An integrated droplet based microfluidic platform for high throughput, multi-parameter screening of photosensitiser activity

With rapid advances in the field of cellomics, genomics, and proteomics, the demands for development of enabling technologies for performing high throughput biological experimentation are ever increasing. Droplet based microfluidic systems have recently been developed to perform high throughput experimentations. With the ability to generate droplets over 1 kHz frequency and perform combinatorial experiments via various passive and active manipulating techniques, microdroplet technology provides an ideal platform for combinatorial biological experiments whilst consuming minimal amount of reagent. As it is possible to generate droplets, manipulate them, and characterise droplets using highly sensitive on-line detection systems, it is now crucial to bring various functionalities together to create a micro total analysis system capable of performing complex biological experiments within microfluidic devices. As such, an integrated droplet based microfluidic platform was developed to assess the efficacy of photodynamic therapy against microbial organisms. Photodynamic therapy is an alternative efficacious treatment method for the treatment of localized microbial infections with several favourable features such as broad spectrum of action, efficient inactivation of multidrug-resistant bacteria, and low mutagenic potential. In order to perform the photosensitiser cytotoxicity screening, various microfluidic modules such as droplet generation, chamber based microdroplet storage and light irradiation, droplet reinjection, electrocoalescence and on-chip viability scoring of cells within droplets using a combination of carboxyfluorescein diacetate and propidium iodide were developed and integrated within the microfluidic platform. The microfluidic system was then used to screen the cytotoxicity of TBO against E.coli cells and the results were validated against conventional colony forming unit assays. Finally, the integrated system was used to assess the effects of several parameters on E.coli viability such as dark toxicity, photosensitiser concentration, light dose and poor oxygenation condition.Open Acces

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.

Combinatorial microfluidic droplet engineering for biomimetic material synthesis

Although droplet-based systems are used in a wide range of technologies, opportunities for systematically customizing their interface chemistries remain relatively unexplored. This article describes a new microfluidic strategy for rapidly tailoring emulsion droplet compositions and properties. The approach utilizes a simple platform for screening arrays of droplet-based microfluidic devices and couples this with combinatorial selection of the droplet compositions. Through the application of genetic algorithms over multiple screening rounds, droplets with target properties can be rapidly generated. The potential of this method is demonstrated by creating droplets with enhanced stability, where this is achieved by selecting carrier fluid chemistries that promote titanium dioxide formation at the droplet interfaces. The interface is a mixture of amorphous and crystalline phases, and the resulting composite droplets are biocompatible, supporting in vitro protein expression in their interiors. This general strategy will find widespread application in advancing emulsion properties for use in chemistry, biology, materials and medicine

Compartmentalization of electrophoretically separated analytes in a multiphase microfluidic platform

Herein, we describe the monolithic integration of a multiphase microfluidic system to a microcapillary gel electrophoresis (?CGE) architecture for the complete isolation and storage of separated analyte bands. Within this platform, analyte molecules are separated using microchannel gel electrophoresis, and the eluted bands are stored in a sequence of approximately 40?600 encapsulating microdroplets. Importantly, employing such a system allows for total control of droplet size, shape, and composition. This approach is utilized to separate, optically detect, and encapsulate two fluorescent analytes from a composite sample mixture. Further to this, we subsequently investigate the potential of the system to be used as a concentration gradient generator through analysis of the segmented analyte bands and droplet composition

A droplet-based microfluidic platform for high-throughput, multi-parameter screening of photosensitizer activity

We present a fully integrated droplet-based microfluidic platform for the high-throughput assessment of photodynamic therapy photosensitizer (PDT) efficacy on Escherichia coli. The described platform is able to controllably encapsulate cells and photosensitizer within pL-volume droplets, incubate the droplets over the course of several days, add predetermined concentrations of viability assay agents, expose droplets to varying doses of electromagnetic radiation, and detect both live and dead cells online to score cell viability. The viability of cells after encapsulation and incubation is assessed in a direct fashion, and the viability scoring method is compared to model live/dead systems for calibration. Final results are validated against conventional colony forming unit assays. In addition, we show that the platform can be used to perform concurrent measurements of light and dark toxicity of the PDT agents and that the platform allows simultaneous measurement of experimental parameters that include dark toxicity, photosensitizer concentration, light dose, and oxygenation levels for the development and testing of PDT agent

3D Droplet Microfluidic Systems for High-Throughput Biological Experimentation

Herein, we describe the development of a multilayer droplet microfluidic system for creating concentration gradients and generating microdroplets of varying composition for high-throughput biochemical and cell-based screening applications. The 3D droplet-based microfluidic device consists of multiple PDMS layers, which are used to generate logarithmic concentration gradient reagent profiles. Parallel flow focusing structures are used to form picoliter-sized droplets of defined volumes but of varying composition. As proof of concept, we demonstrate rapid enzymatic activity assays and drug cytotoxicity assays on bacteria. The 3D droplet-based microfluidic platform has the potential to allow for high-efficiency and high-throughput analysis, overcoming the structural limitations of single layer microfluidic systems

Droplet-Based Microfluidic Platform for High-Throughput, Multi-Parameter Screening of Photosensitizer Activity

We present a fully integrated droplet-based microfluidic platform for the high-throughput assessment of photodynamic therapy photosensitizer (PDT) efficacy on <i>Escherichia coli</i>. The described platform is able to controllably encapsulate cells and photosensitizer within pL-volume droplets, incubate the droplets over the course of several days, add predetermined concentrations of viability assay agents, expose droplets to varying doses of electromagnetic radiation, and detect both live and dead cells online to score cell viability. The viability of cells after encapsulation and incubation is assessed in a direct fashion, and the viability scoring method is compared to model live/dead systems for calibration. Final results are validated against conventional colony forming unit assays. In addition, we show that the platform can be used to perform concurrent measurements of light and dark toxicity of the PDT agents and that the platform allows simultaneous measurement of experimental parameters that include dark toxicity, photosensitizer concentration, light dose, and oxygenation levels for the development and testing of PDT agents