Self-quenching Mechanism: the Influence of Quencher and Spacer on Quencher-fluorescein Probes

Abstract

Fluorescent chemosensors provide an extremely sensitive optical method for the real-time monitoring of molecular recognition events. An understanding of the fluorescent on/ off mechanism is essential for the rational design of fluorescent chemosensors. The majority of fluorescent chemosensors working in aqueous media operate by one of three mechanisms: (1) the suppression of photoinduced electron transfer (PET); (2) alteration of the microenvironment of a solvatochromic fluorophore; and (3) modulation of the fluorescence resonance energy transfer (FRET) between two fluorophores. The first two mechanisms are suitable for small molecule sensing (e.g., cations, small organic compounds, etc.), but FRET is very useful in the biopolymer state, for its distance information can be obtained in the 10 to 100 A region. In spite of the excellent applications of FRET for biomolecule sensing (c-AMP, insulin, etc.), the operations depend on a complicated biological pathway. Peptides with an FRET pair have been used as monitoring probes for a given enzyme activity. However, in various fluorophore pairs that have been examined, FRET peptide probes did not operate properly i.e., they were self-quenched. This self-quenching can be explained in terms of an intramolecular ground-state dimer complex between the FRET fluorophore pair in an aqueous solution. The strength of the aggregation between the two dye molecules depends on the molecular structure, solvent, temperature, and the presence or absence of electrolytes. Our aim is to systematically study the self-quenching mechanism for covalently linked fluorophore pairs. For simplicity, the factors influencing the efficiency of selfquenching are disassembled into four components (Figure 1). A FRET pair was replaced by a quencher-fluorophore pair as the reporting group. This overcomes the restriction for the FRET donor-acceptor resonance condition. From a practical viewpoint, water and fluorescein were chosen as the solvent and fluorophore, respectively; water is a crucial solvent for physiological systems; furthermore, fluorescein has a high fluorescence quantum yield in aqueous solution and its synthetic handling has been well established. As the quencher part, carbazole, methyl red and 1,8naphthalimide were selected. The effect of the spacer can be controlled by varying the alkyl chain length (-(CH2)n-, n = 5, 11, 15) and a flexible tetraethylene glycol unit (Figure 2). At pH 7.5 (50 mM HEPES buffer, I = 0.1 (NaNO3)), the fluorescence emission of C-n (n = 5, 11, 15) and C-TE ([probes] = 1 μM) was measured (Figure 3). It has been reported that there are two opposing factors determining the stability of aggregates of the derivatives containing two Rose Bengal moieties: the longer the chain, the easier it is to