Development of an immunosensor for the determination of rabbit IgG using streptavidin modified screen-printed carbon electrodes

Voltammetric enzyme immunosensors based on the employment of streptavidin modified screen-printed carbon electrodes (SPCEs) for the detection of rabbit IgG, as a model analyte, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate (3-IP) were used as the enzymatic label and substrate, respectively. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 °C on the pre-oxidized surface of the SPCEs. The analytical characteristics of these sensors were evaluated using biotin conjugated to AP.The immunosensor devices were based on a specific reaction of rabbit IgG with its biotinylated antibodies, which were immobilised on the modified screen-printed carbon electrodes through the streptavidin:biotin reaction. The immunosensors were used for a direct determination of AP labelled rabbit IgG, and for free rabbit IgG detection using a sequential competitive immunoassay. A calibration curve in the range of 5 × 10⁻¹¹ to 1 × 10⁻⁹ M of rabbit IgG was obtained with a estimated detection limit of 5 × 10⁻¹¹ M (7.0 ng/ml). These immunosensors were stable for 5 months if they were stored at 4 °C.     

Biocatalytic precipitation induced by an affinity reaction on dendrimer-activated surfaces for the electrochemical signaling from immunosensors

We have developed a strategy of signal generation for immunosensors that transduces biospecific affinity recognition reactions into electrochemical signals. The cyclic voltammetric method, tracking the precipitation of insoluble products onto the sensing surface and the subsequent decrement in the electrode area, was chosen for signal registration. Precipitation of insolubilities was induced by the catalytic reaction of enzymes, which were labeled to the biospecifically attached protein or antibody molecules. As a model system for affinity recognition, we have investigated the functionalization of biotin groups to the sensing monolayer and their biospecific interactions with anti-biotin antibody molecules. The immunosensing interface was developed onto the dendrimer-activated self-assembled monolayers (SAMs), as the base template for the functionalization of the antigen moiety and signal generation. The advantages of using dendrimer-activated SAMs in comparison to the plain modified thiolate SAMs for the sensing surface were shown in terms of sensing performances, and the analytical characteristics of the resulting immunosensor were examined. Additionally, the sensing system was applied for biotin/(strept)avidin couples, extending the applicability of the developed strategy.     

Towards the fabrication of label-free amperometric immunosensors using SWNTs

A label-free immunosensor based on the modulation of the electrochemistry of a surface bound redox species, to detect the presence of antibodies, is demonstrated. In this proof of concept study the model epitope was biotin and the model antibody was anti-biotin IgG. Glassy carbon (GC) electrode surfaces were first modified with 4-nitrophenyl groups by electrochemical reductive adsorption of the corresponding aryl diazonium salt. Subsequently, the nitro group was reductively converted into an amine, giving 4-aminophenyl groups. Oxidatively shortened single walled carbon nanotubes (SWNTs) were then covalently attached to the electrode via self-assembly; a procedure that has previously been shown to give SWNTs aligned normal to the surface. 1,1-Di(aminomethyl)ferrocene was attached to the carboxylic acid terminated SWNTs followed by attachment of biotin to the remaining free amine of the ferrocene derivative. Binding of anti-biotin IgG to the surface bound epitope resulted in attenuation of the ferrocene electrochemistry. This label-free immunosensor was successfully able to detect anti-biotin between 30 and 450 ng mL⁻¹.     

Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B

An immunosensor based on surface plasmon resonance (SPR) with a mixed self-assembled monolayer (SAM) was developed to determine staphylococcal enterotoxin B (SEB). The SAM on a gold surface was fabricated by adsorbing a mixture of 16-mercapto-1-hexadecanoic acid (16-MHA) and hexanethiol at various molar ratios. Initially, full-length anti-SEB was randomly immobilized onto the SAM to form the immunosensing surface. Through optimization of surface functionalization and anti-SEB immobilization, the SPR sensors can be applied to the determination of SEB in a linear range of 0.01?~?1.0 μg.mL^?1. Furthermore, a smaller antibody fragment (F(ab)’) was generated and immobilized randomly (via amino groups) or in an oriented manner (via ?SH groups) to form the immunosensing surface. The oriented immobilization of F(ab)’ led to a 50% increase in the antigen binding efficiency compared to randomly immobilized covalent F(ab’) fragments. The resulting calibration curve showed higher sensitivity. In addition, the specificity and applicability of the proposed immunosensor to milk samples were also demonstrated. Furthermore, the sensor can be regenerated using 0.1 M HCl, and 70% of the initial response was maintained over 3 cycles.     

