Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling.

Detection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens.

Gold-sputtered microelectrodes with built-in gold reference and counter electrodes for electrochemical DNA detection.

Gold-sputtered microelectrodes with built-in gold reference and counter electrodes represent a promising platform for the development of disposable DNA sensors. Pretreating gold electrode surfaces and immobilization of DNA thereon is commonly employed in biosensing applications. However, with no scientific or practical guidelines to prepare a DNA sensor using these miniature gold-sputtered microelectrodes, cleaning and immobilization steps need to be systematically optimized and updated. In this work, we present efficient cleaning and modification of miniaturized gold-sputtered microelectrodes with thiolated DNA probes for DNA detection. Additional discussions on subtleties and nuances involved at each stage of pretreating and modifying gold-sputtered microelectrodes are included to present a robust, well-founded protocol. It was evident that the insights on cleaning polycrystalline gold disk electrodes with a benchmark electrode surface for DNA sensors, cannot be transferred to clean these miniature gold-sputtered microelectrodes. Therefore, a comparison between five different cleaning protocols was made to find the optimal one for gold-sputtered microelectrodes. Additionally, two principally different immobilization techniques for gold-sputtered microelectrode modification with thiolated ssDNA were compared i.e., immobilization through passive chemisorption and potential perturbation were compared in terms of thiol-specific attachment and thiol-unspecific adsorption through nitrogenous bases. The hybridization performance of these prepared electrodes was characterized by their sensitive complementary DNA capturing ability, detected by a standard alkaline phosphatase assay. Immobilization through passive chemisorption proved to be efficient in capturing the complementary target DNA with a detection limit of 0.14 nM and sensitivity of 9.38 A M-1 cm2. In general, this work presents a comprehensive understanding of cleaning, modification and performance of gold-sputtered microelectrodes with built-in gold reference and counter electrodes for both fundamental investigations and practical DNA sensing applications.