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ACS Nano Aug 2023A high-efficiency drug screening method is urgently needed due to the expanding number of potential targets and the extremely long time required to assess them. To date,...
A high-efficiency drug screening method is urgently needed due to the expanding number of potential targets and the extremely long time required to assess them. To date, high throughput and high content have not been successfully combined in image-based drug screening, which is the main obstacle to improve the efficiency. Here, we establish a high-throughput and high-content drug screening method by preparing a superhydrophobic microwell array plate (SMAP) and combining it with protein-retention expansion microscopy (proExM). Primarily, we described a flexible method to prepare the SMAP based on photolithography. Cells were cultured in the SMAP and treated with different drugs using a microcolumn-microwell sandwiching technology. After drug treatment, proExM was applied to realize super-resolution imaging. As a demonstration, a 7 × 7 image array of microtubules was successfully collected within 3 h with 68 nm resolution using this method. Qualitative and quantitative analyses of microtubule and mitochondria morphological changes after drug treatment suggested that more details were revealed after applying proExM, demonstrating the successful combination of high throughput and high content.
Topics: Microscopy; Drug Evaluation, Preclinical; Microtubules; High-Throughput Screening Assays
PubMed: 37548636
DOI: 10.1021/acsnano.3c01865 -
Analytical and Bioanalytical Chemistry Oct 2023A novel method for direct high-throughput analysis of multi-elements in cerebrospinal fluid (CSF) samples by laser ablation inductively coupled plasma mass spectrometry...
A novel method for direct high-throughput analysis of multi-elements in cerebrospinal fluid (CSF) samples by laser ablation inductively coupled plasma mass spectrometry with an aerosol local extraction cryogenic ablation cell (ALEC-LA-ICP-MS) was developed. Microliter-level CSF samples were frozen by a designed cryogenic ablation cell and directly analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) without time-consuming pretreatment. Compared with the precision obtained at room temperature (20℃), that obtained at low temperature (- 25℃) was significantly improved; the RSDs were reduced from 8.3% (Zn) to 32.6% (Mn) to 2.2% (Pb) to 6.5% (Mn) with six times parallel determination. To meet the analytical requirement of the micro-volume CSF samples, the laminar flow aerosol local extraction strategy was adopted to improve the transmission efficiency of aerosols, and the signal intensity was increased by four times compared with the standard commercial ablation cell. The standard solution with 0.4% bovine serum albumin (BSA) matrix was used as matrix-match external standard, and Rh was added into the samples as internal standard. The limits of detection (LODs) ranged from 0.17 μg·L (Mn) to 8.67 μg·L (Mg). Standard addition recovery experiments and the determination of CRM serum L-1 and L-2 were carried out to validate the accuracy of the method; all results indicated there were excellent accuracy and precision in the proposed method. The matrix-scanning function in the GeoLas software combined with the microwell plate realizes the high-throughput automatic analysis. Twenty-four CSF samples from different patients were determined; the results showed that there might be a correlation between the metal elements in CSF and the diseases, which means that the proposed method has potential in the diagnosis of neurological diseases.
PubMed: 37541973
DOI: 10.1007/s00216-023-04878-2 -
Small (Weinheim An Der Bergstrasse,... Nov 2023Droplet array is widely applied in single cell analysis, drug screening, protein crystallization, etc. This work proposes and validates a method for rapid formation of...
Droplet array is widely applied in single cell analysis, drug screening, protein crystallization, etc. This work proposes and validates a method for rapid formation of uniform droplet array based on microwell confined droplets electro-coalescence of screen-printed emulsion droplets, namely electro-coalescence droplet array (ECDA). The electro-coalescence of droplets is according to the polarization induced electrostatic and dielectrophoretic forces, and the dielectrowetting effect. The photolithographically fabricated microwells are highly regular and reproducible, ensuring identical volume and physical confinement to achieve uniform droplet array, and meanwhile the microwell isolation protects the paired water droplets from further fusion and broadens its feasibility to different fluidic systems. Under optimized conditions, a droplet array with an average diameter of 85 µm and a throughput of 10 in a 10 cm × 10 cm chip can be achieved within 5 s at 120 Vpp and 50 kHz. This ECDA chip is validated for various microwell geometries and functional materials. The optimized ECDA are successfully applied for digital viable bacteria counting, showing comparable results to the plate culture counting. Such an ECDA chip, as a digitizable and high-throughput platform, presents excellent potential for high-throughput screening, analysis, absolute quantification, etc.
PubMed: 37449335
DOI: 10.1002/smll.202302998 -
Molecules (Basel, Switzerland) Jul 2023In this study, a new green microwell spectrofluorimetric assay (MW-SFA) with high throughput was developed and validated, for the first time, for the determination of...
Development of a Green Microwell Spectrofluorimetric Assay with High Analytical Throughput for the Determination of Selective Serotonin Reuptake Inhibitors in Pharmaceutical Dosage Forms and Plasma.
