A novel, sensitive electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a single-use carbon-based screen-printed electrode (SPE). The use of disposable electrodes allows for simplified measurement in 35 microL of solution. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) and a concentration as low as 3 x 10(-12) M was determined, which is competitive with colorimetric ELISA or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is related to the sensitive ASV determination of gold(III) at a SPE (detection limit of 5 x 10(-9) M) and to the release of a large number of gold(III) ions from each gold particle anchored on the immunocomplex (e.g., 1.7 x 10(5) gold atoms are theoretically contained in a 18-nm spherical gold particle).

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A carbon-based ink composed of graphite particles and polystyrene was used in association with a manual screenprinter to prepare electrodes on a flexible polyester film. The screen-printing step was followed by a drying step which was achieved within 1 h at room temperature. The screen-printed electrode (SPE) was coated by a polyanionic Nafion film in which electroactive cationic species could be accumulated. A detection limit of 10 -9 M was thus obtained by cyclic voltammetric (CV) determination of [[(4-hydroxyphenyl)amino]carboxyl]cobaltocenium (P + ) after accumulation for 60 min. Since this cationic phenol derivative P + could be generated from the corresponding anionic ester phosphate (S -) by alkaline phosphatase (AP) hydrolysis, the new Ssubstrate was synthesized and the sensitive indirect CV determination of AP was performed at a Nafion-coated SPE. The Ssubstrate did not interfere on the electrochemical response of P + owing to the permselectivity of Nafion. An AP detection of 4 × 10 -16 M was thus achieved in Tris buffer (pH 9) after hydrolysis of S -(10 -4 M) to P + (Michaelis constant K m ) 48 µM) and simultaneous accumulation of P + within Nafion for 1 h. The Nafion-SPE was stuck successfully to the bottom of a microwell, making it possible to work with solution volumes ranging from 50 to 250 µL, well adapted to enzyme immunoassays.The ultrasensitive detection of biological substances using affinity assays, such as immunoassays or nucleic acid hybridizations, is extremely important in many fields, e.g., biological and medical research, diagnostic medicine, genetics, and drug and pesticide testing. Highly sensitive immunoassays are expected to facilitate research for new diagnostic markers of diseases, better monitoring of tumor-specific products, and early detection of antigens at very low level concentrations in biological fluids. In addition, the development of highly sensitive methods allows assays to be conducted with smaller sample volumes and/or shorter analysis times. Therefore, considerable efforts have been focused on the improvement of the affinity assay sensitivity. [1][2][3][4] It depends mainly on the ability to detect the label, and so the most commonly used strategy involves an enzyme label, which provides a signal amplification through the rapid conversion of a substrate to a detectable product. The alkaline phosphatase enzyme (AP) is of particular interest because of its high turnover number and broad substrate specificity. Thus some of the most sensitive enzyme affinity assays developed to date (detection limit close to the zeptomole level) have used the AP label coupled to an ultrasensitive chemiluminescent detection method 5 or with a cascade enzyme reaction monitored by fluorescence. 6 These techniques are, however, sophisticated and costly, and so there is a continuing effort to develop new devices which are not only sensitive but also fast, reliable, low cost, and low size.AP is able to rapidly hydrolyze a large variety of orthophosphoric monoesters into detectable alcohols. Fr...

2)4^2'(42)X COS [(62 -40«23] x d®3(43)42U43)f'i3M3(7«)(424(^34))2(42;(4F))2 (A5b) The ß angles and b¡ summation indexes are as in the free diffusion model, eq Ala and Alb. The functions 44-"( ) are given in ref 1, eq 3.12b and 3.12c, and the summation indexes used in this study, n" can be accurately truncated at 0 < n¡ < 5 for the amplitudes, y, used in this study. The angles a2F and a23 specify the torsional angles between the Ca-C& bond and the C7-H or C7-C< bonds, respectively, when viewed along the C^-C7 bond with 7 = 0. For fluctuations about a trans C"-C{-C7-Ci configuration, <*2f = ±120°and a23 = 180°. As one proceeds away from the "stationary" C" end of the side chain, the torsional angle becomes meaningless if the internal rotations are assumed to be independent. Consequently the torsional angles specifying the angle between the Cs-C7 and C6-H or Cs-Cf bonds, a3F and a34, are averaged and do not appear. The angles a12 and a4F rigorously do not appear since the motion of C" is isotropic and that of C is unrestricted.