Multiplexed assay for proteins based on sequestration electrochemistry using the protein binding electroactive magnetic microbeads

The paramagnetic microbead-based electrochemical binding assay was demonstrated for detecting two kinds of protein simultaneously. The principle of this assay is based on the sequestration electrochemistry. The protein binding electroactive magnetic microbeads which are conjugated with an electroactive compound and a ligand to bind specifically with a target protein were prepared. The avidin-biotin and soybean agglutinin (SBA)-galactosamine were chosen as model protein-ligand systems. The avidin binding electroactive magnetic microbead (ABEMMb) and SBA binding electroactive magnetic microbead (SBEMMb) are constructed by biotin/thionine and galactosamine/ferrocene modified on paramagnetic microbeads. The voltammetric response for these functionalized microbeads was measured by the Nd-Fe-B magnet-incorporating carbon paste rotating disk electrode. The measurements were performed in a microliter droplet using a rotating disk electrode system. Avidin and SBA were simultaneously detected by the decrease in the current responses from the reduction of ABEMMb and SBEMMb that was caused by the binding with target proteins. The limits of detection for avidin and SBA were 4 × 10(-10) and 2 × 10(-10) M, respectively.     

Enhanced electrochemical activity of redox-labels in multi-layered protein films on indium tin oxide nanoparticle-based electrode

Facile electrical communication between redox-active labeling molecules and electrode is essential in the electrochemical detection of bio-affinity reactions. In this report, nanometer-sized indium tin oxide (ITO) particles were employed in the fabrication of porous thick film electrodes to enhance the otherwise impeded electrochemical activity of redox labels in multi-layered protein films, and to enable quantitative detection of avidin/biotin binding interaction. To carry out the affinity reaction, avidin immobilized on an ITO electrode was reacted with mouse IgG labeled with both biotin and ruthenium Tris-(2,2′-bipyridine) (Ru-bipy). The binding reaction between avidin and biotin was detected by the catalytic voltammetry of Ru-bipy in an oxalate-containing electrolyte. On sputtered ITO thin film electrode, although a single layer of Ru-bipy labeled avidin exhibited substantial anodic current, attaching the label to the outer IgG layer of the avidin/biotin-IgG binding pair resulted in almost complete loss of the signal. However, electrochemical current was recovered on ITO film electrodes prepared from nanometer-sized particles. The surface of the nanoparticle structured electrode was found by scanning electron microscopy to be very porous, and had twice as much surface binding capacity for avidin as the sputtered electrode. The results were rationalized by the assumption of different packing density of avidin inner layer on the two surfaces, and consequently different electron transfer distance between the electrode and Ru-bipy on the IgG outer layer. A linear relationship between electrochemical current and IgG concentration was obtained in the range of 40-4000 nmol L⁻¹ on the nanoparticle-based electrode. The approach can be employed in the electrochemical detection of immunoassays using non-enzymatic redox labels.     

Molecular recognition of avidin on biotin-functionalized gold surfaces detected by FT-IRRAS and use of metal carbonyl probes

Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) was successively used to monitor the covalent immobilization of biotin molecules onto a planar gold substrate covered with a self-assembled monolayer of cystamine and to transduce the molecular recognition of avidin and biotin. This detection was greatly facilitated and made selective by the labeling of avidin and of biotin with various transition metal carbonyl probes. The binding of avidin to the surface was optimized by blocking the nonspecific binding sites by adsorption of an unrelated protein, bovine serum albumin. This work exemplifies the feasibility of detecting biomolecular associations involving molecules of any size at a liquid/solid interface by using a simple and accessible surface analysis technique.     

Trinuclear ruthenium clusters as bivalent electrochemical probes for ligand-receptor binding interactions

