Droplet digital microfluidic system for screening filamentous fungi based on enzymatic activity.

Fungal cell-wall-degrading enzymes have great utility in the agricultural and food industries. These cell-wall-degrading enzymes are known to have functions that can help defend against pathogenic organisms. The existing methods used to discover these enzymes are not well adapted to fungi culture and morphology, which prevents the proper evaluation of these enzymes. We report the first droplet-based microfluidic method capable of long-term incubation and low-voltage conditions to sort filamentous fungi inside nanoliter-sized droplets. The new method was characterized and validated in solid-phase media based on colloidal chitin such that the incubation of single spores in droplets was possible over multiple days (2-4 days) and could be sorted without droplet breakage. With long-term culture, we examined the activity of cell-wall-degrading enzymes produced by fungi during solid-state droplet fermentation using three highly sensitive fluorescein-based substrates. We also used the low-voltage droplet sorter to select clones with highly active cell-wall-degrading enzymes, such as chitinases, ß-glucanases, and ß-N-acetylgalactosaminidases, from a filamentous fungi droplet library that had been incubated for >4 days. The new system is portable, affordable for any laboratory, and user-friendly compared to classical droplet-based microfluidic systems. We propose that this system will be useful for the growing number of scientists interested in fungal microbiology who are seeking high-throughput methods to incubate and sort a large library of fungal cells.

Community-led risk analysis of direct-to-consumer whole-genome sequencing.

Direct-to-consumer (DTC) genetic testing is cheaper and more accessible than ever before; however, the intention to combine, reuse, and resell this genetic information as powerful data sets is generally hidden from the consumer. This financial gain is creating a competitive DTC market, reducing the price of whole-genome sequencing (WGS) to under 300 USD. Entering this transition from single-nucleotide polymorphism-based DTC testing to WGS DTC testing, individuals looking for access to their whole-genomic information face new privacy and security risks. Differences between WGS and other methods of consumer genetic tests are left unexplored by regulation, leading to the application of legal data anonymization methods on whole-genome data, and questionable consent methods. Large representative genomic data sets are important for research and improve the standard of medicine and personalized care. However, these data can also be used by market players, law enforcement, and governments for surveillance, population analyses, marketing purposes, and discrimination. Here, we present a summary of the state of WGS DTC genetic testing and its current regulation, through a community-based lens to expose dual-use risks in consumer-facing biotechnologies.

Digital Microfluidics Chips for the Execution and Real-Time Monitoring of Multiple Ribozymatic Cleavage Reactions.

In this paper, we describe the design and performance of two digital microfluidics (DMF) chips capable of executing multiple ribozymatic reactions, with proper controls, in response to short single-stranded DNA inducers. Since the fluorescence output of a reaction is measurable directly from the chip, without the need for gel electrophoresis, a complete experiment involving up to eight reactions (per chip) can be carried out reliably, relatively quickly, and efficiently. The ribozymes can also be used as biosensors of the concentration of oligonucleotide inputs, with high sensitivity, low limits of quantification and of detection, and excellent signal-to-noise ratio. The presented chips are readily usable devices that can be used to automate, speed up, and reduce the costs of ribozymatic reaction experiments.

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation.

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in-house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knockout transfections.

An integrated droplet-digital microfluidic system for on-demand droplet creation, mixing, incubation, and sorting.

Droplet microfluidics is a technique that has the ability to compartmentalize reactions in sub nano- (or pico-) liter volumes that can potentially enable millions of distinct biological assays to be performed on individual cells. In a typical droplet microfluidic system, droplets are manipulated by pressure-based flows. This has limited the fluidic operations that can be performed in these devices. Digital microfluidics is an alternative microfluidic paradigm with precise control and manipulation over individual droplets. Here, we implement an integrated droplet-digital microfluidic (which we call 'ID2M') system in which common fluidic operations (i.e. droplet generation, cell encapsulation, droplet merging and mixing, droplet trapping and incubation, and droplet sorting) can be performed. With the addition of electrodes, we have been able to create droplets on-demand, tune their volumes on-demand, and merge and mix several droplets to produce a dilution series. Moreover, this device can trap and incubate droplets for 24 h that can consequently be sorted and analyzed in multiple n-ary channels (as opposed to typical binary channels). The ID2M platform has been validated as a robust on-demand screening system by sorting fluorescein droplets of different concentration with an efficiency of ∼96%. The utility of the new system is further demonstrated by culturing and sorting tolerant yeast mutants and wild-type yeast cells in ionic liquid based on their growth profiles. This new platform for both droplet and digital microfluidics has the potential to be used for screening different conditions on-chip and for applications like directed evolution.