Enhanced and controlled droplet ejection on magnetic responsive polydimethylsiloxane microarrays.

Efficient removal of droplets from solid surfaces is significant in various fields, including fog collection and condensation heat transfer. However, droplets removal on common surfaces with static structures often occurs passively, which limits the possibility of increasing removal efficiency and lacks intelligent controllability. In this paper, an active strategy based on extrusion ejection is proposed and demonstrated on the magnetic responsive polydimethylsiloxane (PDMS) superhydrophobic microplates (MPSM). The MPSM can reversibly transit between the upright and tilted state as the external magnetic field is alternately applied and removed. Under the magnetic field, the direction and trajectories of droplets departure can be intelligently controlled, demonstrating excellent controllability. More importantly, compared with the static structure where the droplet must reach a certain size before departure, droplets can be ejected at smaller sizes as the MPSM is tilted. These advantages are of great significance in many fields, such as a highly efficient fog harvesting system. This strategy of extrusion ejection based on dynamic surface structure control reported in this work may provide fresh ideas for efficient droplet manipulation.

Ultra-high-throughput mass spectrometry in drug discovery: fundamentals and recent advances.

INTRODUCTION: Ultra-high-throughput mass spectrometry, uHT-MS, is a technology that utilizes ionization and sample delivery technologies optimized to enable sampling from well plates at > 1 sample per second. These technologies do not need a chromatographic separation step and can be utilized in a wide variety of assays to detect a broad range of analytes including small molecules, lipids, and proteins. AREAS COVERED: This manuscript provides a brief historical review of high-throughput mass spectrometry and the recently developed technologies that have enabled uHT-MS. The report also provides examples and references on how uHT-MS has been used in biochemical and chemical assays, nuisance compound profiling, protein analysis and high throughput experimentation for chemical synthesis. EXPERT OPINION: The fast analysis time provided by uHT-MS is transforming how biochemical and chemical assays are performed in drug discovery. The potential to associate phenotypic responses produced by 1000's of compound treatments with changes in endogenous metabolite and lipid signals is becoming feasible. With the augmentation of simple, fast, high-throughput sample preparation, the scope of uHT-MS usage will increase. However, it likely will not supplant LC-MS for analyses that require low detection limits from complex matrices or characterization of complex biotherapeutics such as antibody-drug conjugates.

The Evaluation and Exploration of Piezoelectric Parameter Optimization for Droplet Ejection in Binder Jet 3D Printing Drugs.

Since the first three-dimensional (3D) printed drug was approved by the Food and Drug Administration in 2015, there has been a growing interest in using binder jet 3D printing (BJ-3DP) technology for pharmaceuticals. However, most studies are still at an exploratory stage, lacking micromechanism research, such as the droplet ejection mechanism, the effect of printhead piezoelectric parameters on inkjet smoothness and preparation formability. In this study, based on the inkjet printing and observation platform, the Epson I3200-A1 piezoelectric printhead matched to the self-developed BJ-3DP was selected to analyze the droplet ejection state of self-developed ink at the microlevel with different piezoelectric pulse parameters. The results showed that there was a stable inkjet state with an inkjet pulse width of 3.5 µs, an ink supply pulse width of 4.5 µs, and a jet frequency in the range of 5000-19,000 Hz, ensuring both better droplet pattern and print accuracy, as well as high ejection efficiency. In conclusion, we performed a systematic evaluation of the inkjet behavior under different piezoelectric pulse parameters and provided a good idea and case study for the optimization of printhead piezoelectric parameters when BJ-3DP technology was used in pharmaceuticals.

Acoustic droplet ejection facilitates cell-based high-throughput screenings using natural products.

Natural Products (NPs) are one of the main sources for drug discovery. Many clinical drugs are NPs or NP-inspired compounds, and recently discovered New Chemical Entities (NCEs) of NPs are emerging as promising new drugs. High-Throughput Screening (HTS) of large sample sets or libraries has grown to be vital for the drug discovery field. Industrial-scale HTS of NP libraries can be limited due to the difficulties entailed in working with tiny extract volumes and the variability in viscosity of NP extracts. For these reasons, the implementation of new technologies to miniaturize different reagent volumes grows to be fundamental. Since Acoustic Droplet Ejection (ADE) emerged as a helpful tool in HTS campaigns for the transference of compound libraries. The aim of this work was to test the effectiveness of ADE for the dispensation of NP extract libraries in cell-based HTS assays.

