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Journal of Chromatography. A Jul 2024A novel azo-linked porous organic polymer (AL-POP) was synthesized from caffeic acid and benzidine via an azo-coupling reaction and characterized by FTIR, SEM-EDS, BET,...
Melamine sponges incorporated azo-linked porous organic polymer as adsorbent for extraction and determination of six B vitamins using pipette tip micro solid-phase extraction.
A novel azo-linked porous organic polymer (AL-POP) was synthesized from caffeic acid and benzidine via an azo-coupling reaction and characterized by FTIR, SEM-EDS, BET, TGA, XRD and zeta potential analysis. AL-POPs were incorporated into melamine sponges and used for pipette tip micro solid-phase extraction (PT-MSPE) of six types of B vitamins (including thiamine, riboflavin, nicotinamide, pyridoxine, folic acid, and cyanocobalamin). After extraction, the samples were analyzed using high performance liquid chromatography-diode array detection (HPLC-DAD) system. The effect of AL-POP composition on the extraction efficiency (EE) of vitamins was investigated and benzidine to caffeic acid mol ratio of 1.5, 3.35 mmol of NaNO and reaction time of 8 h were selected as optimum conditions. The efficiency of the extraction process was improved by optimizing various parameters such as the amount of sorbent, pH and ionic strength of the sample, sample volume, number of sorption and desorption cycles, type of wash solvent, and type and volume of eluent solvent. Linearity (R≥0.9987), Limit of detection (LOD) (11.88-18.97 ng/mL), limit of quantification (LOQ) (39.62-63.23 ng/mL), and enrichment factor (EF) (1.27-4.31) were obtained using calibration curves plotted under optimum conditions. Recovery values of these six B vitamins in the spiked multivitamin syrup samples varied from 80.01% to 108.35%, with a relative standard deviation (RSD) below 5.44%. Eventually, the optimized method was successfully used to extract and quantify the B vitamins in multivitamin syrup and non-alcoholic beer.
Topics: Triazines; Limit of Detection; Porosity; Chromatography, High Pressure Liquid; Vitamin B Complex; Adsorption; Polymers; Azo Compounds; Solid Phase Microextraction; Solid Phase Extraction; Hydrogen-Ion Concentration
PubMed: 38788401
DOI: 10.1016/j.chroma.2024.464978 -
Talanta Aug 2024This paper presents a simple method for the surface modification of polypropylene pipette tips by adsorbing a photo-initiator, 2,2-dimethoxy-2-phenylacetophenone...
This paper presents a simple method for the surface modification of polypropylene pipette tips by adsorbing a photo-initiator, 2,2-dimethoxy-2-phenylacetophenone (DMPAP), to create reactive sites for the formation of a layer of ethylene dimethacrylate (EDMA) and subsequent monolith polymerization. The types of monomers and the degree of crosslinking dramatically affected the monolith shrinkage and detachment in unmodified tips. Effective surface modification for anchoring monolithic materials to pipette tips was achieved using 15 wt% DMPAP and 10 wt% EDMA in methanol with UV irradiation at 365 nm. The extraction of 5-hydroxyindoleacetic acid, serotonin, and bisphenol A (BPA) using methacrylate and activated charcoal composite monoliths was investigated in terms of breakthrough capacity. The application of monolithic pipette tip micro-solid-phase extraction followed by HPLC-UV was demonstrated for determining BPA leaching from baby-feeding bottles and canned foods. Wide linearity ranging from 0.1 to 100 ng mL (R = 0.9998) with good repeatability (% RSD = 3.9 %) and accuracy (% recovery = 93-106 %) was obtained. The limit of detection and limit of quantification were 0.084 and 0.280 ng mL, respectively. By varying the sample loading volume from 0.50 to 10.00 mL with eluting volume of 150 μL, a 2-to-52-fold pre-concentration factor was observed.
PubMed: 38781917
DOI: 10.1016/j.talanta.2024.126294 -
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 -
Lab on a Chip Jun 2024Microfluidic dispensing technologies often require additional equipment, posing challenges for their integration into point-of-care testing (POCT) applications. In...
