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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.

Recently graphene has been drawing tremendous attention mainly due to its potential contributions to applications in biology, information technology and energy. Among these applications graphene-based biosensors have been particularly progressed caused in part by development of diverse derivatives of graphene such as graphene oxides (GOs) and graphene quantum dots (GQDs). In this chapter preparation and functionalization of the graphene and GOQs are described together with their optoelectronic properties. Recent progresses in graphene and GQD-based biosensors are also highlighted with emphasis on immunoassay which utilizes unique interaction between antigen and antibody, and oligonucleotide assay which utilizes hybridization process. Since electrical and optical features are the most prominent characteristics of graphene-based nanomaterials, biosensor systems will be focused on electrochemical and fluorescence-based detection scheme.

Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min.

Clinical applicability of a label-free electrochemical method was investigated, where prostate cancer (CaP) biomarkers miR-107 and miR-375 were examined in two different groups of prostate cell lines, metastatic CaP cells (PC-3, DU145, and LNCaP) and immortalized prostate epithelial cells (267B1, X/267B1, and Ki/267B1). First, the expression levels of miR-107 and miR-375 in these cells were analyzed by quantitative real-time polymerase chain reaction (PCR), resulting in useful discernable expression patterns from miR-375. Choosing miR-375 as a target CaP biomarker, a label-free electrochemical analysis was performed based on the optimized conditions of 100 nmol/mL miR-375 capture probe for surface immobilization and 30-minute hybridization. Standard curves were prepared both in buffer and in 100-fold diluted serum resulting in lower detection limit of 11.7 aM and 12.4 aM respectively. The regression equations for buffer and diluted serum were I = −15.1349 log c + 250.9735 and I = −11.8179 log c + 193.1639, respectively, where I is the peak current in μA and c is the concentration of miR-375 in fM. The selectivity of the sensor was confirmed by a one-base mismatch test in 3 different concentrations of miR-375 under diluted serum, 10 aM, 10 fM, and 10 pM. Finally, miR-375 expression in the metastatic CaP cells was quantified to range between 10 and 30 fM, while its expression in the prostate epithelial cells was negligible, demonstrating close agreement with the results from quantitative real-time PCR, and thereby potential viability of the electrochemical evaluation method in clinical environment.