Use Of Magnetic Beads Research Articles

Synthesis and active manipulation of magnetic liquid beads

We report the fabrication and characterisation of magnetic liquid beads with a solid magnetic shell and liquid core using microfluidic techniques. The liquid beads consist of a fluorinated oil core and a polymer shell with magnetite particles. The beads are generated in a flow-focusing polydimethylsiloxane (PDMS) device and cured by photo polymerisation. We investigated the response of the liquid beads to an external magnetic field by characterising their motion towards a permanent magnet. Magnetic sorting of liquid beads in a channel was achieved with 90% efficiency. The results show that the liquid beads can be controlled magnetically and have potential applications in digital microfluidics including nucleic acid amplification, drug delivery, cell culture, sensing, and tissue engineering. The present paper also discusses the magnetophoretic behaviour of the liquid bead by varying its mass and magnetite concentration in the shell. We also demonstrated the two-dimensional self-assembly of magnetic liquid beads for potential use in digital polymerase chain reaction and digital loop mediated isothermal amplification.Graphical abstract

Manipulation of magnetic beads for actively capturing Vibrio parahaemolyticus and nucleic acid based on microfluidic system.

Rapid biological detection of pathogen micro-organisms has attracted much attention for practical biomedical applications. Despite the development in this field, it is still challenging to achieve simple and rapid biological detection using the microfluidic method. Herein, we propose a novel strategy of biological detection that combines precise detection control of the capillary microfluidic chip and versatile manipulation of magnetic beads. The microfluidic chip was fabricated via laser cutting, which utilized capillary pressure to realize rapid passive injection of liquid samples. Under an external magnetic field, the aptamer-modified magnetic beads were actuated to mix with Vibrio parahaemolyticus (V. parahaemolyticus) and its nucleic acid in the capillary microfluidic chip for rapid selective capture and detection, which could be achieved within 40 min. The experimental results demonstrated that V. parahaemolyticus could be captured using on-chip immunomagnetic beads with a high efficiency and significantly enhanced detection value. Due to these superior performances, the capillary microfluidic system, based on the manipulation of magnetic beads, demonstrated great potential for automatic biological detection.

Fully Integrated Microfluidic Device for Magnetic Bead Manipulation to Assist Rapid Reaction and Cleaning.

Methods to manipulate magnetic beads are essential factors to determine the efficiency and dimension of an in vitro diagnostic system. Currently, using movable permanent magnets and planar electromagnets is still the major approach to achieve magnetic bead control, causing significant constraint in the miniaturization of in vitro diagnostic systems. Here, we propose techniques to construct a fully integrated microfluidic device that can conduct automatic magnetic bead manipulation as well as rapid chemical reaction and cleaning in a minimized dimension similar to a USB disk. The device combines the precision control of multiple electromagnetic coils with the compactness of microfluidic channels, leading to one of the smallest automatic magnetic bead manipulation systems that can complete several major magnetic bead-based operation steps such as sample injection, reaction, cleaning, and collection. The influencing factors such as coil driving parameters, surface treatment of the microchannels, and properties of magnetic particles have also been investigated to optimize the device performance. The device can drive mixtures of Fe3O4 microparticles and polymer magnetic beads (PMBs) with a weight ratio of 1:1 at a maximum speed of 0.5 cm·s-1 and reduce the time for DNA binding and dissociation reactions from 20 min to only 48 s. This device has significantly advanced the conventional manipulation methods of magnetic beads and has demonstrated the possibility to construct an automatic and ultraminiaturized in vitro diagnostic system that may facilitate portable or even wearable chemical analysis.

SDR-assisted multi-primers triggering functional signal hairpin probe for miRNA detection with enzyme-free and high sensitivity

MiRNA can exist in body fluids and its changes directly affect human life and health, which has been a research hotspot in recent years. In this work, we designed an enzyme-free sensing system for highly sensitive detection of miRNA-21 based on strand displacement reaction (SDR) that can recycle the target and functional hairpin probes (HP) that modified on upconversion nanomaterials (UCNPs). The target gene circulates through the SDR process and completes the initial signal amplification process. Then, the C2 sequences and targets released by the SDR process can continue to be combined with functional hairpin probes modified with fluorophore and quenching groups. After infrared irradiation, HP is broken, and C2 or the target can take the fluorophores away from the quenching group, so that the fluorescence signal can be detected to achieve highly sensitive detection of the target. In sensing system, the use of magnetic beads can reduce the background signal during the reaction process, and the functional hairpin probe can accurately turn the signal on and off. The sensor system not only shows excellent detection performance (detection limit of 30.3 fM) in buffer and actual detection, but also has excellent selectivity, which can provide a new detection method for miRNA-21 detection.

