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Upconversion nanoparticles (UCNPs) can provide a vehicle for chemical imaging by coupling chemically sensitive dyes and quenchers. The mechanism for coupling of two anthraquinone dyes, Calcium Red and Alizarin Red S, was investigated as a function of pH. The green emission band of the UCNPs was quenched by a pH-dependent inner filter effect (IFE) while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. Contrary to previous expectation, there was little evidence for a resonance energy transfer (RET) mechanism even when the anthraquinones were attached onto the UCNPs through electrostatic attraction. Since the UCNPs are point emitters, only emitters close to the surface of the UCNP are within the expected Förster distance and UC-RET is <10%. The theoretical and experimental analysis of the interaction between UCNPs and pH-sensitive quenchers will allow the design of UCNP pH sensors for determination of pH via IFE. [ABSTRACT FROM AUTHOR]

Taking advantage of BRET, a mutant firefly luciferase with higher pH- and thermo-stability than the wild-type could be coupled with the red-emitting fluorescent protein of mCherry in both a fused and unfused format. The BRET pair allows >40% of the light emitted to be red shifted over 600 nm to the mCherry acceptor wavelength. Taking the expected quantum yield for mCherry (0.22), a good fit to predicted light transfer is shown, with no other losses. Two measurements are considered for ATP determination: (a) a ratiometric technique for ATP measurement using both donor and acceptor emission intensities, making the calibration slope independent of protein concentration in a broad range. This measurement was limited by the BRET efficiency and the low quantum yield of the mCherry acceptor, but this detection limit might be improved with other fluorescent proteins with higher quantum yield. The fused BRET pair also resulted in a small increase in the BRET ratio. (b) An ATP dependent shift in the wavelength maximum using just the acceptor mCherry emission was also proposed for ATP determination. This did not require a high BRET efficiency and only uses emission above 600 nm to obtain the acceptor emission maximum, but not its intensity; it is independent of protein concentration across a broad range. This offers a novel and robust method for determination of ATP between 10−11 to 10−5 M with an easy baseline calibration with ATP concentration >10−4 M. [ABSTRACT FROM AUTHOR]

Gold nanoparticle labelling has been shown to produce a remarkable improvement in sensitivity for small molecule detection based on Surface Plasmon Resonance (SPR) bio-sensing. The LSPR (localised SPR)-SPR coupling effect and size/mass-material properties associated with gold nanoparticles are the two main factors to change the SPR resonance condition and cause the enhancement. In this paper we examine the separation of these factors in the context of a classical SPR bio-interaction assay format, and consider the implications on the design of biodetection systems to maximise response. The coupling effect plays a distance dependent role, which allows it to be mapped. The dominant enhancement associated with this factor only occurs within ∼8 nm for a 20 nm gold nanoparticle and is changing by ∼7 nm wavelength shift/nm distance from the Au SPR thin film. Beyond this distance, the size/mass associated with the nanoparticle itself dominates the enhancement. This is demonstrated in a 20-mer DNA sequence sandwich detection, where the enhancement ratio between the coupling effect and the mass is ∼1.5 : 1. This simple method for deconvolution of the mass and coupling effects allows consideration of formats with LSPR nanoparticle labelling for small molecule detection and the best design of the labelling geometry.

In this work, the first CdSe/ZnS quantum dot (QD) photoluminescence lifetime based chloride ion nanosensor is reported. The acridinium dication lucigenin was self-assembled on the surface of negatively charged mercaptopropionic acid capped QDs to achieve QD-lucigenin conjugates. Upon attachment, a drastic decrease of the photoluminescence lifetime of both QD nanoparticles and lucigenin is observed by virtue of a charge transfer mechanism. Since lucigenin is a chloride-sensitive indicator dye, the photoluminescence decay of QD-lucigenin conjugates changes by adding chloride ion. The photoluminescence lifetime of the QDs in the conjugate increases after reacting with Cl(-), but also shows a concomitant decrease in the lucigenin lifetime immobilized on the surface. The photoluminescence lifetime of QD-lucigenin nanosensors shows a linear response in the Cl(-) concentration range between 0.5 and 50 mM. Moreover, the ratio τ(ave)(QD)/τ(ave)(luc) can be used as an analytical signal since the lifetime ratio presents a linear response in the same Cl(-) concentration range. The system also shows good selectivity towards most of the main anions and molecules that can be found in biological fluids. These nanosensors have been satisfactorily applied for Cl(-) determination in simulated intracellular media with high sensitivity and high selectivity. Finally, we demonstrate the potential application of the proposed nanosensor in confocal fluorescence lifetime imaging (FLIM). These results show the promising application of the QD-lucigenin nanosensors in FLIM, particularly for intracellular sensing, with the invaluable advantages of the time-resolved fluorescence techniques.

A hollow 3 μm sensing microcapsule containing chromoionophores within a 100 nm organosilica shell is reported. This shows a response to an ion step two orders of magnitude faster than 'filled' sensing particles of similar diameter. Incorporation of chromoionophores ETH 5294 and ETH 7061 in the capsule shell is shown with a t(90) response <2 s compared with >15 min for filled particles of similar diameters. This ultrafast geometry is also extended to dual ionophore dye incorporation and preliminary exploration of a FRET-based ratiometric method is examined to extend the pH response range, using a single excitation wavelength.

A silica based analytical nanosphere sensor (ANSor) containing quantum dots (QDs) is reported, which can measure local pH in a ratiometric fashion. A silane modified reference QD was incorporated into a silica matrix by the Stöber method hydrolysis and polycondensation of tetraethoxysilane, giving highly fluorescent QD-embedded silica particles with high yield. A further QD was then bonded onto the silica particle surface and modified with Nile Blue to render it pH responsive. These two populations of QDs were excited simultaneously and gave out well-separated emission peaks which could be taken as a ratio to yield a ratiometric estimate of pH. The sensors are stable, robust and capable of measuring pH in the physiologically relevant range.

