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Semiconductor quantum dots (QDs) have been shown to be highly sensitive to electron or charge transfer processes, which may alter their optical properties. This feature can be exploited for different sensing applications. Here, we demonstrate that QD-anthraquinone conjugates can function as electron transfer-based pH nanosensors. The attachment of the anthraquinones on the surface of QDs results in the reduction of electron hole recombination, and therefore a quenching of the photoluminescence intensity. For some anthraquinone derivatives tested, the quenching mechanism is simply caused by an electron transfer process from QDs to the anthraquinone, functioning as an electron acceptor. For others, electron transfer and energy transfer (FRET) processes were found. A detailed analysis of the quenching processes for CdSe/ZnS QD of two different sizes is presented. The photoluminescence quenching phenomenon of QDs is consistent with the pH sensitive anthraquinone redox chemistry. The resultant family of pH nanosensors shows pKa ranging ∼5-8, being ideal for applications of pH determination in physiological samples like blood or serum, for intracellular pH determination, and for more acidic cellular compartments such as endosomes and lysosomes. The nanosensors showed high selectivity towards many metal cations, including the most physiologically important cations which exist at high concentration in living cells. The reversibility of the proposed systems was also demonstrated. The nanosensors were applied in the determination of pH in samples mimicking the intracellular environment. Finally, the possibility of incorporating a reference QD to achieve quantitative ratiometric measurements was investigated.

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.

Semiconductor nanocrystals, namely, quantum dots (QDs), present a set of unique photoluminescence properties, which has led to increased interest in using them as advantageous alternatives to conventional organic dyes. Many applications of QDs involve surface modification to enhance the solubility or biocompatibility of the QDs. One of the least exploited properties of QDs is the very long photoluminescence lifetime that usually has complex kinetics owing to the effect of quantum confinement. Herein, we describe the effect of different surface modifications on the photoluminescence decay kinetics of QDs. The different surface modifications were carefully chosen to provide lipophilic or water-soluble QDs with either positive or negative surface net charges. We also survey the effect on the QD lifetime of several ligands that interact with the QD surface, such as organic chromophores or fluorescent proteins. The results obtained demonstrate that time-resolved fluorescence is a useful tool for QD-based sensing to set the basis for the development of time-resolved-based nanosensors., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The first CdSe/ZnS quantum dot photoluminescence lifetime-based pH nanosensor has been developed. The average lifetime of mercaptopropionic acid-capped QD nanosensors showed a linear response in the pH range of 5.2-6.9. These nanosensors have been satisfactorily applied for pH estimation in simulated intracellular media, with high sensitivity and high selectivity toward most of the intracellular components.

Herein we report the synthesis of a new chelating dendritic ligand (CDL) and its use in the preparation a compact, stable and water-soluble quantum dot (QD), and further development of specific DNA sensor. The CDL, which contains a chelative dihydrolipoic acid moiety for strong QD surface anchoring and four dendritic carboxylic acid groups, provides a stable, compact and entangled hydrophilic coating around the QD that significantly increases the stability of the resulting water-soluble QD. A CDL-capped CdSe/ZnS core/shell QD (CDL-QD) has stronger fluorescence than that capped by a monodendate single-chain thiol, 3-mercapto-propionic acid (MPA-QD). In addition, the fluorescence of the CDL-QD can be enhanced by 2.5-fold by treatments with Zn2+ or S2- ions, presumably due to effective passivation of the surface defects. This level of fluorescence enhancement obtained for the CDL-QD is much greater than that for the MPA-QD. Further, by coupling a short single-stranded DNA target to the QD via the CDL carboxylic acid group, a functional QD-DNA conjugate that can resist non-specific adsorption and hybridize quickly to its complementary DNA probe has been obtained. This functional QD-DNA conjugate is suitable for specific quantification of short, labelled complementary probes at the low DNA probe:QD copy numbers via a QD-sensitised dye fluorescence resonance energy transfer (FRET) response with 500 pM sensitivity on a conventional fluorimeter.

An enzyme-linked analytical nanosphere sensor (ANSor) is described, responding to enzyme-substrate turnover in the vicinity of a quantum dot (QD) due to coimmobilized enzyme and pH sensitive ligand. QD capping by mercapto-alkanoic acids were rejected as a pH sensitive ligand, but with the use of a layer-by-layer assembly on mercaptopropionic capped QDs and an intermediate poly(allylamine hydrochloride) layer, anthraquinone sulfonate (calcium red, CaR) was introduced to modify the pKa in the immobilized system > 8. QD-CaR absorption shows spectral overlap with QD530 emission at all pHs and gives a complex pH dependent fluorescence resonance energy transfer (FRET) efficiency, due to excited state proton transfer (λ(ex) = 540 nm; λ(em) = 585 nm). In contrast QD615-CaR with spectral overlap between the QD and CaR gave a strong and reproducible pH response. QD-urease and QD-creatinine deiminase conjugates could be linked with pH changes produced by enzyme degradation of urea and creatinine, respectively. Close coupling between the pH sensitive QD and enzyme conjugate maximized signal compared with solution based assays: QD-urease and QD-CD bioconjugates were tested in model biological media (Dulbecco's modified Eagle's Medium and fetal calf serum) and in urine, showing a response in 3-4 min.

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.

A new fluorescent nanosensor family for Zn (2+) determination is reported based on azamacrocycle derivatization of CdSe/ZnS core/shell quantum dot nanoparticles. They are the first zinc ion sensors using QD nanoparticles in a host-guest and receptor-fluorophore system. Three azamacrocycles are demonstrated as receptors: TACN (1,4,7-triazacyclononane), cyclen (1,4,7,10-tetraazacyclododecane), and cyclam (1,4,8,11-tetraazacyclotetradecane). Azamacrocycles conjugated to QDs via an amide link interact directly with one of the photoinduced QD charge carriers, probably transferring the hole in the QD to the azamacrocycle, thereby disrupting the radiative recombination process. When zinc ion enters the aza-crown, the lone pair electrons of the nitrogen atom become involved in the coordination and the energy level is no longer available for the hole-transfer mechanism, switching on the QD emission and a dramatic increase of the fluorescence intensity results, allowing the detection of low concentrations of zinc ions. Using this operating principle, three zinc ion sensors based on CdSe-ZnS core-shell QD nanoparticles showed a very good linearity in the range 5-500 microM, with detection limits lower than 2.4 microM and RSDs approximately 3% ( n = 10). In addition, the versatility of the sensors was demonstrated, since different sizes (and colors) of QDs can be employed and will respond to zinc in a similar way. In a study of interferences, the zinc-sensitive QDs showed good selectivity in comparison with other physiologically important cations and other transition metals tested. The results from fetal calf serum and samples mimicking physiological conditions suggested very good applicability in the determination of zinc ion in physiological samples.

The first sub-micron polyacrylic sensor containing two independent ion-sensing systems is shown, that uses a single excitation wavelength and separates signals by using quantum dot donors to form FRET pairs with other fluorophores.

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]