An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

Successively amplified electrochemical immunoassay based on biocatalytic deposition of silver nanoparticles and silver enhancement

A successively signal-amplified electrochemical immunoassay has been reported on the basis of the biocatalytic deposition of silver nanoparticles with their subsequent enlargement by nanoparticle-promoted catalytic precipitation of silver from the silver-enhancer solution. The immunoassay was carried out based on a heterogeneous sandwich procedure using polystyrene microwells to immobilize antibody. After all the processes comprising the formation of immunocomplex, biocatalytic deposition of silver nanoparticles and following silver enhancement were completed, the silver on polystyrene microwells was dissolved and quantified by anodic stripping voltammetry (ASV). The effect of relevant experimental conditions, including the concentration of ascorbic acid 2-phosphate (AA-p) substrate and Ag(I) ions, the biocatalytic deposition time, and of crucial importance, the silver enhancement time, were investigated and optimized. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1-10 ng ml(-1) with a detection limit of 0.03 ng ml(-1). Scanning electron microscope (SEM) was applied to characterize the silver nanoparticles before and after silver enhancement on the surface of polystyrene microplates. By coupling the highly catalytic effect of enzyme and nanoparticles to successively amplify the analytical signal, the sensitivity of immunoassay was enhanced so dramatically that this approach would be a promising strategy to achieve a lower detection limit for bioassays.     

Immunoassay Comparison of Haplosporidan Spores from Teredo navalis and Haplosporidium nelsoni Spores from Crassostrea virginica

Spores of a haplosporidan infecting Teredo navalis Linnaeus have been described as morphologically indistinguishable from spores of Haplosporidium nelsoni . To test the hypothesis that these organisms are conspecific, a colloidal gold immunoassay was used to compare antigenic characteristics of the spores from both hosts. Rabbit antibody to formalin-fixed spores from T. navalis was tested against paraffin sections of Crassostrea virginica infected with spores of H. nelsoni and against paraffin sections of infected 7". navalis . Application of primary antibody was followed by addition of affinity purified goat anti-rabbit IgG coaled on 5-nm colloidal gold particles. The reaction was enhanced by precipitation of metallic silver; a positive reaction appeared as a dark brown to black signal at the site of each antigen-antibody complex. Haplosporidium nelsoni spores did not react when assayed with the antibody made to spores from T. navalis . Spores from infected T. navalis tissue reacted positively with rabbit antibody. This result indicates that the spores from the 2 hosts are antigenicaliy distinct and suggests that they are different species.     

Ag/SiO2 core-shell nanoparticle-based surface-enhanced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools

A simple, sensitive and highly specific immunoassay has been developed based on surface-enhanced Raman scattering for human alpha-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma. This strategy combines the Ag/SiO2 core-shell nanoparticles embedded with rhodamine B isothiocyanate dye molecules as Raman tags and the amino group modified silica-coated magnetic nanoparticle as immobilization matrix and separation tool. In the proposed system, a sandwich-type immunoassay was performed between polyclonal antibody functionalized Ag/SiO2 nanoparticle-based Raman tags and monoclonal antibody modified silica-coated magnetic nanoparticles. The presence of the analyte and the reaction between the antigen and antibody can be monitored by the Raman spectra of the Ag/SiO2 tags. Compared to the previous surface-enhanced Raman immunoassays, the main advantage of this strategy lies in two aspects. One is the high stability of Raman tags derived from the silica shell-coated silver core-shell nanostructure. The other is the use of silica-coated magnetic nanoparticles as immobilization matrix and separation tool, thus avoiding complicated pretreatment and washing steps. We have studied in detail the experimental parameters such as the effects of the antibody concentration modified on the Raman tags and on the magnetic particles, and the immunoreaction time. Using this strategy, concentration of human AFP up to 0.12 microg/ml was detected with a detection limit of 11.5 pg/ml.     

One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles

The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H₂O₂ chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H₂O₂ CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10⁻¹¹ g ml⁻¹. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive.     

