Modeling analyte transport and capture in porous bead sensors

Porous agarose microbeads, with high surface to volume ratios and high binding densities, are attracting attention as highly sensitive, affordable sensor elements for a variety of high performance bioassays. While such polymer microspheres have been extensively studied and reported on previously and are now moving into real-world clinical practice, very little work has been completed to date to model the convection, diffusion, and binding kinetics of soluble reagents captured within such fibrous networks. Here, we report the development of a three-dimensional computational model and provide the initial evidence for its agreement with experimental outcomes derived from the capture and detection of representative protein and genetic biomolecules in 290 μm porous beads. We compare this model to antibody-mediated capture of C-reactive protein and bovine serum albumin, along with hybridization of oligonucleotide sequences to DNA probes. These results suggest that, due to the porous interior of the agarose bead, internal analyte transport is both diffusion and convection based, and regardless of the nature of analyte, the bead interiors reveal an interesting trickle of convection-driven internal flow. On the basis of this model, the internal to external flow rate ratio is found to be in the range of 1:170 to 1:3100 for beads with agarose concentration ranging from 0.5% to 8% for the sensor ensembles here studied. Further, both model and experimental evidence suggest that binding kinetics strongly affect analyte distribution of captured reagents within the beads. These findings reveal that high association constants create a steep moving boundary in which unbound analytes are held back at the periphery of the bead sensor. Low association constants create a more shallow moving boundary in which unbound analytes diffuse further into the bead before binding. These models agree with experimental evidence and thus serve as a new tool set for the study of bioagent transport processes within a new class of medical microdevices.     

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.     

Rapid,High‐Resolution Magnetic Microscopy of Single Magnetic Microbeads

Magnetic microparticles or “beads” are used in a variety of research applications from cell sorting through to optical force traction microscopy. The magnetic properties of such particles can be tailored for specific applications with the uniformity of individual beads critical to their function. However, the majority of magnetic characterization techniques quantify the magnetic properties from large bead ensembles. Developing new magnetic imaging techniques to evaluate and visualize the magnetic fields from single beads will allow detailed insight into the magnetic uniformity, anisotropy, and alignment of magnetic domains. Here, diamond‐based magnetic microscopy is applied to image and characterize individual magnetic beads with varying magnetic and structural properties: ferromagnetic and superparamagnetic/paramagnetic, shell (coated with magnetic material), and solid (magnetic material dispersed in matrix). The single‐bead magnetic images identify irregularities in the magnetic profiles from individual bead populations. Magnetic simulations account for the varying magnetic profiles and allow to infer the magnetization of individual beads. Additionally, this work shows that the imaging technique can be adapted to achieve illumination‐free tracking of magnetic beads, opening the possibility of tracking cell movements and mechanics in photosensitive contexts.     

Interfacial effects in the flow-bead test for vitreous enamels

Evidence is presented that the rate of flow of a liquid enamel bead down a vertical plate depends on the nature of the plate surface. Two surfaces were compared. One was an enamel surface of the same composition as the bead, and therefore molten during the test; the other was a porous refractory coat that was solid at the test temperature. The rate of flow was greater over the refractory surface. Measurement of contact angles for the flowing beads and for sessile drops led to the conclusion that the surface tension forces per unit width were similar for the drops on the two surfaces. However, the gravitational force per unit width was higher for the drop on the refractory surface. This is responsible for the higher rate of flow over the refractory surface, and arises from the lower rate of lateral spreading of the bead caused by viscous resistance to the flow of the enamel through microchannels in the rough surface.     

Direct manipulation and observation of the rotational motion of single optically trapped microparticles and biological cells in microvortices

This paper describes a method for manipulating and monitoring the rotational motion of single, optically trapped microparticles and living cells in a microvortex. To induce rotation, we placed the microparticle at the center of rotation of the vortex and used the recirculating fluid flow to drive rotation. We have monitored the rotation of single beads (which ranged in diameter from a few micrometers to tens of micrometers) and living cells in a microvortex. To follow the rotation of a smooth and symmetrically shaped bead, we first ablated a small region ( approximately 1 microm) on the bead. An Ar(+) laser was then tightly focused ( approximately 0.5-microm spot size) onto the bead, and rotation was tracked by recording changes in the level of backscattered laser light as the ablated region repeatedly transited the laser focus. Using this method, we have followed bead rotation that varied in frequency from 0.15 to 100 Hz and have studied the effect of bead diameter on the rate of rotation at a given fluid flow rate. To monitor the rotation of single living cells, we selectively stained portions of B-lymphocytes with the fluorescent dye DiOC(6). We observed rotation by following changes in the fluorescence signal as the dye-stained region transited the laser focal volume. This technique provides a simple and sensitive method for controlling and monitoring the rotational motion of microparticles in a microfluidic environment.     

