Evaluating spectral overlap with the degree of quenching in UCP luminescence energy transfer systems.

The use of organic based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limits their effectiveness and longevity bothin vitroandin vivo. As an alternative, lanthanide doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and RET sensing; they exhibit high photo- and chemical stability and utilise near infrared excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd2SO4:Yb:Er (PTIR545) and Gd2SO4:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.

Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates.

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface-accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide-thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state.

Assessing the Covalent Attachment and Energy Transfer Capabilities of Upconverting Phosphors With Cofactor Containing Bioactive Enzymes.

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Examples of biosensors utilizing the apparent energy transfer of UCPs and nanophosphors (UCNPs) with biomolecules have started to appear in the literature but very few exploit the covalent anchoring of the biomolecule to the surface of the UCP to improve the sensitivity of the systems. Here, we demonstrate a robust and versatile method for the covalent attachment of biomolecules to the surface of a variety of UCPs and UCNPs in which the UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate biomolecules with surface-accessible cysteines. Variants of BM3Heme, cytochrome C, glucose oxidase, and glutathione reductase were then attached via maleimide-thiol coupling. BM3Heme, glucose oxidase, and glutathione reductase were shown to retain their activity when coupled to the UCPs potentially opening up opportunities for biosensing applications.

Evaluating spectral overlap with the degree of quenching in UCP luminescence energy transfer systems.

The use of organic-based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic-based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limit their effectiveness and longevity both in vitro and in vivo. As an alternative, lanthanide-doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and FRET sensing; they exhibit high photo- and chemical stability and utilise near infrared (nIR) excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd2SO4:Yb:Er (PTIR545) and Gd2SO4:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.

Imaging redox activity and Fe(II) at the microbe-mineral interface during Fe(III) reduction.

Dissimilatory iron-reducing bacteria (DIRB) play an important role in controlling the redox chemistry of Fe and other transition metals and radionuclides in the environment. During bacterial iron reduction, electrons are transferred from the outer membrane to poorly soluble Fe(III) minerals, although the precise physiological mechanisms and local impact on minerals of these redox processes remain unclear. The aim of this work was to use a range of microscopic techniques to examine the local environment of Geobacter sulfurreducens grown on thin films of Fe(III)-bearing minerals, to provide insight into spatial patterns of Fe(III) reduction and electron transfer. Confocal fluorescence microscopy showed that sparse biofilms formed on the mineral coatings, while the selective Fe(II) probe RhoNox-1 revealed Fe(II) patches on the minerals sometimes co-located with cells. Atomic force microscopy highlighted thin filamentous structures extending radially from the cell surface. Further analysis using fluorescent redox dyes showed redox-active, linear nanowires that formed cell to cell connections, although they were not implicated in playing a dominant role in direct electron transfer to the Fe(III) minerals. Overall this paper provides new methods and insights on studying Fe(III) reduction and other redox transformations in situ.