Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Enhanced mixing efficiency and reduced droplet size with novel droplet generators.

Nowadays, droplet microfluidics has become widely utilized for high-throughput assays. Efficient mixing is crucial for initiating biochemical reactions in many applications. Rapid mixing during droplet formation eliminates the need for incorporating micromixers, which can complicate the chip design. Furthermore, immediate mixing of substances upon contact can significantly improve the consistency of chemical reactions and resulting products. This study introduces three innovative designs for droplet generators that achieve efficient mixing and produce small droplets. The T-cross and cross-T geometries combine cross and T junction mixing mechanisms, resulting in improved mixing efficiency. Numerical simulations were conducted to compare these novel geometries with traditional T and cross junctions in terms of mixing index, droplet diameter, and eccentricity. The cross-T geometry exhibited the highest mixing index and produced the smallest droplets. For the flow rate ratio of 0.5, this geometry offered a 10% increase in the mixing index and a decrease in the droplet diameter by 10% compared to the T junction. While the T junction has the best mixing efficiency among traditional droplet generators, it produces larger droplets, which can increase the risk of contamination due to contact with the microchannel walls. Therefore, the cross-T geometry is highly desirable in most applications due to its production of considerably smaller droplets. The asymmetric cross junction offered a 8% increase in mixing index and around 2% decrease in droplet diameter compared to the conventional cross junction in flow rate ratio of 0.5. All novel geometries demonstrated comparable mixing efficiency to the T junction. The cross junction exhibited the lowest mixing efficiency and produced larger droplets compared to the cross-T geometry (around 1%). Thus, the novel geometries, particularly the cross-T geometry, are a favorable choice for applications where both high mixing efficiency and small droplet sizes are important.

Fabricating and Laminating Films with Through-Holes and Engraved/Protruding Structures for 3D Micro/Nanofluidic Platforms.

Micro/nanofluidic devices have become popular for delicately processing biological, material, and chemical samples. However, their reliance on 2D fabrication schemes has hindered further innovation. Here, a 3D manufacturing method is proposed through the innovation of laminated object manufacturing (LOM), which involves the selection of building materials as well as the development of molding and lamination techniques. Fabrication of interlayer films is demonstrated with both multi-layered micro-/nanostructures and through-holes, using an injection molding approach and establishing strategic principles of film design. Utilization of the multi-layered through-hole films in LOM allows reducing the number of alignments and laminations by at least two times compared to conventional LOM. Using a dual-curing resin for film fabrication, a surface-treatment-free and collapse-free lamination technique is shown for constructing 3D multiscale micro/nanofluidic devices with ultralow aspect ratio nanochannels. The 3D manufacturing method enables the development of a nanochannel-based attoliter droplet generator capable of 3D parallelization for mass production, which implies the remarkable potential to extend numerous existing 2D micro/nanofluidic platforms into a 3D framework.

Coupling a droplet generator with conventional ESI-MS for quantitative analysis of small-volume samples.

Quantitative mass spectrometric analysis of small-volume samples (e.g., < 1 µL) has been a challenge mainly due to the difficulties with sample handling and its injection into the system for analysis. Herein we report a microfluidic analytical platform coupling a droplet generator with conventional electrospray ionization-mass spectrometry (ESI-MS) that enables multiple analyses of a µL-sized sample with sensitivity and repeatability. In an analysis by droplet generator-assisted ESI-MS (DG-ESI-MS), a sample of µL volume is pulled into a sampling capillary and its equal nL-sized portions are generated by a droplet generator and analyzed by ESI-MS at time intervals of choice. The droplet generator is made of PMMA sheets by laser engraving conveniently and at a low cost. In a study to achieve effective ESI-MS detection of water-in-oil droplets, it's found that the problem of MS signal suppression by oil can be solved by using an appropriate organic carrier with ESI-enhancing additives. The proposed DG-ESI-MS method has linear calibration curves for both adenine and phenylalanine with LODs at the sub-µM level. Application of the present analytical platform for monitoring substrate concentration changes in an enzymatic reaction solution of 3 µL is demonstrated.

Simultaneous Droplet Generation with In-Series Droplet T-Junctions Induced by Gravity-Induced Flow.