Despite their popularity, electrochemical biosensors often suffer from low sensitivity. One possible approach to overcome low sensitivity in protein biosensors is to utilize multivalent ligand-receptor interactions. Controlling the spatial arrangement of ligands on surfaces is another crucial aspect of electrochemical biosensor design. We have synthesized and characterized five biotinylated trinuclear ruthenium clusters as potential new biosensor platforms: [Ru(3)O(OAc)(6)CO(4-BMP)(py)](0) (3), [Ru(3)O(OAc)(6)CO(4-BMP)(2)](0) (4), [Ru(3)O(OAc)(6)L(4-BMP)(py)](+) (8), [Ru(3)O(OAc)(6)L(4-BMP)(2)](+) (9), and [Ru(3)O(OAc)(6)L(py)(2)](+) (10) (OAc = acetate, 4-BMP = biotin aminomethylpyridine, py = pyridine, L = pyC16SH). HABA/avidin assays and isothermal titration calorimetry were used to evaluate the avidin binding properties of 3 and 4. The binding constants were found to range from (6.5-8.0) × 10(6) M(-1). Intermolecular protein binding of 4 in solution was determined by native gel electrophoresis. QM, MM, and MD calculations show the capability for the bivalent cluster, 4, to intramolecularly bind to avidin. Electrochemical measurements in solution of 3a and 4a show shifts in E(1/2) of -58 and -53 mV in the presence of avidin, respectively. Self-assembled monolayers formed with 8-10 were investigated as a model biosensor system. Diluent/cluster ratio and composition were found to have a significant effect on the ability of avidin to adequately bind to the cluster. Complexes 8 and 10 showed negligible changes in E(1/2), while complex 9 showed a shift in E(1/2) of -43 mV upon avidin addition. These results suggest that multivalent interactions can have a positive impact on the sensitivity of electrochemical protein biosensors.     

Biotin ligands labeled with daunomycin as an electrochemical probe for avidin and biotin interaction

Electroactive biotin ligands were prepared by the reaction of daunomycin with biotinylating reagents with a different spacer. These biotin ligands exhibited similar electrochemical properties to those of daunomycin, but the adsorptivity of the ligands on the electrode increased with increasing length of the spacer. The electrode response of these ligands decreased when specifically bound with avidin. This made it possible to detect electroinactive avidin indirectly. Biotin was detected by observing the competitive reaction between biotin and the ligands for the limited binding sites of avidin. The binding strength of the labeled biotins with avidin was compared with that of unlabeled biotin by using an enzyme assay.     

Observation of noncovalent complexes to the avidin tetramer by electrospray ionization mass spectrometry

Intact avidin-biotin and avidin-biotin maleimide noncovalent complexes have been observed by electrospray ionization mass spectrometry (ESI-MS) by using an extended mass range quadrupole mass spectrometer. By utilizing mild ES1 interface conditions, the expected solution behavior of four biotin or biotin maleimide molecules noncovalently binding to each avidin tetramer can be preserved in the gas phase. The ESI-MS results show the appropriate mass additions of 973 ± 60 Da for biotin and 1802 ± 40 Da for biotin maleimide to the avidin tetramer species. These results support the hypothesis that substantial retention of higher order structure is possible in the gas phase by using gentle ESI conditions.     

Photocleavage of avidin by a new pyrenyl probe

In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py-biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py-biotin were demonstrated. Binding of Py-biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py-biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.     

Electrochemical Evaluation of the Interaction between Avidin and Biotin at Biotinylated Polypyrrole Electrode Using a Redox Marker

The interaction between avidin and biotin was evaluated electrochemically by monitoring the change in the electrode response of redox markers. Biotin was immobilized on the electrode surface by means of the electrochemical polymerization of biotinylated pyrrole and pyrrole. When avidin was introduced onto the biotinylated polypyrrole electrode surface, the large change in the electrode response of the redox marker was detected. The fact that the change in the electrode response of a marker ion could be attributed to the electrostatic interaction between avidin on the electrode surface and the redox marker ion present in a solution was verified by replacing avidin with NutrAvidin. At a pH lower than the isoelectric point of avidin, the electrode response of ferrocyanide as an anionic marker ion increased linearly within the range of 5.0×10^?9 ?3.0×10^?8 M avidin. The relative standard deviation at 1.5×10^?8 M avidin was about 5.4% (n=5). The detection of biotin was also performed using a competitive reaction between biotin in solution and biotin that had been immobilized on the electrode surface in the form of the biotinylated polypyrrole.     

