Acoustic Droplet Ejection Research Articles

Liquid Handling Technologies: A Study through Major Discoveries and Advancements

Liquid handling is an essential process in laboratories involving automatically transferring accurate amounts of liquids for various task completions such as sample preparation, pipetting, dilution, and mixing. Automated liquid handling systems (ALHS) help to minimize the errors, complications, and difficulties that are faced with manual methods of liquid handling by efficiently enhancing the accuracy, precision, and reproducibility of experimental workflows and have transformed life sciences aiding research in various fields. Processes like Nuclear Magnetic Resonance (NMR) spectrometry, mass spectrometry, and acoustic droplet ejection with ALHS showcase tremendous potential. Outdated manual machinery is being replaced rapidly with automated tools. We have observed that these laboratory setups are sophisticated and costly. It is challenging to access such technologies, especially in poor or developing countries. At the nanoscale level, handling and evaporation of biomaterials becomes a great issue. Multitudinous devices can combat the cost issue along with utilization of single equipment without the enhancement of add-on modules. Liquid handling devices enhance the quality of experiments contributing to efficient dealing of circumstances like COVID-19. They aid in genomics and proteomics and play a pivotal role in liquid manipulation, drug screening, quantifications, forensic studies and many more. Liquid handling devices with robotic equipment can perform multiple tasks and can assist in a wide range of fields. We anticipate that in the coming years, our laboratories will be furnished with advanced, robotic technologies and cause a revolution in the scientific scenery.

Investigation of nanostructured lipid carriers for fast intracellular localization screening using the Echo liquid handler

In the field of precision medicine, therapy is optimized individually for each patient, enhancing efficacy while reducing side effects. This involves the identification of promising drug candidates through high-throughput screening on human derived cells in culture. However, screening of drugs which have poor solubility or permeability remains challenging, especially when targeting intracellular components. Therefore, encapsulation of drugs into advanced delivery systems such as nanostructured lipid carries (NLC) becomes necessary. Here we show that the cellular uptake of NLC with different matrix compositions can be assessed in a high-throughput screening system based on acoustic droplet ejection (ADE) technology (Echo liquid handler). Our findings indicate that surface tension and viscosity of the NLC dispersions need to be tailored to enable a reliable ADE transfer. The automated NLC uptake studies indicated that the composition of the matrix, more specifically the amount of oleic acid, significantly influenced cellular uptake. The data obtained were corroborated by imaging based and spectral flow cytometry cellular uptake studies. These findings thus not only provide the basis for a screening tool to rapidly identify the efficacy of NLC uptake but also enable a next step toward precision high-throughput drug screening under consideration of an optimized drug delivery system.

Fast acoustic droplet ejection based on annular array transducer

Acoustic droplet ejection (ADE) has become the preferred method for liquid transfer in a variety of applications including synthetic biology, genotyping and drug discovery.Comparing with traditional pipetting techniques, the accuracy and data reproducibility of ADE based liquid transfer are improved, waste and cost are reduced, and cross-contamination is eliminated. The key component in the ADE system is the ultrasound transducer, which is responsible for generating focused ultrasound beam for droplet ejection. However, current ADE systems commonly utilize a single-element focused transducer with a fixed focal length that require mechanical movement to focus on the liquid surface, resulting in reduced liquid transfer efficiency. In this study, we first present a high-frequency annular array transducer for the ADE technology, which enables rapid and dynamic axial focusing to the liquid surface without mechanically moving the transducer, thereby accelerating liquid transfer. Experimental results show that the proposed 10 MHz, 5-element annular array transducer has good dynamic axial focusing ability, and can achieve accurate and stable droplet ejection of nanoliter volume at the designed focal length of 26–32 mm. Our results highlight the potential of the annular array transducer in advancing ADE system for rapid liquid transfer. This technology is expected to be useful in a variety of applications where precise and high-throughput liquid transfer is crucial.

Contactless Single Cell Extraction from Monolayer Cell Culture Using A MEMS Acoustic Droplet Ejector.

