Sensitive bondforce measurements of ligand-receptor pairs with magnetic beads

The bondforces between biotinylated surfaces and streptavidin or avidin coated beads are investigated by a magnetic field based manipulation system for magnetic microbeads. The magnetic field is generated by currents through a set of conducting lines, and its gradient exerts a force onto the magnetic beads. The force can be increased until the bond between the bead and the surface breaks. Consistent with other groups we found two conformations for both investigated bonds. The measured bondforces for the two conformations are for Streptavidin-Biotin: 55.9 and 244.7 fN and for Avidin-Biotin: 15.9 and 58.4 fN. These very low bondforces (10-100 times smaller than earlier measurements) match to the extremely low loading rate of about 1 fN/s. This new technique thus allows to investigate biomolecular bonds by extremely low forces.     

Detection and manipulation of biomolecules by magnetic carriers

The detection and manipulation of single molecules on a common platform would be of great interest for basic research of biological or chemical systems. A promising approach is the application of magnetic carriers. The principles are demonstrated in this contribution. It is shown that paramagnetic beads can be detected by highly sensitive magnetoresistive sensors yielding a purely electronic signal. Different configurations are discussed. The capability of the sensors to detect even single markers is demonstrated by a model experiment. In addition, the paramagnetic beads can be used as carriers for biomolecules. They can be manipulated on-chip via currents running through specially designed line patterns. Thus, magnetic markers in combination with magnetoresistive sensors are a promising choice for future integrated lab-on-a-chip systems.     

Numerical technique of blood flow through catheterized arteries with overlapping stenosis

This study is concerned with the surgical technique for the injection of a catheter through arteries with overlapping stenosis in the presence of externally applied magnetic field and Hall currents influences. The nature of blood is analyzed mathematically by considering it as a micropolar fluid. The analysis is carried out for an artery with a mild stenosis. The governing equations with the corresponding boundary conditions solved numerically using Crank–Nicolson implicit finite difference scheme. The numerical technique give excellent agreement for axial velocity of the fluid, the circumferential microrotation, the wall shear stress distribution and the contour plots of stream lines. The obtained results show that the value of axial velocity is higher for a Newtonian fluid than that for a micropolar fluid model, the effect of suitable moving magnetic field (Hall currents influences) accelerates the speed of blood, the size of trapped bolus for the stream lines decrease if the spinning movement of the fluid molecules have considerable value regardless of small or large size of the fluid molecules and the flow of fluid is better with increasing the Hall current effect and the size of trapping bolus increase clearly by increasing the maximum height of stenosis where the fluid moves as a bulk.     

Acoustic wave based MEMS devices for biosensing applications

This paper presents a review of acoustic-wave based MEMS devices that offer a promising technology platform for the development of sensitive, portable, real-time biosensors. MEMS fabrication of acoustic wave based biosensors enables device miniaturization, power consumption reduction and integration with electronic circuits. For biological applications, the biosensors are integrated in a microfluidic system and the sensing area is coated with a biospecific layer. When a bioanalyte interacts with the sensing layer, mass and viscosity variations of the biospecific layer can be detected by monitoring changes in the acoustic wave properties such as velocity, attenuation, resonant frequency and delay time. Few types of acoustic wave devices could be integrated in microfluidic systems without significant degradation of the quality factor. The acoustic wave based MEMS devices reported in the literature as biosensors and presented in this review are film bulk acoustic wave resonators (FBAR), surface acoustic waves (SAW) resonators and SAW delay lines. Different approaches to the realization of FBARs, SAW resonators and SAW delay lines for various biochemical applications are presented. Methods of integration of the acoustic wave MEMS devices in the microfluidic systems and functionalization strategies will be also discussed.     