High-Throughput Generation, Manipulation, and Degradation of Magnetic Nanoparticleladen Alginate Core-shell Beads for Single Bacteria Culturing Analysis.

Microbes could be found almost everywhere around us and have significant impacts on our human society. The treatment of microorganisms has long been seen as a complex problem. Till now, most of the genetic and phenotypic information regarding rare species is buried in the bulk microbial colony due to a lack of efficient tools to screen live bacteria. Droplet microfluidics offers a powerful approach to address this problem. However, the interactions among bacteria and their living environment are entirely restricted by the water/oil interfaces in conventional water/oil single emulsion-based microfluidic systems. Here, we demonstrate an oil-mediated all-aqueous microfluidic workflow that can overcome this drawback. In contrast to the previous works, our all-aqueous culturing environment allows cell-cell and cell-environment interactions, thus facilitating the growth of bacteria. Fe3O4 magnetic nanoparticles added into the alginate beads enables on-chip manipulation of the microcapsules. The core-shell structure separately encapsulates bacteria and magnetic particles in the core and shell to avoid contamination. We demonstrate the feasibility of this approach by single bacterium culturing in droplet-templated alginate beads. Finally, a new approach is proposed to degrade the alginate beads for post-treatment. This novel microfluidic workflow can create new opportunities for microbial applications, such as bacteria culturing and screening.

On‐chip purification of tetracycline from food matrices

AbstractAntibiotics are widespread both to treat human and animal diseases and to improve growth in food animals. However, their overuse in food‐producing animals has led to critical issues for human health, such as the direct toxicity and the development of antibiotic‐resistant bacterial strains. Therefore, the identification of traces of antibiotic in food before entering the market has became extremely important and brought out the need for novel bioanalytical methods and protocols. To meet this need, here, a microfluidic system was set up for the purification of tetracycline (TC) from raw milk, honey, and eggs. The system is based on the use of magnetic beads exposing copper ions, which are loaded in a microfluidic chamber and actuated by a ready‐made device. Tetracycline is captured by copper ions present on the microbeads, purified from the unwanted raw material present in the initial sample and recovered by a suitable elution solution. Different elution solutions were tested and results were evaluated by X‐ray photoelectron spectroscopy (XPS) and visible spectrometry. The microfluidic system was successfully employed for the purification of TC from raw milk, honey, and eggs after an initial dilution in buffer. The overall protocol was, therefore, demonstrated to efficiently purify tetracycline, laying the bases for a future implementation of in‐field on‐chip tests.

Development and validation of novel automatable assay for cholesterol efflux capacity.

During the past decade, evaluation of high-density lipoprotein (HDL) functionality has been well studied for predicting cardiovascular disease risk. Cholesterol efflux capacity (CEC) is the strongest candidate as the biomarker out of various HDL anti-atherosclerotic functions. However, CEC has not yet been introduced clinically because of several technical issues, including the use of radioactive materials and differentiated cells in the assay. Previously, our laboratory developed a radioisotope- and cell-free CEC assay called the immobilized liposome-bound gel beads (ILGs) method to replace the conventional method. However, the separation process of the supernatant was not suitable for installation in an automatic analyzer. This study aims to develop a new method that is easier to operate. We assumed that the use of magnetic beads instead of gel beads would enable the skip of the centrifugal process. First, similar to the ILG method, porous magnetic beads were treated with liposomes containing fluorescently labeled cholesterol. Fluorescence was observed inside the magnetic beads, and almost the same amount of liposomes as in the ILG method was immobilized successfully. These immobilized liposome-bound magnetic beads (ILMs) were available for CEC assay when HDL and apolipoprotein B-100 depleted serum were used as cholesterol acceptors. The ILM method showed sufficient basic performance and a good correlation with the ILG method. Furthermore, when the CEC of 15 serum samples from healthy subjects was measured, a good correlation between HDL-cholesterol level and the ILG method was confirmed. Thus, it was confirmed that the ILM method was successfully developed and could be automated.

Magnetic Bead Manipulation in Microfluidic Chips for Biological Application.

Magnetic beads manipulation in microfluidic chips is a promising research field for biological application, especially in the detection of biological targets. In this review, we intend to present a thorough and in-depth overview of recent magnetic beads manipulation in microfluidic chips and its biological application. First, we introduce the mechanism of magnetic manipulation in microfluidic chip, including force analysis, particle properties, and surface modification. Then, we compare some existing methods of magnetic manipulation in microfluidic chip and list their biological application. Besides, the suggestions and outlook for future developments in the magnetic manipulation system are also discussed and summarized.

