Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.     

Selective sensitivity of ellipsometry to magnetic nanostructures

Magneto-optic (MO) ellipsometry of ferromagnetic materials is extremely sensitive to ultra-thin films, multilayers, and nanostructures. It gives a possibility to measure all components of the magnetization vector in the frame of the magneto-optic vector magnetometry and enables us to separate magnetic contributions from different depths and materials in nanostructures, which is reviewed in this article. The method is based on ellipsometric separation using the selective MO Kerr effect. The figure of merit used to quantify the ellipsometric selectivity to magnetic nanostructures is defined on the basis of linear matrix algebra. We show that the method can be also used to separate MO contributions from areas of the same ferromagnetic materials deposited on different buffer layers. The method is demonstrated using both: (i) modeling of the MO ellipsometry response and (ii) MO measurement of ultra-thin Co islands epitaxially grown on self-organized gold islands on Mo/Al₂O₃ buffer layer prepared using the molecular beam epitaxy at elevated temperatures. The system is studied using longitudinal (in-plane) and polar (perpendicular) MO Kerr effects.     

Latticing vertically aligned Ag nanorods to enhance its SERS sensitivity

Vertically aligned silver nanorods were good substrates for surface-enhanced Raman scattering. It was found that by organizing these nanorods into hexagonal lattice with a pattern size of 400 nm, the Raman sensitivity can be further enhanced by several times. It was also found that this enhancement was dependent on the separation distance of the lattice patterns, which reached a maximum at a separation distance of 200 nm. This study provides an alternative idea to further enhance the Raman scattering using nanostructures.     

Three-dimensional mesoporous gold film to enhance the sensitivity of electrochemical detection

Cell-cell and cell-extracellular matrix (ECM) adhesion are fundamental and important in the development of a cell-based chip. In this study, a novel, simple, rapid, and one-step technique was developed for the fabrication of a uniform three-dimensional mesoporous gold thin film (MPGF) onto a gold (Au) coated glass plate based on an electrochemical deposition method. Scanning electron microscopy images demonstrated that the resulting MPGF electrode had uniformly distributed pores with diameters of about 20 nm. The cyclic voltammetric behavior of [Fe(CN)(6)](4-/3-) coupled onto MPGF and Au electrodes demonstrated that the MPGF electrode had a higher electrocatalytic sensitivity and reversibility than the bare Au electrode. The Arg-Gly-Asp (RGD) sequence containing the peptide was immobilized on the MPGF and bare Au substrates. HeLa cancer cells were then cultured on the RGD peptide layer. The successful immobilization of the peptide and cells was confirmed by atomic force microscopy. The cell proliferation and viability were evaluated by cyclic voltammetry and Trypan blue dyeing assay. These results indicated that the RGD/MPGF modified electrodes showed an electrochemical sensitivity in the detection of cancer cells which is approximately three times higher, especially at low cell density, than RGD/Au electrodes. This much improved sensitivity of the MPGF modified electrode demonstrates the potential for the fabrication of a highly sensitive and low-cost cell-based chip for rapid cancer detection.     

Cell toxicity studies of albumin-based nanosized magnetic beads

The aim of this study was to prepare bovine serum albumin-based beads containing maghemite nanoparticles incorporated via ionic magnetic fluid and to evaluate the cell toxicity of this biocompatible system using the J774-A1 cell line. Transmission electron micrographs obtained from the magnetic fluid sample were used to estimate the average particle diameter around 7.6 nm and diameter dispersion of 0.22. The BSA-based magnetic beads were prepared using the heat protein denaturation route. The nanoparticle concentration in the magnetic fluid sample used for the synthesis of the magnetic beads was in the range of 1.2 x 10(16) to 2.3 x 10(17) particle/ml. The methodology used to investigate the cell toxicity of the magnetic beads was the classical MTT assay. Our observation showed that the toxicity against the J774-A1 cell line depends upon the amount of magnetic material incorporated into the magnetic nanobeads and was found to be 14, 11, 9, 5, and 3% for 2.3 x 10(17), 1.2 x 10(17), 4.6 x 10(16), 2.3 x 10(16), and 1.2 x 10(16) particle/ml, respectively.     

