In Situ Live Monitoring of Extracellular Acidosis near Cancer Cells Using Digital Microfluidics with an Integrated Optical pH Sensor Film.

We demonstrate the live monitoring of extracellular acidification on digital microfluidics using a chip-integrated fluorescent pH sensor film. The metabolism of various types of live cells including cancer and healthy cells were investigated through recording the extracellular pH (pHe) change. An optical pH sensor array was integrated onto a digital microfluidic (DMF) interface with a diameter of 2 mm per pH-sensing spot. Miniaturized, label-free, and noninvasive monitoring of extracellular acidosis on DMF was realized within a pH range of 5.0-8.0 with good sensitivity and rapid response. The pH sensitive probe fluorescein-5-isothiocyanate was covalently bound to poly-2-hydroxyethyl methacrylate and immobilized on a circularly exposed indium tin oxide interface on the DMF top plate. The surface of the fabricated pH sensor spots was modified with polydopamine via self-polymerization. Direct cell attachment on the sensor surfaces enabled rapid pH detection near the cell membranes. Automatic medium exchange on cell-attached pH sensing sites was achieved though solution passive dispensing on DMF. The developed DMF platform was used to monitor the pHe decrease during MCF-7 and A549 cancer cell proliferation due to abnormal glycolysis metabolism. A rapid pH decrease at the pH sensing area in the presence of cancer cells could be detected within 2 min after fresh medium exchange, while no obvious pHe change was observed with HUVEC healthy cells. Real-time detection of cell acidification and cellular response to different metabolic conditions such as higher glucose levels or administered anticancer drugs was possible.

Reversibly-bonded microfluidic devices for stable cell culture and rapid, gentle cell extraction.

Microfluidic chips have emerged as significant tools in cell culture due to their capacity for supporting cells to adopt more physiologically relevant morphologies in 3D compared with traditional cell culture in 2D. Currently, irreversible bonding methods, where chips cannot be detached from their substrates without destroying the structure, are commonly used in fabrication, making it challenging to conduct further analysis on cells that have been cultured on-chip. Although some reversible bonding techniques have been developed, they are either restricted to certain materials such as glass, or require complex processing procedures. Here, we demonstrate a simple and reversible polydimethylsiloxane (PDMS)-polystyrene (PS) bonding technique that allows devices to withstand extended operations while pressurized, and supports long-term stable cell cultures. More importantly, it allows rapid and gentle live cell extraction for downstream manipulation and characterization after long-term on-chip culturing, and even further subculturing. Our new approach could greatly facilitate microfluidic chip-based cell and tissue cultures, overcoming current analytical limitations and opening up new avenues for downstream uses of on-chip cultures, including 3D-engineered tissue structures for biomedical applications.

Optical pH Monitoring in Microdroplet Platforms for Live Cell Experiments Using Colloidal Surfactants.

Droplet microfluidic technology facilitates the development of high-throughput screening applications in nanoliter volumes. Surfactants provide stability for emulsified monodisperse droplets to carry out compartmentalization. Fluorinated silica-based nanoparticles are used; they can minimize crosstalk in microdroplets and provide further functionalities by surface labeling. Here we describe a protocol for monitoring pH changes in live single cells by fluorinated silica nanoparticles, for their synthesis, chip fabrication, and optical monitoring on the microscale. The nanoparticles are doped with ruthenium-tris-1,10-phenanthroline dichloride on the inside and conjugated with fluorescein isothiocyanate on the surface. This protocol may be used more generally to detect pH changes in microdroplets. The fluorinated silica nanoparticles can also be used as droplet stabilizers with an integrated luminescent sensor for other applications.

A microfluidic chip for rapid analysis of DNA melting curves for BRCA2 mutation screening.

A microfluidic chip integrated with a microheater and a luminescent temperature sensor for rapid, spatial melting curve analysis was developed and applied for the screening of a breast cancer gene fragment. The method could detect genetic differences in around 3 minutes total for the whole procedure, which is much faster than established procedures. A microfabrication technique was developed to allow for bonding of a temperature sensing thin film and a Pt microheater with PDMS and the chips could be employed to generate and measure thermal gradients and the fluorescence intensity of stained DNA through multispectral optical imaging. The sensing layer consisting of poly(styrene-co-acrylonitrile) and a tris(1,10-phenanthroline)ruthenium(ii) temperature probe was generated by blade coating on a glass substrate with an attached Pt microheater. Calibration of the temperature between 20 and 90 °C yielded an overall resolution of around 0.13 K. The chip was employed for the screening of the BRCA 2 breast cancer gene; BRCA2 exon 5 was differentiated by its mutant rs80359463 by a 1.1 K difference in melting temperature and two fragments of BRCA2 exon 11 were differentiated by their mutants rs276174826 and rs876660311 by 0.7 K and 2.0 K, respectively. The standard deviations were between 0.1 and 0.5 K. Capable of detecting fluorescence in the DNA and temperature simultaneously and being imaged in a customized assembly, this microchip can be used to screen for mutations in a variety of DNA samples in disease diagnosis and prognosis.