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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 -
Nature Communications Jul 2018For decades, scientists have pursued the goal of performing automated reactions in a compact fluid processor with minimal human intervention. Most advanced fluidic...
For decades, scientists have pursued the goal of performing automated reactions in a compact fluid processor with minimal human intervention. Most advanced fluidic handling technologies (e.g., microfluidic chips and micro-well plates) lack fluid rewritability, and the associated benefits of multi-path routing and re-programmability, due to surface-adsorption-induced contamination on contacting structures. This limits their processing speed and the complexity of reaction test matrices. We present a contactless droplet transport and processing technique called digital acoustofluidics which dynamically manipulates droplets with volumes from 1 nL to 100 µL along any planar axis via acoustic-streaming-induced hydrodynamic traps, all in a contamination-free (lower than 10% diffusion into the fluorinated carrier oil layer) and biocompatible (99.2% cell viability) manner. Hence, digital acoustofluidics can execute reactions on overlapping, non-contaminated, fluidic paths and can scale to perform massive interaction matrices within a single device.
Topics: Acoustics; Humans; Hydrodynamics; Microfluidic Analytical Techniques; Microfluidics
PubMed: 30050088
DOI: 10.1038/s41467-018-05297-z -
RSC Advances Oct 2022Herein, we report a sensitive and selective enzyme-linked aptamer-based sandwich assay (ELASA) to detect lactate dehydrogenase (LDH), which is an attractive biomarker...
Herein, we report a sensitive and selective enzyme-linked aptamer-based sandwich assay (ELASA) to detect lactate dehydrogenase (LDH), which is an attractive biomarker for malaria diagnosis and antimalarial medication. We performed the sandwich assay with a single aptamer sequence, called 2008s, owing to the structural properties of the LDH tetramer instead of using a conventional sandwich assay with two different aptamers (or antibodies) for capturing and probing a target molecule. First, the biotinylated LDH aptamer was linked with immobilized streptavidin on a microwell plate for binding flexibility, and then LDH was bound to the aptamer. Next, a horseradish peroxidase-conjugated aptamer of the same sequence was used to analyze LDH quantitatively. Using this approach, the limit of detection (LOD) of LDH with the naked eye was 100 ng mL, and the LOD and limit of quantification from the absorbance measurements were 34.9 ng mL and 95.5 ng mL, respectively, based on LDH spiked blood samples. Our proposed method selectively binds LDH, not human lactate dehydrogenase. Therefore, this method may be a valuable tool for diagnosing, monitoring, and quarantining malaria cases easily and rapidly.
PubMed: 36320752
DOI: 10.1039/d2ra03796c -
Biotechnology Letters Nov 2016To compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes. (Comparative Study)
Comparative Study
OBJECTIVES
To compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes.
RESULTS
Pellets cultured in concave microwell plates had a significantly higher level of GAG per DNA content and greater proteoglycan content than those cultured in tubes at day 7 and 14. Three chondrogenic markers, SOX-9, COL2A1 and aggrecan, showed significantly higher expression in pellets cultured in concave microwell plates than those cultured in tubes at day 7 and 14. At day 21, there was not a significant difference in the expression of these markers. COL10A1, the typical hypertrophy marker, was significantly lower in concave microwell plates during the whole culture period. Runx-2, a marker of hypertrophy and osteogenesis, was significantly lower at day 7 in pellets cultured in concave microwell plates than those cultured in tubes.
CONCLUSION
Concave microwell plates provide a convenient and effective tool for the study of in vitro chondrogenesis and may replace the use of propylene culture tube.
Topics: Aged; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Chondrogenesis; DNA; Gene Expression Regulation; Humans; Mesenchymal Stem Cells; Middle Aged
PubMed: 27534541
DOI: 10.1007/s10529-016-2170-8 -
In Vivo (Athens, Greece) 2020In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously...
BACKGROUND
In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously X-ray-irradiated cells was newly established.
MATERIALS AND METHODS
PC12 cell suspension was irradiated by X-ray (0.5 Gy) in serum-supplemented medium, immediately inoculated into 96-microwell plates and incubated overnight. The medium was replaced with fresh serum-depleted medium containing 50 ng/ml nerve growth factor to induce differentiation toward nerve-like cells with characteristic neurites according to the overlay method without changing the medium. The differentiated cells were treated by anticancer drugs as well as antioxidants, oxaliplatin or bortezomib, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.
