Magnetic Microbeads Research Articles

Investigation and Demonstration for immunoassay technology based on impedance Amplifies of magnetic and dielectric microbeads

BackgroundBead-based immunoassays typically require researchers to perform the analysis in a laboratory setting, involving complex processing procedures, most of the time, constant potential or current instruments are used to measure the bio-impedance change. However, almost all biological materials are decorated on the electrode surface to obtain the maximum sensitivity, resulting in difficulty to clean, separate, and other applications of fixed bead-based immunoassay. ResultWe present an innovative unfixed bead-based immunoassay technology that utilizes electrical impedance characteristics, offering the advantages of being rapid, portable, miniaturized, and cost-effective. This technique which is known as Impedance-enhanced electrochemical immunoassay (IE-ECIA) employs antibody-coated Magnetic beads (MBs) and Dielectric beads (DBs) linked to antigens for quantitative with miniaturization electronic devices, which has been successfully applied in an immune impedance sensor for detecting the Dengue virus (DENV), which is an affordable test with a low-cost microfluidic impedance biosensor combined with immune magnetic and dielectric micro/nanobeads for antigen detection. HighlightThe proposed immunoassay technology introduces a novel microfluidic platform incorporating magnetic and dielectric beads alongside with an electrochemical impedance biosensor, therefore facilitating continuous-fluid quantitative analysis. This innovative and unconventional approach leverages free-motion multiplex beads housing magnetic and dielectric bead-bound antigens within a simple linear microchannel of microfluidic chip architecture, making the high-through measurements more efficient, enhanced Electrochemical immunoassay (ECIA) signals of Bio-on-magnetic-beads (BOMB). This advancement holds significant potential for enhancing diagnostic capabilities for emerging infectious diseases within clinical environments, thereby bolstering rapid detection capabilities and aiding in the advancement of novel pharmaceuticals, benefiting all the stakeholders.

Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen.

Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determinationof cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0pg·mL-1, representing two orders of magnitude improvement compared withequivalent microtiter plate-based assay (LOD of 460pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.

Monolayer-Fluorescence Counting for Ultrasensitive Detection of Tumour Cell-Derived Extracellular Vesicles Using a Step-wedge Microfluidic Platform

Extracellular vesicles have emerged as significant noninvasive tumour biomarkers, and ultrasensitive detection methods are important for early cancer diagnosis. Here, a monolayer-fluorescence counting strategy was developed for the ultrasensitive detection of extracellular vesicles using a microfluidic platform. Extracellular vesicles were specially captured and separated from complex matrix by immune magnetic microbeads (IMBs). With an enhancing acoustic wave microfluidic channel, the magnetic-extracellular vesicles immune complex was adequately mixed and reacted with antibody-modified fluorescence microbeads (IFBs). Using a newly designed step-wedge microfluidic structure, micrometer-size particles were trapped in large numbers with monolayer in the step zone, and the fluidic resistance was reduced through the wedge structure. This detection signal of the fluorescence-labelled immune sandwich complex could be read by counting, avoiding background interference and achieving an ultrasensitive detection with a detection limit of 850 particles/mL. Moreover, this method showed strong reliability and specificity in clinic samples. In addition, a miniaturized detection device was developed based on this microfluidic platform, improving this detection automation.

Development of Nitric Oxide-Donating Netarsudil Derivatives as a Synergistic Therapy for Glaucoma with Reduced Ocular Irritation.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a series of NO-donating Netarsudil derivatives were designed, synthesized, and their activities in vitro and in vivo were evaluated. Among them, (S)-10e released an appropriate amount of NO in aqueous humor in vitro and displayed potent ROCK inhibition. Topical administration of (S)-10e significantly lowered intraocular pressure in an acute ocular hypertension rabbit model and protected retinal ganglion cells in a magnetic microbead occlusion mouse model. A metabolism investigation revealed that (S)-10e released 7a, a metabolite after NO releasing, and 13, an active metabolite of (S)-Netarsudil, in rabbit eyes. Notably, introducing an NO donor moiety attenuated ROCK inhibition-induced ocular irritation in an sGC-independent manner, suggesting that the attenuated conjunctival hyperemia effect of (S)-10e is related to the NO-induced protein S-nitrosation of phosphodiesterase 3A (PDE3A). Overall, (S)-10e is a promising candidate for glaucoma treatment.

