Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance

A direct human ferritin immunosensor was developed using anti-human ferritin monoclonal antibodies (MAbs) immobilized on the gold surface of a self-assembled surface plasmon resonance (SPR) apparatus. A kind of self-assembled monolayer (SAM) prepared by cystamine-glutaraldehyde method was applied to immobilize the MAbs. The reusability of the sensor chip adopting the SAM was found to be better than the other immobilization methods including adsorption, protein A, concanavalin A method. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1M HCl solution. A linear relationship existed between the angle shifts (millidegrees) and the log values of ferritin concentrations in the range from 0.2 to 200 ng/ml in buffer and human serum. When used for 15 days, the angle shifts were all >95% of those on the response at the first day. A 10 M NaOH solution was used for clearing nonspecific binding in human serum. Correlation coefficient was 0.991 between this SPR method and chemiluminescent immunoassay for determination of ferritin in clinical human serum samples. The SPR sensor offers advantages of simplicity of immobilization, high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.     

An amperometric immunosensor for osteoproteogerin based on gold nanoparticles deposited conducting polymer

An amperometric immunosensor was fabricated for the detection of osteoproteogerin (OPG) by covalently immobilizing a monoclonal OPG antibody (anti-OPG) onto the gold nanoparticles (AuNPs) deposited functionalized conducting polymer (5,2′:5′,2″-terthiophene-3′-carboxylic acid). AuNPs were electrochemically deposited onto the conducting polymer using cyclic voltammetry. The particle size of deposited AuNPs was controlled by varying the scan rate and was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The immobilization of anti-OPG was also confirmed using XPS. The principle of immunosensor was based on a competitive immunoassay between free-OPG and labeled-OPG for the active sites of anti-OPG. HRP was used as a label that electrochemically catalyzes the H₂O₂ reduction. The catalytic reduction was monitored amperometrically at −0.4 V vs. Ag/AgCl. The immunosensor showed a linear range between 2.5 and 25 pg/ml and the detection limit was determined to be 2 pg/ml. The proposed immunosensor was successfully applied for real human samples to detect OPG.     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

Amperometric immunosensor for nonylphenol determination based on peroxidase indicating reaction

Novel immunosensor for nonylphenol (NP) determination has been developed by immobilization of specific antibodies together with horseradish peroxidase on the surface of carbon screen-printed electrode. The signal of the immunosensor is generated by the involvement of NP accumulated in the peroxidase oxidation of mediator (Methylene Blue, hydroquinone or iodide). This results in the increase of the signal recorded by linear-sweep voltammetry. The sensitivity of the detection depends on the nature of mediator, its concentration and incubation period. Cross-selectivity of the response toward readily oxidized phenolic compounds has been determined. The immunosensor developed makes it possible to detect from 20 microgL(-1) to 44 mgL(-1) of NP with detection limit 10 microgL(-1) of NP.     

An amplified mass piezoelectric immunosensor for Schistosoma japonicum

An ultrasensitive piezoelectric immunosensor using an amplification path based on an insoluble biocatalyzed precipitation product has proposed for Schistosoma japonicum. A mercapto Schistosoma japonicum antigen was self-assembled onto the quartz crystal surface via an Au nanoparticle mediator monolayer to sense the Schistosoma japonicum antibody (SjAb). And the horseradish peroxidase labeled protein A conjugate which was bounded to the SjAb by a "sandwich" format was used as a biocatalyst for the oxidative precipitation of 4-chloro-1-naphthol by H(2)O(2) to yield the insoluble product benzo-4-chlorohexadienone, resulting in an amplified mass sensing of antigen-antibody interaction. The amount of the precipitate accumulated on the quartz crystal is controlled by the antibody concentration. The SjAb can be linearly determined in the range of 10-200 ngml(-1) and the detection limit reaches as low as 5 ngml(-1).     

