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Optical nanosensors have been designed to utilise the sensitivity of fluorescence for making quantitative measurements in the intracellular environment, using devices that are small enough to be inserted into living cells with a minimum of physical perturbation. Advantages over widely used fluorescence dye based methods are observed because the nanosensor matrix imparts two key benefits; (1) protection of the sensing component from interfering species within the intracellular environment and (2) protection of the intracellular environment from any toxic effects of the sensing component. This Highlight article discusses the recent developments in nanosensor technology and investigates the use of more complex sensing schemes that will expand the range of analytes that can be detected and quantified.

The development and characterisation of a fluorescent optical PEBBLE (Probe Encapsulated By Biologically Localised Embedding) nanosensor for the detection of zinc is detailed. A ratiometric sensor has been fabricated that incorporates two fluorescent dyes; one is sensitive to zinc and the other acts as a reference. The sensing components are entrapped within a polymer matrix by a microemulsion polymerisation process that produces spherical sensors that are in the size region of 20 to 200 nm. Cellular measurements are made possible by the small sensor size and the biocompatibility of the matrix. The effects of reversibility, photobleaching and leaching have been examined, as well as the selectivity towards zinc over other cellular ions such as Na+, Ca2+, K+, and Mg2+. The dynamic range of these sensors was found to be 4 to 50 microM Zn2+ with a linear range from 15 to 40 microM. The response time for the PEBBLE is less than 4 s and the sensor is reversible. In addition, the nanosensors are photostable and leaching from the matrix, determined using a novel method, is minimal. These sensors are capable of real-time inter- and intra-cellular imaging and are insensitive to interference from proteins.

The metalloprotein cytochrome c′ was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO). The metalloprotein was encapsulated in a porous silicate sol–gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements. Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c′ protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre. Such conformational changes were also observed when the cytochrome c′ was stored in solution, although over a 2–3 month period. The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c′ and appear to change the binding affinity of the protein towards NO. However, the encapsulated (reconformed) cytochrome c′ was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%). An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c′ with an approximate working range of 10–400 µmol l–1.

Three heme proteins; horse heart cytochrome c, myoglobin (Mb) and hemoglobin (Hb), have been encapsulated in a sol–gel matrix and investigated for their potential use as biological recognition centres for the biosensing of NO and CO. It has been shown that the encapsulation of these metalloproteins in sol–gels has no effect on the formation of nitrosyl or carbonyl complexes as observed using UV/VIS spectrophotometry. The metalloproteins in sol–gels could be readily reduced to the FeII state, using excess sodium dithionite, and re-oxidized to the FeIII state on the addition of excess potassium ferricyanide. The observed redox capability within sol–gels enabled formation of the nitrosyl complex in both the FeIII and FeII states of Mb and Hb and in the FeIII state of cytochrome c. The formation of these nitrosyl complexes was shown to be reversible. Similarly, the reversible formation of the carbonyl complex with Mb and Hb in the reduced form has been achieved. Titration of a saturated solution of CO with Hb contained in a sol–gel showed a linear increase in intensity of the α(568 nm) and β(540 nm) absorption bands of the metalloprotein until saturation of the iron centres of the Hb occurred. A similar increase in intensity of the α(562 nm) absorption band of cytochrome c was observed when a saturated solution of NO was titrated against this metalloprotein contained in a sol–gel. The results show that sol–gels can be readily used as a host for the encapsulation of cytochrome c, Mb and Hb and that these metalloproteins can be used as the biological recognition centre for the biosensing of NO and CO at the micromolar level.

Nanoparticulate systems in various unique configurations are highly effective at detecting protease activity both in vivo and in vitro. In this article, we have summarised the conventional modern methods for monitoring protease activity, and critically appraised recent advances in protease-responsive nanosensors.

Sol–gel nanoprobes, also known as Photonic Explorer for Bioanalysis with Biologically Localised Embedding (PEBBLE), capable of performing in-vitrointracellular monitoring of reactive oxygen species have been developed using a modified form of 5(6)-carboxyfluorescein diacetate. A sol–gel matrix was selected for the design of the probes as it is photostable, optically transparent and chemically inert, and to minimise leaching of the dye from the porous matrix it was covalently immobilised to silica nanoparticles (15 nm). Using this approach, 0.1% of the dye was found to leach over a typical analysis time of 5 h and minimal photobleaching was observed. In addition, the nanoprobes were shown to respond to hydrogen peroxide, hydroxyl anions, nitric oxide, peroxynitrile and superoxide anions, obtaining limits of detection of 2.2, 1.1, 3.2, 1.1 and 1.1 nM respectively. The nanoprobes were subsequently introduced into bovine oviducts using a lipid transfection reagent (Escort IV™) and fluorescence was observed.

Whilst the authentication of Scotch whisky brands using laboratory based gas chromatography is well established, there is a need for authentication methods for use under field test conditions. The UV/visible absorbance spectra of specific brands of Scotch whisky were found to produce consistent absorbance ranges which allowed the development of a faster, cheaper and more mobile test method. Test samples with spectra outside the acceptable ranges for genuine reference samples were therefore deemed suspect, enabling their authenticity to be later confirmed in the laboratory by GC. This novel application allowed brand authenticity analyses to be undertaken at remote sites where gas chromatography was unavailable and has also allowed the introduction of field testing using a small portable hand held spectrophotometer.

