Impact of Silanization Parameters and Antibody Immobilization Strategy on Binding Capacity of Photonic Ring Resonators

Abstract

Ring resonator-based biosensors have found widespread application as the transducing principle in “lab-on-a-chip” platforms due to their sensitivity, small size and support for multiplexed sensing. Their sensitivity is, however, not inherently selective towards biomarkers, and surface functionalization of the sensors is key in transforming the sensitivity to be specific for a particular biomarker. There is currently no consensus on process parameters for optimized functionalization of these sensors. Moreover, the procedures are typically optimized on flat silicon oxide substrates as test systems prior to applying the procedure to the actual sensor. Here we present what is, to our knowledge, the first comparison of optimization of silanization on flat silicon oxide substrates to results of protein capture on sensors where all parameters of two conjugation protocols are tested on both platforms. The conjugation protocols differed in the chosen silanization solvents and protein immobilization strategy. The data show that selection of acetic acid as the solvent in the silanization step generally yields a higher protein binding capacity for C-reactive protein (CRP) onto anti-CRP functionalized ring resonator sensors than using ethanol as the solvent. Furthermore, using the BS3 linker resulted in more consistent protein binding capacity across the silanization parameters tested. Overall, the data indicate that selection of parameters in the silanization and immobilization protocols harbor potential for improved biosensor binding capacity and should therefore be included as an essential part of the biosensor development process.

Rapid detection of an Ebola biomarker with optical microring resonators

“…The steps of the developed immunoassay on the microring platform consist of sample introduction followed by secondary recognition and amplification steps, highlighted in Figure 2 A. Antibodies for EBOV sGP and Sudan virus (SUDV) sGP were functionalized in a multiplex fashion on the surface of the microrings, with each capture agent spanning 8–12 microrings to provide technical replicates. The sample of interest was introduced to the sensor surface through microfluidic fittings and automated with pumps as previously described ( Figure 2 A, step 1) ( Arnfinnsdottir et al., 2020 ; Medfisch et al., 2020 ; Mordan et al., 2020 ; Robison and Bailey, 2017 ; Robison et al., 2019 ; Wetzler et al., 2020 ). As the antigen is flowed across the surface of the sensor, the primary binding occurred as EBOV sGP was captured by the anti-EBOV sGP antibodies on the sensor surface.…”

Whispering-gallery-mode sensors for biological and physical sensing

“…These can include antibodies 145,151 , antibody fragments 152 , complementary nucleic acid aptamers 19 or other specialized recognition molecules. Most microring resonators are constructed of materials that can be functionalized using straightforward silane and bioconjugate chemistries [153][154][155] . Microring resonators have been used for label-free detection of targets including nucleic acids 18,19 , viruses 156 , proteins 145,151 , nanodiscs 157 , and telomerase activity 158 .…”

Substrate Materials for Biomolecular Immobilization within Electrochemical Biosensors

“…While silanization has the potential to create biointerfaces that are more stable than those obtained using Au-thiol chemistry, silanization has some shortcomings that suggest it may not be generally applicable to biofunctionalization. Silanization protocols are not well standardized, so results often vary with time, temperature, and solvent, and the choice of silane reagents and concentrations varies [34,117]. In addition, silanization depends critically on the hydration state, so the precursor may copolymerize in the presence of trace water, forming aggregates and disordered surface layers on the substrate.…”

“…In addition, silanization depends critically on the hydration state, so the precursor may copolymerize in the presence of trace water, forming aggregates and disordered surface layers on the substrate. The silane layer may also be unstable in aqueous media, undergoing hydrolysis of siloxane bonds [34,117].…”