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Analytical Chemistry Jun 2024We describe micro- and nanoelectrode array analysis with an automated version of the array microcell method (AMCM). Characterization of hundreds of electrodes, with...
We describe micro- and nanoelectrode array analysis with an automated version of the array microcell method (AMCM). Characterization of hundreds of electrodes, with diameters ranging from 100 nm to 2 μm, was carried out by using AMCM voltammetry and chronoamperometry. The influence of solvent evaporation on mass transport in the AMCM pipette and the resultant electrochemical response were investigated, with experimental results supported by finite element method simulations. We also describe the application of AMCM to high-throughput single-entity electrochemistry in measurements of stochastic nanoparticle impacts. Collision experiments recorded 3270 single-particle events from 671 electrodes. Data collection parameters were optimized to enable these experiments to be completed in a few hours, and the collision transient sizes were analyzed with a U-Net deep learning model. Elucidation of collision transient sizes by histograms from these experiments was enhanced due to the large sample size possible with AMCM.
PubMed: 38780285
DOI: 10.1021/acs.analchem.4c01092 -
Journal of the Mechanical Behavior of... Feb 2023The characterization of soft tissues remains a vital need for various bioengineering and medical fields. Developing areas such as regenerative medicine, robot-aided... (Review)
Review
The characterization of soft tissues remains a vital need for various bioengineering and medical fields. Developing areas such as regenerative medicine, robot-aided surgery, and surgical simulations all require accurate knowledge about the mechanical properties of soft tissues to replicate their mechanics. Mechanical properties can be characterized through several different characterization techniques such as atomic force microscopy, compression testing, and tensile testing. However, many of these methods contain considerable differences in ability to accurately characterize the mechanical properties of soft tissues. As a result of these variations, there are often discrepancies in the reported values for numerous studies. This paper reviews common characterization methods that have been applied to obtain the mechanical properties of soft tissues and highlights their advantages as well as disadvantages. The limitations, accuracies, repeatability, in-vivo testing capability, and types of properties measurable for each method are also discussed.
Topics: Microscopy, Atomic Force; Regenerative Medicine
PubMed: 36470112
DOI: 10.1016/j.jmbbm.2022.105575 -
Cells Oct 2023The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell...
The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for the cell analysis by combination with surface-enhanced Raman spectroscopy (SERS) and scanning ion-conducting microscopy (SICM). Unlike the majority of other designs, the pipette opening in our case remains uncovered, which is important for SICM. SERS-active Ag nanoparticles on the pipette surface are formed by vacuum-thermal evaporation followed by annealing. An array of nanoparticles had a diameter on the order of 36 nm and spacing of 12 nm. A two-particle model based on Laplace equations is used to calculate a theoretical enhancement factor (EF). The surface morphology of the samples is investigated by scanning electron microscopy while SICM is used to reveal the surface topography, to evaluate Young's modulus of living cells and to control an injection of the SERS-active pipettes into them. A Raman microscope-spectrometer was used to collect characteristic SERS spectra of cells and cell components. Local Raman spectra were obtained from the cytoplasm and nucleus of the same HEK-293 cancer cell. The EF of the SERS-active pipette was 7 × 10. As a result, we demonstrate utilizing the silver-coated pipette for both the SICM study and the molecular composition analysis of cytoplasm and the nucleus of living cells by SERS. The probe localization in cells is successfully achieved.
Topics: Humans; Silver; Metal Nanoparticles; HEK293 Cells; Microscopy, Electron, Scanning; Single-Cell Analysis; Ions
PubMed: 37947599
DOI: 10.3390/cells12212521 -
Diagnostics (Basel, Switzerland) Feb 2022Nuclear magnetic resonance (NMR) metabolomics is currently popular enough to attract both specialized and non-specialized NMR groups involving both analytical trained...
