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Medicina (Kaunas, Lithuania) Feb 2023This study presents the development and validation of the 96-microwell-based spectrofluorimetric (MW-SFL) and high performance liquid chromatography (HPLC) with...
Development of Novel Microwell-Based Spectrofluorimetry and High-Performance Liquid Chromatography with Fluorescence Detection Methods and High Throughput for Quantitation of Alectinib in Bulk Powder and Urine Samples.
This study presents the development and validation of the 96-microwell-based spectrofluorimetric (MW-SFL) and high performance liquid chromatography (HPLC) with fluorescence detection (HPLC-FD) methods for the quantitation of alectinib (ALC) in its bulk powder form and in urine samples. The MW-SFL was based on the enhancement of the native fluorescence of ALC by the formation of micelles with the surfactant cremophor RH 40 (Cr RH 40) in aqueous media. The MW-SFL was executed in a 96-microwell plate and the relative fluorescence intensity (RFI) was recorded by utilizing a fluorescence plate reader at 450 nm after excitation at 280 nm. The HPLC-FD involved the chromatographic separation of ALC and ponatinib (PTB), as an internal standard (IS), on a C column and a mobile phase composed of methanol:potassium dihydrogen phosphate pH 7 (80:20, /) at a flow rate of 2 mL min. The eluted ALC and PTB were detected by utilizing a fluorescence detector set at 365 nm for excitation and 450 nm for emission. Validation of the MW-SFL and HPLC-FD analytical methods was carried out in accordance with the recommendations issued by the International Council for Harmonization (ICH) for the process of validating analytical procedures. Both methods were efficaciously applied for ALC quantitation in its bulk form as well as in spiked urine; the mean recovery values were ≥86.90 and 95.45% for the MW-SFL and HPLC-FD methods, respectively. Both methodologies are valuable for routine use in quality control (QC) laboratories for determination of ALC in pure powder form and in human urine samples.
Topics: Humans; Chromatography, High Pressure Liquid; Powders; Piperidines; Carbazoles
PubMed: 36984441
DOI: 10.3390/medicina59030441 -
Analytical Chemistry Nov 2021Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and...
Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and the Most Probable Number assays analyze such large volumes, while other assays for bacteria detection (ELISA, lateral flow assays, etc.) typically analyze volumes 1000 times smaller. This study describes flow-through direct immunoassays (FTDI), a new methodology for the targeted detection of bacteria in liquid samples of theoretically any volume. Flow-through direct immunoassays are performed in fluid-permeable microwells (e.g., wells of a filter well plate) that have a membrane on their bottom where the bacteria are trapped before their detection using a direct immunoassay. Two versions of FTDI assays for the detection of in 10 mL of sample were developed. A rapid FTDI assay that can be completed in less than 2.5 h can detect bacteria in levels down to 17 CFU/mL, and an ultrasensitive FTDI assay that employs an additional bacteria culturing step to boost the sensitivity can detect bacteria in levels lower than 1 CFU/mL in less than 5.5 h. All the steps of the assays, including the immunoassay steps, the culturing step, and the analytical signal measurement step are performed inside the well plate to decrease the chance of contamination and ensure a safe, easy process for the user. The assays were assessed and validated in tap water, river water, and apple juice samples, and the results suggests that the assays are robust, precise, and accurate. When the assays are performed in 96-well filter plates, a filter well plate vacuum manifold and a multichannel peristaltic pump are also used, so multiple samples can be analyzed in parallel to allow high-throughput analysis of samples.
Topics: Bacteria; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Immunoassay; Malus
PubMed: 34714057
DOI: 10.1021/acs.analchem.1c02867 -
Cellular and Molecular Bioengineering Dec 2022The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling...
OBJECTIVE
The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling these systems up for high-throughput testing is technically challenging and wasteful given the necessary cell numbers and reagent volumes. However, adopting microscale spheroid cultures for this purpose shows promise. Spheroid systems work with only thousands of cells and microliters of medium.
METHODS
Molded agarose microwells were fabricated using 2% w/v molten agarose and then equilibrated in medium prior to introducing cells. ASCs were seeded at 50, 500, 5k cells/microwell; 5k, 50k, cells/well plate; and 50k and 250k cells/15 mL centrifuge tube to compare chondrogenic responses across spheroid and micromass sizes. Cells were cultured in control or chondrogenic induction media. ASCs coalesced into spheroids/pellets and were cultured at 37 °C and 5% CO for 21 days with media changes every other day.
RESULTS
All culture conditions supported growth of ASCs and formation of viable cell spheroids/micromasses. More robust growth was observed in chondrogenic conditions. Sulfated glycosaminoglycans and collagen II, molecules characteristics of chondrogenesis, were prevalent in both 5000-cell spheroids and 250,000-cell micromasses. Deposition of collagen I, characteristic of fibrocartilage, was more prevalent in the large micromasses than small spheroids.