Panning for gold: Purifying mesencephalic dopaminergic progenitors differentiated from human pluripotent stem cells.

In a recently published study, Xu et al. used two surface markers, CLSTN2 and PTPRO, to generate highly purified donor dopaminergic neurons and achieved stable and predictable therapeutic outcomes by transplantation into the brain of PD animal models (Xu et al., 2022).

Characterization of a Droplet Containing the Clustered Magnetic Beads Manipulation by Magnetically Actuated Chips

A magnetically actuated chip was successfully developed in this study to perform the purpose of transportation for a droplet containing clustered magnetic beads. The magnetic field gradient is generated by the chip of the two-layer 4 × 4 array micro-coils, which was commercially fabricated by printing circuit board (PCB) technology. A numerical model was first established to investigate the magnetic field and thermal field for such a micro-coil. Consequently, the numerical simulations were in reasonable agreement with the experimental results. Moreover, a theoretical analysis was derived to predict the dynamic behaviors of the droplets. This analysis will offer the optimal operation for such a magnetically actuated chip. This study aims to successfully implement the concept of “digital microfluidics” in “point-of-care testing” (POCT). In the future, the micro-coil chip will be of substantial benefit to genetic analysis and infectious disease detection.

The Utilization of Tunable Transducer Elements Formed by the Manipulation of Magnetic Beads with Different Sizes via Optically Induced Dielectrophoresis (ODEP) for High Signal-to-Noise Ratios (SNRs) and Multiplex Fluorescence-Based Biosensing Applications.

Magnetic beads improve biosensing performance by means of their small volume and controllability by magnetic force. In this study, a new technique composed of optically induced dielectrodphoresis (ODEP) manipulation and image processing was used to enhance the signal-to-noise ratio of the fluorescence for stained magnetic beads. According to natural advantages of size-dependent particle isolation by ODEP manipulation, biomarkers in clinical samples can be easily separated by different sizes of magnetic beads with corresponding captured antibodies, and rapidly distinguished by separated location of immunofluorescence. To verify the feasibility of the concept, magnetic beads with three different diameters, including 21.8, 8.7, and 4.2 μm, were easily separated and collected into specific patterns in the defined target zone treated as three dynamic transducer elements to evaluate fluorescence results. In magnetic beads with diameter of 4.2 μm, the lowest signal-to-noise ratio between stained and nonstained magnetic beads was 3.5. With the help of ODEP accumulation and detection threshold setting of 32, the signal-to-noise ratio was increased to 77.4, which makes this method more reliable. With the further optimization of specific antibodies immobilized on different-size magnetic beads in the future, this platform can be a potential candidate for a high-efficiency sensor array in clinical applications.

Prostate-Specific Membrane Antigen (PSMA)-Positive Extracellular Vesicles in Urine-A Potential Liquid Biopsy Strategy for Prostate Cancer Diagnosis?

Simple SummaryProstate cancer is the second most commonly diagnosed cancer and the fifth leading cause of cancer-related death in men. It is a generally slow-growing cancer that—when detected in its early stages—has high chances of successful treatment. Just like all cells, cancerously degenerated cells release extracellular vesicles (EVs) to communicate with other cells. The aim of our research was to specifically isolate prostate cancer-derived EVs from urine, characterize the EV surface markers of a prostate cancer cohort, and assess the potential value of the prostate-specific membrane antigen (PSMA) as a biomarker for liquid biopsy in early cancer diagnostics. Our findings demonstrate that the automated isolation of EVs allows for an overall improvement of the precision in sample purification in comparison to manual isolation, thus optimizing the further characterization of EV surface markers as well as evaluating their use in clinical application.All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.

Use of uncoated magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and real-time-PCR

Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 107 CFU/mL, 1.68 × 106 CFU/mL and 1.68 × 105 CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 104 CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 105 and 2 × 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease.

M R.N.A. Purifications Process: Affinity Chromato-Graphy, Magnetic Beads and Graphene Coated Large Scale Production and Toxicological Aspects

Aim of this work is to verify the state of the art related m R.N.A. purification process , the technology used as well as the materials employed. New methods vs classic methods: the use of affinity cromato-graphy or and high‐gradient magnetic separation (H.G.M.S.). In particular the use of magnetic beads since introduction of the graphene coated ones. Various producers provide many kind of magnetic beads with various grade of efficiency in separation. Becasue today there is a great debate around graphene derivates peresence (or not) in vials of m R.N.A. VACCINE it is relevant to better clarify the production process as well and the materials used.

