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Biosensors & Bioelectronics Aug 2024Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a...
Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.
Topics: Colistin; Deep Learning; Microbial Sensitivity Tests; Anti-Bacterial Agents; Humans; Biosensing Techniques; Escherichia coli; Microfluidics; Klebsiella pneumoniae; Equipment Design; Lab-On-A-Chip Devices
PubMed: 38663322
DOI: 10.1016/j.bios.2024.116301 -
Scientific Reports Apr 2024Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor...
Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay. It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure. A novel assay makes use of photosensitizers doped into a polystyrene matrix. A thin layer of this composite material is coated on the bottom of regular cell culture ware showing perfect biocompatibility. When adherent cells are grown on this coating, resonant excitation of the photosensitizer induces a very local generation of O, which kills the cells residing at the site of illumination. Cells outside the site of illumination are not harmed. When excitation of the photosensitizer is conducted by microscopic illumination, high-precision wounding in any size and geometry is available even in microfluidic channels. Besides proof-of-concept experiments, this study gives further insight into the mechanism of photosensitizer-mediated cell wounding.
Topics: Photosensitizing Agents; Wound Healing; Cell Culture Techniques; Microfluidics; Cell Movement
PubMed: 38643292
DOI: 10.1038/s41598-024-59564-9 -
European Journal of Pharmaceutical... Jun 2024Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that...
Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that concomitantly administered pharmaceuticals inhibit one another's CYP metabolism. The most severe form of CYP interactions is irreversible inhibition, which results in permanent inactivation of the critical CYP pathway and is only restored by de novo synthesis of new functional enzymes. In this study, we conceptualize a microfluidic approach to mechanistic CYP inhibition studies using human liver microsomes (HLMs) immobilized onto the walls of a polymer micropillar array. We evaluated the feasibility of these HLM chips for CYP inhibition studies by establishing the stability and the enzyme kinetics for a CYP2C9 model reaction under microfluidic flow and determining the half-maximal inhibitory concentrations (IC) of three human CYP2C9 inhibitors (sulfaphenazole, tienilic acid, miconazole), including evaluation of their inhibition mechanisms and nonspecific microsomal binding on chip. Overall, the enzyme kinetics of CYP2C9 metabolism on the HLM chip (K = 127 ± 55 µM) was shown to be similar to that of static HLM incubations (K = 114 ± 14 µM) and the IC values toward CYP2C9 derived from the microfluidic assays (sulfaphenazole 0.38 ± 0.09 µM, tienilic acid 3.4 ± 0.6 µM, miconazole 0.54 ± 0.09 µM) correlated well with those determined using current standard IC shift assays. Most importantly, the HLM chip could distinguish between reversible (sulfaphenazole) and irreversible (tienilic acid) enzyme inhibitors in a single, automated experiment, indicating the great potential of the HLM chip to simplify current workflows used in mechanistic CYP inhibition studies. Furthermore, the results suggest that the HLM chip can also identify irreversible enzyme inhibitors, which are not necessarily resulting in a time-dependent inhibition (like suicide inhibitors), but whose inhibition mechanism is based on other kind of covalent or irreversible interaction with the CYP system. With our HLM chip approach, we could identify miconazole as such a compound that nonselectively inhibits the human CYP system with a prolonged, possibly irreversible impact in vitro, even if it is not a time-dependent inhibitor according to the IC shift assay.
Topics: Humans; Microsomes, Liver; Cytochrome P-450 CYP2C9; Kinetics; Cytochrome P-450 Enzyme Inhibitors; Miconazole; Enzymes, Immobilized; Cytochrome P-450 CYP2C9 Inhibitors; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Sulfaphenazole; Microfluidics
PubMed: 38641124
DOI: 10.1016/j.ejps.2024.106773 -
Journal of Nanobiotechnology Apr 2024Pathological conditions linked to shear stress have been identified in hematological diseases, cardiovascular diseases, and cancer. These conditions often exhibit...