Composite Assembly of Silver Nanoparticles with Avidin and Biotinylated AChE on Gold for the Pesticidal Electrochemical Sensing

An amperometric pesticide biosensor has been devised by the composite assembly of silver nanoparticles with avidin and biotinylated acetylcholinesterase (AChE) on gold electrodes modified with a biotin‐terminated self assembly monolayer (SAM). This composite assembly strategy takes use of the biospecific recognition avidin with the biotin from the SAM‐terminals and biotinylated AChE, as well as the electrostatic interaction between silver nanoparticles with negatively charged citrate shell and avidin with encounter charge at pH 7.2. The construction process of the composite interface on gold was monitored by surface plasmon resonance (SPR), and its structure was characterized by attenuated total reflection Fourier‐transform infrared spectra, atomic force microscopy and UV‐vis spectra. The composite interface shows excellent electron transfer ability, as characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimum conditions a quantitative measurement of organophosphate pesticide dimethoate was achieved with the linear range of 0.05 μM to10 μM and the detection limit 0.01 μM, taken as the concentration equivalent to a 10% decrease in signal. Silver nanoparticles conjugated biotin‐avidin system represents a simple and functional approach to the integration of electrode sensing interface with improved biocompatibility and electron transfer ability, which may provide an analytical access to a large group of enzymes for bioelectrochemical application.     

Quantitative determinations of SiC and SiO2 in new ceramic materials by Fourier transform infrared spectroscopy

In this paper, we have critically evaluated the electrochemical behavior of the products of seven substrates of the enzyme label, alkaline phosphate, commonly used in electrochemical immunosensors. These products (and the corresponding substrates) include indigo carmine (3-indoyl phosphate), hydroquinone (hydroquinone diphosphate), 4-nitrophenol (4-nitrophenol phosphate), 4-aminophenol (p-aminophenyl phosphate), 1-naphthol (1-naphthyl phosphate), phenol (phenyl phosphate), and L-ascorbic acid (2-phospho-L-ascorbic acid). Cyclic voltammetry and amperometry of these products were carried out at glassy carbon (GC), screen-printed carbon (SPC) and gold (Au) electrodes, respectively. Among the products, L-ascorbic acid showed the most sensitive (24.8 microA cm(-2), 12.0 microA cm(-2), and 48.0 microA cm(-2) of 100 microM ascorbic acid at GC, SPC, and Au electrodes, respectively) and well-defined amperometric response at all electrodes used, making 2-phospho-l-ascorbic acid the best substrate in electrochemical detection involving an alkaline phosphatase (ALP) enzyme label. The 2-phospho-L-ascorbic acid is also commercially available and inexpensive. Therefore, it was the best choice for electrochemical detection using ALP as label. Using mouse IgG as a model, an ALP enzyme-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was designed by electropolymerization of o-aminobenzoic acid (o-ABA) conductive polymer on the surface of GC, SPC, and Au electrodes. The anti-mouse IgG was subsequently attached on the electrode surface through covalent bonding between IgG antibody and the carboxyl groups from poly(o-ABA). Using 2-phospho-L-ascorbic acid as a substrate, the poly(o-ABA)/Au immunosensor produced the best signal (about 297 times of current density response ratio between 1000 ng mL(-1) and 0 ng mL(-1) of mouse IgG), demonstrating that amperometric immunosensors based on a conducting polymer electrode system were sensitive to concentrations of the mouse IgG down to 1 ng mL(-1), with a linear range of 3-200 ng mL(-1) (S.D.<2; n=3), and very low non-specific adsorption.     

Photoelectrochemical immunosensor for label-free detection and quantification of anti-cholera toxin antibody

We demonstrate herein a newly developed photoelectrochemical immunosensor for the determination of anti-cholera toxin antibody by using a photosensitive biotinylated polypyrrole film. The latter was generated by electro-oxidation of a biotinylated tris(bipyridyl) ruthenium(II) complex bearing pyrrole groups. The photoexcitation of this modified electrode potentiostated at 0.5 V vs SCE, in the presence of an oxidative quencher, pentaaminechloro cobalt(III) chloride (15 mM), led to a cathodic photocurrent. As a result of the affinity interactions, a layer of biotinylated cholera toxin was firmly bound to the functionalized polypyrrole film via avidin bridges. The resulting modified electrodes were tested as immunosensors for the detection of the corresponding antibody from 0 to 200 microg mL(-)(1). The antibody concentration was measured through the decrease in photocurrent intensity resulting from its specific binding onto the polymeric coating, the detection limit being 0.5 microg mL(-)(1).     

