Abstract
Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory.
Highlights
In situ hybridization (ISH) is a molecular technique in which a nucleic acid sequence of interest within an intact cell or a tissue section is hybridized with a labeled probe to give a measurable signal
The diverse microfluidic platforms described in the literature so far have addressed, to some extent, the key challenges of conventional Fluorescence in situ hybridization (FISH) protocols, namely, improving on the long time to result, the labor-intensive procedures, the lack of automation, as well as the relatively high cost of reagents, especially the hybridization probes
Building on recent advances in FISH techniques, the field is poised to evolve into novel directions, which in turn impose a new set of challenges
Summary
In situ hybridization (ISH) is a molecular technique in which a nucleic acid sequence of interest within an intact cell or a tissue section is hybridized with a labeled probe to give a measurable signal. Lee et al fabricated a 16 × 6 array of PDMS wells, each of 1.5 mm diameter, onto a gold functionalized glass slide, which was utilized for cell attachment and removed to carry out the FISH assay in a conventional manner [51] Neither of these systems featured microfluidic flow channels. The first publications on microfluidics-based FISH by Sieben et al in 2007–2008 [37, 63] featured a very high level of integration and automation They presented a device with a circular channel to allow recirculation of probe over immobilized cells (Fig. 3a) based on pneumatic pumps and valves inspired by the work from Mathies’ group [64]. A fairly wide range of design and engineering approaches are available to trap and immobilize the cells and tissue sections, introduce FISH reagents, control temperature and carry out the fluorescence microscopy readout. Commercialization has been pursued for some of the approaches, such as the microFIND device with a straight channel for cell trapping on the nanostructure surface and the Celsee device with cells being trapped in a regular array with holes
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