Use of negative dielectrophoresis for selective elution of protein-bound particles

In this paper with the aid of negative dielectrophoresis force in conjunction with shear force and at an optimal sodium hydroxide concentration we demonstrated a switchlike functionality to elute specifically bound beads from the surface. At an optimal flow rate and sodium hydroxide concentration, negative dielectrophoresis turned on results in bead detachment, whereas when negative dielectrophoresis is off, the beads remain attached. This platform offers the potential for performing a bead-based multiplexed assay where in a single channel various regions are immobilized with a different antibody, each targeting a different antigen. To develop the proof of concept and to demonstrate the switchlike functionality in eluting specifically bound beads from the surface we looked at two different protein interactions. We chose interactions that were in the same order of magnitude in strength as typical antibody-antigen interactions. The first was protein G-IgG interaction, and the second was the interaction between anti-IgG and IgG.     

Magnetic levitation as a platform for competitive protein-ligand binding assays

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater than approximately 65 kDa.     

Chitosan-Alginate Multilayer Beads for Gastric Passage and Controlled Intestinal Release of Protein

Abstract

Chitosan-alginate beads loaded with a model protein, bovine serum albumin (BSA) were investigated to explore the temporary protection of protein against acidic and enzymatic degradation during gastric passage. Optimum conditions were established for preparation of homogenous, spherical, and smooth chitosan-alginate beads loaded with BSA. Multilayer beads were prepared by additional treatment with either chitosan or alginate or both. The presence of chitosan in the coagulation bath during bead preparation resulted in increased entrapment of BSA. During incubation in simulated gastric fluid (SGF pH 1.2), the beads showed swelling and started to float but did not show any sign of erosion. Inclusion of pepsin in the gastric fluid did not show a further effect on the properties of the beads. Release studies were done in simulated gastric fluid (SGF pH 1.2) and subsequently in simulated intestinal fluid (SIF pH 7.5) to mimic the physiological gastrointestinal conditions. After transfer to intestinal fluid, the beads were found to erode, burst, and release the protein. Microscopic and macroscopic observations confirmed that the release of protein was brought about by the burst of beads. Chitosan-reinforced calcium-alginate beads showed delay in the release of BSA. The multilayer beads disintegrated very slowly. The enzymes pepsin and pancreatin did not change the characteristics of BSA-loaded chitosan-alginate beads. Single layer chitosan-alginate beads released 80–90% of the model protein within 12 h while multilayer beads released only 40–50% in the same period of time. The release from chitosan-alginate beads and multilayer beads in SIF was further delayed without prior incubation in SGF. It is concluded that alginate beads reinforced with chitosan offer an excellent perspective for controlled gastrointestinal passage of protein drugs.     

Chitosan-alginate multilayer beads for gastric passage and controlled intestinal release of protein

Chitosan-alginate beads loaded with a model protein, bovine serum albumin (BSA) were investigated to explore the temporary protection of protein against acidic and enzymatic degradation during gastric passage. Optimum conditions were established for preparation of homogenous, spherical, and smooth chitosan-alginate beads loaded with BSA. Multilayer beads were prepared by additional treatment with either chitosan or alginate or both. The presence of chitosan in the coagulation bath during bead preparation resulted in increased entrapment of BSA. During incubation in simulated gastric fluid (SGF pH 1.2), the beads showed swelling and started to float but did not show any sign of erosion. Inclusion of pepsin in the gastric fluid did not show a further effect on the properties of the beads. Release studies were done in simulated gastric fluid (SGF pH 1.2) and subsequently in simulated intestinal fluid (SIF pH 7.5) to mimic the physiological gastrointestinal conditions. After transfer to intestinal fluid, the beads were found to erode, burst, and release the protein. Microscopic and macroscopic observations confirmed that the release of protein was brought about by the burst of beads. Chitosan-reinforced calcium-alginate beads showed delay in the release of BSA. The multilayer beads disintegrated very slowly. The enzymes pepsin and pancreatin did not change the characteristics of BSA-loaded chitosan-alginate beads. Single layer chitosan-alginate beads released 80-90% of the model protein within 12 h while multilayer beads released only 40-50% in the same period of time. The release from chitosan-alginate beads and multilayer beads in SIF was further delayed without prior incubation in SGF. It is concluded that alginate beads reinforced with chitosan offer an excellent perspective for controlled gastrointestinal passage of protein drugs.     