In this study, a new green microwell spectrofluorimetric assay (MW-SFA) with high throughput was developed and validated, for the first time, for the determination of three selective serotonin reuptake inhibitors (SSRIs) in pharmaceutical dosage forms and plasma. These SSRIs were fluoxetine (FLX), fluvoxamine (FXM), and paroxetine (PXT), which are commonly prescribed drugs for depression treatment. The MW-SFA is based on the condensation reaction of SSRIs with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in alkaline media to form highly fluorescent derivatives. The MW-SFA procedures were conducted in 96-microwell white opaque assay plates with a flat bottom and the fluorescence signals were measured using a microplate reader at their maximum excitation and emission wavelengths. The calibration curves were generated with good correlation coefficients (0.9992-0.9995) between the relative fluorescence intensity (RFI) and the SSRI concentrations in the range of 35-800 ng/mL. The limits of detection were in the range of 11-25 ng/mL, and the precision and accuracy were satisfactory. The proposed MW-SFA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms. The statistical analysis for the comparison between the MW-SFA assay results and those of pharmacopeial assays showed no significant differences between the assays in terms of their accuracy and precision. The application of the proposed MW-SFA was extended to successfully analyze SSRIs in plasma samples. The greenness of the assay was confirmed using three different metric tools. The assay was characterized with high throughput properties, enabling the sensitive simultaneous analysis of many samples in a short time. This assay is valuable for rapid routine applications in pharmaceutical quality control units and clinical laboratories for the determination of SSRIs.
Topics: Selective Serotonin Reuptake Inhibitors; Spectrometry, Fluorescence; Fluvoxamine; Plasma; Pharmaceutical Preparations
PubMed: 37446883
DOI: 10.3390/molecules28135221 -
Methods in Molecular Biology (Clifton,... 2023High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems....
High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. Here we describe a modular collection of assays adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. These include protocols for RNA fluorescent in situ hybridization (RNA FISH) as well as immunocytochemical protocols for quantifying proliferating cells targeting phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine (BrdU) incorporated into the nuclear DNA. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single-cell suspension before fixation and staining. By sharing many reagents with established planarian whole-mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment.
Topics: Animals; In Situ Hybridization, Fluorescence; RNA; Planarians; Immunohistochemistry; In Situ Hybridization; Microscopy, Fluorescence; Bromodeoxyuridine
PubMed: 37428375
DOI: 10.1007/978-1-0716-3275-8_8 -
Journal of Visualized Experiments : JoVE Jun 2023Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment....
Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 µm diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.
Topics: Humans; Pluripotent Stem Cells; Islets of Langerhans; Cell Differentiation; Insulin-Secreting Cells; Insulins
PubMed: 37427943
DOI: 10.3791/64840 -
Biomaterials Oct 2023Reprogramming of somatic cells into the pluripotent state is stochastic and inefficient using the conventional culture plates. Novel micro-culture systems employing...
Reprogramming of somatic cells into the pluripotent state is stochastic and inefficient using the conventional culture plates. Novel micro-culture systems employing precisely controlled biophysical cues can improve the reprogramming efficiencies dramatically. Here we perform iPSC induction on our previously developed superhydrophobic microwell array chip (SMAR-chip) where cells undergo distinctive morphology change, switching from 2D monolayers to 3D clumps, and develop into bona fide colonies in more than 90% of the microwells. The PDMS substrate, together with the microwell structure and the superhydrophobic layer constitute a well-controlled microenvironment favorable for the morphogenesis and pluripotency induction. Investigation of the molecular roadmap demonstrates that the SMAR-chip promotes the transition from the initiation phase to the maturation phase and overcomes the roadblocks for reprogramming. In addition, the SMAR-chip also promotes the reprogramming of human cells, opening our method for translational applications. In summary, our study provides a novel platform for efficient cell reprogramming and emphasizes the advantages of employing the insoluble microenvironmental cues for the precise control of cell fate conversion.
Topics: Humans; Cellular Reprogramming; Cell Differentiation; Induced Pluripotent Stem Cells; Hydrophobic and Hydrophilic Interactions
PubMed: 37406601
DOI: 10.1016/j.biomaterials.2023.122215 -
Heliyon Jun 2023Durvalumab (DUR) is a human monoclonal antibody used for the immunotherapy of lung cancer. It is a novel immune-checkpoint inhibitor, which blocks the programmed death 1...
Development of a highly sensitive chemiluminescence immunoassay using a novel signal-enhanced detection system for quantitation of durvalumab, an immune-checkpoint inhibitor monoclonal antibody used for immunotherapy of lung cancer.