To achieve a contactless and damage-less extraction of a single (or a few) human retinal pigment epithelial (RPE) cell(s) from a cell monolayer with acoustic droplet ejection. An acoustic droplet ejector based on a self-focusing acoustic transducer (SFAT) is designed, microfabricated, and placed on a precision movable stage that aligns it to the targeted cell(s) in a Petri dish. The device delivers 20.1 MHz focused ultrasound (FUS) with a focal diameter of 100 μm, which ejects droplets capable of extracting and transferring only the targeted cell(s) from a monolayer. The extraction and collection of 1-10 cells are successfully demonstrated. The number of ejected cells can be controlled by the FUS pulse width. As confirmed by fluorescence-based cell viability assays and re-culture experiments, the ejected cell(s) and remaining monolayer cells are intact without damage after cell ejection. Furthermore, real-time reverse transcription polymerase chain reaction (RT-PCR) tests for both housekeeping genes (GAPDH and β -actin) and RPE-specific genes (MITF, PEDF, and PMEL17) show no significant difference between the acoustically ejected cells and those collected manually with a micropipette. The proposed technology realizes a contactless, damage-free extraction of cells with high spatial resolution and precise control of the number of cells ejected, with a simple setup. This powerful technology not only enables efficient, high-precision cell extraction for quality check applications but also opens new avenues for other advanced biotechnologies such as bioprinting.

A novel ADE system with tunable droplet size and ejection height

Acoustic droplet ejection (ADE) has been proven to move liquids in droplet form from a container to mid-air. However, the droplet sizes produced using the traditional ADE setups are proportional to their heights if the physical framework is unchanged. This limitation obstructs further employment of ADE technology since having the exact sizes of droplets at a specific location or different sizes at different locations is required for many applications. To overcome this limitation and enable more possible applications of ADE, this study proposed an innovative ADE configuration that could manipulate the size and height of the ADE droplets with only electrical signals. To achieve this, a low-voltage driving period and a pinhole structure were added to create a water mound before ejection. First, simulations were conducted to validate the proposed method and find the parameters of the novel ADE setup. After that, a driving circuit featuring a high-voltage pulser and a field-programmable gate array was built. However, a 3D spherical resin model was printed to focus the acoustic wave on the water surface, and a cover with a pinhole was added to create water mound. To observe the behavior of the droplets, a recording system and detection algorithm were developed to capture and identify the dimension/height of the droplets, respectively. Finally, the proposed ADE configuration successfully manipulated the droplet size at the same ejecting height under three driving voltages (65, 70, and 75 V) and pinhole diameters (3.7, 4, and 4.4 mm).

Abstract 3094: Biophysics in kinase drug discovery programs: Spectral shift technology from fragment screening to mechanism of action

Abstract Using Eurofins Discovery’s state-of-the art Hit-Finding platform, our experts designed an optimized screening cascade combining cutting-edge High-throughput Screening (HTS) technologies with the latest Biophysics techniques. With advanced technologies, such as the Echo® MS and RapidFire MS for HTS, and SPR, MST, TSA, nanoDSF and ITC for Biophysics, the cascade is effective for primary screening, orthogonal assays and evaluating Mechanism of Action (MoA). There are many biophysical approaches which bring value to drug discovery programs, and it is the target properties which drive the selection of the most suitable technology. In this presentation, we will highlight the novel Spectral Shift technology, one of the techniques employed by the Dianthus (well-known for the MST-TRIC). The goal was to rapidly implement an assay that monitors the ability of PIM3 to bind to ATP, as well as to compounds and fragments in solution. Using the Echo MS’ acoustic-droplet ejection, nanoliter dispensing in 394-well plates, robust data by Spectral Shift were quickly generated for the fragment-based screening campaign. Hits were confirmed in Dose-response with µM-range affinity values (KD), and TSA was used as an orthogonal assay for the identification of stabilizers among the selected PIM3 binders. For a defined set of 826 proprietary fragments, a single dose screening at 600µM using Spectral Shift was run, leading to the identification of 11% of potential binders. The 100 fragments were then confirmed in dose-response titration and 80 binders were confirmed. The top 30 binders were selected for orthogonal characterization using TSA. This led to the selection of the top 6 confirmed fragments, on which a MoA study in MST-TRIC was performed. Following this evaluation, 3 allosteric binders were identified, with KD from 355 to 550µM (affinity of orthosteric binders from 55 to 333µM). Biophysics plays an important role in drug discovery by qualifying valuable assets and increasing the success rates of future Hit-finding programs. The fragment hits selected in this PIM3 Kinase project, which show ADP-Glo™ activities, are now ready for crystallography studies (NMR & X-ray co-crystallization). Citation Format: Celine Legros, Vanessa Porkolab, Maud Sigoillot, Olivier Mirguet. Biophysics in kinase drug discovery programs: Spectral shift technology from fragment screening to mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3094.