Preparation and characterization of magnetic alginate-chitosan hydrogel beads loaded matrine

The aim of this study was to use alginate-chitosan (Alg-CS) hydrogel beads for developing an oral water-soluble drug delivery system, occupying pH-sensitive property and superparamagnetic. Matrine as a model drug was loaded in Alg-CS hydrogel beads to study the release character of the delivery system. The amount of matrine released from the beads was relatively low in pH 2.5 over 8?h (34.90%), but nearly all of the initial drug content was released in simulated intestinal fluid (SIF, pH 6.8) within 8?h. The results demonstrated that Alg-CS hydrogel beads possess unique pH-dependent swelling behaviors. In addition, the magnetic beads were characterized by Fourier transform infrared spectroscopy, scanning electron microscope, X-ray diffractometry and vibrating-sample magnetometry. Magnetometer measurements data suggested that Alg-CS beads also had superparamagnetic property as well as fast magnetic response. It can be expected that the beads can deliver and release encapsulated anticancer agent at the tumor by the weak magnetic field, and hence could be potential candidates as an orally administered drug delivery system.     

Preparation and characterization of magnetic alginate-chitosan hydrogel beads loaded matrine

The aim of this study was to use alginate-chitosan (Alg-CS) hydrogel beads for developing an oral water-soluble drug delivery system, occupying pH-sensitive property and superparamagnetic. Matrine as a model drug was loaded in Alg-CS hydrogel beads to study the release character of the delivery system. The amount of matrine released from the beads was relatively low in pH 2.5 over 8?h (34.90%), but nearly all of the initial drug content was released in simulated intestinal fluid (SIF, pH 6.8) within 8?h. The results demonstrated that Alg-CS hydrogel beads possess unique pH-dependent swelling behaviors. In addition, the magnetic beads were characterized by Fourier transform infrared spectroscopy, scanning electron microscope, X-ray diffractometry and vibrating-sample magnetometry. Magnetometer measurements data suggested that Alg-CS beads also had superparamagnetic property as well as fast magnetic response. It can be expected that the beads can deliver and release encapsulated anticancer agent at the tumor by the weak magnetic field, and hence could be potential candidates as an orally administered drug delivery system.     

A new method to destruct targeted cells using magnetizable beads and pulsed magnetic force

We investigated a new method to destruct targeted cells using magnetizable beads and pulsed magnetic force. The cells were combined with the beads by an antigen-antibody reaction (cell/bead/antibody complex), aggregated by a magnet, and stimulated by a magnetic stimulator. The viability of the aggregated and stimulated cell/bead/antibody complexes was significantly decreased, and the cells were destructed by the penetration of the beads into the cells or rupturing of the cells by the beads. These results suggest that magnetic aggregation and pulsed magnetic stimulation can effectively damage only the cells targeted by an antigen-antibody reaction.     

Manipulation of self-assembled structures of magnetic beads for microfluidic mixing and assaying

We present an original concept of manipulation of magnetic microbeads in a microchannel. It is based on the dynamic motion of a self-assembled structure of ferrimagnetic beads that are retained within a microfluidic flow using a local alternating magnetic field. The latter induces a rotational motion of the magnetic particles, thereby strongly enhancing the fluid perfusion through the magnetic structure that behaves as a dynamic random porous medium. The result is a very strong particle-liquid interaction that can be controlled by adjusting the magnetic field frequency and amplitude, as well as the liquid flow rate, and is at the basis of very efficient liquid mixing. The principle is demonstrated using a microfluidic chip made of poly(methyl methacrylate) with integrated soft ferromagnetic plate structures. The latter are part of an electromagnetic circuit and serve to locally apply a magnetic field over the section of the microchannel. Starting from a laminar flow pattern of parallel fluorescein dye and nonfluorescent liquid streams, we demonstrate a 95% mixing efficiency using a mixing length of only 400 microm and at liquid flows of the order of 0.5 cm/s. We anticipate that the intense interaction between the fluid and magnetic particles with functionalized surfaces holds large potential for the development of future bead-based assays.     

