Elizabeth A. H. Hall

Fluorescent nanoparticles for intracellular sensing: a review.

Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

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.

Dipicolinic acid (DPA) assay revisited and appraised for spore detection

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.

Electrochemical response of an enzyme-loaded polyaniline film

Abstract A method is reported for the electrochemical preparation of a glucose oxidase loaded polyaniline film. The current response of the film in the presence of glucose was recorded at a constant potential and under cyclic linear potential sweep. An aerobic current due to hydrogen peroxide could be recorded and an anaerobic signal, which was a function of time and glucose concentration, could be measured at +0·65 V as a current increasing linearly with time. Attempts to elucidate the source and nature of this behaviour through the study of the response of the enzyme-loaded film at different thicknesses indicated that the charge transport limitation present in glucose-free solutions diminished with time in the presence of glucose. The ‘species’ resulting in this mediation could not be unambiguously identified.

Ion-transport and diffusion coefficients of non-plasticised methacrylic–acrylic ion-selective membranes

The ion-transport behaviour of methacrylic-acrylic-based polymers for ion-selective electrode (ISE) membranes was investigated by a spectrophotometric method to determine the apparent diffusion coefficient. By observing the degree of deprotonation of the chromoionophore or chromogenic ionophore, the extent of penetration of cations into the polymer films was determined. The transport of the cations into the optode films depended on the stoichiometry of complexation by the ionophores. The apparent diffusion coefficients, estimated from the deprotonation data were of the order of 10(-12) to 10(-11)cm(2)s(-1). These values indicate that the apparent ion mobility in the methacrylic-acrylic ISE membranes is approximately a thousand times lower than that in plasticised PVC ISE membranes. For some ionophores, the value of the apparent diffusion coefficient could be modulated according to the ionophore content in the membrane and the data obtained for a calixarene containing membrane were tested against a model for facilitated diffusion with chained carriers. The data did not fit a model where intramolecular diffusion was limiting, but were consistent with a first-order rate-limiting mechanism involving an intermediate 1:2 complex between ion and ionophore. In this instance, the lowest values for D(app) were thus not necessarily obtained for lowest ionophore loading and in the range examined, a trend of decreasing D(app) with increasing ionophore was noted.

Electrochemical enzyme sensor for formaldehyde operating in the gas phase

An electrochemical cell for the detection of formaldehyde vapour is described containing the enzyme formaldehyde dehydrogenase [EC 1.2.1.46]. The cell operates directly in the gas phase with an almost linear response to formaldehyde up to 6 vppm. The detection limit is 0·3 vppm. At 2 vppm and 6 vppm CH2O the cell could be operated for at least 7 h without loss of activity.

The Emerging Use of Quantum Dots in Analysis

Abstract The optical properties of quantum dots (QDs) have made them attractive materials in diverse fields of application. Since water‐soluble derivatives were obtained, quantum dots have attracted intensive research interest in sensing, diagnosis, imaging, and optical tracking. The analytes that have been targeted span ions, small chemical molecules, proteins, nucleic acids, and cells. The fluorescence of functionalized QDs can be quenched, enhanced, or even ‘switch’ on and off in different cases. However, the mechanisms behind these various responses are not yet all fully understood. This review gives an overview of the emerging use of QDs in analysis. Typical examples, in particular in relation to the discussion on mechanisms are highlighted.

An electrochemically grown polymer as an immobilisation matrix for whole cells: Application in an amperometric dopamine Sensor

Abstract A method is described for the entrapment of whole banana cells in a polypyrrole film electrochemically deposited on a gold electrode. The polyphenol oxidase activity of the incorporated whole cells in the polymer was studied using dopamine as the major substrate. This amperometric-based probe was compared for the measurement of dopamine and related catecholamines. It exhibited high biocatalytic activity, good response time, favourable selectivity and reusability.

Methacrylic-acrylic polymers in ion-selective membranes : achieving the right polymer recipe

The polymer characteristics of the methacrylic‐acrylic copolymers which have been successful in producing membranes for potassium ion-selective electrodes were investigated. Measurement of copolymer glass transition temperature ( Tg )b y differential scanning calorimetry indicated that the Tg influenced the amount of plasticiser required for workable ion selective electrode membranes. A Tg below 20C was required, which could be achieved with or without the use of a plasticiser; without using plasticiser the copolymer should contain more than 80 wt.% of n-butyl acrylate. Under the conditions for the free radical solution polymerisation used, proton NMR spectroscopy studies on the copolymers showed that the incorporation of n-butyl acrylate into the copolymer was lower than expected when the methacrylate content was high. However, when the amount of methacrylate used in the feed was low, n-butyl acrylate incorporation could reach almost 100% (relative to the n-butyl acrylate in the feed). Therefore, for an efficient incorporation of n-butyl acrylate into the copolymer, the methacrylate content must be kept below 30 wt.%. A high concentration of both methacrylate and acrylate monomers should also be used during polymerisation to ensure that the copolymer produced has a molecular weight distribution ( N Mw) of greater than 80 000: this is required to provide physical strength to the ion-selective membrane. Potentiometric studies on some of the high acrylate membranes using valinomycin as potassium-ion selective ionophore showed that these non-plasticised membranes gave performance similar to that of a plasticised poly(vinyl chloride) membrane using the same ionophore and could be readily deployed more widely due to their ease of producing and ionophore incorporation. ©2000 Elsevier Science B.V. All rights reserved.

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2-2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1-2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.