The use of bacteriocins is a promising approach for addressing the immense threat of food-borne and drug-resistant pathogens. In recent years screening platforms for novel bacteriocins using whole-cell biosensors have been established. During screening cell-to-cell heterogeneity is currently neglected but might play a crucial role in signal development of the whole-cell biosensor after bacteriocin exposure. In this study, we explored the temporal dynamics of the signal heterogeneity of the biosensor Listeria innocua LMG2785/pNZpHin2 Lm after nisin exposure using microfluidic single-cell analysis. The results provided novel and detailed insights into the dynamics of cell-to-cell heterogeneity in L. innocua LMG2785/pNZpHin2 Lm at different nisin concentrations with a high spatio-temporal resolution. Furthermore, the formation of subpopulations during bacteriocin exposure was observed. In-depth single-cell tracking even revealed the regeneration of disrupted cells and recovery of pH homeostasis in rare instances. These findings are highly important for the future design and execution of bacteriocin assays and for the interpretation of fluorescence signal development at the population level after exposure to different concentrations of bacteriocins (here, nisin), as well as for obtaining deeper insights into single-cell persistence strategies to quantify the efficacy and efficiency of novel bacteriocins.

OBJECTIVE: Increased levels of glial fibrillary acidic protein (GFAP) in blood have been identified as a valuable biomarker for some neurological disorders, such as Alzheimer\'s disease and multiple sclerosis. However, most blood GFAP quantifications so far were performed using the same bead-based assay, and to date a routine clinical application is lacking.
METHODS: In this study, we validated a novel second-generation (2nd gen) Ella assay to quantify serum GFAP. Furthermore, we compared its performance with a bead-based single molecule array (Simoa) and a homemade GFAP assay in a clinical cohort of neurological diseases, including 210 patients.
RESULTS: Validation experiments resulted in an intra-assay variation of 10 %, an inter-assay of 12 %, a limit of detection of 0.9 pg/mL, a lower limit of quantification of 2.8 pg/mL, and less than 20 % variation in serum samples exposed to up to five freeze-thaw cycles, 120 h at 4 °C and room temperature. Measurement of the clinical cohort using all assays revealed the same pattern of GFAP distribution in the different diagnostic groups. Moreover, we observed a strong correlation between the 2nd gen Ella and Simoa (r=0.91 (95 % CI: 0.88-0.93), p<0.0001) and the homemade immunoassay (r=0.77 (95 % CI: 0.70-0.82), p<0.0001).
CONCLUSIONS: Our results demonstrate a high reliability, precision and reproducibility of the 2nd gen Ella assay. Although a higher assay sensitivity for Simoa was observed, the new microfluidic assay might have the potential to be used for GFAP analysis in daily clinical workups due to its robustness and ease of use.

Accurate sensing and responding to physical microenvironment are crucial for cell function and survival, but the underlying molecular mechanisms remain elusive. Pollen tube (PT) provides a perfect single-cell model for studying mechanobiology since it\'s naturally subjected to complex mechanical instructions from the pistil during invasive growth. Recent reports have revealed discrepant PT behaviors between in vivo and flat, two-dimensional in vitro cultures. Here, we established the Stigma-style-transmitting tract (TT) Physical microenvironment Assay (SPA) to recapitulate pressure changes in the pistil. This biomimetic assay has enabled us to swiftly identify highly redundant genes, GEF8/9/11/12/13, as new regulators for maintaining PTs integrity during style-to-TT emergence. In contrast to normal growth on solid medium, SPA successfully phenocopied gef8/9/11/12/13 PT in vivo growth-arrest deficiency. Our results suggest the existence of distinct signaling pathways regulating in vivo and in vitro PT integrity maintenance, underscoring the necessity of faithfully mimicking the physical microenvironment for studying plant cell biology.

Introduction: A rapid and reliable detection of glial fibrillary acidic protein (GFAP) in biological samples could assist in the diagnostic evaluation of neurodegenerative disorders. Sensitive assays applicable in the routine setting are needed to validate the existing GFAP tests. This study aimed to develop a highly sensitive and clinically applicable microfluidic immunoassay for the measurement of GFAP in blood. Methods: A microfluidic GFAP assay was developed and validated regarding its performance. Subsequently, serum and cerebrospinal fluid (CSF) of Alzheimer\'s disease (AD), Multiple Sclerosis (MS) and control patients were analyzed with the established assay, and levels were compared to the commercial GFAP Simoa discovery kit. Results: The developed GFAP assay showed a good performance with a recovery of 85% of spiked GFAP in serum and assay variations below 15%. The established assay was highly sensitive with a calculated lower limit of quantification and detection of 7.21 pg/mL and 2.37 pg/mL, respectively. GFAP levels were significantly increased in AD compared to control patients with advanced age (p = 0.002). However, GFAP levels revealed no significant increase in MS compared to control patients in the same age range (p = 0.140). Furthermore, serum GFAP levels evaluated with the novel microfluidic assay strongly correlated with Simoa concentrations (r = 0.88 (95% CI: 0.81-0.93), p < 0.0001). Conclusion: We successfully developed a sensitive and easy-to-use microfluidic assay to measure GFAP in blood. Furthermore, we could confirm previous findings of elevated GFAP levels in AD by applying the assay in a cohort of clinically characterized patients.

Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of Salmonella enterica serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define \"stimulation score\" as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions.IMPORTANCE Our findings provide insights into the previously unexplored effects of Salmonella envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains\' immunogenicity in vitro This approach could be particularly beneficial for rapid in vitro screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by \"stimulation score,\" agrees with in vivo cytokine response trends reported in the literature.

Environmental hazards typically are encountered in the gaseous phase; however, selective sensing modalities for identifying and quantitating compounds of interest in an inexpensive, pseudo-real-time format are severely lacking. Here, we present a novel proof-of-concept that combines an Air2Liquid sampler in conjunction with an oil-in-water microfluidic assay for detection of organophosphates. We believe this proof-of-concept will enable development of a new platform technology for semivolatile detection that we have demonstrated to detect 50 pmoles (2 ppb) of neurotoxic organophosphates.

Cell adhesion is implicated in many physiological settings such as the retention of hematopoietic stem cells (HSCs) in their bone marrow niches or their migration into the bloodstream. During HSC mobilization these adhesion sites are cleaved and have to be newly formed during HSC homing and engraftment. To determine the adhesive properties of HSCs on different extracellular matrix (ECM) molecules, we present a microfluidic shear force assay, where a laminar flow is used to detach a semi-adherent cell population, the HSC model cell line KG-1a, from an ECM protein-coated substrate. This technique combines the high throughput of population-based assays with the ability to observe cell detachment in real time. Additionally, it is suitable for weakly adherent cells, as the setup allows cell incubation on various substrates and application of shear stress ranging several orders of magnitude in one setup without additional washing or transfer steps. As a measure for the adhesion strength of the studied cell population on the substrate, the critical shear force τ50 is determined which is required to remove 50% of the initially adherent cell fraction.

We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.

BACKGROUND: Neuroinflammation often develops in sepsis leading to activation of cerebral endothelium, increased permeability of the blood-brain barrier (BBB), and neutrophil infiltration. We have identified protein kinase C-delta (PKCδ) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKCδ by a peptide inhibitor (PKCδ-i) protected endothelial cells, decreased sepsis-mediated neutrophil influx into the lung, and prevented tissue damage. The objective of this study was to elucidate the regulation and relative contribution of PKCδ in the control of individual steps in neuroinflammation during sepsis.
METHODS: The role of PKCδ in mediating human brain microvascular endothelial (HBMVEC) permeability, junctional protein expression, and leukocyte adhesion and migration was investigated in vitro using our novel BBB on-a-chip (B3C) microfluidic assay and in vivo in a rat model of sepsis induced by cecal ligation and puncture (CLP). HBMVEC were cultured under flow in the vascular channels of B3C. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF-α (10 U/ml) for 4 h in the absence or presence of PKCδ-i (5 μM) to quantify neutrophil adhesion and migration in the B3C. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo.
RESULTS: During sepsis, PKCδ is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKCδ-i. Similarly, TNF-α-mediated activation of PKCδ and its translocation in HBMVEC are attenuated by PKCδ-i in vitro. PKCδ inhibition significantly reduced TNF-α-mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24 h after CLP induced sepsis. TNF-α-treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKCδ-i-treated cells. PKCδ inhibition also reduced TNF-α-mediated neutrophil adhesion and migration across HBMVEC in B3C. Interestingly, while PKCδ inhibition decreased the number of adherent neutrophils to baseline (no-treatment group), it significantly reduced the number of migrated neutrophils below the baseline, suggesting a critical role of PKCδ in regulating neutrophil transmigration.
CONCLUSIONS: The BBB on-a-chip (B3C) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKCδ activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKCδ-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage.

Therapeutic potential of mesenchymal stem cells (MSCs) has been reported consistently in animal models of stroke, with mechanism mainly through immunomodulation and paracrine activity. Intravenous injection has been a prevailing route for MSCs administration, but cell quantities needed when scaling-up from mouse to human are extremely high putting into question feasibility of that approach. Intra-arterial delivery directly routes the cells to the brain thus lowering the required dose. Cell engineering may additionally improve cell homing, further potentiating the value of intra-arterial route. Therefore, our goal was to create microfluidic platform for screening and fast selection of molecules that enhance the docking of stem cells to vessel wall. We hypothesized that our software will be capable of detecting distinct docking properties of naïve and ITGA4-engineered MSCs. Indeed, the cell flow tracker analysis revealed positive effect of cell engineering on docking frequency of MSCs (42% vs. 9%, engineered vs. control cells, p < 0.001). These observations were then confirmed in an animal model of focal brain injury where cell engineering resulted in improved homing to the brain. To conclude, we developed a platform to study the docking of cells to the vessel wall which is highly relevant for intraarterial cell targeting or studies on neuroinflammation.