Use of magnetic beads in Next-Generation Sequencing in the Construction of Low Cost 16S rRNA Gene Genomic Libraries

This work aimed to test the new Mag-Bind® TotalPure magnetic beads in the construction of a genomic library of corn and sugarcane endophytes. The technique Next-Generation Sequencing (NGS) is very expensive and for that, one of the most expensive reagents are the magnetics beads that serve to purify the amplified DNA and separate by size depending on their proportion, as shown in the Illumina protocol. We observed that the use of these beads, which are five times cheaper and have the same capacity and quality as those on the market, are good for a satisfactory study of metagenomics and microbiomes. We conclude that the use of omega brand beads has an excellent result in the construction of a genomic library for NGS.

TAMI-33. DYNAMICS OF MICROGLIAL ACTIVATION AND EXPRESSION OF CYTOKINES IN THE SITE OF GLIOBLASTOMA TUMOR RESECTION

Abstract Glioblastoma (GBM) is the most aggressive type of brain cancer. GBM tumors are made up of a large percentage of microgliawhich support the invasive nature of tumor and the resistance to therapy. In the tumor, microglia cells can polarize into M1 and M2 phenotypes. M1 phenotype is distinguished by its ability to eliminate tumor cells, and secrete proinflammatory cytokines, whileM2 phenotype is associated with tumor cell survival and secrete anti-inflammatory cytokines. Tumor resection is one of the primary steps to treat GBM, thus, resulting in tissue damage and causing microglia activation in the surgical area. The activation state of microglia in the site of resection and the impact of microglia on glioma regrowth is unclear. The purpose of this study is to investigate the state of microglia in the tumor resection area and kinetics of microglial activation.C57BL/6/GL261 mouse glioma implantation model was used. 14 days after in-brain implantation of GL261 cells tumors were surgically resected and then re-grown tumors were analyzed at 0 hours,1, 4, 7, 14 and 21 days after tumor resection, together with the originally resected primary tumors. Microglia were purified from tumors with use of magnetic beads. Western blot analysis of microglia, purified from primary and re-grown tumors, demonstrated increase of CD86 (M1) during the first 7 days after resection and then up-regulation of Arginase (M2) in a period from 14thto 21st days after tumor resection. The cytokine expression analysis revealed significant up-regulation of Vascular Endothelial Growth Factor (VEGF) and Monocyte Chemoattractant Protein 1 (MCP1) in microglia purified from re-grown tumors. PCR analysis confirmed these data, indicating increase of VEGF, MCP1 gene expression in tumor infiltrating microglia in re-grown tumor. We can state that microglia infiltrating primary tumor and tumor re-grown after surgical resection, represent significantly different cytokines expression patterns, favorable to increased vascularization and tumor progression.

A MEMS room-temperature resettable thermomagnetic-track-guided magnetic-bead manipulation integrated with magnetoresistive sensing for bead-motion monitoring

In this paper, we report a MEMS room-temperature resettable thermomagnetic-track-guided magnetic-bead manipulation integrated with magnetoresistive sensing for bead-motion monitoring on a silicon wafer. The manipulation/monitoring approach consists of a magnetic bead, a chip (including thermomagnetic NiCu micro-disk tracks, Pt micro heating wires, and Ni magnetoresistive sensing elements, all fabricated on a silicon wafer), pulse-generation circuit, magnetoresistive measuring circuit, thermoelectric generator (TEG), and four external electromagnets. Because Curie temperature of the NiCu micro-disk tracks is in room-temperature range, when a NiCu track is cooled by TEG to lower than the room temperature, the track becomes ferromagnetic and thus is able to attract the bead. When the electromagnets are used to apply magnetic fields to move the bead, the bead motion is guided by the ferromagnetic track. Oppositely, when the heating-wire is used to heat the track higher than the room temperature, the track switches to paramagnetic-like and thus cannot attract the bead. When the magnetic field is applied to move the bead, the bead motion is not-guided by the paramagnetic-like track. By using these approaches, we successfully demonstrated guiding the bead moving around a track and across different tracks. By using magnetoresistive sensing, location of the bead is obtained and consequently monitoring of the bead-motion is achieved. The most important feature is that our approach can easily reset any track anytime during the bead-manipulation in room-temperature range, when comparing to conventional resetting approach which is complicated and requires to several hundred °C. In addition, our approach is the first one to combine the magnetic-track guided magnetic-bead manipulation and magnetoresistive-sensing-element based magnetic-bead motion monitoring on a silicon chip (toward a fully on-chip actuating/sensing MEMS system for magnetic bead manipulation). The results are important and alternative solutions for magnetic-track-guided magnetic bead manipulation for biomedical MEMS.

Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α.