An electrochemical immunosensor using ferrocenyl-tethered dendrimer

We report here an enzyme-amplified, sandwich-type immunosensor for detecting the biospecific interaction between an antibody and antigen using redox mediation. We employed biotin/anti-biotin IgG as a model immunosensing pair. Partially ferrocenyl-tethered dendrimer (Fc-D), whose ferrocene moiety acts as a redox mediator, was immobilized to the electrode surface by covalent binding between the dendrimer amines and the carboxylic acids of a self-assembled monolayer. The unreacted amines of the immobilized Fc-D were modified with biotin groups to allow the specific binding of goat anti-biotin IgG. Rabbit anti-goat IgG-conjugated alkaline phosphatase was bound to goat anti-biotin IgG to catalyze conversion of p-aminophenyl phosphate monohydrate to p-aminophenol. This product is oxidized to quinoimide by the reduction of ferrocenium back to ferrocene, producing an electrocatalytic anodic current. Cyclic voltammograms and surface plasmon resonance experiments showed that the binding of nonspecific proteins is not significant on the biotinylated Fc-D surface. We also examined the change in peak current according to the concentration of anti-biotin IgG and found that the detection range of this immunosensing scheme is between 0.1 and 30 microg mL(-1).     

基于金属离子螯合的压电免疫传感器新型固定化方法

提出了一种基于金属离子螯合作用的压电免疫传感器新型固定化方法. 先在压电石英晶振表面沉积正丁胺等离子体聚合膜(BA-PPF), 再在BA-PPF表面修饰可与金属离子螯合的氨三乙酸基团, 用金属铜离子活化后, 修饰了二乙三胺五乙酸基团的IgG抗体蛋白质分子即可螯合固定于BA-PPF上. 将固定了抗体的压电石英传感器用于正常人免疫球蛋白IgG (NHIgG)的测定, 其频率响应与NHIgG浓度在0.36～63.8 μg/mL范围内存在良好的线性关系. 这种新型压电免疫传感器固定化方法简单快速, 具有良好的通用性.     

FT-IRRAS spectroscopic studies of the interaction of avidin with biotinylated dendrimer surfaces

The interaction of avidin with biotin on a functional Au surface containing fourth generation amine-terminated polyamidoamine (G4-NH₂ PAMAM) dendrimers was investigated through the use of Fourier transform infrared reflection–adsorption spectroscopy (FT-IRRAS). The first step in the fabrication of the functional surfaces used was the construction of an aldehyde-terminated self-assembled monolayer (SAM) through the treatment of Au-coated glass slides with ethanol solutions of self-synthesized 2-hydroxypentamethylene sulfide (HPMS). The as-formed aldehyde-terminated monolayer was subsequently immersed in methanol solutions of G4-NH₂ PAMAM dendrimer to obtain well-organized primary amine-terminated surfaces. Biotinylation of the amine-terminated layers thus obtained was accomplished by use of the N-succinimidyl ester of biotin. Each step of the synthetic process, as well as the performance of final surface for protein recognition was monitored by FT-IRRAS. In particular, the molecular recognition ability was examined and quantified by use of an alkyne dicobalt hexacarbonyl probe coupled with avidin. Non-specific adsorption of avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to solutions of biotin-saturated avidin. The results indicate that the biotinylated G4-NH₂ PAMAM dendrimer layers formed according to this procedure have a high capacity for binding avidin with relatively high specificity. The performance of these layers (i.e. both binding capacity and specificity) improve substantially when 6-mercapto-1-hexanol (MH) is present as a co-adsorbent during the formation of the initial aldehyde-terminated layers. This effect can be attributed to the dilution of the initial aldehyde-terminated SAM, leading to a more favorable spatial arrangement of the subsequent biotinylated surfaces.     