Magnetic Metalloimmunoassays: Influence of Particle Shape and Size on Galvanic Exchange-Based Electrochemical Detection

The electrooxidation of silver (Ag) offers improved signal sharpness and intensity, enabling precise measurements and enhanced resolution in electrochemical sensing. Leveraging silver nanoparticles (AgNPs) as indirect labels in electrochemical assays provides a distinct advantage for detecting various biomarkers at clinically relevant concentrations. In our recent work, we have introduced an electrochemical approach that involves a galvanic exchange (GE) reaction between electrodeposited gold and AgNPs, followed by anodic stripping voltammetry (ASV), with potential applications in immunosensing. In this assay protocol, specific monoclonal antibodies immobilize the biomarker of interest separately on AgNPs and magnetic beads. Subsequently, magnetic bead-AgNP conjugates are attracted to the electrode surface by a magnetic force. However, to achieve the expected analytical performance of the bioassay, it is imperative to characterize the composition, size, shape, and surface modifications of the AgNPs. Consequently, our research focuses on investigating the electrochemical properties of a model bioconjugate formed between magnetic microbeads (MB) and AgNPs through Streptavidin-biotin interactions under various assay conditions. Notably, our recent studies reveal that conducting the GE cycle for three repetitions significantly enhances the Ag signal, yielding an improvement of approximately two-fold or greater compared to previously reported methods. Previous studies have shown that altering the shape of AgNPs, transitioning from nanospheres to other forms like nanocubes and nanodisks enhances both the limit of detection and the range of analyte detection. However, despite these enhancements, spherical AgNPs remain the most stable and easily synthesized option, characterized by excellent homogeneity. Recently, we compared AgNPs with diameters ranging from 20 to 100 nm paired with magnetic microbeads with diameters spanning from 50 nm to 3 µm to identify the optimal combination. Preliminary results indicate that the Mb (3 µm)-AgNP (50 nm) conjugate exhibits the highest Ag charge and Ag collection efficiency. Nonetheless, critical knowledge gaps persist regarding the fundamental understanding of the electrogenerated GE process and we are continually working on refining the electrochemical and assay parameters to develop a robust universal platform for GE-based electrochemical immunosensing.

Magnetically-Enhanced Electrochemical ELISA for the Detection of Sars-Cov-2 Nucleoprotein

Here we report on the development of a novel, magnetically-enhanced electrochemical (MagEC) bioassay targeting SARS-CoV-2 nucleoprotein. The sensing strategy is similar to microbead-based enzyme linked immunosorbent assays (ELISA) with optical detection. The target analyte is captured using a magnetic microbead functionalized with an antibody specific to SARS-CoV-2 nucleoprotein. Once SARS-CoV-2 nucleoprotein has been captured a detection construct, consisting of polystyrene microbeads functionalized with horseradish peroxidase (HRP) and antibodies specific to SARS-CoV-2 nucleoprotein are attached to form the full microbead construct.Once the full microbead constructs are assembled, they are injected into an electrochemical cell containing an assay solution where they are magnetically enriched at the electrode surface. This magnetic localization brings all of the reactants close to their point of detection at the electrode surface and also creates a positive feedback loop, drastically enhancing signal. The electrochemical assay was optimized using model constructs to emulate the fully assembled bead constructs and identify the optimal electrode configuration. Using the model constructs, we found a 60-fold decrease in limit of detection using the MagEC ELISA compared to optical detection with model bead constructs. We compared performance of a bare gold electrode to several geometries of gold microelectrode arrays. We obtained preliminary results for the detection of SARS-CoV-2 nucleoprotein. Additionally, the assay was expanded to incorporate a fluidic manifold to enhance signal, enhance throughput and enable operation in microgravity environments.