Control of specific attachment of proteins by adsorption of polymer layers

This study concerns the design of protein-resistant polymer adsorbed layers for the control of surface binding of biospecific recognition entities. Polymer surface layers were prepared using the adsorption of poly(allylamine hydrochloride) (PAH), poly(l-lysine) (PL), and branched and linear polyethyleneimine (PEI) and further modified by the covalent attachment of biotin for specific avidin attachment. The adsorption of PAH, PL, and PEI on silicon substrates was studied as a function of pH and ionic strength using ellipsometry. Average dry layer thicknesses of approximately 10, approximately 5, approximately 9, and approximately 3 A (+/-1 A) were obtained when polymer adsorption occurred from solutions at pH 9.5 that contained 0.5 M NaCl for PAH, PL, branched PEI, and linear PEI, respectively. These polymers showed significant differences in their efficiency to suppress nonspecific avidin adsorption. At low ionic strength, avidin adsorption occurred on all polymer-coated surfaces at basic pH values, despite the same positive electrostatic charge for protein globules and the surface. Though the net electrostatic repulsion between avidin molecules and branched PEI was efficiently screened in a protein solution of pH 7 and 0.15 M NaCl, branched-PEI coatings of high molecular weight were unique in their ability to provide avidin-resistant surfaces as a result of steric hindrance from the branched architecture of adsorbed polymer chains. All polymers studied were effective in suppressing avidin adsorption at pH 3 as a result of protonation of the avidin surface functional groups at this pH. Branched-PEI-coated surfaces were also effective for the suppression of smaller positively charged proteins such as lysozyme and ribonuclease A at pH 7 and 0.15 M NaCl. They were also resistant to the adsorption of negatively charged proteins such as BSA and fibrinogen at pH 7 and 0.75 M NaCl. Furthermore, by using PEI-modified protein-repellent surfaces, selective binding of avidin was achieved to surface-bound silver nanoparticles, which should provide a promising application for the label-free detection of biological species using surface-enhanced Raman scattering (SERS).     

A Multivalent Structure‐Specific RNA Binder with Extremely Stable Target Binding but Reduced Interaction with Nonspecific RNAs

By greatly enhancing binding affinities against target biomolecules, multivalent interactions provide an attractive strategy for biosensing. However, there is also a major concern for increased binding to nonspecific targets by multivalent binding. A range of charge‐engineered probes of a structure‐specific RNA binding protein PAZ as well as multivalent forms of these PAZ probes were constructed by using diverse multivalent avidin proteins (2‐mer, 4‐mer, and 24‐mer). Increased valency vastly enhanced the binding stability of PAZ to structured target RNA. Surprisingly, nonspecific RNA binding of multivalent PAZ can be reduced even below that of the PAZ monomer by controlling negative charges on both PAZ and multivalent avidin scaffolds. The optimized 24‐meric PAZ showed nearly irreversible binding to target RNA with negligible binding to nonspecific RNA, and this ultra‐specific 24‐meric PAZ probe allowed SERS detection of intact microRNAs at an attomolar level.     

Voltammetric detection of avidin and biotin by biotin labeled with cysteine

An avidin-biotin assay was developed from a voltammetric procedure using biotin labeled with cysteine. Mercury(II) as a marker was used to detect avidin and biotin, because the oxidation wave of mercury decreases when the cysteine part of labeled biotin(LB) complexes with mercury(II).The formation of the mercury(II)-cysteine complex is suppressed when the LB binds to the biotin site of avidin. Accordingly, the concentration of avidin can be estimated from the increasing mercury peak current. Detection of biotin is also carried out by a competitive reaction of biotin and the LB to the binding site on avidin, where the addition of biotin decreases the peak current of mercury. Limits of detection for avidin and biotin were in the 10^–9 mol/L range. The length of the spacer between the cysteine and biotin was investigated. It was observed that the strength of binding increased with increasing length of spacer. Size considerations rules out steric influences, so it is suggested that the binding constant depends on hydrophobic interactions in the binding site.     

