A Quantum Dot-Based FLIM Glucose Nanosensor.

In the last few years, quantum dot (QD) nanoparticles have been employed for bioimaging and sensing due to their excellent optical features. Most studies have used photoluminescence (PL) intensity-based techniques, which have some drawbacks, especially when working with nanoparticles in intracellular media, such as fluctuations in the excitation power, fluorophore concentration dependence, or interference from cell autofluorescence. Some of those limitations can be overcome with the use of time-resolved spectroscopy and fluorescence lifetime imaging microscopy (FLIM) techniques. In this work, CdSe/ZnS QDs with long decay times were modified with aminophenylboronic acid (APBA) to achieve QD-APBA conjugates, which can act as glucose nanosensors. The attachment of the boronic acid moiety on the surface of the nanoparticle quenched the PL average lifetime of the QDs. When glucose bonded to the boronic acid, the PL was recovered and its lifetime was enhanced. The nanosensors were satisfactorily applied to the detection of glucose into MDA-MB-231 cells with FLIM. The long PL lifetimes of the QD nanoparticles made them easily discernible from cell autofluorescence, thereby improving selectivity in their sensing applications. Since the intracellular levels of glucose are related to the metabolic status of cancer cells, the proposed nanosensors could potentially be used in cancer diagnosis.

pH sensitive quantum dot-anthraquinone nanoconjugates.

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.

Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase.

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.

Ratiometric pH-dot ANSors.

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.

Multiplexed energy transfer mechanisms in a dual-function quantum dot for zinc and manganese.

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.

Azamacrocycle activated quantum dot for zinc ion detection.

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.

A quantum dot-lucigenin probe for Cl-.

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 multi-ion particle sensor.

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.

Pharmaceutical powders analysis using FT-Raman spectrometry: simultaneous determination of sulfathiazole and sulfanilamide.

A procedure for rapid quantitative analysis of pharmaceutical powders is described. Powdered samples were measured in a rotating cell in order to avoid sub-sampling problems by increasing the irradiated area. Quantitative determination of sulfathiazole and sulfanilamide, using a simple univariate calibration model is proposed. Even though both antibacterials are of the same chemical family (sulfonamides), the richness of structural information contained in the Raman spectra allowed their determination using the area of two selected bands (1255 and 1629 cm(-1) for sulfathiazole and sulfanilamide, respectively). Relative standard deviation (R.S.D.) values (n=10) of 3.35% and 3.46% for sulfathiazole and sulfanilamide, respectively, demonstrate the good reproducibility of the measurement technique with the rotating cell. The method was successfully applied to the analysis of synthetic mixtures and commercial pharmaceutical powders. The procedure is suitable to be applied to pharmacopoeial uniformity of content testing of batches.

Ultrasonic trapping of microparticles in suspension and reaction monitoring using Raman microspectroscopy.

An ultrasonic standing wave around 2 MHz has been used for trapping and concentration of suspended micrometer-size particles in a flow cell, whereas Raman microspectroscopy was used as a nondestructive technique to provide molecular information about the trapped particles. With this approach, detection and discrimination of different polymer microparticles based on their characteristic Raman spectra was performed. Dextran, poly(vinyl alcohol), and melamine resin-based beads, with and without functionalization, were used for this purpose. Furthermore, taking advantage of the flow-through characteristics of the cell and the versatility of the employed flow system, full control over the media surrounding the trapped particles was achieved. This allowed us to perform chemical reactions on the trapped particles and to monitor spectral changes in real time. Here retention of cation-exchanger beads loaded with silver ions and subsequent reduction of the silver ions was demonstrated. In this way, surface-enhanced Raman (SER) active beads were prepared and retained in the focus of the Raman microscope by means of the ultrasonic field. Injection of analytes in the flow system thus allowed recording of their SER spectra. Using 9-aminoacridine, a linear dependence of the found SER signal in the range from 1 to 10 microM has been achieved. The repeatability in the recorded SER intensities was on the order of 4-5%. This included bead retention, surface-enhanced Raman layer synthesis, and analyte detection.

K+-selective nanospheres: maximising response range and minimising response time.

Cross-linked K(+) ion-selective copolymer nanospheres have been prepared by free-radical photo-initiated polymerization of n-butyl acrylate (nBA) with hexanedioldiacrylate (HDDA). Nanospheres (<200 nm) containing H(+)-chromoionophore (ETH 5294) and lipophilic salt (KTClPB) for H(+)-sensors, or ETH 5294, a K(+)-selective ionophore (valinomycin) and anionic sites for K(+)-sensors were compared, and the effect of varying the normalised concentrations for beta (R(T)(-)/L(T)) and gamma (C(m)(T)/L(T)) was studied. Experimental data were fitted to theoretical curves for the dynamic response range, based on the effect of changes in the concentration of these lipophilic sensing components incorporated into the spheres, and conditions identified for maximising the response range. A complex valinomycin-K(+) formation constant, log K(IL) = 13.13 +/- 2.22, was obtained in the nBA matrix, and from the calibration curves the apparent acid-dissociation equilibrium constant (pK(a) = 12.92 +/- 0.03) was extracted for the H(+)-sensing system, and the equilibrium exchange constant (pK(exch) = 6.16 +/- 0.03, at pH 7) calculated for the K(+)-sensing nanospheres. A basis for establishing optimum performance was identified, whereby response range and response time were balanced with maximum fluorescence yield. Parameters for achieving nanospheres with a response time <5 minutes, covering 2-3 orders of magnitude change in activity were identified, demanding nanospheres with radius <300 nm and beta(crit) approximately 0.6. An RSD(%) approximately 3% was obtained in a study of the reproducibility of the response of the proposed nanospheres, and selectivity was also evaluated for a K(+)-selective nanosensor using several cations as interfering agents. In most cases, the fluorescent emission spectra showed no response to the cations tested, confirming the selectivity of nanospheres to potassium ion. The nanosensors were satisfactorily applied to the determination of K(+) in samples mimicking physiological conditions.

Flow-through sensor with Fourier transform Raman detection for determination of sulfonamides.

A flow-through sensor system with Fourier transform (FT) Raman spectroscopy as detection technique is described. The molecular and structural information contained in Raman spectra together with the selective retention of the species of interest on the sorbent make the proposed methodology highly selective. The flow-through sensor allowed the direct quantitative determination of sulfathiazole and sulfamethoxazole in the presence of other species that are normally encountered with these analytes. The system used Sephadex QAE A-25 resin as packing material of a flow-through cell on which sulfonamides were temporarily retained. Samples were transported by a carrier solution of NaOH 10(-2) mol l(-1) (pH = 12), and 2 ml of a [NaCl (0.10 mol l(-1))/NaOH (10(-2) mol l(-1))] solution was employed as eluent. Using a sample volume of 1 ml, the analytical signal was linear in the range 0.5-7 g l(-1) and 0.5-10 g l(-1), for sulfathiazole and sulfamethoxazole, respectively. RSDs (%) lower than 4% were obtained for both analytes. The sensor was satisfactorily applied to several commercial pharmaceutical preparations for human and animals in different physical presentations, including capsules, syrup, tablets, powders, injectables and suspensions.