A renewable electrochemical magnetic immunosensor based on gold nanoparticle labels

A particle-based renewable electrochemical magnetic immunosensor was developed by using magnetic beads and gold nanoparticle labels. Anti-IgG antibody-modified magnetic beads were attached to a renewable carbon paste transducer surface by magnet that was fixed inside the sensor. Gold nanoparticle labels were capsulated to the surface of magnetic beads by sandwich immunoassay. Highly sensitive electrochemical stripping analysis offers a simple and fast method to quantify the capatured gold nanoparticle tracers and avoid the use of an enzyme label and substrate. The stripping signal of gold nanoparticles is related to the concentration of target IgG in the sample solution. A transmission electron microscopy image shows that the gold nanoparticles were successfully capsulated to the surface of magnetic beads through sandwich immunoreaction events. The parameters of immunoassay, including the loading of magnetic beads, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.02 microg ml(-1) of IgG was obtained under optimum experimental conditions. Such particle-based electrochemical magnetic immunosensors could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for disease diagnostics and biosecurity.     

Development of Matrix Controlled Release Beads by Extrusion-Spheronization Technology Using a Statistical Screening Design

The objective of this study was to develop beads with inherent modified release characteristics requiring no subsequent controlled release coating. Extrusion-spheronization was chosen to accomplish this goal. The model drug was a zwitterion (isoelectric point ∼pH 5.2) with poor solubility and low bulk density. Preliminary studies indicated that matrix beads with modified micro-environmental pH would result in sustained release of the drug. A Nica extruder and spheronizer were used to manufacture the beads. A Plackett-Burman screening design was employed to isolate critical variables influencing the bead characteristics and drug release. Variables studied included the type of polymeric dispersion, polymer concentration, acid type, acid concentration, plasticizer content, drug concentration and spheronizing time. Responses evaluated included capsule fill weight, drug release rate, micro-environmental pH, yield and friability. Beads were successfully manufactured according to the screening design and exhibited different dissolution characteristics. Polymer type (Eudragit RS 30 D over Aquacoat ECD-30), polymer concentration and acid concentration significantly retarded drug release. However, increasing acid concentration increased bead friability. In addition to drug concentration, higher polymer concentration, appropriate acid selection and longer residence times afforded maximum capsule fill weights and increased bead density.     

Integrated inductive sensors for the detection of magnetic microparticles

In this paper, we deal with novel inductive microsensors, realized by using standard CMOS microelectronic technologies, for the detection of small amounts of magnetic beads that are placed in selected regions over the surface of the microsensor. The sensor proposed here represents a step toward the development of integrated inductive biosensors for application in the area of magnetic immunoassay where magnetic markers, carrying specific antibodies that selectively bind to the cells or molecules to be detected, are used; the measurement of the analyte concentration is therefore accomplished by determining the concentration of magnetic particles tied to it. A planar differential transformer structure is proposed here as part of the measurement strategy. The analysis, simulation, and design of the proposed device are reported, and its sensitivity to the quantity of micromagnetic beads deposited over its surface has been also demonstrated through experiments.     

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.     

Two Phase Flow in Stirred Ball Mills

ABSTRACT

Comminution mechanisms in stirred ball mills are not known in detail. Therefore, the multi-phase flow is investigated to improve the understanding of the comminution process, focusing on grinding beads and fluid motion. In order to describe the fluid flow field, an Eularian approach is applied, using the time averaged Navier-Stokes equations, in combination with the standard k-?-Model to model turbulence correlations. For the dispersed phase, a Lagrangian approach is applied. Grinding bead collisions are simulated with a statistical model. Using the energy dissipation distribution, the fluid areas of higher comminution activity in the grinding chamber are identified.     

Evaluation of the sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate coupling chemistry for attachment of oligonucleotides to magnetic nanobeads

The sulfo-SMCC (Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) coupling chemistry was evaluated for immobilization of oligonucleotides onto 130 nm sized magnetic nanobeads aimed for bio-detection in a magnetic readout assay. The chemistry was found to produce a high surface coverage of approximately 93 +/- 10 oligonucleotides per bead whereas stability tests showed that about 50% of the oligonucleotides detached from the bead surfaces after eight weeks of storage in a buffer solution. It was shown that bead aggregation prior to magnetic readout could be suppressed by incubating the samples at 70 degrees C for 30 min. The same temperature was also shown to be the most favorable for hybridization between the oligonucleotide functionalized beads and rolling circle amplified DNA molecules. This should simplify the heating procedure in a biosensor in which hybridization and magnetic readout is performed in the same compartment.     