RESULTS
Antioxidants and anticancer drugs were cytotoxic to differentiating PC12 cells. Combination of anticancer drugs and X-ray irradiation slightly reduced cell viability.
CONCLUSION
The present 'population irradiation method' may be useful for the investigation of the combination effect of X-ray irradiation and any pharmaceutical drug.
Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Immunohistochemistry; Mice; Nervous System; Phosphorylation; Radiation, Ionizing; Signal Transduction; X-Rays
PubMed: 32354886
DOI: 10.21873/invivo.11869 -
Journal of AOAC International Mar 2024Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the...
Development of Green and High Throughput Microwell Spectrophotometric Methods for Determination of Galidesivir in Bulk Drug and Dosage Forms Based on Simple Oxidimetric Reactions with Inorganic Agents.
BACKGROUND
Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form.
OBJECTIVE
The objective of this study was the investigation of oxidation reactions of GDV with five inorganic oxidizing reagents and the employment of the reactions in the development of five green microwell spectrophotometric methods (MW-SPMs) with simple procedure and high throughputs for determination of GDV in its bulk and dosage forms (capsules).
METHODS
The reactions were carried out in 96-well plates and the absorbances of reaction solutions were measured by an absorbance microplate reader. Variables influencing the reactions were carefully investigated and optimized.
RESULTS
Under the refined optimum conditions, Beer's law with excellent correlation coefficients (0.9992-0.9997) was followed in GDV concentrations in a general range of 5-700 µg/mL, and the limits of detection were ≥1.8 µg/mL. All validation parameters of all methods were acceptable. The methods were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision; the recovery percentages were 98.6-101.2 ± 0.58-1.14%. The greenness of MW-SPMs was evaluated by three comprehensive metric tools, which demonstrated the adherence of MW-SPMs to the principles of the green analytical chemistry approach.
CONCLUSIONS
The proposed MW-SPMs combined the advantages of microwell-based practice and the use of common laboratory reagents for the analysis. The advantages of microwell analysis were the high throughput, readily available for semi-automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of using common laboratory reagents were the availability, consistency, compatibility, safety, and cost-effectiveness.
HIGHLIGHTS
Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.
PubMed: 38521540
DOI: 10.1093/jaoacint/qsae026 -
ACS Applied Bio Materials May 2021Prostate-specific membrane antigen (PSMA) is a viable diagnostic biomarker for the detection and treatment of prostate cancer. Although numerous imaging techniques and...
Prostate-specific membrane antigen (PSMA) is a viable diagnostic biomarker for the detection and treatment of prostate cancer. Although numerous imaging techniques and fluorescent probes have been developed, targeted imaging and intraoperative surgery continue to remain as a proof-of-concept with a severe lack of tools having high affinity and penetrative capacity. three-dimensional cell culture has gained immense interest in cancer research and drug discovery programs as it yields important physiological information and serves an excellent model for bioimaging and penetration analysis studies. Current techniques employed in spheroid formation include liquid overlay and hanging drop methods, both of which are low-yielding and technically demanding. We describe for the first time a simple-to-use platform, μSpherePlatform, an inexpensive, high-throughput method yielding morphologically homogeneous spheroids in bulk for analyzing penetrative capacity and imaging ability of PCa diagnostics. Microwell arrays made of agarose have been fabricated using a commercial hairbrush as a master template. This procedure has been described in detail, and arrays of spheroids (100-120 spheroids/6-well plate) with >95% success rates have been produced from PCa cell lines (LNCaP and DU-145). A PSMA-targeted fluorescent conjugate was synthesized and evaluated in the spheroids developed using μSpherePlatform by multiphoton imaging. A synthetic 3D scaffold strategy is reported herein, which (1) correlates perfectly with the model, (2) is amenable for automated analysis, (3) shows a negligible lot to lot variation, (4) is simplistic, (5) is useful for high-throughput assays, (6) is extremely compatible with imaging techniques, (7) generates PCa spheroids within 48 h, and (8) forms large size-controllable spheroids of diameter 500-1300 μm. The μSpherePlatform thus provides a significant contribution to multimodal analyses of cancer diagnostics and deep-tissue imaging studies.