Synthesis of flexible magnetic nanowires of permanently linked core-shell magnetic beads tethered to a glass surface patterned by microcontact printing

We have developed an efficient, one-step method to create magnetic nanowires consisting of permanently linked chains of magnetic beads of varying flexibility tethered to a patterned glass surface using simple amidation chemistry. The flexibility of the nanowire was governed by the molecular weight of the molecule used to covalently link the beads and its length by the height of the microchannel in which it was synthesized. The nanowire diameter was determined both by the bead size and by the number of beads adhering to each dot in the microstamped, patterned array. Longer nanowires can form loops attached at two points on the glass surface. Both single flexible chains and flexible loops can adopt different configurations (straight, hairpin, S-shaped, etc.) when subjected to magnetic fields, the configurations depending on the directions of these fields. Shorter, less flexible nanowires align with the field always and do not exhibit the more exotic configurations seen for long, flexible chains and loops. These magnetic nanowires can have potential use in microfluidic pumping and mixing processes and in microparticle manipulation.     

Three-dimensional magnetic focusing of superparamagnetic beads for on-chip agglutination assays

We present a new method for three-dimensional (3D) magneticfocusing of magnetic microparticles in a microfluidic system. On-chip magnetic particle manipulation in the microchannel is achieved by a high-gradient magnetic field generated by means of a micromachined field concentrator. The system allows retention of functionalized beads in a dense plug while flowing through buffer or analyte. Slowly reducing the magnetic retention force in the presence of a flow results in controlled release of the particles into a fine streamline with regular longitudinal interparticle spacing. Alignment at half-height of the channel is readily obtained through the symmetry of the magnetic field. A single lateral sheath flow is required to provide full 3D focusing of the microparticles in the middle of the microchannel with a maximum deviation of ±5 μm from the center position. With the use of this system, a new approach for performing an immunoagglutination assay on-chip has been implemented. Three-dimensional focusing allowed reliable counting of singlets and agglutinated doublets. We demonstrate the potential of the agglutination assay in a microfluidic format using a streptavidin/biotinylated bovine serum albumin (bBSA) model system. A detection limit of about 400 pg/mL (6 pM) is achieved.     

Size effects of magnetic beads in circulating tumour cells magnetic capture based on streptavidin–biotin complexation

Circulating tumour cells (CTCs) draw significant attention as a promising biomarker for cancer prognosis, status monitoring, and metastasis diagnosis. However, the concentration of CTCs in peripheral blood is usually extremely low, thereby requiring enrichment followed by isolation of CTCs prior to detection. An immunomagnetic separation is a promising tool for CTCs enrichment. In this study, a cost‐effective magnetic separation method, based on streptavidin–biotin complexation, was developed and the effects of magnetic beads’ size in CTCs capture were compared. Magnetic nanobeads which were 25 nm in diameter lead to highest capture efficiency (82.2%) compared with 150 nm magnetic beads and 1 µm microbeads. Based on the streptavidin–biotin system, 25 nm magnetic nanobeads could capture model CTCs over 80% efficiency even at concentrations as low as ∼25 cells/mL that may represent the actual level of CTCs in peripheral blood of cancer patients. Furthermore, the isolated cells remained robust and healthy showing insignificant changes in morphology and behaviour when cultured for 24 h immediately after capture and isolation. The magnetic nanobeads based on streptavidin–biotin complexation showed promise for the easy and efficient capture and isolation of healthy CTCs for further diagnosis and analysis.Inspec keywords: cancer, magnetic separation, nanomedicine, nanomagnetics, proteins, biomagnetism, tumours, cellular biophysics, magnetic particles, molecular biophysics, blood, nanoparticlesOther keywords: streptavidin–biotin complexation, cancer prognosis, peripheral blood, immunomagnetic separation, CTCs capture, streptavidin–biotin system, circulating tumour cells, CTC enrichment, magnetic separation method, magnetic nanobeads, magnetic capture, size 25.0 nm, size 150.0 nm, time 24.0 hour     

