Digital E. coli Counter: A Microfluidics and Computer Vision-Based DNAzyme Method for the Isolation and Specific Detection of E. coli from Water Samples

Biological water contamination detection-based assays are essential to test water quality; however, these assays are prone to false-positive results and inaccuracies, are time-consuming, and use complicated procedures to test large water samples. Herein, we show a simple detection and counting method for E. coli in the water samples involving a combination of DNAzyme sensor, microfluidics, and computer vision strategies. We first isolated E. coli into individual droplets containing a DNAzyme mixture using droplet microfluidics. Upon bacterial cell lysis by heating, the DNAzyme mixture reacted with a particular substrate present in the crude intracellular material (CIM) of E. coli. This event triggers the dissociation of the fluorophore-quencher pair present in the DNAzyme mixture leading to a fluorescence signal, indicating the presence of E. coli in the droplets. We developed an algorithm using computer vision to analyze the fluorescent droplets containing E. coli in the presence of non-fluorescent droplets. The algorithm can detect and count fluorescent droplets representing the number of E. coli present in the sample. Finally, we show that the developed method is highly specific to detect and count E. coli in the presence of other bacteria present in the water sample.

Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly

Mesenchymal stem cells (MSCs)-based therapies have been widely used to promote tissue regeneration and to modulate immune/inflammatory response. The therapeutic potential of MSCs can be further improved by forming multi-cellular spheroids. Meanwhile, hydrogels with macroporous structures are advantageous for improving mass transport properties for the cell-laden matrices. Herein, we report the fabrication of MSC-laden macroporous hydrogel scaffolds through incorporating rapidly dissolvable spherical cell-laden microgels. Dissolvable microgels were fabricated by tandem droplet-microfluidics and thiol-norbornene photopolymerization using a novel fast-degrading macromer poly(ethylene glycol)-norbornene-dopamine (PEGNB-Dopa). The cell-laden microgels were subsequently encapsulated within another bulk hydrogel matrix, whose porous structure was generated efficiently by the rapid degradation of the PEGNB-Dopa microgels. The cytocompatibility of this in situ pore-forming approach was demonstrated with multiple cell types. Furthermore, adjusting the stiffness and cell adhesiveness of the bulk hydrogels afforded the formation of solid cell spheroids or hollow spheres. The assembly of solid or hollow MSC spheroids led to differential activation of AKT pathway. Finally, MSCs solid spheroids formed in situ within the macroporous hydrogels exhibited robust secretion of HGF, VEGF-A, IL-6, IL-8, and TIMP-2. In summary, this platform provides an innovative method for forming cell-laden macroporous hydrogels for a variety of future biomedical applications.

Electrochemical pH regulation in droplet microfluidics

The pH of droplets moving through a microchannel is regulated in real time by water electrolysis. Resulting droplet pHs are within ±0.1 pH units of the predicted values.

Assessment of chimeric antigen receptor T (CAR-T) cytotoxicity by droplet microfluidics in vitro

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-gamma; (IFNgamma) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cell-kill-cell experiment with varying solvent composition.

In vitro cytokine expression analysis by droplet microfluidics

Droplet microfluidics provides a miniaturized platform to conduct biological assays. We previously developed a droplet microfluidic chip assay for screening cancer cells against chemical drugs and chimeric antigen receptor T (CAR-T) cells, respectively. In this study, we investigated chip application on a cytokine expression assay using MCF7 breast cancer reporter cells engineered by fusing green fluorescent protein (GFP) to the C-terminus of endogenous interleukin-6 (IL6) gene. Combined tumor necrosis factor alpha (TNFalpha) treatment and serum-free medium starvation stimulated IL6-GFP expression and enhanced GFP fluorescence. Our data showed that on-chip assay recapitulates the cellular response in vitro, although absolute quantification of IL6 induction could not be accomplished. The demonstration of multi-timepoint IL6 expression analysis paves the way for our future study on tumor response to immune attack via cytokine signaling.

Phenotyping single-cell motility in microfluidic confinement

At all scales, the movement patterns of organisms serve as dynamic read-outs of their behaviour and physiology. We devised a novel droplet microfluidics assay to encapsulate single algal microswimmers inside closed arenas, and comprehensively studied their roaming behaviour subject to a large number of environmental stimuli. We compared two model species, Chlamydomonas reinhardtii (freshwater alga, 2 cilia), and Pyramimonas octopus (marine alga, 8 cilia), and detailed their highly-stereotyped behaviours and the emergence of a trio of macroscopic swimming states (smooth-forward, quiescent, tumbling or excitable backward). Harnessing ultralong timeseries statistics, we reconstructed the species-dependent reaction network that underlies the choice of locomotor behaviour in these aneural organisms, and discovered the presence of macroscopic non-equilibrium probability fluxes in these active systems. We also revealed for the first time how microswimmer motility changes instantaneously when a chemical is added to their microhabitat, by inducing deterministic fusion between paired droplets - one containing a trapped cell, and the other, a pharmacological agent that perturbs cellular excitability. By coupling single-cell entrapment with unprecedented tracking resolution, speed and duration, our approach offers unique and potent opportunities for diagnostics, drug-screening, and for querying the genetic basis of micro-organismal behaviour.

Light-Addressable Potentiometric Sensors in Microfluidics

Light-addressable potentiometric sensor (LAPS) is an electrochemical sensor based on the field-effect principle of semiconductor. It is able to sense the change of Nernst potential on the sensor surface, and the measuring area can be controlled by the illumination. Due to the unique light-addressable ability of LAPS, the chemical imaging system constructed with LAPS can realize the two-dimensional image distribution detection of chemical/biomass. In this paper, the advantages of LAPS as sensing unit of microelectrochemical analysis system are summarized. Then, the greatest development of LAPS analysis system is explained and discussed. Especially, this paper focused on the research of ion diffusion, enzymatic reaction, microbial metabolism and droplet microfluidics by using LAPS analysis system. Finally, the development trends and prospects of LAPS analysis system are illustrated.