Microfluidic dispensing technologies often require additional equipment, posing challenges for their integration into point-of-care testing (POCT) applications. In response to this challenge, we have developed a pipette-operable microfluidic device fabricated using 3D printing technology for precise liquid dispensing. This device features three reaction chambers and three distinct hydrophobic valves to control the flow direction of liquids. Through these valves, the pipette-operable microfluidic device can sequentially dispense and isolate the liquid into the three reaction chambers, allowing for the individual conduction of three distinct reactions. These hydrophobic valves, with optimized flow resistance and burst pressure, can sustain a volumetric flow rate of up to 25 μL s, making them compatible with a standard pipette, a syringe, or a dropper operation. Furthermore, the device is successfully used to operate with various liquids, including BSA, DMEM, FBS, plasma, and blood, representing that the device has the potential to be used for various applications. Additionally, distinct RT-LAMP primer sets have been incorporated for diagnosing SARS-CoV-2, influenza A, and influenza B within each chamber through lyophilization. This pipette-operable microfluidic device serves as a versatile tool for diagnosing these three diseases using a single loading process, with results readable by the naked eye or image assay within 30 minutes of incubation. Finally, the design concepts are extended to engineer a microfluidic device with 20 reaction chambers, offering significant potential for multi-disease diagnostics.
Topics: Lab-On-A-Chip Devices; Humans; Hydrophobic and Hydrophilic Interactions; SARS-CoV-2; Equipment Design; COVID-19; Point-of-Care Testing; Nucleic Acid Amplification Techniques; Microfluidic Analytical Techniques; Printing, Three-Dimensional
PubMed: 38758131
DOI: 10.1039/d4lc00209a -
Channels (Austin, Tex.) Dec 2024PIEZO1 and PIEZO2 are mechanically activated ion channels that confer mechanosensitivity to various cell types. PIEZO channels are commonly examined using the so-called...
PIEZO1 and PIEZO2 are mechanically activated ion channels that confer mechanosensitivity to various cell types. PIEZO channels are commonly examined using the so-called poking technique, where currents are recorded in the whole-cell configuration of the patch-clamp technique, while the cell surface is mechanically stimulated with a small fire-polished patch pipette. Currently, there is no gold standard for mechanical stimulation, and therefore, stimulation protocols differ significantly between laboratories with regard to stimulation velocity, angle, and size of the stimulation probe. Here, we systematically examined the impact of variations in these three stimulation parameters on the outcomes of patch-clamp recordings of PIEZO1 and PIEZO2. We show that the inactivation kinetics of PIEZO1 and, to a lesser extent, of PIEZO2 change with the angle at which the probe that is used for mechanical stimulation is positioned and, even more prominently, with the size of its tip. Moreover, we found that the mechanical activation threshold of PIEZO2, but not PIEZO1, decreased with increasing stimulation speeds. Thus, our data show that two key outcome parameters of PIEZO-related patch-clamp studies are significantly affected by common variations in the mechanical stimulation protocols, which calls for caution when comparing data from different laboratories and highlights the need to establish a gold standard for mechanical stimulation to improve comparability and reproducibility of data obtained with the poking technique.
Topics: Ion Channels; Humans; Kinetics; Patch-Clamp Techniques; HEK293 Cells; Mechanotransduction, Cellular
PubMed: 38754025
DOI: 10.1080/19336950.2024.2355123 -
Analytical Methods : Advancing Methods... May 2024This study outlines the development and optimization of an analytical method using Disposable Pipette Extraction (DPX) followed by high performance liquid...
This study outlines the development and optimization of an analytical method using Disposable Pipette Extraction (DPX) followed by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to determine NAs in medicines. HPLC-MS analysis utilized a reversed-phase and positive mode electrospray ion source. DPX parameters were optimized through univariate and multivariate analyses, including extraction phase, desorption solvent, sample pH, equilibrium time, and extraction/desorption cycles. The optimized conditions included a C18 extraction phase, methanol desorption solvent, pH at 7, an equilibrium time of 30 seconds, 2 extraction cycles, and 5 desorption cycles. Considering this method, it was possible to achieve a sample preparation step for the analysis of NAs in medicines using a minimal amount of extraction phase, sample, and desorption solvent. Furthermore, the total extraction procedure enables the extraction of NAs in around 4 minutes with NA recovery up to 98%. Analytical performance demonstrated precision and accuracy below 15% and a quantification limit of 1 ng mL, meeting validation requirements set by regulations worldwide. Thus, the DPX/HPLC-MS technique offers a faster and cost-effective method for analyzing NAs in medicines compared to traditional approaches. Besides, this method reduces solvent consumption and residue generation, enhancing environmental sustainability according to green chemistry principles.
