Monodisperse Au(25)L(18)(0) (L = S(CH(2))(2)Ph) and [n-Oct(4)N(+)][Au(25)L(18)(-)] clusters were synthesized in tetrahydrofuran. An original strategy was then devised to oxidize them: in the presence of bis(pentafluorobenzoyl) peroxide, the neutral or the negatively charged clusters react as efficient electron donors in a dissociative electron-transfer (ET) process, in the former case yielding [Au(25)L(18)(+)][C(6)F(5)CO(2)(-)]. As opposed to other reported redox methods, this dissociative ET approach is irreversible, easily controllable, and clean, particularly for NMR purposes, as no hydrogen atoms are introduced. By using this approach, the -1, 0, and +1 charge states of Au(25)L(18) could be fully characterized by (1)H and (13)C NMR spectroscopy, using one- and two-dimensional techniques, in various solvents, and as a function of temperature. For all charge states, the NMR results and analysis nicely match recent structural findings about the presence of two different ligand populations in the capping monolayer, each resonance of the two ligand families displaying distinct NMR patterns. The radical nature of Au(25)L(18)(0) is particularly evident in the (1)H and (13)C NMR patterns of the inner ligands. The NMR behavior of radical Au(25)L(18)(0) was also simulated by DFT calculations, and the interplay between theory and experiments revealed a fundamental paramagnetic contribution coming from Fermi contact shifts. Interestingly, the NMR patterns of Au(25)L(18)(-) and Au(25)L(18)(+) were found to be quite similar, pointing to the latter cluster form as a diamagnetic species.
a b s t r a c tIn this study we demonstrate the possibility to prepare highly sensitive nanostructured electrochemical immunosensors by immobilizing biorecognition elements on nanoelectrode ensembles (NEEs) prepared in track-etch polycarbonate membranes. The gold nanodisk electrodes act as electrochemical transducers while the surrounding polycarbonate binds the antibody-based biorecognition layer. The interaction between target protein and antibody is detected by suitable secondary antibodies labelled with a redox enzyme. A redox mediator, added to the sample solution, shuttles electrons from the nanoelectrodes to the biorecognition layer, so generating an electrocatalytic signal. This allows one to fully exploit the highly improved signal-to-background current ratio, typical of NEEs. In particular, the receptor protein HER2 was studied as the target analyte. HER2 detection allows the identification of breast cancer that can be treated with the monoclonal antibody trastuzumab. NEEs were functionalized with trastuzumab which interacts specifically with HER2. The biorecognition process was completed by adding a primary antibody and a secondary antibody labelled with horseradish peroxidase. Hydrogen peroxide was added to modulate the label electroactivity; methylene blue was the redox mediator generating voltammetric signals. NEEs functionalized with trastuzumab were tested to detect small amounts of HER2 in diluted cell lysates and tumour lysates.
Here, a colloidal templating procedure for generating high‐density arrays of gold macroporous microwells, which act as discrete sites for surface‐enhanced Raman scattering (SERS), is reported. Development of such a novel array with discrete macroporous sites requires multiple fabrication steps. First, selective wet‐chemical etching of the distal face of a coherent optical fiber bundle produces a microwell array. The microwells are then selectively filled with a macroporous structure by electroless template synthesis using self‐assembled nanospheres. The fabricated arrays are structured at both the micrometer and nanometer scale on etched imaging bundles. Confocal Raman microscopy is used to detect a benzenethiol monolayer adsorbed on the macroporous gold and to map the spatial distribution of the SERS signal. The Raman enhancement factor of the modified wells is investigated and an average enhancement factor of 4 × 104 is measured. This demonstrates that such nanostructured wells can enhance the local electromagnetic field and lead to a platform of ordered SERS‐active micrometer‐sized spots defined by the initial shape of the etched optical fibers. Since the fabrication steps keep the initial architecture of the optical fiber bundle, such ordered SERS‐active platforms fabricated onto an imaging waveguide open new applications in remote SERS imaging, plasmonic devices, and integrated electro‐optical sensor arrays.
Nanostructured electrochemical biosensors are prepared by immobilizing the biorecognition elements on the polycarbonate (PC) surrounding gold nanodisks (approximately, 30 nm in diameter) in nanoelectrode ensembles (NEEs) made in track-etched commercial membranes. A suitable redox mediator is added to the sample solution to shuttle electrons from the nanoelectrodes to the biorecognition layer, both elements being in strict spatial proximity. In this way one can exploit the highly improved signal-to-background current ratio which is peculiar of NEEs with respect to other electrochemical transducers. Two detection schemes were tested: one based on the direct immobilization of the target protein on the PC of the NEE (approach A) and the other based on the immobilisation on PC of an antibody to capture the target protein (approach B). In both cases, the biorecognition process was completed by adding a primary antibody and a secondary antibody with horse radish peroxidase (HRP) as enzyme label; methylene blue was the redox mediator added to the electrolyte solution. Typical target analytes were single chain fragment variable proteins, for approach A, and trastuzumab (also known as Herceptin), for approach B. NEE-based capture sensors were tested successfully to detect small amounts of the receptor protein HER2 in biological samples.
Abstract. Nanoelectrochemical immunosensors fabricated by templated electrodeposition of gold nanoelectrodes inside the pores of polycarbonate (PC) track-etched membranes, followed by the immobilization of the biorecognition elements on the surrounding PC, have proven high sensitivity and specificity for protein detection. The signal transduction scheme involves a suitable redox mediator added to the sample solution to shuttle electrons from the gold nanoelectrodes to the biorecognition layer, both elements being in strict spatial proximity. Highly improved signal-to-background current ratio, which are peculiar of NEEs with respect to other electrochemical transducers, can be exploited in this way. Two detection schemes were tested: one based on the direct immobilization of the target protein on the PC of the NEE (approach A) and the other based on the immobilisation on PC of an antibody to capture the target protein (approach B). The biorecognition process was completed by adding a primary antibody and a secondary antibody with horse radish peroxidase (HRP) as enzyme label; methylene blue was the redox mediator added to the electrolyte solution. Typical target analytes were single chain fragment variable proteins, for approach A, and trastuzumab (also known as Herceptin®), for approach B. NEE-based capture sensors were tested successfully to detect small amounts of the receptor protein HER2 in biological samples. Finally, motivated by the target of a better control of the geometrical characteristics of ensembles of nanoelectrodes (size, density, geometrical arrangement, and degree of recession), and by the positive results obtained with track-etch membranes of PC from the standpoint of protein immobilization, we demonstrated the fabrication of nanobiosensors by patterning ordered arrays of nanoelectrodes (NEAs) by electron beam lithography (EBL) on polycarbonate. EBL results perfectly suitable for the top-down fabrication of arrays of nanobiosensors on thin PC films deposited on gold coated silicon.
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