Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter ~50 nm which contain a large concentration of Cy5 dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free dye. They do not photo bleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other dyes will be doped into the silica NPs and their use in multiplexing will be investigated.
We describe the synthesis of optical quality thin film materials with high refractive index, employing zirconium based
hybrid sol-gel precursors. As the zirconium propoxide precursor is unstable in the presence of a strong nucleophilic agent
such as water, two synthesis routes have been performed employing a chelating agent and an organosilane precursor to
avoid the formation of any undesired ZrO2 agglomerates, leading to organo-zirconate complexes and silicato-zirconate
copolymers, respectively. The prepared hybrid sol-gel materials were deposited by spin-coating to form a transparent
thin film on silicon substrates, and heat treated at 100 °C for the final stabilisation of the layer.
The effect of the two synthesis routes on the optical properties of zirconium based hybrid sol-gel material is discussed. It
was found that the nature and concentration of the organosilane precursor can significantly affect the structural properties
of the deposited films. A correlation was also demonstrated between the concentration of the organosilane precursor and
the refractive index of the material. By reducing the concentration of organosilane precursor, high refractive index
materials were obtained. Similar behaviour was observed for the materials synthesised via chelating agent. The synthesis
employing an organosilane precursor produces films with higher refractive index. A maximum refractive index of 1.746
was measured at 635nm for the deposited thin films.
Surface plasmon–coupled emission (SPCE) is a phenomenon whereby the light emitted from a fluorescent molecule can couple into the surface plasmon of an adjacent metal layer, resulting in highly directional emission in the region of the surface plasmon resonance (SPR) angle. In addition to high directionality of emission, SPCE has the added advantage of surface selectivity in that the coupling diminishes with increasing distance from the surface. This effect can be exploited in bioassays whereby a fluorescing background from the sample can be suppressed. We have investigated, both theoretically and experimentally, the SPCE effect for a Cy5-spacer-Ag layer system. Both the angular dependence of emission and the dependence of SPCE emission intensity on Cy5-metal separation were investigated. It is demonstrated that SPCE leads to lower total fluorescence signal than that obtained in the absence of a metal layer. This is the first experimental verification of the reduction in SPCE intensity compared to the metal-free case. Our results are in a good agreement with theoretical models. The validation of the theoretical model provides a basis for optimizing biosensor platform performance, particularly in the context of the advantages offered by SPCE of highly directional emission and surface selectivity.
The major trends driving optical chemical sensor technology are miniaturisation and multi-parameter functionality on a single platform (so-called multi-analyte sensing). A multi-analyte sensor chip device based on miniature waveguide structures, porous sensor materials and compact optoelectronic components has been developed. One of the major challenges in fluorescence-based optical sensor design is the efficient capture of emitted fluorescence from a fluorophore and the effective detection of the signal. In this work, the sensor platform has been fabricated using poly(methyl methacrylate), PMMA, as the waveguide material. These platforms employ a novel optical configuration along with rapid prototyping technology, which facilitates the production of an effective sensor platform.
Sensing films for oxygen, carbon dioxide and humidity have been developed. These films consist of a fluorescent indicator dye entrapped in a porous immobilisation matrix. The analyte diffuses through the porous matrix and reacts with the indicator dye, causing changes in the detected fluorescence. The reaction between the dye and the analyte is completely reversible with no degradation of the signal after detection of different concentrations of the analyte. A single LED excitation source is used for all three analytes, and the sensor platform is housed in a compact unit containing the excitation source, filters and detector.
The simultaneous detection of several analytes is a major requirement for fields such as food packaging, environmental quality control and biomedical diagnostics. The current sensor chip is designed for use in indoor air-quality monitoring.
Current sensor trends, such as multianalyte capability, miniaturisation and patternability are important drivers for materials requirements in optical chemical sensors. In particular, issues such as enhanced sensitivity and printablity are key in developing optimised sensor materials for smart windows for bioprocessing applications. This study focuses on combining novel sol-gel-based hybrid matrices with engineered luminescent complexes to produce stable luminescence-based optical sensors with enhanced sensitivity for a range of analytes including oxygen, pH and carbon dioxide. As well as optimising sensor performance, issues such as surface modification of the plastic substrate and compatibility with different deposition techniques were addressed. Hybrid sol-gel matrices were developed using a range of precursors including tetraethoxysilane (TEOS), methyltriethoxysilane (MTEOS), ethyltriethoxysilane (ETEOS), n-propyltriethoxysilane (PTEOS), phenyltriethoxysilane (PhTEOS), and n-octyltriethoxysilane (C8TEOS). Oxygen sensing, based on luminescence quenching of ruthenium phenanthroline complexes, has been realised with each of these hybrid materials. Furthermore, the possibility of immobilising pH-indicators for pH and carbon dioxide sensing has been investigated with some success. In the context of in-situ monitoring of bioprocesses, issues such as humidity interference as well as the chemical robustness of the multianalyte platform, were addressed.