Droplet microfluidics offers a wide range of applications, including high-throughput drug screening and single-cell DNA amplification. However, these platforms are often limited to single-input conditions that prevent them from analyzing multiple input parameters (e.g., combined cellular treatments) in a single experiment. Droplet multiplexing will result in higher overall throughput, lowering cost of fabrication, and cutting down the hands-on time in number of applications such as single-cell analysis. Additionally, while lab-on-a-chip fabrication costs have decreased in recent years, the syringe pumps required for generating droplets of uniform shape and size remain cost-prohibitive for researchers interested in utilizing droplet microfluidics. This work investigates the potential of simultaneously generating droplets from a series of three in-line T-junctions utilizing gravity-driven flow to produce consistent, well-defined droplets. Implementing reservoirs with equal heights produced inconsistent flow rates that increased as a function of the distance between the aqueous inlets and the oil inlet. Optimizing the three reservoir heights identified that taller reservoirs were needed for aqueous inlets closer to the oil inlet. Studying the relationship between the ratio of oil-to-water flow rates (Φ) found that increasing Φ resulted in smaller droplets and an enhanced droplet generation rate. An ANOVA was performed on droplet diameter to confirm no significant difference in droplet size from the three different aqueous inlets. The work described here offers an alternative approach to multiplexed droplet microfluidic devices allowing for the high-throughput interrogation of three sample conditions in a single device. It also has provided an alternative method to induce droplet formation that does not require multiple syringe pumps.

3D printed alginate bead generator for high-throughput cell culture.

Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates of alginate (QAlg) and air (QA), the diameter of device nozzle (d) and the concentration of alginate gel solution (C). We identified that the diameter of beads (D = 0.9 -2.8 mm) can be precisely controlled by changing QA and d. Also the bead generation frequency (f) can be tuned by changing QAlg. Finally, we demonstrated that viability and biological function (pericellular matrix deposition) of chondrocytes were not adversely affected by high f using this bead generator. Because 3D printing is becoming a more accessible technique, our unique design will allow greater access to average biomedical research laboratories, STEM education and industries in cost- and time-effective manner.

An Interface-Particle Interaction Approach for Evaluation of the Co-Encapsulation Efficiency of Cells in a Flow-Focusing Droplet Generator.

Droplet-based microfluidics offers significant advantages, such as high throughput and scalability, making platforms based on this technology ideal candidates for point-of-care (POC) testing and clinical diagnosis. However, the efficiency of co-encapsulation in droplets is suboptimal, limiting the applicability of such platforms for the biosensing applications. The homogeneity of the bioanalytes in the droplets is an unsolved problem. While there is extensive literature on the experimental setups and active methods used to increase the efficiency of such platforms, passive techniques have received less attention, and their fundamentals have not been fully explored. Here, we develop a novel passive technique for investigating cell encapsulation using the finite element method (FEM). The level set method was used to track the interfaces of forming droplets. The effects of walls and the droplet interfaces on relatively large cells were calculated to track them more accurately during encapsulation. The static surface tension force was used to account for the effects of the interfaces on cells. The results revealed that the pairing efficiency is highly sensitive to the standard deviation (SD) of the distance between the cells in the entrance channel. The pairing efficiency prediction error of our model differed by less than 5% from previous experiments. The proposed model can be used to evaluate the performance of droplet-based microfluidic devices to ensure higher precision for co-encapsulation of cells.

Droplet-Based Screening for the Investigation of Microbial Nonlinear Dose-Response Characteristics System, Background and Examples.

Droplet-based microfluidics is a versatile tool to reveal the dose-response relationship of different effectors on the microbial proliferation. Traditional readout parameter is the temporal development of the cell density for different effector concentrations. To determine nonlinear or unconventional dose-response relationships, data with high temporal resolution and dense concentration graduation are essential. If microorganisms with slow microbial growth kinetics are investigated, a sterile and evaporation-free long-term incubation technique is required. Here, we present a modular droplet-based screening system which was developed to solve these issues. Beside relevant technical aspects of the developed modules, the procedural workflow, and exemplary dose-response data for 1D and 2D dose-response screenings are presented.

A micrometer head integrated microfluidic device for facile droplet size control and automatic measurement of a droplet size.

A novel microfluidic droplet generator is proposed, which can control the droplet size through turning an integrated micrometer head with ease, and the size of the produced micro-droplet can be automatically and real-time monitored by an open-sourced software and off-the-shelf hardware.

The Effect of Oil Viscosity on Droplet Generation Rate and Droplet Size in a T-Junction Microfluidic Droplet Generator.

