Facilitated crystal handling using a simple device for evaporation reduction in microtiter plates.
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ABSTRACT:
SUBMITTER: Barthel T
PROVIDER: S-EPMC7941301 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Formulation screening for biotherapeutics can cover a vast array of excipients and stress conditions. These studies consume quantities of limited material and, with higher concentrated therapeutics, more material is needed. Here, we evaluate the use of crystal zenith (CZ) microtiter plates in conjunction with FluoroTec-coated butyl rubber mats as a small-volume, high-throughput system for formulation stability studies. The system was studied for evaporation, edge effects, and stability with comparisons to type 1 glass and CZ vials for multiple antibodies and formulations. Evaporation was minimal at 4°C and could be reduced at elevated temperatures using sealed, mylar bags. Edge effects were not observed until 12 weeks at 40°C. The overall stability ranking as measured by the rate of change in high molecular weight and percent main peak species was comparable across both vials and plates at 4°C and 40°C out to 12 weeks. Product quality attributes as measured by the multi-attribute method were also comparable across all containers for each molecule formulation. A potential difference was measured for subvisible particle analysis, with the plates measuring lower particle counts than the vials. Overall, CZ plates are a viable alternative to traditional vials for small-volume, high-throughput formulation stability screening studies.
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Project description:Interfacial water evaporation technology by using solar energy provides one of the promising pathways for freshwater shortage management. However, current research mainly focuses on the improvement of evaporation efficiency by macro or microregulations, ignoring the steam temperature, which is a manifestation of the quality of water. Herein not only is a high-rate solar evaporation achieved but also steam temperature is enhanced by a simple three-tier (wet absorber-air gap-dry absorber) device. In a routine interfacial evaporation test, the evaporator achieves a stable evaporation rate up to 2.15 kg m-2 h-1 under one sun, demonstrating a competitive evaporation rate compared with other reports. With the three-tier device, the steam temperature can increase 33.7%, 41.13%, and 47% without dry absorber under one sun, two sun, and three sun illumination, respectively. At the same time, the steam temperature can be as high as 95.5 °C under three sun intensities. This work provides the possibility of using a simple three-tier device for high-temperature steam generation without extra energy input, which contributes to an idea for future research on the production high-quality water.
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Project description:BackgroundLight transmission aggregometry (LTA) can be performed with microtiter plates (96-well LTA). When conducting LTA, an agonist is added to platelet-rich plasma and the sample is shaken for minutes after which absorbance readings are done. Platelet aggregation is detected as decrease in absorbance. However, the classical method is cumbersome and therefore microtiter plates can be used for concomitant testing of multiple samples. Furthermore, it would be convenient to prepare the plate in advance of platelet aggregation testing. Aim: The aim of the present study was to establish a simplified 96-well LTA protocol, where plates were pre-coated with agonists and stored at -80 C until use.ResultsWe developed and validated a protocol for 96-well LTA using a Victor X5 plate reader and pre-coated microtiter plates. The minimum requirement of platelet-rich plasma was 45 ?L per sample and the sample platelet count should not be below 100 x109/L. Optimal absorbance reading was 595 nm wavelengths. Platelet aggregation results were higher at 37°C than at room temperature. Platelet adherence to wells after stimulation was observed; it was not avoided by pre-coating of the wells with gelatin. A range of up to 7 concentrations for each agonist (collagen, arachidonic acid, adenosine diphosphate, thrombin receptor-activating peptide and protease-activated receptor-4) was tested concomitantly. A transient rise in platelet aggregation was observed after 2 minutes of shaking in some samples with low agonist concentration, and platelet aggregation was optimal after 10 minutes of shaking for samples with high agonist concentration. Plates could be stored at -80°C for 15 days without significant change in the platelet aggregation results.ConclusionThe 96-well LTA is suitable for platelet aggregation testing and a range of agonist concentrations can be concomitantly tested.
