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.

Direct Analysis from Phase-Separated Liquid Samples using ADE-OPI-MS: Applicability to High-Throughput Screening for Inhibitors of Diacylglycerol Acyltransferase 2.

The primary goal of high-throughput screening (HTS) is to rapidly survey a broad collection of compounds, numbering from tens of thousands to millions of members, and identify those that modulate the activity of a therapeutic target of interest. For nearly two decades, mass spectrometry has been used as a label-free, direct-detection method for HTS and is widely acknowledged as being less susceptible to interferences than traditional optical techniques. Despite these advantages, the throughput of conventional MS-based platforms like RapidFire or parallel LC-MS, which typically acquire data at speeds of 6-30 s/sample, can still be limiting for large HTS campaigns. To overcome this bottleneck, the field has recently turned to chromatography-free approaches including MALDI-TOF-MS and acoustic droplet ejection-MS, both of which are capable of throughputs of 1 sample/second or faster. In keeping with these advances, we report here on our own characterization of an acoustic droplet ejection, open port interface (ADE-OPI)-MS system as a platform for HTS using the membrane-associated, lipid metabolizing enzyme diacylglycerol acyltransferase 2 (DGAT2) as a model system. We demonstrate for the first time that the platform is capable of ejecting droplets from phase-separated samples, allowing direct coupling of liquid-liquid extraction with OPI-MS analysis. By applying the platform to screen a 6400-member library, we further demonstrate that the ADE-OPI-MS assay is suitable for HTS and also performs comparably to LC-MS, but with an efficiency gain of >20-fold.

Acoustic Mist Ionization-Mass Spectrometry: A Comparison to Conventional High-Throughput Screening and Compound Profiling Platforms.

Drug discovery usually begins with a high-throughput screen (HTS) of thousands to millions of molecules to identify starting points for medicinal chemistry. Conventional HTS platforms require expensive reagents and typically have complex assay formats. HTS platforms based on radioactivity are expensive, both in terms of reagent cost and disposal. Furthermore, nonspecific interferences common to these technologies result in an extensive attrition of hits during validation experiments. Mass spectrometry (MS) is a highly selective, label-free technology that can quantify multiple analytes in a single experiment. However, most commercial MS platforms typically involve a separation or cleanup prior to analysis and are too slow for large-scale screening campaigns. Recently, an MS platform (AMI-MS) was introduced that uses acoustically generated droplets to deliver analyte molecules directly from microtiter plates into the mass spectrometer at subsecond per well sampling rates. Here, we demonstrate the application of AMI-MS by developing an HTS-compatible assay that measures the inhibition of histone acetyltransferase activity. Real-time kinetic measurements from a single well were used to determine enzyme Km and Vmax values. We compare the AMI-MS readout with conventional platforms in single-shot screening and multipoint profiling modes. The AMI-MS assay identified 86% of hits previously identified, with a pIC50 ≥ 5.0, in a scintillation proximity assay (SPA) HTS at a lower hit rate and with a significantly reduced cost per well compared to the SPA-based readout. Furthermore, pIC50s, as measured by AMI-MS, showed a good correlation with values generated by RapidFire-MS. AMI-MS has the potential to provide significant improvements to high-throughput bioassays.

Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform.

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform's utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient's response to therapy and show the patient's achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

Acoustic Mist Ionization Platform for Direct and Contactless Ultrahigh-Throughput Mass Spectrometry Analysis of Liquid Samples.

Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector. Installing simple plate handling automation allowed us to reach a throughput of 100 000 samples per day on a single mass spectrometer. We applied AMI-MS to identify inhibitors of a human histone deacetylase from AstraZeneca's collection of 2 million small molecules and measured their half-maximal inhibitory concentration. The speed, sensitivity, simplicity, robustness, and consumption of nanoliter volumes of sample suggest that this technology will have a major impact across many areas of basic and applied research.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Novel Acoustic Loading of a Mass Spectrometer: Toward Next-Generation High-Throughput MS Screening.

