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RSC Advances Jan 2023This work proposes a highly specific method of Cr determination based on the double reactions of two metals, Co with dithizone to form a (DTZ)-Co complex, and the...
This work proposes a highly specific method of Cr determination based on the double reactions of two metals, Co with dithizone to form a (DTZ)-Co complex, and the replacement of Co in the formed complex with Cr. The fast degradation of DTZ in solution in wet analysis was overcome by preparing dithizone functionalized polyurethane nanofibers that were electrospun into a membrane (DTZ/PU-NF) and a microwell plate film (DTZ/PU-MPF). For comparison, the performance of diphenylcarbazide (DPC), a currently used complexing agent for Cr, was also investigated. Colour changes were detected as red-green-blue values. The DTZ/PU-NF was smooth, with an average diameter of 384.09 nm and no bead appeared. A dense network structure was formed. The best formulation of DTZ, PU and Co was also applied as a microwell plate film. In the presence of Cr, the colour of DTZ-Co changed from red to magenta. Among the three studied methods, the colorimetric DTZ-Co/PU-NF presented the best results. Its linearity range was 0.001-1.0 mg L, with a regression equation of Cr = -0.189 + (0.0056 × red) + (0.0086 × green) - (0.0129 × blue), of 0.990. The limit of detection was 0.001 mg L and the precision was 1.7%. The applicability of DTZ/PU-NF was validated for Cr in vegetable oils with recoveries of 89.5-116.8%. The sensitivity of DTZ/PU-NF was ten times higher than that of DTZ/PU-MPF. The methods based on DTZ-Co/PU-NF and DTZ-Co/PU-MPF proved to be highly selective, rapid, user-friendly, simple and reliable.
PubMed: 36756414
DOI: 10.1039/d2ra07636e -
Nature Communications Jan 2023Sensing small biomolecules in biofluids remains challenging for many optical chemosensors based on supramolecular host-guest interactions due to adverse interplays with...
Sensing small biomolecules in biofluids remains challenging for many optical chemosensors based on supramolecular host-guest interactions due to adverse interplays with salts, proteins, and other biofluid components. Instead of following the established strategy of developing alternative synthetic binders with improved affinities and selectivity, we report a molecular engineering approach that addresses this biofluid challenge. Here we introduce a cucurbit[8]uril-based rotaxane chemosensor feasible for sensing the health-relevant biomarker tryptophan at physiologically relevant concentrations, even in protein- and lipid-containing human blood serum and urine. Moreover, this chemosensor enables emission-based high-throughput screening in a microwell plate format and can be used for label-free enzymatic reaction monitoring and chirality sensing. Printed sensor chips with surface-immobilized rotaxane-microarrays are used for fluorescence microscopy imaging of tryptophan. Our system overcomes the limitations of current supramolecular host-guest chemosensors and will foster future applications of supramolecular sensors for molecular diagnostics.
Topics: Humans; Serum; Rotaxanes; Tryptophan; Body Fluids
PubMed: 36720875
DOI: 10.1038/s41467-023-36057-3 -
Frontiers in Veterinary Science 2022The Chinese traditional medicinal plants , and in a ratio of 108:65:27 form a compound named Dahuang Qinyu San (DQS), which inhibits and kills and to a certain extent...
The Chinese traditional medicinal plants , and in a ratio of 108:65:27 form a compound named Dahuang Qinyu San (DQS), which inhibits and kills and to a certain extent in fish and shrimp aquaculture environments. The active ingredients quercetin, emodin, baicalin, and aloe-emodin are obtained from the semi-biomimetic extract of DQS (SEDQS). However, the antibacterial mechanism of SEDQS against is still unclear. This study used the microwell-plate method to determine the Minimum Inhibitory Concentration (MIC) of SEDQS against () isolated from geese. In addition, the effect of SEDQS on the growth curve, respiratory metabolic system, cell wall, soluble protein, and nucleic acid in bacterial liquid of was detected by spectrophotometer and reagent kit. The effects of SEDQS on DNA, binding gel blocking, virulence gene expression, and pathogenicity-related proteins were determined by gel electrophoresis, SDS-PAGE, and fluorescence quantitative PCR. The study found that a concentration of 1/4 MIC-2 MIC (2.27-18.2 mg/ml) SEDQS can significantly inhibit the normal growth of , destroy the cell membrane structure of bacteria resulting in the leak of nucleic acid, protein, and other contents ( < 0.01). It also significantly inhibited the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH; < 0.01) in a concentration-dependent manner. When the concentration of SEDQS was 1/2 MIC to 2 MIC (4.55-18.2 mg/ml), the expression levels of , and virulence genes ( < 0.01) all decreased by more than 31, 11, 18, 30, 34, and 21% respectively compared with the control group. SEDQS could significantly inhibit the expression of six virulence genes and play an important role in the pathogenicity of infected host. The SEDQS could exert antibacterial pharmacological effects by inhibiting the growth and metabolism of and inhibiting the expression of major virulence factors. It has potential application value as an antibiotic alternative.
PubMed: 36713859
DOI: 10.3389/fvets.2022.1083223 -
Transplant International : Official... 2022Neonatal porcine islet-like cell clusters (NPICCs) are a promising source for islet cell transplantation. Excellent islet quality is important to achieve a cure for type...
Neonatal porcine islet-like cell clusters (NPICCs) are a promising source for islet cell transplantation. Excellent islet quality is important to achieve a cure for type 1 diabetes. We investigated formation of cell clusters from dispersed NPICCs on microwell cell culture plates, evaluated the composition of re-aggregated porcine islets (REPIs) and compared function by transplantation into diabetic NOD-SCID IL2rγ (NSG) mice with native NPICCs. Dissociation of NPICCs into single cells and re-aggregation resulted in the formation of uniform REPI clusters. A higher prevalence of normoglycemia was observed in diabetic NSG mice after transplantation with a limited number ( = 1500) of REPIs (85.7%) versus NPICCs ( = 1500) (33.3%) ( < 0.05). Transplanted REPIs and NPICCs displayed a similar architecture of endocrine and endothelial cells. Intraperitoneal glucose tolerance tests revealed an improved beta cell function after transplantation of 1500 REPIs (AUC glucose 0-120 min 6260 ± 305.3) as compared to transplantation of 3000 native NPICCs (AUC glucose 0-120 min 8073 ± 536.2) ( < 0.01). Re-aggregation of single cells from dissociated NPICCs generates cell clusters with excellent functionality and improved function as compared to native NPICCs.
Topics: Swine; Animals; Mice; Insulin; Endothelial Cells; Diabetes Mellitus, Experimental; Mice, Inbred NOD; Mice, SCID; Islets of Langerhans; Islets of Langerhans Transplantation; Glucose; Transplantation, Heterologous; Blood Glucose
PubMed: 36685665
DOI: 10.3389/ti.2022.10697 -
Microsystems & Nanoengineering 2023In point-of-care testing (POCT), tests are performed near patients and results are given rapidly for timely clinical decisions. Immunodiagnostic assays are one of the...
In point-of-care testing (POCT), tests are performed near patients and results are given rapidly for timely clinical decisions. Immunodiagnostic assays are one of the most important analyses for detecting and quantifying protein-based biomarkers. However, existing POCT immunodiagnostics mainly rely on the lateral flow assay (LFA), which has limited sensitivity or quantification capability. Although other immunodiagnostic assays, such as enzyme-linked immunosorbent assays (ELISAs), offer more sensitive and quantitative results, they require complex liquid manipulations that are difficult to implement in POCT settings by conventional means. Here, we show the development of DropLab, an automated sample-in-answer-out POCT immunodiagnostic platform based on magnetic digital microfluidic (MDM) technology. DropLab performs microbead-based ELISA in droplets to offer more sensitive and quantitative testing results. The intricate liquid manipulations required for ELISA are accomplished by controlling droplets with magnetic microbeads using MDM technology, which enables us to achieve full automation and easy operations with DropLab. Four ELISAs (the sample in triplicates and a negative control) can be run in parallel on the thermoformed disposable chip, which greatly improves the throughput and accuracy compared to those of other POCT immunodiagnostic devices. DropLab was validated by measuring two protein targets and one antibody target. The testing results showed that the limit of detection (LOD) of DropLab matched that of the conventional ELISA in a microwell plate. DropLab brings MDM one step closer to being a viable medical technology that is ready for real-world POCT applications.
PubMed: 36644334
DOI: 10.1038/s41378-022-00475-y -
Frontiers in Microbiology 2022In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is...
In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 μm × 600 μm × 700 μm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.
