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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 -
MethodsX Jun 2024Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a...
Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a molecule of interest. MCP is a relatively low-cost and accessible technique that uses commercially available reagents and common cell culture equipment. However, it can be technically challenging, slow, and incompatible with microwell cell culture plates that are widely used for screening and other applications. Here, we describe a novel protocol using medical biopsy punches to transfer patterns into standard 96-well plates via polydimethylsiloxane (PDMS) cutouts. We demonstrate that this method can be used to deposit patterns of poly-D-lysine (PDL) into the microwells of glass-bottom plates. As a proof-of-concept, we show that cultured rodent glial cells preferentially grow and extend processes on the pattern. This method will allow larger scale MCP experiments in which different patterns, proteins, or other factors can be assayed in parallel.•Biopsy punches enable both cutting out small circular stamps and plunging them into tissue culture microwells to transfer proteins.•Compared to standard MCP, this method offers a more rapid workflow to pattern proteins onto substrates, and allows use of microwell plates that permits larger-scale experiments.
PubMed: 38524307
DOI: 10.1016/j.mex.2024.102665 -
RSC Advances Mar 2024This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures...
A prototype of ultrasensitive time-resolved fluoroimmunoassay for the quantitation of lead in plasma using a fluorescence-enhanced europium chelate label for the detection system.
This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures were conducted in 96-microwell plates and involved the competitive binding format. The assay used a mouse monoclonal antibody, designated as 2C33, that specifically recognized the diethylenetriamine pentaacetic acid chelate of Pb (Pb-DTPA) but did not recognize Pb-free DTPA chelator. The antigen used for coating onto the inner surfaces of assay plate microwells was Pb-DTPA conjugated with bovine serum albumin protein (Pb-DTPA-BSA). The competitive binding reaction occurred between Pb-DTPA chelates, formed in the sample solutions by treating the samples with an excess DTPA, and the coated Pb-DTPA-BSA for a limited quantity of 2C33 antibody binding sites. The antigen-antibody complex formed in the plate wells was quantified by a europium-DTPA-labeled secondary antibody and a fluorescence enhancement solution. The conditions of the assay were refined, and its optimum procedures were established. The TRFIA was validated following the immunoassay validation guidelines, and all of the validation criteria were acceptable. The working range of the assay was 20-300 pg mL and its limit of quantitation was 20 pg mL. Metals that are commonly encountered in blood plasma did not interfere with Pb in the analysis by the proposed TRFIA. The assay was applied to the quantitation of Pb in plasma samples with satisfactory accuracy and precision. The results were compared favorably with those obtained by atomic emission spectroscopy. In conclusion, the present study represents the first TRFIA for the quantitation of Pb in plasma. The assay is superior to the existing atomic spectrometric methods and other immunoassays for Pb in terms of sensitivity, convenience, and analysis throughputs. The proposed TRFIA is anticipated to effectively contribute to assessing Pb concentrations and controlling the exposure of humans to its potential toxicity.
PubMed: 38495999
DOI: 10.1039/d3ra07673c -
PLoS Biology Mar 2024Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture...
Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.
Topics: Microtechnology; Cell Culture Techniques; Microfluidics; Printing, Three-Dimensional; Lab-On-A-Chip Devices
PubMed: 38478490
DOI: 10.1371/journal.pbio.3002503 -
Biomicrofluidics Jan 2024Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture...
Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens.
PubMed: 38434239
DOI: 10.1063/5.0188270 -
Molecules (Basel, Switzerland) Nov 2023Ruxolitinib (RUX) is a potent drug that has been approved by the Food and Drug Administration for the treatment of myelofibrosis, polycythemia vera, and...
Spectrophotometric Study of Charge-Transfer Complexes of Ruxolitinib with Chloranilic Acid and 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone: An Application to the Development of a Green and High-Throughput Microwell Method for Quantification of Ruxolitinib in Its Pharmaceutical Formulations.
Ruxolitinib (RUX) is a potent drug that has been approved by the Food and Drug Administration for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. This study describes the formation of colored charge-transfer complexes (CTCs) of RUX, an electron donor, with chloranilic acid (CLA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), the π-electron acceptors. The CTCs were characterized using UV-visible spectrophotometry. The formation of CTCs in methanol was confirmed via formation of new absorption bands with maximum absorption at 530 and 470 nm for CTCs with CLA and DDQ, respectively. The molar absorptivity and other physicochemical and electronic properties of CTCs were determined. The molar ratio was found to be 1:1 for both CTCs with CLA and CTCs with DDQ. The site of interaction on RUX molecules was assigned and the mechanisms of the reactions were postulated. The reactions were employed as basis for the development of a novel green and one-step microwell spectrophotometric method (MW-SPM) for high-throughput quantitation of RUX. Reactions of RUX with CLA and DDQ were carried out in 96-well transparent plates, and the absorbances of the colored CTCs were measured by an absorbance microplate reader. The MW-SPM was validated according to the ICH guidelines. The limits of quantitation were 7.5 and 12.6 µg/mL for the methods involving reactions with CLA and DDQ, respectively. The method was applied with great reliability to the quantitation of RUX content in Jakavi tablets and Opzelura cream. The greenness of the MW-SPM was assessed by three different metric tools, and the results proved that the method fulfills the requirements of green analytical approaches. In addition, the one-step reactions and simultaneous handling of a large number of samples with micro-volumes using the proposed method enables the high-throughput analysis. In conclusion, this study describes the first MW-SPM, a valuable analytical tool for the quality control of pharmaceutical formulations of RUX.