Nuclear magnetic resonance (NMR) metabolomics is currently popular enough to attract both specialized and non-specialized NMR groups involving both analytical trained personnel and newcomers, including undergraduate students. Recent interlaboratory studies performed by established NMR metabolomics groups demonstrated high reproducibility of the state-of-the-art NMR equipment and SOPs. There is, however, no assessment of NMR reproducibility when mixing both analytical experts and newcomers. An interlaboratory assessment of NMR quantitation reproducibility was performed using two NMR instruments belonging to different laboratories and involving several operators with different backgrounds and metabolomics expertise for the purpose of assessing the limiting factors for data reproducibility in a multipurpose NMR environment. The variability induced by the operator, automatic pipettes, NMR tubes and NMR instruments was evaluated in order to assess the limiting factors for quantitation reproducibility. The results estimated the expected reproducibility data in a real-life multipurpose NMR laboratory to a maximum 4% variability, demonstrating that the current NMR equipment and SOPs may compensate some of the operator-induced variability.
PubMed: 35328113
DOI: 10.3390/diagnostics12030559 -
MedRxiv : the Preprint Server For... Jul 2021There is an urgent need to expand testing for SARS-CoV-2 and other respiratory pathogens as the global community struggles to control the COVID-19 pandemic. Current...
There is an urgent need to expand testing for SARS-CoV-2 and other respiratory pathogens as the global community struggles to control the COVID-19 pandemic. Current diagnostic methods can be affected by supply chain bottlenecks and require the assistance of medical professionals, impeding the implementation of large-scale testing. Self-collection of saliva may solve these problems, as it can be completed without specialized training and uses generic materials. In this study, we observed thirty individuals who self-collected saliva using four different collection devices and analyzed their feedback. Two of these devices, a funnel and bulb pipette, were used to evaluate at-home saliva collection by 60 individuals. All devices enabled the safe, unsupervised self-collection of saliva. The quantity and quality of the samples received were acceptable for SARS-CoV-2 diagnostic testing, as determined by RNase P detection. Here, we demonstrate inexpensive, generic, buffer free collection devices suitable for unsupervised and home saliva self-collection.
PubMed: 33564787
DOI: 10.1101/2021.02.01.21250946 -
Neuron Sep 2017Neuronal membrane potential is of fundamental importance for the mechanistic understanding of brain function. This review discusses progress in whole-cell patch-clamp... (Review)
Review
Neuronal membrane potential is of fundamental importance for the mechanistic understanding of brain function. This review discusses progress in whole-cell patch-clamp recordings for low-noise measurement of neuronal membrane potential in awake behaving animals. Whole-cell recordings can be combined with two-photon microscopy to target fluorescently labeled neurons, revealing cell-type-specific membrane potential dynamics of retrogradely or genetically labeled neurons. Dual whole-cell recordings reveal behavioral modulation of membrane potential synchrony and properties of synaptic transmission in vivo. Optogenetic manipulations are also readily integrated with whole-cell recordings, providing detailed information about the effect of specific perturbations on the membrane potential of diverse types of neurons. Exciting developments for future behavioral experiments include dendritic whole-cell recordings and imaging, and use of the whole-cell recording pipette for single-cell delivery of drugs and DNA, as well as RNA expression profiling. Whole-cell recordings therefore offer unique opportunities for investigating the neuronal circuits and synaptic mechanisms driving membrane potential dynamics during behavior.
Topics: Animals; Behavior, Animal; Membrane Potentials; Microscopy, Fluorescence, Multiphoton; Neurons; Optogenetics; Patch-Clamp Techniques
PubMed: 28910617
DOI: 10.1016/j.neuron.2017.06.049 -
Nature Communications Feb 2021Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological...
Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.
