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

Abstract

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 ( 1 O 2 ) 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.

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum

“…Nanobodies, a novel small molecule antibody, are the great alternatives to avoid the deficiency of traditional antibodies but can achieve the matching performance with monoclonal antibody [ 21 ]. In the recent years, nanobodies were widely applied to detect the specific targets including the bacteria [ 11 , 22 , 23 ], viruses [ 24 , 25 , 26 ], parasite [ 27 ], toxin [ 28 ], insecticide [ 29 ] and other target antigens [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ].…”

Recent Advances in the Development and Characterization of Electrochemical and Electrical Biosensors for Small Molecule Neurotransmitters

“…Although antibodies are important in the field of biosensing and have been used, for example, to detect protein biomarkers via electrochemical impedance spectroscopy, they have gained little popularity in the electronic detection of neurotransmitters, typically due to size constraints. Their miniaturized counterparts also have yet to be widely adopted as a preferred method for neurotransmitter detection and have been used primarily in other fields of biosensing (i.e., for other bioanalytes) but may hold promise for future applications. − …”

Electrochemical Affinity Biosensors: Pervasive Devices with Exciting Alliances and Horizons Ahead

“…Attractive affinity bioplatforms have been also developed involving EIS as a transduction technique, highlighting the need of a thoughtful design of the receptive interface and electrical properties of the interface to achieve both sensitive and reliable biosensing . Moreover, mixed electrochemical techniques, such as electrochemiluminescence (ECL), have made strong inroads by providing advantages in terms of low background noise, high sensitivity and selectivity, fast response, wide range of detection, low cost, and ease of use. , …”

“…126 Moreover, mixed electrochemical techniques, such as electrochemiluminescence (ECL), have made strong inroads by providing advantages in terms of low background noise, high sensitivity and selectivity, fast response, wide range of detection, low cost, and ease of use. 127,128 Numerous bioplatforms have been developed by exploiting ratiometric approaches based on the differential measurement and/or the ratio of two electrochemical signals generally with opposite trends and only one of them target-dependent, to ensure the reliability and robustness of the analytical results. 129 The use of new tools to correct for the baseline drift experienced by in vivo electrochemical responses 106 and the feasibility of establishing a universal slope for direct calibrationfree PON electrochemical immunoassays in serum 130 brings us a step closer to the development of substantially shorter decentralized electrochemical affinity-based bioassays.…”