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Pharmaceutics Jan 2021High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing...
High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of was dependent on the plate position of the sample replicates and value. Positive values (57.0 mL/g, coefficients of variation () 8.9%) showed a lower variability compared to negative values (-14.8 mL/g, 13.4%). The variability of was not reduced using more data points (120 vs. 30). A analysis exclusively based on center wells showed a lower (<2%) compared to edge wells (5-12%) or a combination of edge and center wells (2-5%). We present plate designs for analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).
PubMed: 33514069
DOI: 10.3390/pharmaceutics13020172 -
Frontiers in Bioengineering and... 2021Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce...
Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce an electrochemical microwell plate (ec-MP) composed of a 96 electrochemical deepwell plate and a recently developed 96-channel multipotentiostat. Using the ec-MP we investigated the electrochemical and metabolic properties of the EAM models and with acetate and lactate as electron donor combined with an individual genetic analysis of each well. Electrochemical cultivation of pure cultures achieved maximum current densities ( ) and coulombic efficiencies () that were well in line with literature data. The co-cultivation of and led to an increased current density of of 88.57 ± 14.04 µA cm (lactate) and of 99.36 ± 19.12 µA cm (lactate and acetate). Further, a decreased time period of reaching and biphasic current production was revealed and the microbial electrochemical performance could be linked to the shift in the relative abundance.
PubMed: 35242754
DOI: 10.3389/fbioe.2021.821734 -
Lab on a Chip Apr 2024Automated high-throughput liquid handling operations in biolabs necessitate miniaturised and automatised equipment for effective space utilisation and system...
Automated high-throughput liquid handling operations in biolabs necessitate miniaturised and automatised equipment for effective space utilisation and system integration. This paper presents a thermal segment microwell plate control unit designed for enhanced microwell-based experimentation in liquid handling setups. The development of this device stems from the need to move towards geometry standardization and system integration of automated lab equipment. It incorporates features based on Smart Sensor and Sensor 4.0 concepts. An enzymatic activity assay is implemented with the developed device on a liquid handling station, allowing fast characterisation a high-throughput approach. The device outperforms other comparable devices in certain metrics based on automated liquid handling requirements and addresses the needs of future biolabs in automation, especially in high-throughput screening.
PubMed: 38456212
DOI: 10.1039/d3lc00714f -
Biotechnology Letters Nov 2016To compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes. (Comparative Study)
Comparative Study
OBJECTIVES
To compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes.
RESULTS
Pellets cultured in concave microwell plates had a significantly higher level of GAG per DNA content and greater proteoglycan content than those cultured in tubes at day 7 and 14. Three chondrogenic markers, SOX-9, COL2A1 and aggrecan, showed significantly higher expression in pellets cultured in concave microwell plates than those cultured in tubes at day 7 and 14. At day 21, there was not a significant difference in the expression of these markers. COL10A1, the typical hypertrophy marker, was significantly lower in concave microwell plates during the whole culture period. Runx-2, a marker of hypertrophy and osteogenesis, was significantly lower at day 7 in pellets cultured in concave microwell plates than those cultured in tubes.
CONCLUSION
Concave microwell plates provide a convenient and effective tool for the study of in vitro chondrogenesis and may replace the use of propylene culture tube.
Topics: Aged; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Chondrogenesis; DNA; Gene Expression Regulation; Humans; Mesenchymal Stem Cells; Middle Aged
PubMed: 27534541
DOI: 10.1007/s10529-016-2170-8 -
Molecules (Basel, Switzerland) Mar 2023This study describes the development of a one-step microwell spectrofluorimetric assay (MW-SFA) with high sensitivity and throughput for the determination of four...
One-Step Microwell Plate-Based Spectrofluorimetric Assay for Direct Determination of Statins in Bulk Forms and Pharmaceutical Formulations: A Green Eco-Friendly and High-Throughput Analytical Approach.
This study describes the development of a one-step microwell spectrofluorimetric assay (MW-SFA) with high sensitivity and throughput for the determination of four statins in their pharmaceutical and formulations (tablets). These statins were pitavastatin (PIT), fluvastatin (FLU), rosuvastatin (ROS) and atorvastatin (ATO). The MW-SFA involves the measurement of the native fluorescence of the statin aqueous solutions. The assay was conducted in white opaque 96-microwell plates, and the fluorescence intensities of the solutions were measured by using a fluorescence microplate reader. The optimum conditions of the assay were established; under which, linear relationships with good correlation coefficients (0.9991-0.9996) were found between the fluorescence intensity and the concentration of the statin drug in a range of 0.2-200 µg mL with limits of detection in a range of 0.1-4.1 µg mL. The proposed MW-SFA showed high precision, as the values of the relative standard deviations did not exceed 2.5%. The accuracy of the assay was proven by recovery studies, as the recovery values were 99.5-101.4% (±1.4-2.1%). The assay was applied to the determination of the investigated statins in their tablets. The results were statistically compared with those obtained by a reference method and the results proved to have comparable accuracy and precision of both methods, as evidenced by the t- and F-tests, respectively. The green and eco-friendly feature of the proposed assay was assessed by four different metric tools, and all the results proved that the assay meets the requirements of green and eco-friendly analytical approaches. In addition, ever-increasing miniaturization as handling of large numbers of micro-volume samples simultaneously in the proposed assay gave it a high-throughput feature. Therefore, the assay is a valuable tool for the rapid routine application in the pharmaceutical quality control units for the determination of statins.