Durvalumab (DUR) is a human monoclonal antibody used for the immunotherapy of lung cancer. It is a novel immune-checkpoint inhibitor, which blocks the programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) proteins and works to promote the normal immune responses that attack the tumour cells. To support the pharmacokinetic (PK) studies, therapeutic drug monitoring (TDM) and refining the safety profile of DUR, an efficient assay is required, preferably immunoassay. This study describes, for the first time, the development of a highly sensitive chemiluminescence immunoassay (CLIA) for the quantitation of DUR in plasma samples with enhanced chemiluminescence detection system. The CLIA protocol was conducted in 96-microwell plates and involved the non-competitive binding reaction of DUR to its specific antigen (PD-L1 protein). The immune complex of DUR with PD-L1 formed onto the inner surface of the assay plate wells was quantified by a chemiluminescence (CL)-producing horseradish peroxidase (HRP) reaction. The reaction employed 4-(1,2,4-triazol-1-yl)phenol (TRP) as an efficient enhancer of the HRP-luminol-hydrogen peroxide (HO) CL reaction. The optimum protocol of the proposed CLIA was established, and its validation parameters were assessed as per the guidelines for the validation of immunoassays for bioanalysis. The working dynamic range of the assay was 10-800 pg mL with a limit of detection (LOD) of 10.3 pg mL. The assay enables the accurate and precise quantitation of DUR in human plasma at a concentration as low as 30.8 pg mL. The CLIA protocol is simple and convenient; an analyst can analyse several hundreds of samples per working day. This high throughput property enables the processing of many samples in clinical settings. The proposed CLIA has a significant benefit in the quantitation of DUR in clinical settings for assessment of its PK, TDM and refining the safety profile.
PubMed: 37389074
DOI: 10.1016/j.heliyon.2023.e15782 -
TheScientificWorldJournal 2023There is an increase in mortality and morbidity in the health facilities due to nosocomial infections caused by multidrug-resistant nosocomial bacteria; hence, there is...
There is an increase in mortality and morbidity in the health facilities due to nosocomial infections caused by multidrug-resistant nosocomial bacteria; hence, there is a need for new antibacterial agents. has been found to possess medicinal value. Plant phytochemicals may have antimicrobial activity against some resistant pathogens. The antibacterial efficacy of root extracts against and was investigated using the microbroth dilution method. All extracts from the roots had an inhibitory effect on the growth of both bacteria, with the most susceptible being . The most potent extract was the ethyl acetate extract which caused a percentage inhibition of 86% against . The toxicity of the extract was determined on sheep erythrocytes, and its effect on membrane integrity was determined by quantifying the amount of protein and nucleic acid leakage from the bacteria. The lowest concentration of extract used, which was 100 g/ml, did not cause haemolysis of the erythrocytes, while at 1 mg/ml of the extract, 21% haemolysis was observed. The ethyl acetate extract caused membrane impairment of leading to protein leakage. The effect of the extract on the biofilms of was determined in 96-microwell plates using crystal violet. In the concentration range of 0-100 g/ml, the extract inhibited the formation of biofilms and decreased the attachment efficiency. The phytochemical constituents of the extract were determined using gas chromatography-mass spectrometry. Results of analysis showed the presence of 3-methylene-15-methoxy pentadecanol, 2-acetyl-6-(t-butyl)-4-methylphenol, 2-(2,2,3,3-tetrafluoropropanoyl) cyclohexane-1,4-dione, E,E,Z-1,3,12-nonadecatriene-5,14-diol, and stigmasta-5,22-dien-3-ol. Fractionation and purification will elucidate the potential antimicrobial compounds which are present in the roots of .
Topics: Animals; Sheep; Pseudomonas aeruginosa; Vernonia; Hemolysis; Plant Extracts; Microbial Sensitivity Tests; Anti-Bacterial Agents; Anti-Infective Agents; Bacteria; Biofilms; Phytochemicals
PubMed: 37324654
DOI: 10.1155/2023/5782656 -
Methods in Molecular Biology (Clifton,... 2023The field of oncology increasingly focuses on strategies to predict effectiveness of a given therapy on a patient-by-patient basis. Such precision or personalized...
The field of oncology increasingly focuses on strategies to predict effectiveness of a given therapy on a patient-by-patient basis. Such precision or personalized oncology has the potential of significantly extending patient survival time. Patient-derived organoids are seen as the main source of patient tumor tissue that may be used for therapy testing in personalized oncology. The gold standard approach for culturing cancer organoids is in standard multi-well plates coated with Matrigel. Despite their effectiveness, these standard organoid cultures have drawbacks, namely, requirement of a large starting cell population and polydispersity of cancer organoid sizes. The latter drawback makes it challenging to monitor and quantify changes in organoid size in response to therapy. Microfluidic devices with integrated arrays of microwells may be used to both decrease the amount of starting cellular material required to form organoids and to standardize organoid size to make therapy assessment easier. Herein, we describe methodology for making microfluidic device as well as for seeding patient-derived cancer cells, culturing organoids, and testing therapies using these devices.
Topics: Humans; Microfluidics; Neoplasms; Precision Medicine; Organoids
PubMed: 37300619
DOI: 10.1007/978-1-0716-3271-0_15