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An in-depth understanding of biochemical processes occurring within biological systems is key for early diagnosis of disease and identification of appropriate treatments. Nanobiophotonics offers huge potential benefits for intracellular diagnostics and therapeutics. Intracellular sensing using fluorescent nanoparticles is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. This work is focused on synthesis of optical chemical nanosensors for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly silica matrix with co-encapsulated Texas Red, acting as a reference dye, and pH-sensitive fluorescein isothiocyanate enabling ratiometric quantitative environmental detection. In order to obtain silica-based nanoparticles -70 nm in size, a modified sol-gel-based Stöber method was employed. The potential of these nanosensors for intracellular pH monitoring is demonstrated inside a live human embryonic kidney cell line whereby a significant change in fluorescence is observed when the cell pH is switched from acidic to basic. High loading efficiencies of nanoparticles into the cells is seen, with little effect on cell morphology even following extended nanoparticle exposure (up to 72 h). Nanoparticle incubation time and the fast response of the nanosensor (-2 s) make it a very powerful tool in monitoring the processes occurring within the cytosol.

There is increasing interest in the use of nanoparticles (NPs) for biomedical applications. In particular, nanobiophotonic approaches using fluorescence offers the potential of high sensitivity and selectivity in applications such as cell imaging and intracellular sensing. In this review, we focus primarily on the use of fluorescent silica NPs for these applications and, in so doing, aim to enhance and complement the key recent review articles on these topics. We summarize the main synthetic approaches, namely the Stöber and microemulsion processes, and, in this context, we deal with issues in relation to both covalent and physical incorporation of different types of dyes in the particles. The important issue of NP functionalization for conjugation to biomolecules is discussed and strategies published in the recent literature are highlighted and evaluated. We cite recent examples of the use of fluorescent silica NPs for cell imaging in the areas of cancer, stem cell and infectious disease research, and we review the current literature on the use of silica NPs for intracellular sensing of oxygen, pH and ionic species. We include a short final section which seeks to identify the main challenges and obstacles in relation to the potential widespread use of these particles for in vivo diagnostics and therapeutics.

We present a polymer biochip with embedded optics which allows the detection of supercritical angle fluorescence (SAF) without losses due to total internal reflection within the substrate. The chip design comprises structured spherical and aspherical optical elements on the bottom, while the top is chemically functionalized for direct binding of biomolecules. Furthermore, this design facilitates integration in lab-on-a-chip systems with appropriate microfluidics. In the confocal optical setup an ellipsoidal mirror is used for collection of SAF light above the critical angle of the water-polymer interface, which is detected by a photon-counting detector. The work presented here represents a proof of concept for performing sensitive and rapid point-of-care testing, using this low-cost, robust and disposable optical biochip platform. The performance of the platform was validated using direct binding DNA and human IgG assays which yielded low limits of detection 10 pM for DNA and 10 pg/ml for human IgG.

We have carried out a human IgG immunoassay on a novel disposable optical array biochip using surface plasmon-coupled emission (SPCE) detection. The work successfully combines the advantages of the highly directional SPCE emission profile and enhanced surface plasmon excitation with the high light collection efficiency achieved using supercritical angle fluorescence (SAF). This is achieved using an array of transparent paraboloid polymer elements which have been coated with a thin gold layer to facilitate SPCE. Moreover, since only the emission of molecules which are close to the metal surface couple into the surface plasmon, the detection is highly surface-specific leading to background suppression and increased signal-to-noise ratio. Theoretical calculations have been carried out in order to match the surface plasmon resonance angles and SPCE emission angles to the paraboloid array features for light collection. A sandwich assay format was used and a dose response curve was obtained in the concentration range 2 ng/ml to 200 microg/ml yielding a limit of detection of 20 ng/ml. This is the first demonstration of an SPCE-based assay on a disposable biochip platform and indicates the potential of SPCE-based arrays for high-throughput analysis of biomolecular interactions., (Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.)

This article describes strategies for achieving fluorescence enhancement in optical biochips. Two strategies are discussed: plasmonic enhancement, which is due to the localized surface plasmon resonance of metal nanostructures that are adjacent to the fluorescent labels in optical immunoassays; and the use of high-brightness silica nanoparticles as enhanced labels. We present a review of the state-of-the-art in both areas, including synthesis techniques for the metal and silica nanoparticles and the use of the nanoparticles in optical immunoassays. Data are presented that highlight the key design parameters which influence the level of enhancement and model assay data are presented that illustrate potential enhancements in assay performance.