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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 -
Journal of Assisted Reproduction and... Jul 2022Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual...
PURPOSE
Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection.
METHODS
The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes).
RESULTS
Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection.
CONCLUSION
This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.
Topics: Animals; Blastocyst; Male; Mice; Microinjections; Oocytes; Polymerization; Semen; Sperm Injections, Intracytoplasmic
PubMed: 35552947
DOI: 10.1007/s10815-022-02485-1 -
Cold Spring Harbor Protocols Aug 2017This protocol describes an example of complete zygote enucleation and transplantation of male and female pronuclei; however, single pronuclei can also be removed and...
This protocol describes an example of complete zygote enucleation and transplantation of male and female pronuclei; however, single pronuclei can also be removed and transplanted. In this method, pronuclei are removed without penetrating the plasma membrane of the zygote. Instead, they are withdrawn individually or together into a membrane-bound karyoplast that can then be fused with a recipient enucleated zygote using inactivated Sendai virus or electrofusion. Preincubation of the embryos in the presence of the cytoskeletal inhibitors cytochalasin B and colcemid is critical for the survival of the embryos during this microsurgical procedure. The protocol is divided into five parts: (1) isolating embryos, (2) making an enucleation/injection pipette, (3) enucleating a zygote, (4) preparing inactivated Sendai virus, and (5) introducing pronuclei into enucleated zygotes.
Topics: Animals; Cell Fusion; Cell Nucleus; Female; Male; Mice; Microinjections; Nuclear Transfer Techniques; Zygote
PubMed: 28765300
DOI: 10.1101/pdb.prot094417 -
Journal of Visualized Experiments : JoVE Jul 2019The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as...
The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.
Topics: Animals; Cost-Benefit Analysis; Drosophila melanogaster; Female; Laboratories; Male
PubMed: 31403618
DOI: 10.3791/59613 -
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 -
Analytical and Bioanalytical Chemistry May 2018An accurate and precise 3 μL blood collection and dispensing system is presented for the preparation of dried blood spot (DBS) samples. Using end-to-end glass...
An accurate and precise 3 μL blood collection and dispensing system is presented for the preparation of dried blood spot (DBS) samples. Using end-to-end glass capillaries in conjugation with pre-punched DBS pads, a blood micro collection system was developed to eliminate the haematocrit dispersion, widely associated with DBS technology, while providing better levels of accuracy and precision during sample preparation. This methodology is compared to traditional micro-volume blood collection systems, such as a pipette and a digitally controlled analytical syringe. Results showed that % of recovery for the capillary methodology was closer to 100% across the three haematocrit (HCT) levels tested and when prepared by two users (98 to 100% for capillaries, 78 to 104% for pipette and 93 to 97% for digital syringe) attesting a higher accuracy. Additionally, by taking advantage of the capillary action mechanism to collect and dispense autonomously the desired volume of blood onto the DBS pad, coefficients of variation between two individuals were significantly lower than with standard methodologies (capillaries-0.05 to 0.77%, pipette-12.71 to 18.53% and digital syringe-0.72 to 1.77%). This alternate aspiration and dispensing methodology could be used by different users without compromising accuracy or precision when handling low volumes of blood during the pre-analytical steps. Graphical abstract Comparison of novel capillary dispensing methodology for dried blood spot sample preparation with pipette and digital syringe methodologies through accuracy and precision measurements of caffeine.
Topics: Blood Specimen Collection; Caffeine; Dried Blood Spot Testing; Equipment Design; Hematocrit; Humans; Reproducibility of Results; Sample Size
PubMed: 29623384
DOI: 10.1007/s00216-018-0993-y -
BMC Biotechnology Aug 2023The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by...
BACKGROUND
The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by mechanically removing cells through manual scraping with a sharp-edged tool. This step is traditionally done with pipette tips and is unsuitable for high-throughput assays, as the created scratches are highly variable in width and position. Commercially available solutions are often expensive, and require specific cultureware which might not be suitable for all studies.
RESULTS
In this study, we have developed a flexible cell scratch device comprising a single wounding tool, a guide and an imaging template for consistent and reproducible scratch assays in 96-well plates. Our results showed that the device produced a more consistent scratch profile compared to the conventional method of using pipette tips. The imaging template also allowed operators to easily locate and image the same region of interest at different time points, which potentially could be used for other assays.
CONCLUSIONS
Our flexible yet effective scratch device thus enables robust scratch assays that can be applied to different experimental needs, providing researchers with an easy and reliable tool for their studies.
Topics: Research Design; Biological Assay; High-Throughput Screening Assays; Wound Healing
PubMed: 37641063
DOI: 10.1186/s12896-023-00806-5 -
Journal of Separation Science Jul 2022The liquid extraction surface analysis technique is a new high-throughput instrument for ambient mass spectrometry. The benefits of the liquid extraction surface... (Review)
Review
The liquid extraction surface analysis technique is a new high-throughput instrument for ambient mass spectrometry. The benefits of the liquid extraction surface analysis-mass spectrometry approach are the high throughput screening of samples and the absence of sample preparation. liquid extraction surface analysis-mass spectrometry also consumes less solvent for extraction, making it more environmentally friendly and there is no substrate restriction. It utilizes advanced instrumentation like the use of robotic pipettes, nanoelectrospray systems, electronspray ionization chips which makes it highly efficient. In recent years, liquid extraction surface analysis-mass spectrometry has seen widespread use in a variety of analytical fields including drug metabolite analysis, mapping drug distribution in tissues, protein and lipid characterization, etc. In this review, we have summarized the basic working principles of the liquid extraction surface analysis-mass spectrometry approach in detail along with a detailed description of the recently reported applications in the analysis of proteins, lipids, drugs and foods. The investigated analytes along with detection methodologies and significant outcomes of various research reports have been presented with the help of tables. This tool has also been utilized in clinical investigations of biological fluids, fingerprint analysis and authentication of agarwood.
Topics: Mass Spectrometry; Proteins; Spectrometry, Mass, Electrospray Ionization
PubMed: 35579471
DOI: 10.1002/jssc.202100996 -
Journal of Research of the National... 2017
PubMed: 34877090
DOI: 10.6028/jres.122.002 -
BioRxiv : the Preprint Server For... Jan 2023Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular...
Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses ∼50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10 cm /s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, ∼20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.
PubMed: 36712045
DOI: 10.1101/2023.01.02.522313