Pathological conditions linked to shear stress have been identified in hematological diseases, cardiovascular diseases, and cancer. These conditions often exhibit significantly elevated shear stress levels, surpassing 1000 dyn/cm in severely stenotic arteries. Heightened shear stress can induce mechanical harm to endothelial cells, potentially leading to bleeding and fatal consequences. However, current technology still grapples with limitations, including inadequate flexibility in simulating bodily shear stress environments, limited range of shear stress generation, and spatial and temporal adaptability. Consequently, a comprehensive understanding of the mechanisms underlying the impact of shear stress on physiological and pathological conditions, like thrombosis, remains inadequate. To address these limitations, this study presents a microfluidic-based shear stress generation chip as a proposed solution. The chip achieves a substantial 929-fold variation in shear stress solely by adjusting the degree of constriction in branch channels after PDMS fabrication. Experiments demonstrated that a rapid increase in shear stress up to 1000 dyn/cm significantly detached 88.2% cells from the substrate. Long-term exposure (24 h) to shear stress levels below 8.3 dyn/cm did not significantly impact cell growth. Furthermore, cells exposed to shear stress levels equal to or greater than 8.3 dyn/cm exhibited significant alterations in aspect ratio and orientation, following a normal distribution. This microfluidic chip provides a reliable tool for investigating cellular responses to the wide-ranging shear stress existing in both physiological and pathological flow conditions.
Topics: Humans; Microfluidics; Endothelial Cells; Cell Line; Thrombosis; Stress, Mechanical
PubMed: 38632623
DOI: 10.1186/s12951-024-02334-6 -
Advanced Science (Weinheim,... Jun 2024The ability to precisely control in vitro enzymatic reactions in synthetic cells plays a crucial role in the bottom-up design of artificial cell models that can...
The ability to precisely control in vitro enzymatic reactions in synthetic cells plays a crucial role in the bottom-up design of artificial cell models that can recapitulate the key cellular features and functions such as metabolism. However, integration of enzymatic reactions has been limited to bulk or microfluidic emulsions without a membrane, lacking the ability to design more sophisticated higher-order artificial cell communities for reconstituting spatiotemporal biological information at multiple length scales. Herein, droplet microfluidics is utilized to synthesize artificial cell-like polymersomes with distinct molecular permeability for spatiotemporal control of enzymatic reactions driven by external signals and fuels. The presence of a competing reverse enzymatic reaction that depletes the active substrates is shown to enable demonstration of fuel-driven formation of sub-microcompartments within polymersomes as well as realization of out-of-equilibrium systems. In addition, the different permeability characteristics of polymersome membranes are exploited to successfully construct a programmable enzymatic reaction network that mimics cellular communication within a heterogeneous cell community through selective molecular transport.
Topics: Artificial Cells; Polymers; Microfluidics; Enzymes
PubMed: 38627986
DOI: 10.1002/advs.202305760 -
International Journal of Nanomedicine 2024Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose...
PURPOSE
Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration.
METHODS
Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis.
RESULTS
The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models.
CONCLUSION
Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.
Topics: Humans; Liposomes; Tumor Suppressor Protein p53; Cell Line, Tumor; Microfluidics; Proto-Oncogene Proteins c-mdm2; Apoptosis; Eye Diseases; Imidazoles; Piperazines
PubMed: 38623081
DOI: 10.2147/IJN.S452134 -
Scientific Reports Apr 2024Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a...
Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.
Topics: Microfluidics; Operating Rooms; Ciliophora
PubMed: 38622246
DOI: 10.1038/s41598-024-59286-y -
Scientific Reports Apr 2024Rapid mixing and precise timing are key for accurate biomedical assay measurement, particularly when the result is determined as the rate of a reaction: for example...
Rapid mixing and precise timing are key for accurate biomedical assay measurement, particularly when the result is determined as the rate of a reaction: for example rapid immunoassay in which the amount of captured target is kinetically determined; determination of the concentration of an enzyme or enzyme substrate; or as the final stage in any procedure that involves a capture reagent when an enzyme reaction is used as the indicator. Rapid mixing and precise timing are however difficult to achieve in point-of-care devices designed for small sample volumes and fast time to result. By using centrifugal microfluidics and transposing the reaction surface from a chamber to a single mm-scale bead we demonstrate an elegant and easily manufacturable solution. Reagents (which may be, for example, an enzyme, enzyme substrate, antibody or antigen) are immobilised on the surface of a single small bead (typically 1-2 mm in diameter) contained in a cylindrical reaction chamber subjected to periodically changing rotational accelerations which promote both mixing and uniform mass-transfer to the bead surface. The gradient of Euler force across the chamber resulting from rotational acceleration of the disc, dΩ/dt, drives circulation of fluid in the chamber. Oscillation of Euler force by oscillation of rotational acceleration with period, T, less than that of the hydrodynamic relaxation time of the fluid, folds the fluid streamlines. Movement of the bead in response to the fluid and the changing rotational acceleration provides a dynamically changing chamber shape, further folding and expanding the fluid. Bead rotation and translation driven by fluid flow and disc motion give uniformity of reaction over the surface. Critical parameters for mixing and reaction uniformity are the ratio of chamber radius to bead radius, r/r, and the product Tr(dΩ/dt), of oscillation period and Euler force gradient across the fluid. We illustrate application of the concept using the reaction of horse radish peroxidase (HRP) immobilised on the bead surface with its substrate tetramethylbenzidine (TMB) in solution. Acceleration from rest to break a hydrophobic valve provided precise timing for TMB contact with the bead. Solution uniformity from reaction on the surface of the bead in volumes 20-50 uL was obtained in times of 2.5 s or less. Accurate measurement of the amount of surface-bound HRP by model fitting to the measured kinetics of colour development at 10 s intervals is demonstrated.