Accelerating Pharmaceutical Process Development with an Acoustic Droplet Ejection-Multiple Reaction Monitoring-Mass Spectrometry Workflow.

Fast-paced pharmaceutical process developments (e.g., high-throughput experimentation, directed evolution, and machine learning) involve the introduction of fast, sensitive, and accurate analytical assays using limited sample volumes. In recent years, acoustic droplet ejection (ADE) coupled with an open port interface has been invented as a sampling technology for mass spectrometry, providing high-throughput nanoliter analytical measurements directly from the standard microplates. Herein, we introduce an ADE-multiple reaction monitoring-mass spectrometry (ADE-MRM-MS) workflow to accelerate pharmaceutical process research and development (PR&D). This systematic workflow outlines the selection of MRM transitions and optimization of assay parameters in a data-driven manner using rapid measurements (1 sample/s). The synergy between ADE sampling and MRM analysis enables analytical assays with excellent sensitivity, selectivity, and speed for PR&D reaction screenings. This workflow was utilized to develop new ADE-MRM-MS assays guiding a variety of industrial processes, including (1) screening of Ni-based catalysts for C-N cross-coupling reaction at 1 Hz and (2) high-throughput regioisomer analysis-enabled enzyme library screening for peptide ligation reaction. ADE-MRM-MS assays were demonstrated to deliver accurate results that are comparable to conventional liquid chromatography (LC) experiments while providing >100-fold throughput enhancement.

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.

Effect of Wettability on the Collision Behavior of Acoustically Excited Droplets.

Acoustic droplet ejection (ADE) is a noncontact technique for micro-liquid handling (usually nanoliters or picoliters) that is not restricted by nozzles and enables high-throughput liquid dispensing without sacrificing precision. It is widely regarded as the most advanced solution for liquid handling in large-scale drug screening. Stable coalescence of the acoustically excited droplets on the target substrate is a fundamental requirement during the application of the ADE system. However, it is challenging to investigate the collision behavior of nanoliter droplets flying upward during the ADE. In particular, the dependence of the droplet's collision behavior on substrate wettability and droplet velocity has yet to be thoroughly analyzed. In this paper, the kinetic processes of binary droplet collisions were investigated experimentally for different wettability substrate surfaces. Four states occur as the droplet collision velocity increases: coalescence after minor deformation, complete rebound, coalescence during rebound, and direct coalescence. For the hydrophilic substrate, there are wider ranges of Weber number (We) and Reynolds number (Re) in the complete rebound state. And with the decrease of the substrate wettability, the critical Weber and Reynolds numbers for the coalescence during rebound and the direct coalescence decrease. It is further revealed that the hydrophilic substrate is susceptible to droplet rebound because the sessile droplet has a larger radius of curvature and the viscous energy dissipation is greater. Besides, the prediction model of the maximum spreading diameter was established by modifying the droplet morphology in the complete rebound state. It is found that, under the same Weber and Reynolds numbers, droplet collisions on the hydrophilic substrate achieve a smaller maximum spreading coefficient and greater viscous energy dissipation, so the hydrophilic substrate is prone to droplet bounce.

