Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

/pdf/reactions-in-droplets-in-microfluidic-channels-22iig0bcxm.pdf

Reactions in Droplets in Microfluidic Channels

Droplet microfluidics generates and manipulates discrete droplets through immiscible multiphase flows inside microchannels Due to its remarkable advantages, droplet microfluidics bears significant value in an extremely wide range of area In this review, we provide a comprehensive and in-depth insight into droplet microfluidics, covering fundamental research from microfluidic chip fabrication and droplet generation to the applications of droplets in bio(chemical) analysis and materials generation The purpose of this review is to convey the fundamentals of droplet microfluidics, a critical analysis on its current status and challenges, and opinions on its future development We believe this review will promote communications among biology, chemistry, physics, and materials science

Emerging Droplet Microfluidics

Asymmetric transformations catalyzed by enzymes in organic solvents

Motility is a fundamental property of mammalian cells that normally is observed in tissue culture by time lapse microscopy where resolution is limited by the wavelength of light. This paper examines a powerful electrical technique by which cell motion is quantitatively measured at the nanometer level. In this method, the cells are cultured on small evaporated gold electrodes carrying weak ac currents. A large change in the measured electrical impedance of the electrodes is observed when cells attach and spread on these electrodes. When the impedance is tracked as a function of time, fluctuations are observed that are a direct measure of cell motion. Surprisingly, these fluctuations continue even when the cell layer becomes confluent. By comparing the measured impedance with a theoretical model, it is clear that under these circumstances the average motions of the cell layer of 1 nm can be inferred from the measurements. We refer to this aspect of cell motility as micromotion.

https://www.pnas.org/content/pnas/88/17/7896.full.pdf

Micromotion of mammalian cells measured electrically.

http://chemotaxis.semmelweis.hu/CHTXhpg/ECIS/WegenerJ_ExpCellRes_2000.pdf

Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces.

Mammalian cells can be cultured and therefore studied in vitro. Normally, the cells' morphology and other static properties are observed with the aid of a light microscope. A method is described here that allows observation of the dynamical aspects of cultured cells. Mammalian fibroblasts are cultured in polystyrene dishes that contain evaporated gold electrodes. As the cells attach to the electrodes, their presence and their motion is directly reflected in the measured impedance.

Use of Electric Fields to Monitor the Dynamical Aspect of Cell Behavior in Tissue Culture

We investigated the ability of several hydrolases to catalyze reactions with an abiotic water-insoluble substrate, carbonic acid diphenyl ester, also known as diphenyl carbonate (DPC). In single-phase water/organic systems, turnover numbers (TN) of greater than 2 x 10(4) min(-1)have been achieved for the hydrolysis of DPC. The K(m) values for the hydrolytic reaction were measured to be 200 microM and 330 microM for Candida cylindracea lipase and Porcine liver esterase, respectively. In addition to hydrolysis, we observed transesterification of carbonates with a wide variety of alcohol and phenol species. Transesterifications of DPC with bifunctional alcohols resulted in the synthesis of polycarbonates. We investigated the stability and transesterification activity of these enzymes in several water-restricted environments to limit competing hydrolysis reactions. We find that, with the removal of water, hydrolysis is reduced more than four orders of magnitude while transesterification is diminished only 10-fold (turnover numbers of 600 min(-1) in water-miscible systems to 60 min(-1) in water-restricted environments with pure Candida lipase). Stability of the Candida lipase in these water-restricted environments (half-life of longer than 3 days) is much greater than in water/organic single phase systems (5 h in 20% methanol). In addition, the Candida lipase displayed enantiomeric selectivity in transesterifications of DPC with racemic 2-butanol (greater than 80% ee).

Enzymatic transesterifications of carbonates in water-restricted environments.

Anchorage-dependent cell growth is demonstrated on microcarriers of fluorocarbon fluid formed by emulsification and stabilized with polylysine.

https://science.sciencemag.org/content/sci/219/4591/1448.full.pdf

Cell growth on liquid microcarriers

Test method and apparatus for determining the presence of, the concentration of, or the absence of, immunologically-active substances in liquid media by measuring any change of electrical impedance of an electrode due to the presence of, or the absence of, the reaction of a product of enzyme linked immunologically-active substance and a proper enzyme substrate.

Electrical detection of the imune reaction

Anchorage-dependent fibroblasts can be cultured by using as a substrate the protein layer that spontaneously forms at the liquid-liquid interface between fluorocarbon fluids and tissue culture medium. For this novel substrate to be effective in supporting confluent cell layers, the protein monolayer must support the stresses exerted by spreading fibroblasts. The composition of the fluorocarbon fluid has a significant effect on the strength of the protein layer and, thus, on the patterns of cell growth. Evidence is presented demonstrating that a protein film, sufficiently strong to support cell growth, does not occur on purified fluorocarbon fluids but requires the presence of trace amounts of polar, surface active compounds. By the addition of small quantities of pentafluorobenzoyl chloride to alumina-treated fluorocarbon fluids, excellent interfacial substrates can be produced. We have applied this understanding to produce a liquid microcarrier system capable of general use with a variety of cells, including human fibroblasts. A microcarrier in which the fluorocarbon is replaced with polydimethyldiphenyl siloxane is also described.

https://www.pnas.org/content/pnas/80/18/5622.full.pdf

Charles R. Keese

Papers

Use of Electric Fields to Monitor the Dynamical Aspect of Cell Behavior in Tissue Culture

Enzymatic transesterifications of carbonates in water-restricted environments.

Cell growth on liquid microcarriers

Electrical detection of the imune reaction

Cell growth on liquid interfaces: Role of surface active compounds