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A novel highly sensitive dendrimer coupled impedimetric immunosensor was developed for the label-free and reagent-free detection of cardiac troponin I (TnI) in serum samples. The immunosensor probe was fabricated by covalently attaching carboxylic acid-functionalized third generation (G3) poly (amidoamine) (PAMAM) dendrimer (Den) on the 3, 3', 5, 5'-tetramethyl benzidine (TMB) modified 6-mercaptohexanoic acid (MHA) self-assembled monolayer (SAM) on a gold (Au) electrode. Monoclonal anti-TnI antibody was then covalently immobilized on the Den and TMB attached MHA SAM modified surface. TMB was used as an internal surface redox couple for generating signal which also allowed to avoid the use of an external one (i.e. ferricyanide couple) in solution during the impedance measurement for monitoring the antibody-antigen binding. On the other hand, Den was used as a signal enhancer by immobilizing more anti-body on the immunosensor probe. The immunosensor probe was characterized using X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance (QCM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) techniques. The TnI detection in diluted serum was based on the measurement of charge transfer resistance (Rct) of the electron transfer process of the surface-attached TMB before and after immunobinding. Under the optimized condition, the proposed immunosensor could detect human TnI in diluted serum samples as low as 11.7 fM with a wide linear dynamic range, good stability, and excellent specificity. The validity of the proposed method was tested in various TnI spiked human undiluted serum samples and was compared with the enzyme-linked immunosorbent assay (ELISA). The results suggested that the proposed immunosensor could be a useful tool for practical applications in clinical diagnosis.

An ultrasensitive electrochemical nanostructured immunosensor for a breast cancer biomarker carbohydrate antigen 15-3 (CA 15-3) was fabricated using non-covalent functionalized graphene oxides (GO/Py-COOH) as sensor probe and multiwalled carbon nanotube (MWCNTs)-supported numerous ferritin as labels. The immunosensor was constructed by immobilizing a monoclonal anti-CA 15-3 antibody on the GO modified cysteamine (Cys) self-assembled monolayer (SAM) on an Au electrode (Au/Cys) through the amide bond formation between the carboxylic acid groups of GO/Py-COOH and amine groups of anti-CA 15-3. Secondary antibody conjugated MWCNT-supported ferritin labels (Ab2-MWCNT-Ferritin) were prepared through the amide bond formation between amine groups of Ab2 and ferritin and carboxylic acid groups of MWCNTs. The detection of CA 15-3 was based on the enhanced bioelectrocatalytic reduction of hydrogen peroxide mediated by hydroquinone (HQ) at the GO/Py-COOH-based sensor probe. The GO/Py-COOH-based sensor probe and Ab2-MWCNT-Ferritin labels were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscope (SEM), transmission electron microscope (TEM), and x-ray photoelectron spectroscopy (XPS) techniques. Using differential pulse voltammetry (DPV) technique, CA 15-3 can be selectively detected as low as 0.01 ± 0.07 U/mL in human serum samples. Additionally, the proposed CA 15-3 immunosensor showed excellent selectivity and better stability in human serum samples, which demonstrated that the proposed immunosensor has potentials in proteomic researches and diagnostics.

A highly sensitive quartz crystal microbalance (QCM) immunosensor based on magnetic bead-supported bienzyme catalyzed mass enhanced strategy was developed for the detection of human immunoglobulin G (hIgG) protein. The high sensitive detection was achieved by increasing the deposited mass on the QCM crystal through the enhanced precipitation of 4-chloro-1-naphthol (CN) using higher amounts of horseradish peroxidase (HRP) and glucose oxidase (GOx) bienzymes attached on the magnetic beads (MB). The protein A (PA) and capture antibody (monoclonal anti-human IgG antibody produced in mouse, Ab 1 )-based QCM probe and the detection antibody (anti-human IgG antibody produced in goat, Ab 2 )-based MB/HRP/GO x bienzymatic bioconjugates were characterized using scanning electron microscope, transmission electron microscope, cyclic voltammetry, and electrochemical impedance spectroscopy techniques. Under the optimized experimental condition, the linear range and the detection limit of hIgG immunosensor were determined to be 5.0 pg/mL–20.0 ng/mL and 5.0±0.18 pg/mL, respectively. The applicability of the present hIgG immunosensor was examined in hIgG spiked human serum samples and excellent recoveries of hIgG were obtained.

An ultrasensitive electrochemical immunosensor for a protein using a Ag (I)-cysteamine complex (Ag-Cys) as a label was fabricated. The low detection of a protein was based on the electrochemical stripping of Ag from the adsorbed Ag-Cys complex on the gold nanoparticles (AuNPs) conjugated human immunoglobulin G (anti-IgG) antibody (AuNPs-anti-IgG). The electrochemical immunosensor was fabricated by immobilizing anti-IgG antibody on a poly-5,2':5',2''-terthiophene-3'-carboxylic acid (polyTTCA) film grown on the glassy carbon electrode through the covalent bond formation between amine groups of anti-IgG and carboxylic acid groups of polyTTCA. The target protein, IgG was sandwiched between the anti-IgG antibody that covalently attached onto the polyTTCA layer and AuNPs-anti-IgG. Using square wave voltammetry, well defined Ag stripping voltammograms were obtained for the each target concentration. Various experimental parameters were optimized and interference effects from other proteins were checked out. The immunosensor exhibited a wide dynamic range with the detection limit of 0.4 ± 0.05 fg/mL. To evaluate the analytical reliability, the proposed immunosensor was applied to human IgG spiked serum samples and acceptable results were obtained indicating that the method can be readily extended to other bioaffinity assays of clinical or environmental significance.

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.

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2':5',2"-terthiophene-3'-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H(2)O(2) in a choline solution at +0.6V. The other one modified with ChO/HRP utilized the reduction process of H(2)O(2) in a choline solution at -0.2V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0 x 10(-6) to 8.0 x 10(-5) M and the other based on ChO/CPME from 1.0 x 10(-6) to 5.0 x 10(-5) M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0 x 10(-7) and 4.0 x 10(-7) M, respectively. The response time of sensors was less than 5s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.