Two-photon fluorescence lifetime for label-free microfluidic droplet sorting.

Microfluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm.

Analysis of double-emulsion droplets with ESI mass spectrometry for monitoring lipase-catalyzed ester hydrolysis at nanoliter scale.

Microfluidic double-emulsion droplets allow the realization and study of biphasic chemical processes such as chemical reactions or extractions on the nanoliter scale. Double emulsions of the rare type (o1/w/o2) are used here to realize a lipase-catalyzed reaction in the non-polar phase. The surrounding aqueous phase induces the transfer of the hydrophilic product from the core oil phase, allowing on-the-fly MS analysis in single double droplets. A microfluidic two-step emulsification process is developed to generate the (o1/w/o2) double-emulsion droplets. In this first example of microfluidic double-emulsion MS coupling, we show in proof-of-concept experiments that the chemical composition of the water layer can be read online using ESI-MS. Double-emulsion droplets were further employed as two-phase micro-reactors for the hydrolysis of the lipophilic ester p-nitrophenyl palmitate catalyzed by the Candida antarctica lipase B (CalB). Finally, the formation of the hydrophilic reaction product p-nitrophenol within the double-emulsion droplet micro-reactors is verified by subjecting the double-emulsion droplets to online ESI-MS analysis.

Semaphorin 7A coordinates neutrophil response during pulmonary inflammation and sepsis.

ABSTRACT: Pulmonary defense mechanisms are critical for host integrity during pneumonia and sepsis. This defense is fundamentally dependent on the activation of neutrophils during the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet function, yet its role on neutrophil function within the lung is not well understood. This study aimed to identify the role of Sema7A during pulmonary inflammation and sepsis. In patients with acute respiratory distress syndrome (ARDS), we were able to show a correlation between Sema7A and oxygenation levels. During subsequent workup, we found that Sema7A binds to the neutrophil PlexinC1 receptor, increasing integrins, and L-selectin on neutrophils. Sema7A prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/-animals in the lung during pulmonary inflammation. This effect resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pulmonary sepsis. This finding was associated with significantly worse outcome of Sema7A-/- animals in a model of pulmonary sepsis. Sema7A has an immunomodulatory effect in the lung, affecting pulmonary sepsis and ARDS. This effect influences the response of neutrophils to external aggression and might influence patient outcome. This trial was registered at www.ClinicalTrials.gov as #NCT02692118.

Calibration of the Chemcatcher® passive sampler and derivation of generic sampling rates for a broad application in monitoring of surface waters.

We determined sampling rates for 34 pesticides, five pesticide transformation products, and 34 pharmaceutical compounds with the Chemcatcher (CC) passive sampler in a laboratory-based continuous-flow system at 40 cm/s and ambient temperature. Three different sampling phases were used: styrene divinylbenzene disks (SDB-XC), styrene divinylbenzene reversed phase sulfonate disks (SDB-RPS), and hydrophilic lipophilic balance disks (HLB), in all cases covered with a diffusion-limiting polyethersulfone membrane. The measured sampling rates range from 0.007 L/d to 0.193 L/d for CC with SDB-XC (CC-XC), from 0.055 L/d to 0.796 L/d for CC with SDB-RPS (CC-RPS), and from 0.018 L/d to 0.073 L/d for CC equipped with HLB (CC-HLB). Comparison with sampling rates from literature enabled to derive generic sampling rates that can be used for compounds with unknown uptake kinetics such as transformations products and new compounds of interest. Field trial results demonstrate that the presently derived generic sampling rates are suitable for estimating time-weighted average concentrations within reasonable uncertainty limits. In this way, Chemcatcher passive sampling can be applied approximately to a broad range of solutes without the need for deriving compound-specific sampling rates, which enable compliance checks against environmental quality standards and further risk assessment.

Fluorescence lifetime activated droplet sorting (FLADS) for label-free sorting of Synechocystis sp. PCC6803.

This study presents the label-free sorting of cyanobacterial cells in droplets with single-cell sensitivity based on their fluorescence lifetime. We separated living and dead cyanobacteria (Synechocystis sp. PCC6803) using fluorescence lifetime signals of the photopigment autofluorescence to indicate their photosynthetic activity. We developed a setup and a chip design to achieve live/dead sorting accuracies of more than 97% at a droplet frequency of 100 Hz with a PDMS-based chip system and standard optics using fluorescence lifetime as the sorting criterion. The obtained sorting accuracies could be experimentally confirmed by cell plating and observing the droplet sorting process via a high-speed camera. The herein presented results demonstrate the capabilities of the developed system for studying the effects of stressors on cyanobacterial physiology and the subsequent deterministic sorting of different stress-response phenotypes. This technology eliminates the need for tedious staining of cyanobacterial cells, which makes it particularly attractive for its application in the field of phototrophic microbial bio(techno)logic and in the context of cell secretion studies.

Integration of Impedimetric Sensors for In Situ Electrochemical Impedance Spectroscopy in Free-Flow Electrophoresis Applications in Lab-on-Chip Systems.

Miniaturization and integration of chemical reactions into fluidic systems in combination with product purification or buffer exchange can reduce the amount of solvents and reactants required while increasing synthesis efficiency. A critical step is the regulation of flow rates to realize optimal synthesis conditions and high purification rates, so real-time, label-free monitoring is required in methods such as free-flow electrophoresis. Optical detection methods are widely used, but they often have complex excitation and detection setups that are disadvantageous for point-of-care applications. The method we have chosen is electrochemical impedance spectroscopy for detecting charged compounds in aqueous buffers with low ionic strength. Propranolol was selected for proof of concept and was separated from the organic solvent and the precursor oxirane by free-flow electrophoresis. For this purpose, electrode structures were fabricated in microfluidic channels by photolithographic lift-off technique and optimized in terms of positioning, electrode size and distance for sensitive detection, and quantification of propranolol in the nanomolar range. It is also noteworthy that the organic solvent dimethyl sulfoxide (DMSO) could be detected and quantified by an increased impedance magnitude. Subsequently, the optimized interdigital electrode structures were integrated into the outlet channels of the electrophoretic separation chamber to monitor the various outgoing fluidic streams and provide in-line control of the fluidic flows for the purification step. In conclusion, we can provide a microfluidic chip to monitor the separation efficiency of a substance mixture during free-flow electrophoresis without the need of complex analytical techniques using electrochemical impedance spectroscopy.