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.
Dwight
Stephen J
SJ
Department of Chemistry, and Institute for Polymers and Organic Solids, University of California, Santa Barbara, CA 93106, USA.
Gaylord
Brent S
BS
Hong
Janice W
JW
Bazan
Guillermo C
GC
D001360:Avidin / Q000032:analysis / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D002414:Cations, Monovalent; D005029:Ethylenediamines / Q000737:chemistry; D008094:Lithium / Q000737:chemistry; D010434:Pepsin A / Q000032:analysis / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D014867:Water / Q000737:chemistry
D002414:Cations, Monovalent; D005029:Ethylenediamines; D011145:Polyvinyls; D014867:Water; D001360:Avidin; D012710:Serum Albumin, Bovine; D008094:Lithium; D012964:Sodium; D010434:Pepsin A