Label-free SERS for rapid identification of interleukin 6 based on intrinsic SERS fingerprint of antibody­gold nanoparticles conjugate.

A label-free surface-enhanced Raman scattering (SERS) was designed for sensitive detection of interleukin-6 (IL-6). The sensing element composed of anti-IL-6 antibodies adsorbed on the surface of spherical gold nanoparticles (AuNPs) as SERS-active surface. The principle of detection was probing antibody conformational changes using its intrinsic SERS fingerprint after binding to IL-6. Comparison of SERS spectra of antibody before and after binding to IL-6 showed that secondary structure of antibody does not change upon binding to IL-6. Vibrational information from disulfide bonds ν(SS) in antibody structure indicated some changes of geometry around SS bridges as a consequence of the immunocomplex formation. Transmission electron microscopy (TEM) and UV-Vis spectroscopy were used to confirm AuNPs conjugation with antibody as well as IL-6 binding to antibody on the surface of AuNPs. The SERS-based immunoassay showed a wide linear range (2.0-1000 pg mL-1) and a high sensitivity with a limit of detection (LOD) as low as 0.91 pg mL-1 (0.04 pM) without using any extrinsic Raman label. UV-Vis spectroscopy was employed as a conventional method for IL-6 detection based on observation of any change in the position of localized surface plasmon resonance (LSPR) band of AuNPs-antibody conjugates with LOD of 10 ng mL-1.

Selective generation of gold nanostructures mediated by flavo-enzymes to develop optical biosensors.

This paper explores, for the first time, the use of flavo-enzymes for the enzymatic generation of gold nanomaterials. It has been demonstrated that when the oxidation of glucose by GOx is carried out in the presence of Au(III), the in-situ formation of gold nanomaterials is observed. Moreover, depending on the experimental conditions, either nanoparticles (AuNPs) or nanoclusters (AuNCs) are better observed, whose spectroscopical properties can be related to the concentration of glucose. Working at pH 6, only AuNCs with fluorescence at 420 nm (λex=335 nm) are obtained (linear relationship from 6.0·10-5 M to 1.5·10-3 M glucose). However, when the enzymatic reaction is performed at pH 8, AuNPs (λmax=580 nm) are also obtained (linear relationship from 5.5·10-4 M to 2.0·10-3 M glucose). Mathematical equations describing the variation of fluorescence and absorbance values during the reaction have been proposed. The results obtained suggest that AuNCs are formed using GOx as nucleation seeds. Since AuNPs belong to the branched-type, it is suggested that they are obtained by AuNC coalescence. From these models the AuNP molar absorptivity per atom was obtained (2.0(±0.3)·103 M-1cm-1). Finally, the method has been applied to the determination of glucose in orange juice and human plasma samples. Comparing the results with the GOx/HRP/TMB method and a commercial glucometer, no significant differences (P=0.05) are obtained.

Antibody-Directed Synthesis of Catalytic Nanoclusters for Bioanalytical Assays.

Synthesis of atomic nanoclusters (NCs) using proteins as a scaffold has attracted great attention. Usually, the synthetic conditions for the synthesis of NCs stabilized with proteins require extreme pH values or temperature. These harsh reaction conditions cause the denaturation of the proteins and end up in the loss of their biological functions. Until now, there are no examples of the use of antibodies as NC stabilizers. In this work, we present the first method for the synthesis of catalytic NCs that uses antibodies for the stabilization of NCs. Anti-BSA IgG was used as a model to demonstrate that it is possible to use an antibody as a scaffold for the synthesis of semiconductor and metallic NCs with catalytic properties. The synthesis of antibodies modified with NCs is carried out under nondenaturing conditions, which do not affect the antibody structure. The resulting antibodies still maintain the affinity for target antigens and protein G. The catalytic properties of the anti-BSA IgG modified with NCs can be used to the quantification of bovine serum albumin (BSA) in a direct sandwich enzyme-linked immunosorbent assay (ELISA).