Streptavidin-coated Microspheres Research Articles

Microfluidic system to detect DNA amplicons using agglutination technique

This research investigates the design, fabrication and testing of an easy-to-use and disposable microfluidic system for DNA amplification detection. This is suitable for point-of-care testing (POCT) applications. The microfluidic system utilizes biotin-labelled DNA to agglutinate streptavidin-coated microspheres. The microfluidic system is designed to retain aggregates of cross-linked microspheres as opposed to single microspheres, indicating the detection of biotin-labelled DNA. The microfluidic platform is composed of a filter design and inlet/outlet reservoirs, which was fabricated using microfabrication techniques. This research demonstrates that the microfluidic system is an improvement on the current DNA detection technique that uses particle agglutination. Such a system may, in turn, form the basis of future hand-held, compact, point-of-care biosensors for disease screening and identification.

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O(f)) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O(f) and the cantilever, the fsLP-IF generated at O(f) was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte-endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O(f) and the adhesive interface, were on the order of 10(-13) and 10(-12)N-s, respectively. When the total impulse at O(f) is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.

Characterization of molecular interactions of an immobilized biotinylated monolayer and streptavidin-coated microspheres by bond-rupture scanning

Bond-rupture approach has been used in the understanding of biomolecular interactions of highly specific recognition, e.g., an antibody and its antigen, by a functionalized and self-assembled monolayer (SAM). One of the most challenging issues of diagnostics is to distinguish between true binding and the ever-present non-specific binding in which a species gives false results in conventional affinity methods. In this study, bond-rupture scanning was proposed to characterize bindings by introducing energy mechanically through displacement of a resonant quartz crystal. This system was able to measure the resonant frequency difference, due to mass changes and bond breakages between supramolecular interaction of biotinylated SAM and streptavidin-coated polystyrene microsphere (SCPM). Both 2-μm and 4-μm of SCPMs revealed two recognized desorption patterns at 4 V and 2 V amplitudes respectively. It rapidly provided confirmation of the presence of a target analyte. From this study, it can be shown that an established approach of dynamic bond-rupture scanning can be adopted as a promising diagnostic tool for investigating various interactions of bacteria or virus on an immobilized biomolecular surface by measuring the characteristic level of mechanical energy required to break bonds.

Modulation of immune response by combined targeting of complement receptors and low-affinity Fcγ receptors

Immune complexes (ICs) induce effective pathogen-specific innate and humoral immune response via immunecomplex-binding receptors, such as murine complement receptor type 1 and 2 (mCR1/2) and murine low-affinity Fc receptors for IgG (mFcγRII and III). The exact function of mCR1/2 in cooperation with mFcγRII/III in modulation of humoral immunity has not yet been adequately clarified. The aim of this study was to target these receptors by specific single-chain fragments of antibody (scFv), either individually or in combination, thus modelling the action of IC. For targeting, we used scFv derived from the well-characterized 7g6 and 2.4g2 monoclonal antibodies recognizing mCR1/2 and mFcγRII/III, respectively. These scFvs were monobiotinylated and conjugated to streptavidin or streptavidin-coated microspheres. Such complexes were investigated with respect to target receptor recognition and in vivo localization. Antibody response against the constructs was measured by ELISA and ELISPOT. Results show that targeting streptavidin complexes to mFcγRII/III induces stronger IgG1 response than targeting to mCR1/2 yet both strategies enhance the antibody response compared to the control group immunized with non-targeted peptide–streptavidin complexes. Moreover, the immunogenicity of coupled antigens increased using microspheres as carrier, instead of using soluble streptavidin. In summery, our in vivo experiments reveal that mFcγRII/III is more potent a target than CR1/2 and show that combined targeting of CR1/2 and FcγRII/III receptors does not result in cumulative enhancement of the antigen specific immune response. In addition, microparticle-mediated enhancement of immunization can be further improved by FcγRII/III targeting.

Liquid-Based Fluorescence In Situ Hybridization Assay for Detection of ERBB2 Gene Amplification in Patients with Breast Cancer3

Current reference methods for evaluating gene amplification and expression of ERBB2 (also known as HER-2)--cell-based fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC)--are subjective and influenced by methods of tissue preparation and fixation. We developed and evaluated a novel, quantitative liquid-based FISH (L-FISH) assay that uses flow cytometry to detect ERBB2 gene amplification in breast cancer patients. DNA was extracted from serum or tissue, biotinylated, hybridized to differentially labeled probes for ERBB2 and a chromosome 17-specific single-copy sequence (17-SSC), and immobilized to streptavidin-coated microspheres. The ERBB2/17-SSC signal ratio measured by flow cytometry was used to evaluate ERBB2 amplification. We used L-FISH to test 122 stored formalin-fixed, paraffin-embedded (FFPE) tissue samples and 22 serum samples from randomly selected breast cancer patients; results were compared with those obtained with conventional FISH and IHC. The inter- and intraassay imprecisions were 3.7%-18.9% for FFPE tissue and 2.8%-6.3% for serum. Overall, L-FISH analyses of FFPE tissues demonstrated 84.4% concordance with results obtained with conventional FISH (P < 0.001) and 78.8% concordance with IHC results (P < 0.001). L-FISH analyses of serum samples showed 91% concordance with tissue-based IHC/FISH results (P = 0.038). Our data indicate that this PCR-free L-FISH method can be used to evaluate ERBB2 amplification in both cell-containing (paraffin-embedded tissue) and cell-free (serum) samples. This approach provides more objective results and is amenable to automation and quantitative measurement.

Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to Hydrophilic Microspheres: Development of a Mobile Microscale UV Light Actinometer

( E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine was biotinylated through a diisopropylsilylacetal linkage and attached to the surface of hydrophilic streptavidin-coated microspheres through the high-affinity noncovalent interaction between biotin and streptavidin. The functionalized microspheres form a stable suspension in water. Upon UV irradiation, the nonfluorescent ( E)-5-[2-(methoxycarbonyl)ethenyl]cytidine on the microspheres undergoes photocyclization to produce highly fluorescent 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine. The fluorescence intensity of the microspheres can be correlated to the particle-specific UV doses applied at different suspension concentrations. The microspheres allow one to measure the UV dose (fluence) distribution in high-throughput water disinfection systems.

Forced Agglutination as a Tool to Improve the Sensory Response of a Carboxylated Poly(p-phenyleneethynylene)

A water-soluble biotinylated poly(p-phenyleneethynylene) (PPE, 3) was synthesized by postfunctionalization of an amine−PPE precursor 2a and investigated for sensory responses in aqueous buffer at physiological pH. Polymer 3 shows similar sensitivity (KSV = 1.1 × 104 M-1) to Hg2+ ions when compared to PPE 1 (KSV = 1.3 × 104 M-1); 3 was exposed to streptavidin-coated microspheres (SCM) to form a self-assembled complex, which exhibits an enhanced sensitivity toward Hg2+ ions. If 3 was exposed to either avidin, a streptavidin−rhodamine conjugate, or a streptavidin−Texas Red conjugate, fluorescence of 3 was quenched with KSV values of 33 × 106, 28 × 106, and 9 × 106 M-1, respectively. When lightly cross-linked polymer arrays 3−avidin were exposed to Hg2+ ions, they were more sensitive than 3 alone. The complex was quenched by Hg2+ ions with a KSV of 1.1 × 105 M-1. The negatively charged PPE 1 was incubated with the positively charged avidin to form an electrostatic 1−avidin complex. The 3−avidin complex was ab...

Quantitative Analysis of Tethered Vesicle Assemblies by Quartz Crystal Microbalance with Dissipation Monitoring: Binding Dynamics and Bound Water Content

To implement the molecular recognition properties of membrane proteins for applications including biosensors and diagnostic arrays, the construction of a biomimetic platform capable of maintaining protein structure and function is required. In this paper, we describe a tethered phospholipid vesicle assembly that overcomes the major limitations of planar supported lipid bilayers and alternative biomimetic membrane platforms and characterize it using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy. We provide evidence of a one-step mechanism for bilayer formation and monitor the subsequent adsorption and binding of streptavidin, vesicles, and streptavidin-coated microspheres. For all three species, we identify a critical surface density above which a significant amount of coupled interstitial water contributes to the response of the quartz resonator in a phenomenon similar to dynamic coupling due to surface roughness. A Sauerbrey-type analysis is sufficient to accurately interpret the QCM-D results for streptavidin binding if water is treated as an additional inertial mass, but viscoelastic models must be invoked for vesicle and microsphere binding. Additionally, we present evidence of vesicle flattening, possibly enhanced by a biotin-mediated membrane-membrane interaction.

Bacteriorhodopsin conjugates as anchors for supported membranes.

The sophistication of supported lipid bilayer membranes has increased steadily as new applications are being explored. In general, tethered lipids are used to anchor the lipid bilayer to the substrate. Here we describe a new type of anchoring system for supported lipid bilayers that is based on biotin-PEG3400-bacteriorhodopsin conjugates. Amine-based coupling was used to construct the polymer conjugates, followed by fluorophore labeling to enable confocal imaging. The bacteriorhodopsin-based anchoring system was used to construct solid-supported vesicles from streptavidin-coated microspheres. This method could provide a new route for the stability enhancement of supported lipid bilayer membrane assemblies.

Detection of PCR amplicons from bacterial pathogens using microsphere agglutination

For rapid and inexpensive detection of polymerase chain reaction (PCR) amplicons, a novel microsphere agglutination assay has been developed. PCR is carried out using biotinylated forward and reverse primers, and the amplified DNA fragments are able to agglutinate streptavidin-coated microspheres (5.7 μm in diameter). Purification of PCR amplicons is unnecessary when initial primer concentrations are 250 nM. Agglutination can be identified visually within 2 min without any additional equipment or reagents. Using listeriolysin ( lisA)-specific biotinylated primers, we have successfully detected and identified Listeria monocytogenes lisA + cells among Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7 cells. The simplicity of this protocol considerably reduces the time and cost of diagnostic PCR experiments. This procedure is potentially useful for various studies and field applications.