Jonathan W. Aylott

Dual fluorescent labelling of cellulose nanocrystals for pH sensing.

Cellulose nanocrystals were converted into ratiometric pH-sensing nanoparticles by dual fluorescent labelling employing a facile one-pot procedure. A simple and versatile three-step procedure was also demonstrated extending the number of fluorophores available for grafting. In this method an amine group was introduced via esterification followed by a thiol-ene click reaction.

A fluorescent PEBBLE nanosensor for intracellular free zinc

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.

Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging

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.

Optical nanosensors—an enabling technology for intracellular measurements

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.

Sol–gel encapsulation of metalloproteins for the development of optical biosensors for nitrogen monoxide and carbon monoxide

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.

Integrated organic light-emitting device'fluorescence-based chemical sensors

A fluorescent chemical sensor platform, integrating an organic light-emitting device (OLED) light-source with a fluorescent probe, is demonstrated for a subsecond-fast oxygen sensor. The integration results in strong light coupling and negligible heating of the sensor film or analyte. The potential in vivo operation of compact, stand-alone, battery-powered, OLED-based miniaturized sensor arrays for chemical and biological applications is discussed.

Optical Biosensing of Nitrate Ions Using a Sol–Gel Immobilized Nitrate Reductase

The coupling of enzymes with sol–gel technology creates exciting possibilities for biosensing. Enzymes can be highly selective and will only respond to specific analytes. Sol–gels provide a unique matrix in which various biomaterials can be immobilized without any loss of enzyme activity. These two components have been combined for the optical biosensing of nitrate ions. The periplasmic nitrate reductase (Nap) extracted from the denitrifying bacterium Thiosphaera pantotropha reacts specifically with the nitrate (NO 3 - ) anion. The encapsulation of this enzyme in a sol–gel structure for the optical biosensing of nitrate ions is reported. The reduction of nitrate by periplasmic nitrate reductase results in a characteristic change in the UV/VIS absorption spectrum of the nitrate reductase. This spectroscopic change has been quantitatively calibrated against nitrate concentration. The nitrate biosensing system is fully reversible and is highly sensitive and selective to nitrate ions. The results obtained show that the activity of the enzyme is not affected by the sol–gel matrix, even after a storage period of up to six months. As no leaching of the Nap from the sol–gel matrix was observed, it is clear that the encapsulation of this nitrate sensitive enzyme in a sol–gel medium represents an ideal anionic recognition element of an optical biosensor for the detection of nitrate ions in the µmol l - 1 range.

Thermoresponsive Polymer Colloids for Drug Delivery and Cancer Therapy

Many difficulties in treating cancer arise from the problems in directing highly cytotoxic agents to the deseased tissues, cells and intracellular compartments. Many drug delivery systems have been devised to address this problem, including those that show a change in properties in response to a temperature stimulus. In particular, colloidal materials based on thermoresponsive polymers offer a means to transport drugs selectively into tumour tissues that are hyperthermic, either intrinsically or through the application of clinical procedures such as localised heating. In this paper, the key attributes of thermoresponsive polymer colloids are considered, a number of important recent examples are discussed and the possible future developments of these materials are evaluated.

Enhanced uptake of nanoparticle drug carriers via a thermoresponsive shell enhances cytotoxicity in a cancer cell line

Polymer particles consisting of a biodegradable poly[lactide-co-glycolide] (PLGA) core and a thermoresponsive shell have been formulated to encapsulate the dye rhodamine 6G and the potent cytotoxic drug paclitaxel. Cellular uptake of these particles is significantly enhanced above the thermal transition temperature (TTT) of the polymer shells in the human breast carcinoma cell line MCF-7 as determined by flow cytometry and fluorescence microscopy. Paclitaxel-loaded particles display reduced and enhanced cytotoxicity below and above the TTT respectively compared to unencapsulated drug. The data suggests a potential route to enhanced anti-cancer efficacy through temperature-mediated cell targeting.

Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p < 0.05). The nanosensors were also found to remain in the C. elegans lumen >24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.