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Molecules (Basel, Switzerland) May 2023This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three...
Development and Validation of Green and High-Throughput Microwell Spectrophotometric Assay for the Determination of Selective Serotonin Reuptake Inhibitors in Their Pharmaceutical Dosage Forms.
This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three selective serotonin reuptake inhibitors (SSRIs) in their pharmaceutical dosage forms. These SSRIs are fluoxetine, fluvoxamine, and paroxetine, the most prescribed drugs for the treatment of depression. The proposed assay was based on the formation of orange-colored -substituted naphthoquinone derivatives upon the reaction of SSRIs with 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline media. The assay was conducted in 96-microwell assay plates, and the absorbances of the reaction products were measured by a microplate reader at their maximum absorbance wavelengths. The optimum conditions of the reaction were refined and established. Under these conditions, calibration curves were generated, and linear regression equations were computed. The linear relations between the absorbances and drug concentrations were linear with good correlation coefficients (0.9992-0.9997) in the range of 2-80 µg/mL. The assay limits of detection were in the range of 1.5-4.2 µg/mL. The precision was satisfactory as the values of relative standard deviation did not exceed 1.70%. The accuracy of the assay was ≥98.2%. The proposed MW-SPA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms with acceptable accuracy and precision; the label claims were 99.2-100.5% (±0.96-1.35%). The results of the proposed MW-SPA were compared with those of the official/pre-validated assays by statistical analysis with respect to the accuracy (by -test) and precision (by F-test). No significant differences were found between the calculated and theoretical values of the t- and F-tests at the 95% confidence level, proving similar accuracy and precision in the determination of SSRIs by both assays. The greenness of the proposed assay was confirmed by two metric tools. In addition, the assay is characterized with a high-throughput property which enables the simultaneous analysis of many samples in a short time. Therefore, the assay is a valuable tool for rapid routine application in pharmaceutical quality control units for the determination of the investigated SSRIs.
Topics: Selective Serotonin Reuptake Inhibitors; Spectrophotometry; Fluoxetine; Fluvoxamine; Pharmaceutical Preparations
PubMed: 37241961
DOI: 10.3390/molecules28104221 -
ELife Apr 2021Powered by flagella, many bacterial species exhibit collective motion on a solid surface commonly known as swarming. As a natural example of active matter, swarming is...
Powered by flagella, many bacterial species exhibit collective motion on a solid surface commonly known as swarming. As a natural example of active matter, swarming is also an essential biological phenotype associated with virulence, chemotaxis, and host pathogenesis. Physical changes like cell elongation and hyper-flagellation have been shown to accompany the swarming phenotype. Less studied, however, are the contrasts of collective motion between the swarming cells and their counterpart planktonic cells of comparable cell density. Here, we show that confining bacterial movement in circular microwells allows distinguishing bacterial swarming from collective swimming. On a soft agar plate, a novel bacterial strain sp. SM3 in swarming and planktonic states exhibited different motion patterns when confined to circular microwells of a specific range of sizes. When the confinement diameter was between 40 μm and 90 μm, swarming SM3 formed a single-swirl motion pattern in the microwells whereas planktonic SM3 formed multiple swirls. Similar differential behavior is observed across several other species of gram-negative bacteria. We also observed 'rafting behavior' of swarming bacteria upon dilution. We hypothesize that the rafting behavior might account for the motion pattern difference. We were able to predict these experimental features via numerical simulations where swarming cells are modeled with stronger cell-cell alignment interaction. Our experimental design using PDMS microchip disk arrays enabled us to observe bacterial swarming on murine intestinal surface, suggesting a new method for characterizing bacterial swarming under complex environments, such as in polymicrobial niches, and for in vivo swarming exploration.
Topics: Animals; Bacterial Load; Cluster Analysis; Colitis; Computer Simulation; Dextran Sulfate; Disease Models, Animal; Enterobacter; Female; Flagella; Gastrointestinal Microbiome; Host-Pathogen Interactions; Intestinal Mucosa; Mice, Inbred C57BL; Models, Theoretical; Movement; Numerical Analysis, Computer-Assisted; Phenotype; Mice
PubMed: 33884952
DOI: 10.7554/eLife.64176 -
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 -
Advanced Science (Weinheim,... Apr 2024Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls...
Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c-Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate-based systematic evolution of ligands by exponential enrichment (microwell-SELEX), and identify the specific aptamer (MA9C1) against c-Myc. The multifunctional aptamer-based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c-Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c-Myc by the ubiquitin-proteasome system, but also reduces the Max protein, synergistically inhibiting c-Myc transcriptional activity. Combination of the artificial cyclization and anti-PD-L1 aptamer (PA1)-based delivery system, circular PA1-ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c-Myc degrader for the clinic. Therefore, this aptamer-based degrader provides an invaluable potential degrader in drug discovery and anti-tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets.
