Development of electrochemical immunosensors for detection of Tau protein : computational and experimental studies

Abstract

Tau protein is a microtubule-associated protein (MAP) found in neuronal cells of the central nervous system. In recent years it has become an important biomarker for neurodegeneration and pathologies of the nervous system, thereby necessitating novel approaches for its detection. This study involves the development of two immunosensors for the detection of tau protein. The study makes use of nanomaterials and antibody transducers as signal enhancing strategies. Both sensors rely on indirect detection of tau protein as a copper(II) complex using a Cu(II)/Cu (I) redox probe. The electrochemical immunoassay is based on the immobilisation of anti-tau antibodies onto a gold working electrode that has been modified with nanomaterials using N- Hydroxysuccinimide (NHS) binder. The first sensor makes use of gold nanoparticles (AuNPs) and the second utilises a nanocomposite of graphene oxide (GO) decorated with silver nanoparticles (AgNPs). Cyclic voltammetry (CV) was used to optimise the electrochemical signal of the tau protein, while quantitative analyses were achieved by differential pulse voltammetry (DPV) and square wave voltammetry (SWV) under the established optimised conditions. Results for the quantitative experimental studies revealed relatively low detection limits for both sensors. The lowest of these detection limits were obtained for DPV analysis of using sensor 1 which produced an LOD of 3.31 nM and an LOQ of 11.04 nM. For sensor 2 the SWV analysis produced the lowest LOD and LOQ of 1.73 nM and 5.76 nM respectively. Computational chemistry methods implemented at the DFT level were used to support the developed electrochemical sensor. The molecular docking results showed relatively good binding affinity of -4.72 kcal/mol between the NHS and the antibody. A 100 ns MD simulation showed a good free binding energy value of -20.51 kcal/mol at pH 7, in accordance with the optimum pH implemented in the experimental work. Furthermore, adsorption studies were performed between the citrate coated nanoparticles on the Au electrode and NHS/anti-tau antibody/tau complex. The energy adsorption simulations revealed the energy favoured interaction between the designed layers with the stabilizing energy changes from -- 23.74 to -142.96 kcal/mol for sensor 1 and for sensor 2 it changed from -7.6 to -127.82 kcal/mol. Overall the computational data correlated well with experimental work. The two novel immunosensors developed in this work, give new insights into electrochemical and computational methods for the detection of proteins, and could lead to the fabrication of a device for point-of-applications in early diagnosis of neurodegenerative disorders.