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Methods in Molecular Biology (Clifton,... 2023Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector-carbon fiber...
Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector-carbon fiber electrode (CFE)-is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high-resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance, and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here, we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.
Topics: Carbon Fiber; Catecholamines; Catechols; Chromaffin Cells; Electrochemistry; Exocytosis
PubMed: 36205899
DOI: 10.1007/978-1-0716-2671-9_17 -
Journal of Separation Science Mar 2016Disposable pipette tips extraction consists of a solid-phase extraction in which the sorbent is poorly dispersed in a pipette tip, which allows a quick and dynamic... (Review)
Review
Disposable pipette tips extraction consists of a solid-phase extraction in which the sorbent is poorly dispersed in a pipette tip, which allows a quick and dynamic contact between the aspirated analyte from the sample and the solid phase. It is a technique used particularly in food and forensic analysis, since it requires a small amount of sample and solvent. This article highlights the principles, advantages and disadvantages of the disposable pipette tips extraction method and reviews recent applications.
Topics: Disposable Equipment; Food Analysis; Forensic Toxicology; Humans; Pesticide Residues; Solid Phase Extraction
PubMed: 27027593
DOI: 10.1002/jssc.201500932 -
Cell Calcium Nov 2021Sea urchin sperm swimming is regulated by speract, a decapeptide released from egg jelly that induces chemotaxis and triggers membrane potential (Em) changes,...
Sea urchin sperm swimming is regulated by speract, a decapeptide released from egg jelly that induces chemotaxis and triggers membrane potential (Em) changes, intracellular increases in cyclic nucleotides (cGMP, cAMP), pH (pH) and calcium concentration ([Ca]). The identity of the ionic transporters associated with the [Ca] changes required for chemotaxis is not fully known. CatSper, a sperm exclusive Ca channel has been detected by proteomic analysis and immunofluorescence in sea urchin sperm and there is evidence for its involvement in chemotaxis. This work presents an electrophysiological characterization of a CatSper channel in sea urchin sperm. By swelling sperm suspending them in 10-fold diluted artificial sea water (ASW) we achieve on-cell patch-clamp recordings that document a mildly voltage and pH dependent Na permeable channel (in absence of divalent ions in the pipette), sensitive to speract, and blocked by Mibefradil (Mibe), NNC55-0396 (NNC) and RU1968 (RU) resembling CatSper. We also recorded a voltage dependent Cl channel inhibited by Niflumic Acid and the TMEM16A blocker.
Topics: Animals; Calcium; Calcium Channels; Male; Proteomics; Sea Urchins; Sperm Motility; Spermatozoa
PubMed: 34509139
DOI: 10.1016/j.ceca.2021.102466 -
Ultrasonics Sonochemistry Dec 2023This article proposes a substantive scope and scenario of a laboratory class that introduces students to the field of sonochemistry. The class requires only basic...
This article proposes a substantive scope and scenario of a laboratory class that introduces students to the field of sonochemistry. The class requires only basic laboratory equipment - typical laboratory glassware like graduated pipettes and conical flasks, as well as simple inorganic chemicals. It is designed to acquaint students with fundamental aspects of sonochemistry. In the qualitative aspect, they will conduct and observe some sonochemical reactions like a synthesis of hydrogen peroxide and ultrasound-assisted degradation of toxic chromates(VI) which will demonstrate the indirect consequences of water sonolysis which is the most basic sonochemical reaction, as well as they will illustrate the applications of sonochemistry. In the quantitative aspect, students will learn about how to measure the power of ultrasound and the sonochemical efficiency of the reaction and will conduct experiments allowing for the calculation of these parameters. Finally, an introduction to and demonstration of the sonocatalytic effect is planned. An evaluation system, consisting of a report and test, is also proposed.
PubMed: 37976564
DOI: 10.1016/j.ultsonch.2023.106691 -
Micromachines May 2022Programmed mini-pumps play a significant role in various fields, such as chemistry, biology, and medicine, to transport a measured volume of liquid, especially in the...
Programmed mini-pumps play a significant role in various fields, such as chemistry, biology, and medicine, to transport a measured volume of liquid, especially in the current detection of COVID-19 with PCR. In view of the cost of the current automatic pipetting pump being higher, which is difficult to use in a regular lab, this paper designed and assembled a three-dimensional programmed mini-pump with the common parts and components, such as PLC controller, motor, microinjector, etc. With the weighting calibration before and after pipetting operation, the error of the pipette in 10 μL (0.2%), 2 μL (1.8%), and 1 μL (5.6%) can be obtained. Besides, the contrast test between three-dimensional programmed mini-pump and manual pipette was conducted with the ORF1ab and pGEM-3Zf (+) genes in qPCR. The results proved that the custom-made three-dimensional programmed mini-pump has a stronger reproducibility compared with manual pipette (ORF1ab: 24.06 ± 0.33 vs. 23.50 ± 0.58, = 0.1014; pGEM-3Zf (+): 11.83.06 ± 0.24 vs. 11.50 ± 0.34, = 0.8779). These results can lay the foundation for the functional, fast, and low-cost programmed mini-pump in PCR or other applications for trace measurements.