A new chemically amplified electrochemical system for the detection of biological affinity reactions: direct and competitive biotin assay

Quantitation of biological affinity reactions by a newly developed chemically amplified electrochemical detection method was demonstrated with the biotin-avidin binding pair. In the method, ruthenium tris(2,2'-bipyridine)(Ru-bipy) was used as an electrochemical signal-generating tag. Its oxidation current on an indium tin oxide (ITO) electrode was amplified with a sacrificial electron donor, oxalate. Because oxalate itself produced negligible current on the electrode, the signal-to-background ratio was greatly enhanced in comparison with other chemical amplification systems. Although the Ru-bipy/oxalate redox couple has been employed previously in electrochemiluminescent and photoelectrochemical detection, its use in a catalytic amperometric detection of biological binding assays has not been reported. To implement the method in the detection of biotin-avidin recognition, avidin was immobilized on an ITO electrode, and was reacted with biotin in solution. Immobilization of avidin by passive adsorption was found to be relatively stable under the condition of the affinity reaction. In the direct assay, biotin labelled with Ru-bipy was recognized by avidin and accumulated on the electrode surface, which was then detected electrochemically in the presence of oxalate. A linear relationship between electrochemical current and biotin concentration was obtained in the range of 1-300 ng mL(-1). In the competitive assay, a mixed solution of unlabelled biotin (the analyte) of various concentrations and 100 ng mL(-1) labelled biotin was reacted with avidin on the surface. As the concentration of the unlabelled biotin increased, less labelled biotin bound to avidin, leading to a reduction in the electro-catalytical response of Ru-bipy. A detection limit of 1 ng mL(-1) biotin was obtained in the competitive assay, which is close to the sensitivity of some enzyme-labelled amperometric assays.     

Coupling artificial actin cortices to biofunctionalized lipid monolayers

We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.     

Electrical properties of diamond surfaces functionalized with molecular monolayers

Recent studies have shown that semiconductor surfaces such as silicon and diamond can be functionalized with organic monolayers, and that these monolayer films can be used to tether biomolecules such as DNA to the surfaces. Electrical measurements of these interfaces show a change in response to DNA hybridization and other biological binding processes, but the fundamental nature of the electrical signal transduction has remained unclear. We have explored the electrical impedance of polycrystalline and single-crystal diamond surfaces modified with an organic monolayer produced by photochemical reaction of diamond with 1-dodecene. Our results show that, by measuring the impedance as a function of frequency and potential, it is possible to dissect the complex interfacial structure into frequency ranges where the total impedance is controlled by the molecular monolayer, by the diamond space-charge region, and by the electrolyte. The results have implications for understanding the ability to use molecularly modified semiconductor surfaces for applications such as chemical and biological sensing.     

Analysis of protein complexes with hydrogen exchange and mass spectrometry

Analysis of protein complexes using hydrogen exchange (HX) combined with high resolution electrospray mass spectrometry (MS) is demonstrated. HX MS offers the possibility to analyze the strength of binding in protein complexes, to identify regions that undergo binding induced structural changes, and to study the nature (hydrophobic, electrostatic, etc.) of binding between two or more proteins. In the current work, a heteromeric complex containing UBC9 (an E2 conjugating enzyme) and SUMO-1 (a ubiquitin-like modifier) was investigated by incubating the complex in D2O and measuring the amount of deuterium incorporation with MS. SUMO-1 had significant changes in deuterium levels when bound to UBC9. In contract, few or no changes in deuterium levels were detected in UBC9 when part of the complex, even at the binding interface. Titrations were used to estimate the binding constant for the complex. The nature of the interface was probed by creating a site-directed mutant form of UBC9. The mutant form showed no detectable binding to SUMO-1 and thereby suggested that binding between these two proteins is primarily electrostatically driven. This application of HX MS demonstrates its value in the study of protein complexes and protein machinery.     

Transport of Na2SO4 and MgSO4 solutions through a composite membrane

Retention curves with a composite membrane (HR 95) have been measured for different solutions of Na₂SO₄ and MgSO₄. Salt permeabilities in the skin and the porous layer of the composite membrane have been calculated. The results show that the salt permeability in the skin layer is 15% of that corresponding to the porous layer. Electrical resistances for the composite membrane and another membrane similar to the supported membrane (without skin layer) have been measured using both direct and alternating current. From the a.c. values, assuming that no concentration polarization exists at the skin-porous layer interface, the electrical resistance for the skin layer at different concentrations was estimated. The membrane resistance values were also determined by impedance spectroscopy measurements, and the results agree with those previously found by a.c. measurements.     

