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.     

Voltammetric detection of ovalbumin using a peptide labeled with an electroactive compound

For this study, a new method was developed to electrochemically detect ovalbumin via its binding with the peptide-1(RNRCKGTDVQAW) in lysozymes. The peptide that exists at the C-terminal of a lysozyme was combined with ovalbumin. When an electroactive compound was introduced to the N-terminal side of the peptide through ethylene gycolbis(sulfosuccinimidyl succinate), the labeled peptide-1 served as a probe for the detection of ovalbumin. The electrode responses of labeled peptide-1 were measured after the labeled peptide-1 and ovalbumin were incubated in a 0.1 M phosphate buffer (pH 5.6). As a result, the electrode response decreased as the concentration of ovalbumin increased. The detection limit of ovalbumin was 2.3 × 10⁻¹¹ M as estimated at 3-fold the standard deviation (3σ) (n = 5). Because the steric structure of the peptide and some of the amino acid residues were related to the binding, we prepared a peptide-2, to which the N- and C-terminals of peptide-1 were alternated. The decrease in the response for the labeled peptide-2 was less than that for the labeled peptide-1. In addition, the peak current of a peptide-3, for which the D of peptide-1 was replaced with S, was hardly changed with or without ovalbumin. Therefore, it was clear that the binding was influenced by the steric factors and by the sequence of the peptide. However, a peptide-1 with bis(sulfosuccinimidyl) suberate was designed to investigate the hydrophobic influences on the probe. The change in the peak current was smaller than that of peptide-1 with ethylene gycolbis(sulfosuccinimidyl succinate), which was due to the hydrophobic properties of the alkyl chain between the peptide and the ovalbumin. The proposed method could be applied to the determination of ovalbumin in egg whites. Consequently, the concept becomes an electrochemical sensing method for proteins based on the protein–peptide interaction.     

Voltammetric homogeneous binding assay of biotin without a separation step using iminobiotin labeled with an electroactive compound.

Avidin, which is one type of glycoprotein, has a strong affinity with biotin (Ka = 10(15) M(-1)). Iminobiotin also forms a complex with avidin (Ka = 10(8) M(-1) at pH 9.5). The avidin-iminobiotin complex changes to the avidin-biotin complex in the presence of biotin because of the difference of the binding constant to avidin. In this study, the interaction between avidin and iminobiotin labeled with an electroactive compound was investigated by voltammetry. After avidin and the labeled iminobiotin (LI) were incubated in 0.1 M phosphate buffer (pH 7.0), the peak currents of LI were measured in various concentrations of biotin. The peak currents increased with increasing the concentration of biotin. Thus, this observation indicates the formation of avidin-biotin complex. On the other hand, the formation of avidin-iminobiotin complex depended on the pH of the solution. LI combines with the avidin at pH 5.6-8.9 and dissociates at pH 4.6.     

Voltammetric detection of lectin using sugar labeled with electroactive substance.

The voltammetric detection of soybean agglutinin (SBA) was investigated on the basis of an interaction between the lectin and a sugar. Because galactose and lactose combined with SBA, the sugars were labeled by a Schiff base with an electroactive daunomycin. After the labeled sugar and SBA were mixed, measurements were carried out by voltammetry. When SBA-sugar binding occurs, a part of daunomycin of the labeled sugar is taken to the binding sites. As a result, SBA is detected by a change in the peak current of daunomycin, and the SBA-sugar interaction is evaluated. The length of the alkyl chain between daunomycin and the sugar was also considered. The electrode response to the concentration of SBA was linear over the range of 0.04-0.8 microg min(-1). The merits of this procedure are the convenient preparation of labeled sugar and a rapid measurement without separation. On the other hand, the detection of sugar at the 10(-9) mol dm(-3) level was achieved by a competitive reaction to limited binding sites of the lectin between the sugar and the labeled sugar.     

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.     

Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes

A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL⁻¹ CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.     

Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV–Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.     