Removal of lead and cadmium ions from water using primary-secondary amine modified magnetic microbeads: Equilibrium, kinetic, and thermodynamic studies

This study developed novel magnetic microbeads based on trimethoxysilylpropylethylenediamine (TSD) as a primary-secondary amine source through a one-pot process. The process involves coating magnetic iron oxide nanoparticles (MNPs) with primary-secondary amine (TSD), followed by cross-linking with alginate (Alg). The ternary magnetic microbeads (Alg@MTSD) were used to remove Pb2+ and Cd2+ ions from aqueous media. The nanocomposite was characterized using various techniques, including Fourier-transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). Adsorption experiments were conducted under optimal conditions, and isothermal models, including Langmuir and Freundlich, as well as kinetic models, were applied to evaluate the experimental data. The findings indicated that the Langmuir model best fit the adsorption data for Pb2+ and Cd2+ ions, with R2 > 0.99. The maximum adsorption capacities were 68 mg·g−1 for Pb2+ and 51 mg·g−1 for Cd2+. Kinetic studies showed that the adsorption process followed the pseudo-second-order model. Thermodynamic analysis suggested that the adsorption mechanism was physisorption.

Effect of magnetic microbeads on sustained and targeted delivery of transforming growth factor-beta-1 for rotator cuff healing in a rat rotator cuff repair model

Structural failure is a well-established complication of rotator cuff repair procedures. To evaluate the effect of magnetic microbeads, designed for precise drug delivery via magnetic force, on sustained transforming growth factor-beta-1 (TGF-β1) release and rotator cuff healing in a rat rotator cuff repair model. TGF-β1 laden microbeads were prepared, and baseline in vitro experiments included the magnetization of the microbeads and TGF-β1 release tests. In an in vivo experiment using a rat rotator cuff repair model on both shoulders, 72 rats were randomly assigned to three groups (24 per group): group A, conventional repair; group B, repair with and simple TGF-β1 injection; and group C, repair with magnet insertion into the humeral head and TGF-β1 laden microbead injection. Delivery of TGF-β1 was evaluated at 1 and 7 days after the intervention using PCR, Western blot, and immunohistochemistry. At 6 weeks post-intervention, rotator cuff healing was assessed using biomechanical and histological analysis. The in vitro experiments confirmed the magnetization property of the microbeads and sustained delivery of TGF-β1 for up to 10 days. No difference in the TGF-β1 expression was found at day 1 in vivo. However, at day 7, group C exhibited a significantly elevated expression of TGF-β1 in both PCR and Western blot analyses compared to groups A and B (all P < 0.05). Immunohistochemical analysis revealed a higher expression of TGF-β1 at the repair site in group C on day 7. At 6 weeks, biomechanical analysis demonstrated a significantly higher ultimate failure load in group C than in groups A and B (P < 0.05) and greater stiffness than in group A (P = 0.045). In addition, histological analysis showed denser and more regular collagen fibers with complete continuity to the bone in group C than in groups A and B, a statistically significant difference according to the semi-quantitative scoring system (all P < 0.05). The use of the TGF-β1 laden magnetic microbeads demonstrated sustained delivery of TGF-β1 to the repair site, improving rotator cuff healing.

Selection of functional sperm by using hyaluronic acid modified magnetic microbeads and an electromagnetic manipulation system.

In recent years, the incidence of infertility has increased year by year. Assisted reproductive technology (ART) is one of the effective strategies to treat infertility. In the process of ART, commonly used methods for sperm separation have shortcomings and there is still room for improvement. In this study, a functional sperm selection strategy was established based on hyaluronic acid (HA) modified magnetic microbeads (MBs) and a supporting two-dimensional electromagnetic manipulation device system. Due to the modification of HA on the surface of MBs, the HA-MBs have the ability to target and bind to specific receptors on the sperm membrane to form a sperm-MB complex. A disulfide linker was introduced to connect HA and MBs. After modifying HA and connecting it with the disulfide linker, the sperm of sperm-MBs can be released under the combined effect of hyaluronidase and reduced GSH with the disulfide bond broken and HA degraded. A two-dimensional electromagnetic manipulation system was introduced to generate a magnetic field and control the directional movement of the sperm-MB complex under the guidance of an inverted microscope. The free MBs can also be removed after the sperm is released. Furthermore, the mouse sperm selected through this strategy can achieve normal insemination via ICSI and the obtained blastocysts have normal morphology and developmental milestones. This strategy has potential to be developed into an automated screening solution for the screening of functional sperm for assisted reproductive technology.