High-throughput, direct measurement of substrate-to-product conversion by label-free detection, without the need for engineered substrates or secondary assays, could be considered the "holy grail" of drug discovery screening. Mass spectrometry (MS) has the potential to be part of this ultimate screening solution, but is constrained by the limitations of existing MS sample introduction modes that cannot meet the throughput requirements of high-throughput screening (HTS). Here we report data from a prototype system (Echo-MS) that uses acoustic droplet ejection (ADE) to transfer femtoliter-scale droplets in a rapid, precise, and accurate fashion directly into the MS. The acoustic source can load samples into the MS from a microtiter plate at a rate of up to three samples per second. The resulting MS signal displays a very sharp attack profile and ions are detected within 50 ms of activation of the acoustic transducer. Additionally, we show that the system is capable of generating multiply charged ion species from simple peptides and large proteins. The combination of high speed and low sample volume has significant potential within not only drug discovery, but also other areas of the industry.

Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.

Acoustic liquid handling uses high-frequency acoustic signals that are focused on the surface of a fluid to eject droplets with high accuracy and precision for various life science applications. Here we present a multiwell source plate, the Echo Qualified Reservoir (ER), which can acoustically transfer over 2.5 mL of fluid per well in 25-nL increments using an Echo 525 liquid handler. We demonstrate two Labcyte technologies-Dynamic Fluid Analysis (DFA) methods and a high-voltage (HV) grid-that are required to maintain accurate and precise fluid transfers from the ER at this volume scale. DFA methods were employed to dynamically assess the energy requirements of the fluid and adjust the acoustic ejection parameters to maintain a constant velocity droplet. Furthermore, we demonstrate that the HV grid enhances droplet velocity and coalescence at the destination plate. These technologies enabled 5-µL per destination well transfers to a 384-well plate, with accuracy and precision values better than 4%. Last, we used the ER and Echo 525 liquid handler to perform a quantitative polymerase chain reaction (qPCR) assay to demonstrate an application that benefits from the flexibility and larger volume capabilities of the ER.

Acoustically mounted microcrystals yield high-resolution X-ray structures.

We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 Ǻ resolution crystal structures.

Gradient, contact-free volume transfers minimize compound loss in dose-response experiments.

More accurate dose-response curves can be constructed by eliminating aqueous serial dilution of compounds. Traditional serial dilutions that use aqueous diluents can result in errors in dose-response values of up to 4 orders of magnitude for a significant percentage of a compound library. When DMSO is used as the diluent, the errors are reduced but not eliminated. The authors use acoustic drop ejection (ADE) to transfer different volumes of model library compounds, directly creating a concentration gradient series in the receiver assay plate. Sample losses and contamination associated with compound handling are therefore avoided or minimized, particularly in the case of less water-soluble compounds. ADE is particularly well suited for assay miniaturization, but gradient volume dispensing is not limited to miniaturized applications.

Mixed alkanethiol self-assembled monolayers as substrates for microarraying applications.

In current microarraying experiments, data quality is in large part determined by the quality of the spots that compose the microarray. Since many microarrays are made with contact printing techniques, microarray spot quality is fundamentally linked to the surface characteristics of the microarray substrate. In this work, surface coatings, consisting of self-assembled monolayers (SAMs) of mixed alkanethiol molecules, were used to control the surface properties of the microarray substrate. X-ray photoelectron spectroscopy and equilibrium contact angle measurements were performed in order to confirm the chemical content and wettability of these surface coatings. To test their performance in microarraying applications, sample microarrays were printed on these mixed alkanethiol films and then characterized with a noncontact visual metrology system and a fluorescence scanner. This work demonstrates that utilizing mixed alkanethiol SAMs as a surface coating provides spatially homogeneous surface characteristics that are reproducible across multiple microarray substrates as well as within a substrate. In addition, this paper demonstrates that these films are stable and robust as they can maintain their surface characteristics over time. Overall, it is demonstrated that SAMs of mixed alkanethiols serve as a useful surface coating, which enhances spot and therefore data quality in microarraying applications.