Magnetic Particle-based Immunoassay Research Articles

Droplet-based microfluidic washing module for magnetic particle-based assays.

In this paper, we propose a continuous flow droplet-based microfluidic platform for magnetic particle-based assays by employing in-droplet washing. The droplet-based washing was implemented by traversing functionalized magnetic particles across a laterally merged droplet from one side (containing sample and reagent) to the other (containing buffer) by an external magnetic field. Consequently, the magnetic particles were extracted to a parallel-synchronized train of washing buffer droplets, and unbound reagents were left in an original train of sample droplets. To realize the droplet-based washing function, the following four procedures were sequentially carried in a droplet-based microfluidic device: parallel synchronization of two trains of droplets by using a ladder-like channel network; lateral electrocoalescence by an electric field; magnetic particle manipulation by a magnetic field; and asymmetrical splitting of merged droplets. For the stable droplet synchronization and electrocoalescence, we optimized droplet generation conditions by varying the flow rate ratio (or droplet size). Image analysis was carried out to determine the fluorescent intensity of reagents before and after the washing step. As a result, the unbound reagents in sample droplets were significantly removed by more than a factor of 25 in the single washing step, while the magnetic particles were successfully extracted into washing buffer droplets. As a proof-of-principle, we demonstrate a magnetic particle-based immunoassay with streptavidin-coated magnetic particles and fluorescently labelled biotin in the proposed continuous flow droplet-based microfluidic platform.

A Magnetic Particle-Based Competitive Enzyme Immunoassay for Rapid Determination of Ciprofloxacin: A Potential Method for the General Detection of Fluoroquinolones

Fluoroquinolones are effective antimicrobial agents, but their residues in food may cause serious health issues. Hence, it is necessary to develop a rapid and simple assay for fluoroquinolones. In this study, monoclonal antibodies against ciprofloxacin with broad specificity to fluoroquinolones were prepared. The newly developed magnetic particle-based competitive enzyme immunoassay was performed in a homogeneous sample, and ciprofloxacin was quantitatively detected in the range of 0.3–24.3 ng mL−1. The IC50 and LOD values of the method were 2.27 ng mL−1 and 0.25 ng mL−1. In chicken muscle, the recoveries of spiked ciprofloxacin at 8, 20, and 40 ng mL−1 were all higher than 79%. Additionally, the performance of the developed magnetic particle-based immunoassay exhibited an excellent correlation with commercial ELISA kits (r = 0.997). The anti-ciprofloxacin monoclonal antibodies provided high cross-reactivity with eight fluoroquinolones analogues: enrofloxacin (110.1%), sarafloxacin (99.7%), difloxacin (90.2%), danofloxacin (89.8%), norfloxacin (110.3%), lomefloxacin (65.2%), ofloxacin (75.1%), and flumequine (45.0%). This protocol provides an alternative immunoassay for ciprofloxacin with the advantages of simple separation, a homogeneous reaction, rapid detection (within 30 min), and stable results. Furthermore, it may be employed for the rapid general determination of fluoroquinolones in animal products.

Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay.

This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems) was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead-protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.

Accelerated Immunoassays Based on Magnetic Particle Dynamics in a Rotating Capillary Tube with Stationary Magnetic Field

A rapid and simple magnetic particle-based immunoassay has been demonstrated in a capillary mixing system. Antibody-coated micrometer size superparamagnetic polystyrene (SPP) particles were used in an assay for rabbit IgG in a sandwich (noncompetitive) format. The kinetics of the assay was compared between a plate-based system and a single capillary tube. The interaction between the antigen (R-IgG) and the antibody (anti-R-IgG) that was carried by the SPP particles in a rotating capillary was tested under a stationary magnetic field. Competing magnetic and viscous drag forces helped to enhance the interaction between the analyte and the capture antibodies on the particles. The dimensionless Mason number (Mn) was employed to characterize the magnetic particle dynamics; a previously determined critical Mason number (Mn(c)) was employed as a guide to the appropriate experimental conditions of magnetic field strength and rotational speed of the capillary. The advantage of the rotating capillary system included a short assay time and a reduced reactive volume (20 μL). The results show that the immunoassay kinetics were improved by the formation of chains of the SPP particles for the conditions that corresponded to the critical Mason number.

Magnetic particle-based immunoassay of phosphorylated p53 using protein cage templated lead phosphate and carbon nanospheres for signal amplification

Phosphorylated p53 at serine 15 (phospho-p5315) is a potential biomarker of gamma-radiation exposure. In this paper, we described a new magnetic particle (MP)-based electrochemical immunoassay of human phospho-p5315 using carbon nanospheres (NS) and protein cage nanoparticles (PCN) for signal amplification. Greatly enhanced sensitivity was achieved for three reasons: 1) PCN and the p5315 signal antibody (p5315 Ab2) are linked to the carbon NS (PCN-p5315 Ab2-NS) as a label; 2) PCN increases the amount of metal ions in the cavity of each apoferritin; 3) MPs capture a large amount of primary antibodies. Protein cage templated metallic phosphates, instead of enzymes, as multi-labels have the advantage of eliminating the addition of mediator or immunoreagents and, thus, makes the immunoassay system simpler. Subsequent stripping voltametric analysis, detected olead ions on a disposable screen-printed electrode. The response current was proportional to the phospho-p5315 concentration in the range of 0.02 to 20 ng mL−1 with a detection limit of 0.01 ng mL−1, which was 30-fold lower than that of the ELISA measurement of phospho-p5315. This method shows an acceptable stability and reproducibility and the assay results for phospho-p5315-spiked human serum presented good recovery rates.