We report the development of enhanced optical platforms for fluorescence-based biosensors. A previous analysis by us has shown that the emission of fluorescence in such a system is highly anisotropic and is preferentially emitted into the substrate over a well-defined angular range, with the result that the light is guided along the substrate via total internal reflection. However, conventional optical biosensors based on fluorescence detection typically employ a detector that is positioned either directly above or directly below the biochip. As a consequence, only a small fraction of the total emitted fluorescence is detected, which impacts adversely on sensor performance. The enhanced biosensor presented here is based on a novel, generic platform specifically designed to overcome the inherent limitations of planar substrates. The platform incorporates custom-designed optical elements, the purpose of which is to redirect the emitted fluorescence onto a detector positioned beneath the biochip. Platforms were fabricated using the polymer processing technique of microinjection moulding. In this paper we demonstrate the ability of this optical system to achieve a 80-fold luminescence capture enhancement. We also demonstrate its effectiveness as an enhanced biosensor platform by carrying out a proof of principle BSA/antiBSA competitive assay. This work has significant implications for the development of mass-producible, highly efficient optical biosensors.
In this paper, we report on a strategy, which produces enhancement of fluorescence using the so-called plasmonic effect whereby the presence of adjacent metallic nanoparticles can dramatically alter the fluorescence emission and absorption properties of a fluorophore. The effect, which is a result of the surface plasmon resonance of the metal surface, can lead to increases in quantum efficiency, radiative decay rates and photostability of the fluorophore, and depends very sensitively on parameters such as geometry of the nanoparticles, nanoparticle-fluorophore separation and fluorophore type. The work is aimed at improving the efficiency of optical biochips. Key benefits from this enhancement include lower limits of detection, reduced reagent requirements and better resolution. This study is part of a comprehensive investigation of plasmonic enhancement using a range of metal nanoparticle (NP) fabrication techniques and a range of measurement configurations. The focus here is on the fabrication of chemically prepared silver-gold alloy spherical NP with a variable thickness silica shell on the surface of which is immobilised a layer of fluorescent dye molecules. The variable thickness shell serves to control the dye-NP separation, which plays a key role in the enhancement mechanism. Transmission electron microscopy (TEM) was used to characterise the NP. The dye used here was the ruthenium polypyridyl complex [Ru(II)-tris(4,7-diphenyl-1,10-phenanthroline)], abbreviated to [Ru(dpp)3]2+. This paper reports the tuning of the NP plasmon resonance via NP size and alloy composition. The wavelength of the plasmon peak as a function of NP size and composition correlated very well with theoretical predictions based on the Mie scattering theory. Preliminary fluorescence enhancement measurements on this system yielded an enhancement factor of approximately 5.
It is well established that the presence of metallic surfaces or particles in the vicinity of a fluorophore can dramatically increase the radiative decay rate, and consequently the quantum efficiency, of the fluorophore. This effect, which depends on parameters such as metal particle size and fluorophore-particle separation, is manifest as a substantial enhancement in fluorescence emission intensity. This presentation will focus on optimisation strategies to maximise the enhancement for important applications such as fluorescence-based biochip platforms.
Ordered arrays of metallic nano-islands were fabricated on a range of substrates by a process of natural lithography using monodisperse polystyrene nanospheres. The metal particle dimensions were tailored in order to match the plasmon resonance wavelength to the spectral absorption of the fluorophore. The fluorophore Cy5 dye, which is widely used in optical immunoassays and has a medium quantum efficiency (~0.3), was used in this study of the plasmonic enhancement effect.
The morphology of the metallic arrays was investigated using scanning electron microscope (SEM) and atomic force microscope (AFM). Absorption and emission spectroscopies were used to elucidate the enhancement effect and its dependence on metal island morphology. Results were correlated with existing theoretical models. The applicability of this important technique to sensor platforms, such as fluorescence-based biochips, will be discussed.