A Comprehensive Review of Surface Acoustic Wave-Enabled Acoustic Droplet Ejection Technology and Its Applications.

This review focuses on the development of surface acoustic wave-enabled acoustic drop ejection (SAW-ADE) technology, which utilizes surface acoustic waves to eject droplets from liquids without touching the sample. The technology offers advantages such as high throughput, high precision, non-contact, and integration with automated systems while saving samples and reagents. The article first provides an overview of the SAW-ADE technology, including its basic theory, simulation verification, and comparison with other types of acoustic drop ejection technology. The influencing factors of SAW-ADE technology are classified into four categories: fluid properties, device configuration, presence of channels or chambers, and driving signals. The influencing factors discussed in detail from various aspects, such as the volume, viscosity, and surface tension of the liquid; the type of substrate material, interdigital transducers, and the driving waveform; sessile droplets and fluid in channels/chambers; and the power, frequency, and modulation of the input signal. The ejection performance of droplets is influenced by various factors, and their optimization can be achieved by taking into account all of the above factors and designing appropriate configurations. Additionally, the article briefly introduces the application scenarios of SAW-ADE technology in bioprinters and chemical analyses and provides prospects for future development. The article contributes to the field of microfluidics and lab-on-a-chip technology and may help researchers to design and optimize SAW-ADE systems for specific applications.

Structure Design and Characterization of 3D Printing System of Thermal Battery Electrode Ink Film.

In this paper, a 3D printing system for a thermal battery electrode ink film is set up and investigated based on the on-demand microdroplet ejection technology. The optimal structural dimensions of the spray chamber and metal membrane of the micronozzle are determined via simulation analysis. The workflow and functional requirements of the printing system are set up. The printing system includes a pretreatment system, piezoelectric micronozzle, motion control system, piezoelectric drive system, sealing system, and liquid conveying system. Different printing parameters are compared to obtain optimized printing parameters, which can be attributed to the optimal pattern of the film. The feasibility and controllability of 3D printing methods are verified by printing tests. The size and output speed of the droplets can be controlled by adjusting the amplitude and frequency of the driving waveform acting on the piezoelectric actuator. So, the required shape and thickness of the film can be achieved. An ink film in terms of nozzle diameter = 0.6 mm, printing height = 8 mm, wiring width = 1 mm, input voltage = 3 V and square wave signal frequency = 35 Hz can be achieved. The electrochemical performance of thin-film electrodes is crucial in thermal batteries. The voltage of the thermal battery reaches its peak and tends to flatten out at around 100 s when using this printed film. The electrical performance of the thermal batteries using the printed thin films is found to be stable. This stabilized voltage makes it applicable to thermal batteries.

Identification of the Dominant Factor for Droplet Ejection from a Tungsten Electrode during AC Tungsten Inert Gas Welding by Visualisation of Electrode Phenomena.

Droplet ejections from a molten tungsten electrode during alternating current tungsten inert gas (AC TIG) welding were observed successfully by a high-speed video captured at 75,000 fps. The welding conditions and timings that were likely to occur were investigated. The electrode surface temperature was also measured. A crater was formed on the surface of the electrode, and a droplet ejection occurred following the separation of the tip of the ridge growing from the centre of the crater. A series of droplet ejections occurred on a time scale of approximately 0.4 ms. Our results showed that the high temperature of the electrode surface was the common factor for droplet ejections. The dominant force for droplet ejection was discussed by estimating the balance of forces acting on the molten electrode surface. The pressure due to surface tension was the largest pressure at any time during the AC cycle, which decreased in the second half of the EP period. Our findings suggest that the surface tension was the dominant force for changing the electrode shape and that droplet ejections occurred when the surface tension decreased due to the increase in the electrode surface temperature.

Advances in ionisation techniques for mass spectrometry-based omics research.

Omics analysis by mass spectrometry (MS) is a vast field, with proteomics, metabolomics and lipidomics dominating recent research by exploiting biological MS ionisation techniques. Traditional MS ionisation techniques such as electrospray ionisation have limitations in analyte-specific sensitivity, modes of sampling and throughput, leading to many researchers investigating new ionisation methods for omics research. In this review, we examine the current landscape of these new ionisation techniques, divided into the three groups of (electro)spray-based, laser-based and other miscellaneous ionisation techniques. Due to the wide range of new developments, this review can only provide a starting point for further reading on each ionisation technique, as each have unique benefits, often for specialised applications, which promise beneficial results for different areas in the omics world.