Direct electrochemistry and electrocatalysis of heme-proteins entrapped in agarose hydrogel films

Three heme-proteins, including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), were immobilized on edge-plane pyrolytic graphite (EPG) electrodes by agarose hydrogel. The proteins entrapped in the agarose film undergo fast direct electron transfer reactions, corresponding to FeIII = e- --> FeII. The formal potential (E degrees'), the apparent coverage (Gamma), the electron transfer coefficient (alpha) and the apparent electron transfer rate constant (ks) were calculated by integrating cyclic voltammograms or performing nonlinear regression analysis of square wave voltammetric (SWV) experimental data. The E degrees's are linearly dependent on solution pH (redox Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet visible (UV-Vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the agarose film are little different from that proteins alone, and the conformation changes reversibly in the range of pH 3.0-10.0. Atomic force microscopy (AFM) images of the agarose film indicate a stable and crystal-like structure formed possibly due to the synergistic interaction of hydrogen bonding between N,N-dimethylformamide (DMF), agarose hydrogel and heme-proteins. This suggests a strong interaction between the heme-proteins and the agarose hydrogel. DMF plays an important role in immobilizing proteins and enhancing electron transfer between proteins and electrodes. The mechanisms for catalytic reduction of hydrogen peroxide and nitric oxide (NO) by proteins entrapped in agarose hydrogel were also explored.     

An ISFET-based immunosensor for the detection of beta-Bungarotoxin

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).     

Ultrasensitive electrochemical immunosensor for HE4 based on rolling circle amplification

A novel electrochemical immunoassay system for the detection of human epididymis-specific protein 4 (HE4) was developed. A chitosan-titanium carbide (TiC) nanocomposition film was first electrodeposited onto a tin-doped indium oxide (ITO) electrode at a constant potential. Gold (Au) nanoparticles were then electrodeposited on the surface of the chitosan-TiC film by cyclic voltammetry (CV). The capture antibody (anti-HE4) was adsorbed onto the Au and TiC nanoparticles. After a specific sandwich immunoreaction among the capture antibody, HE4, and biotinylated secondary antibody, biotinylated primer DNA was immobilized on the secondary antibody by biotin-streptavidin system. Appropriate amounts of circular template DNA and biotinylated primer DNA were used for rolling circle amplification (RCA) under optimal conditions. The RCA products provided a large number of sites to link DNA detection probes. Doxorubicin hydrochloride intercalated the CG-GC steps between the RCA products and the DNA detection probes, which was monitored by differential pulse voltammetry (DPV) based on the current signal of doxorubicin hydrochloride. With the above-mentioned amplification factors, the current responded to HE4 linearly in the concentration range of 3-300 pM under optimal detection conditions, with a detection limit of 0.06 pM. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical impedance spectroscopy (EIS). This system was successfully employed for the detection of HE4 with good accuracy and renewable ability.     

Development of an immunosensor based on pressure transduction

Traditional strategies for signal transduction in immunosensors are based on piezoelectric, thermometric, electrochemical, magnetic and optical methods. The use of pressure as a signal transduction method in immunosensors has not been reported previously. An immunosensor incorporating the detection of a change in pressure as the signal-transducing mechanism was investigated. A commercially available ultra-low pressure sensor was used in conjunction with a sealed chamber to assess the feasibility of this strategy. A key feature of the current approach is the use of a thin membrane (or film) in which to perform an immunoassay and subsequently to detect production of gas. The thinness contributes to efficient gas evolution and minimizes the effect of liquid acting as a "sink" for gas molecules. This feature also simplifies measurement of evolved gas, which traditionally was based on the use of bulk solutions, shaking and pH changes to "release" dissolved gas (especially carbon dioxide). Gas generation in the current approach is achieved by the coupling of catalase to haptens or antibodies for use in competitive or sandwich immunoassays, respectively. Hydrogen peroxide is used as the substrate. Performance characteristics of the sensor apparatus were assessed in several ways. Injection of various volumes of air from a gas-tight syringe produced an essentially linear relationship from 0.2 to 2.0 microl of injected volume, with a slope of approximately 5 V/microl. Depending on the duration of the sampling period, specific signals in excess of 2 V have been obtained for 0.01 units of catalase (approximately 0.4 ng of protein). Development and use of this sensing apparatus will be described for both competitive and sandwich immunoassays.     

