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Biosensors May 2023Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The...
Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The development of LoCs, where the components for performing the nucleic acid amplification are all integrated, can be an issue. In this work, we present a LoC-PCR device where thermalization, temperature control and detection elements are all integrated on a single glass substrate named System-on-Glass (SoG) obtained using metal thin-film deposition. By using a microwell plate optically coupled with the SoG, real-time reverse transcriptase PCR of RNA extracted from both a plant and human virus has been carried out in the developed LoC-PCR device. The limit of detection and time of analysis for the detection of the two viruses by using the LoC-PCR were compared with those achieved by standard equipment. The results showed that the two systems can detect the same concentration of RNA; however, the LoC-PCR performs the analysis in half of the time compared to the standard thermocycler, with the advantage of the portability, leading to a point-of-care device for several diagnostic applications.
Topics: Humans; Lab-On-A-Chip Devices; Nucleic Acid Amplification Techniques; Viruses; Real-Time Polymerase Chain Reaction; RNA, Viral
PubMed: 37232905
DOI: 10.3390/bios13050544 -
RSC Advances May 2024Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene...
Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.
PubMed: 38741966
DOI: 10.1039/d4ra01317d -
Annals of Plastic Surgery Oct 2019Biofilms represent a complex milieu of matrix-enclosed microorganisms, which can significantly contribute to the pathology of chronic wounds. In this study, we compare... (Comparative Study)
Comparative Study Review
BACKGROUND
Biofilms represent a complex milieu of matrix-enclosed microorganisms, which can significantly contribute to the pathology of chronic wounds. In this study, we compare the activity of 3 commercial antimicrobial wound care solutions, Vashe (HOCl based), PhaseOne (HOCl based), and Sulfamylon (mafenide acetate), for their in vitro activity against bacterial and fungal biofilms.
METHODS
Reference and clinical isolates of 6 Gram-negative bacterial species (36 total strains), 3 Gram-positive bacteria (21 strains), and 3 Candida species (9 strains) were used to create biofilms. Various working concentrations of the 3 antiseptic agents were incubated with the biofilms in microwell plates; they were monitored from 1 minute to 24 hours to compare bacterial and fungal viability through colony forming unit analysis.
RESULTS
Vashe and PhaseOne displayed excellent bactericidal and fungicidal activity, whereas Sulfamylon demonstrated minimal activity against the biofilms tested. With the exception of Candida albicans, all biofilms were eliminated at either 1 or 10 minutes using Vashe and PhaseOne solutions. In most cases, mafenide was unable to eliminate both bacterial and fungal biofilms, even with 24 hours of treatment.
CONCLUSIONS
Biofilms represent a major clinical challenge, with no clear consensus for treatment of chronic wounds or prosthetic devices. Our results suggest that hypochlorous acid-based wound solutions such as Vashe and PhaseOne are more efficacious than mafenide in eliminating bacterial and fungal biofilms. Further studies are necessary to investigate and compare the in vivo efficacy of these products in clinical care.
Topics: Administration, Topical; Anti-Infective Agents; Antifungal Agents; Biofilms; Chronic Disease; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Sensitivity and Specificity; Solutions; Wounds and Injuries
PubMed: 31524733
DOI: 10.1097/SAP.0000000000001996 -
Medicina (Kaunas, Lithuania) Oct 2023Ceritinib (CER) is a potent drug of the third-generation tyrosine kinase inhibitor class. CER has been approved for the treatment of patients with non-small-cell lung...
Development of Two Novel One-Step and Green Microwell Spectrophotometric Methods for High-Throughput Determination of Ceritinib, a Potent Drug for Treatment of Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer.