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Project description:Droplet-based microfluidic screening techniques can benefit from interfacing established microtiter plate-based screening and sample management workflows. Interfacing tools are required both for loading preconfigured microtiter-plate (MTP)-based sample collections into droplets and for dispensing the used droplets samples back into MTPs for subsequent storage or further processing. Here, we present a collection of Digital Microfluidic Pipetting Tips (DMPTs) with integrated facilities for droplet generation and manipulation together with a robotic system for its operation. This combination serves as a bidirectional sampling interface for sample transfer from wells into droplets (w2d) and vice versa droplets into wells (d2w). The DMPT were designed to fit into 96-deep-well MTPs and prepared from glass by means of microsystems technology. The aspirated samples are converted into the channel-confined droplets' sequences separated by an immiscible carrier medium. To comply with the demands of dose-response assays, up to three additional assay compound solutions can be added to the sample droplets. To enable different procedural assay protocols, four different DMPT variants were made. In this way, droplet series with gradually changing composition can be generated for, e.g., 2D screening purposes. The developed DMPT and their common fluidic connector are described here. To handle the opposite transfer d2w, a robotic transfer system was set up and is described briefly.
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Project description:We have developed a methodology that enables for the rapid measurement of ionization constants (pK a) of series of compounds by UV spectrophotometry. This protocol, which is straightforward to set up, takes advantage of the sensitivity of UV spectroscopy and the throughput enabled by the 96-well microplate (as opposed to the use of 1 cm quartz cuvette). The compounds, in stock solutions in DMSO, are dissolved in several aqueous buffer solutions directly in the microtiter plate, allowing the simultaneous determination of the UV spectra as a function of pH. Further treatment of the data provides the pK a values in a medium-throughput manner. The pK a values of 11 new antitrypanosomal dibasic compounds were determined using this methodology.
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Project description:Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.
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Project description:Microtiter plate-based bacterial biofilm assay is frequently used to study bacterial biofilm development and growth. While this assay is simple and relatively high-throughput, it frequently shows difficulty in establishing robust biofilm attachment in the wells. We report that the consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a Pseudomonas fluorescens Pf0-1 model. Scanning electron microscopy imaging suggests that the treated surfaces could provide points of attachment to facilitate the recruitment of bacteria in the initial phase of biofilm colony establishment. The sandblast treated polypropylene, but not polystyrene, plates were found suitable in studying the impact of flavonoid quercetin on the biofilm formation in Bacillus subtilis FB17. Further investigation revealed that due to the hydrophobicity of the polystyrene surfaces, a greater amount of quercetin was adsorbed on the plate surface, effectively lowering the concentration of the flavonoid in solution.
| S-EPMC9086168 | biostudies-literature
Project description:Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of captured protein from sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g. immunoassays) but hardly ever prior to LC-MS-based detection, and here we describe some applications, opportunities, and challenges of corresponding workflows.
2021-11-25 | PXD027530 | Pride
Project description:Extracellular vesicles (EVs) are promising biomarkers for several diseases, however, no simple and robust methods exist to characterize EVs in a clinical setting. The EV Array analysis is based on a protein microarray platform, where antibodies are printed onto a solid surface that enables the capture of small EVs (sEVs) by their surface or surface-associated proteins. The EV Array analysis was transferred to an easily handled microtiter plate (MTP) format and a range of optimization experiments were performed within this study. The optimization was performed in a comprehensive analytical setup where the focus was on the selection of additives added to spotting-, blocking-, and incubation buffers as well as the storage of printed antibody arrays under different temperatures from one day to 12 weeks. After ending the analysis, the stability of the fluorescent signal was investigated at different storage conditions for up to eight weeks. The various parameters and conditions tested within this study were shown to have a high influence on each other. The reactivity of the spots was found to be preserved for up to 12 weeks when stored at room temperature and using blocking procedure IV in combination with trehalose in the spotting buffer. Similar preservation could be obtained using glycerol or sciSPOT D1 in the spotting buffers, but only if stored at 4 °C after blocking procedure I. Conclusively, it was found that immediate scanning of the MTPs after analysis was not critical if stored dried, in the dark, and at room temperature. The findings in this study highlight the necessity of performing optimization experiments when transferring an established analysis to a new technological platform.
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Project description:Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development.
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