Microfluidic devices for high-throughput plant phenotyping and bioenergy harvesting from microbes and living plants

Microfluidics and micro/nanofabrication techniques provide powerful technological platforms to develop miniature bioassay devices for studying cellular and multicellular organisms. Microfluidic devices have many advantages over traditional counterparts, including good throughput due to parallel experiments, low infrastructural cost, fast reaction, reduced consumption of agent and reagent, and avoidance of contamination. This thesis is focused on the development of a microfluidic toolkit with several miniature devices to tackle important problems that the fields of plant phenotyping and bioenergy harvesting are facing. The ultimate goal of this research is to realize high-throughput screening methods for studying environment-genomics of plants through phenomics, and understanding microbial and plant metabolisms that contribute to harvesting bioenergy from microbes and living plants in different environments. First, we develop vertical microfluidic plant chips and miniature greenhouses for high throughput phenotyping of Arabidopsis plants. The vertical design allows for gravitropic growth of multiple plants and continuous monitoring of seed germination and plant development at both the whole-plant and cellular levels. An automatic seed trapping method is developed to facilitate seed loading process. Also, electrospun nanofibrous membranes are incorporated with a seed germination chip to obtain a set of incubation temperatures on the device. Furthermore, miniature greenhouses are designed to house the plant and seed chips and to flexibly change temperature and light conditions for high-throughput plant phenotyping on a multi-scale level. Second, to screen bacteria and mutants for elucidating mechanisms of electricity generation, we develop two types of miniature microbial fuel cells (µMFCs) using conductive poly(3,4-ethylenedioxythiophene) nanofibers and porous graphene foam (GF) as three-dimensional (3D) anode materials. It is demonstrated that in the nanofiber-based µMFC, the nanofibers are suitable for rapid electron transfer and Shewanella oneidensis can fully colonize the interior region of the nanofibers. The GF-based µMFC is featured with a porous anolyte chamber formed by embedding a GF anode inside a microchannel. The interconnected pores of the GF provide 3D scaffolds favorable for cell attachment, inoculation and colonization, and more importantly, allow flowing nutritional and bacterial media throughout the anode with minimal waste. Therefore, the nutrients in bio-convertible substrates can be efficiently used by microbes for sustainable production of electrons. Last, we develop a first miniature plant-MFC or µPMFC device as a technological interface to study bioenergy harvesting from microbes and living plants. A pilot research is conducted to create the µPMFC device by sandwiching a hydrophilic semi-permeable membrane between a µMFC and a plant growth chamber. Mass transport of carbon-containing organic exudates from the plant roots to the µMFC is quantified. This work represents an important step towards screening plants, microbes, and their mutants to maximize energy generation of PMFCs

High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms

Droplet-based microfluidic analysis and screening of single plant cells.

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.BBSRC and EPSRC OpenPlant BB/L014130/

Whole-organism phenotypic screening methods used in early-phase anthelmintic drug discovery

Diseases caused by parasitic helminths (worms) represent a major global health burden in both humans and animals. As vaccines against helminths have yet to achieve a prominent role in worm control, anthelmintics are the primary tool to limit production losses and disease due to helminth infections in both human and veterinary medicine. However, the excessive and often uncontrolled use of these drugs has led to widespread anthelmintic resistance in these worms - particularly of animals - to almost all commercially available anthelmintics, severely compromising control. Thus, there is a major demand for the discovery and development of new classes of anthelmintics. A key component of the discovery process is screening libraries of compounds for anthelmintic activity. Given the need for, and major interest by the pharmaceutical industry in, novel anthelmintics, we considered it both timely and appropriate to re-examine screening methods used for anthelmintic discovery. Thus, we reviewed current literature (1977-2021) on whole-worm phenotypic screening assays developed and used in academic laboratories, with a particular focus on those employed to discover nematocides. This review reveals that at least 50 distinct phenotypic assays with low-, medium- or high-throughput capacity were developed over this period, with more recently developed methods being quantitative, semi-automated and higher throughput. The main features assessed or measured in these assays include worm motility, growth/development, morphological changes, viability/lethality, pharyngeal pumping, egg hatching, larval migration, CO2- or ATP-production and/or enzyme activity. Recent progress in assay development has led to the routine application of practical, cost-effective, medium- to high-throughput whole-worm screening assays in academic or public-private partnership (PPP) contexts, and major potential for novel high-content, high-throughput platforms in the near future. Complementing this progress are major advances in the molecular data sciences, computational biology and informatics, which are likely to further enable and accelerate anthelmintic drug discovery and development

