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'-pyridyl pyridinium 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.