Numerical analysis of transport and binding of a target analyte and functionalized magnetic microspheres in a microfluidic immunoassay

In situ binding of a target analyte on functionalized magnetic microspheres in a straight microchannel, representing a microfluidic immunoassay, is investigated numerically using an Eulerian–Lagrangian approach. Magnetic particles in the microfluidic channel are transported, using an externally imposed magnetic field, in such a manner that the particle–analyte collision is facilitated. The effects of both-ways momentum coupling between the dispersed and fluid phases on the fluid flow, particle trajectories and the analyte concentration profiles have been investigated. The particle–analyte collision is assumed to result in chemical binding between the analyte and the functionalized magnetic particles. Analyte concentration and fluid velocity fields are found to be influenced by dipole strength (P) and particle loading. In situ target analyte binding on magnetic microspheres has been quantified in terms of binding efficiency (BE), which is found to be functions of the particle loading, particle radius, dipole strength, flow velocity and the fluid viscosity. While particle loading increases, the BE also increases, but the analyte binding per particle slightly decreases. With increase in dipole strength, the BE first increases and then decreases. If the particle loading is increased, the maximum BE occurs at a higher value of P, but at a lower value of viscosity. An increase in flow velocity is found to have strong adverse effect on the BE. The study is important for the selection of optimum operating parameters so that the analyte BE of a magnetic particle-based immunoassay can be maximized.

Triclosan and methyl-triclosan monitoring study in the northeast of Spain using a magnetic particle enzyme immunoassay and confirmatory analysis by gas chromatography–mass spectrometry

For the first time, the occurrence of triclosan and its metabolite methyl-triclosan was investigated in a typical Mediterranean area using a two-step methodology based on screening using a magnetic particle immunoassay (IA) and confirmatory analysis by solid phase extraction (SPE) followed by gas chromatography–mass spectrometry (GC–MS). In this study, 95 environmental samples were analyzed. A commercial immunoassay was assessed for use in the different types of water selected for this study. A large monitoring study was performed on the influent and the effluent of eight wastewater treatment plants (WWTPs), water samples from Ebro and Llobregat rivers, and drinking water. All wastewater samples tested in this study (influents and effluents) showed the presence of triclosan, with concentrations for raw influents being high (10 μg/L as average value). The percentages of triclosan removal for the WWTPs were evaluated (30–70%) along the different treatment processes showing that the best removal rates were obtained by the processes equipped with membrane bioreactors (MBRs). However, important concentrations of triclosan were detected even after treatment by MBRs. The presence of this biocide was confirmed in 50% of the river samples analyzed. Twenty two drinking water samples from the Barcelona city area were investigated, and in this case no triclosan was detected. Due to its properties and the widespread usage of triclosan, there is a need for monitoring and controlling the amounts present in wastewater effluents, river water, drinking water catchments areas, and drinking water. To this end, we present a feasible methodology using a magnetic particle-based immunoassay as a screening, followed by confirmatory analysis using solid phase extraction–gas chromatography–mass spectrometry (SPE–GC–MS).

High-Throughput Automated Luminescent Magnetic Particle-Based Immunoassay to Monitor Human Exposure to Pyrethroid Insecticides

We have developed a sensitive, automated, competitive chemiluminescent immunoassay for the detection of 3-phenoxybenzoic acid (3-PBA), a metabolite common to many pyrethroid insecticides. The system uses a competitive hapten-protein conjugate that has been labeled with an acridinium ester as the chemiluminescent probe and secondary antibody-coated paramagnetic particles for the separation. After the immunoassay reagents and samples are combined for the competitive incubation step, a fully automated system is used to load the postincubation mixture into a delivery cuvette, facilitating the subsequent magnetic separation of the immunocomplex and the measurement of chemiluminescent signal for quantification. The immunoassay format described here supports the requirement for high throughput necessary for monitoring large numbers of samples in population-based studies. The optimized immunoassay was more sensitive than the conventional enzyme immunoassay in buffer (IC(50) = 0.1 and 2 microg/L, respectively). The mixed-mode solid-phase extraction used for sample preparation to reduce possible urinary matrix effects allowed the accurate measurement of 3-PBA levels as low as 1 microg/L. The automated chemiluminescent immunoassay described here is sensitive, simple to use, and more rapid than the previously reported standard microplate assay.