Time evolution of the plasma spatial structure during the formation of a current sheet in argon according to holographic interferometry

The spatiotemporal dynamics of plasma sheets formed in argon plasma in two- and three-dimensional magnetic configurations with an X-type singular line was studied using holographic interferometry. An analogy is revealed between the development of plasma sheets and the time evolution of the current sheet structure, which was previously studied using magnetic measurements. In the late stage of the sheet evolution, a change in the rotation direction of plasma sheets formed in 3D magnetic configurations was observed. Apparently, this is caused by a change in the direction of the Hall currents at the side edges of the current sheet.     

High sensitivity detection of molecular recognition using magnetically labelled biomolecules and magnetoresistive sensors

Small magnetoresistive spin valve sensors (2 x 6 microm(2)) were used to detect the binding of single streptavidin functionalized 2 microm magnetic microspheres to a biotinylated sensor surface. The sensor signals, using 8 mA sense current, were in the order of 150-400 microV for a single microsphere depending on sensor sensitivity and the thickness of the passivation layer over the sensor surface. Sensor saturation signals were 1-2 mV representing an estimated 6-20 microspheres, with a noise level of approximately 10 microV. The detection of biomolecular recognition for the streptavidin-biotin model was shown using both single and differential sensor architectures. The signal data compares favourably with previously reported signals for high numbers of magnetic microspheres detected using larger multilayered giant magnetoresistance sensors. A wide range of applications is foreseen for this system in the development of biochips, high sensitivity biosensors and the detection of single molecules and single molecule interactions.     

Formation of a cylindrical ion beam in the field of an axisymmetric system of ring currents at a low Hall current

A study is made of the structure of an accelerating layer with a closed Hall current and the geometry of an ion beam in an external magnetic field created by an arbitrary axisymmetric system of ring currents under conditions such that the Hall current can be ignored. It is shown that the ion trajectories are perpendicular to the magnetron cutoff surface for electrons and that the cathode plasma boundary coincides with a magnetic field line. A magnetic field configuration is found in which the cutoff surface is a plane surface perpendicular to the axis of the system. It is shown that, for a small ratio of the gyroradius of the electrons (in terms of the maximum energy acquired by them in the layer) to the characteristic size of the structure, such a configuration provides sufficient means to ensure the formation of slightly converging ion beams or those that are essentially parallel to the system axis.     

SPR生物传感器及其应用进展

基于表面等离子体共振 (SPR)技术的光学生物传感器是进行生物分子相互作用分析的一种先进手段。与传统的超速离心、荧光法等相比 ,它具有实时检测、无需标记、耗样最少等特点 ,在药物筛选、临床诊断、食物及环境监控和膜生物学等领域中的新兴应用日益扩大 ,并且已成为生命科学和制药研究的一种标准的生物物理学工具。综述了近几年国际上生物传感器的应用进展情况 ,并简要展望了该技术的发展和应用前景     

Molecular charge contact biosensing based on the interaction of biologically modified magnetic beads with an ion-sensitive field effect transistor

In this article, we report a novel method of biomolecular recognition based on the molecular charge contact (MCC). As one of the MCC biosensing method, the interaction between DNA-coated magnetic beads and a silicon-based semiconductor, an ion-sensitive field effect transistor (ISFET) could be detected for DNA molecular recognition events using the principle of the field effect, which enables detecting ionic or molecular charges. After DNA-coated magnetic beads had been introduced and brought in contact with the gate surface by a magnet, the threshold voltage of the ISFET was shifted in the positive direction by immobilization, hybridization and extension reaction of DNA molecules on magnetic beads. This positive shift was based on the increase in negative charges of the phosphate groups in them. Then, the ISFET device could be reused a couple of dozen times continuously and cost-effectively because the oligonucleotide probes were tethered to the magnetic beads, but this was not done directly on the gate surface of the ISFET. Moreover, the MCC biosensing method enabled discrimination of a single nucleotide polymorphism. By creating an interaction of magnetic beads with the semiconductor, we can expect enhancement of the reaction efficiency in a solution and reuse of the device by separating the reaction field from the sensing substrate.     