Shape sensitivity analysis of magnetic forces

This paper presents a shape sensitivity analysis of magnetic forces evaluated using the Maxwell stress tensor and the finite element method. The formulation is based upon a discrete approach which takes the analytical derivatives of the finite element equations with respect to the shape variables and also on the adjoint variable method in order to carry out the derivation procedure. Sensitivity analysis is developed in the context of the axisymmetrical nonlinear magnetostatic field problem with a modified magnetic vector potential as state variable. Numerical results are presented to validate this methodology. Shape sensitivity analysis is then applied to the optimization of the force-displacement characteristic of a linear actuator. A sequential quadratic programming method is used in the optimization process     

Ligand and protein fishing with heat shock protein 90 coated magnetic beads

Heat shock protein 90alpha (Hsp90alpha) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90alpha has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90alpha ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90alpha coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.     

Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: horseradish peroxidase immobilized on magnetic beads

Horseradish peroxidase was immobilized on the magnetic poly(glycidylmethacrylate-co-methylmethacrylate) (poly(GMA-MMA)), via covalent bonding and used for the treatment of phenolic wastewater in continuous systems. For this purposes, horseradish peroxidase (HRP) was covalently immobilized onto magnetic poly(GMA-MMA) beds using glutaraldehyde (GA) as a coupling agent. The maximum HRP immobilization capacity of the magnetic poly(GMA-MMA)-GA beads was 3.35 mg g(-1). The immobilized HRP retained 79% of the activity of the free HRP used for immobilization. The immobilized HRP was used for the removal of phenol and p-chlorophenol via polymerization of dissolved phenols in the presence of hydrogen peroxide (H(2)O(2)). The effect of pH and temperature on the phenol oxidation rate was investigated. The results were compared with the free HRP, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP. The optimum pH value for free and immobilized HRP was observed at pH 7.0. The optimum temperature for phenols oxidation with immobilized HRP was between 25 and 35 degrees C and the immobilized HRP has more resistance to temperature inactivation than that of the free form. Finally, the immobilized HRP was operated in a magnetically stabilized fluidized bed reactor, and phenols were successfully removed in the enzyme reactor.     

Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na₂SO₄. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na₂SO₄ was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity.     

C18磁性微球在有机污染物分析检测中的应用

环境水的有机物污染已经给人类健康带来了极大的危害,有机污染物监测已引起了广泛的重视。C18磁性微球作为有机污染物分析的新兴方法,与传统的离心、过滤、固相萃取等方法相比具有良好的优势。本文主要阐述了C18磁性微球的结构及其特性,并详细介绍了疏水性C18键合硅胶及活性基团修饰的C18键合硅胶在分离富集有机污染物中的应用。最后对C18磁性微球的未来发展进行了展望。     

Dynamics of capturing process of multiple magnetic nanoparticles in a flow through microfluidic bioseparation system

A mathematical model based on finite-element technique is developed for predicting the transport and capture of multiple magnetic nanoparticles in a microfluidic system that consists of a microfluidic channel enclosed by a permanent magnet. The trajectories and trapping efficiencies are calculated for multiple magnetic nanoparticles when released in the microsystem. It is demonstrated that not only the size but also the point of release of nanoparticles within the microchannel affects the capturing process. Influence of three important parameters, inlet velocities of fluid containing magnetic nanoparticles, diameter of magnetic nanoparticles and magnetic field strength on the trapping efficiency are investigated and optimised values of inlet velocity and magnetic field strength for completely trapping 50 nm magnetic nanoparticles are predicted. It is further demonstrated that the angular position of magnet around the microchannel is also critical in dictating the resulting bioseparation performance. Furthermore, combination of these analyses using the mathematical model will be very useful in the design and development of novel microfluidic bioseparation microsystems.