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PloS One 2012Hybrid combinatorial chemistry strategies that use DNA as an information-carrying medium are proving to be powerful tools for molecular discovery. In order to extend...
Hybrid combinatorial chemistry strategies that use DNA as an information-carrying medium are proving to be powerful tools for molecular discovery. In order to extend these efforts, we present a highly parallel format for DNA-programmed chemical library synthesis. The new format uses a standard microwell plate footprint and is compatible with commercially available automation technology. It can accommodate a wide variety of combinatorial synthetic schemes with up to 384 different building blocks per chemical step. We demonstrate that fluidic routing of DNA populations in the highly parallel format occurs with excellent specificity, and that chemistry on DNA arrayed into 384 well plates proceeds robustly, two requirements for the high-fidelity translation and efficient in vitro evolution of small molecules.
Topics: Blotting, Southern; Combinatorial Chemistry Techniques; DNA; Gene Library; Nucleic Acid Hybridization; Reproducibility of Results; Small Molecule Libraries
PubMed: 22479318
DOI: 10.1371/journal.pone.0032299 -
Communications Biology Jul 2022Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are...
Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are incompatible with indiscriminate single cell sequencing because they are rare, transient, or can only be identified by imaging. Existing methods for isolating cells based on imaging for single cell sequencing are technically challenging, time-consuming, and prone to loss because of the need to physically transport single cells. Here, we developed See-N-Seq, a method to rapidly screen cells in microwell plates in order to isolate RNA from specific single cells without needing to physically extract each cell. Our approach involves encapsulating the cell sample in a micropatterned hydrogel with spatially varying porosity to selectively expose specific cells for targeted RNA extraction. Extracted RNA can then be captured, barcoded, reverse transcribed, amplified, and sequenced at high-depth. We used See-N-Seq to isolate and sequence RNA from cell-cell conjugates forming an immunological synapse between T-cells and antigen presenting cells. In the hours after synapsing, we found time-dependent bifurcation of single cell transcriptomic profiles towards Type 1 and Type 2 helper T-cells lineages. Our results demonstrate how See-N-Seq can be used to associate transcriptomic data with specific functions and behaviors in single cells.
Topics: High-Throughput Nucleotide Sequencing; Hydrogels; Microscopy; Porosity; RNA; Sequence Analysis, RNA
PubMed: 35908100
DOI: 10.1038/s42003-022-03703-3 -
Scientific Reports Oct 2020The enzyme-linked immunosorbent assay (ELISA) is widely used in various fields to detect specific biomarkers. However, ELISA tests have limited detection sensitivity...
The enzyme-linked immunosorbent assay (ELISA) is widely used in various fields to detect specific biomarkers. However, ELISA tests have limited detection sensitivity (≥ 1 pM), which is insufficiently sensitive for the detection of small amounts of biomarkers in the early stages of disease or infection. Herein, a method for the rapid and highly sensitive detection of specific antigens, using temperature-responsive liposomes (TLip) containing a squaraine dye that exhibits fluorescence at the phase transition temperature of the liposomes, was developed. A proof-of-concept study using biotinylated TLip and a streptavidin-immobilized microwell plate showed that the TLip bound to the plate via specific molecular recognition could be distinguished from unbound TLip within 1 min because of the difference in the heating time required for the fluorescence emission of TLip. This system could be used to detect prostate specific antigen (PSA) based on a sandwich immunosorbent assay using detection and capture antibodies, in which the limit of detection was as low as 27.6 ag/mL in a 100-μL PSA solution, 0.97 aM in terms of molar concentration. The present temperature-responsive liposome-linked immunosorbent assay provides an advanced platform for the rapid and highly sensitive detection of biomarkers for use in diagnosis and biological inspections.
Topics: Antibodies; Biomarkers; Enzyme-Linked Immunosorbent Assay; Humans; Immunosorbents; Limit of Detection; Liposomes; Prostate-Specific Antigen; Temperature
PubMed: 33093468
DOI: 10.1038/s41598-020-75011-x -
Molecules (Basel, Switzerland) Jul 2022A sustainable downscaled procedure using smartphone-based colorimetric determination of manganese (Mn(II)) was developed. This novel Mn(II) determination procedure is...