Topics: Biocompatible Materials; Cell Culture Techniques, Three Dimensional; Cell Line, Tumor; Humans; Male; Materials Testing; Molecular Structure; Particle Size; Prostate-Specific Antigen; Prostatic Neoplasms
PubMed: 35006838
DOI: 10.1021/acsabm.1c00086 -
High-throughput organo-on-pillar (high-TOP) array system for three-dimensional ex vivo drug testing.Biomaterials May 2023The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy....
The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix-cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.
Topics: Female; Humans; Cell Culture Techniques; Reproducibility of Results; Drug Evaluation, Preclinical; Drug Discovery; Organoids; Ovarian Neoplasms; High-Throughput Screening Assays
PubMed: 36924663
DOI: 10.1016/j.biomaterials.2023.122087 -
Veterinary Journal (London, England :... Nov 2017Unbalanced coagulation and fibrinolysis leads to hemorrhage or thrombosis. Thromboelastography has been used to characterize hypo- and hyper-fibrinolysis in dogs,...
Unbalanced coagulation and fibrinolysis leads to hemorrhage or thrombosis. Thromboelastography has been used to characterize hypo- and hyper-fibrinolysis in dogs, however the technique requires specialized instrumentation and proprietary reagents that limit its availability. The aim of this study was to develop a simple microplate method for assessment of fibrinolysis in canine plasma. Plasma from healthy dogs was mixed in a microwell plate with tissue factor, calcium, phospholipid and tissue plasminogen activator. Light absorbance was measured at regular intervals until return to baseline. Peak optical density (milli-absorption units, mAU), formation velocity (mAU/s), lysis velocity (mAU/s) and area under the curve (mAU.s) were calculated. The influence of potential interferents, variation in fibrinogen and ex vivo addition of heparin and aminocaproic acid on assay performance was determined. Inter-day coefficients of variation were ≤15% for all variables. Bilirubin≤1.88mg/dL and hemoglobin≤0.09mg/dL did not interfere with assay variables. Aminocaproic acid (40μg/mL) and heparin (0.125U/mL) caused almost complete inhibition of fibrinolysis and coagulation, respectively. All variables except lysis velocity (R=0.08) were associated with fibrinogen concentration (R>0.8). This assay showed acceptable performance characteristics for measurement of fibrinolysis in normal canine plasma. The assay utilizes small volume citrate plasma samples and readily available instrumentation and reagents, is not influenced by mild to moderate hemolysis or icterus and detects the presence of fibrinolysis inhibitors.
Topics: Animals; Blood Coagulation Tests; Dog Diseases; Dogs; Female; Fibrinogen; Fibrinolysis; Male; Plasma; Predictive Value of Tests; Thrombelastography; Thrombosis
PubMed: 29183569
DOI: 10.1016/j.tvjl.2017.10.020 -
Analytical Chemistry Mar 2016We report cross-interface emulsification (XiE), a simple method for the generation of monodisperse droplets of controllable volumes from picoliter to nanoliter. A device...
We report cross-interface emulsification (XiE), a simple method for the generation of monodisperse droplets of controllable volumes from picoliter to nanoliter. A device is set up in which a fused-silica capillary is vibrating across the surface of the continuous phase (mineral oil) in a reservoir, and the flow of the dispersed phase (aqueous solution) in the capillary is segmented into monodisperse droplets at the air/oil interface. We find that the volume of droplets is mainly dominated by the flow rate and vibrating frequency and not significantly influenced by other factors, such as the viscosity of the continuous phase and dispersed phase, the inner diameter of the capillary (20-100 μm), or the shape of the tip (tapered or flat). These features reflect high robustness, flexibility, and precision of XiE for on-demand volume control of droplets. The droplets automatically assemble into planar monolayer droplet arrays (PMDA) in flat-bottomed microwells of 96-well plates, offering excellent convenience for imaging of droplets. As a representative application, we carry out digital loop-mediated isothermal amplification using PMDAs with multivolume droplets for the absolute quantification of nucleic acids. Our results demonstrate that XiE is simple and controllable for the production of monodisperse size-tunable droplets, and it offers opportunities for common laboratories, even without microfabrication facilities, to perform digital quantification, single cell analysis, and other biochemical assays with high throughput.
PubMed: 26849419
DOI: 10.1021/acs.analchem.5b04510