A high-throughput screening assay for mutant isocitrate dehydrogenase 1 using acoustic droplet ejection mass spectrometry.

Acoustic droplet ejection mass spectrometry (ADE-MS) has recently emerged as a promising label-free, MS-based readout method for high throughput screening (HTS) campaigns in early pharmaceutical drug discovery, since it enables high-speed analysis directly from 384- or 1536-well plates. In this manuscript we describe our characterization of an ADE-MS based high sample content enzymatic assay for mutant isocitrate dehydrogenase 1 (IDH1) R132H with a strong focus on assay development. IDH1 R132H has become a very attractive therapeutic target in the field of antitumor drug discovery, and several pharmaceutical companies have attempted to develop novel small molecule inhibitors against mutant IDH1. With the development of an mIDH1 ADE-MS based HTS assay and a detailed comparison of this new readout technique to the commonly used fluorescence intensity mIDH1 assay, we demonstrated good correlation of both methods and were able to identify new potent inhibitors of mIDH1.

Multiscale 3D Bioprinting by Nozzle-Free Acoustic Droplet Ejection.

Bioprinting allows the manufacture of complex cell-laden hydrogel constructs that can mature into tissue replacements in subsequent cell culture processes. The nozzles used in currently available bioprinters limit the print resolution and at dimensions below 100 µm clogging is expected. Most critically, the reduction of nozzle diameter also increases shear stress during printing. At critical shear stress, mechanical damage to printed cells triggers cell death. To overcome these limitations, a novel 3D bioprinting method based on the principle of acoustic droplet ejection (ADE) is introduced here. The absence of a nozzle in this method minimizes critical shear stress. A numerical simulation reveals that maximum shear stress during the ADE process is 2.7 times lower than with a Ø150 µm microvalve nozzle. Printing of cell clusters contained in droplets at the millimeter length scale, as well as in droplets the size of a single cell, is feasible. The precise 3D build-up of cell-laden structures is demonstrated and evidence is provided that there are no negative effects on stem cell morphology, proliferation, or differentiation capacities. This multiscale acoustic bioprinting technique thus holds promise for cell-preserving creation of complex and individualized cell-laden 3D hydrogel structures.

Picomole-Scale Synthesis and Screening of Macrocyclic Compound Libraries by Acoustic Liquid Transfer.

Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets, such as protein-protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale by using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced the reaction volumes, reagent consumption, and synthesis time. As a proof-of-concept, we assembled a 2700-member target-focused macrocyclic library that we could subsequently assay in the same microtiter synthesis plates, saving the need for additional transfers and deconvolution schemes. We screened the library against the MDM2-p53 protein-protein interaction and generated micromolar and sub-micromolar inhibitors. Our approach based on acoustic liquid transfer provides a general strategy for the development of macrocycle ligands.

Busting Myths in Compound Handling Practices for Assay Developers.

Since the advent of modern-day screening collections in the early 2000s, various aspects of our knowledge of good handling practices have continued to evolve. Some early practices, however, continue to prevail due to the absence of defining data that would bust the myths of tradition. The lack of defining data leads to a gap between plate-based screeners, on the one hand, and compound sample handling groups, on the other, with the latter being the default party to blame when an assay goes awry.In this paper, we highlight recommended practices that ensure sample integrity and present myth busting data that can help determine the root cause of an assay gone bad. We show how a strong and collaborative relationship between screening and sample handling groups is the better state that leads to the accomplishment of the common goal of finding breakthrough medicines.

Acoustic Droplet Vitrification Method for High-Efficiency Preservation of Rare Cells.

