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Scientific Reports Aug 2022Cross-contamination of biological samples during handling and preparation, is a major issue in laboratory setups, leading to false-positives or false-negatives. Sample...
Cross-contamination of biological samples during handling and preparation, is a major issue in laboratory setups, leading to false-positives or false-negatives. Sample carryover residue in pipette tips contributes greatly to this issue. Most pipette tips on the market are manufactured with hydrophobic polymers that are able to repel high surface tension liquids, yet they lack in performance when low surface tension liquids and viscous fluids are involved. Moreover, hydrophobicity of pipette tips can result in hydrophobic adsorption of biomolecules, causing inaccuracies and loss in precision during pipetting. Here we propose the use of lubricant-infused surface (LIS) technology to achieve omniphobic properties in pipette tips. Using a versatile and simple design, the inner lumen of commercially available pipette tips was coated with a fluorosilane (FS) layer using chemical vapor deposition (CVD). The presence of FS groups on the tips is confirmed by x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) tests. After lubrication of the tips through a fluorinated lubricant, the omniphobicity and repellent behaviour of the tips drastically enhanced which are revealed via static and hysteresis contact angle measurements. The repellency of the lubricant-infused pipette tips against physical adsorption is investigated through pipetting a food coloring dye as well as human blood samples and are compared to the untreated tips. The results show significantly less amount carryover residue when the lubricant-infused tips are utilized compared to commercially available ones. We also demonstrate the lubricant-infused tips reduce bacteria contamination of the inner lumen by 3 to 6-log (over 99%, depending on the tip size) after pipetting up and down the bacteria solution.
Topics: Humans; Adsorption; Hydrophobic and Hydrophilic Interactions; Lubricants; Lubrication; Surface Properties; Complex Mixtures
PubMed: 36008518
DOI: 10.1038/s41598-022-18756-x -
Theriogenology Jan 2020The safety of assisted reproductive technology (ART) is of frequent concern. Unfortunately, animal models for studying the safety of intracytoplasmic sperm injection...
The safety of assisted reproductive technology (ART) is of frequent concern. Unfortunately, animal models for studying the safety of intracytoplasmic sperm injection (ICSI) have limitations in mimicking human ICSI manipulations. As reported herein, we invented a modified holding pipette for mouse oocyte injection that resulted in the delivery of live pups. A modified holding pipette was prepared for mouse oocyte injection and was compared with the conventional pipette for human use and a trumpet-shaped pipette. After ICSI, the oocytes were cultured to cleavage embryos until fallopian transfer. The use of the trumpet-shaped holding pipette and the new modified holding pipette for mouse oocyte injection achieved comparable and satisfactory oocyte survival rates (83.44% and 85.71%, respectively) and embryo cleavage rates (41.98% and 42.42%, respectively), which were significantly higher than those obtained with the human egg-holding pipette (oocyte survival rate: 65.85%; embryo cleavage rate: 27.78%). After 13 embryos were transferred using each type of pipette, three live pups were produced with the new modified holding pipette, one was produced with the holding pipette for human use, and none were produced with the trumpet-shaped holding pipette. The modified holding pipette for oocyte injection is effective and very easy to prepare. Moreover, using this new method, we produced live pups, which will contribute to a useful animal model for safety studies of ICSI in the future.
Topics: Animals; Embryo Culture Techniques; Escherichia coli Proteins; Female; Membrane Transport Proteins; Mice; Pregnancy; Sperm Injections, Intracytoplasmic
PubMed: 31541783
DOI: 10.1016/j.theriogenology.2019.09.008 -
Analytica Chimica Acta Jul 2024The convenient preparation and application of functionalized organic-inorganic hybrid monolithic materials have obtained substantial interest in the pretreatment of...
BACKGROUND
The convenient preparation and application of functionalized organic-inorganic hybrid monolithic materials have obtained substantial interest in the pretreatment of complex samples by solid-phase extraction (SPE). Compared to the in-tube solid-phase microextraction in fused-silica capillaries, micro SPE in plastic pipette tips have fascinating merits for the easily operated enrichment of trace target analytes from biological samples. However, the poor compatibility of organic-inorganic hybrid monoliths with plastics leads to the rare appearance of commercial hybrid monolithic pipette tips (HMPTs). Therefore, how to synthesize the organic-inorganic hybrid monolithic materials with better extraction performance in plastic pipette tips becomes a challenge.