Recent advances on electroactive CNT-based membranes for environmental applications: The perfect match of electrochemistry and membrane separation

Global climate change, growing population, and environmental pollution underscore the need for a greater focus on providing advanced water treatment technologies. Although electrochemical based-processes are becoming promising solutions, they still face challenges owing to mass transport and upscaling which hinder the exploitation of this technology. Electrode design and reactor configuration are key factors for achieving operational improvements. The electroactive membrane has proven to be a breakthrough technology integrating electrochemistry and membrane separation with an enhanced mass transport by convection. In this review article, we discuss recent progress in environmental applications of electroactive membranes with particular focus on those composed of carbon nanotubes (CNT) due to their intriguing physicochemical properties. Their applications in degradation of refractory contaminants, detoxification and sequestration of toxic heavy metal ions, and membrane fouling alleviations are systematically reviewed. We then discuss the existing limitations and opportunities for future research. The development of advanced electroactive systems depends on interdisciplinary collaborations in the areas of materials, electrochemistry, membrane development, and environmental sciences.     

Synthesis of novel polydiazocine for electroactive materials based on diazocine

In this study, we successfully designed and synthesized a novel class of ladder‐type polymer polydi‐benzoyl[b,f][1,5]diazocine ( PBDA‐a ) and polydimethoxybenzoyl[b,f][1,5]diazocines ( PBDA‐b ) for novel electrochemical actuators via one‐pot polymerization. The structure of polymers was characterized using ¹³C NMR, FTIR and MALDI‐TOF. Both of the polydiazocines exhibited high thermal stability and excellent solubility in common solvent. Molecular conformation studies using the density functional theory method revealed that the conformational structure of PBDA‐a preferred to display zigzag conformation while PBDA‐b display helix conformation. The mechanism of the polydiazocine synthesis is assumed to condensation polymerization proceeding by an unprecedented cyclization of the isocyanate with the neighboring acyl group, followed by the dimerization to form diazocine ring. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4694–4701     

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B‐subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1‐oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene‐dextran sulfate‐polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (K_d) determination. The K_d values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC₅₀ values of an ELISA‐type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen‐induced secretory diarrhea.     

Water quality monitoring using an enhanced chemiluminescent assay based on peroxidase-catalyzed peroxidation of luminol

Enhanced chemiluminescence (ECL) describes the phenomenon of the light output increase in the reaction of oxidation of luminol catalyzed by horseradish peroxidase (HRP) in the presence of certain phenolic compounds. This work summarizes the effects of preincubation of certain substances with HRP on the chemiluminescent reaction intensity. Preincubation of herbicide, detergent, surfactants (Brij-96 and Tween-20), phenol, metal ions (mercury, cobalt, and nickel), and bactericide with HRP had an inhibitory effect on the enzyme activity. HRP-preincubation with metal ions (cadmium, magnesium, and zinc), as well as with some insecticides, stimulated the chemiluminescent intensity. Calibration graphs were obtained to demonstrate the possibility to determine the pollutant concentration. Light emission from the peroxidase catalyzed enhanced chemilum inescence is affected by a wide number of chemicals and, therefore, the method can beused for on-site monitoring of water quality. A rapid and simple assay to detect water contamination has been developed.     

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptoundecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN)₆^3−/4−were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (R_ct) increased with increasing SRB concentration. A linear relationship between R_ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 × 10⁷ cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost, and time-saving monitoring of microbial populations.     

Functional lipid microstructures immobilized on a gold electrode for voltammetric biosensing of cholera toxin

Redox functionalized microstructures of diacetylene lipids containing cell surface ligand GM1 have been prepared for the construction of an electrochemical biosensor for cholera toxin from Vibrio cholerae. Incorporation of lipid molecules with disulfide functionality into the microstructures allows for firm attachment of the microstructures on a gold surface to form a sensing interface. The observed morphology of the microstructures is platelet, with size around 240 nm as determined by dynamic light scattering and transmission electron microscopy. The electrochemical response stems from electron transfer between the electrode and the redox sites on the microstructures, and the Faradaic current is influenced by the binding events of protein toxins to the ligands displayed on the crystalline surface. Electrochemical characterization indicates that electron transfer of surface ferrocene on the gold electrode is facile. Differential pulse voltammetry was used to measure the current magnitude as a function of toxin concentration, and a working range expanding from 1.0 x 10(-8) to 5.0 x 10(-7) M was obtained. Bovine serum albumin (BSA) was used as a control agent with which no interference to Faradaic response was found in the same concentration range. Atomic force microscopy (AFM) was used to characterize the morphology and distribution of microstructures on the gold surface. The effectiveness of the design for bypassing surface fouling of proteins in electrochemical detection has been demonstrated, and a binding regulated electron hopping mechanism for the observed electrochemical behavior has been proposed.     

