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Talanta Jul 2024The 96 laser-induced multigraphene electrode (96L-MGE) integrated microwell plate (96 L-MGE-MP) is described. Each cell includes separate working, auxiliary, and...
The 96 laser-induced multigraphene electrode (96L-MGE) integrated microwell plate (96 L-MGE-MP) is described. Each cell includes separate working, auxiliary, and reference electrodes, and the array sits on a poly-methyl methacrylate (PMMA) well. The 96 electrochemical cells were fabricated by laser ablation of polyimide adhesive tape, which created laser-induced graphene electrodes (L-GE). The microwell was produced using laser ablation of the PMMA sheet as well. The morphology and electrochemical characterization of L-GE were controlled by tuning the laser processing. L-GE fabricated at laser power-laser speed ratios of 0.008-0.02 W s mmdisplayed good electrochemical behaviors. Under the optimal condition of L-GE fabrication, the measured L-GE surface roughness was 475.47 nm. The 96 L-MGE can be fabricated in 24.2 min and is compatible with various analytes. 10 benchmark redox compounds were shown as electrocatalytic examples. The performance of each analyte was investigated by voltammetry. As proof of concept, 96 L-MGE-MP was connected to a 96× connector for multichannel detection. The RSD of the 96 L-MGE-MPwas below 5.3%, which demonstrated good fabrication reproducibility.
PubMed: 38547843
DOI: 10.1016/j.talanta.2024.125912 -
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 -
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 -
Journal of the American Chemical Society Apr 2024Recent advances have demonstrated the promise of complex multicomponent polymeric supports to enable supra-biological enzyme performance. However, the discovery of such...
Recent advances have demonstrated the promise of complex multicomponent polymeric supports to enable supra-biological enzyme performance. However, the discovery of such supports has been limited by time-consuming, low-throughput synthesis and screening. Here, we describe a novel combinatorial and high-throughput platform that enables rapid screening of complex and heterogeneous copolymer brushes as enzyme immobilization supports, named combinatorial high-throughput enzyme support screening (CHESS). Using a 384-well plate format, we synthesized arrays of three-component polymer brushes in the microwells using photoactivated surface-initiated polymerization and immobilized enzymes in situ. The utility of CHESS to identify optimal immobilization supports under thermally and chemically denaturing conditions was demonstrated usingLipase A (LipA). The identification of supports with optimal compositions was validated by immobilizing LipA on polymer-brush-modified biocatalyst particles. We further demonstrated that CHESS could be used to predict the optimal composition of polymer brushes priori for the previously unexplored enzyme, alkaline phosphatase (AlkP). Our findings demonstrate that CHESS represents a predictable and reliable platform for dramatically accelerating the search of chemical compositions for immobilization supports and further facilitates the discovery of biocompatible and stabilizing materials.
Topics: Enzymes, Immobilized; High-Throughput Screening Assays; Polymers
PubMed: 38500441
DOI: 10.1021/jacs.3c14273 -
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 -
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 -
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 -
Biotechnology and Bioengineering Jun 2024The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high...
The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell-specific perfusion rate (CSPR). To obtain a cost-productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor-intensive and time-consuming experiments, as investigations are performed in lab-scale perfusion bioreactors. Small-scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi-perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi-perfusion. The CSPR-based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d. The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 10 cells mL were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR-based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations.
Topics: Bioreactors; CHO Cells; Cricetulus; Animals; Perfusion; Cell Culture Techniques; High-Throughput Screening Assays; Cell Count; Batch Cell Culture Techniques
PubMed: 38433473
DOI: 10.1002/bit.28685 -
Biotechnology and Bioengineering Jun 2024The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative....
The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative. Developing and optimizing perfusion operations requires screening various parameters, which is expensive and time-consuming when using benchtop bioreactors. Scale-down models (SDMs) are the most feasible option for high-throughput data generation and condition screening. However, new SDMs mimicking perfusion are required, enabling experiments to be run in parallel. In this study, a method using microwell plates (MWP) operating in semi-perfusion mode with an implemented cell bleed step is presented. A CHO cell line was cultivated in a 24-well MWP (V = 1.2 mL) and grown at four high cell density (HCD) setpoints. Quasi steady-state condition was obtained by manually performing cell bleeds followed by a total medium exchange after centrifugation. Further, two HCD setpoints were scaled up (V = 30 mL), comparing a squared six-well deepwell plate (DWP) to shake flasks (SF). This evaluation showed comparable results between systems (DWP vs. SF) and scales (MWP vs. DWP + SF). The results show that the well-plate-based methods are suitable to perform HCD and quasi steady-state cultivations providing a robust solution to industrially relevant challenges such as cell clone and media selection.
Topics: CHO Cells; Cricetulus; Animals; High-Throughput Screening Assays; Bioreactors; Cell Culture Techniques; Cell Count
PubMed: 38393309
DOI: 10.1002/bit.28682