RESULTS
We develop a facile and cheap strategy to immobilize organic-inorganic hybrid monoliths in pipette tips. Melamine sponge was employed as the supporting skeleton to in situ assemble amine- and thiol-bifunctionalized hybrid monolithic material via "one pot" in a pipette tip, and gold nanoparticles (GNPs) and thiol-modified aptamer against human α-thrombin were sequentially attached to the hybrid monolith within the HMPTs. The average coverage density of the aptamer with GNPs as an intermediary reached as high as 818.5 pmol μL. The enriched thrombin concentration was determined by a sensitive enzymatic chromogenic assay with the limit of detection of 2 nM. The extraction recovery of thrombin at 10 nM in human serum was 86.1 % with a relative standard deviation of 6.1 %. This proposed protocol has been applied to the enrichment and determination of thrombin in real serum sample with strong anti-interference ability, low limit of detection and high recovery.
SIGNIFICANCE
The amine- and thiol-bifunctionalized HMPTs prepared with sponge as the skeleton frame provided a novel substrate material to decorate aptamers for efficient enrichment of proteins. This enlightens us that we can take advantage of the tunability of sponge assisted HMPTs to produce and tailor a variety of micro SPE pipette tips for broader applications on the analysis of trace targets in complex biological, clinic and environmental samples.
Topics: Triazines; Aptamers, Nucleotide; Humans; Thrombin; Gold; Metal Nanoparticles; Solid Phase Extraction
PubMed: 38834272
DOI: 10.1016/j.aca.2024.342780 -
Ultramicroscopy May 2021Scanning ion conductance microscopy (SICM) as an emerging non-contact scanning probe microscopy technique and featuring its strong in-situ detectability for soft and...
Scanning ion conductance microscopy (SICM) as an emerging non-contact scanning probe microscopy technique and featuring its strong in-situ detectability for soft and viscous samples, is increasingly used in biomedical and materials related studies. In SICM measurements, employing theta pipette as SICM probe to scan sample is an effective method to extend the applications of SICM for multi-parameter measurement. There are two crucial but still unclear issues that influence the reliability and accuracy of the usage of theta pipette in the SICM measurements, which are the safe feedback threshold and the horizontal measurement offset. In this work, aiming at the theta pipette configuration of SICM, we systematically investigated the two issues of the theta pipette by both finite element method (FEM) simulation and SICM experiments. The FEM analysis results show that the safe feedback threshold of the one side barrel of the theta pipette is above 99.5%, and the horizontal measurement offset is ~0.53 times of the inner radius of the probe tip. Based on this, we proposed an improved scanning method used by the theta pipette to solve the reliability and accuracy problems caused by the feedback threshold too close to the reference current (100%) and the measurement offset error at the tip radius level. Then through testing the polydimethylsiloxane (PDMS) samples with different embossed patterns with the improved method of SICM, we can conclude that the improved method can enhance the scanning reliability by adding the double barrels approaching process and increase the positioning accuracy by compensating an offset distance. The theoretical analysis and the improved scanning method in this work demonstrate more property and usage details of the theta pipette, and further improve the reliability and accuracy of the diversified multifunctional applications of the theta pipette for SICM to meet the increasingly complex and precise research needs.
PubMed: 33689886
DOI: 10.1016/j.ultramic.2021.113240 -
Methods in Enzymology 2019Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique... (Review)
Review
Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique features, address key limitations of conventional cell-based assays with their ensemble average response. While many different single cell techniques exist for suspension cultures, which can process and characterize large numbers of individual cells in rapid succession, the access to surface-immobilized cells in typical 2D and 3D culture environments remains challenging. Open space microfluidics has created new possibilities in this area, allowing for exclusive access to single cells in adherent cultures, even at high confluency. In this chapter, we briefly review new microtechnologies for the investigation of protein function in single adherent cells, and present an overview over related recent applications of the multifunctional pipette (Biopen), a microfluidic multi-solution dispensing system that uses hydrodynamic confinement in open volume environments in order to establish a superfusion zone over selected single cells in adherent cultures.