Topics: Adult; Aged; Animals; Automation; Brain; Deep Learning; Electrophysiology; Female; Humans; Image Processing, Computer-Assisted; Male; Middle Aged; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Software; Video Recording
PubMed: 33568670
DOI: 10.1038/s41467-021-21291-4 -
Journal of Mass Spectrometry and... Nov 2023Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as... (Review)
Review
Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as it is generally accepted that variations exist, but thus far, there have been limited studies on the extent of these variations in practice. In this mini-review, we highlight how manual pipetting is a key technique in the laboratory, and, although simple, inaccuracy and imprecision exist. If variations are not adequately addressed, errors can be compounded and consequently compromise data quality. Determination of the accuracy and precision of manual pipetting is straightforward, and here we review two common approaches that use gravimetry and spectrophotometry as readouts. We also provide detailed protocols for determination of accuracy and precision using manual single and multi-channel pipettes. These simple-to-use methods can be used by any laboratory for competency training and regular checks. Having a common protocol for evaluation of variation will also enable cross-laboratory comparison and potentially facilitate establishment of a reference value of acceptable ranges for operator error. Such a value could be of relevance to the scientific community for benchmarking and assuring good laboratory practice.
PubMed: 37841753
DOI: 10.1016/j.jmsacl.2023.09.001 -
BMC Infectious Diseases Mar 2022There is an urgent need to expand testing for SARS-CoV-2 and other respiratory pathogens as the global community struggles to control the COVID-19 pandemic. Current...
BACKGROUND
There is an urgent need to expand testing for SARS-CoV-2 and other respiratory pathogens as the global community struggles to control the COVID-19 pandemic. Current diagnostic methods can be affected by supply chain bottlenecks and require the assistance of medical professionals, impeding the implementation of large-scale testing. Self-collection of saliva may solve these problems, as it can be completed without specialized training and uses generic materials.
METHODS
We observed 30 individuals who self-collected saliva using four different collection devices and analyzed their feedback. Two of these devices, a funnel and bulb pipette, were used to evaluate at-home saliva collection by 60 individuals. SARS-CoV-2-spiked saliva samples were subjected to temperature cycles designed to simulate the conditions the samples might be exposed to during the summer and winter seasons and sensitivity of detection was evaluated.
RESULTS
All devices enabled the safe, unsupervised self-collection of saliva. The quantity and quality of the samples received were acceptable for SARS-CoV-2 diagnostic testing, as determined by human RNase P detection. There was no significant difference in SARS-CoV-2 nucleocapsid gene (N1) detection between the freshly spiked samples and those incubated with the summer and winter profiles.
CONCLUSION
We demonstrate inexpensive, generic, buffer free collection devices suitable for unsupervised and home saliva self-collection.
Topics: COVID-19; Humans; Nucleocapsid Proteins; Pandemics; SARS-CoV-2; Saliva
PubMed: 35337266
DOI: 10.1186/s12879-022-07285-7 -
Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay.Biological & Pharmaceutical Bulletin 2018Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To... (Review)
Review
Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To understand this complex and dynamic system, artificial lipid bilayer membranes have served as unprecedented tools for experimental examination, in which some aspects of biomembrane features have been extracted, and to which various methodologies have been applied. Among the lipid bilayers involving liposomes, planar lipid bilayers and nanodiscs, recent developments of lipid bilayer methods and the results of our channel studies are reviewed herein. Principles and techniques of bilayer formation are summarized, which have been extended to the current techniques, where a bilayer is formed from lipid-coated water-in-oil droplets (water-in-oil bilayer). In our newly developed method, termed the contact bubble bilayer (CBB) method, a water bubble is blown from a pipette into a bulk oil phase, and monolayer-lined bubbles are docked to form a bilayer through manipulation by pipette. An asymmetric bilayer can be readily formed, and changes in composition in one leaflet were possible. Taking advantage of the topological configuration of the CBB, such that the membrane's hydrophobic interior is contiguous with the surrounding bulk organic phase, oil-dissolved substances such as cholesterol were delivered directly to the bilayer interior to perfuse around the membrane-embedded channels (membrane perfusion), and current recordings in the single-channel allowed detection of immediate changes in the channels' response to cholesterol. Chemical and mechanical manipulation in each monolayer (monolayer technology) allows the examination of dynamic channel-membrane interplay.
Topics: Animals; Cell Membrane; Humans; Hydrophobic and Hydrophilic Interactions; Ion Channels; Lipid Bilayers; Membranes
PubMed: 29491206
DOI: 10.1248/bpb.b17-00708