Contraction process of an electroactive actuator based on a one microsecond atomistic molecular dynamics simulation

The contraction process of an electroactive actuator constituted by calix[4]arene units and quaterthiophene segments has been investigated at the microscopic level by using atomistic molecular dynamics simulations in dichloromethane solution using explicit solvent molecules. Results derived from a 1 mus trajectory of the oxidized and deprotonated actuator indicate that the contraction occurs through a non-concerted mechanism in which each actuating units present in the system behave independently. The efficiency of the contraction process can be reduced by the presence of secondary conformational transitions in the calix[4]arene scaffolds. Accordingly, the drastic reduction of the molecular length expected during the contraction process can be limited by such transitions, which involve the rotational isomerism of a phenolate ring. However, such type of conformational transitions does not compromise the actuator power due to its intrinsic capacity to adopt compact molecular arrangements. On the other hand, the rate of the contraction process is influenced by the presence of solvent molecules, which have been found to reduce it by a factor of about 1000.     

Solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with tetramethylrhodamine B isothiocyanate

Solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) based on an antibody labeled with tetramethylrhodamine B isothiocyanate (TRITC) was described. The SS-RTP properties of rabbit anti-goat antibody labeled with TRITC (RAGAb-TRITC) and its immune complex with goat-immunoglobulin G (G-IgG) were studied on a polyamide membrane (PM). The results showed that RAGAb-TRITC can react specifically with G-IgG on a PM, and retain the excellent SS-RTP property of TRITC, λ_ex^max/λ_em^max=558 nm/700 nm. The dependence of the phosphorescence intensity on the amount of antigen G-IgG (in a sample volume of 0.4 μl) was linear. Compared with enzyme-linked immunosorbent assay (ELISA), this assay showed a lower detection limit (0.37 pg per spot), a better linear relationship and a wider dynamic range (0.62–30 pg). The method was applied directly to determination of G-IgG in goat serum and the results agreed well with those obtained by ELISA. Therefore, this study shows the high sensitivity in SS-RTP and specificity of the immunological reaction. Moreover, it is a simple but powerful procedure.     

Detection of thrombin using an excimer aptamer switch labeled with dual pyrene molecules

We constructed an excimer aptamer probe containing one pyrene molecule at each end of a DNA aptamer to achieve the detection of thrombin, which binds to the heparin-binding site of thrombin with high binding affinity. The specific binding of thrombin to the excimer aptamer probe brought the two pyrene molecules at the termini of the duplex of the aptamer into close proximity, generating an excimer. The excimer emitted a distinct fluorescence peak, and fluorometric measurement of excimer allowed the sensitive detection of thrombin. The effects of experimental conditions like pH, ionic strength, and cations were investigated and optimized. The detection limit for thrombin was about 42 pM. This aptamer switch has potential in the study of molecular interactions and protein sensing with other switch-based detection strategy.

A novel polynuclear donor complex based on helical peptides with aligned electroactive moieties

Two newly synthesised 21-amino acid peptides substituted with ferrocene groups preserve the helical structure of the peptide and behave as multi-centre donor systems with six electroactive reversible redox centres per molecule. The formal potential of the hexaferrocene compound is slightly less positive than that of its single centre analogue.     

Electrochemical immunosensor for Forest-Spring encephalitis based on protein A labeled with colloidal gold

An electrochemical immunosensor for diagnosis of Forest-Spring encephalitis has been proposed. It comprises a screen-printed thick-film graphite electrode serving as the transducer and a layer of the Forest-Spring encephalitis antigen immobilized on the electrode and functioning as the biorecognition substance. The procedure includes formation of an antigen-antibody immune complex, localization of colloidal gold-labeled protein A on the complex, and recording of gold oxidation voltammogram, which provides information about the presence and the concentration of antibodies in blood serum. The response is proportional to the concentration of antibodies over the interval from 10(-7) to 10(-2) mg mL(-1). The detection limit is 10(-7) mg mL(-1).     

Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, Remazol brilliant blue R, as a substrate.

Micrococcus luteus (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.