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BioTechniques Jan 1993The number of PCR samples that can be simultaneously processed has been dramatically increased over existing practices by using a new polycarbonate 864-well microwell...
The number of PCR samples that can be simultaneously processed has been dramatically increased over existing practices by using a new polycarbonate 864-well microwell plate and a modified air cycling oven. In thirty 9-min cycles, four plates containing 3455 samples can be amplified in 4.5 h. Amplification is rapid, uniform and reliable from sample to sample and run to run. This PCR method can satisfy the Human Genome Project's need for high-throughput sample analysis using PCR.
Topics: Biotechnology; Evaluation Studies as Topic; Hot Temperature; Polymerase Chain Reaction
PubMed: 8424866
DOI: No ID Found -
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 -
Scientific Reports Jan 2018Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major...
Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.
Topics: Antineoplastic Agents; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Docetaxel; Drug Screening Assays, Antitumor; High-Throughput Screening Assays; Humans; Male; Prostatic Neoplasms; Spheroids, Cellular; Taxoids; Tumor Cells, Cultured
PubMed: 29321576
DOI: 10.1038/s41598-017-18050-1 -
Biosensors & Bioelectronics Nov 2022Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection....
Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-β-D-glucuronide by the β-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h-2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis.
Topics: Biosensing Techniques; Escherichia coli; Escherichia coli Infections; Humans; Luminescence; Microfluidics
PubMed: 35932553
DOI: 10.1016/j.bios.2022.114594 -
Talanta Aug 2014A method for rapid, inexpensive and sensitive simultaneous analysis of glucose, creatinine, triglycerides, total cholesterol and total protein is needed to analyze...
A method for rapid, inexpensive and sensitive simultaneous analysis of glucose, creatinine, triglycerides, total cholesterol and total protein is needed to analyze blood. The proposed method is based on the production of a specific color after reaction. The method was adapted to a 64-microwell plate format, and it uses the transparency scanner feature of a commercially available desktop scanner. Each microwell plate had an 8×8 array of flat-bottomed 250μL microwells, and these microwells were used to simultaneously house the solutions for clinical assay. The scanned image was saved in TIFF format in a portable computer and then processed using a Graphic User Interface (GUI) designed in our laboratory to obtain analytical curves and to automate the mathematics and statistics calculations. This automation improved the analytical frequency of the method. The results showed that it is possible to measure a few microliters of solution with exactitude and precision better than 5.30%. The measured concentration ranges of glucose, triglycerides, creatinine, total cholesterol and total protein were 0.781 to 100, 1.56 to 200, 0.031 to 4.0, 1.56 to 200mg dL(-1) and 0.031 to 4.0g dL(-1), respectively. The limits of detection were 16.2, 51.7, 0.12, 41.5mg dL(-1) and 0.62g dL(-1) for glucose, triglycerides, creatinine, total cholesterol and total protein, respectively. The recoveries were from 98.7% to 101.3% for total cholesterol, 98.7% to 124.9% for triglycerides, 54.2% to 98.3% for total protein, 89.6% to 101% for glucose and 65.7% to 115.4% for creatinine. The results provided by the scanner were compared with those obtained with a commercial photometer and did not show significant differences at a confidence level of 95%. Good results were obtained for the correlation coefficient and Root Mean Square Error of Prediction (RMSEP) values for the five parameters, especially the total cholesterol and creatinine. The RMSEP values for glucose, creatinine, triglyceride, total cholesterol and total protein were 8.05, 0.28, 7.69, 1.41mg dL(-1) and 2.2g dL(-1), respectively.
Topics: Blood Chemical Analysis; Blood Glucose; Blood Proteins; Cholesterol; Colorimetry; Creatinine; Humans; Image Processing, Computer-Assisted; Reproducibility of Results; Signal Processing, Computer-Assisted; Triglycerides
PubMed: 24881545
DOI: 10.1016/j.talanta.2014.03.066 -
Advanced Healthcare Materials Sep 2022For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell...
For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell arrays are commonly anchored in non-oxygen-permeable culture plates, leading to limited oxygen supply for avascular spheroids. Herein, a polydimethylsiloxane (PDMS)-based oxygen-permeable microwell device is introduced for generating highly viable and functional hepatocellular carcinoma (HCC) spheroids. The PDMS sheets at the bottom of the microwell device provide a high flux of oxygen like in vivo neighboring hepatic sinusoids. Owing to the better oxygen supply, the generated HepG2 spheroids are larger in size and exhibit higher viability and proliferation with less cell apoptosis and necrosis. These spheroids also exhibit lower levels of anaerobic cellular respiration and express higher levels of liver-related functions. In anti-cancer drug testing, spheroids cultured in PDMS plates show a significantly stronger resistance against doxorubicin because of the stronger stem-cell and multidrug resistance phenotype. Moreover, higher expression of vascular endothelial growth factor-A produces a stronger angiogenesis capability of the spheroids. Overall, compared to the spheroids cultured in conventional non-oxygen-permeable plates, these spheroids can be used as a more favorable model for early-stage HCCs and be applied in high-throughput anti-cancer drug screening.
Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Culture Techniques; Dimethylpolysiloxanes; Doxorubicin; Humans; Liver Neoplasms; Oxygen; Spheroids, Cellular; Vascular Endothelial Growth Factor A
PubMed: 35841538
DOI: 10.1002/adhm.202200863 -
Archives of Microbiology May 2022The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate...