PubMed: 36590440
DOI: 10.3389/fmicb.2022.1031439 -
Antibiotics (Basel, Switzerland) Dec 2022Fungal infections are often consequent to prolonged antibiotic treatments. Vancomycin (Van) is the first-choice antibiotic in the treatment of infections associated...
Fungal infections are often consequent to prolonged antibiotic treatments. Vancomycin (Van) is the first-choice antibiotic in the treatment of infections associated with colonization of catheter surfaces. We demonstrate the direct effect of Van in promoting the formation of the biofilm of the emergent yeast pathogen developed in the conventional polystyrene microwell plate model, as well as on silicone surfaces (22 and 28% increase in total biomass, respectively) and on an biofilm, residual after vancomycin treatment, where achieved 99% of the mixed biofilm population. The effect of Van was assessed also in vivo, in the infection model, which showed higher mortality when infected with the yeast pathogen in the presence of the antibiotic. This evidence enhances awareness of the potential risk associated with prolonged antibiotic use in promoting fungal infections.
PubMed: 36551428
DOI: 10.3390/antibiotics11121771 -
Nature Communications Dec 2022The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular...
The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.
Topics: Biomolecular Condensates; Intracellular Space; Microfluidics; Phase Transition
PubMed: 36543777
DOI: 10.1038/s41467-022-35265-7 -
Cellular and Molecular Bioengineering Dec 2022The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling...
OBJECTIVE
The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling these systems up for high-throughput testing is technically challenging and wasteful given the necessary cell numbers and reagent volumes. However, adopting microscale spheroid cultures for this purpose shows promise. Spheroid systems work with only thousands of cells and microliters of medium.
METHODS
Molded agarose microwells were fabricated using 2% w/v molten agarose and then equilibrated in medium prior to introducing cells. ASCs were seeded at 50, 500, 5k cells/microwell; 5k, 50k, cells/well plate; and 50k and 250k cells/15 mL centrifuge tube to compare chondrogenic responses across spheroid and micromass sizes. Cells were cultured in control or chondrogenic induction media. ASCs coalesced into spheroids/pellets and were cultured at 37 °C and 5% CO for 21 days with media changes every other day.
RESULTS
All culture conditions supported growth of ASCs and formation of viable cell spheroids/micromasses. More robust growth was observed in chondrogenic conditions. Sulfated glycosaminoglycans and collagen II, molecules characteristics of chondrogenesis, were prevalent in both 5000-cell spheroids and 250,000-cell micromasses. Deposition of collagen I, characteristic of fibrocartilage, was more prevalent in the large micromasses than small spheroids.
CONCLUSIONS
Chondrogenic differentiation was consistently induced using high-throughput spheroid formats, particularly when seeding at cell densities of 5000 cells/spheroid. This opens possibilities for highly arrayed experiments investigating tissue repair and remodeling during or after exposure to drugs, toxins, or other chemicals.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s12195-022-00746-8.
PubMed: 36531862
DOI: 10.1007/s12195-022-00746-8 -
Micromachines Nov 2022In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep...
In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep Eutectic Solvent (DES)-based media. 3D-printed polylactic acid (PLA) microwell plates or tubular microfluidic reactors were modified with polyethylenimine (PEI) and lipase from (CALB) was covalently immobilized in the interior of each structure. DESs were found to have a negligible effect on the activity and stability of CALB, and the system proved highly stable and reusable in the presence of DESs for the hydrolysis of p-nitrophenyl butyrate (p-NPB). A kinetic study under flow conditions revealed an enhancement of substrate accessibility in the presence of Betaine: Glycerol (Bet:Gly) DES, while the system was not severely affected by diffusion limitations. Incubation of microreactors in 100% Bet:Gly preserved the enzyme activity by 53% for 30 days of storage at 60 °C, while the buffer-stored sample had already been deactivated. The microfluidic enzyme reactor was efficiently used for the trans-esterification of ethyl ferulate (EF) with glycerol towards the production of glyceryl ferulate (GF), known for its antioxidant potential. The biocatalytic process under continuous flow conditions exhibited 23 times higher productivity than the batch reaction system. This study featured an effective and robust biocatalytic system with immobilized lipase that can be used both in hydrolytic and synthetic applications, while further optimization is expected to upgrade the microreactor system performance.
PubMed: 36422383
DOI: 10.3390/mi13111954