Sustainable Downscaled Catalytic Colorimetric Determination of Manganese in Freshwater Using Smartphone-Based Monitoring Oxidation of 3,3',5,5'-Tetramethylbenzidine by Periodate.
A sustainable downscaled procedure using smartphone-based colorimetric determination of manganese (Mn(II)) was developed. This novel Mn(II) determination procedure is proposed using a simple, available microwell-plate platform and a smartphone as a detector. This approach is based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by periodate using Mn(II) as a catalyst. The catalytic kinetics of Mn(II) under different conditions was investigated to determine the optimum condition where the different catalytic activities of various concentrations of Mn(II) evince. Under the optimum condition, the bluish-green product of oxidized TMB, proportioned to the concentration of Mn(II), was monitored using a smartphone camera, and the color signals were processed using ImageJ Software. The developed procedure showed great selectivity and sensitivity as linearity ranged from 1.8 × 10 to 4.6 × 10 M (0.1 to 2.5 μg/mL). The limits of detection and quantitation were 3.6 × 10 and 1.1 × 10 M (0.2 and 0.6 μg/mL), respectively. The determination of Mn(II) in freshwater samples was demonstrated to assess environmental water quality as an initial model to more easily promote water management according to the United Nations Sustainable Development Goals (UN-SDGs). The intensity of the red could be successfully applied to evaluate Mn(II) in canals and river water with no significant differences compared with the reference method of Inductively Coupled Plasma Optical Emission Spectrometry at a confidence level of 95%.
Topics: Benzidines; Colorimetry; Fresh Water; Manganese; Periodic Acid; Smartphone
PubMed: 35956794
DOI: 10.3390/molecules27154841 -
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 -
PloS One 2021To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In...
To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100μm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.
Topics: Antineoplastic Agents; Astrocytes; Cells, Cultured; Drug Screening Assays, Antitumor; Glioblastoma; High-Throughput Screening Assays; Humans; Primary Cell Culture; Spheroids, Cellular
PubMed: 34855773
DOI: 10.1371/journal.pone.0251998 -
Bioactive Materials Sep 2022While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic...
While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D. Herein, we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip. The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers. To resolve the problem, we designed and assembled a fast aligner that is cost-effective and convenient to operate. To enable quasi-3D microenvironment of cells, we fabricated polydimethylsiloxane (PDMS) microwell arrays (formed by micropillars of a few microns in diameter) with the microwell diameters close to the cell sizes. An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS. We examined three types of cells in 2D, quasi-3D, and 3D microenvironments; the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells. We also constructed transgenic human mesenchymal stem cells (hMSCs); under cyclic stretching, the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate. This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies, but also inspires further studies of the topological effects of biomaterials on cells.
PubMed: 35356817
DOI: 10.1016/j.bioactmat.2021.12.010 -
Journal of Analytical Methods in... 2013A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal...
A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25-50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40 μ L. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.
PubMed: 24000318
DOI: 10.1155/2013/584964 -
Frontiers in Veterinary Science 2022The Chinese traditional medicinal plants , and in a ratio of 108:65:27 form a compound named Dahuang Qinyu San (DQS), which inhibits and kills and to a certain extent...