Topics: Microfluidics; Antibodies; Antigens; Point-of-Care Systems; Hydrophobic and Hydrophilic Interactions
PubMed: 38622241
DOI: 10.1038/s41598-024-58720-5 -
International Journal of Molecular... Apr 2024The implantation of human embryos is a complex process involving various cytokines and receptors expressed by both endometrium and embryos. However, the role of...
Detection of Interleukin-1 β (IL-1β) in Single Human Blastocyst-Conditioned Medium Using Ultrasensitive Bead-Based Digital Microfluidic Chip and Its Relationship with Embryonic Implantation Potential.
The implantation of human embryos is a complex process involving various cytokines and receptors expressed by both endometrium and embryos. However, the role of cytokines produced by a single embryo in successful implantation is largely unknown. This study aimed to investigate the role of IL-1β expressed in a single-embryo-conditioned medium (ECM) in embryo implantation. Seventy samples of single ECM were analyzed by a specially designed magnetic-beads-based microfluidic chip from 15 women. We discovered that IL-1β level increased as the embryo developed, and the difference was significant. In addition, receiver operator characteristic (ROC) curves analysis showed a higher chance of pregnancy when the IL-1β level on day 5 ECM was below 79.37 pg/mL and the difference between day 5 and day 3 was below 24.90 pg/mL. Our study discovered a possible association between embryonic proteomic expression and successful implantation, which might facilitate single-embryo transfer in the future by helping clinicians identify the embryo with the greatest implantation potential.
Topics: Pregnancy; Humans; Female; Culture Media, Conditioned; Interleukin-1beta; Microfluidics; Proteomics; Blastocyst; Embryo Implantation; Cytokines
PubMed: 38612816
DOI: 10.3390/ijms25074006 -
International Journal of Molecular... Mar 2024Long COVID (LongC) is associated with a myriad of symptoms including cognitive impairment. We reported at the beginning of the COVID-19 pandemic that neuronal-enriched...
Long COVID (LongC) is associated with a myriad of symptoms including cognitive impairment. We reported at the beginning of the COVID-19 pandemic that neuronal-enriched or L1CAM+ extracellular vesicles (nEVs) from people with LongC contained proteins associated with Alzheimer's disease (AD). Since that time, a subset of people with prior COVID infection continue to report neurological problems more than three months after infection. Blood markers to better characterize LongC are elusive. To further identify neuronal proteins associated with LongC, we maximized the number of nEVs isolated from plasma by developing a hybrid EV Microfluidic Affinity Purification (EV-MAP) technique. We isolated nEVs from people with LongC and neurological complaints, AD, and HIV infection with mild cognitive impairment. Using the OLINK platform that assesses 384 neurological proteins, we identified 11 significant proteins increased in LongC and 2 decreased (BST1, GGT1). Fourteen proteins were increased in AD and forty proteins associated with HIV cognitive impairment were elevated with one decreased (IVD). One common protein (BST1) was decreased in LongC and increased in HIV. Six proteins (MIF, ENO1, MESD, NUDT5, TNFSF14 and FYB1) were expressed in both LongC and AD and no proteins were common to HIV and AD. This study begins to identify differences and similarities in the neuronal response to LongC versus AD and HIV infection.
Topics: Humans; Post-Acute COVID-19 Syndrome; COVID-19; Alzheimer Disease; HIV Infections; Microfluidics; Pandemics; Extracellular Vesicles
PubMed: 38612641
DOI: 10.3390/ijms25073830