Characterization of Covalently Bound Anti-Human Immunoglobulins on Self-Assembled Monolayer Modified Gold Electrodes (original) (raw)

The attachment of biological recognition sites is essential for the successful development of reliable biosensors being able to detect target analytes at low concentrations. In addition, the immobilization strategy can affect biological systems and the processes associated with key relevant events such as biorecognition itself. The selected biofunctionalization protocol depends on several factors involving biomolecules properties, nature of the immobilization surface, sample matrix, and buffer medium. It also impacts on the sensor's analytical performance metrics such as the sensitivity, selectivity, and reproducibility. Ideally, the bioactive binding sites should be immobilized onto surfaces with controlled density and proper orientation in order to be accessible for reacting with the ligand. For a reproducible assay, bioreceptors activity and surface coverage should remain unchanged from one batch to the other. Up to date biomolecule immobilization involving silanized layers, polymer membranes, Langmuir-Blodgett films, protein A, and self-assembled monolayer (SAM), has been achieved on various substrates, ensuring also the correct orientation of the recognition site without loss of activity and high loading of the sensor surface in order to gain maximum signal as well as selective analyte detection. [1] All the above reveal the importance of investigating the surface chemistry of biomodified materials, including noble metals, nanoparticles, metal oxides, polymers etc., for the optimization of the incorporation of biomolecules in miniaturized electronic sensors. Especially, the incorporation of biorecognition sites on inert metal surfaces such as gold gives rise to biosensors taking advantage of different physical quantifiable signals including electrochemical sensing, [2-4] surface plasmon resonance (SPR), [5-8] or the lately gaining significant attention electrolyte gated thin film transistors (EG-TFTs) [9-13] based biosensors. Biorecognition elements (e.g., proteins, DNA, cells, etc.) have been largely immobilized on active gold sensor surfaces via weak physical (ionic, hydrophobic) or strong chemical (through thiol chemistry, bifunctional linkers or adapter mole cules) interactions. [14] Herein, a Bioconjugated gold surfaces constitute interesting platforms for biosensing applications. The immobilization of antibodies such as anti-immunoglobulin G and M (anti-IgG and anti-IgM) on gold electrodes via self-assembled monolayers (SAMs) is here studied as a model system for further immunoassays development. The biolayer is characterized by means of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), a dedicated thin-film transistor (TFT)-based platform and electrochemical surface plasmon resonance (EC-SPR). XPS analysis confirms the presence of all the chemical species involved in the fabrication process as well as the covalent attachment of the antibodies with high reproducibility. Visualization of the biolayer topography by AFM shows nanostructures with a thickness consistent with the actual size of the protein, which is also verified by SPR measurements. EC-SPR allows taking advantage of complementary electrochemical and optical signals during the functionalization steps. Moreover, the functionalization of gold leads to a change in the work function, which is demonstrated in an electrolyte gated thin-film transistor configuration. Such configuration enables also to evaluate the electrostatic changes occurring on the gate that are connected with the threshold voltage shifts. The data support that functional biomodified gold surfaces can be reproducibly prepared, which is a prerequisite for further biosensor development.