An optical tweezers integrated microfluidic platform for the identification and retrieval of antigen-specific B cells

status: accepte

KreaSensa: een innovatieve biosensor voor de detectie van creatinine

Veertien KU Leuven-studenten hebben een functioneel diagnosetoestel ontwikkeld, gebouwd en geproduceerd dat het bloed van nierpatiënten in vijf minuten kan analyseren voor de aanwezigheid van creatinine. Dit compact, snel en kostefficiënt apparaat bestaat uit: (i) een enzymatisch systeem om een signaal in de vorm van een kleurverandering te produceren, (ii) een SIMPLE cartridge om autonoom alle componenten van de biologische reactie te transporteren naar de uitleesplek en (iii) een detector om het signaal te meten. Met hun ontwerp kaapten de studenten drie prijzen weg op de SensUs-wedstrijd (‘Analytical Performance’, ‘Public Inspiration’ en ‘Translation Potential’), een internationale competitie voor de ontwikkeling van nieuwe oplossingen en innovatie in het veld van moleculaire biosensoren voor de gezondheidszorg. Momenteel gebeuren creatinine analyses met spectrofotometers: grote en dure laboratoriumtoestellen waarvan de resultaten niet direct beschikbaar zijn voor de patiënt. Er bestaan al wel snellere sensoren die buiten het labo te gebruiken zijn, maar het prijskaartje daarvan is te hoog voor particulieren. Een goedkoop, draagbaar en snel alternatief voor de huidige dure en trage toestellen werd gebouwd door de KU Leuven studenten, gecoördineerd door het onderzoeksteam van professor Jeroen Lammertyn. Het apparaat, ontwikkeld en gevalideerd door de studenten, kan met wat aanpassingen een heuse lab-on-a-chip worden. Dit kan voorgesteld worden als een klein plastic plaatje, met daarop microkanaaltjes met de dikte van een menselijk haar. Door die kanaaltjes kan bloed of urine vloeien, waarbij de chip dan zeer precies en snel biomerkers van ziektes – hier dus nierfalen – kan opmeten. Momenteel wordt er gewerkt aan een wetenschappelijke publicatie hierover en de aanvraag van een patent voor de technologie Het prototype kan momenteel maar één molecule testen, maar dit kan uitgebreid worden naar andere moleculen, zodat alle nierziektes getest kunnen worden. Er kan eveneens met urine gewerkt worden in plaats van met bloed, of andere biomerkers kunnen opmeten worden die andere aandoeningen aanduiden. Een draagbaar toestel als dit kan ook interessant zijn voor artsen in rusthuizen en voor thuisverplegers – hun patiënten kunnen zich niet of moeilijk verplaatsen. Zelfs voor dierenartsen kan deze biosensor zijn nut hebben. Het meettoestel kan ook verkleind worden tot een apparaat dat op een smartphone geklikt kan worden.status: publishe

(i)SIMPLE, microfluidic devices for POC applications

status: publishe

Multiplex Analysis to Unravel the Mode of Antifungal Activity of the Plant Defensin HsAFP1 in Single Yeast Cells

Single cell analyses have gained increasing interest over bulk approaches because of considerable cell-to-cell variability within isogenic populations. Herein, flow cytometry remains golden standard due to its high-throughput efficiency and versatility, although it does not allow to investigate the interdependency of cellular events over time. Starting from our microfluidic platform that enables to trap and retain individual cells on a fixed location over time, here, we focused on unraveling kinetic responses of single Saccharomyces cerevisiae yeast cells upon treatment with the antifungal plant defensin HsAFP1. We monitored the time between production of reactive oxygen species (ROS) and membrane permeabilization (MP) in single yeast cells for different HsAFP1 doses using two fluorescent dyes with non-overlapping spectra. Within a time frame of 2 min, only <0.3% cells displayed time between the induction of ROS and MP. Reducing the time frame to 30 s did not result in increased numbers of cells with time between these events, pointing to ROS and MP induction as highly dynamic and correlated processes. In conclusion, using an in-house developed continuous microfluidic platform, we investigated the mode of action of HsAFP1 at single cell level, thereby uncovering the close interdependency between ROS induction and MP in yeast

Integrated Microwell Array Technologies for Single Cell Analysis

status: Published onlin

A microfluidic platform for single B-cell isolation to rapidly discover human monoclonal antibodies

status: accepte

Tuning the surface interactions between single cells and an OSTE+ microwell array for enhanced single cell manipulation

Retrieving single cells of interest from an array of microwells for further off-chip analysis is crucial in numerous biological applications. To this end, several single cell manipulation strategies have been developed, including optical tweezers (OT). OT represent a unique approach for contactless cell retrieval, but their performance is often suboptimal due to nonspecific cell adhesion to the microwell surface. In this study, we focused on improving the surface chemistry of microwell arrays to ensure efficient single cell manipulation using OT. For this purpose, the surface of an off-stoichiometry thiol-ene-epoxy (OSTE+) microwell array was grafted with polyethylene glycol (PEG) molecules with different molecular weights: PEG 360, PEG 500, PEG 2000, and a PEG Mix (an equimolar ratio of PEG 500 and PEG 2000). Contact angle measurements showed that the PEG grafting process resulted in an increased surface energy, which was stable for at least 16 weeks. Next, cell adhesion of two cell types, baker's yeast (Saccharomyces cerevisiae) and human B cells, to surfaces treated with different PEGs was evaluated by registering the presence of cellular motion inside microwells and the efficiency of optical lifting of cells that display motion. Optimal results were obtained for surfaces grafted with PEG 2000 and PEG Mix, reaching an average fraction of cells with motion of over 93% and an average lifting efficiency of over 96% for both cell types. Upon the integration of this microwell array with a polydimethylsiloxane (PDMS) microfluidic channel, PEG Mix resulted in proper washing of non-seeded cells. We further demonstrated the wide applicability of the platform by manipulating non-responding yeast cells to antifungal treatment and B cells expressing surface IgG antibodies. The combination of the optimized microwell surface with continuous microfluidics results in a powerful and versatile platform, allowing high-throughput single cell studies and retrieval of target cells for off-chip analysis

Reaction injection molding of hydrophilic-in-hydrophobic femtolitre-well arrays

Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally.status: publishe

Using high-amplitude and focused transcranial alternating current stimulation to entrain physiological tremor

Transcranial alternating current stimulation (tACS) is a noninvasive neuromodulation method that can entrain physiological tremor in healthy volunteers. We conducted two experiments to investigate the effectiveness of high-amplitude and focused tACS montages at entraining physiological tremor. Experiment 1 used saline-soaked sponge electrodes with an extra-cephalic return electrode and compared the effects of a motor (MC) and prefrontal cortex (PFC) electrode location. Average peak-amplitude was 1.925 mA. Experiment 2 used gel-filled cup-electrodes in a 4 × 1 focused montage and compared the effects of MC and occipital cortex (OC) tACS. Average peak-amplitude was 4.45 mA. Experiment 1 showed that unfocused MC and PFC tACS both produced phosphenes and significant phase entrainment. Experiment 2 showed that focused MC and OC tACS produced no phosphenes but only focused MC tACS caused significant phase entrainment. At the group level, tACS did not have a significant effect on tremor amplitude. However, with focused tACS there was a significant correlation between phase entrainment and tremor amplitude modulation: subjects with higher phase entrainment showed more tremor amplitude modulation. We conclude that: (1) focused montages allow for high-amplitude tACS without phosphenes and (2) high amplitude focused tACS can entrain physiological tremor.status: publishe