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[Methods in Molecular Biology] Calcium Signalling Volume 1925 (Methods and Protocols) || High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels
摘要： Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.
关键词： Cytosol,Calcium probes,Calcium,High-throughput screening,Aequorin,Mitochondria,ER
更新于2025-11-21 11:20:42
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Peptic Fluorescent “Signal-On” and “Signal-Off” Sensors Utilized for the Detection Protein Post-Translational Modifications
摘要： Protein post-translational modifications (PTMs) are typically enzyme-catalyzed events generating functional diversi?cation of proteome; thus, multiple PTM enzymes have been validated as potential drug targets. We have previously introduced energy-transfer-based signal-modulation method called quenching resonance energy transfer (QRET), and utilize it to monitor PTM addition or removal using the developed peptide-break technology. Now we have reinvented the QRET technology, and as a model, we introduced the tunable ?uorescent “signal-on” and “signal-o?” detection scheme in the peptide-break PTM detection. Taking the advantage of time-resolved ?uorescence-based single-label detection technology, we were able to select the signal direction upon PTM addition or removal by simply introducing di?erent soluble Eu3+-signal-modulating molecule. This enables the selection of positive signal change upon measurable event, without any additional labeling steps, changes in assay condition or Eu3+-reporter. The concept functionality was demonstrated with four Eu3+-signal modulators in a high-throughput compatible kinase and phosphatase assays using signal-on and signal-o? readout at 615 nm or time-resolved Fo?rster resonance energy transfer at 665 nm. Our data suggest that the introduced signal modulation methodology provides a transitional ?uorescence-based single-label detection concept not limited only to PTM detection.
关键词： time-resolved fluorescence,signal-off,high-throughput screening,peptide-break technology,protein post-translational modifications,FRET,signal-on
更新于2025-11-19 16:56:35
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Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening
摘要： The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure–activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure–activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.
关键词： c-di-GMP,structure–activity relationships,FRET,diguanylate cyclase inhibitors,high-throughput screening
更新于2025-09-23 15:23:52
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Modification of AlphaLISA Excitation Wavelength Leads to Improved Assay Sensitivity for Photosynthetic Tissue Samples
摘要： For the purposes of high-throughput immunoassay screening, PerkinElmer’s AlphaLISA technology offers many benefits over traditional enzyme-linked immunosorbant assay (ELISA). However, its 680 nm excitation wavelength coincides with a wavelength of peak photosynthetic pigment absorbance, hindering the technology’s utility within the plant biotechnology industry. In assays containing photosynthetic matrices, it is proposed that excitation of chlorophyll leads to the production of singlet oxygen, which initiates a pigment-associated background signal, reducing assay sensitivity. A customized donor bead, modified for excitation outside the range of photosystem absorbance, was tested for its capacity to improve assay sensitivity with extracts containing photosynthetic pigments. In three assays designed against crystalline domain insecticidal protein targets, use of the customized donor bead along with its altered excitation wavelength led to the elimination of pigment-associated signal and improved separation between target-positive and null samples. Reduction in null photosynthetic extract signal led to a 16× sensitivity improvement in a quantitative assay. The customized donor bead was also found to be photostable under ambient laboratory lighting, potentially improving the overall utility of AlphaLISA technology. The customized donor bead enables sensitive, high-throughput immunoassay screening of photosynthetic tissues within the plant biotechnology industry using a convenient, photostable protocol.
关键词： homogenous,HTS,photosynthetic,Alpha,plant,high-throughput screening,automated biology
更新于2025-09-23 15:22:29
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Fluorescence-based high throughput screening technologies for natural chloride ion channel blockers
摘要： Chloride channels represent a group of potential drug targets, their blockers showed significant protecting effect on impaired cells by modulating apoptosis, autophagy and other cell signals. However, clinical drugs with chloride channel inhibitory properties have not yet been developed. Natural product extract becomes an underlying candidate satisfied the clinical requirements for its low toxicity, low cost and abundant sources. Here, a fluorescence-based EYFP-H148Q/I153L-HeLa cell line model was constructed by molecular cloning and verified by Real-time PCR and Western Blotting assay. By using this chloride channel blocker screening model, 7 hit compounds chosen from 6988 natural compounds showed the channel blocking activity. Then the hit compounds were further validated by electrophysiological patch-clamp analysis. Our study preliminarily identified PC-4 as a new chloride channel inhibitors and demonstrated the reliability and sensitivity of fluorescence-based high throughput screening technologies for discovery of biologically active compounds from natural herbal compounds.
关键词： natural compound,high throughput screening,chloride channel blocker,patch clamp
更新于2025-09-23 15:21:21
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Luminescent nanomaterials for droplet tracking in a microfluidic trapping array
摘要： The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped β-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform.
关键词： Rare earth elements,Single-cell analysis,Nanoparticles,Microfluidics,high-throughput screening
更新于2025-09-23 15:21:21
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Discovery of Novel Two-Dimensional Photovoltaic Materials Accelerated by Machine Learning
摘要： Searching for novel, high-performance, two-dimensional photovoltaic (2DPV) materials is an important pursuit for solar cell applications. In this work, an efficient method based on the machine learning algorithm combined with high-throughput screening is developed. Twenty-six 2DPV candidates are successfully ruled out from 187093 experimentally identified inorganic crystal structures, whose conversion efficiencies are predicted by density functional theory calculations. Our results indicate that Sb2Se2Te, Sb2Te3, and Bi2Se3 exhibit conversion efficiencies that are much higher than those of others, which make them promising 2DPV candidates for further applications. The superior photovoltaic performance is then analyzed, and the hidden structure-related relationships with photovoltaic properties are established, thus providing important information for the further examination of 2DPV materials. Given the rapid development of the database of materials, this approach not only provides an efficient way of searching for novel 2DPV materials but also can be applied to exploration of a broad range of functional materials.
关键词： high-throughput screening,machine learning,solar cell applications,density functional theory,two-dimensional photovoltaic materials
更新于2025-09-23 15:19:57
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Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors
摘要： The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor “all” kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.
关键词： high-throughput screening,ATP-binding site,protein kinases,fluorescence polarization,BODIPY ATP-y-S
更新于2025-09-23 15:19:57
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Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry
摘要： Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) – a form of ambient laser desorption ionisation mass spectrometry – to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.
关键词： synthetic biology,metabolic engineering,REIMS,ambient mass spectrometry,high throughput screening
更新于2025-09-19 17:13:59
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Probing Specificity of Protein–Protein Interactions with Chiral Plasmonic Nanostructures
摘要： Protein?protein interactions (PPIs) play a pivotal role in many biological processes. Discriminating functionally important well-defined protein?protein complexes formed by specific interactions from random aggregates produced by nonspecific interactions is therefore a critical capability. While there are many techniques which enable rapid screening of binding affinities in PPIs, there is no generic spectroscopic phenomenon which provides rapid characterization of the structure of protein?protein complexes. In this study we show that chiral plasmonic fields probe the structural order and hence the level of PPI specificity in a model antibody?antigen system. Using surface-immobilized Fab′ fragments of polyclonal rabbit IgG antibodies with high specificity for bovine serum albumin (BSA), we show that chiral plasmonic fields can discriminate between a structurally anisotropic ensemble of BSA-Fab′ complexes and random ovalbumin (OVA)-Fab′ aggregates, demonstrating their potential as the basis of a useful proteomic technology for the initial rapid high-throughput screening of PPIs.
关键词： specificity,Protein?protein interactions,structural order,chiral plasmonic nanostructures,high-throughput screening
更新于2025-09-16 10:30:52