The Chinese traditional medicinal plants , and in a ratio of 108:65:27 form a compound named Dahuang Qinyu San (DQS), which inhibits and kills and to a certain extent in fish and shrimp aquaculture environments. The active ingredients quercetin, emodin, baicalin, and aloe-emodin are obtained from the semi-biomimetic extract of DQS (SEDQS). However, the antibacterial mechanism of SEDQS against is still unclear. This study used the microwell-plate method to determine the Minimum Inhibitory Concentration (MIC) of SEDQS against () isolated from geese. In addition, the effect of SEDQS on the growth curve, respiratory metabolic system, cell wall, soluble protein, and nucleic acid in bacterial liquid of was detected by spectrophotometer and reagent kit. The effects of SEDQS on DNA, binding gel blocking, virulence gene expression, and pathogenicity-related proteins were determined by gel electrophoresis, SDS-PAGE, and fluorescence quantitative PCR. The study found that a concentration of 1/4 MIC-2 MIC (2.27-18.2 mg/ml) SEDQS can significantly inhibit the normal growth of , destroy the cell membrane structure of bacteria resulting in the leak of nucleic acid, protein, and other contents ( < 0.01). It also significantly inhibited the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH; < 0.01) in a concentration-dependent manner. When the concentration of SEDQS was 1/2 MIC to 2 MIC (4.55-18.2 mg/ml), the expression levels of , and virulence genes ( < 0.01) all decreased by more than 31, 11, 18, 30, 34, and 21% respectively compared with the control group. SEDQS could significantly inhibit the expression of six virulence genes and play an important role in the pathogenicity of infected host. The SEDQS could exert antibacterial pharmacological effects by inhibiting the growth and metabolism of and inhibiting the expression of major virulence factors. It has potential application value as an antibiotic alternative.
PubMed: 36713859
DOI: 10.3389/fvets.2022.1083223 -
World Journal of Gastroenterology Mar 2019The bacteria () is commonly associated with Guillane-Barré syndrome (GBS) and irritable bowel syndrome (IBS), but studies have also linked it with Miller Fisher...
BACKGROUND
The bacteria () is commonly associated with Guillane-Barré syndrome (GBS) and irritable bowel syndrome (IBS), but studies have also linked it with Miller Fisher syndrome, reactive arthritis and other disorders, some of which are autoimmune. It is possible that and its toxins may be cross-reactive with some human tissues and food antigens, potentially leading to autoimmune responses.
AIM
To measure the immune reactivity of and cytolethal distending toxin (Cdt) antibodies with tissue and food antigens to examine their role in autoimmunities.
METHODS
Using enzyme-linked immunosorbent assay (ELISA) methodology, specific antibodies made against and Cdt were applied to a variety of microwell plates coated with 45 tissues and 180 food antigens. The resulting immunoreactivities were compared to reactions with control wells coated with human serum albumin (HSA) which were used as negative controls and with wells coated with lysate or Cdt which served as positive controls.
RESULTS
At 3 SD above the mean of control wells coated with HSA or 0.41 OD, the mouse monoclonal antibody made against showed moderate to high reactions with zonulin, somatotropin, acetylcholine receptor, β-amyloid and presenilin. This immune reaction was low with an additional 25 tissue antigens including asialoganglioside, and the same antibody did not react at all with another 15 tissue antigens. Examining the reaction between antibody and 180 food antigens, we found insignificant reactions with 163 foods but low to high immune reactions with 17 food antigens. Similarly, we examined the reaction of Cdt with the same tissues and food antigens. The strongest reactions were observed with zonulin, intrinsic factor and somatotropin. The reaction was moderate with 9 different tissue antigens including thyroid peroxidase, and reaction was low with another 10 different antigens, including neuronal antigens. The reaction of Cdt antibody with an additional 23 tissue antigens was insignificant. Regarding the reaction of Cdt antibody with different food antigens, 160 out of 180 foods showed insignificant reactions, while 20 foods showed reactions ranging from low to high.
CONCLUSION
Our findings indicate that and its Cdt may play a role in inflammation and autoimmunities beyond the gut.
Topics: Antibodies, Bacterial; Autoimmune Diseases; Autoimmunity; Bacterial Toxins; Campylobacter Infections; Campylobacter jejuni; Cholera Toxin; Cross Reactions; Dietary Proteins; Haptoglobins; Host Microbial Interactions; Human Growth Hormone; Humans; Intrinsic Factor; Protein Precursors
PubMed: 30862994
DOI: 10.3748/wjg.v25.i9.1050