Porous silicon as functionalized material for immunosensor application

Recently, for sensor application, porous silicon has received a great deal of attention due to the high specific surface area and the easy fabrication using some established processes of the usual silicon technology. We herein, report the development of a novel immunosensors based on porous silicon for antigen detection. The multilayer immunosensor structure was fabricated following the successive steps: APTS self-assembled monolayer (SAM) layer, glutaaldehyde linker, anti-rabbit IgG binding. The insulating properties of the aminopropyl-triethoxysilane (APTS) monolayer were studied with cyclic voltammetry and the molecular structure was characterized with Fourier-transform infrared (FTIR) technique. The binding between antibody and different antigen concentration (rabbit IgG) was monitored by measuring the capacitance-voltage curve of the antibody functionalized EIS structure. A detection limit of 10 ng/ml of antigen can be detected.     

Stimuli-sensitive thin films prepared by a layer-by-layer deposition of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

Layered thin films composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) were prepared by a layer-by-layer deposition of avidin and ib-PEI on a solid surface, and the disintegration induced by changing environmental pH and adding biotin in the solution was studied. The avidin/ib-PEI layered film could be deposited only from the solutions of pH 10-12. The film did not form in pH 9 or more acidic media because of a low affinity of protonated 2-iminobiotin residues in ib-PEI to avidin. The avidin/ib-PEI layered films were stable in pH 8-12 solutions, while in pH 5-7 media the film decomposed spontaneously as a result of the protonation to 2-iminobiotin residues in ib-PEI. The avidin/ib-PEI films were disintegrated also upon addition of biotin and analogues in the solution owing to the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI films were disintegrated rapidly by addition of 10(-)(5) M of biotin or desthiobiotin, while the rate was slower upon adding the same concentration of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. On the other hand, the film was fully decomposed within 1 min in the 10(-)(3) M lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid solution. Thus, the decomposition rate is highly dependent on the concentration of the stimulants. It was observed that the stimuli-induced decomposition of the films is slow at pH 12, in contrast to a rapid decomposition in pH 8 medium due to a low affinity of the protonated 2-iminobiotin to avidin. The present system may be useful for constructing stimuli-sensitive devices that can release drug or other functional molecules.     

金表面自组装化学发光免疫传感器

用Ｎ－乙酰半胱氨酸金表面自组装技术及ＥＤＣ、ＮＨＳ偶联剂将兔ＩｇＧ固定于金表面制成免疫传感器探头，用碱性磷酸酯酶标记山羊抗兔ＩｇＧ加入待测的含有山羊抗兔ＩｇＧ的样品中，采用竞争头测定山羊抗兔ＩｇＧ含量，标记的碱性磷酸酯酶可催化底物ＡＭＰＰＤ产生化学发光，其发光强度与样品浓度成反比。测得山羊抗兔ＩｇＧ的最低检出限为０．８ｍｇ／Ｌ，线性测定范围为０．８－４０ｍｇ／Ｌ。     

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.     

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.     

Studies on biotin–avidin indirect conjugated technology for a piezoelectric DNA sensor

Because of their high sensitivity, piezoelectric sensor techniques are extremely useful for environmental or clinical analysis. We developed a piezoelectric crystal DNA biosensor for the detection of the hybridization reaction based on the self-assembled monolayer technology and biotin–avidin system. 3,3′-Dithiopropionic acid was applied to form a self-assembled monolayer (SAM) on the gold surface of the quartz crystal. Avidin was coated on the gold electrode conjugated with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and then biotinylated nucleotide acids were immobilized on the gold electrode surface through the specific interaction of biotin and avidin. Our results indicated that, using this immobilization method, the piezoelectric DNA sensor shows a higher sensitivity and specificity in detecting the hybridization reaction. The sensor can be used repeatedly by electrode regeneration.     

