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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 -
Analytical Sciences : the International... Dec 2023Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to...
Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments.
Topics: Humans; Microfluidics; Nucleic Acid Amplification Techniques; DNA; Real-Time Polymerase Chain Reaction; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques
PubMed: 37710081
DOI: 10.1007/s44211-023-00425-2 -
STAR Protocols Sep 2023The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time...
The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.
Topics: Humans; Organoids; Brain; Head
PubMed: 37715950
DOI: 10.1016/j.xpro.2023.102502 -
Analytical and Bioanalytical Chemistry Oct 2023A novel method for direct high-throughput analysis of multi-elements in cerebrospinal fluid (CSF) samples by laser ablation inductively coupled plasma mass spectrometry...
A novel method for direct high-throughput analysis of multi-elements in cerebrospinal fluid (CSF) samples by laser ablation inductively coupled plasma mass spectrometry with an aerosol local extraction cryogenic ablation cell (ALEC-LA-ICP-MS) was developed. Microliter-level CSF samples were frozen by a designed cryogenic ablation cell and directly analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) without time-consuming pretreatment. Compared with the precision obtained at room temperature (20℃), that obtained at low temperature (- 25℃) was significantly improved; the RSDs were reduced from 8.3% (Zn) to 32.6% (Mn) to 2.2% (Pb) to 6.5% (Mn) with six times parallel determination. To meet the analytical requirement of the micro-volume CSF samples, the laminar flow aerosol local extraction strategy was adopted to improve the transmission efficiency of aerosols, and the signal intensity was increased by four times compared with the standard commercial ablation cell. The standard solution with 0.4% bovine serum albumin (BSA) matrix was used as matrix-match external standard, and Rh was added into the samples as internal standard. The limits of detection (LODs) ranged from 0.17 μg·L (Mn) to 8.67 μg·L (Mg). Standard addition recovery experiments and the determination of CRM serum L-1 and L-2 were carried out to validate the accuracy of the method; all results indicated there were excellent accuracy and precision in the proposed method. The matrix-scanning function in the GeoLas software combined with the microwell plate realizes the high-throughput automatic analysis. Twenty-four CSF samples from different patients were determined; the results showed that there might be a correlation between the metal elements in CSF and the diseases, which means that the proposed method has potential in the diagnosis of neurological diseases.
PubMed: 37541973
DOI: 10.1007/s00216-023-04878-2 -
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 -
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 -
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 -
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 -
Contact Lens & Anterior Eye : the... May 2024Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown....
PURPOSE
Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown. This study examines the in vitro pro-inflammatory effect of the FR on human corneal epithelial cells in varying degrees of MDF.
METHODS
Normal SL neophytes were recruited to wear SL 8 h daily for 4 days. Following 8 h on days 1 and 4, optical coherence tomography (OCT) images were acquired for MDF quantification using ImageJ, and the FR was collected. FR samples from the same eye were later pooled, diluted 2-fold and applied on human telomerase-immortalized corneal epithelial (hTCEpi) cells cultured on Terasaki microwell plates. Tumor necrosis factor (TNF)-α and culture media were used as positive and negative controls, respectively. After a 30-minute treatment, the nuclear factor-kappa B (NF-κB) pathway was measured by NF-κB-p65 immunofluorescence and images were analyzed with ImageJ. Pearson's correlation was conducted to determine the association between median nuclear fluorescence and MDF.
RESULTS
Fourteen FR samples with a mean volume of 22 ± 16 µl were tested. Mean MDF severity following 8 h of SL wear was 25 ± 17 units (range 7 - 64). The median nuclear fluorescence (NF-κB-p65 translocation) in cultured hTCEpi cells ranged from 31.43 to 45.16 while the negative and positive controls were 44.71 ± 1.72 and 108.77 ± 68.38, respectively. Although a potential positive trend between MDF and median nuclear fluorescence was observed, Pearson's correlation analysis revealed no significant association (r = +0.48, P = 0.09).
CONCLUSIONS
The results suggest that the FR can trigger NF-κB-p65 translocation in hTCEpi cells, which may be associated with MDF severity. This study introduces the use of Terasaki microwell plates for immunofluorescence studies of the FR. The technique is simple, minimizes sample usage, and does not require expensive instrumentation.
PubMed: 38762441
DOI: 10.1016/j.clae.2024.102187 -
ACS Biomaterials Science & Engineering Jan 2024Cell distribution is one of the primary factors that can affect cell morphology and behaviors, as it determines cell-cell interactions. Despite the importance of cell...
Cell distribution is one of the primary factors that can affect cell morphology and behaviors, as it determines cell-cell interactions. Despite the importance of cell distribution, the seeding process of in vitro cell culture still highly relies on the traditional method using manual pipetting. Because manual pipetting cannot ensure a uniform cell distribution and has the possibility of compromising experimental reproducibility, an accurate and systemic seeding method that enables uniform cell seeding over versatile culture substrates is required. Here, we developed a perforated plate-based cell seeding device called the CellShower, which enabled uniform cell seeding over a large area of cell culture substrates. The working principles of the CellShower are based on the laminar filling flow and capillary force in microfluidics, and the design of the CellShower was optimized with numerical simulations. The versatility of the CellShower in view of uniform cell seeding was demonstrated by applying it to various types of culture substrates from a conventional culture dish to culture substrates having nanotopography, porous structures, and 3D concave structures. The CellShower and its operating principles are expected to contribute to enhancing the accuracy and reproducibility of biological experiments.
Topics: Reproducibility of Results; Cell Culture Techniques; Porosity
PubMed: 38048415
DOI: 10.1021/acsbiomaterials.3c01203