There have been growing interests in droplet-based microfluidics due to its capability to outperform conventional biological assays by providing various advantages, such as precise handling of liquid/cell samples, fast reaction time, and extremely high-throughput analysis/screening. The droplet-based microfluidics utilizes the interaction between the interfacial tension and the fluidic shear force to break continuous fluids into uniform-sized segments within a microchannel. In this paper, the effect of different viscosities of carrier oil on water-in-oil emulsion, particularly how droplet size and droplet generation rate are affected, has been investigated using a commonly used T-junction microfluidic droplet generator design connected to a pressure-controlled pump. We have tested mineral oils with four different viscosities (5, 7, 10, and 15 cSt) to compare the droplet generation under five different flow pressure conditions (i.e., water flow pressure of 30-150 mbar and oil flow pressure of 40-200 mbar). The results showed that regardless of the flow pressure levels, the droplet size decreased as the oil viscosity increased. Average size of the droplets decreased by approximately 32% when the viscosity of the oil changed from 5 to 15 cSt at the flow pressure of 30 mbar for water and 40 mbar for oil. Interestingly, a similar trend was observed in the droplet generation rate. Droplet generation rate and the oil viscosity showed high linear correlation (R2 = 0.9979) at the water flow pressure 30 mbar and oil flow pressure 40 mbar.

A High Temperature Drop-On-Demand Droplet Generator for Metallic Melts.

In this study we present the design and functionality of a pneumatic drop-on-demand droplet generator that produces metallic micro particles with a size range of 300 µm to 1350 µm at high temperatures of up to 1600 °C. Molten metal droplets were generated from an EN 1.3505 (AISI 52100) steel which solidified during a falling distance of 6.5 m. We analyzed the resulting particle size and morphology using static image analysis. Furthermore, the droplet formation mode was analyzed using high-speed recordings and the pressure oscillation was measured in the crucible. The system is meant to be reproducible in all aspects and therefore the in-situ measurements are set to control the droplet size and trajectory during the run. Additionally, the ex-situ measurements are done on the particles in order to characterize them in size and morphology aspects.

Faraday Waves-Based Integrated Ultrasonic Micro-Droplet Generator and Applications.

An in-depth review on a new ultrasonic micro-droplet generator which utilizes megahertz (MHz) Faraday waves excited by silicon-based multiple Fourier horn ultrasonic nozzles (MFHUNs) and its potential applications is presented. The new droplet generator has demonstrated capability for producing micro droplets of controllable size and size distribution and desirable throughput at very low electrical drive power. For comparison, the serious deficiencies of current commercial droplet generators (nebulizers) and the other ultrasonic droplet generators explored in recent years are first discussed. The architecture, working principle, simulation, and design of the multiple Fourier horns (MFH) in resonance aimed at the amplified longitudinal vibration amplitude on the end face of nozzle tip, and the fabrication and characterization of the nozzles are then described in detail. Subsequently, a linear theory on the temporal instability of Faraday waves on a liquid layer resting on the planar end face of the MFHUN and the detailed experimental verifications are presented. The linear theory serves to elucidate the dynamics of droplet ejection from the free liquid surface and predict the vibration amplitude onset threshold for droplet ejection and the droplet diameters. A battery-run pocket-size clogging-free integrated micro droplet generator realized using the MFHUN is then described. The subsequent report on the successful nebulization of a variety of commercial pulmonary medicines against common diseases and on the experimental antidote solutions to cyanide poisoning using the new droplet generator serves to support its imminent application to inhalation drug delivery.

Spray Droplet Characterization from a Single Nozzle by High Speed Image Analysis Using an In-Focus Droplet Criterion.

Accurate spray characterization helps to better understand the pesticide spray application process. The goal of this research was to present the proof of principle of a droplet size and velocity measuring technique for different types of hydraulic spray nozzles using a high speed backlight image acquisition and analysis system. As only part of the drops of an agricultural spray can be in focus at any given moment, an in-focus criterion based on the gray level gradient was proposed to decide whether a given droplet is in focus or not. In a first experiment, differently sized droplets were generated with a piezoelectric generator and studied to establish the relationship between size and in-focus characteristics. In a second experiment, it was demonstrated that droplet sizes and velocities from a real sprayer could be measured reliably in a non-intrusive way using the newly developed image acquisition set-up and image processing. Measured droplet sizes ranged from 24 µm to 543 µm, depending on the nozzle type and size. Droplet velocities ranged from around 0.5 m/s to 12 m/s. The droplet size and velocity results were compared and related well with the results obtained with a Phase Doppler Particle Analyzer (PDPA).