Cryopreservation is a key step for current translational medicine including reproductive medicine, regenerative medicine, and cell therapy. However, it is challenging to preserve rare cells for practical applications due to the difficulty in handling low numbers of cells as well as the lack of highly efficient and biocompatible preservation protocols. Here, we developed an acoustic droplet vitrification method for high-efficiency handling and preservation of rare cells. By employing an acoustic droplet ejection device, we can encapsulate rare cells into water-in-air droplets with a volume from ∼pL to ∼nL and deposit these cell-containing droplets into a droplet array onto a substrate. By incorporating a cooling system into the droplet array substrate, we can vitrify hundreds to thousands of rare cells at an ultrafast speed (about ∼2 s) based on the high surface to volume ratio of the droplets. By optimizing this method with three different cell lines (a human lung cancer cell line, A549 cells, a human liver cell line, L02 cells, and a mouse embryonic fibroblast cell line, 3T3-L1 cells), we developed an effective protocol with excellent cell viability (e.g., >85% for days, >70% for months), proliferation, and adhesion. As a proof-of-concept application, we demonstrated that our method can rapidly handle and efficiently preserve rare cells, highlighting its broad applications in species diversity, basic research, and clinical medicine.

Integration of Acoustic Liquid Handling into Quantitative Analysis of Biological Matrix Samples.

Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.

High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology.

The Labcyte Echo acoustic liquid handler allows accurate droplet ejection at high speed from a source well plate to a destination plate. It has already been used in various miniaturized biological assays, such as quantitative PCR (q-PCR), quantitative real-time PCR (q-RT-PCR), protein crystallization, drug screening, cell dispensing, and siRNA transfection. However, no plasmid DNA transfection assay has been published so far using this dispensing technology. In this study, we evaluated the ability of the Echo 550 device to perform plasmid DNA transfection in 384-well plates. Due to the high throughput of this device, we simultaneously optimized the three main parameters of a transfection process: dilution of the transfection reagent, DNA amount, and starting DNA concentration. We defined a four-step protocol whose optimal settings allowed us to transfect HeLa cells with up to 90% efficiency and reach a co-expression of nearly 100% within transfected cells in co-transfection experiments. This fast, reliable, and automated protocol opens new ways to easily and rapidly identify optimal transfection settings for a given cell type. Furthermore, it permits easy software-based transfection control and multiplexing of plasmids distributed on wells of a source plate. This new development could lead to new array applications, such as human ORFeome protein expression or CRISPR-Cas9-based gene function validation in nonpooled screening strategies.

Using sound pulses to solve the crystal-harvesting bottleneck.

Crystal harvesting has proven to be difficult to automate and remains the rate-limiting step for many structure-determination and high-throughput screening projects. This has resulted in crystals being prepared more rapidly than they can be harvested for X-ray data collection. Fourth-generation synchrotrons will support extraordinarily rapid rates of data acquisition, putting further pressure on the crystal-harvesting bottleneck. Here, a simple solution is reported in which crystals can be acoustically harvested from slightly modified MiTeGen In Situ-1 crystallization plates. This technique uses an acoustic pulse to eject each crystal out of its crystallization well, through a short air column and onto a micro-mesh (improving on previous work, which required separately grown crystals to be transferred before harvesting). Crystals can be individually harvested or can be serially combined with a chemical library such as a fragment library.

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.

Gentle, fast and effective crystal soaking by acoustic dispensing.

The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.

High Throughput siRNA Screening Using Reverse Transfection.

RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis.

Precision Cancer Medicine in the Acoustic Dispensing Era: Ex Vivo Primary Cell Drug Sensitivity Testing.

Cancer therapy is increasingly becoming individualized, but there are also big gaps between the molecular knowledge of individual cancers we can generate today and what can be applied in the clinic. In an attempt to bridge this knowledge gap between cancer genetic and molecular profiling and clinically useful information, an individualized systems medicine program has been established at the Institute for Molecular Medicine Finland (FIMM), University of Helsinki, and the Helsinki University Hospital. Central to this program is drug sensitivity and resistance testing (DSRT), in which responses of primary cancer cells to a comprehensive clinical oncology and signal transduction drug collection are monitored. The drug sensitivity information is used with molecular profiling to establish hypotheses on individual cancer-selective targeting drug combinations and their predictive biomarkers, which can be explored in the clinic. Here, we describe how acoustic droplet ejection is enabling DSRT in our cancer individualized systems medicine program to (1) generate consistent but configurable assay-ready plates and determine how this affects data quality, (2) flexibly prepare drug combination testing plates, (3) dispense reagents and cells to the assay plates, and (4) perform ultra-miniaturized follow-up assays on the cells from DSRT plates.