Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

We present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 ± 9 µm. By investigating different oil–surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for four-color fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5–35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R² = 0.99) across all targets in the LabDisk.

Gas Crosstalk between PFPE–PEG–PFPE Triblock Copolymer Surfactant-Based Microdroplets and Monitoring Bacterial Gas Metabolism with Droplet-Based Microfluidics

The PFPE–PEG–PFPE (Perfluoropolyether-polyethylene glycol-perfluoropolyether) surfactant has been used in droplet-based microfluidics and is known to provide high droplet stability and biocompatibility. Since this surfactant ensures the stability of droplets, droplet-based microfluidic systems have been widely used to encapsulate and analyze various biological components at the single-molecule scale, including viruses, bacteria, nucleic acids and proteins. In this study, we experimentally confirmed that gas crosstalk occurred between droplets formed by fluorinated oil and the PFPE–PEG–PFPE surfactant. E. coli K-12 bacterial cells were encapsulated with Luria–Bertani broth within droplets for the cultivation, and gas crosstalk was identified with neighboring droplets that contain phenol red. Since bacteria produce ammonia gas during its metabolism, penetration of ammonia gas initiates a color change of phenol red-containing droplets. Ammonia gas exchange was also confirmed by reacting ammonium chloride and sodium hydroxide within droplets that encapsulated. Herein, we demonstrate the gas crosstalk issue between droplets when it is formed using the PFPE–PEG–PFPE surfactant and also confirm that the density of droplet barrier has effects on gas crosstalk. Our results also suggest that droplet-based microfluidics can be used for the monitoring of living bacteria by the determination of bacterial metabolites during cultivation.

Galactic cosmic ray induced ionisation on Venus and its effect on cloud droplet stability

<p>Galactic cosmic rays are ubiquitous in solar system atmospheres. On Venus, the altitude of peak ion production due to cosmic rays (the Pfotzer-Regener maximum) occurs at ~63 km, within the optically thick region of the upper clouds. This indicates the possibility of electrical effects on droplets within Venusian clouds. Motivated by this, our VENI (Venusian Electricity, Nephology, and Ionisation) project explores effects of galactic cosmic ray (GCR) induced ionisation on cloud droplets in circumstances with relevance to Venus’ atmosphere. Charge is known to lower the critical supersaturation required for cloud droplets to form; slightly larger droplets are stable at lower saturation ratios if sufficiently charged. Condensation of gas directly onto ions is also potentially possible on Venus if the atmosphere is sufficiently supersaturated. GCRs and the secondary charged particles they produce are therefore anticipated to affect cloud droplet behaviour on Venus.</p><p>Experiments have been conducted using electrically isolated droplets, through levitation in a standing acoustic wave. The droplets are monitored with a high-magnification CCD camera to determine their evaporation rate and charge. The charge is measured both by the deflection in an electric field and by passing the droplet through a custom-built induction ring. A relationship between the evaporation rate and charge of the droplets is found to be consistent with theory, allowing droplet lifetime to be predicted for a given charge. Further experiments using sulphuric acid droplets in a carbon dioxide environment offer more direct relevance to the Venusian environment and cosmic ray enhancement due to solar energetic particles (SEPs) in space weather events will be simulated using a corona source.</p>

Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules

Fluorosurfactant-stabilized microfluidic droplets are widely used as pico- to nanoliter volume reactors in chemistry and biology. However, current surfactants cannot completely prevent inter-droplet transfer of small organic molecules encapsulated or produced inside the droplets. In addition, the microdroplets typically coalesce at temperatures higher than 80 °C. Therefore, the use of droplet-based platforms for ultrahigh-throughput combination drug screening and polymerase chain reaction (PCR)-based rare mutation detection has been limited. Here, we provide insights into designing surfactants that form robust microdroplets with improved stability and resistance to inter-droplet transfer. We used a panel of dendritic oligo-glycerol-based surfactants to demonstrate that a high degree of inter- and intramolecular hydrogen bonding, as well as the dendritic architecture, contribute to high droplet stability in PCR thermal cycling and minimize inter-droplet transfer of the water-soluble fluorescent dye sodium fluorescein salt and the drug doxycycline.

Collagen peptide-loaded W1/O single emulsions and W1/O/W2 double emulsions: influence of collagen peptide and salt concentration, dispersed phase fraction and type of hydrophilic emulsifier on droplet stability and encapsulation efficiency

Collagen peptide-loaded double emulsions are developed by using various formulation parameters to utilize as food-grade functional ingredients with excellent droplet stability and encapsulation efficiency of collagen peptide.

Analysis of droplet stability after ejection from an inkjet nozzle

Inkjet technology is a commendable tool in many applications including graphics printing, bioengineering and micro-electromechanical systems (MEMS). Droplet stability is a key factor influencing inkjet performance. The stability can be analysed using dimensionless numbers that usually combine thermophysical properties and system dimensions. In this paper, a drop-on-demand (DOD) inkjet experimental system is established. A numerical model is developed to investigate the influence of the operating conditions on droplet stability, including nozzle dimensions, driving parameters (the pulse amplitude and width used to drive droplet formation) and fluid properties. The results indicate that the stability can be improved by decreasing the pulse amplitude and width, decreasing the fluid density and viscosity or increasing the nozzle diameter and fluid surface tension. Based on case analysis and modelling, a dimensionless number ($Z$), the reciprocal of the Ohnesorge number, is numerically determined for a stable droplet to lie in a range between 4 and 8. To explicitly combine the driving parameters, a new stability criterion, $Pj$, is further proposed. A general rule taking into account both $Pj$ and $Z$ is proposed for choosing appropriate driving parameters to eject stable droplets for a known nozzle and fluid, which is further validated by experiments.