Avidin Molecules Research Articles

Establishment of self-organization system in rapidly formed multicellular heterospheroids

Multicellular heterospheroids including two or more cell types have some tissue/organ properties and can be used in cell-to-cell interaction studies. However, the spheroid formation is difficult to control because the adhesion efficacy is different in each cell type. To solve this, we applied a rapid cell-to-cell adhesion method, avidin–biotin (AB) binding, to spheroid formation. Introduction of avidin or biotin molecules to the cell surfaces of Mile Sven 1 (MS1) cells promoted formation of spheroid in minutes. This method allowed the construction of heterospheroids having homogenous distributions of different cell types. Interestingly, cells showed self-organization and MS1 cells formed networks with Hep G2 cells. NIH3T3 cells also remodeled when mixed with Hep G2 cells. In contrast, a combination of MS1 and NIH3T3 cells failed to show pattern formation, indicating that self-organization was based on the composition of cell types. Actin polymerization not cell proliferation was the dominant factor in remodeling of heterospheroids in the first 24 h. We also demonstrated the self-organization of spheroids comprising three different cell types. The new technology to assemble cells is important not only to study cell-to-cell interaction but also to make three-dimensional complicated tissues.

Selective adsorption of protein molecules on phase-separated sapphire surfaces

Site-selective adsorption of protein molecules was found on sapphire surfaces that exhibit a phase separation into two domains: weakly charged hydrophobic domain and negatively charged hydrophilic one. Ferritin and bovine serum albumin molecules, which are negatively charged in a buffer solution, are adsorbed to the hydrophobic domains. Avidin molecules, which are positively charged, are adsorbed to the other domain. Fibrinogen molecules, which consist of both negative and positive modules, are adsorbed to the whole sapphire surface. Hemoglobin molecules, whose net charge is almost zero, are also adsorbed to the whole surfaces. These results indicate that electrostatic double layer interaction is the primary origin of the observed selectivity. Dependence of protein adsorption or desorption behaviors on the pH value can also be interpreted by the proposed model.

Conductive AFM patterning on oligo(ethylene glycol)-terminated alkyl monolayers on silicon substrates: proposed mechanism and fabrication of avidin patterns.

Micro- and nanopatterns of biomolecules on inert, ultrathin platforms on nonoxidized silicon are ideal interfaces between silicon-based microelectronics and biological systems. We report here the local oxidation nanolithography with conductive atomic force microscopy (cAFM) on highly protein-resistant, oligo(ethylene glycol) (OEG)-terminated alkyl monolayers on nonoxidized silicon substrates. We propose a mechanism for this process, suggesting that it is possible to oxidize only the top ethylene glycol units to generate carboxylic acid and aldehyde groups on the film surface. We show that avidin molecules can be attached selectively to the oxidized pattern and the density can be varied by altering the bias voltage during cAFM patterning. Biotinylated molecules and nanoparticles are selectively immobilized on the resultant avidin patterns. Since one of the most established methods for immobilization of biomolecules is based on avidin-biotin binding and a wide variety of biotinylated biomolecules are available, this approach represents a versatile means for prototyping any nanostructures presenting these biomolecules on silicon substrates.

Surface‐engineered hydroxyapatite nanocrystal/ poly(ε‐caprolactone) hybrid scaffolds for bone tissue engineering

AbstractTo achieve novel polymer/bioceramic composite scaffolds for use in materials for bone tissue engineering, we prepared organic/inorganic hybrid scaffolds composed of biodegradable poly(ε‐caprolactone) (PCL) and hydroxyapatite (HA), which has excellent biocompatibility with hard tissues and high osteoconductivity and bioactivity. To improve the interactions between the scaffolds and osteoblasts, we focused on surface‐engineered, porous HA/PCL scaffolds that had HA molecules on their surfaces and within them because of the biochemical affinity between the biotin and avidin molecules. The surface modification of HA nanocrystals was performed with two different methods. Using Fourier transform infrared, X‐ray diffraction, and thermogravimetric analysis measurements, we found that surface‐modified HA nanocrystals prepared with an ethylene glycol mediated coupling method showed a higher degree of coupling (%) than those prepared via a direct coupling method. HA/PCL hybrid scaffolds with a well‐controlled porous architecture were fabricated with a gas‐blowing/particle‐leaching process. All HA/PCL scaffold samples exhibited approximately 80–85% porosity. As the HA concentration within the HA/PCL scaffolds increased, the porosity of the HA/PCL scaffolds gradually decreased. The homogeneous immobilization of biotin‐conjugated HA nanocrystals on a three‐dimensional, porous scaffold was observed with confocal microscopy. According to an in vitro cytotoxicity study, all scaffold samples exhibited greater than 80% cell viability, regardless of the HA/PCL composition or preparation method. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Spectral dependence of gold nanorods on the optical properties of substrates and adsorption of polypeptides