Topics: Animals; Cell Adhesion; Enzyme Assays; Equipment Design; Humans; Hydrodynamics; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Proteins; Single-Cell Analysis
PubMed: 31668227
DOI: 10.1016/bs.mie.2019.07.038 -
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 -
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 -
Sensors (Basel, Switzerland) Nov 2023A pipette-free and fully integrated device that can be used to accurately recognize the presence of infectious pathogens is an important and useful tool in point-of-care...
Pipette-Free and Fully Integrated Paper Device Employing DNA Extraction, Isothermal Amplification, and Carmoisine-Based Colorimetric Detection for Determining Infectious Pathogens.
A pipette-free and fully integrated device that can be used to accurately recognize the presence of infectious pathogens is an important and useful tool in point-of-care testing, particularly when aiming to decrease the unpredictable threats posed by disease outbreak. In this study, a paper device is developed to integrate the three main processes required for detecting infectious pathogens, including DNA extraction, loop-mediated isothermal amplification (LAMP), and detection. All key reagents, including sodium dodecyl sulfate (SDS), NaOH, LAMP reagents, and carmoisine, are placed on the paper device. The paper device is operated simply via sliding and folding without using any bulky equipment, and the results can be directly observed by the naked eye. The optimized concentrations of sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), and carmoisine were found to be 0.1%, 0.1 M, and 0.5 mg/mL, respectively. The paper device was used to detect at concentrations as low as 10 CFU/mL within 60 min. Also, spiked in milk was successfully detected using the paper device, demonstrating the feasible application in real sample analysis.
Topics: Colorimetry; Sodium Dodecyl Sulfate; Sodium Hydroxide; Nucleic Acid Amplification Techniques; DNA
PubMed: 38005500
DOI: 10.3390/s23229112 -
Sensors (Basel, Switzerland) Sep 2023A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on...
A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on experimenter experience, more and more researchers are focusing on patch-clamp automation. The existing automated patch-clamp system focuses on the process of completing the experiment; the detection method in each step is relatively simple, and the robustness of the complex brain film environment is lacking, which will increase the detection error in the microscopic environment, affecting the success rate of the automated patch clamp. To address these problems, we propose a method that is suitable for the contact between pipette tips and neuronal cells in automated patch-clamp systems. It mainly includes two key steps: precise positioning of pipettes and contact judgment. First, to obtain the precise coordinates of the tip of the pipette, we use the Mixture of Gaussian (MOG) algorithm for motion detection to focus on the tip area under the microscope. We use the object detection model to eliminate the encirclement frame of the pipette tip to reduce the influence of different shaped tips, and then use the sweeping line algorithm to accurately locate the pipette tip. We also use the object detection model to obtain a three-dimensional bounding frame of neuronal cells. When the microscope focuses on the maximum plane of the cell, which is the height in the middle of the enclosing frame, we detect the focus of the tip of the pipette to determine whether the contact between the tip and the cell is successful, because the cell and the pipette will be at the same height at this time. We propose a multitasking network CU-net that can judge the focus of pipette tips in complex contexts. Finally, we design an automated contact sensing process in combination with resistance constraints and apply it to our automated patch-clamp system. The experimental results show that our method can increase the success rate of pipette contact with cells in patch-clamp experiments.
Topics: Robotic Surgical Procedures; Robotics; Brain; Automation; Neurons
PubMed: 37836974
DOI: 10.3390/s23198144 -
Journal of Neuroscience Methods Jan 2021Whole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory...
BACKGROUND
Whole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode's aperture and a cell's membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane.
NEW METHOD
We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette's tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings deep in the brain of the living mouse.
RESULTS
This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26 % (n = 17/64) to 48 % (n = 32/66). Whole-cell recording yield improved from 10 % (n = 9/88) to 24 % (n = 18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex.
COMPARISON WITH EXISTING METHOD(S)
Current methods of in vivo whole-cell patch clamping do not synchronize the position of the pipette to motion of the cell.
CONCLUSIONS
This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.
Topics: Animals; Brain; Cell Membrane; Electrophysiological Phenomena; Mice; Neurons; Patch-Clamp Techniques
PubMed: 33242530
DOI: 10.1016/j.jneumeth.2020.109008