An Inert Continuous Microreactor for the Isolation and Analysis of a Single Microbial Cell

Abstract

Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC) device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP) was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular L-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level.

Heterogeneity in Pure Microbial Systems: Experimental Measurements and Modeling

“…A complementary way to evaluate the microbial phenotypic heterogeneity under realistic bioprocess conditions is by employing scale down single cell micro-cultivation devices in which large scale reactors conditions are mimicked. Single cells in lab-on-a-chip microfluidic devices allow parallelization and high throughput experiments ( Grünberger et al, 2014 ; Dusny and Schmid, 2015 ; Rosenthal et al, 2015 ; Oliveira et al, 2016 ), contributing to large-scale bioprocess improvement ( Grünberger et al, 2012 ; Ladner et al, 2017 ). Sorting of different subpopulations of cells in order to understand the physiological responses in fluctuating microenvironments was also performed by microarray analysis ( Hewitt et al, 2007 ).…”

Beyond the bulk: disclosing the life of single microbial cells

“…Quantitative knowledge on the magnitude of heating is necessary for the effective compensation of joule heating with temperature control systems (Rosenthal et al. 2015 ). Joule heating compensation has already allowed long-term single cell cultivations of several yeast and bacteria species in negative dielectrophoresis traps without technology-borne effects on microbial physiology (Dusny et al.…”

“… 2013 ; Rosenthal et al. 2015 ). The Envirostat concept enabled the determination of direct connections between the phenotypes of individual cells with their environmental conditions (Dusny et al.…”

“… 2009b ; Rosenthal et al. 2015 ). However, single cell ecosystems are artificial, and possible biases caused by the specific retention method or by interference of the materials used for constructing the microfluidic device have to be considered carefully.…”

Editorial for Special Issue: Advances in Microfluidic Devices for Cell Handling and Analysis

“…Jo and coauthors have been developing a simple and robust single cell sorting platform based on a magnetophoretic method using monodisperse magnetic nanoparticles (MNPs) and droplet microfluidics with >94% purity [ 3 ]. An interesting technique is presented by Katrin Rosenthal et al [ 4 ] towards a continuous microreactor for the isolation and analysis of a single microbial cell.…”