Topics: Hydroxymethylglutaryl-CoA Reductase Inhibitors; Drug Compounding; Spectrometry, Fluorescence; Tablets
PubMed: 36985779
DOI: 10.3390/molecules28062808 -
SLAS Discovery : Advancing Life... Jan 2017Zebrafish ( Danio rerio) is an important vertebrate model organism in biomedical research, especially suitable for morphological screening due to its transparent body...
Zebrafish ( Danio rerio) is an important vertebrate model organism in biomedical research, especially suitable for morphological screening due to its transparent body during early development. Deep learning has emerged as a dominant paradigm for data analysis and found a number of applications in computer vision and image analysis. Here we demonstrate the potential of a deep learning approach for accurate high-throughput classification of whole-body zebrafish deformations in multifish microwell plates. Deep learning uses the raw image data as an input, without the need of expert knowledge for feature design or optimization of the segmentation parameters. We trained the deep learning classifier on as few as 84 images (before data augmentation) and achieved a classification accuracy of 92.8% on an unseen test data set that is comparable to the previous state of the art (95%) based on user-specified segmentation and deformation metrics. Ablation studies by digitally removing whole fish or parts of the fish from the images revealed that the classifier learned discriminative features from the image foreground, and we observed that the deformations of the head region, rather than the visually apparent bent tail, were more important for good classification performance.
Topics: Animals; Camptothecin; Deep Learning; Neural Networks, Computer; Zebrafish
PubMed: 27613194
DOI: 10.1177/1087057116667894 -
Analytical Methods : Advancing Methods... Dec 2023This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct...
This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct enzyme-linked immunosorbent assay (ELISA) technology. The polyester microplate was designed to contain 96 zones with a 3 mm diameter each, and a volume of 2-3 μL. The experimental conditions including reagent concentration and reaction time were optimized. The microplate image was digitized and analyzed using graphical software. The linear range obtained between protein S concentrations and pixel intensity was 0-10 μg mL, with a correlation coefficient of 0.99 and a limit of detection of 0.44 μg mL. The developed methodology showed satisfactory intraplate and interplate repeatability with RSD values lower than 7.8%. The results achieved through immunoassay performed on polyester microplates were consistent with those of the RT-PCR method and showed a sensitivity of 100% and 90% and specificity of 85.71% and 100% for saliva and nasopharyngeal samples, respectively. The proposed direct immunoassay on polyester microplates emerges as an alternative to conventional immunoassays performed on commercial polystyrene plates, given the low cost of the device, low consumption of samples and reagents, lower waste generation, and shorter analysis time. Moreover, the immunoassay has shown great potential for diagnosing COVID-19 with precision and accuracy.
Topics: Humans; Saliva; Spike Glycoprotein, Coronavirus; Colorimetry; COVID-19; Immunoassay
PubMed: 38073521
DOI: 10.1039/d3ay01755a -
Scientific Reports Mar 2022Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of...
Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of uniform cell clusters can be produced using a conventional microwell plate, numerous obstacles need to be overcome for its clinical use. In this study, we aimed to develop a novel bag culture method for the production of uniform cell clusters on a large scale (10-10 clusters). We prepared small-scale culture bags (< 10 clusters) with microwells at the bottom and optimized the conditions for producing uniform-sized clusters in the bag using undifferentiated induced pluripotent stem cells (iPSCs). Subsequently, we verified the suitability of the bag culture method using iPSC-derived pancreatic islet cells (iPICs) and successfully demonstrate the production of 6.5 × 10 uniform iPIC clusters using a large-scale bag. In addition, we simplified the pre- and post-process of the culture-a degassing process before cell seeding and a cluster harvesting process. In conclusion, compared with conventional methods, the cluster production method using bags exhibits improved scalability, sterility, and operability for both clinical and research use.
Topics: Cell Differentiation; Diabetes Mellitus, Type 1; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells
PubMed: 35338209
DOI: 10.1038/s41598-022-09124-w -
Analytical Chemistry May 2019The microwell plate/microtiter plate is among the most widely used tools in immune assays. In this paper, we report on a sensitive method for enhancing fluorescence...
The microwell plate/microtiter plate is among the most widely used tools in immune assays. In this paper, we report on a sensitive method for enhancing fluorescence emission detection by simply adding several droplets of an immiscible organic compound into the microwells before detection. To prove the concept, human IgA was determined on a microwell plate using this droplet enhanced fluorescence (DEF) detection method. An obvious enhancement in fluorescence was observed. The detection limit (LOD) was about 1/20 times and the sensitivity was 4 times greater than that without droplets. To prove the use of the method for disease diagnosis, the IgG of measles in human plasma was determined using the proposed DEF method. A LOD of around 1/5 times and a sensitivity of 4 times the DEF were easily achieved compared to ELISA with a conventional fluorescence detection.
Topics: Fluorescence; Fluoroimmunoassay; Humans; Immunoglobulin G; Limit of Detection; Measles; Measles virus
PubMed: 30973223
DOI: 10.1021/acs.analchem.8b05668 -
Methods in Molecular Biology (Clifton,... 2021Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual...
Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual sample all at once has been useful in developing a proteomics view of biology. This in turn has enabled the development of disease-associated immunodiagnostic panels for better prognosis and well-being. Moreover, it is well understood that such multiplexing approaches lend themselves to automation, thereby reducing labor while providing the ability to dramatically conserve both reagent and sample all of which will reduce the cost per test. Here we describe various methods to create and use multiplex immunoassays in the wells of microtiter plates or similar formats.
Topics: Animals; Automation, Laboratory; Humans; Immunoenzyme Techniques; Immunologic Tests; Protein Array Analysis; Sensitivity and Specificity
PubMed: 33237405
DOI: 10.1007/978-1-0716-1064-0_2