An amperometric immunosensor based on an electrochemically pretreated carbon-paraffin electrode for complement III (C3) assay

An electrochemical immunosensor based on the adsorption of anti-complement III antibody onto an electrochemical pretreated carbon-paraffin electrode has been proposed for the detection of complement III (C(3)). The competitive immunoassay format was adopted with horseradish peroxide-C(3) (HRP-C(3)) as a tracer, 3,3'5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide as the enzyme substrates. In order to measure the amount of HRP-C(3) binding onto the electrode surface, the product of the enzyme catalytic reaction was detected at 100 mV (vs. Ag/AgCl reference electrode). The system was optimized to realize a reliable determination of C(3) in the range of 0.06-10 microg/ml. It exhibits some advantages, such as simplicity of fabrication, rapidity of measurement, and satisfactory sensitivity and reproducibility.     

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.     

A novel label-free amperometric immunosensor for carcinoembryonic antigen based on redox membrane

A label-free immunosensor was developed to detect the presence of an antigen. This immunosensor was based on the modulation of the electrochemistry of the surface bound redox species K(3)Fe(CN)(6) (FC). The model antigen was carcinoembryonic antigen (CEA) and the model epitope was the antibody of CEA (anti-CEA). Glassy carbon (GC) electrode surfaces were first drop-coated with a mixture of FC and chitosan and air-dried. The electrode surface was then covered with nafion membrane, which contained gold nanoparticles. After binding with polyethyleneimine (PEI), glutaraldehyde (GA) was used to cross-link PEI and anti-CEA. Binding of CEA to the surface bound epitope resulted in attenuation of the FC electrochemistry. Under optimal conditions, the response of the label-free immunosensor had a linear range of 0.01-150 ng mL(-1) with a detection limit of 3 pg mL(-1) (S/N = 3). Its response was better than those of radioimmunoassays, enzyme-linked immunosorbent assays, and chemiluminescence assays.     

Label-free immunosensor for prostate-specific antigen based on single-walled carbon nanotube array-modified microelectrodes

We have fabricated a label-free electrochemical immunosensor using microelectrode arrays modified with single-walled carbon nanotubes (SWNTs). Label-free detection of a cancer marker, total prostate-specific antigen (T-PSA), was carried out using differential pulse voltammetry (DPV). The current signals, derived from the oxidation of tyrosine (Tyr), and tryptophan (Trp) residues, increased with the interaction between T-PSA on T-PSA-mAb covalently immobilized on SWNTs. The selectivity of our biosensor was challenged using bovine serum albumin (BSA) as the target protein. The detection limit for T-PSA was determined as 0.25 ng/mL. Since the cut-off limit of T-PSA between prostate hyperplasia and cancer is 4 ng/mL, the performance of our label-free electrochemical immunosensor seems promising for further clinical applications.     

An ultrasensitive and stable potentiometric immunosensor

We describe a novel quantitative polypyrrole based potentiometric biosensor that provides broad-spectrum assay capability. The biosensor allows for capture of analytes of interest from complex real samples such as serum and whole blood, and subsequent measurement in a controlled matrix environment. The technology is rapid (<15 min), ultrasensitive (<50 fM) and reproducible (CV<5% at 0.1 ng/ml). In addition the system has shown a wide dynamic range (four to five orders of magnitude), and good stability, 37 degrees C for at least 4 months. This potentiometric biosensor detects enzyme labelled immuno-complexes formed at the surface of a polypyrrole coated, screenprinted gold electrode. Detection is mediated by a secondary reaction that produces charged products (a 'charge-step' procedure). A shift in potential is measured at the sensor surface, caused by local changes in redox state, pH and/or ionic strength. The magnitude of the difference in potential is related to the concentration of the formed receptor-target complex. The potentiometric sensing technology has been demonstrated in assays for hepatitis B surface antigen (HBsAg) (Mw>300 kDa), Troponin I (Mw approximately 23 kDa), Digoxin (Mw 780 Da) and tumour necrosis factor (hTNF-alpha) (Mw approximately 23 kDa). These model targets were chosen to represent analytes of a range of molecular weights, and because of their requirement for assays of high analytical sensitivity and precision. All these assays were performed using complex fluid samples and the presence of any non-specific binding has no significant effect on the final measurement. New assays can be transferred and optimised readily.     

