An Electrochemical DNA Biosensor Developed on a Nanocomposite Platform of Gold and Poly(propyleneimine) Dendrimer.

An electrochemical DNA nanobiosensor was prepared by immobilization of a 20mer thiolated probe DNA on electro-deposited generation 4 (G4) poly(propyleneimine) dendrimer (PPI) doped with gold nanoparticles (AuNP) as platform, on a glassy carbon electrode (GCE). Field emission scanning electron microscopy results confirmed the codeposition of PPI (which was linked to the carbon electrode surface by C-N covalent bonds) and AuNP ca 60 nm. Voltammetric interrogations showed that the platform (GCE/PPI-AuNP) was conducting and exhibited reversible electrochemistry (E°' = 235 mV) in pH 7.2 phosphate buffer saline solution (PBS) due to the PPI component. The redox chemistry of PPI was pH dependent and involves a two electron, one proton process, as interpreted from a 28 mV/pH value obtained from pH studies. The charge transfer resistance (Rct) from the electrochemical impedance spectroscopy (EIS) profiles of GCE/PPI-AuNP monitored with ferro/ferricyanide (Fe(CN)63-/4-) redox probe, decreased by 81% compared to bare GCE. The conductivity (in PBS) and reduced Rct (in Fe(CN)63-/4-) values confirmed PPI-AuNP as a suitable electron transfer mediator platform for voltammetric and impedimetric DNA biosensor. The DNA probe was effectively wired onto the GCE/PPI-AuNP via Au-S linkage and electrostatic interactions. The nanobiosensor responses to target DNA which gave a dynamic linear range of 0.01 - 5 nM in PBS was based on the changes in Rct values using Fe(CN)63-/4- redox probe.

Electrochemical Immunosensor Based on Polythionine/Gold Nanoparticles for the Determination of Aflatoxin B1.

An aflatoxin B1 (AFB1) electrochemical immunosensor was developed by the immobilisation of aflatoxin B1-bovine serum albumin (AFB1-BSA) conjugate on a polythionine (PTH)/gold nanoparticles (AuNP)-modified glassy carbon electrode (GCE). The surface of the AFB1-BSA conjugate was covered with horseradish peroxidase (HRP), in order to prevent non-specific binding of the immunosensors with ions in the test solution. The AFB1 immunosensor exhibited a quasi-reversible electrochemistry as indicated by a cyclic voltammetric (CV) peak separation (ΔEp) value of 62 mV. The experimental procedure for the detection of AFB1 involved the setting up of a competition between free AFB1 and the immobilised AFB1-BSA conjugate for the binding sites of free anti-aflatoxin B1 (anti-AFB1) antibody. The immunosensor's differential pulse voltammetry (DPV) responses (peak currents) decreased as the concentration of free AFB1 increased within a dynamic linear range (DLR) of 0.6 - 2.4 ng/mL AFB1 and a limit of detection (LOD) of 0.07 ng/mL AFB1. This immunosensing procedure eliminates the need for enzyme-labeled secondary antibodies normally used in conventional ELISA-based immunosensors.

Citrate-capped silver nanoparticles as a probe for sensitive and selective colorimetric and spectrophotometric sensing of creatinine in human urine.

Urinary creatinine concentration is a critical physiological parameter that enables reliable assessment of patient renal function and diagnosis of a broad spectrum of diseases. In this study, a simple and inexpensive sensor comprising monodisperse, citrate-capped silver nanoparticles (cc-AgNPs) was developed, which enabled rapid, sensitive and selective quantitation of creatinine directly in unprocessed urine. The mechanism of this sensor entails the creatinine-mediated aggregation of the cc-AgNPs (within 1 min) under alkaline conditions (pH 12). This is attributed to the tautomerization of creatinine to its amino anionic species at alkaline pH, which cross-link the cc-AgNPs via hydrogen bond networks with the negatively charged citrate caps. Creatinine elicited visibly-discernable color changes of the cc-AgNPs colloids in a concentration-dependent manner up to 10 µM. UV-visible spectroscopic analyses of the cc-AgNPs revealed that creatinine elicited a concentration-dependent decrease in intensity of the localized surface plasmon resonance (LSPR) band centered around 403 nm, with a concomitant increase in intensity of the red-shifted LSPR band at 670 nm. This observation denotes a creatinine-mediated increase in cc-AgNP particle size via aggregation, as confirmed by transmission electron microscopy analysis. The cc-AgNP sensor exhibited a linear correlation between the A670/A403 extinction ratio and creatinine concentration range of 0-4.2 µM in aqueous solutions (R2 = 0.996), and a low detection limit of 53.4 nM. Hence, the simplicity, short assay time, and high sensitivity and selectivity of our cc-AgNP sensor affirms its utility as a creatinine monitoring assay for low-resource, point-of-care settings.

Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro.

While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5'-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O(2) and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner.