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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 -
Environmental Health Perspectives Dec 2023The rapid evolution of electronic cigarette (e-cigarette) products warrants surveillance of the differences in exposure across device types-modifiable devices (MODs),...
BACKGROUND
The rapid evolution of electronic cigarette (e-cigarette) products warrants surveillance of the differences in exposure across device types-modifiable devices (MODs), cartridge ("pod")-containing devices (PODs), disposable PODs (d-PODs)-and flavors of the products available on the market.
OBJECTIVE
This study aimed to measure and compare metal aerosol concentrations by device type and common flavors.
METHODS
We collected aerosol from 104 MODs, 67 PODs (four brands: JUUL, Bo, Suorin, PHIX), and 23 d-PODs (three brands: ZPOD, Bidi, Stig) via droplet deposition in a series of conical pipette tips. Metals and metalloids [aluminum (Al), arsenic (As), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), antimony (Sb), tin (Sn), and zinc (Zn)] were measured using inductively coupled plasma mass spectrometry (ICP-MS), results were log-transformed for statistical analysis, and concentrations are reported in aerosol units ().
RESULTS
Of the 12 elements analyzed, concentrations were statistically significantly higher in MOD devices, except for Co and Ni, which were higher in PODs and d-PODs. Of the POD brands analyzed, PHIX had the highest median concentrations among four metals (Al, Ni, Pb, and Sn) compared to the rest of the POD brands. According to POD flavor, seven metals were three to seven orders of magnitude higher in tobacco-flavored aerosol compared to those in mint and mango flavors. Among the d-POD brands, concentrations of four metals (Al, Cu, Ni, and Pb) were higher in the ZPOD brand than in Bidi Stick and Stig devices. According to d-POD flavor, only Cr concentrations were found to be statistically significantly higher in mint than tobacco-flavored d-PODs.
DISCUSSION
We observed wide variability in aerosol metal concentrations within and between the different e-cigarette device types, brands, and flavors. Overall, MOD devices generated aerosols with higher metal concentrations than PODs and d-PODs, and tobacco-flavored aerosols contained the highest metal concentrations. Continued research is needed to evaluate additional factors (i.e., nicotine type) that contribute to metal exposure from new and emerging e-cigarette devices in order to inform policy. https://doi.org/10.1289/EHP11921.
Topics: Electronic Nicotine Delivery Systems; Lead; Aluminum; Aerosols; Copper; Chromium
PubMed: 38048100
DOI: 10.1289/EHP11921 -
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 -
Fertility and Sterility Nov 2023To present a novel trophectoderm biopsy method, independent of laser pulses, using innovatively designed micropipettes on blastocysts at different stages and show...
OBJECTIVE
To present a novel trophectoderm biopsy method, independent of laser pulses, using innovatively designed micropipettes on blastocysts at different stages and show variable characteristics.
DESIGN
A step-by-step demonstration of this method with narrated video.
SETTING
In vitro laboratory fertilization.
PATIENTS
Individuals whose embryos underwent preimplantation genetic testing.
INTERVENTIONS
Trophectoderm biopsy is accomplished using micropipettes that contain a set of innovative designs. Both biopsy and holding pipettes are characterized by sharp, flat opening ends. The holding pipette is designed with an inclined plane on the outer wall surface of its opening end. It is used to help the biopsy pipette make contact with the holding pipette with increased stability, preventing slipping during the detachment of the trophectoderm cells. There is a narrow structure inside the biopsy pipette, which is designed to trap released fragments and prevent sample loss. A trophectoderm biopsy for fully expanded blastocysts starts from artificial shrinkage, followed by zona pellucida drilling. Then, 5-10 trophectoderm cells are aspirated into a biopsy pipette. The blastocyst is released from the holding pipette, the edge of the opening end of the biopsy pipette is tightly pressed onto the inclined plane of the holding pipette, and the biopsy pipette is flicked directly without laser pulses or pulling off the trophectoderm cells. The aspirated trophectoderm cells are subsequently detached by mechanical friction between the edges of the biopsy and holding pipettes. Apart from drilling the zona pellucida for fully expanded blastocysts, the remaining steps do not require lasers. For hatching (including peanut-shaped and 8-shaped) and hatched blastocysts, a trophectoderm biopsy is accomplished by aspirating the cells without securing the blastocyst with a holding pipette, followed by detachment using the direct flicking method.
MAIN OUTCOME MEASURES
The biopsy time, sample loss rate, successful DNA amplification rate, and survival rate.
RESULTS
The innovatively designed micropipettes facilitate the successful detachment of trophectoderm cells through a single direct flicking procedure. This eliminates thermal damage caused by laser pulses, notably simplifying operational steps and shortening the biopsy time. Significant differences were noted between the direct flicking and conventional methods, wherein laser pulses and pulling of trophectoderm cells are prerequisites for cell detachment. When comparing the average biopsy time of fully expanded blastocyst (61 ± 8s vs. 104 ± 9s, P<.05), peanut-shaped hatching blastocyst (35 ± 6s vs. 113 ± 13s, P<.05), 8-shaped hatching blastocyst (32 ± 4s vs. 59 ± 6s, P<.05), and hatched blastocyst (34 ± 4s vs. 67 ± 8s, P<.05), the direct flicking method shows a significantly decreased biopsy time. The narrow structure inside the biopsy pipette effectively prevents sample loss, showing a significantly reduced sample loss rate (0%) compared with the conventional biopsy method (18%) for trainees. Moreover, a satisfactory survival rate (100%) and successful DNA amplification rate (99.5%) were achieved using the direct flicking method.
CONCLUSIONS
This innovative trophectoderm biopsy method, independent of laser pulses, has wide applicability and a satisfactory, stable performance. Moreover, the simplicity of the method makes it easy to master.
Topics: Humans; Biopsy; Blastocyst; DNA; Fertilization in Vitro; Lasers
PubMed: 37487821
DOI: 10.1016/j.fertnstert.2023.07.010 -
Nature Protocols Aug 2023Neural circuits are assembled from an enormous variety of neuronal cell types. Although significant advances have been made in classifying neurons on the basis of... (Review)
Review
Neural circuits are assembled from an enormous variety of neuronal cell types. Although significant advances have been made in classifying neurons on the basis of morphological, molecular and electrophysiological properties, understanding how this diversity contributes to brain function during behavior has remained a major experimental challenge. Here, we present an extension to our previous protocol, in which we describe the technical procedures for performing juxtacellular opto-tagging of single neurons in freely moving mice by using Channelrhodopsin-2-expressing viral vectors. This method allows one to selectively target molecularly defined cell classes for in vivo single-cell recordings. The targeted cells can be labeled via juxtacellular procedures and further characterized via post-hoc morphological and molecular analysis. In its current form, the protocol allows multiple recording and labeling attempts to be performed within individual animals, by means of a mechanical pipette micropositioning system. We provide proof-of-principle validation of this technique by recording from Calbindin-positive pyramidal neurons in the mouse hippocampus during spatial exploration; however, this approach can easily be extended to other behaviors and cortical or subcortical areas. The procedures described here, from the viral injection to the histological processing of brain sections, can be completed in ~4-5 weeks.This protocol is an extension to: Nat. Protoc. 9, 2369-2381 (2014): https://doi.org/10.1038/nprot.2014.161.
Topics: Mice; Animals; Neurons; Pyramidal Cells; Brain
PubMed: 37420087
DOI: 10.1038/s41596-023-00842-7 -
Talanta Sep 2023An efficient sample preparation based on pipette tip microextraction that can be used for the analysis of retinol in human serum has been developed. Altogether, nine...
An efficient sample preparation based on pipette tip microextraction that can be used for the analysis of retinol in human serum has been developed. Altogether, nine commercial pipette tips were compared based on recovery, sample volume, use of organic solvent, handling difficulty, duration of the preparation process, price, and greenness of the method. Retinol acetate was used as the internal standard. The extraction efficiency for both compounds was evaluated to optimize and select the best pipette tip for sample preparation, which was the WAX-S XTR pipette tip containing an ion exchanger and salt. This tip combined solid phase extraction and salting-out assisted liquid‒liquid extraction. Satisfying recoveries of 100 and 80% for retinol and retinol acetate, respectively, and good repeatability were demonstrated. The action of this pipette tip was based on the clean-up workflow in which the interferences were retained on the sorbent. The presence of residual interferences in the extracted samples did not affect the HPLC separation of compounds of interest. The simplicity of the clean-up workflow reduced the time of the sample preparation compared to the bind-wash-elute counterpart workflow. The advantages of our technique are its environmental friendliness and cost effectiveness. The selected pipette tip with an excellent microextraction efficiency enables sample preparation in both clinical research and practice.
Topics: Humans; Vitamin A; Solid Phase Extraction; Diterpenes; Retinyl Esters; Sodium Chloride
PubMed: 37220691
DOI: 10.1016/j.talanta.2023.124689 -
Molecules (Basel, Switzerland) Jun 2023This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based... (Review)
Review
This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based materials are promising for analytical chemistry, including applications as sorbents in liquid chromatography. These sorbents can be functionalized to produce unique extraction or stationary phases. Additionally, graphene-based sorbents can be supported in various materials and have consequently been applied to produce various devices for sample preparation. Graphene-based sorbents are employed in diverse applications, including food and environmental LC analysis. This review summarizes the application of graphene-based materials in food and environmental water analysis in the last five years (2019 to 2023). Offline and online sample preparation methods, such as dispersive solid phase microextraction, stir bar sorptive extraction, pipette tip solid phase extraction, in-tube solid-phase microextraction, and others, are reviewed. The review also summarizes the application of the columns produced with graphene-based materials in separating food and water components and contaminants. Graphene-based materials have been reported as stationary phases for LC columns. Graphene-based stationary phases have been reported in packed, monolithic, and open tubular columns and have been used in LC and capillary electrochromatography modes.
Topics: Graphite; Chromatography, Liquid; Solid Phase Extraction; Solid Phase Microextraction; Water
PubMed: 37446796
DOI: 10.3390/molecules28135134 -
Nature Microbiology Dec 2023Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across...
Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it is time-intensive and resource-consuming. Here we describe the geometric viability assay (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing over 10-fold the time and consumables required. GVA computes a sample's viable cell count on the basis of the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with Gram-positive and Gram-negative planktonic bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis), biofilms and fungi (Saccharomyces cerevisiae). Laborious CFU experiments such as checkerboard assays, treatment time-courses and drug screens against slow-growing cells are simplified by GVA. The ease and low cost of GVA evinces that it can replace existing viability assays and enable viability measurements at previously impractical scales.
Topics: Colony Count, Microbial; Escherichia coli; Biofilms; Gram-Negative Bacteria; Pseudomonas aeruginosa
PubMed: 37919425
DOI: 10.1038/s41564-023-01513-9 -
The Review of Scientific Instruments Sep 2023Liquid handling is a necessary act to deal with liquid samples from scientific labs to industry. However, existing pipetting devices suffer from inaccuracy and low...
Liquid handling is a necessary act to deal with liquid samples from scientific labs to industry. However, existing pipetting devices suffer from inaccuracy and low precision when dealing with submicroliter liquids, which significantly affect their applications in low-volume quantitation. In this article, we present an automated liquid pipetting device that can aspirate liquid from microplates and dispense nanoliter droplets with high precision. Liquid aspiration is realized by using a micropump and a solenoid valve, and on-demand nanoliter droplet printing is realized by using a low-cost and interchangeable pipette tip combined with a piezoelectric actuator. Based on the microfluidic printing technology, the volumetric coefficient of variation of the dispensed liquid is less than 2% below 1 µl. A demonstration of concentration dilution for quantitative analysis has been successfully performed using the automated liquid pipetting device, demonstrating its potential in low-volume liquid handling for a wide range of biomedical applications.
PubMed: 37728420
DOI: 10.1063/5.0139565