Hot electron induced cathodic electrochemiluminescence at AuSb alloy electrode for fabricating immunosensor with self-assembled monolayers.

Thin antimony oxide covered AuSb alloy electrode was firstly found to be an excellent cold cathode for generating hot electrons during cathodic pulse polarization. Owing to the injection of hot electrons and the subsequent generation of hydrated electrons, fluorescein iso-thiocyanate (FITC) that cannot be excited in common ECL was cathodically excited at the alloy electrode. Self-assembled thiol monolayers were formed on the electrode surface due to the presence of Au in the alloy, to which strepavidin was covalently bound, and then biotinylated antibody was immobilized through the strepavidin-biotin interaction. As a simple model, an immunosensor for the detection of human IgG (hIgG) using FITC as labeling agent was fabricated. ECL signals were responsive to the amount of hIgG bounded to the immunosensor. The ECL intensity was linearly changed with the logarithm of hIgG concentration in the range of 1.0-1000 ng mL(-1), and the detection limit was ca. 0.3 ng mL(-1) (S/N=3). The proposed immunosensor showed a broad linear range (three magnitudes), good reproducibility and stability, which is promising in detecting FITC-based labels in various types of bioaffinity assays.

Cathodic electrochemiluminescence at C/CxO(1-x) electrodes for the fabrication of label-free biosensors.

Cathodic electrochemiluminescence (ECL) is firstly observed at a carbon oxide covered glassy carbon (C/C(x)O(1-x)) electrode as a large cathodic pulse polarization is applied. This insulating carbon oxide (C(x)O(1-x)) film is constructed on a glassy carbon (GC) substrate by electrochemical oxidization in basic media. The film properties, such as the composition of carbon and oxygen, and the thickness as well, can be controlled by the potential and the duration in the oxidizing process. X-Ray photoelectron spectroscopy (XPS) studies show that carbonyl and carboxyl dominate at the oxidized surface, to which antibodies can be covalently bound. The specific immunoreaction between antigen (Ag) and antibody (Ab) resulted in a decrease in the ECL intensity, thus creating an interesting basis for the development of a label-free cathodic ECL immunosensor. As an example, human IgG (hIgG) was sensitively determined in the concentration range of 0.01-100 ng mL(-1), and the detection limit was ca. 1.0 pg mL (-1) (S/N = 3). In addition, the content of hIgG in human serum has been assayed by the developed immunosensor and a commercially available immune turbidimetry method, respectively, and consistent results were obtained. The prepared immunosensor provides a promising approach for the clinical determination of IgG levels in human serum, because it is simple, rapid, highly sensitive, specific, and without the need of tedious labeling operations.

A reagentless DNA biosensor based on cathodic electrochemiluminescence at a C/C(x)O(1-x) electrode.

A reagentless signal-on electrochemiluminescence (ECL) biosensor for DNA hybridization detection was developed based on the quenching effect of ferrocene (Fc) on intrinsic cathodic ECL at thin oxide covered glassy carbon (C/C(x)O(1-x)) electrodes. To construct the DNA biosensor, molecular beacon (MB) modified with ferrocene (3'-Fc) was attached to a C/C(x)O(1-x) electrode via the covalent bound between labeled amino (5'-NH(2)) and surface functional groups. It was found that the immobilization of the probe on the electrode surface mainly depended on the fraction of surface carbonyl moiety. When a complementary target DNA (cDNA) was present, the stem-loop of MB on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the moving away of Fc from the electrode surface, and the restoring of the cathodic ECL signal. The restoration of the ECL intensity was linearly changed with the logarithm of cDNA concentration in the range of 1.0x10(-11) to 7.0x10(-8)M, and the detection limit was ca. 5.0pM (S/N=3). Additionally, single-base mismatched DNA can be effectively discriminated from the cDNA. The great advantage of the biosensor lies in its simplicity and cost-effective with ECL generated from the electrode itself, and no adscititious luminophore is required.

Catalytic electrogenerated chemiluminescence and nitrate reduction at CdS nanotubes modified glassy carbon electrode.

Electrochemical and electrogenerated chemiluminescence (ECL) properties of a glassy carbon electrode modified with CdS nanotubes (CdS-GCE) are investigated in neutral media. The cyclic voltammogram (CV) shows two cathodic peaks (P(C1) and P(C2)) at -0.76 and -0.97 V and an anodic peak (P(A)) at -0.8 V, while two ECL peaks around -0.76 V are observed. Similar mechanisms of both ECLs are supposed and possibly related to the capture of an electron at a surface trap, that is, the surface sulfide vacancy (V(S)(2+)) of CdS nanotubes and its electrocatalytic reduction to H(2)O(2) generated from the dissolved oxygen. P(C2) and P(A) are ascribed to the two-electron redox at V(S)(2+). Moreover, electrocatalysis to nitrate reduction is also found at P(C2), with a good linear relationship between nitrate concentration and electrocatalytic peak current in CV.

Determination of trace mercury by solid substrate room temperature phosphorescence quenching method based on lead carboxymethyl cellulose (Pb(CMC)(2)) particles containing luminescent salicyl fluorones molecules.

Luminescent particles of lead carboxymethyl cellulose (Pb(CMC)(2)), which contains salicyl fluorones (THBF), Pb(CMC)(2)-THBF, were synthesized by sol-gel method. Pb(CMC)(2)-THBF can emit intense and stable solid substrate room temperature phosphorescence (SS-RTP) on filter paper. EDTA can chelate the Pb(2+) in Pb(CMC)(2)-THBF, causing it decompose into aqueous soluble components PbY(2-), CMC(-) and THBF, and these components can react with Hg(2+) to form (CMC)(2)Hg-THBF, causing decrease of phosphorescence intensity. Based on the facts above, a new method for the determination of trace mercury by SS-RTP quenching method was established. The linear range of this method is 2.0-40.0fgspot(-1) (5.0-100.0pgml(-1)) of Hg(2+), with a detection limit (LD) of 0.26fgspot(-1), and the regression equation of working curve is [Formula: see text] (fgspot(-1), 0.4mul spot(-1)), r = 0.9994. This method has been applied to the determination of trace mercury in water sample with satisfactory results. The mechanism of SS-RTP emission is also discussed.

Determination of human IgG by solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing rhodamine 6G luminescent molecules.

Luminescent 50-nm silicon dioxide nanoparticles containing both types of rhodamine 6G (R; particles denoted R-SiO2) were synthesized by the sol-gel method. In the presence of Pb(Ac)2 as a heavy atom perturber the particle can emit the intense and stable room-temperature phosphorescence (RTP) signal of R on a polyamide membrane, with lambda(ex)max/ lambda(em)max=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO2 and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was enhanced after the immunoreaction. Thus a new method for solid-substrate room-temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established on the basis of antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624-20.0 pg spot(-1) of human IgG (corresponding to a concentration range of 0.156-50.0 ng mL(-1), sample volume 0.40 microL spot(-1)). The regression equations of the working curves are DeltaIp = 71.27+7.208 m(IgG) (pg spot(-1)) (r = 0.9996). Detection limits calculated as 3 Sb/k are 0.022 pg spot(-1). Compared with the same IA using fluorescein isothiocyanate (FITC) as the marker the new method was more sensitive and had a wider linear range. After elevenfold replicate measurement RSD are 4.5 and 3.6% for samples containing 0.156 and 50.0 ng mL(-1) IgG, respectively. This method is sensitive, accurate, and of high precision.