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Angewandte Chemie (International Ed. in... Jul 2023Within the realm of drug discovery, high-throughput experimentation techniques enable the rapid optimization of reactions and expedited generation of drug compound...
Within the realm of drug discovery, high-throughput experimentation techniques enable the rapid optimization of reactions and expedited generation of drug compound libraries for biological and pharmacokinetic evaluation. Herein we report the development of a segmented flow mass spectrometry-based platform to enable the rapid exploration of photoredox reactions for early-stage drug discovery. Specifically, microwell plate-based photochemical reaction screens were reformatted to segmented flow format to enable delivery to nanoelectrospray ionization-mass spectrometry analysis. This approach was demonstrated for the late-stage modification of complex drug scaffolds, as well as the subsequent structure-activity relationship evaluation of synthesized analogs. This technology is anticipated to expand the robust capabilities of photoredox catalysis in drug discovery by enabling high-throughput library diversification.
Topics: Mass Spectrometry; Drug Discovery; Catalysis; Spectrometry, Mass, Electrospray Ionization; High-Throughput Screening Assays
PubMed: 36940229
DOI: 10.1002/anie.202301664 -
Lab on a Chip Apr 2024Automated high-throughput liquid handling operations in biolabs necessitate miniaturised and automatised equipment for effective space utilisation and system...
Automated high-throughput liquid handling operations in biolabs necessitate miniaturised and automatised equipment for effective space utilisation and system integration. This paper presents a thermal segment microwell plate control unit designed for enhanced microwell-based experimentation in liquid handling setups. The development of this device stems from the need to move towards geometry standardization and system integration of automated lab equipment. It incorporates features based on Smart Sensor and Sensor 4.0 concepts. An enzymatic activity assay is implemented with the developed device on a liquid handling station, allowing fast characterisation a high-throughput approach. The device outperforms other comparable devices in certain metrics based on automated liquid handling requirements and addresses the needs of future biolabs in automation, especially in high-throughput screening.
PubMed: 38456212
DOI: 10.1039/d3lc00714f -
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 -
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 -
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 -
Annual International Conference of the... Aug 2015A major impediment to anti-cancer drug development is the lack of a reliable and inexpensive tumor model to test the efficacy of candidate compounds. This need has...
A major impediment to anti-cancer drug development is the lack of a reliable and inexpensive tumor model to test the efficacy of candidate compounds. This need has emerged due to the insufficiency of widely-used monolayer cultures to predict drug efficacy in vivo. Spheroids, 3D compact clusters of cancer cells, mimic important characteristics of tumors and provide a tissue analog for drug testing. Here we present a novel spheroid formation microtechnology that is simple to use and allows high throughput drug screening in 384-microwell plates. This approach is based on a polymeric aqueous two-phase system. The denser aqueous phase is mixed with cancer cells at a desired density. Using a robotic liquid handler, a drop of this cell suspension is dispensed into each well of a 384-microwell plate containing the second, immersion aqueous phase. Cancer cells remain contained in the drop, which rests on the well bottom, and form a spheroid during incubation. The use of liquid handling robotics ensures precise dispensing of a single drop, resulting in a single spheroid per well and homogenously sized spheroids within each plate. We confirmed the consistency of production of spheroids and demonstrated their biological relevance to tumors. A proof of concept study with spheroids of triple negative breast cancer cells treated with a standard chemotherapeutic compound, doxorubicin, showed the potential of this method for drug testing. This spheroid culture microtechnology presents key advantages over existing methods such as the ease of drug and viability reagent addition, ability to analyze spheroids without transferring them to a new plate, and the elimination of the need for specialized plates or devices to form spheroids. Incorporating this technology in anti-cancer drug development pipeline will help examine the efficacy of drug candidates more effectively and expedite discovery of novel drugs.
Topics: Antineoplastic Agents; Doxorubicin; Drug Screening Assays, Antitumor; Equipment Design; Female; High-Throughput Screening Assays; Humans; Polymers; Robotics; Spheroids, Cellular; Triple Negative Breast Neoplasms
PubMed: 26736723
DOI: 10.1109/EMBC.2015.7318823 -
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