Antibody-Immobilized Cellulose Nanocrystals for the Detection of Cancer Biomarkers

Abstract

Naturally derived anisotropic nanomaterials such as cellulose nanocrystals (CNC) are an intriguing material for biosensors due to their exceptional mechanical properties, nanoscale structure, and sustainability. However, relatively little is known about antibody immobilization on CNC; this is particularly true for the commonly available CNC that are produced by sulfuric acid extraction from woody biomass. The main objective of this research was to develop a multi-step reaction scheme to immobilize model cancer antibodies on the surfaces of CNC in a liquid dispersion and compare the results to a previously established method for immobilizing antibodies on solid CNC films. An additional objective was to understand whether the modified CNC could be used as probe for the cancer detection of the corresponding antigens. All of the surface modifications were performed using 3-aminopropyl-triethoxysilane (APTES) and glutaric anhydride (GA) chemistry. This was followed by the immobilization of the following primary monoclonal antibodies: alpha-fetoprotein (AFP) antibody, which is used in liver cancer detection, prostate-specific antigen (PSA) antibody which is used in prostate cancer detection, and carcinoembryonic antigen (CEA) antibody which is used in ovarian cancer detection. To the best of author’s knowledge, quartz crystal microbalance with dissipation monitoring (QCM-D) has never been used before for characterizing or analyzing the sensing performance of the antibody-immobilized CNC. In this work, QCM-D was used to follow each step of the immobilization scheme and antigen-binding in real time. QCM-D also enabled quantification of the amounts of antibody and antigen-binding. In addition, thermal gravimetric analysis (TGA) and Fourier Transform Infrared Spectroscopy (FTIR), coupled TGA-FTIR, and microscopy methods were used to characterize the results of each step in the protocol. In addition, fluorescence anisotropy (FA) was used to explore the selectivity of the antibody-antigen binding. All antibodies were successfully immobilized on both dispersed CNC and CNC films. While immobilization on the films was the simpler, greater immobilization and antigen-binding were achieved on dispersed CNC. This is attributed to the accessibility of the entire CNC surface area for functionalization in the dispersions; in contrast only the outermost CNC surfaces were accessible in the solid films. However, APTES functionalization of dispersed CNC tended to result in cross-linking and aggregation; this was mitigated by running the reaction at relatively dilute conditions. The extent of aggregation was characterized using optical microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS). In terms of differences between the model antibodies, CEA antibody showed the highest affinity to its immunogen (CEA) whereas the antibodies PSA and AFP showed cross-reactivities with other antigens from the QCM-D and FA results. This research provides novel insights into the challenges and benefits of immobilizing antibodies on CNC using different approaches and provides a framework for detailed analysis of the process using QCM-D in addition to more standard analytical techniques. This pioneering research study also highlights the promise of using antibody immobilization on CNC for biomarker detection using piezo-based methods such as QCM-D and microcantilever arrays.