Gold nanorods (NRs) wrapped with poly(styrene sulfonate) were fixed on glass or indium tin oxide (ITO) substrates that had been modified with polycationic polymer or 3-aminopropyltrimethoxysilane layers. The spectral properties of the NRs were affected by the refractive indices of the substrates and their surface conditions. Surface modification with poly(styrene sulfonate) and avidin molecules shifted the longitudinal surface plasmon (SP) bands to longer wavelength when the substrates were placed in air. In water and a buffered solution, red shifts of the SP bands were also observed, but the shift shifts were not as large as that in the air. It was found that refractive indices around the polymer-wrapped NRs were sensitive to the adsorption of organic molecules even in the aqueous media.

Fast ferritin immunoassay on PDMS microchips

AbstractA heterogeneous sandwich immunoassay of ferritin on a poly(dimethylsiloxane) microfluidic chip is proposed. An undemanding “prepolymerization technique” based on wet treatment of a phosphor bronze substrate was used for the microchip fabrication. Receptor rabbit antibodies were immobilized via passive sorption directly on microchannel walls. After the incubation of ferritin samples, secondary biotinylated antibodies were introduced. A solution of avidin molecules labeled by fluorescein isothiocyanate was finally added into the microchannels. Lamp-based fluorescence detection of the immunocomplex was then carried out. Dynamic detection range of the method was in the interval from 100 ng mL−1 to 10 μg mL−1.

Facile Fabrication of Two‐Dimensionally Ordered Macroporous Silver Thin Films and Their Application in Molecular Sensing

AbstractLarge‐area, freestanding, two‐dimensionally ordered macroporous (2DOM) Ag thin films with adjustable periodic spacings and good mechanical robustness were fabricated via nanosphere lithography at the solution surface (NSLSS) combined with interfacial reactions. The obtained 2DOM Ag thin films exhibited typical properties of plasmonic crystals with well‐resolved reflectivity dips and vivid colors. The facile NSLSS method enabled the 2DOM Ag thin films to be readily transferred to arbitrary substrates to realize application as thin film sensors. Their application as both a surface plasmon resonance (SPR) sensor and a surface‐enhanced Raman scattering (SERS) sensor in the detection of both small molecules and biological macromolecules were explored. As SERS substrates for the detection of 4‐aminothiophenol (4‐ATP) molecules, the 2DOM Ag thin films showed enhancement factors as high as the 107 order, which made them a promising sensor for the detection of trace amount of analyte adsorbed on the surface. As SPR sensors, the 2DOM Ag thin films modified with a self‐assembled monolayer of recognizing molecules as binding sites showed remarkable shift in the reflectivity dips responsive to the chemical environments. When used for the specific detection of avidin molecules, the 2DOM Ag thin film biosensors exhibited excellent performance with low detection limit (≈100 pM) and broad working range (100 pM–200 nM), indicating that they may be a promising candidate for high‐performance biosensors.

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1–4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1–2°. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

Synthesis of a New NIR Fluorescent Nd Complex Labeling Agent

Fluorescent analysis has been widely used in biological, chemical and analytical research. A useful fluorescent labeling agent should include NIR emission, a large Stoke's shift, and good labeling ability without interfering with the pharmacological profile of the labeled compound. Thus, we planned to develop an M-AMF-DOTA(Nd) derivative composed of an NIR fluorescent moiety and a maleimide conjugating moiety as a new NIR fluorescent labeling agent which fulfills these requirements. M-AMF-DOTA(Nd) was synthesized from 4-amino-fluorescein and was conjugated with an avidin molecule (Avidin-AMF-DOTA(Nd)) through Lys-side chains by reaction with 2-iminothiolane. The fluorescent features of M-AMF-DOTA(Nd) and Avidin-AMF-DOTA(Nd) were comparatively evaluated. A binding assay of Avidin-AMF-DOTA(Nd) with D-biotin and a tumor cell-uptake study were performed to estimate the effects of conjugation on the biological and physicochemical features of the protein. M-AMF-DOTA(Nd) was obtained in 22% overall yield. M-AMF-DOTA(Nd) had a typical NIR fluorescence from the Nd ion (880 nm and 900 nm from 488 nm excitation). Avidin-AMF-DOTA(Nd) was easily synthesized and also had typical NIR fluorescence from the Nd ion without loss of fluorescent intensity. The binding affinity of Avidin-AMF-DOTA(Nd) to D-biotin was equivalent to naive avidin. Avidin-AMF-DOTA(Nd) was taken up by tumor cells in the same manner as avidin conjugated with fluorescein isothiocyanate, an established, widely used fluorescent avidin. Results from this study indicate that M-AMF-DOTA(Nd) is a potential labeling agent for routine NIR fluorescent analysis.