Modified Atmosphere Packaged (MAP) food employs a protective gas mixture, which normally contains selected amounts of carbon dioxide (CO2) and oxygen (O2), in order to extend the shelf life of food. Conventional MAP analysis of package integrity involves destructive sampling of packages followed by carbon dioxide and oxygen detection. For quality control reasons, as well as to enhance food safety, the concept of optical on-pack sensors for monitoring the gas composition of the MAP package at different stages of the distribution process is very attractive. The objective of this work was to develop printable formulations of oxygen and carbon dioxide sensors for use in food packaging. Oxygen sensing is achieved by detecting the degree of quenching of a fluorescent ruthenium complex entrapped in a sol-gel matrix. In particular, a measurement technique based on the quenching of the fluorescence decay time, phase fluorometric detection, is employed. A scheme for detecting CO2 has been developed which is compatible with the oxygen detection scheme. It is fluorescence-based and uses the pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) indicator dye encapsulated in an organically modified silica (ORMOSIL) glass matrix. Dual Luminophore Referencing (DLR) has been employed as an internal referencing scheme, which provides many of the advantages of lifetime-based fluorometric methods. Oxygen cross-sensitivity was minimised by encapsulating the reference luminophore in dense sol-gel microspheres. The sensor performance compared well with standard methods for both oxygen and carbon dioxide detection. The results of preliminary on-pack print trials are presented and a preliminary design of an integrated dual gas optical read-out device is discussed.
Pressure-sensitive paint (PSP) technology is an important new technique, which allows pressure mapping of surfaces under aerodynamic conditions. The principle used is that oxygen in the airflow around the surface quenches luminescence from an oxygen-sensitive dye in the paint and by imaging the illuminated surface with a gated CCD camera, the oxygen profile, and hence the pressure profile of the surface is mapped. The temperature dependence of these paints is a problem. Temperature correction of the surface pressure measurement is often carried out by incorporating a second temperature-sensitive luminophore to provide a surface temperature profile. This usually complicates the measurement process by requiring a second camera or sophisticated filtering to distinguish between the two luminescent processes. In this work, a new oxygen permeable sol-gel coating containing both a temperature (manganese doped magnesium fluorogermanate(MFG) and pressure (Ru(dpp)32+) sensitive luminophore is described. The luminescent lifetimes of each luminophore are separated by several orders of magnitude, allowing pressure and temperature-dependent lifetime measurements to be separated in the time domain. The luminophores were selected such that their absorption bands overlap and their emission spectra occur in adjacent spectral regions thus enabling the use of a single excitation source and single camera. Both lifetimes were measured with a single camera with gated image intensifier. The system thus provides a temperature-corrected surface pressure profile.
The concept of fully- or quasi-distributed chemical sensing using OTDR techniques has attracted much research interest. Much of the work to date in this area has employed large core non-telecom grade optical fiber. Typically, this has been PCS fiber from which the cladding has been removed and replaced with sections of doped polymers. Evanescent wave interactions between the guided light and the coatings provided the sensor modulation. In this paper we report the use of telecom-grade optical fibers to which analyte- sensitive reagents are attached via a novel configuration at the fiber tip. This approach enables the use of low attenuation fibers, good quality directional couplers and direct, as opposed to evanescent wave, interactions. In order to exploit fully the low attenuation and commercially available OTDR units, it is necessary to operate at one of the telecom windows, usually 850 nm. This requires the use of appropriate NIR dyes. We report some initial results for pH sensing using this approach. We also present results for O2 single point sensing using blue-excited fluorescence and comment on factors concerning the use of fluorescence quenching in distributed systems.
A dissolved oxygen sensor, based on sol-gel-derived silica thin films impregnated with an oxygen-sensitive ruthenium complex, is reported. Porous sol-gel silica films, dipcoated onto either planar glass substrates or declad optical fibers, are doped with the complex [RuII-tris(4,7-diphenyl-1,10-phenanthroline)], whose fluorescence emission is quenched by oxygen. The complex is entrapped in the cage-like structure of the sol-gel matrix, but is accessible to oxygen via the microporous channels. This work compares the difference in oxygen quenching response between gas phase and aqueous phase measurements. Optimization of dissolved oxygen response by tailoring of the film fabrication parameters is reported. Using a high-brightness blue LED, combined with a miniature photodiode-based detection system, these results establish the viability of a low-cost, high-performance, portable optical dissolved oxygen sensor.
