Surface Of Microbeads Research Articles

Microbead QD-ELISA: Microbead ELISA Using Biocatalytic Formation of Quantum Dots for Ultra High Sensitive Optical and Electrochemical Detection.

Electrochemical detection strategies employing semiconductor quantum dots (QDs) open up new opportunities for highly sensitive detection of biological targets. We designed a new assay based on microbead linked enzymatic generation of CdS QDs (Microbead QD-ELISA) and employed it in optical and electrochemical affinity assays for the cancer biomarker superoxide dismutase 2 (SOD2). Biotinylated antibodies against SOD2 were immobilized on the surface of polyvinyl chloride microbeads bearing streptavidin. In order to prevent any non-specific adsorption the microbeads were further blocked with bovine serum albumin. The analyte, SOD2 was captured on microbeads and labeled with alkaline phosphatase-conjugated antibody linked with mouse antibody against SOD2. Hydrolysis of para-nitrophenylphosphate by immobilized alkaline phosphatase triggered the rapid formation of phosphate-stabilized CdS QDs on the surface of microbeads. The resulting semiconductor nanoparticles were detected by fluorescence spectroscopy, microscopy, and square-wave voltammetry (SWV). The electrochemical assay based on the detection with square-wave voltammograms of Cd2+ ions originating from immobilized CdS QDs showed linearity up to 45 ng mL-1, and the limit of SOD2 detection equal to 0.44 ng mL-1 (1.96 × 10-11 M). This detection limit is lower by 2 orders of magnitude in comparison with that of other previously published assays for superoxide dismutase. The electrochemical assay was validated with HepG2 (Human hepatocellular carcinoma) cell lysate containing SOD2.

A microchip for integrated single-cell genotoxicity assay

With the development of large-scale biologic databases, precision medicine is becoming a frontier in biomedical research. As a main focus of precision medicine study, cancer has been widely accepted as a disease born out of inherited genetic variations or accumulating genomic damage. At the single-cell level, microfluidics or lab-on-a-chip technology for cancer study is an emerging tool for improving risk assessment, diagnostic categories and therapeutic strategies. This work presents a multi-layer microchip for single-cell gene expression profiling. Treated by three drug reagents (i.e. methyl methanesulfonate, docetaxel and colchicine) with varied concentrations and time lengths, individual human breast cancer cells (MCF-7) are then lysed on-chip, and the released mRNA templates are captured and reversely transcribed into cDNA on microbead surface. Three genes (GAPDH, CDKN1A, AURKA) are amplified and quantified simultaneously through triplex real-time polymerase chain reactions (qPCR). Readout per run is set to be eighteen, and can be further improved following same approach. The microchip is able to integrate all steps of single-cell gene expression profiling, and provide precision study of drug induced genotoxicity with reduced reagents consumption per reaction and instrumental cost.

Continuous Modular Biomimetic Utilization of Carbon Dioxide toward Multi- and Chemoenzymatic Systems

This study shows the preparation and application of modular biomimetic carbon dioxide utilization of multi- and chemoenzymatic systems for long-term stable continuous operation. The system is assembled by enzyme immobilization on the silica-shell surface of the hybrid microbeads forming stabilized carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPCase) microbeads, respectively. The CA and PEPCase microbeads were very stable, preserving 85% of their initial activity over more than 30 days. In addition, both hybrid microbeads were repeatedly used successfully for more than 20 cycles of reaction, and they still remained active with facile magnetic separability at room temperature. In addition, the CA and PEPCase microbeads were employed within a modular enzyme reactor system to show its long-term and stable use in the continuous and/or simultaneous production of oxaloacetate (OAA) and CaCO3 from a continuously supplied CO2 solution. It was found that the production of OAA and CaCO3 was stable f...

A Liquid-Handling Robot for Automated Attachment of Biomolecules to Microbeads.

