Luciferase‐free Luciferin Electrochemiluminescence

Abstract

Luciferin is one of Nature's most widespread luminophores, and enzymes that catalyze luciferin luminescence are the basis of successful commercial "glow" assays for gene expression and metabolic ATP formation. Herein we report an electrochemical method to promote firefly's luciferin luminescence in the absence of its natural biocatalyst-luciferase. We have gained experimental and computational insights on the mechanism of the enzyme-free luciferin electrochemiluminescence, demonstrated its spectral tuning from green to red by means of electrolyte engineering, proven that the colour change does not require, as still debated, a keto/enol isomerization of the light emitter, and gained evidence of the electrostatic-assisted stabilization of the charge-transfer excited state by double layer electric fields. Luciferin's electrochemiluminescence, as well as the in situ generation of fluorescent oxyluciferin, are applied towards an optical measurement of diffusion coefficients.

Separating Convective from Diffusive Mass Transport Mechanisms in Ionic Liquids by Redox Pro-fluorescence Microscopy

“…In addition to natural convection, it is possible that a lateral heterogeneity in current density (across the Pt mesh) may lead to material transport via diffusioosmotic fluid flow, , which in turn may lead to the observed asymmetry. The average D obtained in [EMIM]­[EtSO 4 ] is 1 order of magnitude smaller than in DMSO, as expected based on the higher viscosity of the former (∼100 vs 2.0 cP) …”

“…d -Luciferin sodium salt (≥95%, Cayman Chemical Company, Michigan) was used as starting material for the synthesis of firefly luciferyl adenylate (hereafter AMP–luc ). AMP–luc was prepared and titrated as described previously. , Pyridine (≥99.5%) and acetonitrile (≥99.9%, MeCN) were purchased from Honeywell (North Carolina). 1-Butyl-1-methylpyrrolidinium bis-(trifluoromethylsulfonyl)­imide (99.5%, [BMPyrr]­[NTf 2 ]) and 1-ethyl-3-methylimidazolium tetrafluoroborate (>98%, [EMIM]­[BF 4 ]) were purchased from Iolitec (Germany).…”

“…We have applied this optical approachredox pro-fluorescence microscopyto RTILs that have a different propensity for forming compact double layer cation-rich structures and hence a different intrinsic inhibition of adventitious inner-sphere gas-evolving redox reactions. The electrode-generated fluorophore is oxyluciferin ( ox–luc , Figure a), which is the final product of the cathodic ECL (Figure b and Video S1, Supporting Information) light path of non-fluorescent firefly’s luciferin adenylate ester ( AMP–luc ) …”

“…(a) Proposed mechanism for the cathodic electrochemiluminescence (ECL) of AMP–luc . (b) Reaction of AMP–luc (0.4 × 10 –3 M, oxygen-saturated, −2.0 V vs Ag/AgCl) with electrode-generated superoxide leads to the excited state of ox–luc , which relaxes emitting red light.…”

“…The electrode-generated fluorophore is oxyluciferin (ox−luc, Figure 1a), 56 which is the final product of the cathodic ECL (Figure 1b and Video S1, Supporting Information) light path of non-fluorescent firefly's luciferin adenylate ester (AMP− luc). 39 ■ EXPERIMENAL SECTION Materials. Unless stated otherwise, all reagents were of analytical grade and used without further purification.…”