Development of a microfluidic platform for microdroplet generation and analysis

Intenzifikacija procesov na osnovi miniaturizacije, visoka zmogljivost, nizki stroški obratovanja in potrošnega materiala, laminarni tokovni režimi, precizen nadzor in možnost manipulacije toka tekočin, učinkovit prenos toplote in snovi. Vse to in še več so lastnosti, ki definirajo mikrofluidiko, vedo, ki je spremenila paradigmo biokemijskih in kemijskih raziskav ter načrtovanja procesov v zadnjih treh desetletjih. Ena izmed podskupin mikrofluidike je kapljična mikrofluidika, ki deluje na principu ustvarjanja kapljic s prostorninami med 10-9 L in 10-15 L, ki jih lahko uporabimo za pripravo mikrobioreaktorjev za karakterizacijo posameznih celic. Glavna uporabnost takšnih sistemov je izvedba hitrih presejalnih testov, s katerimi povišamo pretočnost eksperimentov za faktor več tisoč in hkrati občutno znižamo stroške njihove izvedbe. Glavni kriteriji za uspešnost takšnega sistema so pravilen izbor posameznih komponent dvofaznega sistema, stabilnost nastale emulzije, uspešna inkapsulacija biološkega materiala v kapljice ter njihova detekcija in sortiranje glede na analizo vsebine. V nalogi je predstavljen razvoj mikropretočne platforme za izvedbo hitrih presejalnih testov na osnovi detekcije vsebine kapljic in nadaljnjega sortiranja. Opisane so različne komponente in pogoji tvorbe kapljic, ki zagotavljajo tvorbo stabilne in monodisperzne emulzije na različnih mikrofluidnih čipih. Opisan je postopek izdelave mikrofluidnega čipa iz polidimetilsiloksana za pripravo kapljic, ki v nadaljevanju omogoča tudi povezavo z ustreznim detektorjem riboflavina in sortiranje kapljic. V okviru predstavljenega dela sem uspel razviti detekcijo vsebine kapljic, ki so vsebovale različne koncentracije standarda riboflavina s sklopljenim sistemom čipa in detektorja absorbance. Razviti sistem bo v nadaljevanju omogočal visokozmogljivostni presejalni test za izbor ustreznega produkcijskega mikroorganizma za proizvodnjo riboflavina.Process intensification by miniaturization, high-throughput, low operating and material costs, laminar fluid flow, precise control, and fluid manipulation, efficient heat and mass transfer. All of the above and more are key properties that define microfluidics, a science that has changed the paradigm of biochemical and chemical research and process development in the last three decades. Droplet-based microfluidics is a subclass of microfluidics, where small droplets from 10-9 L to 10-15 L are generated. Droplets serve as isolated microbioreactors where various biological experiments can be performed. High throughput screening (HTS) experiments are the main and most important use of droplet-based microfluidics. A 1000-fold increase in throughput and a significant reduction of assay reagent cost can be obtained by using microfluidics for screening experiments. The main criteria for successful usage of a droplet-based microfluidics platform are an adequate selection of chemicals of a two-phase system, emulsion stability, successful encapsulation of biological material in droplets, detection and sorting, based on content analysis. Development of a droplet microfluidic platform with intent to use it for HTS experiments is presented. Chemicals that meet the criteria for use in droplet microfluidics are reported. Different droplet generation conditions which ensure the creation of stable and monodisperse emulsion on various microfluidic chips are presented. The process of droplet generation chip made from polydimethylsiloxane fabrication is described. The coupled system of microfluidic chip and optical detector ODcube instant proved to be successful in carrying out detection experiments on droplets based on different concentrations of riboflavin standard laden droplets and has the potential to be used in future in a HTS experiment for selection of the best riboflavin producing microorganism mutant

Droplet Microfluidic Technique for the Study of Fermentation

We demonstrate a technique that uses microdroplets for culturing and selecting bacterial cultures in a model biotechnological application. We propose an assay for determination of ethanol concentration that provides increased dynamic range and is compatible with droplet microfluidic screening technologies. The assay comprises two enzymes—alcohol oxidase (AOX) and horseradish peroxidase (HRP)—and a colorimetric readout system of phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP). The microdroplet method provides high repeatability (a relative error of measured ethanol concentration < 5%), high specificity for ethanol, low consumption of reagents and wide dynamic range (1–70 g·L-1) compared to existing assays. We report the use of this method in a screen of ethanol generation efficiency of Zymomonas mobilis (strain 3881) against the concentration of glucose in the culture media