Topics: Chromatography, High Pressure Liquid; Nitrosamines; Limit of Detection; Mass Spectrometry; Reproducibility of Results; Solid Phase Extraction; Liquid Chromatography-Mass Spectrometry
PubMed: 38747210
DOI: 10.1039/d4ay00554f -
BioRxiv : the Preprint Server For... May 2024Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology...
Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called 'tch and isplay for iquid iopsy (CAD-LB)'. CAD-LB rapidly captures fluorescently labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV-associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LB's clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.
PubMed: 38746341
DOI: 10.1101/2024.04.29.589900 -
Neuroscience Research May 2024The gramicidin-perforated patch-clamp technique is indispensable for recording neuronal activities without changing the intracellular Cl concentration. Conventionally,...
The gramicidin-perforated patch-clamp technique is indispensable for recording neuronal activities without changing the intracellular Cl concentration. Conventionally, gramicidin contained in the pipette fluid is delivered to the cell membrane by passive diffusion. Gramicidin deposited on the pipette orifice sometimes hampers giga-seal formation, and perforation progresses only slowly. These problems may be circumvented by delivering a high concentration of gramicidin from an intra-pipette capillary after a giga-seal is formed. We herein describe the detailed protocol of this improved method. This protocol would greatly facilitate the investigation of Cl gradient-dependent neuronal activities.
PubMed: 38740268
DOI: 10.1016/j.neures.2024.05.002 -
Angewandte Chemie (International Ed. in... May 2024Capturing short-lived intermediates at the molecular level is key to understanding the mechanism and dynamics of chemical reactions. Here, we have developed a...
Capturing short-lived intermediates at the molecular level is key to understanding the mechanism and dynamics of chemical reactions. Here, we have developed a paper-in-tip bipolar electrolytic electrospray mass spectrometry platform, in which a piece of triangular conductive paper incorporated into a plastic pipette tip serves not only as an electrospray emitter but also as a bipolar electrode (BPE), thus triggering both electrospray and electrolysis simultaneously upon application of a high voltage. The bipolar electrolysis induces a pair of redox reactions on both sides of BPE, enabling both electro-oxidation and electro-reduction processes regardless of the positive or negative ion mode, thus facilitating access to complementary structural information for mechanism elucidation. Our method enables real-time monitoring of transient intermediates (such as N,N-dimethylaniline radical cation, dopamine o-quinone (DAQ) and sulfenic acid with half-lives ranging from microseconds to minutes) and transient processes (such as DAQ cyclization with a rate constant of 0.15 s). This platform also provides key insights into electrocatalytic reactions such as Fe (III)-catalyzed dopamine oxidation to quinone species at physiological pH for neuromelanin formation.
PubMed: 38717236
DOI: 10.1002/anie.202318169 -
The Journal of Dermatology May 2024We compared the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of azoles in antifungal drug-susceptible, terbinafine-resistant,...
We compared the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of azoles in antifungal drug-susceptible, terbinafine-resistant, and lowly itraconazole (ITCZ)-susceptible strains of dermatophytes. To assess the MICs of ITCZ, ravuconazole (RVCZ), efinaconazole (EFCZ), and luliconazole (LUCZ) in the isolates, broth microdilution assays were performed based on the Clinical and Laboratory Standards Institute M38-A2 guidelines with modifications. After the assays for determining the MICs, the inoculum suspensions in wells were resuspended, then 10 μL of the growth solution in each well was inoculated onto potato dextrose agar with the use of a pipette. After 7 days of incubation at 28°C, the MFCs were determined as the lowest concentration of a drug that allowed the growth of colonies on the potato dextrose agar. The MICs in the dermatophytes were <0.03 to >32 mg/L for ITCZ, <0.03 to 4 mg/L for RVCZ, <0.03 to 2 mg/L for EFCZ, and <0.03 mg/L for LUCZ. The MFCs in the dermatophytes were 1 to >32 mg/L for ITCZ, 0.06 to >32 mg/L for RVCZ, <0.03 to 4 mg/L for EFCZ, and <0.03 to 2 mg/L for LUCZ. If the drug susceptibility test shows that the fungi are resistant to the drug, the treatment can be changed to a susceptible drug in advance, or if the fungi are low-susceptible, the treatment can be done with the recognition that it may require a longer treatment period than usual.
PubMed: 38712872
DOI: 10.1111/1346-8138.17241