Florian Hollfelder

Microdroplets in Microfluidics: An Evolving Platform for Discoveries in Chemistry and Biology

Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.

Quantitative detection of protein expression in single cells using droplet microfluidics

We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.

Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets

We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.

Development of quantitative cell-based enzyme assays in microdroplets.

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

Cross-talk between Histone Modifications in Response to Histone Deacetylase Inhibitors MLL4 LINKS HISTONE H3 ACETYLATION AND HISTONE H3K4 METHYLATION

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the “degree” of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.

An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets.

Microdroplets have great potential for high‐throughput biochemical screening. We report the design of an integrated microfluidic device for droplet formation, incubation and screening. Picolitre water‐in‐oil droplets can be stored in a reservoir that contains ∼106 droplets. In this reservoir droplets are stable for at least 6 h, which gives an extended timescale for biochemical experiments. We demonstrate the utility of the system by following the in vitro expression of green fluorescent protein. The high efficiency allows protein expression from a single molecule of DNA template, creating “monoclonal droplets” in which genotype and phenotype are combined in one emulsion compartment.

What makes an enzyme promiscuous

Kinetic analyses of promiscuous enzymes reveal rate accelerations, (k(cat)/K(M))/k(2), of up to 10(18) for their secondary activities. Such large values suggest that binding and catalysis can be highly efficient for more than one reaction, challenging the notion that proficient catalysis requires specificity. Growing numbers of reported promiscuous activities indicate that catalytic versatility is an inherent property of many enzymes. The examples discussed here illustrate promiscuous molecular recognition mechanisms that, together with knowledge from structural and computational analysis, might be used for the identification or development of catalysts for new reactions.

Controlling the Retention of Small Molecules in Emulsion Microdroplets for Use in Cell-Based Assays

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%). We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.

Simultaneous determination of gene expression and enzymatic activity in individual bacterial cells in microdroplet compartments.

A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.

Microfluidic droplets: new integrated workflows for biological experiments

Miniaturization of the classical test tube to picoliter dimensions is possible in monodisperse water-in-oil droplets that are generated in microfluidic devices. The establishment of standard unit operations for droplet handling and the ability to carry out experiments with DNA, proteins, cells and organisms provides the basis for the design of more complex workflows to address biological challenges. The emerging experimental format makes possible a quantitative readout for large numbers of experiments with a precision comparable to the macroscopic scale. Directed evolution, diagnostics and compound screening are areas in which the first steps are being taken toward the long-term goal of transforming the way we design and carry out experiments.