Simultaneous sample washing and concentration using a "trapping-and-releasing" mechanism of magnetic beads on a microfluidic chip

Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 μL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 μL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.     

Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody

A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0-500 ppb) under optimum conditions. The time required for analysis is less than 15 min.     

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL⁻¹) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL⁻¹.     

Bubble cell for magnetic bead trapping in capillary electrophoresis

A bubble cell capillary classically used to extend the optical path length for UV–vis detection is employed here to trap magnetic beads. With this system, a large amount of beads can be captured without inducing a strong pressure drop, as it is the case with magnetic beads trapped in a standard capillary, thereby having less effect on the experimental conditions. Using numerical simulations and microscopic visualizations, the capture of beads inside a bubble cell was investigated with two magnet configurations. Pressure-driven and electro-osmotic flow velocities were measured for different amounts of protein-A-coated beads or C18-functionalized beads (RPC-18). Solid-phase extraction of a model antibody on protein-A beads and preconcentration of fluorescein on RPC-18 beads were performed as proof of concept experiments.     

Differential magnetic catch and release: experimental parameters for controlled separation of magnetic nanoparticles

Differential magnetic catch and release (DMCR) has been used as a method for the purification and separation of magnetic nanoparticles. DMCR separates nanoparticles in the mobile phase by magnetic trapping of magnetic nanoparticles against the wall of an open tubular capillary wrapped between two narrowly spaced electromagnetic poles. Using Au and CoFe(2)O(4) nanoparticles as model systems, the loading capacity of the 250 μm diameter capillary is determined to be ～130 μg, and is scalable to higher quantities with larger bore capillary. Peak resolution in DMCR is externally controlled by selection of the release time (R(t)) at which the magnetic flux density is removed, however, longer capture times are shown to reduce the capture yield. In addition, the magnetic nanoparticle capture yields are observed to depend on the nanoparticle diameter, mobile phase viscosity and velocity, and applied magnetic flux. Using these optimized parameters, three samples of CoFe(2)O(4) nanoparticles whose diameters are different by less than 10 nm are separated with excellent resolution and capture yield, demonstrating the capability of DMCR for separation and purification of magnetic nanoparticles.     

Microchip integrating magnetic nanoparticles for allergy diagnosis

We report on the development of a simple and easy to use microchip dedicated to allergy diagnosis. This microchip combines both the advantages of homogeneous immunoassays i.e. species diffusion and heterogeneous immunoassays i.e. easy separation and preconcentration steps. In vitro allergy diagnosis is based on specific Immunoglobulin E (IgE) quantitation, in that way we have developed and integrated magnetic core-shell nanoparticles (MCSNPs) as an IgE capture nanoplatform in a microdevice taking benefit from both their magnetic and colloidal properties. Integrating such immunosupport allows to perform the target analyte (IgE) capture in the colloidal phase thus increasing the analyte capture kinetics since both immunological partners are diffusing during the immune reaction. This colloidal approach improves 1000 times the analyte capture kinetics compared to conventional methods. Moreover, based on the MCSNPs' magnetic properties and on the magnetic chamber we have previously developed the MCSNPs and therefore the target can be confined and preconcentrated within the microdevice prior to the detection step. The MCSNPs preconcentration factor achieved was about 35,000 and allows to reach high sensitivity thus avoiding catalytic amplification during the detection step. The developed microchip offers many advantages: the analytical procedure was fully integrated on-chip, analyses were performed in short assay time (20 min), the sample and reagents consumption was reduced to few microlitres (5 μL) while a low limit of detection can be achieved (about 1 ng mL(-1)).     

Nanoparticle flotation collectors: mechanisms behind a new technology

This is the first report describing a new technology where hydrophobic nanoparticles adsorb onto much larger, hydrophilic mineral particle surfaces to facilitate attachment to air bubbles in flotation. The adsorption of 46 nm cationic polystyrene nanoparticles onto 43 μm diameter glass beads, a mineral model, facilitates virtually complete removal of the beads by flotation. As little as 5% coverage of the bead surfaces with nanoparticles promotes high flotation efficiencies. The maximum force required to pull a glass bead from an air bubble interface into the aqueous phase was measured by micromechanics. The pull-off force was 1.9 μN for glass beads coated with nanoparticles, compared to 0.0086 μN for clean beads. The pull-off forces were modeled using Scheludko's classical expression. We propose that the bubble/bead contact area may not be dry (completely dewetted). Instead, for hydrophobic nanoparticles sitting on a hydrophilic surface, it is possible that only the nanoparticles penetrate the air/water interface to form a three-phase contact line. We present a new model for pull-off forces for such a wet contact patch between the bead and the air bubble. Contact angle measurements of both nanoparticle coated glass and smooth films from dissolved nanoparticles were performed to support the modeling.     

