XPORT ENTRAP: A droplet microfluidic platform for enhanced DNA transfer between microbial species.

A significant hurdle for the widespread implementation and use of synthetic biology is the challenge of highly efficient introduction of DNA into microorganisms. This is especially a barrier for the utilization of non-model organisms and/or novel chassis species for a variety of applications, ranging from molecular biology to biotechnology and biomanufacturing applications. Common approaches to episomal and chromosomal gene editing, which employ techniques such as chemical competence and electroporation, are typically only amenable to a small subset of microbial species while leaving the vast majority of microorganisms in nature genetically inaccessible. To address this challenge, we have employed the previously described B. subtilis broad-host conjugation strain, XPORT, which was modularly designed for loading DNA cargo and conjugating such DNA into recalcitrant microbes. In this current work, we have leveraged and adapted the XPORT strain for use in a droplet microfluidic platform to enable increased efficiency of conjugation-based DNA transfer. The system named DNA ENTRAP (DNA ENhanced TRAnsfer Platform) utilizes cell-encapsulated water-in-oil emulsion droplets as pico-liter-volume bioreactors that allows controlled contacts between the donor and receiver cells within the emulsion bioreactor. This allowed enhanced XPORT-mediated genetic transfer over the current benchtop XPORT process, demonstrated using two different Bacillus subtilis strains (donor and receiver), as well as increased throughput (e.g., number of successfully conjugated cells) due to the automated assay steps inherent to microfluidic lab-on-a-chip systems. DNA ENTRAP paves the way for a streamlined automation of culturing and XPORT-mediated genetic transfer processes as well as future high-throughput cell engineering and screening applications.

Sub-second heat inactivation of coronavirus using a betacoronavirus model.

Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and four proteins, three of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub-second heat inactivation of CoV. The system is composed of a stainless-steel capillary immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log10 reduction in viral titer (starting titer: 5 × 107 PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log10 reduction).

Eliminating air bubble in microfluidic systems utilizing integrated in-line sloped microstructures.

In most microfluidic systems, formation and accumulation of air and other gas bubbles can be detrimental to their operation. Air bubbles in a microfluidic channel induce a pressure profile fluctuation and therefore disturb the stability of the system. Once an air bubble is generated, it is also extremely difficult to remove such bubbles from the microfluidic systems. In tissue and cell culture microfluidic systems, a single air bubble can completely shear off cells that are being cultured. Air bubbles can be especially problematic in microfluidic systems that have to operate for long periods of time, since completely eliminating the generation of air bubbles for prolonged periods of time, where a single air bubble can ruin an entire multi-day/multi-week experiment, is extremely challenging. Several in-line and off-chip bubble traps have been developed so far, but cannot completely eliminate air bubbles from the system or are relatively difficult to integrate into microfluidic systems. Recent advancements in two-photon polymerization (2PP)-based microfabrication method eliminates the restriction in Z-axis control in conventional two-dimensional microfabrication methods, and thus enables complex 3D structures to be fabricated at sub-micrometer resolution. In this work, by utilizing this 2PP technique, we developed a sloped microfluidic structure that is capable of both trapping and real-time removal of air bubbles from the system in a consistent and reliable manner. The novel structures and designs developed in this work present a unique opportunity to overcome many limitations of current methods, bring state-of-the-art solutions in air bubble removal, and enable a multifunctional microfluidic device to operate seamlessly free from air bubble disruption. The microfabricated system was tested in both droplet microfluidics and continuous-flow microfluidics applications, and demonstrated to be effective in preventing air bubble aggregation over time. This simple sloped microstructure can be easily integrated into broad ranges of microfluidic devices to minimize bubble introduction, which will contribute to creating a stable and bubble-free microfluidic platform amenable for long-term operation.

An ultra high-efficiency droplet microfluidics platform using automatically synchronized droplet pairing and merging.

