Investigation of air bubble behaviour after gas embolism events induced in a microfluidic network mimicking microvasculature.

Gas embolism is a medical condition that occurs when gas bubbles are present in veins or arteries, decreasing blood flow and potentially reducing oxygen delivery to vital organs, such as the brain. Although usually reported as rare, gas embolism can lead to severe neurological damage or death. However, presently, only limited understanding exists regarding the microscale processes leading to the formation, persistence, movement, and resolution of gas emboli, as modulated by microvasculature geometrical features and blood properties. Because gas embolism is initially a physico-chemical-only process, with biological responses starting later, the opportunity exists to fully study the genesis and evolution of gas emboli using in vitro microfluidic networks mimicking small regions of microvasculature. The microfluidics networks used in this study, which aim to mimic microvasculature geometry, comprise linear channels with T-, or Y-junction air inlets, with 20, 40, and 60 µm widths (arterial or venous), and a 30 µm width honeycombed network (arterial) with three bifurcation angles (30°, 60°, and 90°). Synthetic blood, equivalent to 46% haematocrit concentrations, and water were used to study the modulation of gas embolism-like events by liquid viscosity. Our study shows that (i) longer bubbles with lower velocity occur in narrower channels, e.g., with 20 µm width; (ii) the resistance of air bubbles to the flow increases with the higher haematocrit concentration; and lastly (iii) the propensity of gas embolism-like events in honeycomb architectures increases for more acute, e.g., 30°, bifurcation angles. A dimensionless analysis using Euler, Weber, and capillary numbers demarcated the conditions conducive to gas embolism. This work suggests that in vitro experimentation using microfluidic devices with microvascular tissue-like structures could assist medical guidelines and management in preventing and mitigating the effects of gas embolism.

A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing.

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4-5 days. With process intensification, RBD-SrtA yields reached 6.4 µg/mL and 13.4 µg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Patterns of bacterial motility in microfluidics-confining environments.

Understanding the motility behavior of bacteria in confining microenvironments, in which they search for available physical space and move in response to stimuli, is important for environmental, food industry, and biomedical applications. We studied the motility of five bacterial species with various sizes and flagellar architectures (Vibrio natriegens, Magnetococcus marinus, Pseudomonas putida, Vibrio fischeri, and Escherichia coli) in microfluidic environments presenting various levels of confinement and geometrical complexity, in the absence of external flow and concentration gradients. When the confinement is moderate, such as in quasi-open spaces with only one limiting wall, and in wide channels, the motility behavior of bacteria with complex flagellar architectures approximately follows the hydrodynamics-based predictions developed for simple monotrichous bacteria. Specifically, V. natriegens and V. fischeri moved parallel to the wall and P. putida and E. coli presented a stable movement parallel to the wall but with incidental wall escape events, while M. marinus exhibited frequent flipping between wall accumulator and wall escaper regimes. Conversely, in tighter confining environments, the motility is governed by the steric interactions between bacteria and the surrounding walls. In mesoscale regions, where the impacts of hydrodynamics and steric interactions overlap, these mechanisms can either push bacteria in the same directions in linear channels, leading to smooth bacterial movement, or they could be oppositional (e.g., in mesoscale-sized meandered channels), leading to chaotic movement and subsequent bacterial trapping. The study provides a methodological template for the design of microfluidic devices for single-cell genomic screening, bacterial entrapment for diagnostics, or biocomputation.

An AgNP-deposited commercial electrochemistry test strip as a platform for urea detection.

We developed an inexpensive, portable platform for urea detection via electrochemistry by depositing silver nanoparticles (AgNPs) on a commercial glucose test strip. We modified this strip by first removing the enzymes from the surface, followed by electrodeposition of AgNPs on one channel (working electrode). The morphology of the modified test strip was characterized by Scanning Electron Microscopy (SEM), and its electrochemical performance was evaluated via Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). We evaluated the performance of the device for urea detection via measurements of the dependency of peak currents vs the analyte concentration and from the relationship between the peak current and the square root of the scan rates. The observed linear range is 1-8 mM (corresponding to the physiological range of urea concentration in human blood), and the limit of detection (LOD) is 0.14 mM. The selectivity, reproducibility, reusability, and storage stability of the modified test strips are also reported. Additional tests were performed to validate the ability to measure urea in the presence of confounding factors such as spiked plasma and milk. The results demonstrate the potential of this simple and portable EC platform to be used in applications such as medical diagnosis and food safety.

Correction to 'Something has to give: scaling combinatorial computing by biological agents exploring physical networks encoding NP-complete problems'.

[This corrects the article DOI: 10.1098/rsfs.2018.0034.].

Dual-phone illumination-imaging system for high resolution and large field of view multi-modal microscopy.