Diagnostics, drug delivery, and other biomedical industries rely on cross-linking ligands to microbead surfaces. Microbead functionalization requires multiple steps of liquid exchange, incubation, and mixing, which are laborious and time intensive. Although automated systems exist, they are expensive and cumbersome, limiting their routine use in biomedical laboratories. We present a small, bench-top robotic system that automates microparticle functionalization and streamlines sample preparation. The robot uses a programmable microcontroller to regulate liquid exchange, incubation, and mixing functions. Filters with a pore diameter smaller than the minimum bead diameter are used to prevent bead loss during liquid exchange. The robot uses three liquid reagents and processes up to 10(7) microbeads per batch. The effectiveness of microbead functionalization was compared with a manual covalent coupling process and evaluated via flow cytometry and fluorescent imaging. The mean percentages of successfully functionalized beads were 91% and 92% for the robot and manual methods, respectively, with less than 5% bead loss. Although the two methods share similar qualities, the automated approach required approximately 10 min of active labor, compared with 3 h for the manual approach. These results suggest that a low-cost, automated microbead functionalization system can streamline sample preparation with minimal operator intervention.

Stimuli-responsive polyaniline coated silica microspheres and their electrorheology

Silica/polyaniline (PANI) core–shell structured microspheres were synthesized by coating the surface of silica micro-beads with PANI and applied as a candidate inorganic/polymer composite electrorheological (ER) material. The silica micro-beads were initially modified using N-[(3-trimethoxysilyl)-propyl] aniline to activate an aniline functional group on the silica surface for a better PANI coating. The morphology of the PANI coating on the silica surface was examined by scanning electron microscopy and the silica/PANI core–shell structure was confirmed by transmission electron microscopy. The chemical structure of the particles was confirmed by Fourier transform infrared spectroscopy. Rotational rheometry was performed to confirm the difference in the ER properties between pure silica and silica/PANI microsphere-based ER fluids when dispersed in silicone oil.

Preparation of Engineered Salmonella Typhimurium-Driven Hyaluronic-Acid-Based Microbeads with Both Chemotactic and Biological Targeting Towards Breast Cancer Cells for Enhanced Anticancer Therapy.

In this study, a new type of targeted bacteriobots is prepared and investigated as a therapeutic strategy against solid tumors. Maleimide-functionalized hyaluronic acid (HA) polymer is synthesized and cross-linked with four-arm-thiolated polyethylene glycol (PEG-SH) to form HA microbeads with diameter of 8 μm through the Michael-type addition. Docetaxel (DTX)-loaded nanoparticles are encapsulated in HA-PEG microbeads and sustained in vitro drug-release pattern of the DTX from the HA-PEG microbeads is observed for up to 96 h. Dual-targeted bacteriobots are prepared using CD 44 receptor-targeted HA microbeads synthesized via microfluidics, followed by the attachment of the flagellar bacterium Salmonella typhimurium, which have been genetically engineered for tumor targeting, onto the surface of the HA microbeads by the specific interaction between streptavidin on the HA beads and biotin on the bacteria. After the attachment of bacteria, the bacteriobots show an average velocity of 0.72 μm s(-1) and high chemotactic migration velocity of 0.43 μm s(-1) towards 4T1 cells lysates. CD 44 receptor-specific cellular uptake is verified through flow cytometry analysis and confocal imaging, demonstrating enhanced intracellular uptake in CD 44 receptor positive tumor cells compared to normal cells. Therefore, the present study suggests that these bacteriobots have dual-tumor-targeting abilities displaying their potential for targeted anticancer therapy.

Microvalve-assisted bead-beating system for selective nucleic acid preparation from bacteria and viruses

Microfluidic chips are versatile tools for automatic nucleic acid extraction and are very important for nucleic acid-based detection of pathogens in aqueous samples because polymerase chain reaction can amplify nucleic acids outside of the cell. In this study, we developed a simple and effective microfluidic chip platform for selectively extracting nucleic acid from bacteria and viruses by physical adsorption and bead beating. The sample was spiked with bacteria and virus, which were pseudo-selectively adsorbed onto the microbead in the first microchamber, whereas viruses in the output solution from the first chamber were adsorbed onto the surface of microbeads in second chamber. The bacteria and viruses were efficiently lysed in each chamber by microvalve-assisted bead beating following optimization of lysis conditions. The nucleic acids of viruses were well-separated in the first chamber and those of bacteria were isolated in the second chamber.