Topics: Drug Compounding; Reproducibility of Results; Benzoquinones; Spectrophotometry; Tablets
PubMed: 38067605
DOI: 10.3390/molecules28237877 -
Communications Biology Nov 2023The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent...
The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
Topics: Optogenetics; Feedback; Escherichia coli; Algorithms; Spectrum Analysis
PubMed: 38001175
DOI: 10.1038/s42003-023-05532-4 -
Molecules (Basel, Switzerland) Oct 2023This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These...
Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex.
This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These platforms were a microwell-based enzyme-linked immunosorbent assay (ELISA) and a kinetic exclusion assay (KinExA) with a KinExA 3200 immunosensor. Both ELISA and KinExA were developed utilizing the same antibody and coating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized Cu(II)-ethylenediamine tetraacetic acid complex (Cu(II)-EDTA) but did not recognize Cu(II)-free EDTA. The 8D66 monoclonal antibody was generated by the fusion of spleen cells of an immunized BALB/c mouse with SP2/0-Ag14 myeloma cells. The immunogen was a protein conjugate of Cu(II)-EDTA with keyhole limpet hemocyanin protein. The coating reagent was Cu(II)-EDTA covalently linked to bovine serum albumin protein (Cu(II)-EDTA-BSA). Both assays involved the competitive binding reaction between Cu(II)-EDTA complexes, formed in the sample solution, and Cu(II)-EDTA-BSA conjugate which has been immobilized onto ELISA plates (in ELISA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of the 8D66 antibody. In ELISA, color signals were generated by a peroxidase-labeled secondary antibody and 3,3',5,5'-tetramethylbenzidine substrate. In KinExA, a fluorescein isothiocyanate-labeled secondary antibody was used to generate KinExAgram (trend-line fluorescence responses vs. time). The conditions of both ELISA and KinExA were investigated, and the optimum procedures were established. Both ELISA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in drinking water did not interfere with the Cu(II) analysis by both ELISA and KinExA. Both assays were applied to the determination of Cu(II) in drinking water with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy in terms of their abilities to accurately and precisely determine Cu(II) in drinking water samples. A comparative evaluation of ELISA and KinExA revealed that KinExA had a higher sensitivity and better precision than ELISA, whereas both assays had comparable accuracy. Both ELISA and KinExA were superior to the existing atomic spectrometric methods for Cu(II) in terms of sensitivity, convenience, and analysis throughputs. The proposed ELISA and KinExA are anticipated to effectively contribute to assessing Cu(II) concentrations and control the exposure of humans to its potential toxicities.
Topics: Humans; Animals; Mice; Copper; Antibodies, Monoclonal; Edetic Acid; Drinking Water; Biosensing Techniques; Immunoassay; Antigens; Indicators and Reagents
PubMed: 37894495
DOI: 10.3390/molecules28207017 -
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 -
Journal of the Indian Society of... 2023Antimicrobial intracanal medicaments play a vital role in successful outcome of any endodontic procedure. One such plant extract Cuminium cyminium, as intracanal...
CONTEXT
Antimicrobial intracanal medicaments play a vital role in successful outcome of any endodontic procedure. One such plant extract Cuminium cyminium, as intracanal medicaments needs to be researched.
AIMS
The purpose of this study was in vitro assessment of the antibacterial activity of ethanol extract of C. Cyminium in comparison to Calcium hydroxide (Ca[OH]) as intracanal medicament against the pathogens of endodontic infection, at an interval 1 h, 24 h, 48 h, and 72 h.
SETTINGS AND DESIGN
The study was conducted in the central research laboratory of our institute. Freshly prepared C. cyminium extract was procured from AYUSH approved laboratory and direct contact test (DCT) was utilized, which is based on turbidometric determination of microbial growth in a 96-well microplate, carrying 6 times for each bacteria.
METHODOLOGY
Three groups were assigned for each material in a 96 microwell plate for DCT. Bacterial growth kinetics was monitored at intervals of 1 h, 24 h, 48 h, and 72 h using spectrophotometer at 595 nm. The optical density of T2 (Test group), P2 (Positive control), and N2 (Negative control) was considered.
STATISTICAL ANALYSIS USED
After compiling the data, based on the normality of data, further statistical analysis was conducted using Kolmogorov-Smirnov test, Paired t-test, and pairwise comparisons by Turkey's multiple post hoc procedures. The level of statistical significance was set at P = 0.05.
RESULTS
The comparison of mean optical density values of C. cyminium in comparison with Ca(OH) against the microorganisms of endodontic origin showed a statically significant decrease in bacterial viability at the end of 24 h, 48 h, and 72 h.
CONCLUSION
Based on the results of the study, it can be concluded that C. cyminium has significant antibacterial action against endodontic origin, at interval of 24 h, 48 h, and 72 h.
Topics: Humans; Anti-Infective Agents; Anti-Bacterial Agents; Calcium Hydroxide; Bacteria; Ethanol; Plant Extracts
PubMed: 37861640
DOI: 10.4103/jisppd.jisppd_303_23