CONCLUSIONS
Chondrogenic differentiation was consistently induced using high-throughput spheroid formats, particularly when seeding at cell densities of 5000 cells/spheroid. This opens possibilities for highly arrayed experiments investigating tissue repair and remodeling during or after exposure to drugs, toxins, or other chemicals.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s12195-022-00746-8.
PubMed: 36531862
DOI: 10.1007/s12195-022-00746-8 -
Data in Brief Aug 2022SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and...
SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.
PubMed: 35789908
DOI: 10.1016/j.dib.2022.108415 -
Communications Biology Nov 2023The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent...
The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
Topics: Optogenetics; Feedback; Escherichia coli; Algorithms; Spectrum Analysis
PubMed: 38001175
DOI: 10.1038/s42003-023-05532-4 -
Lab on a Chip Apr 2024Tumor spheroids are now intensively investigated toward preclinical and clinical applications, necessitating the establishment of accessible and cost-effective methods...
Tumor spheroids are now intensively investigated toward preclinical and clinical applications, necessitating the establishment of accessible and cost-effective methods for routine operations. Without losing the advantage of organ-chip technologies, we developed a rocking system for facile formation and culture of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions. While the rocking is controlled with a step motor, the microfluidic device is made of two plastic plates, allowing plugging directly syringe tubes with Luer connectors. Upon injection of the culture medium into the tubes and subsequent rocking of the chip, the medium flows back and forth in the channel underneath the membrane, ensuring a diffusion-based culture. Our results showed that such a rocking- and diffusion-based culture method significantly improved the quality of the tumor spheroids when compared to the static culture, particularly in terms of growth rate, roundness, junction formation and compactness of the spheroids. Notably, dynamically cultured tumor spheroids showed increased drug resistance, suggesting alternative assay conditions. Overall, the present method is pumpless, connectionless, and user-friendly, thereby facilitating the advancement of tumor-spheroid-based applications.
Topics: Spheroids, Cellular; Humans; Lab-On-A-Chip Devices; Cell Culture Techniques; Diffusion; Microfluidic Analytical Techniques; Hydrogels; Cell Line, Tumor; Tumor Cells, Cultured; Equipment Design
PubMed: 38629978
DOI: 10.1039/d3lc01116j -
The Analyst May 2020We describe a method for the differentiation of carboxylate anions on disposable paper supports (common printer paper, filter paper, chromatography paper), based on...
We describe a method for the differentiation of carboxylate anions on disposable paper supports (common printer paper, filter paper, chromatography paper), based on differential patterns of interactions between carboxylates and a fluorescent sensing system. The sensor was built from commercially available components, namely a polycationic fifth generation amine-terminated poly(amidoamine) dendrimer (PAMAM G5) and a small organic fluorophore (calcein) through non-covalent interactions. The assay's physical dimensions were chosen to conform to the microwell plate standard so detection could be carried out on widely available plate reader instrumentation. The sensing complex was first deposited in spots on a paper support to prepare the sensor strip; a carboxylate solution was then loaded on each spot. Nuanced changes in fluorescence were associated with carboxylate binding to the PAMAM dendrimer, characteristic of the structure and affinity of each carboxylate. Such signal changes, interpreted through Linear Discriminant Analysis (LDA), contained enough information to recognize and successfully discriminate most anions in the panel. Among the substrates we tested, chromatography paper was the most promising. The relationship between the structure of the carboxylates and the patterns giving rise to their differentiation was also discussed. Finally, the long-term stability ("shelf life") of the pre-assembled [calcein·dendrimer] sensing system was found to be excellent when deposited on paper support.
PubMed: 32207473
DOI: 10.1039/d0an00137f -
Methods in Molecular Biology (Clifton,... 2021Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions...
Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions is crucial for delineating the mechanisms governing transcription, genome duplication, and translation. Here we describe a detailed protocol for the quantitative analysis of protein-nucleic acid interactions based on protein-induced fluorescence enhancement (PIFE). While PIFE has mainly been used in single-molecule studies, we modified its application for bulk measurement of protein-nucleic acid interactions in microwell plates using standard fluorescent plate readers. The microwell plate PIFE assay (mwPIFE) is simple, does not require laborious protein labeling, and is high throughput. These properties predispose mwPIFE to become a method of choice for routine applications that require multiple parallel measurements such as buffer optimization, competition experiments, or screening chemical libraries for binding modulators.
Topics: DNA; Microscopy, Fluorescence; Nucleoproteins; Protein Binding; Proteins; RNA
PubMed: 33201465
DOI: 10.1007/978-1-0716-0935-4_7 -
Nature Communications Dec 2022The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular...
The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.
Topics: Biomolecular Condensates; Intracellular Space; Microfluidics; Phase Transition
PubMed: 36543777
DOI: 10.1038/s41467-022-35265-7 -
Open Research Europe 2022Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology...
Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid-structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures.
PubMed: 37645341
DOI: 10.12688/openreseurope.14894.2