Highly dense, optically inactive silica microbeads for the isolation and identification of circulating tumor cells

Efficient isolation of circulating tumor cells (CTCs) from whole blood is a major challenge for the clinical application of CTCs. Here, we report an efficient method to isolate CTCs from whole blood using highly dense and transparent silica microbeads. The surfaces of silica microbeads were fully covered with an antibody to capture CTCs, and blocked by zwitterionic moieties to prevent the non-specific adsorption of blood cells. Owing to the high density of the silica microbeads, the complexation of CTCs with silica microbeads resulted in the efficient sedimentation of CTC-microbead complexes, which enabled their discrimination from other blood cells in density gradient media. Model CTCs (MCF-7, HCC827, and SHP-77) with various levels of epithelial cell adhesion molecule (EpCAM) were isolated efficiently, especially those with low EpCAM expression (SHP-77). Moreover, the transparency of silica microbeads enabled CTCs to be clearly identified without interference caused by microbeads. The improved sensitivity resulted in increased CTC recovery from patient samples compared with the FDA-approved CellSearch system (14/15 using our method; 5/15 using the CellSearch system). These results indicate that the isolation method described in this report constitutes a powerful tool for the isolation of CTCs from whole blood, which has important applications in clinical practice.

Monodisperse ultra-large-pore silica coated polystyrene core-shell microbeads via layer-by-layer assembly for nano-micro composite

Polystyrene(PS)@SiO2 core-shell microbeads with large pore and large particle size were prepared via layer-by-layer(LBL)assembly technique for potential applications in nano-micro composites. Negative silica nanoparticles synthesized via modified Stober method and cationic poly(diallyldimethylammonium chloride)were alternately adsorbed on the surface of microbeads. Zeta potential, size, and morphology of the microbeads were monitored during LBL assembly process to ensure the successful deposition of silica nanoparticles. The porous shell was characterized using nitrogen adsorption and desorption analyses, and the surface area, volume and diameter of the pores were derived. It is found that the porous shell thickness and the pore size can be tuned by changing the coating times of silica nanoparticles. Finally, PS@SiO2 core-shell microbeads with 5,μm PS solid core and 350,nm mesoporous shell(mean BJH pore diameter is ~27,nm)were used to load CdSe/ZnS quantum dots(QDs). The fluorescence microscopic image and the optical amplification of the QDs-embedded microbeads (QDBs)indicate that the as-prepared core-shell microbeads can provide adequate space for QDs and may be useful for further application of nano-micro composites.

Polylactide-based microspheres prepared using solid-state copolymerized chitosan and d,l-lactide

Amphiphilic chitosan-g-poly(d,l-lactide) copolymers have been manufactured via solid-state mechanochemical copolymerization and tailored to design polyester-based microspheres for tissue engineering. A single-step solid-state reactive blending (SSRB) using low-temperature co-extrusion has been used to prepare these copolymers. These materials have been valorized to stabilize microspheres processed by an oil/water emulsion evaporation technique. Introduction of the copolymers either in water or in the oil phase of the emulsion allowed to replace a non-degradable emulsifier typically used for microparticle preparation. To enhance cell adhesion, these copolymers were also tailored to bring amino-saccharide positively charged segments to the microbead surface. Size distribution, surface morphology, and total microparticle yield have been studied and optimized as a function of the copolymer composition.

Mass transfer in the biomolecular binding of a target against probe molecules on the surface of microbeads sequestered in wells in a microfluidic cell.