PubMed: 38682443
DOI: 10.1002/advs.202309639 -
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 -
Journal of Visualized Experiments : JoVE Aug 2023Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the...
Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.
Topics: Saccharomyces cerevisiae; Optogenetics; Saccharomycetales; Automation; High-Throughput Screening Assays
PubMed: 37590537
DOI: 10.3791/65686 -
Journal of Colloid and Interface Science Oct 2022Microscopic self-propelled motors (SPMs) are an area of active research, but very little investigation has been conducted on millimetre-scale or macroscopic SPMs and...
HYPOTHESIS
Microscopic self-propelled motors (SPMs) are an area of active research, but very little investigation has been conducted on millimetre-scale or macroscopic SPMs and exploring their potential in biomedical research. In this study, we tested if 3D reactive inkjet (RIJ) printing could be used for precise fabrication of millimetre-scale self-propelled motors (SPMs) with well-defined shapes from regenerated silk fibroin (RSF) by converting water soluble RSF (silk I) to insoluble silk fibroin (silk II). Secondly, we compared the different propulsion behaviour of the SPMs to put forward the best geometry and propulsion mechanism for potential applications in enhancing the sensitivity of diffusion-rate limited biomedical assays by inducing fluid flow.
EXPERIMENTS
SPMs with four different geometric shapes and propelled by two different mechanisms (catalysis and surface tension gradient) were fabricated by 3D RIJ printing and compared. For bubble propulsion, the structures were selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment in hydrogen peroxide solutions. For surface tension propulsion, PEG-doped structures were propelled through surface tension gradients caused by leaching of PEG surfactant in deionized water.
FINDINGS
The results demonstrated the ability of 3D inkjet printing to fabricate SPMs with desired propulsion mechanism and fine-tune the propulsion by precisely fabricating the different geometric shapes. The resulting 3D structures were capable of generating motion without external actuation, thereby enabling applications in biomedicine such as micro-stirring small fluid volumes to enhance biological assay sensitivity. The surface tension gradient caused by the leaching of surfactant led to faster propulsion velocities with smooth deceleration, whereas, in comparison, catalysis-induced bubble propulsion tended to be jerky and uneven in deceleration, and therefore less suitable for aforementioned applications. Computational fluid dynamic simulations were used to compare the various experimental SPMs ability to enhance mixing when deployed within 96-well plate microwells, to reveal the effect of both SPM shape and motion character on performance, and show viability for small scale mixing applications.
Topics: Fibroins; Printing, Three-Dimensional; Silk; Surface-Active Agents; Water
PubMed: 35576653
DOI: 10.1016/j.jcis.2022.05.011 -
Analytical Chemistry Nov 2022Macromolecular association is crucial to many fields in biomedical sciences, including drug development, gene editing, and diagnostics. In particular, protein-protein...
Macromolecular association is crucial to many fields in biomedical sciences, including drug development, gene editing, and diagnostics. In particular, protein-protein association and dissociation rate constants are typically determined using surface plasmon resonance systems, which require costly instrumentation and cumbersome procedures (e.g., blocking, washing, and separation). Herein, we demonstrate that protein-binding constants can be readily determined using a real-time biosensing platform facilitated by graphene oxide-modified microwell plates and fluorophore-labeled proteins, where the fluorescent probes remain highly fluorescent during protein association, whereas fluorescent bioprobes that are not associated with their counterparts are quenched by graphene oxide. Binding data of three pairs of proteins were systematically determined employing this single-step platform and compared with those data reported by the suppliers or the literature, suggesting that this approach is comparable and consistent with the existing ones. Such pairs include (i) human immunoglobulin G (H-IgG)-fluorophore-labeled anti-H-IgG, (ii) prostate-specific antigen (PSA)-quantum dot-labeled anti-PSA, and (iii) anti-RBD-fluorophore-labeled SARS-CoV-2 spike receptor-binding domain recombinant protein. We also offer an open-source software that automatically determines the binding kinetics constants of proteins. This Technical Note introduces a simple, yet effective, platform to determine relevant information on protein kinetics, which can be performed using a microwell plate reader and economical materials like graphene oxide. We foresee a new generation of diagnostics based on our affordable protein kinetics analysis.
Topics: Humans; Male; COVID-19; SARS-CoV-2; Kinetics; Biosensing Techniques; Fluorescent Dyes; Immunoglobulin G
PubMed: 36253365
DOI: 10.1021/acs.analchem.2c03305 -
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