The use of magnetic beads bio-functionalized by antibodies (Ab) is constantly increasing with a wide range of biomedical applications. However, despite an urgent need for current methods to monitor Ab’s grafting process and orientation, existing methods are still either cumbersome and/or limited. In this work, we propose a new simple and rapid analytical approach to evaluate antibody orientation and density on magnetic beads. This approach relies on the cleavage by IdeS, a highly specific protease for human immunoglobulin G (hIgG), of immobilized antibodies. The F(ab)2 and Fc fragments could be then accurately quantified by size exclusion chromatography (SEC)-coupled to fluorescent detection (FLD), and the ratio of these fragments was used to give insight on the IgG orientation at the bead surface. Four different commercially available magnetic beads, bearing carboxyl groups, tosyl groups, streptavidin, or protein G on their surface have been used in this study. Results obtained showed that this approach ensures reliable information on hIgG orientation and bead surface coverage. Protein G magnetic beads demonstrated an optimal orientation of antibodies for antigen capture (75% of accessible F(ab)2 fragment) compared to tosylactivated, carboxylated, and streptavidin ones. Capture efficiency of the different functionalized beads towards human TNF-α immunocapture, a biomarker of inflammation, has been also compared. Protein G beads provided a more efficient capture compared to other beads. In the future, this approach could be applied to any type of surface and beads to assess hIgG coverage and orientation after any type of immobilization.Graphical abstract A rapid and simple approach to evaluate orientation and density of antibodies immobilized on magnetic beadsSupplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03608-w.

Electrochemical Competitive Magneto-Immunoassay for Determination of Neurotoxin Domoic Acid

Domoic acid is heterocyclic amino acid neurotoxin and the principal toxin associated with amnesic shellfish poisoning (ASP) produced by certain species of marine diatom. It can bio accumulated at high concentrations in the shellfish tissues when the toxic phytoplankton is high in concentration in the surrounding waters. Ingestion of domoic acid contaminated shellfish leads to ASP, which is characterized by both gastrointestinal and neurological symptoms, including severe headache, seizures, tremors, and either temporary or permanent memory loss.Herein, the detection of domoic acid is carried out through an enzymatic immunoassay that uses magnetic beads as support of the immunoreaction and screen-printed electrodes as suitable detector of the enzymatic product. Thereby, the immunological reaction is performed in solution, where the domoic acid, antigen, domoic acid labeled with horseradish peroxidase are incubated with the rabbit IgG antibody against domoic acid. Subsequently, the antibody-antigen complex is captured by the anti-rabbit IgG modified magnetic beads. Then, the magnetic beads are deposited onto the electrode surface with the help of the magnet placed under the screen printed electrode. Finally, the analytical signal is obtained by amperometric detection of the product enzymatically generated from the mixture of H2O2and 3,3,5,5-tetramethylbenzidine (TMB).The use of magnetic beads provides the capability to preconcentrate the sample and avoid the poisoning of the electrode surface through non specific absorptions.The experimental conditions of the magneto-immunological assay has been optimized and a calibration curve has been obtained between 0-2 ng/mL domoic acid, being able to detect down to 100 pg/mL domoic acid. Acknowledgements This work has been carried out in the framework of FLAshMoB project. This project is funded by the MarTERA partners; Italian Ministry of Education, Universities and Research (MIUR), French National Research Agency (ANR) and Spanish Centre for the Development of Industrial Technology (CDTI) and co-funded by the European Union.

Disposable electrochemical microfluidic device for ultrasensitive detection of egg allergen in wine samples

Food allergies have been increasing all over the world. Egg is an important component in the food industries and the second most common cause of food allergy, shortly after milk. In the wine industry, egg white is applied as a fining agent for tannin removal. In this study, a sandwich-based immunoassay for ultrasensitive detection of ovalbumin (OVA) in wine samples was developed. The assay involves the use of magnetic beads (MBs) decorated with a polyclonal anti-OVA antibody (Ab2) and horseradish peroxidase (HRP), used as label for the quantification in a disposable electrochemical microfluidic device (DEμD) here developed. The Ab2-MB-HRP prepared was applied to capture, separate, and pre-concentrate OVA from wine samples. In the DEμD, OVA was immune-magnetically captured (OVA-Ab2-MB-HRP), producing a sandwich structure (GO-Ab1-OVA-Ab2-MB-HRP) on the electrode's surface. This arrangement results in an ultrasensitive device, achieving the ultralow limit of detection of 0.2 fg mL−1 OVA. Five samples of wines were analyzed by using the immuno-magneto-assay which presents excellent accuracy compared with enzyme-linked immunosorbent assay (ELISA).