Diagnostic tools which screen the binding interactions of a protein target against a display of biomolecular probes to identify molecules which bind the target are central to cell proteomic studies, and to diagnostic assays. Here, we study a microfluidic design for screening interactions in which the probe molecules are hosted on microbeads sequestered in wells arranged at the bottom of a microfluidic flow channel. Assays are undertaken by streaming an analyte solution with a fluorescently labelled target through the cell, and identifying the fluorescing beads. Numerical simulations are first constructed for the analyte flow over the microbeads in the well array, and the increase in the target concentration on the microbead surface. The binding profile is expressed as a function of the ratio of the convective to the diffusive transport rates (Peclet number or Pe), and the ratio of the kinetic to the diffusive rates (Damkohler number, Da). For any Pe, as Da becomes small enough, the transport is determined by the intrinsic kinetic binding rate. As Pe increases, a thin concentration boundary layer develops over the top surface of the microbead because of the convective flow, and target binds more rapidly. However, the relatively stagnant layers of liquid in the well provide a diffusion barrier which slows the target transport, and for any Da and Pe the transport is slower than equivalent patches of probes arranged on the channel wall. Experiments are also undertaken at high Pe, using the binding of fluorescently labelled NeutrAvidin as a target to probes of its binding partner, biotin, on the microbead surface. The binding profile is compared to the simulations to measure the kinetic rate constant, and this comparison shows that the transport in the cell is not kinetically limited because of the diffusion barriers created by the stagnant liquid layer in the well. Simulations and experiments on microbeads which are only partially recessed in the well demonstrate an increase in the mass transfer rate as more of the microbead surface intersects the flow and the diffusion limitation due to the stagnant layer of liquid surrounding the bottom part of the microbead is minimized.

Effect of chitosan coating on a bacteria‐based alginate microrobot

To develop an efficient bacteria-based microrobot, first, therapeutic bacteria should be encapsulated into microbeads using biodegradable and biocompatible materials; second, the releasing rate of the encapsulated bacteria for theragnostic function should be regulated; and finally, flagellated bacteria should be attached on the microbeads to ensure the motility of the microrobot. For the therapeutic bacteria encapsulation, an alginate can be a promising candidate as a biodegradable and biocompatible material. Owing to the non-regulated releasing rate of the encapsulated bacteria in alginate microbeads and the weak attachment of flagellated bacteria on the surface of alginate microbeads, however, the alginate microbeads cannot be used as effective cargo for a bacteria-based microrobot. In this paper, to enhance the stability of the bacteria encapsulation and the adhesion of flagellated bacteria in alginate microbeads, we performed a surface modification of alginate microbeads using chitosan coating. The bacteria-encapsulated alginate microbeads with 1% chitosan coating maintained their structural integrity up to 72 h, whereas the control alginate microbead group without chitosan coating showed severe degradations after 24 h. The chitosan coating in alginate microbeads shows the enhanced attachment of flagellated bacteria on the surface of alginate microbeads. The bacteria-actuated microrobot with the enhanced flagellated bacteria attachment could show approximately 4.2 times higher average velocities than the control bacteria-actuated microrobot without chitosan coating. Consequently, the surface modification using chitosan coating enhanced the structural stability and the motility of the bacteria-based alginate microrobots.

Microfluidic-based multiplex immunoassay system integrated with an array of QD-encoded microbeads

Here, we developed a multiplex immunoassay platform within microfluidic devices that combines suspension and the planar microarray format. For the suspension microarray format, QD-embedded polymeric microbeads with an average diameter of 24μm were prepared using the Shirasu Porous Glass (SPG) membrane emulsification technique. To furnish the microbeads with resolvable spectral codes, QDs with two different colors (450nm for blue and 520nm for green) were used and different spectral codes were obtained by changing the ratio of emission intensity of the two different QDs within the microbeads. The surfaces of the QD-encoded microbeads were then functionalized with probe antibodies for immunoassays. The planar microarray format was achieved by an array of microholes fabricated in PDMS. Each microhole was designed to trap a single microbead and eventually generated a microbeads array within the microfluidic system. The feasibility of the microbead array within microfluidic devices for use in a multiplex immunoassay was demonstrated by immunobinding assays between IgG and anti-IgG and/or between IgM and anti-IgM. Furthermore, the resultant microbead-based on-chip assay could be used for a sandwich assay to detect prostate-specific antigen (PSA), a model cancer marker, with a detection limit of 1ng/mL. The combination of suspension and the planar microarray format enabled the spatial location of individual microbeads within physically separated regions and thus facilitated the simultaneous determination of different targets that interacted with the corresponding microbeads.