Fabrication and Characterization of Collagen–Magnetic Particle Composite Microbeads for Targeted Cell Adhesion and Proliferation

The integration of the biocompatibility of collagen and the remote-control ability of magnetic elements serves as both a cell scaffold and an actuator. We studied the preparation, characterization, and potential applications of collagen–magnetic particle composite microbeads (CMPMBs). The interplay among collagen concentration, particle size, and surface roughness was found to influence cell adhesion and proliferation. Adsorption and desorption tests showed the reversible attachment of the particles to magnetic sheets, enabling precise spatial control and targeted cell delivery. The particles demonstrated their utility as cell carriers, supporting cell migration and proliferation. These findings showcase the potential of CMPMBs as a promising platform for advanced cell delivery and tissue regeneration applications. The ability to fine-tune particle properties and manipulate them using magnetic fields offers new possibilities for creating complex tissue constructs and controlling cellular behavior, which could contribute to the development of more effective regenerative therapies and tissue engineering approaches.

Caspase-8-mediated inflammation but not apoptosis drives death of retinal ganglion cells and loss of visual function in glaucomaa.

Glaucoma is a complex multifactorial disease where apoptosis and inflammation represent two key pathogenic mechanisms. However, the relative contribution of apoptosis versus inflammation in axon degeneration and death of retinal ganglion cells (RGCs) is not well understood. In glaucoma, caspase-8 is linked to RGC apoptosis, as well as glial activation and neuroinflammation. To uncouple these two pathways and determine the extent to which caspase-8-mediated inflammation and/or apoptosis contributes to the death of RGCs, we used the caspase-8 D387A mutant mouse (Casp8 DA/DA ) in which a point mutation in the auto-cleavage site blocks caspase-8-mediated apoptosis but does not block caspase-8-mediated inflammation. Intracameral injection of magnetic microbeads was used to elevate the intraocular pressure (IOP) in wild-type, Fas deficient Faslpr, and Casp8 DA/DA mice. IOP was monitored by rebound tonometry. Two weeks post microbead injection, retinas were collected for microglia activation analysis. Five weeks post microbead injection, visual acuity and RGC function were assessed by optometer reflex (OMR) and pattern electroretinogram (pERG), respectively. Retina and optic nerves were processed for RGC and axon quantification. Two- and five-weeks post microbead injection, expression of the necrosis marker, RIPK3, was assessed by qPCR. Wild-type, Faslpr, and Casp8 DA/DA mice showed similar IOP elevation as compared to saline controls. A significant reduction in both visual acuity and pERG that correlated with a significant loss of RGCs and axons was observed in wild-type but not in Faslpr mice. The Casp8 DA/DA mice displayed a significant reduction in visual acuity and pERG amplitude and loss of RGCs and axons similar to that in wild-type mice. Immunostaining revealed equal numbers of activated microglia, double positive for P2ry12 and IB4, in the retinas from microbead-injected wild-type and Casp8 DA/DA mutant mice. qPCR analysis revealed no induction of RIPK3 in wild-type or Casp8 DA/DA mice at two- or five-weeks post microbead injection. Our results demonstrate that caspase-8-mediated extrinsic apoptosis is not involved in the death of RGCs in the microbead-induced mouse model of glaucoma implicating caspase-8-mediated inflammation, but not apoptosis, as the driving force in glaucoma progression. Taken together, these results identify the caspase-8-mediated inflammatory pathway as a potential target for neuroprotection in glaucoma.

Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.Graphical

The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy.

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme's proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease's active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.

The ketogenic diet and hypoxia promote mitophagy in the context of glaucoma.

Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from glaucoma-associated neurodegeneration, with a concomitant increase in mitochondrial biogenesis. This study aimed to determine if the ketogenic diet also promoted mitophagy. MitoQC mice that carry a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane were placed on a ketogenic diet or standard rodent chow for 5 weeks; ocular hypertension (OHT) was induced via magnetic microbead injection in a subset of control or ketogenic diet animals 1 week after the diet began. As a measure of mitophagy, mitolysosomes were quantified in sectioned retina immunolabeled with RBPMS for RGCs or vimentin for Müller glia. Mitolysosomes were significantly increased as a result of OHT and the ketogenic diet (KD) in RGCs. Interestingly, the ketogenic diet increased mitolysosome number significantly higher than OHT alone. In contrast, OHT and the ketogenic diet both increased mitolysosome number in Müller glia to a similar degree. To understand if hypoxia could be a stimulus for mitophagy, we quantified mitolysosomes after acute OHT, finding significantly greater mitolysosome number in cells positive for pimonidazole, an adduct formed in cells exposed to hypoxia. Retinal protein analysis for BNIP3 and NIX showed no differences across groups, suggesting that these receptors were equivocal for mitophagy in this model of OHT. Our data indicate that OHT and hypoxia stimulate mitophagy and that the ketogenic diet is an additive for mitophagy in RGCs. The different response across RGCs and Müller glia to the ketogenic diet may reflect the different metabolic needs of these cell types.

Innovative optimization for maximum magnetic nanoparticles production by Trichoderma asperellum with evaluation of their antibacterial activity, and application in sustainable dye decolorization

A pioneer microbial-based synthesis of various nanoparticles is gaining more attention due to their potent applications in various industries. The magnetic nanoparticles (M-nps) were fabricated for the first-time using the cell free extract of Trichoderma asperellum. The M-nps bioprocess optimization was performed using Response Surface Methodology and Artificial Neural Network.The bioprocess was significantly (P < 0.05) affected by pH, stirring speed, and incubation period. The M-nps displayed a strong antibacterial efficiency against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus in a dose-dependent manner. The minimum inhibitory concentration (MIC) for E. coli, P. aeruginosa, and S. aureus were respectively found to be 25.0, 6.25, and 3.125 µg/ml. While the minimum bactericidal concentration (MBC) was 25.0, 12.5, and 6.25 µg/ml. The highest inhibition zone of 22 mm was recorded for B. subtilis at 80 µg/ml with MIC and MBC of 3.125 µg/ml. The sodium alginate/M-nps microbeads was successfully prepared without any crosslinkers. The microbeads exhibited the highest adsorptive efficiency (84.32%) for malachite green at pH 8.0, dose 0.1 g, and time 60 min. The experimental data were superlative fitted to Langmuir’s isotherm, hinting monolayer adsorption of MG molecules with 117.65 mg g‐−1 adsorptive capacity. The regenerated beads continued up-to six consecutive cycles and maintained more than 90%, indicating the multiple time use of the prepared material. Hence, the green magnetic nanoparticles display promising application in combating bacterial pathogens. The magnetic microbeads can be employed as an alternative eco-friendly, highly efficient, sustainable, and low-cost biosorbent in removing contaminants from wastewater.

Electrochemical immunosensing of walnut and hazelnut allergenic proteins in processed foods

Nuts are a well-known cause of food allergy and, once this has been diagnosed, due to the likelihood of cross-sensitization to multiple tree nut allergens, their strict avoidance from the diet is advisable. In this context, we present electrochemical bioplatforms to detect traces of hazelnut and walnut in processed foods through the determination of their respective allergenic proteins Cor a 9 and Jug r 1 in a fast and sensitive assay. First, the evaluation of the single determination of both proteins was performed by building sandwich immunoconjugates on the surface of magnetic microbeads relying on specific antibodies unmodified or conjugated to horseradish peroxidase. Amperometric transduction was made upon trapping the magnetic bioconjugates on the surface of disposable carbon electrodes, using the hydroquinone/hydrogen peroxide system. The great analytical performance achieved with the individual platforms (detection limits of 0.12 and 0.56 ng mL−1 for Jug r 1 and Cor a 9, respectively), led us to the individual and dual quantification of both proteins in raw dough and baked cookies incurred with ground nuts. The developed method allowed detecting baked cookies incurred with 0.0025% ground walnut and 0.00002% ground hazelnut with results comparable to those provided by ELISA techniques. The feasibility of performing the dual determination of both allergens in a single run was demonstrated.

