This review focuses on the development and use of microfluidic devices within a clinical setting. The underlying theoretical background of microfluidics is briefly elucidated. The materials and techniques used to fabricate the devices and their applicability to the clinical environment are described. The current research in this area is appraised and projections for future applications are discussed.

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.

Cellular introduction of PEBBLEs (photonic explorers for bioanalysis with biologically localized embedding) has been investigated by a wide variety of methods in a range of cell types. These methods include surface functionalization with CPPs (cell-penetrating peptides), pinocytosis, commercial lipid transfection agents, cytochalasin D, picoinjection, and Gene gun bombardment. This paper will overview several of the most popular methods used for the introduction of PEBBLE nanosensors to the cellular environment and discuss the efficacy of the techniques.

This communication reports the development of a microfluidic device capable of maintaining the long-term culture of viable tissue biopsies. Tissue-based models will enable evaluation of cell-cell and cellmatrix interactions within multi-cellular systems. The device demonstrated is a prototype, fabricated with the capacity to receive biopsy samples up to 2 mm 3 , from various tissue sources. Presently, this system has been tested with human colorectal tissue biopsies, for periods in excess of 3 days. The response of normal colorectal tissue and neoplastic biopsies to hypoxia was assayed by the release of vascular endothelial growth factor (VEGF) into the media, which was measured off-chip. As anticipated, the hypoxia induced a greater VEGF response in the tumour biopsies than the nonmalignant tissue.

Sol-gel nanoprobes, also known as Photonic Explorer for Bioanalysis with Biologically Localised Embedding (PEBBLE), capable of performing in-vitro intracellular 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.