镀银磁性PMMA微球的制备

采用化学镀法制得了表面包覆银层的导电磁性聚甲基丙烯酸甲酯(PMMA)微球。对粗糙度不同的微球进行镀银,研究了银在微球上的沉积机理。表面改性使磁性PMMA微球功能化,具有和银强烈结合的能力,从而得到包覆均匀的导电磁性微球。研究了硝酸银和磁性微球的含量对包覆效果及导电性能的影响,并通过SEM和EDS对镀银磁性微球的表面形貌及组成进行了分析。     

Modeling of chromium (VI) biosorption by immobilized Spirulina platensis in packed column

This study describes biosorption of chromium (VI) by immobilized Spirulina platensis, in calcium alginate beads. Three aspects viz. optimization of bead parameters, equilibrium conditions and packed column operation were studied and subsequently modeled. Under optimized bead diameter (2.6 mm), calcium alginate concentration (2%, w/v) and biomass loading (2.6%, w/v) maximum biosorption was achieved. 140 g l⁻¹ loading of optimized beads resulted in 99% adsorption of chromium (VI) ions from an aqueous solution containing 100 mg l⁻¹ of chromium (VI). The quantitative chromium (VI) uptake was effectively described by Freundlich adsorption isotherm. The immobilized S. platensis beads were further used in a packed bed column wherein the effects of bed height, feed flow rate, inlet chromium (VI) ion concentration were studied by assessing breakthrough time. The performance data were tested for various models fitting in order to predict scale up-design parameters such as breakthrough time and column height. Results were encouraging.     

Titration without Mixing or Dilution: Sequential Injection of Chemical Sensing Membranes

A novel, miniaturized titration was developed using beads 35 μm in diameter as semisolid aqueous titrant retained in a nonaqueous sample stream. Agarose beads with internally bound pH indicator served as a pH sensing membrane material swollen with aqueous NaOH titrant. The indicator monitored the remaining titrant within the agarose beads during perfusion with H(2)SO(4) in 1-butanol samples. Irreversible reaction of 2 mg bead layers was made possible by automated packing and disposal in a flow cell. This strategy substituted membrane advantages for the burdens of mixing and unnecessary dilution under laminar flow conditions. The agarose environment was conditioned with NaCl to tolerate dissolved salt in the sample. Transmittance measurements were made via fiber optics through FEP PTFE optical windows. A simple inverse relationship held between endpoint volume and acid concentration so that calibration curves were linear, R(2) = 0.9980.     

An NMR method of measuring the mass of magnetic beads in a lowdensity fluid [biomedical measurement]

A nuclear magnetic resonance (NMR) method of determining the mass of the magnetic particles present in a fluid by observing the relative frequency shift of an NMR signal is proposed. Experimental tests have been carried out to find the ratio of the mass of the beads to the total mass of the liquid with various densities by using this method. The method can be used to measure masses in a fluid containing a low concentration of magnetic particles     

生物传感用巨磁电阻传感器及其磁珠检测性能

本文采用直流磁控溅射沉积了结构为NiFeCo(缓冲层)/[Cu/NiFeCo]×10/ Ta,巨磁电阻(GMR)值为9.8%的多层膜,利用微细加工技术制备了基于此GMR多层膜的生物传感器,GMR电阻线条的线宽分别为3μm和5μm.测试了单个GMR传感器的特性,并将该传感器件和外接可调电阻组成惠斯通电桥,采用该GMR电桥对Dynal公司的MyOne磁珠进行了检测.分别测试了施加变化垂直磁场和施加间歇式恒定垂直磁场时GMR电桥信号对传感器表面覆盖磁珠的响应,研究了GMR电桥信号和磁珠覆盖率的关系.选用器件电阻线宽分别为3μm和5μm的传感器测试了器件线宽对传感器灵敏度的影响.结果表明,GMR传感器能够检测到磁珠的存在,最低能检测的磁珠数量约100个,且GMR电桥信号与磁珠覆盖率基本成正比,器件的灵敏度与传感器线宽基本成反比.