High throughput trapping and arrangement of biological cells using self-assembled optical tweezer

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

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Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211000677 ◽

2021 ◽

pp. 247255522110006

Author(s):

Michael D. Scholle ◽

Zachary A. Gurard-Levin

Keyword(s):

Mass Spectrometry ◽

Drug Discovery ◽

High Throughput ◽

Ionization Mass Spectrometry ◽

Ionization Mass ◽

Label Free ◽

Desorption Ionization ◽

Arginase 1 ◽

Self Assembled ◽

High Throughput Assay

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid–metabolizing enzymes.

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Teflon-on-Glass Molding Enables High-Throughput Fabrication of Hydrophilic-in-Hydrophobic Microwells for Bead-Based Digital Bioassays

Materials ◽

10.3390/ma11112154 ◽

2018 ◽

Vol 11 (11) ◽

pp. 2154

Author(s):

Lisa Tripodi ◽

Karen Ven ◽

Dries Kil ◽

Iene Rutten ◽

Robert Puers ◽

...

Keyword(s):

High Throughput ◽

Optical Tweezer ◽

Glass Molding ◽

Molding Method ◽

Superparamagnetic Beads ◽

Single Target ◽

Scaling Potential ◽

Target Molecules ◽

Lift Off ◽

Up Scaling

In recent years, Teflon-on-glass microwells have been successfully implemented in bead-based digital bioassays for the sensitive detection of single target molecules. Their hydrophilic-in-hydrophobic (HIH) nature enables the isolation and analysis of individual beads, carrying the target molecules, which can be further manipulated accurately through optical tweezer (OT) setups. However, these Teflon HIH-microwell platforms are conventionally fabricated through a complex, time-consuming and labor-intensive dry lift-off procedure which involves a series of major steps, limiting the up-scaling potential of these platforms. Alternative Teflon-based microwell fabrication methods have been extensively explored in literature but they preclude the generation of hydrophobic wells with hydrophilic bottom, thereby hampering the bioassay performance. Here, we present a new Teflon-on-glass molding method for the high throughput fabrication of hydrophilic-in-hydrophobic (HIH) microwell arrays, able to empower bead-based digital bioassays. Microwells 2.95 μm in depth and 3.86 μm in diameter were obtained to host individual beads. In these microwell arrays, sealing of reagents was demonstrated with an efficiency of 100% and seeding of superparamagnetic beads was achieved with an efficiency of 99.6%. The proposed method requires half as many steps when compared to the traditional dry lift-off process, is freely scalable and has the potential to be implemented in different bead-based bioassay applications.

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High-throughput photocapture approach for reaction discovery

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2003347117 ◽

2020 ◽

Vol 117 (24) ◽

pp. 13261-13266 ◽

Cited By ~ 5

Author(s):

Alison A. Bayly ◽

Benjamin R. McDonald ◽

Milan Mrksich ◽

Karl A. Scheidt

Keyword(s):

High Throughput ◽

Rapid Assessment ◽

Ionization Mass ◽

Reductive Coupling ◽

Self Assembled Monolayer ◽

High Throughput Analysis ◽

Reaction Discovery ◽

Assembled Monolayers ◽

Self Assembled ◽

Bioactive Agents

Modern organic reaction discovery and development relies on the rapid assessment of large arrays of hypothesis-driven experiments. The time-intensive nature of reaction analysis presents the greatest practical barrier for the execution of this iterative process that underpins the development of new bioactive agents. Toward addressing this critical bottleneck, we report herein a high-throughput analysis (HTA) method of reaction mixtures by photocapture on a 384-spot diazirine-terminated self-assembled monolayer, and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) analysis. This analytical platform has been applied to the identification of a single-electron-promoted reductive coupling of acyl azolium species.

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Ligand identification of the adenosine A2Areceptor in self-assembled nanodiscs by affinity mass spectrometry

Analytical Methods ◽

10.1039/c7ay01891f ◽

2017 ◽

Vol 9 (40) ◽

pp. 5851-5858 ◽

Cited By ~ 3

Author(s):

Jun Ma ◽

Yan Lu ◽

Dong Wu ◽

Yao Peng ◽

Wendy Loa-Kum-Cheung ◽

...

Keyword(s):

Mass Spectrometry ◽

High Throughput ◽

High Throughput Screening ◽

Ligand Identification ◽

Self Assembled

The combination of nanodisc and affinity LC/MS techniques potentiates the high-throughput screening against GPCRs.

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Volumetric Stress-Strain Analysis of Optohydrodynamically Suspended Biological Cells

Journal of Biomechanical Engineering ◽

10.1115/1.4002939 ◽

2010 ◽

Vol 133 (1) ◽

Cited By ~ 6

Author(s):

Sean S. Kohles ◽

Yu Liang ◽

Asit K. Saha

Keyword(s):

Single Cells ◽

Strain Analysis ◽

Optical Tweezer ◽

Three Dimensions ◽

Shear Stresses ◽

Strain Response ◽

Biological Cells ◽

Two Dimensional ◽

Stress Strain ◽

Volumetric Stress

Ongoing investigations are exploring the biomechanical properties of isolated and suspended biological cells in pursuit of understanding single-cell mechanobiology. An optical tweezer with minimal applied laser power has positioned biologic cells at the geometric center of a microfluidic cross-junction, creating a novel optohydrodynamic trap. The resulting fluid flow environment facilitates unique multiaxial loading of single cells with site-specific normal and shear stresses resulting in a physical albeit extensional state. A recent two-dimensional analysis has explored the cytoskeletal strain response due to these fluid-induced stresses [Wilson and Kohles, 2010, “Two-Dimensional Modeling of Nanomechanical Stresses-Strains in Healthy and Diseased Single-Cells During Microfluidic Manipulation,” J Nanotechnol Eng Med, 1(2), p. 021005]. Results described a microfluidic environment having controlled nanometer and piconewton resolution. In this present study, computational fluid dynamics combined with multiphysics modeling has further characterized the applied fluid stress environment and the solid cellular strain response in three dimensions to accompany experimental cell stimulation. A volumetric stress-strain analysis was applied to representative living cell biomechanical data. The presented normal and shear stress surface maps will guide future microfluidic experiments as well as provide a framework for characterizing cytoskeletal structure influencing the stress to strain response.

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