Antibody Immobilization on Sulfated Cellulose Nanocrystals

Abstract

Exploiting cellulose nanocrystals’ high aspect ratio and tailorable surface for immunological biosensors has been hindered by the relatively limited research on using commonly available sulfated cellulose nanocrystals (CNCs) for antibody immobilization and by the low hydrolytic stability of dried assemblies produced from sulfated CNCs. Herein, we report a reaction scheme that enables both hydrolytic stability and antibody immobilization through 3-aminopropyl-triethoxysilane and glutaric anhydride chemistry. Immobilization was demonstrated using three model antibodies used in the detection of the cancer biomarkers: alpha-fetoprotein, prostate-specific antigen, and carcinoembryonic antigen. Thermogravimetric analysis coupled with Fourier-transform infrared spectroscopy provided evidence of CNC modification. Quartz crystal microbalance with dissipation monitoring was used to monitor binding during each step of the immobilization scheme as well as binding of the corresponding antigens. The general reaction scheme was tested using both aqueous CNC dispersions and CNC films. Film modification is slightly simpler as it avoids centrifugation and washing steps. However, modifying the dispersed CNCs provides access to their entire surface area and results in a greater capacity for antigen binding.

Properties of APTES-Modified CNC Films

“…Some aggregates were large enough to distort cross-polarized optical microscopy images. Previous work showed that CNC-APTES aggregates became larger and more numerous with increasing concentration, which could result in less available surface area for further modification and nonuniform macroscale properties. The CNC and APTES concentrations used in this study were optimized by Nori, to achieve enough colloidal stability for further surface reactions …”

“…Based on a previously developed method, , the as-received CNC dispersion was added in an APTES plus solvent (water–ethanol, 80:20 v/v) mixture at a mass ratio of 1.8:1 after adjusting the pH to 4.0 with the addition of glacial acetic acid. It is noted that running the reaction with a higher APTES ratio increased aggregation, most likely due to APTES cross-linking the CNCs.…”

“…After this, several researchers utilized APTES-modified CNCs for uses such as immobilization supports for nanoparticles, coating agents to alter interphase interactions, and in nanocomposite membranes used for metal removal and dye adsorption . Nori et al used glutaric anhydride to link CNC-APTES that had been modified with antibodies used in cancer biomarker detection . The resulting CNC-APTES-antibody films had sufficient hydrolytic stability to enable prolonged quartz crystal microbalance with dissipation (QCMD) monitoring using phosphate-buffered saline as the medium .…”

“… 29 Nori et al used glutaric anhydride to link CNC-APTES that had been modified with antibodies used in cancer biomarker detection. 30 The resulting CNC-APTES-antibody films had sufficient hydrolytic stability to enable prolonged quartz crystal microbalance with dissipation (QCMD) monitoring using phosphate-buffered saline as the medium. 30 However, to the best of the authors' knowledge, the collective effects of APTES modification on CNC films’ hydrolytic stability, mechanical properties, and ability to act as nonspecific sensors or adsorbents have not previously been studied.…”

Cellulose Nanocrystal Composites with Enhanced Mechanical Properties for Robust Transparent Thin Films

“…Due to their high surface-to-volume ratio, cellulose nanomaterials can be chemically modified to provide surface functionalization and overcome the limits related to their hydrophilic nature, thus broadening the range of materials to which they can be combined and expanding the application possibilities. 15 In this regard, oxidation treatments, such as TEMPO-mediated oxidation (via 2,2,6,6-tetramethylpiperidin-1yl-oxyl), can be used to introduce carboxylic acid moieties in CNCs, which in turn can support additional modification by binding with different biomolecules, such as amino acids, 26 peptides, 27 or antibodies, 28 thus fostering potential biomedical applications.…”