Novel Rapid Protein Coating Technique for Silicon Photonic Biosensor to Improve Surface Morphology and Increase Bioreceptor Density

Silicon photonic devices with either silicon or silicon nitride waveguides have increasingly been used in many applications besides communications, especially as sensors in label-free biosensing, where guided light signals are affected by biorecognition molecules immobilized on the surface. The coating of protein (i.e., bioreceptors) by biochemical process on the waveguide surface is a crucial step in creating a functionalized device that can be used for biosensing. As a conventional method that uses 3-aminopropryltriethoxysilane (APTES) and glutaraldehyde (GA), the APTES-GA method has the limitation of using a GA crosslink, of which the two functional groups can bind to nonspecific proteins, causing irregular binding. In this study, we proposed a new coating technique to avoid such problem by applying APTES silanization with 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC)-N-hydroxysuccinimide (NHS) protein crosslink, denoted by the APTES-(EDC/NHS) method. The EDC/NHS reaction was shown to be able to immobilize protein in ordered orientation due to consistent arrangement between a carboxylic group of protein molecules and an amine group of covalent-linked APTES on surface. By applying APTES silanization, we circumvented the use of hazardous cleaning agent in the conventional EDC/NHS technique. Several surface characterization techniques were carried out to assess and compare the two biocoating techniques, including scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), spectroscopic ellipsometry (SE), and atomic force microscopy (AFM). On silicon, the results of antihuman TNF-alpha antibody coating showed that the proposed APTES-(EDC/NHS) technique has better repeatability in terms of less roughness of the coated protein at 1.5 nm compared with 6.3 nm, due to the ordered arrangement of coated antibody molecules. On a silicon nitride waveguide device, the proposed APTES-(EDC/NHS) technique exhibits dense antibody immobilization on a waveguide in SEM images due to stable amide bond formation via EDC/NHS crosslink mechanism. The specificity of the immobilized antibodies was confirmed by enzyme-linked immunosorbent assays (ELISA), with an average optical density at 450 nm of 0.175 ± 0.01 compared with 0.064 ± 0.009 of negative control. The proposed technique also reduced the overall process time since proteins are crosslinked to the silanized waveguide surface in a single step.