Voltammetric investigation of avidin-biotin complex formation using an electroactive bisbiotinyl compound

Formation of avidin-biotin complex was investigated using bisbiotinyl thionine (BBT) by means of voltammetric techniques. Thionine is an electroactive compound and has two amino groups that are necessary for the reaction with a biotinylation reagent. The biotinylation of thionine produces a new reagent with two biotin moieties at each end of thionine. Three BBTs of different lengths of the spacer that connects the biotin moiety to the thionine moiety were prepared. The avidin-biotin binding assay was achieved by measuring the electrode response of the thionine moiety in BBT. The binding affinity and the conformation of complex, which depended on the length of spacer, are discussed. BBT in which the spacer is shortest (BBT-S, distance between carbonyl group of the two biotin moieties: 11 Å) binds with only one avidin molecule. BBT with medium length of spacer (BBT-M, 28.8 Å) forms the complex with two avidin molecules. BBT with the longest spacer (BBT-L, 46.6 Å) allows binding with two avidin molecules as well as intramolecular binding within one avidin molecule. The affinity constants of BBT-S, BBT-M and BBT-L for avidin were estimated to be 7.0 × 10¹² M⁻¹, 3.2 × 10¹² M⁻¹ and 4.0 × 10¹² M⁻¹, respectively.     

A smart surface in a microfluidic chip for controlled protein separation

The smart surface created in a microfluidic chip has shown the capability of adsorbing and releasing proteins under electrical control. The inner surface of the chip channel was first coated by a thin layer of Au through sputtering and was subsequently modified with loosely packed self-assembled monolayers (SAMs) of thiols with terminal carboxylic or amino groups. Upon application of an external electric potential to the gold substrate, reversible conformational transformation between "bent" and "straight" states for the anchored mercapto chains could be modulated, through the electrostatic effect between the ionized terminal groups and the charged gold substrate. Thus, a hydrophobic or hydrophilic channel surface was established and could be reversibly switched electrochemically. Accordingly, the microchips prepared in this way can reversibly and selectively adsorb and release differently charged proteins under electrical control. Two model proteins, avidin and streptavidin, were demonstrated to be readily adsorbed by the smart chips under negative and positive potential, respectively. Also, more than 90 % of the adsorbed proteins could be released upon an electrical command. Furthermore, these chips were applied to the controlled separation of avidin and streptavidin mixtures with 1:1 and 1:1000 molar ratios. Under specific applied potentials, the chips adsorbed a certain protein from the mixture whereas the other protein was allowed to flow out, after which the adsorbed protein could be released by switching the applied potential. Thus, two eluted protein fractions were obtained and the separation of the two proteins was achieved. For the former mixture, each eluted fraction contained up to approximately 80-90 % avidin or streptavidin. For the latter mixture, the resulting separation efficiency indicated that the molar ratio of avidin and streptavidin could be increased from 1:1000 to about 32:1 after five run separations.     

Nanometric protein arrays on protein-resistant monolayers on silicon surfaces

We present a novel approach for preparation of nanometric protein arrays, based on binding of avidin molecules to nanotemplates generated by conductive AFM lithography on robust oligo(ethylene glycol)-terminated monolayers on silicon (111) surfaces that are protein-resistant. We showed that only biotinated-BSA but not the native BSA bind to the avidin arrays and that the resulting arrays of biotinated BSA could bind avidin to form protein dots with a feature size of approximately 30 nm. This result demonstrates that the avidin array may serve as templates for preparation of nanoarrays of a wide variety of biotin-tagged proteins for studying their interactions with other protein molecules at nanoscale.     

Streaming current, permeability, and microelectrophoresis of polystyrene latices in methanol-ethanol mixtures

The influence of the solvent (methanol-ethanol mixtures) on the electrokinetic behavior of polystyrene latices with sulfate groups was studied (methanol content was increased by 0.2 at a constant KBr concentration of 1 mM). Viscosity, density, and dielectric constant (eta, rho, and epsilon) were determined at experimental conditions. Two latices (with different surface charge densities and sizes) were used. Electrophoresis measurements were used for dilute dispersions. Streaming current and hydrodynamic permeability were measured for porous plugs. Linear trends in the electrokinetic measurements were observed in the whole molar fraction range. The experimental data obtained from different techniques allow determining the zeta potential according to a well-established classical relationship. The results obtained were analyzed on the basis of the solvent mixture properties and the electrical interface behavior. In addition, permeability data provided valuable information to interpret effects at the solid-liquid interface of the porous plug.