The on-chip measurement of absorbing species has proven to be challenging, particularly with respect to the sample pathlengths available in a miniaturised system. This paper demonstrates how the principles of total internal reflection can be utilised to form a liquid-core waveguide along a single microfluidic channel, increasing the sampling pathlength to 5 mm while maintaining a detection volume ofor = 1 microL. This was achieved using the Teflon fluoropolymers PTFE, FEP and AF as cladding for the liquid-core waveguide. In conjunction with a 3D chip architecture, the use of the liquid-core waveguide enables more efficient use of the probing light beam along with easy and effective coupling of the source, microfluidic chip and the detection system. The confirmation that waveguiding was occurring was successfully demonstrated and the subsequent spectrophotometric analysis of crystal violet provided a linear calibration with reproducibility (2.4% RSD) and limits of detection (1.3 microM), comparable to absorbance measurements made with a standard UV-Vis spectrophotometer.

Polyacrylamide-based, ratiometric, spherical, optical nanosensors, or polyacrylamide PEBBLEs (Probes Encapsulated By Biologically Localized Embedding), have been fabricated, aimed at real-time glucose imaging in intact biological systems, i.e. living cells. These nanosensors are prepared using a microemulsion polymerization process, and their average size is about 45 nm in diameter. The sensors incorporate glucose oxidase (GOx), an oxygen sensitive fluorescent indicator (Ru[dpp(SO3Na)2]3)Cl2, and an oxygen insensitive fluorescent dye, Oregon Green 488-dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements. The enzymatic oxidation of glucose to gluconic acid results in the local depletion of oxygen, which is measured by the oxygen sensitive ruthenium dye. The small size and inert matrix of these sensors allows them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. The PEBBLE matrix protects the enzyme and fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a larger variety of dyes can be used in optimal fashion. Furthermore, the PEBBLE matrix enables the synergistic approach in which there is a steady state of local oxygen consumption, and this cannot be achieved by separately introducing free enzyme and dyes into a cell. The work presented here describes the production and characterization of glucose sensitive PEBBLEs, and their potential for intracellular glucose measurements. The sensor response is determined in terms of the linear range, ratiometric operation, response time, sensor stability, reversibility and immunity to interferences.

Ratiometric pH nanosensors with tuneable pK(a) were prepared by entrapping combinations of two pH-sensitive fluorophores (fluorescein isothiocyanate dextran (FITC-D) and Oregon Green(®) dextran (OG-D)) and a reference fluorophore (5-(and-6)-carboxytetramethylrhodamine dextran (TAMRA-D)), in a biocompatible polymer matrix. Dual-fluorophore pH nanosensors permit the measurement of an extended dynamic range, from pH 4.0 to 7.5.

Probes Encapsulated By Biologically Localised Embedding (PEBBLEs) are optical sensors with nanometer dimensions fabricated by microemulsion polymerisation. The most beneficial characteristic of these sensors is the protection offered by the sensor matrix which decreases interaction between the fluorophore and the cell. These sensors have been introduced to the cell by a number of methods; however this paper discusses the development of a generic method to facilitate inclusion of this type of sensor in the cell by a simple incubation step. This was achieved by covalent linkage of a synthetic Cell Penetrating Peptide (CPP) based on the Human Immuno-deficiency Virus (HIV) -1 Tat, to the external sensor matrix. Calcium sensors were used to demonstrate this approach to incorporate the sensors within the cell. Characterisation revealed the calcium sensors were approximately 30 +/- 7 nm in diameter with a slightly negative zeta potential. The sensors demonstrated a linear range of 0-50 microM with negligible interference from a range of cellular ions and protein. Leaching of entrapped dyes from the calcium sensors was determined as 3% in a 24 h period, while photobleaching of the entrapped dye was minimal over a 40 min period. The sensors ability to cross the cell membrane using the covalently attached synthetic Tat peptide is demonstrated. Cellular inclusion of the sensors occurred within a 30 min incubation period.

Whilst the authentication of Scotch whisky brands using laboratory based gas chromatography is well established, there is a need for authentication methods for use under field test conditions. The UV/visible absorbance spectra of specific brands of Scotch whisky were found to produce consistent absorbance ranges which allowed the development of a faster, cheaper and more mobile test method. Test samples with spectra outside the acceptable ranges for genuine reference samples were therefore deemed suspect, enabling their authenticity to be later confirmed in the laboratory by GC. This novel application allowed brand authenticity analyses to be undertaken at remote sites where gas chromatography was unavailable and has also allowed the introduction of field testing using a small portable hand held spectrophotometer.

Analytical methods are required by trading standards and regulatory authorities to confirm the authenticity of Scotch whisky brands. The characteristics of Scotch whisky are strongly influenced by the cereals used in fermentation and by distillation, maturation and blending regimes. This leads to characteristic analytical profiles for various congeners, which can be used as reference points in authenticity analysis. Scotch whisky higher-alcohol profiles for specific brands were shown to be very consistent over many production batches and so their gas-chromatographic determination, together with the presence of other specific congeners, offered an effective approach to authenticity analysis. Pyrolysis–mass spectrometry followed by multivariate analysis of the resulting mass spectra enabled non-authentic samples to be discriminated from the authentic product. Hence, an increasingly sophisticated portfolio of analytical strategies are available in order to confirm brand authenticity.