Hundred-micron droplet ejection by focused ultrasound for genomic applications

Droplet-based microfluidic (DMF) technology is widely used in genetic analysis. Among multiple droplet generation techniques, acoustic droplet ejection (ADE) is a non-contact, high-precision method that utilizes the energy of focused ultrasound. The ejection of a single droplet of controlled size, the performance of multi-droplet ejections and the effects of the ultrasound energy on nucleic acid molecules in the droplets, are the main research interests in droplet-based genomics. In this work, a focused ultrasonic transducer with a frequency of 20 MHz was used, and we achieved droplet ejection successfully. Various experiments were carried out to study ADE and the beneficial results were obtained, including the characterization of ADE conditions, the relationship between droplet volume and parameters (including power, duration and distance), the energy required for ADE, and the effects of ultrasound energy on DNA strands, etc. It was first noticed that the superior limit of the droplet size could be increased by adjusting the height of the transducer. In addition, multi-droplet ejections at the optimal delay time were realized with good linearity. Above all, we demonstrated that the nucleic acid molecules remained their integrity when the exciting power was the maximum and the exciting duration was the longest in our system. In summary, ADE was proved to have an important role and a vast prospect in DMF technology for genomics-related applications.

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target.

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.

Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.

With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.

Controllable Droplet Ejection of Multiple Reagents through Focused Acoustic Beams.

Acoustic droplet ejection (ADE) technology has revolutionized fluid handling with its contactless and fast fluid transfer. For precise droplet ejection and stable droplet coalescence at the target substrates for further detection, the input power of the ADE system needs to be adjusted. Currently, the existing power control method depends on scanning the source fluid wells one by one, which cannot afford precise and highly efficient droplet velocity adjustment, and the complicated operation caused by the repeated power evaluation processes for thousands of fluid transfers will waste much time. We propose a new method, which realizes the controllable ejection of multiple reagents by analyzing the effect of the product of kinematic viscosity and surface tension of the reagents on the droplet initial velocity. The experimental results obtained by ejecting dimethyl sulfoxide coincide well with the predicted results, and the relative error in the droplet initial velocity is mostly less than 8%. On the basis of the input power prediction method proposed in this paper, the ADE system is successfully constructed for continuous dispensing of polystyrene microspheres as cell surrogates, which provided an advanced liquid handling solution for research in biochemistry and other fields.

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.

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

AbstractMacrocyclic 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.

Co-crystallization of hepatitis C virus NS3/4A inhibitors and SARS-CoV-2 main protease using high-density acoustic droplet ejection (ADE)

Co-crystallization of hepatitis C virus NS3/4A inhibitors and SARS-CoV-2 main protease using high-density acoustic droplet ejection (ADE)

Acoustic Ejection Mass Spectrometry for High-Throughput Analysis

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery.

Acoustic droplet ejection (ADE)-open port interface (OPI)-mass spectrometry (MS) has recently been introduced as a versatile analytical method that combines fast and contactless acoustic sampling with sensitive and accurate electrospray ionization (ESI)-MS-based analyte detection. The potential of the technology to provide label-free measurements in subsecond analytical cycle times makes it an attractive option for high-throughput screening (HTS). Here, we report the first implementation of ADE-OPI-MS in a fully automated HTS environment, based on the example of a biochemical assay aiming at the identification of small-molecule inhibitors of the cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). First, we describe the optimization of the method to enable sensitive and accurate determination of enzyme activity and inhibition in miniaturized 1536-well microtiter plate format. Then we show both results from a validation single-concentration screen using a test set of 5500 compounds, and the subsequent concentration-response testing of selected hits in direct comparison with a previously established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) readout. Finally, we present the development of an in-line OPI cleaning procedure aiming to match the instrument robustness required for large-scale HTS campaigns. Overall, this work points to critical method development parameters and provides guidance for the establishment of integrated ADE-OPI-MS as HTS-compatible technology for early drug discovery.