Amplified surface plasmon resonance immunosensor for interferon-gamma based on a streptavidin-incorporated aptamer

Interferon-gamma (IFN-γ) is associated with susceptibility to tuberculosis, which is a major public health problem worldwide. Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, this assay is still labor-intensive and time-consuming. We therefore designed a DNA aptamer hairpin structure for the detection of IFN-γ with high sensitivity and selectivity. A streptavidin DNA aptamer was incorporated into the IFN-γ binding aptamer probe for the amplified detection of the target molecules. Initially, the probe remained in the inactive configuration. The addition of IFN-γ induced the rearrangement of the aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for the streptavidin molecular recognition. Under optimized conditions, the detection limit was determined to be 33 pM, with a dynamic range from 0.3 to 333 nM, both of which were superior to those of corresponding optical sensors. Because combined aptamers are composed of nucleic acids, this optical aptasensor provided the advantages of high sensitivity, simplicity, reusability, and no further labeling or sample pre-treatment.     

An impedimetric immunosensor based on interdigitated microelectrodes (IDmicroE) for the determination of atrazine residues in food samples

A novel impedimetric immunosensor for atrazine detection has been developed. The immunosensor is based on an array of interdigitated micro-electrodes (IDmicroE) and immunoreagents specifically developed to detect this pesticide. Immunochemical determination of atrazine is possible without the use of any label. An atrazine-haptenized protein was covalently immobilized on the surface of the interdigitated mu-electrodes area (interdigits space) previously activated with (3-glycidoxypropyl)trimethoxysilane. Before, the gold electrodes were blocked using N-acetylcysteamine to prevent non-specific adsorptions. All biofunctionalization steps were characterized by chemical affinity methods and impedance spectroscopy. Immunosensors measures are made by exposing the sensor to solutions containing a mixture of the analyte and the specific antibody. With this configuration, the immunosensor detects atrazine with a limit of detection of 0.04 microg L(-1) without the use of any label. The potential of the immunosensor to analyze pesticide residues in complex sample matrices, such as red wine, has been evaluated. The results shown that after solid-phase extraction atrazine can be determined in this type of sample with a limit of detection of 0.19 microg L(-1), far below the Maximum Residue Level (MRL) established by EC for residues of this herbicide in wine.     

An impedimetric immunosensor for the label-free detection of bisphenol A

Label-free detection of bisphenol A based on the impedance measurement was achieved with an impedimetric immunosensor. The immunosensor was fabricated by the covalent bond formation between a polyclonal antibody and a carboxylic acid group functionalized onto a nano-particle comprised conducting polymer. By using a commercial reagent 4,4-bis(4-hydroxyphenyl) valeric acid (BHPVA), which has an analogous structure of BPA, we have prepared the antigen through the conjugation of BHPVA with bovine serum albumin (BSA) and then produced a specific polyclonal antibody. The immobilization of antibody and the interaction between antibody and antigen were studied using quartz crystal microbalance (QCM) and electrochemical impedance spectroscopic (EIS) techniques. The impedance and mass changes due to the specific immuno-interaction at the sensor surface were utilized to detect antigen and bisphenol A (BPA). The immunosensor showed specific recognition of BPA with less interference than 4.5% from other common phenolic compounds. Under an optimized condition, the linear dynamic range of BPA detection was between 1 and 100 ng/ml. The detection limit of bisphenol A was determined to be 0.3+/-0.07 ng/ml. The proposed immunosensor was applied to a human serum sample and the BPA concentration was determined by the standard addition method.     

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.