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This study was the first attempt to optimize a recombinase polymerase amplification (RPA) and lateral flow (LF) assay combined with immunomagnetic separation (IMS) for the detection of Vibrio parahaemolyticus in raw oysters. The newly developed IMS-RPA-LF assay effectively combines sample preparation, amplification, and detection into a single platform. Under optimal conditions, the average capture efficiency (CE) for 104 colony forming units (CFU)/mL of four V. parahaemolyticus strains with 0.4 mg of immunomagnetic beads within 45 min was 80.3%. After optimization, the RPA-LF assay was able to detect V. parahaemolyticus within 15 min, comprising DNA amplification with RPA for 10 min at 37 °C and visualization of the amplicons through LF strips for 5 min. The RPA-LF assay exhibited good specificity by showing a test line for eight V. parahaemolyticus strains with different serotypes but no cross-reaction with 12 non-V. parahaemolyticus bacteria. RPA-LF assay was found to be sensitive and detected as low as 10 pg genomic DNA of V. parahaemolyticus. For spiked oyster samples, the detection sensitivity of V. parahaemolyticus was improved to 2 CFU/g by IMS-RPA-LF after enrichment for 4 h; in contrast, the IMS-PCR method required 8 h. Hence, even when V. parahaemolyticus was present in very low numbers in samples, the IMS-RPA-LF assay could be completed within half a workday. Because of the high sensitivity, specificity, and speed of the IMS-RPA-LF assay, this newly developed method opens a novel pathway for rapid diagnostic screening of V. parahaemolyticus in seafood, which is an increasingly important health issue worldwide.

Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. Despite this, throughputs, yields, viabilities, and processing times of common sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are underreported. In the current study, we set out to quantify the ability of these sorting methods to separate defined mixtures of alkaline phosphatase liver/bone/kidney (ALPL)-expressing and non-expressing cell types. Results showed that initial MACS runs performed using manufacturer’s recommended antibody and microbead concentrations produced inaccurate ALPL+ vs. ALPL− cell splits compared to FACS when ALPL+ cells were present in larger proportions (>~25%). Accuracy at all proportions could be achieved by using substantially higher concentrations of labeling reagents. Importantly, MACS sorts resulted in only 7–9% cell loss compared to ~70% cell loss for FACS. Additionally, MACS processing was 4–6 times faster than FACS for single, low proportion samples but took similar time for single, high-proportion samples. When processing multiple samples, MACS was always faster overall due to its ability to run samples in parallel. Average cell viability for all groups remained high (>83%), regardless of sorting method. Despite requiring substantial optimization, the ability of MACS to isolate increased cell numbers in less time than FACS may prove valuable in both basic science and translational, cell-based applications.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC–MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC–MS analysis. A sensitive and specific LC–MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Cell separation is at the core of current methods in experimental biology and medicine. Its importance is illustrated by the large number of physical and biochemical principles that have been evaluated for application to cell separation. The development of cell separation methods is driven by the needs of biological and medical research, and the ever-increasing demands for sensitivity, selectivity, yield, timeliness and economy of the process. The interdisciplinary nature of research in this area and the volume of information available in research publications and conferences necessitates a basic description of the fundamental processes involved in magnetic cell separation that may help the user in navigating this wealth of information available online and in scientific publications. This book will appeal to researchers in many areas utilizing this technique, including those working in cell biology, clinical research, inorganic chemistry, biochemistry, chemical engineering, materials science, physics and electrical engineering. * Provides examples of how to calculate the volume magnetic susceptibility, a fundamental quantity for calculating the magnetic force acting on a cell, from various types of magnetic susceptibilities available in literature* Introduces the elements of magnetostatics as they apply to cell magnetization and the magnetization of magnetic micro- and nano- particles used for cell separation* Describes the parameters used to determine cell magnetophoresis

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

The food industry requires rapid and simple detection methods for preventing harm from pathogenic bacteria. Until now, various technologies used to detect foodborne bacteria were time-consuming and laborious. Therefore, we have developed an automated immunomagnetic separation combined with a colorimetric assay for the rapid detection of E. coli O157:H7 in food samples. The colorimetric detection method using enzymatic reaction is fascinating because of its simplicity and rapidity and does not need sophisticated devices. Moreover, the proposed procedures for the detection of bacteria in food take less than 3 h including pre-enrichment, separation and detection steps. First, target-specific immunomagnetic beads were introduced to contaminated milk in a pre-enrichment step. Second, the pre-enriched sample solution containing target bacteria bound on immunomagnetic beads was injected into an automated pretreatment system. Subsequently, the immunomagnetic beads along with target bacteria were separated and concentrated into a recovery tube. Finally, released β-galactosidase from E. coli O157:H7 after lysis was reacted with chlorophenol red β-galactopyranoside (CPRG) used as a substrate and the colorimetric change of CPRG was determined by absorbance measuring or the naked eye. By the proposed approach in this study, we could detect 3 × 102 CFU/mL of E. coli O157:H7 from a milk sample within 3 h.

By binding magnetic nanoparticles to the surface of cells, it is possible to manipulate and control cell function with an external magnetic field. The technique of activating cells with magnetic nanoparticles offers a means to isolate and explore cellular mechanics and ion channel activation to gain better understanding of these processes. Here, we go beyond using this technique as an investigative tool and focus on its potential to actively control cellular functions and processes with an eye towards biological and clinical applications. In particular, we focus on applications in tissue engineering and regenerative medicine.

Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31−/CD34+/PDGFRα+/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFRα−/vimentin+) and fibroblasts (CD31−/CD34−/PDGFRα+/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine.

Rapid and accurate food pathogen detection is an essential step to preventing foodborne illnesses. Before detection, removal of bacteria from the food matrix and concentration to detectable levels are often essential steps. Although many reviews discuss rapid concentration methods for foodborne pathogens, the use of glycan-coated magnetic nanoparticles (MNPs) is often omitted. This review seeks to analyze the potential of this technique as a rapid and cost-effective solution for concentration of bacteria directly from foods. The primary focus is the mechanism of glycan-coated MNP binding, as well as its current applications in concentration of foodborne pathogens. First, a background on the synthesis, properties, and applications of MNPs is provided. Second, synthesis of glycan-coated particles and their theorized mechanism for bacterial adhesion is described. Existing research into extraction of bacteria directly from food matrices is also analyzed. Finally, glycan-coated MNPs are compared to the magnetic separation technique of immunomagnetic separation (IMS) in terms of cost, time, and other factors. At its current state, glycan-coated MNPs require more research to fully identify the mechanism, potential for optimization, and extraction capabilities directly in food matrices. However, current research indicates glycan-coated MNPs are an incredibly cost-effective method for rapid food pathogen extraction and concentration.

Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein–peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes , that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide–peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide–peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.