Microtiter plate with built-in oxygen sensors: a novel approach to investigate the dynamics of Pseudomonas aeruginosa growth suppression in the presence of divalent cations and antibiotics.
The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate reader. This technology is appropriate for investigating the effect of antibiotics on cell kinetics because there is a direct correlation between the latter and the amount of dissolved oxygen in the medium of an assay. In this study, the metabolic activity of the opportunistic human pathogen Pseudomonas aeruginosa PA01 was investigated using the OxoPlate OP96U optical sensor technology. The response of P. aeruginosa to aminoglycoside antibiotics when Caand Mg ions are present in the Evans defined synthetic medium was measured. The results revealed that the effect of antibiotics on P. aeruginosa is influenced by the concentration of divalent cations present in the test medium, although the efficiency of Ca in supressing antibiotic activity was found to be greater than that of Mg. By comparison to tobramycin, the effect of amikacin is largely inhibited by the Caand Mgconcentrations. The study results underscore that the reliability of the observation of growth inhibitors is enhanced by the oxygen consumption measurements. Thus, the OxoPlate OP96U system is proven to be an accurate method to test the effectiveness of antibiotic treatments against P. aeruginosa.
Topics: Anti-Bacterial Agents; Cations, Divalent; Humans; Microbial Sensitivity Tests; Oxygen; Pseudomonas aeruginosa; Reproducibility of Results; Tobramycin
PubMed: 35508818
DOI: 10.1007/s00203-022-02877-y -
ACS Applied Bio Materials Mar 2022We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using...
We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized -isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader. While demonstrating the ability to detect a broad range of relevant alcohols and organic acids, we showed that the response of the films correlated strongly with the octanol-water partition coefficient (log ) of the analyte. Differences in the secretion of ethanol and succinic acid from strains of and , respectively, were further detected via optical characterization of the films. These differences, which in some cases were as low as ∼3 g/L, were confirmed by high-performance liquid chromatography, thereby demonstrating the sensitivity of this approach. Our findings highlight the potential utility of this multiplexed approach for the detection of small organic analytes in complex biological media, which overcomes a major challenge in conventional optical sensing methods.
Topics: Acids; Alcohols; Culture Media; Hydrogels; Octanols; Organic Chemicals
PubMed: 35166523
DOI: 10.1021/acsabm.1c01267 -
Journal of Obstetrics and Gynaecology... Jun 2003Historically, the treatment of severe male factor infertility has relied on donor sperm insemination. A decade ago the option of treating severe male factor infertility... (Review)
Review
Historically, the treatment of severe male factor infertility has relied on donor sperm insemination. A decade ago the option of treating severe male factor infertility with partner sperm became a viable alternative. With the introduction of intracytoplasmic sperm injection (ICSI) in conjunction with in vitro fertilization (IVF), only men who produce no sperm are denied the option of fathering their own children. The use of ICSI has been extended to couples with mild male factors. Despite the known genetic risks (both inherent and de novo) of ICSI to offspring, couples with male factors as part of their infertility problem often prefer ICSI to standard IVF, due to apprehension that their sperm might not otherwise succeed in fertilization. This apprehension would be alleviated if an assay for the egg binding capability of human sperm were available. We examine here the possibility that recombinant human zona pellucida 3 (rec hZP3), the primary sperm receptor sulfoglycoprotein of the egg zona pellucida (ZP), be used as a human ZP surrogate for assessing sperm ability to bind to the ZP. Unlike human eggs, which cannot be obtained for this purpose, rec hZP3 can be produced in quantity. An efficient assay can be established by incubating sperm with rec hZP3 coated to a microwell plate. Infertile men with sperm having ability to bind to rec hZP3 can be advised to select standard IVF or intrauterine insemination, which have fewer genetic and medical risks.
Topics: Adult; Female; Fertilization in Vitro; Humans; Infertility, Male; Male; Pregnancy; Sperm Injections, Intracytoplasmic; Sperm-Ovum Interactions; Spermatozoa; Zona Pellucida
PubMed: 12806448
DOI: 10.1016/s1701-2163(16)30308-5 -
Analytica Chimica Acta Dec 2018A strategy to design an immunoassay based on imprinted proteins to detect a foodborne toxin zearalenone (ZEN) has been proposed. Proteins were used as scaffolds for...
A strategy to design an immunoassay based on imprinted proteins to detect a foodborne toxin zearalenone (ZEN) has been proposed. Proteins were used as scaffolds for generation of binding cavities with a high specificity against ZEN. Different proteins and different bioimprinting approaches were tested. The imprinted proteins primarily prepared in a tube and then immobilized on a microwell plate, and directly prepared in the microwell plate were compared for an application in immunosorbent detection of ZEN in naturally contaminated wheat and maize samples. The assay was combined with a rapid extraction technique. Specific interactions between the imprinted proteins and the target in a μg kg linear range was achieved. As a confirmatory method, liquid chromatography coupled to tandem mass spectrometry was used. The obtained results allow using the developed immunoassay for the analysis of ZEN with a sensitivity matching the maximum permitted level for ZEN in unprocessed cereals established by the European Commission (100 μg kg).
Topics: Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Food Contamination; Molecular Imprinting; Proteins; Tandem Mass Spectrometry; Zearalenone
PubMed: 30327118
DOI: 10.1016/j.aca.2018.07.062