Droplet microfluidics systems hold great promise in their ability to conduct high-throughput assays for a broad range of life science applications. Despite their promise in the field and capability to conduct complex liquid handling steps, currently, most droplet microfluidic systems used for real assays utilize only a few droplet manipulation steps connected in series, and are often not integrated together on a single chip or platform. This is due to the fact that linking multiple sequential droplet functions within a single chip to operate at high efficiency over long periods of time remains technically challenging. Considering sequential manipulation is often required to conduct high-throughput screening assays on large cellular and molecular libraries, advancements in sequential operation and integration are required to advance the field. This current limitation greatly reduces the type of assays that can be realized in a high-throughput droplet format and becomes more prevalent in large library screening applications. Here we present an integrated multi-layer droplet microfluidic platform that can handle large numbers of droplets with high efficiency and minimum error. The platform combines two-photon photolithography-fabricated curved microstructures that allow high-efficiency (99.9%) re-flow of droplets and a unique droplet cleaving that automatically synchronizes paired droplets enabling high-efficiency (99.9%) downstream merging. We demonstrate that this method is applicable to a broad range of droplet sizes, including relatively large droplet sizes (hundreds of micrometers in diameter) that are typically more difficult to manipulate with high efficiency, yet are required in many cell assay applications requiring large organisms or multiple incubation steps. The utility of this highly efficient integrated droplet microfluidic platform was demonstrated by conducting a mock antibiotic screening assay against a bacterial pathogen. The approach and system presented here provides new avenues for the realization of ultra-high-efficiency multi-step droplet microfluidic systems with minimal error.

PRESCIENT: platform for the rapid evaluation of antibody success using integrated microfluidics enabled technology.

Identifying antibodies (Abs) that neutralize infectious agents is the first step for developing therapeutics, vaccines, and diagnostic tools for these infectious agents. However, current approaches for identifying neutralizing Abs (nAbs) typically rely on dilution-based assays that are costly, inefficient, and only survey a small subset of the entire repertoire. There are also intrinsic biases in many steps of conventional nAb identification processes. More importantly, conventional assays rely on simple Ab-antigen binding assays, which may not result in identifying the most potent nAbs, as the strongest binder may not be the most potent nAb. Droplet microfluidic systems have the capability to overcome such limitations by conducting complex multi-step assays with high reliability, resolution, and throughput in a pico-liter volume water-in-oil emulsion droplet format. Here, we describe the development of PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated microfluidics ENabled Technology), a droplet microfluidic system that can enable high-throughput single-cell resolution identification of nAb repertoires elicited in response to viral infection. We demonstrate PRESCIENT's ability to identify Abs that neutralize a model viral agent, Murine coronavirus (murine hepatitis virus), which causes high mortality rates in experimentally infected mice. In-droplet infection of host cells by the virus was first demonstrated, followed by demonstration of in-droplet neutralization by nAbs produced from a single Ab-producing hybridoma cell. Finally, fluorescence intensity analyses of two populations of hybridoma cell lines (nAb-producing and non-nAb-producing hybridoma cell lines) successfully discriminated between the two populations. The presented strategy and platform have the potential to identify and investigate neutralizing activities against a broad range of potential infectious agents for which nAbs have yet to be discovered, significantly advancing the nAb identification process as well as reinvigorating the field of Ab discovery, characterization, and development.

Enhancing droplet transition capabilities using sloped microfluidic channel geometry for stable droplet operation.

Droplet-based microfluidics technology allows for the generation and control of droplets that function as independent chemical and biological reactors, enabling broad ranges of high-throughput assays. As more complex multi-step assays are being realized in droplet format, maintaining droplet stability throughout the assay becomes a critical requirement. Unfortunately, as droplets go through multiple manipulation steps, droplet breakage is commonly seen, especially where droplets have to go through sharp transitions in direction and shape. Standard microfabrication techniques typically result in inherent sharp geometry in Z-direction due to their two-dimensional fabrication nature. Recent advancement in micro- and nano- fabrication technology using two-photon polymerization (2PP) is enabling complex 3D microstructures with sub-micrometer resolution to be readily fabricated. Here, utilizing this microfabrication technique, we present a simple solution to the droplet stability challenge by utilizing sloped-geometry microfluidic channels to enable microdroplets to smoothly transition between microfluidic channels having two different heights without breakage. The technique and innovation demonstrated here have the potential to replace conventional droplet microfluidic device fabrication approaches and enable droplet microfluidic platforms to achieve significantly higher level of efficiency, accuracy, and stability never realized before.