Photoexcited quantum dots (QDs) offer a wealth of mechanisms for interactions with the valence band holes or conduction band electrons, influencing electron-hole recombination. The potential to use combinations of these mechanistic pathways to achieve detection of different metal ions with one modified QD system has been tested. Dual-function water-soluble core/shell-modified CdSe/ZnS quantum dot nanoparticles have been created, that exploit two different fluorescence emission pathways for the detection of two heavy metal ions: Zn(2+) and Mn(2+). A QD-zincon system is proposed, which shows a static Perrin-type quenching mechanism, with sphere of action radii 1.1, 1.3 and 1.6 nm respectively, for 500, 540 and 620 nm QD emission. The QD-zincon system was produced using a layer-by-layer approach: mercaptopropionic acid-capped QDs were modified with a positive polyelectrolyte by electrostatic interaction and then a negatively charged chromogenic reagent, zincon, classically used for the determination of metals. QD-zincon is able to coordinate both Zn(2+) and Mn(2+) and, by exploiting two different mechanisms, QD-zincon conjugates can be tailored to respond to Zn(2+) or Mn(2+). Upon coordination of zincon with Mn(2+), a dramatic enhancement of the fluorescence intensity results as the quenching interaction between zincon and QDs is deactivated, thereby 'switching on the fluorescence emission'. The versatility of this system is demonstrated in terms of fluorescent emission wavelength, which could be selected across a wide range, through choice of QDs (examples are shown for lambda(max) = 500, 540 and 620 nm). In contrast, in the case of Zn(2+) detection, the mechanism is based on the radiationless resonance energy transfer (RET) from QDs acting as donor, to the acceptor zincon-Zn(2+), since its absorption spectra offer adequate overlap with the emission spectra of QD(540) and QD(620), producing a useful analytical signal by the RET process. Using these different operating principles, CdSe/ZnS core/shell QD-zincon conjugates showed very good linearity in the range 10-1000 microM and 5-500 microM for Zn(2+) and Mn(2+) nanosensors, respectively, and RSDs about 3% (n = 10). In a study of interferences, the QD-zincon conjugate showed higher selectivity than the corresponding method with zincon in solution. The results from synthetic ionic mixtures suggested very good applicability in the determination of Zn(2+) and Mn(2+) in samples containing other metal ions, with just a small reduction of sensitivity at very high ionic concentration.

In this work, the first chloride ion sensor based on QD-lucigenin nanoparticles is reported. The mechanism uses the ability of semiconductor QDs to engage in short range exchange processes, leading to fluorescence changes. An acridinium dication (lucigenin) which is an electron acceptor, was self-assembled on the surface of negative charged QDs (capped with mercaptopropionic acid). Mutual quenching of the lucigenin and QD were observed. From a sphere of action, Perrin-type model, exchange was estimated to occur over a range of the order of 2 nm. The possibility of spin-orbit coupling (SOC) or electron transfer between the QD and the lucigenin dication (Luc(2+)) is discussed. The radical cation Luc(+*) was not identified, but electron transfer from the QD conduction band to the Luc(2+), then electron transfer back, from the Luc(+*) to the QD valence band, could lead to mutual quenching, without build up of Luc(+*) . SOC between the QD and lucigenin, with or without charge transfer being involved, can also account for the results obtained. Lucigenin is also a chloride-sensitive indicator dye, with a sensing mechanism based on SOC. In the QD-MPA-lucigenin conjugate luminescence is restored by adding chloride ion. Thus, the presence of chloride is transduced into an enhancement of the luminescence of QDs. Using this operating principle, a chloride ion sensor based on CdSe-ZnS core-shell QD nanoparticles, showed a very good linearity in the range 1-250 mM, with a detection limit 0.29 mM and a RSD of 2.5% (n = 10). In a study of interferences, the chloride sensitive QDs showed good selectivity to most of the other anions tested. The versatility of the system was also demonstrated in terms of fluorescent emission wavelength, which could be selected across a wide range through choice of QDs. Examples are shown for lambda(max) = 500, 540 and 620 nm. The results from samples mimicking physiological conditions suggested very good applicability in the determination of chloride ion in physiological samples.

Electrochemical surface plasmon resonance (E-SPR) was used to investigate whether the chromic properties of a polydiacetylene (PDA) vesicle films, adsorbed onto an ultra-thin gold electrode, could be triggered by applied potential. This approach constitutes a preliminary model for a novel approach to the use of a triggered chromic transition, as an indicator of biorecognition headgroup binding in these materials. A PDA chromic blue-red transition was identified in E-SPR against the background Deltaepsilon(e) and Deltaepsilon(m). The latter resulted in a ca. 100 mDeg V(-1) shift in the SPR minimum, in the presence of PDA, with the PDA shielding changes in epsilon(e). Electrochemical charge transfer processes in the pre-oxide/oxide anodic region with adsorbed oxygen and hydroxide, involving a change in Au redox state (Au(0)/Au(+)) were visible in the SPR, due to a change in the gold layer thickness and gold oxide layer. However, the cathodic processes, not involving a change in the Au redox state or a increase/decrease in the surface layer dielectric, did not cause a change in the SPR. Based on this, dramatic changes in the optical properties of the adsorbed PDA film could be triggered at an applied cathodic potential, and were identified using SPR. These correlated with a pH-induced chromic transition. Both protonation and ion binding, linked with headgroup environment, were implicated in causing structural transitions in the adsorbed vesicle layer that may also be linked with their bulk optical properties.