Magnetic bead-based DNA detection with multi-layers quantum dots labeling for rapid detection of Escherichia coli O157:H7

This work demonstrated the feasibility of detecting 250zM Escherichia coli O157:H7 eaeA target DNA by using a magnetic bead-based DNA detection assay with designed labeling strategy within 40-60min. The magnetic beads were used as the solid support for the binding probe and isolated the target DNA from the sample. The detection signals could be amplified from the multi-layers biotin-streptavidin conjugated quantum dots based on binding with specific designed biotinlyted linker. This assay method would provide a simple, rapid, and ultra-sensitive detection method for DNA or other biomolecular analysis.     

An integrated microfluidic platform for magnetic microbeads separation and confinement

An innovative microfluidic platform for magnetic beads manipulation is introduced, consisting of novel microfabricated 3D magnetic devices positioned in a microfluidic chamber. Each magnetic device comprises of an embedded actuation micro-coil in various design versions, a ferromagnetic pillar, a magnetic backside plate and a sensing micro-coil. The various designs of the micro-coils enable efficient magnetic beads trapping and concentration in different patterns. The finite element analysis (FEA) results show a significant increase of the developed force on suspended magnetic beads when the magnetic pillar and backside plate were integrated into the device structure. These simulation results were confirmed experimentally by measuring the magnetic beads trapping ratios for the different designs and structures of the devices under continuous flow conditions. The trapping ratios and profiles were studied using beads counting, measuring the change of inductance with the sensing micro-coil and by image processing. The devices have efficiently demonstrated a controlled and localized magnetic beads trapping and concentration at small spatial locations for the first time. The new results shown in this study demonstrate the feasibility of efficiently using these original devices as key elements in complex bio-analysis systems.     

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.     

LED-based interferometric reflectance imaging sensor for quantitative dynamic monitoring of biomolecular interactions

Label-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the Interferometric Reflectance Imaging Sensor (IRIS) using an inexpensive and durable multi-color LED illumination source to monitor protein-protein and DNA-DNA interactions. We demonstrate the capability of this system to dynamically monitor antigen-antibody interactions with a noise floor of 5.2 pg/mm(2) and DNA single mismatch detection under denaturing conditions in an array format. Our experiments show that this platform has comparable sensitivity to high-end label-free biosensors at a much lower cost with the capability to be translated to field-deployable applications.     

A new set up for multi-analyte sensing: at-line bio-process monitoring

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO?-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO?-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO?-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.     

On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.     

Nanowiring of a redox enzyme by metallized peptides

A molecular assembly consisting of a redox enzyme, NADH peroxidase, a metallized double-helical peptide, and a gold nanoparticle immobilized onto a gold wire derivatized with a benzenedithiol compound, initiated and conducted redox signals in the presence of H(2)O(2) and NADH. The current generated by the binding of NADH, the electron donor, was transduced through the molecular assembly with apparently little loss of signal to the solution. The currents measured correlate to an electron transfer rate constant on the order of 3,000 s(-1) within each assembly. This electron transfer rate is two orders of magnitude higher than the endogenous electron transfer rate from NADH to the native enzyme, 27 s(-1). This rate indicates that the metallized peptide is in a conformation conducive for electron transfer and, in conjunction with the redox enzyme, can form effective conduits of electrical signals. This work demonstrates the feasibility of utilizing designed and highly efficient biomolecular assemblies for the production of ultra-sensitive, in-situ biosensors.     

Configurational Statistics of Magnetic Bead Detection with Magnetoresistive Sensors

Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor. Consequently, the signal from multiple beads also depends on their locations. Thus, a given coverage of the functionalized area with magnetic beads does not result in a given detector response, except on the average, over many realizations of the same coverage. We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively.     

Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production

During the last decades, whole‐cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high‐gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis‐à‐vis pure alginate beads was achieved with alginate beads with embedded Bayoxide® magnetite or MagPrep® silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting® technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.© 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009