Ceritinib (CER) is a potent drug of the third-generation tyrosine kinase inhibitor class. CER has been approved for the treatment of patients with non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma kinase (ALK) mutation gene. In the literature, there is no green and high-throughput analytical method for the quantitation of CER in its dosage form (Zykadia capsules). This study describes, for the first time, the development and validation of two novel one-step and green microwell spectrophotometric methods (MW-SPMs) for the high-throughput quantitation of CER in Zykadia capsules. These two methods were based on an formation of colored derivatives upon the reaction of CER with two different benzoquinone reagents via two different mechanisms. These reagents were -benzoquinone (OBQ) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), and their reactions proceeded via condensation and charge transfer reactions, respectively. The reactions were carried out in 96-well transparent plates, and the absorbances of the colored reaction products were measured with an absorbance microplate reader at 540 and 460 nm for reactions with OBQ and DDQ, respectively. The optimum conditions of reactions were established, their molar ratios were determined, and reaction mechanisms were postulated. Under the refined optimum reaction conditions, procedures of MW-SPMs were established and validated according to the guidelines of the International Council on Harmonization. The limits of quantitation were 6.5 and 10.2 µg/well for methods involving reactions with OBQ and DDQ, respectively. Both methods were applied with great reliability to the determination of CER content in Zykadia capsules and their drug uniformity. Greenness of the MW-SPMs was evaluated using three different metric tools, and the results proved that the two methods fulfil the requirements of green analytical approaches. In addition, the simultaneous handling of a large number of samples with microvolumes in the proposed methods gave them the advantage of a high-throughput analysis. : The two methods are valuable tools for rapid routine application in pharmaceutical quality control units for the quantitation of CER.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Anaplastic Lymphoma Kinase; Reproducibility of Results; Benzoquinones; Indicators and Reagents
PubMed: 37893531
DOI: 10.3390/medicina59101813 -
Biosensors & Bioelectronics Jul 2021Type 2 diabetes is currently one of the most common metabolic diseases, affecting all ages worldwide. As the incidence of type 2 diabetes increases, a growing number of...
Type 2 diabetes is currently one of the most common metabolic diseases, affecting all ages worldwide. As the incidence of type 2 diabetes increases, a growing number of studies focus on islets of Langerhans. A three-dimensional research model that maps islet morphology and maintains hormonal balance in vivo is still needed. In this work, we present an Islet-on-a-chip system, specifically a micropillar-based microfluidic platform for three-dimensional pancreatic islet cell culture and analysis. The microfluidic system consisted of two culture chambers that were equipped with 15 circular microtraps each, which were built with seven round micropillars each. Micropillars in the structure of microtraps supported cell aggregation by limiting the growth surface and minimizing wall shear stress, thereby ensuring proper medium diffusion and optimal culture conditions for cell aggregates. Our system is compatible with microwell plate readers and confocal laser scanning microscopes. Because of optimization of the immunostaining method, the appropriate cell distribution and high viability and proliferation up to 72 h of culture were confirmed. Enzyme-linked immunosorbent assays were performed to measure insulin and glucagon secretion after stimulation with different glucose concentrations. To our knowledge, this is the first Lab-on-a-chip system which enables the formation and three-dimensional culture of cell aggregates composed of commercially available α and β pancreatic islet cells. The specific composition and arrangement of cells in the obtained model corresponds to the arrangement of the cells in rodent pancreatic islets in vivo. This Islet-on-a-chip system may be utilized to test pathogenic effectors and future therapeutic agents.
Topics: Biomimetics; Biosensing Techniques; Cell Culture Techniques; Diabetes Mellitus, Type 2; Glucose; Humans; Insulin; Islets of Langerhans; Lab-On-A-Chip Devices; Microfluidics
PubMed: 33845292
DOI: 10.1016/j.bios.2021.113215 -
Langmuir : the ACS Journal of Surfaces... Jan 2023Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent...
Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent cross-linking. Covalent attachment gives greater stability in complex biological mixtures. However, current multistep chemical activation methods add complexity and cost, require specific functional groups, and can introduce cytotoxic chemicals that affect downstream cellular applications. Here, we show a method for one-step linker-free activation of microplates by energetic ions from plasma for covalent immobilization of DNA and protein. Two types of energetic ion plasma treatment were shown to be effective: plasma immersion ion implantation (PIII) and plasma-activated coating (PAC). This is the first time that PIII and PAC have been reported in microwell plates with nonflat geometry. We confirm that the plasma treatment generates radical-activated surfaces at the bottom of wells despite potential shadowing from the walls. Comprehensive surface characterization studies were used to compare the PIII and PAC microplate surface composition, wettability, radical density, optical properties, stability, and biomolecule immobilization density. PAC plates were found to have more nitrogen and lower radical density and were more hydrophobic and more stable over 3 months than PIII plates. Optimal conditions were obtained for high-density DNA (PAC, 0 or 21% nitrogen, pH 3-4) and streptavidin (PAC, 21% nitrogen, pH 5-7) binding while retaining optical properties required for typical high-throughput biochemical microplate assays, such as low autofluorescence and high transparency. DNA hybridization and protein activity of immobilized molecules were confirmed. We show that PAC activation allows for high-density covalent immobilization of functional DNA and protein in a single step on both 96- and 384-well plates without specific linker chemistry. These microplates could be used in the future to bind other user-selected ligands in a wide range of applications, for example, for solid phase polymerase chain reaction and stem cell culture and differentiation.
Topics: Indicators and Reagents; Wettability; Streptavidin; DNA; Surface Properties
PubMed: 36550613
DOI: 10.1021/acs.langmuir.2c02573 -
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 -
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 -
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