Using an electro-microchip, a nanogold probe, and silver enhancement in an immunoassay

This paper presents a novel immunoassay that uses an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs were introduced into the electro-microchip by the specific binding of the antibodies-ANPs conjugates and then were coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a "bridge" between two electrodes of the electro-microchip allowing electrons to pass. The variation of impedance can be easily measured with a commercial LCR meter. Various gap sizes (20, 50, 100, and 200 microm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 20microm gap has the highest sensitivity. There was a significant difference in impedance between the experiment sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay (ELISA). In addition, it shows a high detection sensitivity (10 microg/mL of 1st antibody (IgG) immobilized on slides and 1 ng/mL of antigen (protein A)). There is a clear distinction between the signal intensity and the logarithm of the sample concentration. The proposed new immunoassay method has potential applications in proteomics research and clinical diagnosis.     

In situ enzymatic silver enhancement based on functionalized graphene oxide and layer-by-layer assembled gold nanoparticles for ultrasensitive detection of thrombin

A highly specific in situ amplification strategy was designed for ultrasensitive detection of thrombin by combining the layer-by-layer (LBL) assembled amplification with alkaline phosphatase (ALP) and gold nanoparticles (Au) mediated silver deposition. High-density carboxyl functionalized graphene oxide (FGO) was introduced as a nanocarrier for LBL assembling of alkaline phosphatase decorated gold nanoparticles (ALP-Au), which was further adopted to label thrombin aptamer II. After sandwich-type reaction, numerous ALP were captured onto the aptasensor surface and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP), which in situ generated ascorbic acid (AA), reducing Ag(+) to Ag nanoparticles (AgNPs) for electrochemical readout. Inspiringly, the in situ amplification strategy with ethanolamine as an effective blocking agent showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal, which was favorable to enhance the sensitivity of aptasensor. Our novel dramatic signal amplification strategy, with a detection limit of 2.7fM, showed about 2-3 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal enzyme-based electrochemical assay.     

A novel bifunctional europium chelate applied in quantitative determination of human immunoglobin G using time-resolved fluoroimmunoassay

The authors demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) protocol for quantification of human IgG with the new bifunctional chelate Eu(TTA)₃(5-NH₂-phen) (ETNP) labeling the goat anti-human IgG. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreactions. Goat anti-human IgG was immobilized on aldehyde-modified glass slides. The human IgG analyte was first captured by the primary antibody and then sandwiched by a secondary antibody labeled with the chelate ETNP. The experimental procedure was simple to follow and gave desirable levels of sensitivity and low limits of detection. To the best of our knowledge, this is the first application of the new chelate, ETNP, in an immunoassay. In comparison to typical organic, fluorescent compounds and other lanthanide fluorescent chelates used in immunoassay, the detection sensitivity of our method using ETNP chelate in the solid phase was greatly improved and a concentration of human IgG about 5 μg/L could be detected under optimal conditions. The main result of this work shows that the new chelate ETNP can be applied as a powerful fluorescent labeling material for constructing ultrasensitive TRFIAs. The detection of human IgG, using ETNP as the chelate, is a model example of the effectiveness of this immunoassay. Many other types of antigen–antibody immunoassays should be possible using the protocol described herein.     

Signal enhancement of surface plasmon-coupled emission (SPCE) with the evanescent field of surface plasmons on a bimetallic paraboloid biochip

We present a novel approach to the enhancement of surface plasmon-coupled emission (SPCE) using surface plasmon excitation in a bimetal (Ag/Au) layer and we validate the enhancement by presenting the results of a model human IgG immunoassay. Theoretical calculations using Fresnel's equations have been carried out to determine the optimum bimetallic composition and the resulting electric field enhancement. Signal enhancement of SPCE was confirmed using a range of bimetallic layers which were deposited on the surface of a high collection efficiency polymer array biochip and subsequently immobilized with Alexa Fluor 647 labeled anti-human IgG. The bimetallic film of Ag/Au (36/10nm) was determined as an optimum substrate for maximum SPCE signal which was a compromise between the long-term stability of the metal layer and the optimized evanescent field enhancement. An enhanced dose-dependent response was also demonstrated which was ～3 times greater than that detected with a pure gold layer. A human IgG immunoassay showed a dose-dependent response yielding a limit of detection of 1pg/ml by the 3σ rule. The improved performance of the bimetal layer compared to that of an assay carried out on a pure gold layer is attributed to the enhanced evanescent field intensity of surface plasmons in the bimetal combination which excites more fluorescence hence producing an enhanced SPCE signal. This result demonstrates the potential of the SPCE-based array biochips as a sensitive and high-throughput analysis platform for biomolecular interactions.     

Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels

A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on horseradish peroxidase-doped nanosilica particles (HRP-SiO(2)) with the conjugation of anti-IgG antibodies (anti-IgG-SiO(2)-HRP). With the sandwich-type immunoassay format, the linear range of the developed immunosensor by using anti-IgG-SiO(2)-HRP as tracer and hydrogen peroxide (H(2)O(2)) as enzyme substrate is 0.01-15 nmol/L IgG with a detection limit of 5.0 pmol/L, while the assay sensitivity by directly using HRP-labeled anti-IgG as secondary antibodies is 1.0-10 nmol/L with a detection limit of 0.1 nmol/L IgG. The reproducibility, stability and specificity of the proposed immunoassay method were acceptable. The IgG concentrations of the clinical serum specimens assayed by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) method.     

A chemiluminescent metalloimmunoassay based on copper‐enhanced gold nanoparticles for quantification of human growth hormone

A chemiluminescence (CL) immunoassay was developed to determine human growth hormone (hGH) based on copper‐enhanced gold nanoparticles. In this method, gold nanoparticles were deposited on polystyrene wells for adsorption of human growth antibodies as well as catalyst for reducing of copper ions from the copper enhancer solution. The reduction of copper ions was prevented where the gold nanoparticles were covered by the antibody–antigen immunocomplex. The deposited copper on Au nanoparticles was then dissolved in HNO₃ solution and quantified using the CL method. The CL intensity response was logarithmically dependent on the hGH concentrations over the range 0.2–50 ng/mL, with a detection limit (3σ) of 0.036 ng/mL. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.     

Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

A simple and sensitive flow injection chemiluminescence immunoassay for chloramphenicol based on gold nanoparticle‐loaded enzyme

A simple and ultrasensitive flow injection chemiluminescence competitive immunoassay based on gold nanoparticle‐loaded enzyme for the detection of chloramphenicol (CAP) residues in shrimp and honey has been developed. Due to their good biocompatibility and large specific surface area, carboxylic resin beads can be used as solid phase carriers to immobilize more coating antigens (Ag). In addition, gold nanoparticles could provide an effective matrix for loading more CAP antibody and horseradish peroxidase, which would effectively catalyze the system of luminol–p‐iodophenol (PIP)–H₂O₂. A competitive immunoassay strategy was used for detection of CAP, in which CAP in the sample would compete with the coating Ag for the limited antibodies, leading to a chemiluminescence (CL) signal decrease with increase in CAP concentration. A wide linear range 0.001–10 ng ml^?1 (R² = 0.9961) was obtained under optimized conditions, and the detection limit (3σ) was calculated to be 0.33 pg ml^?1. This method was also been successfully applied to determine CAP in shrimp and honey samples. The immunosensor proposed in this study not only has the advantages of high sensitivity, wider linear range, and satisfactory stability, but also expands the application of flow injection CL immunoassay in antibiotic detection.     

A chemiluminescent immunosensor based on antibody immobilized carboxylic resin beads coupled with micro-bubble accelerated immunoreaction for fast flow-injection immunoassay

A novel immunoaffinity column used as an immunosensor for flow-injection chemiluminescent (CL) immunoassay was prepared by immobilizing antibody on carboxylic resin beads. The immunosensor could fast recognize and trap the immunocomplex of horseradish peroxidase (HRP)-labeled antibody and antigen, which was firstly formed with a micro-bubble accelerated pre-incubation process, to produce a sandwich immunocomplex. The HRP introduced in the immunoaffinity column could catalyze the CL reaction to produce enzyme-enhanced emission. With alpha-fetoprotein (AFP) as a mode, a flow-injection CL immunoassay was proposed. The whole assay for one sample, including the pre-incubation and the regeneration of immunoaffinity column, could be performed within 16min. The linear range was 1.0-80ng/ml with a correlation coefficient of 0.998 and a detection limit of 0.1ng/ml at a signal/noise ratio of 3. The intra- and inter-assay coefficients of variation at 20ng/ml AFP were 1.2% and 8.5%, respectively. The storage stability of the immunoaffinity column and the accuracy for sample detection were acceptable. This flexible, sensitive, low-cost, and rapid method is valuable for clinical immunoassay.     

Label-free immunosensor based on gold nanoparticle silver enhancement

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.     

Retrograde tracing of neural pathways with a protein-gold complex

Summary In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

Retrograde tracing of neural pathways with a protein-gold complex. I. Light microscopic detection after silver intensification

In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.