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Phosphorus is not only an import nutrient to aquatic habitats, but it also acts as a growth inhibitor in aquatic ecosystems; however, it also aggravates environmental issues, such as eutrophication. There is a growing interest in rapid phosphorus detection to manage and protect water resources. Due to the large molecular structure and high hydration energy of phosphate ions, ion-selective electrodes (ISEs) remain in their infancy for real-time measurements in terms of practical application. In this study, a newly developed ionophore based on a biomimetic nicotinamide functional group was used to detect phosphate selectively, displaying efficient binding through charge interactions and hydrogen bonds. The ISE membrane containing silicone rubber demonstrated an effective detection performance over a long period of time. With a dynamic range between 10−6 and 10−2 M and a limit of detection of 0.85 × 10−6 M (26 μg/L), the newly synthesized ISE membranes demonstrated selectivity for phosphate ions over other ions, including acetate, sulfate, and chloride.

Phosphate sensors have been actively studied owing to their importance in water environment monitoring because phosphate is one of the nutrients that result in algal blooms. As with other nutrients, seamless monitoring of phosphate is important for understanding and evaluating eutrophication. However, field-deployable phosphate sensors have not been well developed yet due to the chemical characteristics of phosphate. In this paper, we report on a luminescent coordination polymer particle (CPP) that can respond selectively and sensitively to a phosphate ion against other ions in an aquatic ecosystem. The CPPs with an average size of 88.1 ± 12.2 nm are embedded into membranes for reusable purpose. Due to the specific binding of phosphates to europium ions, the luminescence quenching behavior of CPPs embedded into membranes shows a linear relationship with phosphate concentrations (3–500 μM) and detection limit of 1.52 μM. Consistent luminescence signals were also observed during repeated measurements in the pH range of 3–10. Moreover, the practical application was confirmed by sensing phosphate in actual environmental samples such as tap water and lake water.

Absence of functional groups on the basal plane of molybdenum disulfide (MoS2) significantly hinders the performance of MoS2 field-effect transistor-based biosensor (bio-FET). We present a novel method for creating nano-scale holes on a MoS2 channel using block copolymer lithography, where the edge areas on the nanoholes were used to form covalent linkage between the capture molecules of cortisol aptamer and the MoS2 channel. The comparative analysis of Raman and XPS spectra well supported the formation of the nanoholes on MoS2 together with the concomitant increase in the edge area. The performance of the nanomesh bio-FET was studied by comparing its detection behavior for cortisol with that of a bio-FET manufactured with pristine MoS2. The nanomesh MoS2 bio-FET detected cortisol 109 times as low as that by the pristine MoS2 bio-FET. The selectivity of the nanomesh bio-FET was validated by detection experiments with other steroid hormones and antigens. Additionally, clinical applicability was demonstrated by performing experiments in artificial saliva and human serum.

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.

A highly sensitive square-wave voltammetric thrombin (TB) aptamer sensor was developed using functional polydopamine (PD) film by doping and depositing gold nanoparticles into the bulk and the surface of PD. The aptamer sensor was fabricated by immobilizing a thiolated TB-binding aptamer (TBA) on the AuNPs-doped/deposited PD film. AuNPs-supported methylene blue labels were used for the detection of human α-TB. Under the optimized conditions, the aptamer sensor’s dynamic range and the detection limit were determined to be 2.0 pM–50 nM and 0.97±0.06 pM, respectively. Finally, the proposed aptamer sensor was successfully examined in human serum samples and satisfactory results were obtained.

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.

A highly sensitive electrochemical carcinoembryonic antigen (CEA) immunosensor was fabricated by covalently immobilizing a monoclonal CEA antibody (anti-CEA, Ab(1)) and a mediator (thionine, Th) on a gold nanoparticle (AuNP)-encapsulated dendrimer (Den/AuNP). Multiwalled carbon nanotube (MWCNT)-supported secondary antibody (Ab(2))-conjugated multiple bienzymes, glucose oxidase (GOx), and horseradish peroxidase (HRP) (Ab(2)/MWCNT/GOx/HRP) were used as electrochemical labels. The highly sensitive detection was achieved by the increased HRP-electrocatalyzed reduction of hydrogen peroxide, which was locally generated by the enzyme GOx. The immunosensor surface was characterized using electrochemical impedance spectroscopy, atomic force microscopy, and quartz crystal microbalance techniques. The Den/AuNP and Ab(2)/MWCNT/GOx/HRP bioconjugates were characterized using high-resolution transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. Cyclic voltammetry and square wave voltammetry techniques were used to monitor the increased electrocatalyzed reduction of hydrogen peroxide by HRP. The linear dynamic range and the detection limit were determined to be 10.0 pg/mL to 50.0 ng/mL and 4.4 ± 0.1 pg/mL, respectively. The validity of the immunosensor response was tested in various CEA-spiked human serum samples, and the results were compared to those of an enzyme-linked immunosorbent assay method.

A stimulated mass enhancement strategy based on enormous biocatalytic precipitation of 4-chloro-1-naphthol using magnetic bead-supported horseradish peroxidase and glucose oxidase bienzymes was developed for the highly sensitive detection of interleukin-6 in serum using a quartz crystal microbalance technique.

Analysis Research Division, Daegu Center, Korea Basic Science Institute, Daegu 702-701, KoreaReceived December 8, 2012, Accepted December 27, 2012Key Words : Gold nanoparticles, Multiple Nonenzymatic Labels, Impedance, Aptamer, ThrombinThrombin (TB) is a coagulation and serine protease thatconverts soluble fibrinogen to insoluble strands of fibrin aswell as catalyzing many other coagulation-related reactionsresponsible for blood clotting. It plays an important role inthrombosis and haemostasis and its detection is very usefulfor early diagnosis of intracerebral hemorrhage