Fluorometric flow-immunoassay for alkylphenol polyethoxylates on a microchip containing a fluorescence detector comprised of an organic light emitting diode and an organic photodiode

A compact fluorescence detector was constructed on a microchip from an organic light emitting diode (OLED) as the light source and an organic photodiode (OPD) as the photo-detector and was used in an immunoassay for alkylphenol polyethoxylates (APE). The OLED based on a terbium complex emitted a sharp light at the main wavelength of 546nm with a full width at half maximum of 9nm. The incident photo-to-current conversion efficiency (IPCE) of the OPD fabricated with Fullerene 70 (C70) and tris[4-(5-phenylthiopen-2-yl)phenyl]-amine (TPTPA) was approximately 44% for light at a wavelength of 586nm. The performance of the fluorescence detector was evaluated for the determination of resorufin (λem=586nm) and the photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the range from 0 to 18µM with a detection limit (S/N=3) of 0.6µM. The fluorescence detector was successfully utilized in a competitive enzyme-linked immunosorbent assay for APE, where an anti-APE antibody was immobilized on the surface of the channel of the Polydimethylsiloxane (PDMS) microchip or on the surface of magnetic microbeads. After an immunoreaction with a sample solution of APE containing a horse radish peroxidase (HRP)-labeled APE, the fluorescence of resorufin generated just after introduction of a mixed solution of Amplex Red and H2O2 was measured using the fluorescence detector. The calibration curve for the photocurrent signals of the OPD due to the fluorescence of resorufin against the logarithmic concentration of APE was sigmoidal in shape. The detection limits defined as IC80 were ca. 1ppb and ca. 2ppb, respectively, for the methods using the anti-APE antibody immobilized on the surface of the microchannel and in the case where the antibody was immobilized on the surface of magnetic microbeads.

Uniform core-shell photonic crystal microbeads as microcarriers for optical encoding.

We demonstrate a rapid and robust method to fabricate uniform core-shell photonic crystal (PC) microbeads by the microfluidic and centrifugation-redispersion technique. Colored crystalline colloidal arrays (CCAs) were first prepared through centrifugation-redispersion approach by self-assembly of polystyrene-poly(N-isopropylacrylamide) (PS-PNIPAm) core/shell nanoparticles (NPs). Different from the conventional NPs (e.g., charged PS or PNIPAm NPs), PS-PNIPAm NPs could easily self-assemble into well-ordered CCAs by only one purification step without laborious pretreatment (e.g., dialysis or ion exchange) or slow solvent-evaporation process. The CCAs is then encapsulated into a transparent polymer shell with functional groups (e.g., copolymer of ETPTA and butyl acrylate (BA)), triggering the formation of core-shell PC microbeads which can be used as optical encoding microcarriers. Importantly, this technique allows us to produce core-shell PC microbeads in a rapid and robust way, and the optical reflections of the PC microbeads appear highly stable to various external stimuli (e.g., temperature, pH value, and ionic strength) relying on the features of the CCAs core and protection of the polymer shell. Moreover, various probe biomolecules (e.g., proteins, antibodies, and so on) can be easily linked on the surface of the core-shell PC microbeads owing to the hydrophilic modification induced by the hydrolysis of BA on the microbead surface, enabling the multiplex biomolecular detection.

Characterization and stability analysis of biopolymeric matrices designed for phage-controlled release.

Alginate and low methoxylated pectin gel matrices emulsified with oleic acid were studied for phage oral delivery. Matrix structural analysis revealed that emulsified pectin (EP) gel microbeads were harder and more cohesive than those of emulsified alginate (EA). EP showed high swelling capacity and slower matrix degradation in aqueous media, suggesting that oleic acid is mainly located on the surface of EP microbeads. EA and EP matrices having p-nitrophenyl palmitate (C-16 ester) as tracer dissolved into oleic acid and in the presence of lipase confirmed this hypothesis which is consistent with EP better phage protective capability. Surface analysis of gel microbeads by scanning electron microscopy revealed strong differences between EP and EA gel microbeads. Phage release kinetics was tested using semi-empirical mathematical models. Experimental curve best fitted the Korsmeyer-Peppas model, predicting transport mechanisms according to the high swelling and degradation of EP. The proposed encapsulation model represents an innovative technology for phage therapy, which can be extrapolated to other therapeutic purposes, using a simple environmentally friendly synthesis procedure and cheap food-grade raw materials.