The sol-gel process has been used to entrap pH indicators in porous glass coatings for sensor applications. This sensor is based on evanescent wave absorption using an unclad optical fiber dipcoated with the pH sensitive coating. The entrapped pH indicators show a broadening of the pH range with respect to the behavior in solution giving accurate measurement over three pH units when one indicator is used (bromophenol blue) and over six pH units (pH 3-9) when two indicators are used (bromophenol blue and bromocresol purple). The response of the pH sensor was monitored by measuring absorption at 590 nm referenced against a nonabsorbing region of the spectrum. This enabled the use of LED sources together with low cost photodiodes. The sensor displayed short response time and good repeatability. The thickness and stability of the pH sensitive coatings can be influenced by modifying the composition of the starting sol mixture. The evanescent absorption, and hence the sensitivity of the sensor, can be increased by selectively launching higher order modes in the fiber. These issues together with a full sensor characterization will be reported.
Two evanescent wave fiber optic sensors for oxygen are reported: one intensity based and the other based on phase fluorimetry. Both sensors employ the quenching by oxygen of the fluorescence from a ruthenium complex trapped in the cagelike structure of a sol-gel-derived porous film on a declad section of multimode optical fiber. The sensors exhibit excellent performance using excitation from new high brightness blue LEDs and establish the viability of low-cost portable sensor devices based on the 501-gel process.
Two evanescent wave fiber optic sensors for oxygen are reported: (i) intensity-based, and (ii) based on phase fluorimetry. Both sensors employ the quenching by oxygen of the fluorescence form a ruthenium complex trapped in the cage-line structure of a sol-gel-derived porous film on a declad section of multimode optical fiber. The sensors exhibit excellent performance using excitation from new high brightness blue LEDs and establish the viability of low-cost portable sensor devices based on the sol-gel process. The data presented for the intensity-based sensor were obtained using an all solid state excitation and detection system. Preliminary results obtained by application of the intensity- based sensor to dissolved oxygen measurement are also reported.
In this paper an overview is presented of the state-of-the-art of optical sensors which employ sol-gel-derived coatings. The technique is particularly suited to the side-coating of optical fibers or waveguides in evanescent-wave sensors because precise control of sensitivity- determining parameters, such as the coating thickness and length, is achievable. Sensors based on entrapped organic and inorganic dyes, enzymes and other biomolecules have been reported. The main features of the process are illustrated by examples of chemical sensors and biosensors from the literature. In particular, the development of an oxygen sensor based on the quenching of fluorescence from a sol-gel-entrappd ruthenium complex is described. This sensor may be operated in intensity or decay-time mode. The latter offers many advantages over intensity-based sensing and may also be used to provide useful diagnostic information concerning the distribution/accessibility of the sensor fluorophore. Issues which require further investigation before this technology can proceed to the stage of industrial development are also highlighted.
The ability to determine oxygen concentration is of great importance for many industhal, medical and
environmental applications. Optical oxygen sensors are attractive because of their many advantages over other
sensor types, in that they offer the possibility of miniaturization, have fast response times, do not consume
oxygen, and are not easily poisoned.
Porous glass coatings produced by the sol-gel process offer a number of advantages in optical sensor applications. In reagent-based chemical sensors they may be used to provide a robust support matrix in which analyte-sensitive dyes may be entrapped and into which the analyte may diffuse. Although distal tip and side coatings are both possible the latter, evanescent wave approach, is more advantageous. These advantages and the benefits of the sol-gel approach are illustrated by preliminary results from an oxygen sensor based on fluorescence quenching of sol-gel-entrapped ruthenium complexes. Declad multimode optical fibers coated with a thin microporous film containing either of two different oxygen-sensitive complexes were investigated. Under evanescent wave excitation the sensors exhibited repeatable quenching behavior when exposed to varying concentrations of oxygen. The oxygen sensitivity was improved by appropriate choice of ruthenium complex and film preparation. The sensors showed good signal-to-noise ratio, fast response time, and low photobleaching.
Eu3+ and U022+ have been used as optical probes iii sol-gel silica. Optical fluorescence and decay times were monitored as a function of processing parameters such as pH and densification temperature, and the optical data were correlated with structural changes in the materials. Fluorescence and decay times of Eu3+ ljO,2+ co-doped materials were studied and energy transfer effects were interpreted in terms of the pore morphology.
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