An Electrochemical Impedance Immunosensor Based on Gold Nanoparticle‐Modified Electrodes for the Detection of HbA1c in Human Blood

A label‐free immunosensor for the detection of HbA1c was developed based on gold nanoparticle (AuNP)‐aryl diazonium salt modified glassy carbon (GC) electrode where transduction is achieved using electrochemical impedance spectroscopy (EIS). GC electrodes were first modified with 4‐aminophenyl (Ph‐NH₂) layers to which AuNPs were attached. Thereafter an oligo(ethylene glycol) (OEG‐COOH) species were covalently attached to the remaining free amine groups on the Ph‐NH₂ surface. The AuNP surfaces were further modified with Ph‐NH₂ followed by attachment of a glycosylated pentapeptide (GPP), an analogon to HbA1c. Exposure of this interface to anti‐HbA1c IgG resulted in a change in charge transfer resistance (R_ct) due to the anti‐HbA1c IgG selectively complexing to the surface bound GPP. To detect the amount of HbA1c, a competitive inhibition assay was employed where the surface bound GPP and HbA1c in solution compete for the anti‐HbA1c IgG antibodies. The higher the concentration of HbA1c, the less antibody binds to the sensing interface and the lower the change of R_ct. The response of the immunosensor is linear with the HbA1c% of total haemoglobin in the range of 0%–23.3%. This competitive inhibition assay can be used for the detection of HbA1c in human blood. The performance of the immunosensor for detection of HbA1c in human blood is comparable to the clinical laboratory method.     

Bioconjugation of Ln3+-doped LaF3 nanoparticles to avidin

The binding of Eu3+-doped LaF3 nanoparticles with biotin moieties at the surface of the stabilizing ligand layer to avidin, immobilized on cross-linked aragose beads, is described. The biotin moieties were attached to the nanoparticles by reaction of an activated ester with the amino groups on the surface of the nanoparticles resulting from the 2-aminoethyl phosphate ligands that were coordinated to the surface through the phosphate end. This strategy of employing the reactions of amines with activated esters provides a general platform to modify the surface of the 2-aminophosphate stabilized Ln3+-doped LaF3 nanoparticles with biologically relevant groups. Significant suppression of nonspecific binding to the avidin modified aragose beads has been realized by the incorporation of poly(ethylene glycol) units via the same reaction of a primary amine with an activated ester. The particle size distribution of the functionalized nanoparticles was within 10-50 nm, with a quantum yield of 19% in H2O for the LaF3 nanoparticles codoped with Ce3+ and Tb3+. A discreet, 4 unit poly(ethylene glycol) spaced heterobifunctional cross-linker, functionalized with biotin and N-hydroxysuccinimide at opposite termini, was covalently linked to the 2-aminoethyl phosphate ligand via the N-hydroxysuccinimide activated ester, making an amide bond, imparting biological activity to the particle. Modification of the remaining unreacted amino groups of the stabilizing ligands was done with Me(OCH2CH2)3CH2CH2(C=O)-NHS (NHS = N-hydroxysuccinimide).     

基于生物素-亲和素系统的压电免疫传感器检测相思子毒素研究

利用生物素-亲和素系统的放大作用和纳米金质量扩增效应,建立了压电免疫传感器检测相思子毒素的新方法.首先在石英晶体的金电极上依次组装二巯基丙酸、EDC和NHS进行表面修饰,然后通过亲和素固定生物素标记相思子毒素多抗来制备敏感膜,利用纳米金的质量扩增效应设计了一种"毒素-纳米金标记单抗"复合物,成功实现了对相思子毒素的检测,提高了传感器灵敏度和重现性.本传感器对相思子毒素响应的线性范围为0.05～5 mg/L; 回归方程为Δf＝50.81CAbrin+67.11(r=0.9903,n＝10,P<0.0001); 检测灵敏度为50.81 Hz · L/mg.     

Electrochemical evaluation of avidin-biotin interaction using N-iodoacetyl-N-biotinylhexylenediamine

The avidin-biotin assay was investigated by an electrochemical procedure based on the chemical reaction between cysteine containing a thiol group and N-iodoacetyl-N-biotinylhexylenediamine (IB). In the presence of avidin and biotin this reaction, whereby the thiol group combines with IB, is controlled because IB has a biotin part; that is, avidin and biotin are detected indirectly by measurement of iodide ion or cysteine. To achieve a high sensitivity of detection, Hg(II) was introduced as a marker that interacts with cysteine and the oxidation peak from Hg(0) to Hg(II) was measured. The sensitivity of detection of avidin was at the level of 10⁻⁹ M. The relative standard deviation at 1 × 10⁻⁸ M avidin was 4.8% (n = 5). On the other hand, a response curve to detect biotin was obtained by the competitive reaction between IB and biotin for the limited binding sites of avidin. The change in peak current enables the detection of biotin at the level of 10⁻⁹ M. This method has the advantage that it is not necessary to separate free IB from bound IB.