PubMed: 35630239
DOI: 10.3390/mi13050772 -
The Analyst Mar 2022Resistive pulse sensors have been used to characterise everything from whole cells to small molecules. Their integration into microfluidic devices has simplified sample...
Resistive pulse sensors have been used to characterise everything from whole cells to small molecules. Their integration into microfluidic devices has simplified sample handling whilst increasing throughput. Typically, these devices measure a limited size range, making them prone to blockages in complex sample matrixes. To prolong their life and facilitate their use, samples are often filtered or prepared to match the sample with the sensor diameter. Here, we advance our tuneable flow resistive pulse sensor which utilises additively manufactured parts. The sensor allows parts to be easily changed, washed and cleaned, its simplicity and versatility allow components from existing nanopore fabrication techniques such as glass pipettes to be integrated into a single device. This creates a multi-nanopore sensor that can simultaneously measure particles from 0.1 to 30 μm in diameter. The orientation and controlled fluid flow in the device allow the sensors to be placed in series, whereby smaller particles can be measured in the presence of larger ones without the risk of being blocked. We illustrate the concept of a multi-pore flow resistive pulse sensor, by combining an additively manufactured tuneable sensor, termed sensor 1, with a fixed nanopore sensor, termed sensor 2. Sensor 1 measures particles as small as 10 μm in diameter, whilst sensor 2 can be used to characterise particles as small as 100 nm, depending upon its dimensions. We illustrate the dual pore sensor by measuring 1 and 10 μm particles simultaneously.
Topics: Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Microfluidics; Nanopores; Particle Size
PubMed: 35244649
DOI: 10.1039/d2an00128d -
Journal of Neuroscience Methods Sep 2019Single cell intracellular recordings in-vivo at deep brain structures are seldomly accompanied by nearby optogenetics or drug application. The use of such tools is... (Review)
Review
Single cell intracellular recordings in-vivo at deep brain structures are seldomly accompanied by nearby optogenetics or drug application. The use of such tools is limited as both light and drugs cannot penetrate deep inside brain tissue. Hence, the optical fiber or drug delivery pipette needs to be placed within the brain close to the recording pipette. So far, however, this has required highly accurate hardware to achieve. These complications have now been solved by new approaches enabling intracellular recordings both for optogenetics and pharmacological application by the use of a single manipulator. In this manuscript we review these technologies - their pros, cons and implications.
Topics: Animals; Brain; Drug Delivery Systems; Electrophysiological Phenomena; Neurosciences; Optogenetics; Patch-Clamp Techniques; Pharmacology
PubMed: 31288037
DOI: 10.1016/j.jneumeth.2019.108324 -
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 -
Micron (Oxford, England : 1993) Apr 2016Scanning ion-conductance microscopy (SICM) belongs to the family of scanning-probe microscopies. The spatial resolution of these techniques is limited by the size of the... (Review)
Review
Scanning ion-conductance microscopy (SICM) belongs to the family of scanning-probe microscopies. The spatial resolution of these techniques is limited by the size of the probe. In SICM the probe is a pipette, obtained by heating and pulling a glass capillary tubing. The size of the pipette tip is therefore an important parameter in SICM experiments. However, the characterization of the tip is not a consolidated routine in SICM experimental practice. In addition, potential and limitations of the different methods available for this characterization may not be known to all users. We present an overview of different methods for characterizing size and geometry of the pipette tip, with the aim of collecting and facilitating the use of several pieces of information appeared in the literature in a wide interval of time under different disciplines. In fact, several methods that have been developed for pipettes used in cell physiology can be also fruitfully employed in the characterization of the SICM probes. The overview includes imaging techniques, such as scanning electron microscopy and atomic Force microscopy, and indirect methods, which measure some physical parameter related to the size of the pipette. Examples of these parameters are the electrical resistance of the pipette filled with a saline solution and the surface tension at the pipette tip. We discuss advantages and drawbacks of the methods, which may be helpful in answering a wide range of experimental questions.
PubMed: 26826619
DOI: 10.1016/j.micron.2016.01.002