Time-resolved energy transfer in DNA sequence detection using water-soluble conjugated polymers: The role of electrostatic and hydrophobic interactions

Abstract

We have investigated the energy transfer processes in DNA sequence detection by using cationic conjugated polymers and peptide nucleic acid (PNA) probes with ultrafast pump-dump-emission spectroscopy. Pump-dump-emission spectroscopy provides femtosecond temporal resolution and high sensitivity and avoids interference from the solvent response. The energy transfer from donor (the conjugated polymer) to acceptor (a fluorescent molecule attached to a PNA terminus) has been time resolved. The results indicate that both electrostatic and hydrophobic interactions contribute to the formation of cationic conjugated polymers͞PNA-C͞ DNA complexes. The two interactions result in two different binding conformations. This picture is supported by the average donor-acceptor separations as estimated from time-resolved and steady-state measurements. Electrostatic interactions dominate at low concentrations and in mixed solvents. C onjugated polymers are novel materials with useful optical and electronic properties (1). Although structural disorder causes the effective localization length (conjugation length) to be significantly shorter than the actual chain length, an excitation can migrate along the chain before it is quenched via electron transfer to a nearby quencher (2-5) or before the excitation energy is transferred to a nearby acceptor (6 -10). Thus, conjugated polymers function as light-harvesting materials and thereby exhibit greatly enhanced quenching efficiencies via electron transfer (2-5) and optical amplification via Förster resonance energy transfer (FRET) (6 -10). Because of these exceptional properties, conjugated polymers offer potential for use in detecting biological and chemical target molecules with high sensitivity (2, 7-10).To develop homogenenous biosensors, water solubility is essential. Water-soluble conjugated polymers (conjugated polyelectrolytes) are obtained by terminating the solubilizing side groups of traditional conjugated chains, e.g., poly(phenylene vinylenes) or polyf luorenes, with charged moieties (8,9,11,12). Positively charged (cationic) conjugated polymers (CCPs) are particularly useful because biopolymers such as DNA and RNA are negatively charged. When these negatively charged biopolymers are labeled with f luorescent molecules, electrostatic interactions bring the f luorescent labels sufficiently close to the CCP to enable fast and efficient FRET.The long-range excitation energy transfer from donor to acceptor via the dipole-dipole interaction was initially described by Förster (6). The rate of energy transfer for donor and acceptor separated by a distance r DA is given by (13):D is the lifetime of donor in the absence of acceptor, Q D is the quantum yield of the donor in the absence of acceptor, N is Avogadro's number, n is the refractive index of the medium (typically assumed to be 1.4 for biomolecules in aqueous solution), and 2 is a factor describing the relative orientation of the transition dipoles of the donor and acceptor. The integral, J(), expresses the degree of spectral o...

Design of fluorescent materials for chemical sensing

“…The use of water-soluble conjugated polymers for biosensors applications has been extensively studied by Bazan and coworkers. [121][122][123][124][125][126] Fluorescent conjugated polymers as 2,29-bipyridyl-phenylene-vinylene-based polymers, 127 terpyridine-based poly(p-phenylene-ethynylene)-alt-(thienylene-ethylene) polymers, 128 poly(p-phenylene-ethynylene) 129 and others 130 have been reported as sensitive probes for the detection of metal ions. 131 ''Turn-on'' chemosensors based on CP have been developed for the sensing of metal ions by Jones and coworkers.…”

Bimolecular Excited-State Electron Transfer with Surprisingly Long-Lived Radical Ions

“…19 An innovative approach utilizing the electrostatic interactions between cationic CPEs with negatively charged electron acceptor moieties has therefore attracted extensive attention. 8,15,[20][21][22][23][24] For example, this approach was used to achieve strong electrostatic interactions between cationic CPEs and DNA base pairs, allowing the detection of DNA based on fluorescence quenching of the cationic CPEs. 5 In addition, efficient energy transfer from photoexcited cationic polyfluorene, a CPE, to porphyrin demonstrated that the cationic polyfluorene can form an electrostatic assembly with small molecules and act as a photosensitizer in the assembly.…”

SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification

“…A recent report from Heeger et al (43) estimated this distance for the same CP structure and a similar PNA-FL͞DNA acceptor complex by using the steady state fluorescence and absorption spectra of the donor-acceptor pair. Experimental separation distances were reported for time-resolved spectroscopy measurements and provided values within the estimated R o of 37.2 Å.…”