AFM imaging of functionalized carbon nanotubes on biological membranes

Multifunctional carbon nanotubes are promising for biomedical applications as theirnano-size, together with their physical stability, gives access into the cell and variouscellular compartments including the nucleus. However, the direct and label-free detection ofcarbon nanotube uptake into cells is a challenging task. The atomic force microscope(AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thusallows following of the docking of carbon nanotubes to biological membranes. Herewe present topographical AFM images of non-covalently functionalized singlewalled (SWNT) and double walled carbon nanotubes (DWNT) immobilized ondifferent biological membranes, such as plasma membranes and nuclear envelopes, aswell as on a monolayer of avidin molecules. We were able to visualize DWNT onthe nuclear membrane while at the same time resolving individual nuclear porecomplexes. Furthermore, we succeeded in localizing individual SWNT at the border ofincubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

Imaging Surface Charges of Individual Biomolecules

Surface charges play a key role in determining the structure and function of proteins, DNA, and larger biomolecular structures. Here we report on the measurement of the electrostatic surface potential of individual DNA and avidin molecules with nanometer resolution using Kelvin probe force microscopy. We also show, for the first time, the surface potential of buffer salts shielding individual DNA molecules, which would not be possible with conventional ensemble techniques.

A Micromachined Quartz Resonator Array for Biosensing Applications

An 8-pixel micromachined quartz crystal resonator array with a fundamental resonance frequency of 66 MHz has been designed, fabricated, and tested. A compact impedance-spectrum-analyzer electronic interface has been developed and combined with the quartz resonator array to form the biosensing system. The sensor array was calibrated using water-glycerol solutions, and the performance was found to be exactly as expected. Measurement of the crosstalk between the sensor pixels showed an isolation of ~ 30 dB. Selective functionalization of the pixels was achieved through the use of aqueous 3, 3 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">'</sup> -Dithiobis (sulfosuccinimidylpropionate) (DTSSP) molecules. The adsorption of avidin on DTSSP gave a frequency signal of 60 kHz in comparison to unfunctionalized pixels. The specific adsorption of avidin on functionalized pixels was confirmed through fluorescence microscopy. Comparing the performance of the micromachined quartz crystal microbalance (QCM) with a commercial 5-MHz device, we found that the micromachined QCM has a 4.25 times higher signal-to-noise ratio. Based on the measurement of the noise and using three times the frequency noise as the limit for the detection of avidin molecules, we expect to resolve a minimum of ~ 1/960 of a monolayer of avidin corresponding to an aerial mass density resolution of 0.7 ng/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> .

Simultaneous topography and recognition imaging: physical aspects and optimal imagingconditions

Simultaneous topography and recognition imaging (TREC) allows for the investigation ofreceptor distributions on natural biological surfaces under physiological conditions.Based on atomic force microscopy (AFM) in combination with a cantilever tipcarrying a ligand molecule, it enables us to sense topography and recognition ofreceptor molecules simultaneously with nanometre accuracy. In this study weintroduce optimized handling conditions and investigate the physical properties of thecantilever–tip–sample ensemble, which is essential for the interpretation of theexperimental data gained from this technique. In contrast to conventional AFMmethods, TREC is based on a more sophisticated feedback loop, which enablesus to discriminate topographical contributions from recognition events in theAFM cantilever motion. The features of this feedback loop were investigatedthrough a detailed analysis of the topography and recognition data obtained on amodel protein system. Single avidin molecules immobilized on a mica substratewere imaged with an AFM tip functionalized with a biotinylated IgG. A simpleprocedure for adjusting the optimal amplitude for TREC imaging is describedby exploiting the sharp localization of the TREC signal within a small range ofoscillation amplitudes. This procedure can also be used for proving the specificity ofthe detected receptor–ligand interactions. For understanding and eliminatingtopographical crosstalk in the recognition images we developed a simple theoretical model,which nicely explains its origin and its dependence on the excitation frequency.

