Multifunctional Catecholate-Modified 3D TiO2 Nanostructures for Highly Sensitive Photoelectrochemical IL-6 Immunosensors

Abstract

Protein immunosensors are valuable analytical tools that allow for the detection of proteins with high sensitivity and specificity based on affinity-based interaction between antigen/antibody pairs.1 These devices are increasingly important in clinical settings due to their roles in detecting inflammatory processes, autoimmune disorders, cardiovascular disorders and cancers.1,2 Interleukin-6 (IL-6) is a protein biomarker that plays key roles in immunomodulation, hematopoiesis and inflammatory processes in the human body.3 Elevated levels of IL-6 can be used as evidence for diagnosis of various cancers and autoimmune disorders and even show the progression of COVID-19.3,4 In this work, we developed multifunctional three dimensional (3D) TiO2 nanostructured photoelectrodes as a sensing platform which combine photoelectrochemical (PEC) transduction with a signal-off direct immunoassay to detect IL-6. Surface modification with catecholate ligands was used to modify the properties of TiO2 nanostructures in a three-pronged approach of nanomorphology tuning, photocurrent signal enhancement, and facilitating bioconjugation.3D TiO2 nanostructures were synthesized via acid-hydrothermal process using titanium (IV) butoxide as a precursor. During the synthesis process 3,4-dihydroxybenzaldehyde (DHBA) was added in-situ into the reaction vessel. DHBA is part of the catecholate family of organic ligands which adsorb readily onto TiO2 surfaces. During the synthesis process, DHBA acted as a surfactant with preferential binding affinity for specific crystal facets of TiO2. The addition of DHBA resulted in anisotropic TiO2 crystal growth during synthesis, leading to globular nanorod cluster morphologies with high surface area-to-volume ratios. The 3D TiO2 nanostructures were further modified with extra DHBA after their synthesis. The 3D morphology combined with the extra DHBA resulted in higher UV/visible light absorption and photocurrent density measurements. The synthesized 3D TiO2 nanostructures were used to fabricate photoelectrodes for PEC signal-off immunoassay.The signal-off PEC IL-6 direct immunoassay was constructed by immobilizing a probe layer of anti-IL-6 on the surface 3D TiO2 photoelectrodes. When solutions containing target IL-6 encounter the photoelectrode, IL-6 is immobilized onto the photoelectrode surface through antibody/antigen complexation. Il-6 immobilization on the photoelectrode results in photocurrent signal decrease due to steric hinderance and this signal decrease can be directly correlated to the target concentration. The 3D nanostructure morphology contributed to increased sensitivity for the immunosensor as the higher electroactive surface area can accommodate a higher probe immobilization efficiency. Higher photocurrent generation also resulted in improved limit of detection (LOD) and dynamic range for the sensor. Functionalization by DHBA not only improves photocurrent generation, but also creates an avenue for biofunctionalization through Schiff-base linking between -COH groups of DHBA and -NH2 groups of anti-IL-6. The resultant immunosensor demonstrated excellent performance with an LOD of 3.6 pg mL-1 in plasma and 1.6 pg mL-1 in buffer solution, while having a log-linear dynamic range of 2-2000 pg mL-1. The 3D TiO2 photoelectrodes also demonstrate excellent short-term and long-term stability with little degradation after repeated use.It is evident from our investigations that combining PEC transduction mechanisms with direct signal-off immunoassays can result in high performance biosensors. In particular, the surface modification of 3D TiO2 nanostructure scaffolds with DHBA facilitates the fabrication of materials that are highly nanoporous and generate high photocurrent, resulting in biosensors with large dynamic ranges and low LOD.(1) Zhao, W. W.; Xu, J. J.; Chen, H. Y. Photoelectrochemical Immunoassays. Anal. Chem. 2018, 90 (1), 615–627. https://doi.org/10.1021/acs.analchem.7b04672.(2) Chikkaveeraiah, B. V.; Bhirde, A. A.; Morgan, N. Y.; Eden, H. S.; Chen, X. Electrochemical Immunosensors for Detection of Cancer Protein Biomarkers. ACS Nano 2012, 6 (8), 6546–6561. https://doi.org/10.1021/nn3023969.(3) Khan, M. A.; Mujahid, M. Recent Advances in Electrochemical and Optical Biosensors Designed for Detection of Interleukin 6. Sensors (Switzerland). MDPI AG February 1, 2020. https://doi.org/10.3390/s20030646.(4) Rubin, E. J.; Longo, D. L.; Baden, L. R. Interleukin-6 Receptor Inhibition in Covid-19 — Cooling the Inflammatory Soup. N. Engl. J. Med. 2021, 384 (16), 1564–1565. https://doi.org/10.1056/nejme2103108.