Viral load quantitation at the point-of-care with shaken digital droplet RT-LAMP.

Viral load quantitation is useful in clinical point-of-care settings to assess the status of patients with infectious disease, track response to treatment, and estimate infectiousness. However, existing methods for quantitating viral loads are complex and difficult to integrate into these settings. Here, we describe a simple, instrument-free approach for viral load quantitation suitable for point-of-care use. We develop a shaken digital droplet assay that can quantitate SARS-CoV2 with sensitivity comparable to gold standard qPCR.

High-throughput cell spheroid production and assembly analysis by microfluidics and deep learning.

3D cell culture models are important tools in translational research but have been out of reach for high-throughput screening due to complexity, requirement of large cell numbers and inadequate standardization. Microfluidics and culture model miniaturization technologies could overcome these challenges. Here, we present a high-throughput workflow to produce and characterize the formation of miniaturized spheroids using deep learning. We train a convolutional neural network (CNN) for cell ensemble morphology classification for droplet microfluidic minispheroid production, benchmark it against more conventional image analysis, and characterize minispheroid assembly determining optimal surfactant concentrations and incubation times for minispheroid production for three cell lines with different spheroid formation properties. Notably, this format is compatible with large-scale spheroid production and screening. The presented workflow and CNN offer a template for large scale minispheroid production and analysis and can be extended and re-trained to characterize morphological responses in spheroids to additives, culture conditions and large drug libraries.

Rapid Production and Recovery of Cell Spheroids by Automated Droplet Microfluidics.

The future of the life sciences is linked to automation and microfluidics. As robots start working side by side with scientists, robotic automation of microfluidics in general, and droplet microfluidics in particular, will significantly extend and accelerate the life sciences. Here, we demonstrate the automation of droplet microfluidics using an inexpensive liquid-handling robot to produce human scaffold-free cell spheroids at high throughput. We use pipette actuation and interface the pipetting tip with a droplet-generating microfluidic device. In this device, we produce highly monodisperse droplets with a diameter coefficient of variation (CV) lower than 2%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard labware containers at a throughput of 85,000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high throughout the process and decreases by >10% (depending on the cell line used) after a 16 h incubation period in nanoliter droplets and automated recovery. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single-cell cultures, but assembly methods for spheroids (e.g., hanging drop microplates) have limited throughput. The increased throughput and decreased cost of our method enable spheroid production at the scale needed for lead discovery drug screening, and approach the cost at which these microtissues could be used as building blocks for organ-scale regenerative medicine.

Micro- and Nanostructured Polyaniline for Instant Identification of Metal Ions in Solution.

The unique properties of nanomaterials enable the creation new analytical devices. Polyaniline (PANI) micro- and nanofiber network, freestanding in the gap between two gold microelectrodes, has been used in a new nanodetector for metal ions in solutions. The gold electrodes were modified with the aid of alkanethiols, forming a self-assembled monolayer (SAM), which is able to block the ion current flow, but also to interact with metal ions when specific functional molecules are incorporated into the layer. The electric field of the trapped metal ions induces change of the electrical conductivity of polyaniline nanofibers in vicinity. A small injected sample (75 µL) of a solution of salt (about 0.5 µg of salt) was enough to induce a reproducible change in the electrical conductivity of polyaniline nano-network, which was registered as a function of time within 10⁻20 s. The response was proportional to the concentration of ions. It also depends on properties of ions, e.g., the ionic radius, which allows for identification of metal ions by analyzing the parameters of the signal: the retention time (RT), half width (HW), amplitude (A) and integral intensity (INT). The advantage of the new device is the instant responsiveness and easy operation, but also the simple construction based on organic (polymer) technology. The system is "open"-when learned and calibrated adequately, other metal ions can be analyzed. The nanodetector can be used in cases where monitoring of the presence and concentration of metal ions is important.

Erratum: Publisher's Note: "Micropipette-powered droplet based microfluidics" [Biomicrofluidics 12, 044106 (2018)].

[This corrects the article DOI: 10.1063/1.5037795.].

Micropipette-powered droplet based microfluidics.

Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.

Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.

Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening.

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Green synthesis of rifampicin-loaded copper nanoparticles with enhanced antimicrobial activity.

The antimicrobial properties of copper and rifampicin-loaded copper nanoparticles were investigated using four strains: Staphylococcus aureus, Escherichia coli, Bacillus pumilis and Pseudomonas fluorescens. Spherical-shaped copper nanoparticles were synthesized via green reduction method from the peppermint extract. It was found that adsorption of rifampicin on the copper nanosurface enhances its biological activity and prevents the development of resistance. The interactions between rifampicin-copper nanoparticles and bacteria cells were monitored using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). It was proven that loaded with rifampicin copper nanoparticles were able to damage the S. aureus cell membrane and facilitate the bacteria biofilm matrix disintegration. Moreover, the DNA decomposition of S. aureus treated with copper and rifampicin-copper nanoparticles was confirmed by agarose gel electrophoresis. The results obtained indicate that adsorption of rifampicin on the copper nanoparticles surface might provide the reduction of antibiotic dosage and prevent its adverse side effects.

An efficient 3D cell culture method on biomimetic nanostructured grids.

Current techniques of in vitro cell cultures are able to mimic the in vivo environment only to a limited extent, as they enable cells to grow only in two dimensions. Therefore cell culture approaches should rely on scaffolds that provide support comparable to the extracellular matrix. Here we demonstrate the advantages of novel nanostructured three-dimensional grids fabricated using electro-spinning technique, as scaffolds for cultures of neoplastic cells. The results of the study show that the fibers allow for a dynamic growth of HeLa cells, which form multi-layer structures of symmetrical and spherical character. This indicates that the applied scaffolds are nontoxic and allow proper flow of oxygen, nutrients, and growth factors. In addition, grids have been proven to be useful in in situ examination of cells ultrastructure.