁺) and [N-(biotinoyl)-N‘-(acetyl 4,4‘-pyridylpyridinium iodide)] ethylenediamine (BPP⁺), which contains a molecular recognition fragment (biotin) attached to a unit that accepts an electron from a CP excited state, and the proteins avidin, tau, BSA, and pepsin A. Fluorescence quenching experiments were examined in a variety of conditions. Experiments carried out in water and in ammonium carbonate buffer (which ensures avidin/biotin complexation) reveal that nonspecific interactions between the CP and the proteins cause substantial perturbations on the CP fluorescence. The overall findings are not consistent with a simple mechanism whereby avidin complexation of BPP⁺ leads to encapsulation of the quencher molecule and recovery of Li⁺-MPS-PPV fluorescence. Instead, we propose that binding of BPP⁺ to avidin results in the quenching unit attaching to a positively charged macromolecule. Electrostatic attraction to the negatively charged conjugated polymer results in closer proximity to the quencher. Therefore, more enhanced fluorescence quenching is observed.