Measurement of Triclosan in Water Using a Magnetic Particle Enzyme Immunoassay

A sensitive magnetic particle-based immunoassay to determine triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-[5-chloro-2-(2,4-dichlorophenoxy)phenoxy]hexanoic acid-keyhole limpet hemocyanin. Horseradish peroxidase was conjugated with 4-[3-bromo-4-(2,4-dibromophenoxy)phenoxy]butyric acid via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The triclosan antibody was coupled to magnetic particles via the NHS/EDC reaction. The antibodies were able to recognize some structurally related polybrominated biphenyl ethers but did not recognize various common pollutants that were less similar to the hapten. The ELISA could detect triclosan in standard solution (25% methanol/H2O v/v) at 20 ppt and its metabolite, methyl-triclosan, at 15 ppt. Water samples from different treatment stages were prepared to contain 25% methanol and analyzed directly without any sample extraction or preconcentration. The results showed that recoveries were >80% and the % CV was <10%, demonstrating the assay was both accurate and precise. Application of the triclosan ELISA to water treatment plants showed that tap water at various purification stages had low concentrations of triclosan (<20 ppt) and required an increased sample size for appropriate detection and measurement. Application of ELISA to the wastewater treatment plants (WWTP) demonstrated high concentrations of triclosan (in general, >3000 ppt in water entering the WWTP) with the levels decreasing as the water proceeded through the processing plant (<500 ppt at outflow sewage). The ELISA measurement was shown to be equivalent to the more specific GC-MS analysis on a number of wastewater treatment samples with a high degree of correlation, with the exception of a few samples with very high triclosan concentrations (>5000 ppt). Measurement of methyl-triclosan (in WWTP) using GC-MS demonstrated the levels of this compound to be low. In summary, a rapid, sensitive, accurate, and precise magnetic particle-based immunoassay has been developed for triclosan analysis, which can serve as a cost-effective monitoring tool for various water samples.

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.

Determination of Spinosad and Its Metabolites in Food and Environmental Matrices. 3. Immunoassay Methods

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.

Quantitative Determination of PCBs in Soil and Water by a Magnetic Particle-Based Immunoassay

A competitive enzyme immunoassay for the quantification of polychlorinated biphenyls (PCBs) in soil and water was developed utilizing amine-terminated superparamagnetic particles as the solid phase. Aroclor 1254 was covalently attached to a bovine serum albumin carrier, and the resulting PCB−protein conjugate was used to immunize rabbits and to produce polyclonal antibodies with reactivity to a broad range of PCBs. Specificity studies indicate that the polyclonal antibody can detect Aroclors 1016, 1232, 1242, 1248, 1254, 1260, 1262, and 1268. The immunoassay has a estimated detection limit of 0.2 ppb (ng/mL) in water and 0.5 ppm (mg/kg) in soil based on Aroclor 1254. The assay compares favorably to GC method 8080 results when soil (r = 0.960) or water samples are evaluated (r = 0.909). The typical within assay % CV is less than 9% in water samples, and recovery studies averaged 99% in water and 85% in soil using a 1-min extraction. This immunochemical method provides quantitative field or laboratory screening results for characterizing and delineating PCB-contaminated sites.

Determination of Captan in Water, Peaches, and Apple Juice by a Magnetic Particle-Based Immunoassay

Abstract A magnetic particle-based enzyme-linked immunosorbent assay (EUSA) was used to determine captan residues in water, peaches, and apple juice. Precision studies of captan-spiked water samples showed coefficients of variation (CVs) of 24% at 0.1 ppm and 15% at 2.0 ppm. Average recoveries from water were 110–118%, with an estimated sensitivity of 0.01 ppm for captan. Low cross-reactivity was seen with the related phthalimide fungicides capta-fol and folpet. A least-detectable dose (LDD) of 1 ppm was estimated for captafol in the immunoassay; an LDD of 8.6 ppm was estimated for folpet. Apple juice samples were analyzed by a 3-step procedure: the sample was concentrated on a Cis bonded silica solid-phase column, the eluate was diluted in buffer, and the sample was assayed by ELISA. The detection limit in apple juice was estimated to be 0.1 ppm on the basis of the detection limit observed in water and the required calculation factors; the average recovery was 115%. Acetone extraction, Cis bonded silica solid-phase extraction column, and the immunoassay were used consecutively to analyze peach samples. The detection limit in peaches was estimated to be 0.15 ppm on the basis of the detection limit observed in water and the required calculation factors. Captan residues were found in all but one of the peach samples and ranged from 0.15 to 1.12 ppm (|ig/g). Recovery of added captan from peaches averaged 98% for both spike levels tested. Results of analysis of captan in peaches by immunoassay and gas chromatography with electron capture detection (GC/ECD) were compared. GC/ECD gave an average recovery of 75%; immunoassay gave an average recovery of 112%.

Determination of benomyl (as carbendazim) and carbendazim in water, soil, and fruit juice by a magnetic particle-based immunoassay

A competitive enzyme-linked immunosorbent assay (ELISA) for the quantiation of benomyl (as carbendazim) and carbendazim in water was developed. This assay utilizes magnetic particles as the solid phase to attach polyclonal rabbit anti-carbendazim antibodies. The ELISA has an estimated detection limit of 0.1 ng/ml of carbendazim in water. It compares favorably with HPLC measurements in the analysis of water samples. Studies indicate that this immunoassay can also be used for the determination of carbendazim in fruit juice and soil