Zinc alginate-carboxymethyl cashew gum microbeads for prolonged drug release: Development and optimization

Isoxsuprine HCl-loaded microbeads using sodium alginate (SA)-carboxymethyl cashew gum (CMCG) polymer-blends were developed through ionotropic-gelation technique using ZnSO4 as cross-linker. Effects of polymer-blend ratio and cross-linker concentration on drug encapsulation efficiency (DEE) and cumulative drug release at 7h (R7h) were optimized by 32 factorial design. Optimized microbeads were of excellent combination of high DEE (79.92±2.51%) and suitable sustained drug release pattern over a prolonged period of 7h (58.67±2.26%). The microbead surface morphology was analyzed by SEM. The physical state of isoxsuprine HCl within the optimized microbead matrix was analyzed by FTIR and DSC. In vitro isoxsuprine HCl release from alginate-CMCG microbeads in phosphate buffer (pH, 6.8) showed prolonged sustained drug release and Korsmeyer-Peppas model (R2=0.9959–0.9992) over 7h.

PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array

Glycan-based suspension array (SGA) is an “in-house” developed multi-target immunoassay, employing commercially available fluorescent microbeads as a solid support for unique chemically synthesized glycopolymers which capture naturally occurring human anti-glycan antibodies. SGA is a sensitive and reliable tool for the high-throughput screening of anti-glycan antibody alterations characteristic for a vast number of human diseases including cancer. However, unspecific background binding, for instance binding of non-target antibodies, is a common obstacle in such immunoassays. In an attempt to reduce unspecific background binding of serum (or plasma) antibodies, we prepared glycosylated microbeads modified with linear poly(ethylene glycols) (PEGs) of different lengths. We compared several kinds of PEG modifications: (a) partial side-chain substitution of glycopolymers by PEGs of different lengths, (b) end-point addition of biotin-linked PEGs to glycopolymer-coupled beads, and (c) linking of heterobifunctional PEGs to the bead surface prior to glycopolymer immobilization. Among the various modifications investigated, the direct modification of the bead surface with linear heterobifunctional PEGs, consisting of 23- and 60PEG-units significantly reduced the background binding. The end-point addition of biotin-linked PEGs, especially in the case of PEG consisting from 50PEG-units, helped to repel non-target binding caused by endogenous biotin. We observed unspecific binding predominantly for antibodies of IgG but of IgM class. The novel design of fluorescent microbeads allows the detection of human anti-glycan antibodies with increased specificity and opens new horizons for practical application of SGA as a diagnostic tool.

Biodegradable polyester-based microcarriers with modified surface tailored for tissue engineering.

Microcarriers have been proposed in tissue engineering, namely for bone, cartilage, skin, vascular, and central nervous system. Although polyester-based microcarriers have been already used for this purpose, their surface properties should be improved to provide better cell growth. The goal of this study was to prepare microbeads based on poly(D,L-lactide) acid, poly(L-lactide) acid, and to study cell behavior (adhesion, spreading, growth, and proliferation) in function of microbead topography and surface chemistry. To improve L-929 fibroblasts adhesion, microbead surface has been modified with three polycations: chitosan, poly(2-dimethylamino ethylmethacrylate) (PDMAEMA), or chitosan-g-oligolactide copolymer (chit-g-OLA). Although modification of the microbead surface with chitosan and PDMAEMA was performed through physical adsorption on the previously prepared microbeads, chit-g-OLA copolymer was introduced directly during microbead processing. This simple approach (1) bypass the use of an emulsifier (polyvinyl alcohol, PVA); (2) avoid surface "contamination" with PVA molecules limiting a control of the surface characteristics. In vitro study of the growth of mouse fibroblasts on the microbeads showed that both surface topography and chemistry affected cell attachment, spreading, and proliferation. Cultivation of L-929 fibroblasts for 7 days resulted in the formation of a 3D cell-scaffold network.

Nanoflaky MnO2grown in situ on carbon microbeads as an anode material for high-performance lithium-ion batteries

A flower-like MnO2/CMB nanocomposite is synthesizedvia in situgrowth of birnessite-type MnO2on the surface of monodisperse carbon microbeads.