Magnetic targeting and cellular uptake of polymer microcapsules simultaneously functionalized with magnetic and luminescent nanocrystals

By using a flow channel system for modeling the bloodstream in the circulatory system and by locally creating a magnetic field gradient caused by a permanent magnet, we demonstrate specific trapping of polymer capsules simultaneously functionalized with two types of nanoparticles--magnetic and luminescent nanocrystals. In the regions where the capsules were trapped by the magnetic field, drastically increased uptake of capsules by cells has been observed. The uptake of capsules by cells could be conveniently monitored with a fluorescence microscope by the luminescence of CdTe nanocrystals that had been embedded into the shells of the capsules. Our experiments envisage the feasibility of magnetic targeting of polymer capsules loaded by pharmaceutical agents to pathogenic parts of a tissue.     

Continuous microfluidic DNA extraction using phase-transfer magnetophoresis

This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 μl s(-1) and an eluate flow rate of 0.35 μl s(-1). DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.     

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.     

Microfluidic magnetophoretic separations of immunomagnetically labeled rare mammalian cells

Immunomagnetic isolation and magnetophoresis in microfluidics have emerged as viable techniques for the separation, fractionation, and enrichment of rare cells. Here we present the development and characterization of a microfluidic system that incorporates an angled permanent magnet for the lateral magnetophoresis of superparamagnetic beads and labeled cell-bead complexes. A numerical model, based on the relevant transport processes, is developed as a design tool for the demonstration and prediction of magnetophoretic displacement. We employ a dimensionless magnetophoresis parameter to efficiently investigate the design space, gain insight into the physics of the system, and compare results across the vast spectrum of magnetophoretic microfluidic systems. The numerical model and theoretical analysis are experimentally validated by the lateral magnetophoretic deflection of superparamagnetic beads and magnetically labeled breast adenocarcinoma MCF-7 cells in a microfluidic device that incorporates a permanent magnet angled relative to the flow. Through the dimensionless magnetophoresis parameter, the transition between regimes of magnetophoretic action, from hydrodynamically dominated (magnetic deflection) to magnetically dominated (magnetic capture), is experimentally identified. This powerful tool and theoretical framework enables efficient device and experiment design of biologically relevant systems, taking into account their inherent variability and labeling distributions. This analysis identifies the necessary beads, magnet configuration (orientation), magnet type (permanent, ferromagnetic, electromagnet), flow rate, channel geometry, and buffer to achieve the desired level of magnetophoretic deflection or capture.     

Microfluidic single-cell cultivation chip with controllable immobilization and selective release of yeast cells

We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.     

Ultra magnetic liposomes for MR imaging, targeting, and hyperthermia

Magnetic liposomes offer opportunities as theranostic systems. The prerequisite for efficient imaging, tissue targeting or hyperthermia is high magnetic load of these vesicles. Here we describe the preparation of Ultra Magnetic Liposomes (UMLs), which may encapsulate iron oxide nanoparticles in a volume fraction of up to 30%. This remarkable magnetic charge provides UMLs with high magnetic mobilities, MRI relaxivities, and heating capacities for magnetic hyperthermia. Moreover, these UMLs are rapidly and efficiently internalized by cultured tumor cells and, when they are administered to mice, they can be vectorized to tumors by an external magnet.     

Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads

A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm of the concentration of APnEOs was plotted against the chemiluminescence intensity as the number of photons in 100 ms using standard APnEOs sample solutions at various concentrations (0–1000 ppb) under optimum conditions. The lower detection limit defined as IC₈₀ is ca 10 ppb. The time required for analysis is less than 15 min per a sample. The present method was successfully applied to the determination of APnEOs in river water.     

A novel high-performance fourier transform ion cyclotron resonance cell for improved biopolymer characterization

A new trapped ion cell design for use with Fourier transform ion cyclotron resonance mass spectrometry is described. The design employs 15 cylindrical ring electrodes to generate trapping potential wells and 32 separately assignable rod electrodes for excitation and detection. The rod electrodes are positioned internal to the ring electrodes and provide excitation fields that are thereby linearized along the magnetic field over the entire trapped ion volume. The new design also affords flexibility in the shaping of the trapping field using the 15 ring electrodes. Many different trapping well shapes can be generated by applying different voltages to the individual ring electrodes, ranging from quadratic to linearly ramped along the magnetic field axis, to a shape that is nearly flat over the entire trap volume, but rises very steeply near the ends of the trap. This feature should be useful for trapping larger ion populations and extension of the useful range of ion manipulation and dissociation experiments since the number of stages of ion manipulation or dissociation is limited in practice by the initial trapped ion population size. Predicted trapping well shapes for two different ring electrode configurations are presented, and these and several other possible configurations are discussed, as are the predicted excitation fields based on the use of rod electrodes internal to the trapping ring electrodes. Initial results are presented from an implementation of the design using a 3.5 T superconducting magnet. It was found that ions can be successfully trapped and detected with this cell design and that selected ion accumulation can be performed with the utilization of four rods for quadrupolar excitation. The initial results presented here illustrate the feasibility of this cell design and demonstrate differences in observed performance based upon different trapping well shapes.     