An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Abstract 39: Preclinical in vitro and in vivo evaluation of CD8αβ+ CD19 CAR iPSCs T cells generated in a TCR signal-independent manner using DLL4/VCAM-coupled microbeads

Abstract The use of clonally-derived, induced pluripotent stem cells (iPSCs) as starting material for therapeutic T cell manufacturing would overcome many limitations of autologous Chimeric Antigen Receptor T cell (CAR-T) therapies. Complex, multi-stage genome engineering, large batch production and rigorous lot testing could provide a consistent source of off-the-shelf, functionally enhanced T cell products. However, this vision cannot be realized using existing T cell differentiation platforms, which fail to present Notch ligands with the precision and intensity required to control T cell differentiation in scalable, suspension culture. We have previously shown that VCAM1 synergizes with DLL4 to enhance Notch signaling and progenitor T cell differentiation. We have extended the utility of this discovery through the creation of DLL4/VCAM1-coated magnetic microbeads, enabling precise and temporal control of Notch signaling in suspension culture. iPSC-derived CD34+ cells cultured with DLL4/VCAM1 beads demonstrate dose-responsive activation of Notch gene expression (e.g., Notch1, DTX1, TCF7), resultant T lineage commitment (e.g. CD5, CD7 expression) and maturation to CD4-CD8+ (single positive) cells in a TCR signal-independent manner. Removal of the endogenous TCR is necessary to prevent graft-versus-host-disease. Therefore, differentiation and maturation in a TCR-independent process presents substantial advantage in the development of safe, off the shelf iPSC T cell products. Using a clonal iPSC line with a CD19 CAR knocked in at the TRAC locus, we apply this approach to generate CAR-expressing, CD8αβ+ functional T cells, capable of multiple rounds of in vitro tumor cell lysis and sustained tumor growth inhibition in vivo comparable to primary T cells. This advancement demonstrates proof-of-concept for generation of highly efficacious, off-the-shelf CAR-T cells using novel, small-footprint manufacturing in order to broaden the applicability and accessibility of T cell therapies for cancer patients. Citation Format: Muluken S. Belew, Richard Carpenedo, Alessia Pallaoro, Hiofan Hoi, Avisek Deyati, Daniel C. Kirouac, Siddarth Chandrasekaran, Valerie Wall, Libin Abraham, Sommer Apelu, Michael H. Cadell, Amanda AuYeung, Omar Subedar, Elisa Martinez, Shri Joshi, Elizabeth Csaszar, Steven Woodside, Emily Titus, Christopher Bond. Preclinical in vitro and in vivo evaluation of CD8αβ+ CD19 CAR iPSCs T cells generated in a TCR signal-independent manner using DLL4/VCAM-coupled microbeads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 39.

Selective visual detection of multiplex PCR amplicon using magnetic microbeads

Nucleic acid amplification tests (NATs), such as genetic tests using polymerase chain reaction (PCR), are sensitive methods for detecting pathogens and food contamination. The rapid, easy, and inexpensive detection of amplicons, DNA, or RNA is key to realizing on-site NATs. We have previously developed a novel amplicon detection method using magnetic microbeads based on the hydrophobicity of DNA in deionized water. In this study, we aimed to expand the method for the detection of multiplex DNA amplicons. Tagged primers and probes for selective attachment were used to detect amplicons from two strawberry pathogens. The amplicon-labeled magnetic microbeads were placed in the round-bottom well of a hydrophilic glass substrate. The attachment of amplicons to the magnetic microbeads changed their surface from hydrophilic to hydrophobic. The magnetized microbeads concentrated at the bottom when the substrate was placed on a permanent magnet, and the concentrated microbeads were easily recognizable by the naked eye. Microbeads without amplicons were adsorbed over a broad area of the bottom of the glass well owing to their hydrophilicity. The appropriate tag probe was attached to specific amplicons for detection, and each amplicon from multiplex PCR was selectively detected within approximately 15 min. Notably, this method requires no electric power and contributes to the realization of on-site NAT detection. This study presents a simple and rapid method for the selective detection of multiplex PCR amplicons using DNA–DNA hybridization.

Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93-98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.