In this paper we present for the first time a system comprised of two mobile phones, one for illumination and the other for microscopy, as a portable, user-friendly, and cost-effective microscopy platform for a wide range of applications. Versatile and adaptive illumination is made with a Retina display of an Apple mobile phone device. The phone screen is used to project various illumination patterns onto the specimen being imaged, each corresponding to a different illumination mode, such as bright-field, dark-field, point illumination, Rheinberg illumination, and fluorescence microscopy. The second phone (a Nokia phone) is modified to record microscopic images about the sample. This imaging platform provides a high spatial resolution of at least 2 µm, a large field-of-view of 3.6 × 2.7 mm, and a working distance of 0.6 mm. We demonstrate the performance of this platform for the visualization of microorganisms within microfluidic devices to gather qualitative and quantitative information regarding microorganism morphology, dimension, count, and velocity/trajectories in the x-y plane.

Something has to give: scaling combinatorial computing by biological agents exploring physical networks encoding NP-complete problems.

On-chip network-based computation, using biological agents, is a new hardware-embedded approach which attempts to find solutions to combinatorial problems, in principle, in a shorter time than the fast, but sequential electronic computers. This analytical review starts by describing the underlying mathematical principles, presents several types of combinatorial (including NP-complete) problems and shows current implementations of proof of principle developments. Taking the subset sum problem as example for in-depth analysis, the review presents various options of computing agents, and compares several possible operation 'run modes' of network-based computer systems. Given the brute force approach of network-based systems for solving a problem of input size C, 2C solutions must be visited. As this exponentially increasing workload needs to be distributed in space, time, and per computing agent, this review identifies the scaling-related key technological challenges in terms of chip fabrication, readout reliability and energy efficiency. The estimated computing time of massively parallel or combinatorially operating biological agents is then compared to that of electronic computers. Among future developments which could considerably improve network-based computing, labelling agents 'on the fly' and the readout of their travel history at network exits could offer promising avenues for finding hardware-embedded solutions to combinatorial problems.

RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase.

The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.

A Hierarchical 3D Nanostructured Microfluidic Device for Sensitive Detection of Pathogenic Bacteria.

Efficient capture and rapid detection of pathogenic bacteria from body fluids lead to early diagnostics of bacterial infections and significantly enhance the survival rate. We propose a universal nano/microfluidic device integrated with a 3D nanostructured detection platform for sensitive and quantifiable detection of pathogenic bacteria. Surface characterization of the nanostructured detection platform confirms a uniform distribution of hierarchical 3D nano-/microisland (NMI) structures with spatial orientation and nanorough protrusions. The hierarchical 3D NMI is the unique characteristic of the integrated device, which enables enhanced capture and quantifiable detection of bacteria via both a probe-free and immunoaffinity detection method. As a proof of principle, we demonstrate probe-free capture of pathogenic Escherichia coli (E. coli) and immunocapture of methicillin-resistant-Staphylococcus aureus (MRSA). Our device demonstrates a linear range between 50 and 104 CFU mL-1 , with average efficiency of 93% and 85% for probe-free detection of E. coli and immunoaffinity detection of MRSA, respectively. It is successfully demonstrated that the spatial orientation of 3D NMIs contributes in quantifiable detection of fluorescently labeled bacteria, while the nanorough protrusions contribute in probe-free capture of bacteria. The ease of fabrication, integration, and implementation can inspire future point-of-care devices based on nanomaterial interfaces for sensitive and high-throughput optical detection.

Comparative analysis for the production of fatty acid alkyl esterase using whole cell biocatalyst and purified enzyme from Rhizopus oryzae on waste cooking oil (sunflower oil).

The petroleum fuel is nearing the line of extinction. Recent research and technology have provided promising outcomes to rely on biodiesel as the alternative and conventional source of fuel. The use of renewable source - vegetable oil constitutes the main stream of research. In this preliminary study, Waste Cooking Oil (WCO) was used as the substrate for biodiesel production. Lipase enzyme producing fungi Rhizopus oryzae 262 and commercially available pure lipase enzyme were used for comparative study in the production of Fatty Acid Alkyl Esters (FAAE). The whole cell (RO 262) and pure lipase enzyme (PE) were immobilized using calcium alginate beads. Calcium alginate was prepared by optimizing with different molar ratios of calcium chloride and different per cent sodium alginate. Entrapment immobilization was done for whole cell biocatalyst (WCB). PE was also immobilized by entrapment for the transesterification reaction. Seven different solvents - methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol and iso-amyl alcohol were used as the acyl acceptors. The reaction parameters like temperature (30°C), molar ratio (1:3 - oil:solvent), reaction time (24 h), and amount of enzyme (10% mass ratio to oil) were also optimized for methanol alone. The same parameters were adopted for the other acyl acceptors too. Among the different acyl acceptors - methanol, whose reaction parameters were optimized showed maximum conversion of triglycerides to FAAE-94% with PE and 84% with WCB. On the whole, PE showed better catalytic converting ability with all the acyl acceptor compared to WCB. Gas chromatography analysis (GC) was done to determine the fatty acid composition of WCO (sunflower oil) and FAAE production with different acyl acceptors.