Magnetoswitchable controlled hydrophilicity/hydrophobicity of electrode surfaces using alkyl-chain-functionalized magnetic particles: application for switchable electrochemistry

Magnetic nanoparticles consisting of undecanoate-capped magnetite (average diameter approximately 4.5 nm; saturated magnetization, M(s), 38.5 emu g(-1)) are used to control and switch the hydrophobic or hydrophilic properties of the electrode surface. A two-phase system consisting of an aqueous buffer solution and a toluene phase that includes the suspended capped magnetic nanoparticles is used to control the interfacial properties of the electrode surface. The magnetic attraction of the functionalized particles to the electrode by means of an external magnet yields a hydrophobic interface that acts as an insulating layer, prohibiting interfacial electron transfer. The retraction of the magnetic particles from the electrode to the upper toluene phase by means of the external magnet generates a hydrophilic electrode that reveals effective interfacial electron transfer. The electron-transfer resistance and double-layer capacitance of the electrode surface upon the attraction and retraction of the functionalized magnetic particles to and from the electrode, respectively, by means of the external magnet were probed by Faradaic impedance spectroscopy (R(et) = 170 Omega and C(dl) = 40 microF sm(-2) in the hydrophilic state of the electrode and R(et) = 22 k Omega and C(dl) = 0.5 microF sm(-2) in the hydrophobic state of the interface). The magnetoswitchable control of the interface enables magnetic switching of the bioelectrocatalytic oxidation of glucose in the presence of glucose oxidase and ferrocene dicarboxylic acid to "ON" and "OFF" states.     

Magnetic domain wall conduits for single cell applications

The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation.     

One-pot synthesis of pyranocoumarins via microwave-assisted pseudo multicomponent reactions and their molecular switching properties

Two new pyranocoumarins were synthesized via one-pot, microwave-assisted pseudo multicomponent condensations of coumarin and 4-methylquinoline to investigate their molecular switching properties. Both are light-sensitive and have a distinct change of color upon UV irradiation. The reaction can be reverted by treating the photogenerated products with imidazoline-functionalized magnetic nanoparticles, which can be swiftly recycled with an external permanent magnet.     

Electrochemical assay of concanavalin A-ovalbumin binding on magnetic beads

To monitor protein-glycoprotein interactions on magnetic beads, the present study developed an electrochemical assay of the binding between concanavalin A (ConA) and ovalbumin (OVA). The system was a powerful model that could be used to evaluate cell junctions. ConA with an electroactive daunomycin was immobilized on 6 different sizes of magnetic beads (diameter: 1.0-8.9 μm) through a cross-linking agent. Six sizes of OVA-beads (diameter: 1.0-8.9 μm) were also prepared using a similar method. The binding was evaluated using an oxidation peak of ConA with daunomycin because ConA recognized OVA with α-mannose residues. When binding took place on the beads' surface, the peak current was decreased due to the electroactive moieties being covered with OVA. When ConA/daunomycin-OVA binding was evaluated, the change of the peak current obtained by the beads (diameter: 8.9 μm) modified with ConA and daunomycin was the greatest in the presence of OVA-modified beads (diameter: 2.5 μm). In contrast, particle agglomeration was observed for the smallest beads (diameter: 1.0 μm) with ConA/daunomycin and OVA. The results suggested that ConA-OVA binding depended on the size of beads. Thus, this method could be applied to measure protein-glycoprotein interactions on the cell surface.     

Dielectrophoretic trapping of nanoparticles with an electrokinetic nanoprobe

A high aspect ratio 3D electrokinetic nanoprobe is used to trap polystyrene particles (200 nm), gold nanoshells (120 nm), and gold nanoparticles (mean diameter 35 nm) at low voltages (<1 V_rms). The nanoprobe is fabricated using room temperature self‐assembly methods, without the need for nanoresolution lithography. The nanoprobe (150–500 nm in diameter, 2–150 μm in length) is mounted on the end of a glass micropipette, enabling user‐specified positioning. The nanoprobe is one electrode within a point‐and‐plate configuration, with an indium–tin oxide cover slip serving as the planar electrode. The 3D structure of the nanoprobe enhances dielectrophoretic capture; further, electro‐hydrodynamic flow enhances trapping, increasing the effective trapping region. Numerical simulations show low heating (1 K), even in biological media of moderate conductivity (1 S/m).