Optimization of Ormosil Films for Optical Sensor Applications

Recent work has indicated that Ormosil films, fabricated from organically modified precursors, produce better sensor performance for some specific applications, compared to films fabricated from conventional sol-gel precursors such as TEOS or TMOS. This paper aims to compare film properties and sensor behavior for films fabricated from tetraethoxysilane (TEOS) and tetramethoxysilane (TMOS) silica precursors and both methyltrimethoxysilane (MTMS) and methyltriethoxysilane (MTES) organically modified precursors. Microstructural differences, for example, porosity changes due to the different precursor backbone structures, are interrogated by monitoring oxygen gas and aqueous-phase sensor response. Oxygen sensing using these films is enabled by incorporating in the films an oxygen-sensitive ruthenium dye whose fluorescence is quenched in the presence of oxygen. Film properties such as thickness, thickness stabilization time, as well as sensor response, are discussed in terms of relative hydrolysis and condensation behavior for the different precursors. Film hydrophobicity, an issue which has been identified as being of crucial importance for optimum dissolved oxygen sensor response, is discussed and contact angle measurements are used to investigate the degree of hydrophobicity for different film types. The main motivation for this work is film optimization for optical gas-phase and dissolved oxygen sensors.     

Demonstration of a surface plasmon-coupled emission (SPCE)-based immunoassay in the absence of a spacer layer

The technique of surface plasmon-coupled emission (SPCE) involves the coupling of light which is emitted from a fluorophore into the surface plasmon of an adjacent thin metal film, giving rise to highly directional emission. We have combined the advantages of SPCE with the high light collection efficiency of supercritical angle fluorescence by carrying out an immunoassay on a paraboloid array biochip in the absence of the conventional SPCE spacer layer normally used to minimize metal quenching of the fluorescence. In this work, we have successfully demonstrated an SPCE-based assay by utilizing the protein assay layer as the spacer layer. A novel 3 × 3 injection molded polymer biochip with paraboloid elements was used. The paraboloid elements served to enhance the light collection efficiency while the top surface was coated with a gold layer to use excitation of surface plasmons and detection of SPCE emission. Theoretical modeling of the gold-protein layer structure showed that the surface plasmon resonance angles were located in the detection range of the paraboloid biochip. The polarization dependence of SPCE emission was also demonstrated. Finally, a human IgG sandwich immunoassay was carried out which exhibited a limit of detection of ~10 ng/ml using 3σ. The results demonstrate the potential of the SPCE-based paraboloid array biochip as a novel platform for high-throughput analysis of biomolecular interactions.     

Development of an integrated optic oxygen sensor using a novel, generic platform

This paper describes the development of a generic platform for enhanced, integrated optic sensors based on fluorescence detection. The platform employs a novel optical configuration in order to achieve enhanced performance and has inherent multianalyte detection capability. The sensor element comprises a multimode ridge waveguide that has been patterned with an analyte-sensitive fluorescent spot, which is excited directly using a LED. The platform was applied to the detection of gaseous oxygen as a proof of principle. The sol-gel-derived sensor spots were doped with an oxygen-sensitive fluorescent dichlororuthenium dye complex and intensity-based calibration data were generated from the oxygen-dependent waveguide output. The sensor achieved a LOD of 0.62% and a resolution of less than 0.96% gaseous oxygen, which compares favourably with a similar, recently reported system. This device highlights the combination of inexpensive rapid prototyping techniques and a dedicated sensor enhancement strategy that together facilitate the production of an effective prototype sensor platform.     

Optical Properties of Micro-patterned Silver Nanoparticle Substrates

In this paper, we describe a novel technique for depositing metal nanoparticles (NPs) on a planar substrate whereby the NPs are micro-patterned on the surface by a simple stamp-printing procedure. The method exploits the attractive force between negatively charged colloidal metal NPs and positively-charged polyelectrolyte layers which have been selectively deposited on the surface. Using this technique, large uniform areas of patterned metal NPs, with different plasmonic properties, were achieved by optimisation of the stamping process. We report the observation of unusual fluorescence emission from these structures. The emission was measured using epifluorescence microscopy. Fluorescence lifetime behaviour was also measured. Furthermore, the μ-patterned NPs exhibited blinking behaviour under 469 nm excitation and the fluorescence spectrum was multi-peaked. It has been established that the fluorescence is independent of the plasmon resonance properties of the NPs. As well as optimising the novel NP μ-patterning technique, this work discusses the origin and characteristics of the anomalous fluorescence behaviour in order to characterise and minimise this unwanted background contribution in the use of metal NPs for plasmonic enhancement of fluorescence for optical biochip applications.     

Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection

The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity.     

Development of a fluorescence lifetime-based sol-gel humidity sensor

A sol-gel-based optical sensor for the measurement of relative humidity has been developed. It is based on the changes in fluorescence intensity and/or lifetime of the ruthenium complex, ruthenium(II)diphenylphenanthroline-dipyridophenazinehexafluorophosphate. Sensitivity to relative humidity has been demonstrated over the range 0-100% relative humidity. This sensor has been developed for application in the field of indoor air-quality monitoring and displays a limit of detection of 0.35% relative humidity and a resolution of 1.13% over the concentration range of interest (0-50% relative humidity). The effects of varying process parameters on the sensor performance were studied along with the effects of cross-sensitivity to molecular oxygen.     

Characterisation of porosity and sensor response times of sol-gel-derived thin films for oxygen sensor applications

In recent years, sol-gel-derived films have played a significant role in optical sensor development. This paper focuses on the characterisation of oxygen-sensitive tetraethoxysilane (TEOS) and methyltriethoxysilane (MTEOS)-based films. Film porosity and sensor response times are reported for a range of films fabricated under different conditions. Porosity data are correlated with predicted film behaviour and also with previous characterisation studies. Oxygen diffusion coefficients are estimated from the response times. The enhanced diffusion coefficient of MTEOS films compared to TEOS films is discussed in terms of the relative oxygen solubility of the films. Comparisons are made with data for oxygen-sensitive polymer films, indicating an enhanced solubility for sol-gel films compared with typical polymer films.     

Synthesis and characterization of monodisperse, mesoporous, and magnetic sub-micron particles doped with a near-infrared fluorescent dye

Recently, multifunctional silica nanoparticles have been investigated extensively for their potential use in biomedical applications. We have prepared sub-micron monodisperse and stable multifunctional mesoporous silica particles with a high level of magnetization and fluorescence in the near infrared region using an one-pot synthesis technique. Commercial magnetite nanocrystals and a conjugated-NIR-dye were incorporated inside the particles during the silica condensation reaction. The particles were then coated with polyethyleneglycol to stop aggregation. X-ray diffraction, N₂ adsorption analysis, TEM, fluorescence and absorbance measurements were used to structurally characterize the particles. These mesoporous silica spheres have a large surface area (1978 m²/g) with 3.40 nm pore diameter and a high fluorescence in the near infrared region at λ=700 nm. To explore the potential of these particles for drug delivery applications, the pore accessibility to hydrophobic drugs was simulated by successfully trapping a hydrophobic ruthenium dye complex inside the particle with an estimated concentration of 3 wt%. Fluorescence imaging confirmed the presence of both NIR dye and the post-grafted ruthenium dye complex inside the particles. These particles moved at approximately 150 μm/s under the influence of a magnetic field, hence demonstrating the multifunctionality and potential for biomedical applications in targeting and imaging.     

Enhanced Fluorescence Sensing Using Sol-Gel Materials

The versatility of the sol-gel process with regard to the design of optimized films for fluorescence-based sensing is treated in detail. Sol-gel films are typically used to provide a microporous support matrix in which analyte-sensitive fluorophores are entrapped and into which smaller analyte species may diffuse and interact. The versatility of the process facilitates tailoring of the physicochemical film properties to optimize sensor performance. General principles of sensor optimization are presented. These include issues such as immobilization properties, refractive index control, and sensitivity enhancement. The particular advantages conferred by the use of ormosils are emphasized. In addition, the contribution made by tailored sol-gel films to recent advances in specific fluorescence-based sensor applications is emphasized. This work focuses mainly on results produced in the laboratory of the authors over the past decade.     

Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol-gel matrix

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM. In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.