Chapter New Opportunities in Metabolomics and Biochemical Phenotyping for Plant Systems Biology

Pollution contro

New Opportunities in Metabolomics and Biochemical Phenotyping for Plant Systems Biology

Pollution contro

High-throughput microfluidic assay devices for culturing of soybean and microalgae and microfluidic electrophoretic ion nutrient sensor

In the past decade, there are significant challenges in agriculture because of the rapidly growing global population. Meanwhile, microfluidic devices or lab-on-a-chip devices, which are a set of micro-structure etch or molded into glass, silicon wafer, PDMS, or other materials, have been rapidly developed to achieve features, such as mix, separate, sort, sense, and control biochemical environment. The advantages of microfluidic technologies include high-throughput, low cost, precision control, and highly sensitive. In particular, they have offered promising potential for applications in medical diagnosis, drug discovery, and gene sequencing. However, the potential of microfluidic technologies for application in agriculture is far from being developed. This thesis focuses on the application of microfluidic technologies in agriculture. In this thesis, three different types of microfluidic systems were developed to present three approaches in agriculture investigation. Firstly, this report a high throughput approach to build a steady-state discrete relative humidity gradient using a modified multi-well plate. The customized device was applied to generate a set of humidity conditions to study the plant-pathogen interaction for two types of soybean beans, Williams and Williams 82. Next, a microfluidic microalgal bioreactor is presented to culture and screen microalgae strains growth under a set of CO2 concentration conditions. C. reinhardtii strains CC620 were cultured and screened in the customized bioreactor to validate the workability of the system. Growth rates of the cultured strain cells were analyzed under different CO2 concentrations. In addition, a multi-well-plate-based microalgal bioreactor array was also developed to do long-term culturing and screening. This work showed a promising microfluidic bioreactor for in-line screening based on microalgal culture under different CO2 concentrations. Finally, this report presents a microchip sensor system for ions separation and detection basing electrophoresis. It is a system owning high potential in various ions concentration analysis with high specificity and sensitivity. In addition, a solution sampling system was developed to extract solution from the soil. All those presented technologies not only have advantages including high-throughput, low cost, and highly sensitive but also have good extensibility and robustness. With a simple modification, those technologies can be expanded to different application areas due to experimental purposes. Thus, those presented microfluidic technologies provide new approaches and powerful tools in agriculture investigation. Furthermore, they have great potential to accelerate the development of agriculture

Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture

Screening for novel producer strains and enhanced therapeutic production at reduced cost has been the focus of most of the biopharmaceutical industries. The obligation to carry out prolonged intensive pilot scale experiments gave birth to micro-scale bioreactor systems. Screening large number of microorganisms using shake flasks and benchtop bioreactors is tedious and consumes resources. Microbioreactors that mimic benchtop bioreactors are capable not only of high throughput screening of producer strains, but also aid in optimizing the growth kinetics and expression of proteins. Modern technology has enabled the collection of precise online data for variables such as optical density (OD), pH, temperature, dissolved oxygen (DO), and adjusting in mixing inside microreactors. Microbioreactors have become an irreplaceable tool for biochemical engineers and biotechnologists to perform a large number of experiments simultaneously. Another aspect that is vital to any industry is the product yield and subsequent downstream processing. Perfusion bioreactors are one of the upcoming advances in bioreactor systems that have the potential to revolutionize biologics production. This chapter intends to take a review of different aspects of microbioreactors and perfusion bioreactors including their potential in high throughput pilot studies and microbial and mammalian cell cultivation technologies