One Pot Synthesis of Surface PEGylated Core–Shell Microparticles by Suspension Polymerization with Surface Enrichment of Biotin/Avidin Conjugation

Core-shell microparticles that consist of poly(vinyl neodecanoate) (VND) crosslinked with poly(ethylene glycol dimethacrylate) (EGDMA) as the core and poly(ethylene glycol methacrylate) (PEGMA) (Mn = 360 or Mn = 526 g . mol(-1)) as the shell have been synthesized using suspension polymerization by a conventional free radical polymerization process. Interfacial tension and stability tests show that PEGMA acts as an amphiphilic macromonomer and is located on the oil/water interface of the suspension system, thus forming an outer layer during the polymerization. Kinetic studies of the monomers' conversion of VND, EGDMA, and PEGMA have been carried out using (1)H NMR spectroscopy. EGDMA and PEGMA were found to have faster reaction rates compared to VND. Moreover, scanning electron microscopy showed that the polymerization of these particles starts from the shell and finishes towards the core. Consequently, the resulting microsphere is found to have a multi-layer structure. Biotin was covalently bound to the surface by the PEGMA hydroxy groups. Conjugation of biotin with streptavidin PE (phycoerythrin) was subsequently carried out. Confocal microscopy was used to confirm the presence of fluorescing streptavidin. The amount of avidin conjugated to the microspheres was calculated by the release of a 2-(4-hydroxyphenylazo)benzoic acid/avidin complex using UV/vis spectroscopy. One avidin molecule was found to occupy 7 nm(2) on the surface of the microspheres.

The application of IgM in an xMAP suspension assay using recombinant Alt a1 as a model target protein

The potential of Immunoglobulin M (IgM) in an Multi Analyte Profiling (xMAP) multiplex suspension assay was determined using Alt a1, a major allergen of Alternaria alternata, as the model target protein. LumAvidin (LAM) and carboxylated (CAM) microspheres, from LUMINEX™ Corporation, were compared. The reproducible standard curves for concentrations between 1 pg/mL and 10 ng/mL and 3 pg/mL and 300 ng/mL of the allergen were generated by the LAM assay, allowing for the quantitation of an unknown test sample. The success of the LAM assay is dependent on avidin molecules, initially bound on the microsphere, along with the use of a phycoerythrin-coupled secondary IgM. The standard curves generated by the CAM assay for the two allergen concentrations were erratic, falling short of quantifying the test sample. This is the first time a monoclonal IgM is successfully applied as a primary and secondary antibody in the xMAP format. The routine application of this assay will be consolidated as new IgM-based xMAP diagnostics become available.

Superporous agarose—Reticulated vitreous carbon electrodes for electrochemical sandwich bioassays

We report on the use of flow-through electrodes fabricated from a composite of superporous agarose (SPA) and reticulated vitreous carbon (RVC) for carrying out sandwich bioassays via a model sandwich assay scheme. The flow-through design of the SPA–RVC electrodes allows for ease in solution handling with the use of micropipettors while allowing sandwich assays to be performed on the SPA matrix inside the RVC. A sandwich bioassay was devised for detecting biotinylated bovine serum albumin (b-BSA) as a proof-of-concept scheme to demonstrate applicability of SPA–RVC electrodes to carry out sandwich assays. In this bioassay scheme, SPA–RVC electrodes with avidin molecules immobilized on the SPA matrix were incubated with low quantities of b-BSA followed by incubation with avidinylated alkaline phosphatase (av-ALP). This construct creates a sandwich bioassay whereby b-BSA is sandwiched between the two avidin complexes. Av-ALP labels captured on the bound b-BSA catalytically hydrolyze conversion of 4-aminophenylphosphate (PAPP) to electrochemically active 4-aminophenol (PAP) which is then voltammetrically detected inside the RVC. The lower concentration detection limit for b-BSA was 0.32 ± 0.1 ng mL −1 and the absolute detection limit was 32 ± 10 pg. Non-specific binding of av-ALP enzyme labels onto the avidin-activated SPA–RVC electrodes was low. Catalytic generation of PAP by non-specifically bound av-ALP occurs at a rate less than 2% of that for PAP generation by av-ALP in [(SPA-av) − (b-BSA-b) − (av-ALP)] sandwich constructs.

Avidin conjugation to up-conversion phosphor NaYF4:Yb3+, Er3+ by the oxidation of the oligosaccharide chains

NaYF4:Yb3+, Er3+ nanoparticles were successfully prepared by a polyol process using diethyleneglycol (DEG) as solvent. After being functionalized with SiO2–NH2 layer, these NaYF4:Yb3+, Er3+ nanoparticles can conjugate with activated avidin molecules (activated by the oxidation of the oligosaccharide chain). The as-formed NaYF4:Yb3+, Er3+ nanoparticles, NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups, avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR), UV/Vis absorption spectra, and up-conversion luminescence spectra, respectively. The biofunctionalization of the NaYF4:Yb3+, Er3+ nanoparticles has less effect on their luminescence properties, i.e., they still show the up-conversion emission (from Er3+, with 4S3/2 → 4I15/2 at ~540 nm and 4F9/2 → 4I15/2 at ~653 nm), indicative of the great potential for these NaYF4:Yb3+, Er3+ nanoparticles to be used as fluorescence probes for biological system.

Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO 2/Si 3N 4 electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10 −4 to 10 −1 M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol −1). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules.

Unbinding Molecular Recognition Force Maps of Localized Single Receptor Molecules by Atomic Force Microscopy

Atomic force microscopy is a technique capable to study biological recognition processes at the single-molecule level. In this work we operate the AFM in a force-scan based mode, the jumping mode, where simultaneous topographic and tip-sample adhesion maps are acquired. This approach obtains the unbinding force between a well-defined receptor molecule and a ligand attached to the AFM tip. The method is applied to the avidin-biotin system. In contrast with previous data, we obtain laterally resolved adhesion maps of avidin-biotin unbinding forces highly correlated with single avidin molecules in the corresponding topographic map. The scanning rate 250 pixel s(-1) (2 min for a 128 x 128 image) is limited by the hydrodynamic drag force. We are able to build a rupture-force distribution histogram that corresponds to a single defined molecule. Furthermore, we find that due to the motility of the polymer used as spacer to anchor the ligand to the tip, its direction at rupture does not generally coincide with the normal to the tip-sample, this introduces an appreciable error in the measured force.

Development of a sensor-array chip with immobilized antibodies and the application of a wireless antigen-screening system

Abstract The detection of antigen–antibody reactions in normal saline solution, measured using a chip-mounted source-drain electrode coated with an insulation layer with immobilized antibody or antigen molecules, was investigated as a function of concentration. Five pairs of source-drain electrodes (×8 arrays) were fabricated on a glass substrate of 20 mm × 30 mm size, in which the individual Au/Cr (1.0/0.1 μm thickness) sensors had widths of 50 μm, and an inter-electrode interval of 100 μm. The fabricated source-drain electrodes were further coated with an insulation layer comprising various porous materials for use as an adsorbent for receiving antibody molecules. The sensor chip was equipped with a handy-type sensor signal analyzer that comprised either an amplifier circuit with a Miniship™, or a system in a packaged LSI device. The weak current received by each sensor electrode was amplified and modulated with a sensor signal analyzer to give a digital signal, which was then wirelessly transmitted to a PC connected to a receiver device. The effects of the immobilized antibody molecules on the detection selectivity and detection limits were examined. The sensing functions were determined by measurement of the source-drain electrode voltage when the surface of the insulator was contacted with antigen solutions of various concentrations. The sensor ability was also evaluated by comparing the relative selectivity of each sensor. When a sensor surface with immobilized biotin was used for avidin sensing, or immobilized with leukotriene B4 for anti-leukotriene sensing, the sensor showed the largest effect for the selective detection of all sensor testing conducted. In contrast, when the sensor surface was immobilized with avidin for biotin sensing, or immobilized with anti-leukotriene for leukotriene sensing, the sensor exhibited low selectivity. Based on the results obtained, an adsorbed antibody on an adsorption particle is effectively expressed as the selective detection ability for the sensor-array chips. When liquid is deposited on the surface of an insulator, an electric double layer arises at the boundary surface. In this respect, the insulator molecules become polarized, resulting in the formation of opposing charges on both the boundary surface between the liquid and solid, and on the reverse side of the insulator. In the same way, when a solution mixture containing antigens is deposited on the surface of an insulator with antibodies immobilized to its surface, specific antigens and antibody reactions occur and the charge on the reverse side of the insulator is increased. The relationship between the source-drain electrode current and the sensor voltage follows Ohm's law. Based on this principle, a source-drain electrode produces a signal in response to changes in current originating from antigen–antibody reactions at the boundary surface on the insulator layer. The avidin molecule is composed of four domain structures, such that, four acceptors per molecule exist on the surface of the adsorbent particle. Biotin preferably interacts with this acceptor, because the molecular size of biotin is smaller that that of avidin. The sensor voltage changes do not suffer in the presence of cation contamination. In contrast, the molecular size of avidin is much larger than that of the cations in solution. In this respect, the cations are considered to interact with the sensor surface in preference to the biotin antibodies, thus promoting polarization of the insulator layer. As a result, the detection voltages increased, while the detection selectivity decreased.