Efficient bioconjugation of protein capture agents to biosensor surfaces using aniline-catalyzed hydrazone ligation

Abstract

Aniline-catalyzed hydrazone ligation between surface-immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method for attaching protein capture agents to model oxide-coated biosensor substrates. Silicon photonic microring resonators are used to directly evaluate the efficiency of this surface bioconjugate reaction at various pHs and in the presence or absence of aniline as a nucleophilic catalyst. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, allowing derivatization of substrates with reduced incubation time and sample consumption. This increase in antibody loading directly results in more sensitive antigen detection when functionalized microrings are employed in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH-dependent noncovalent binding trend that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent.

Figure 1

Surface reaction scheme for conjugating antibodies onto oxide-passivated silicon photonic microring resonators via hydrazone ligation. (a) 3-N-((6-(N′-Isopropylidenehydrazino))-nicotinamide)propyl triethyoxysilane (HyNic silane) is immobilized onto the silicon/silica surface. (Note that the free hydrazine is protected as an acetone hydrazone, 1.) (b) In parallel, proteins are modified with succinimidyl 4-formylbenzoate (S-4FB) via their primary amines, 2; and, if added, aniline forms a Schiff base, 3. (c) The 4FB-modified protein is conjugated to the HyNic-modified silicon/silica surface via the transimination of the Schiff base with the hydrazine to form the hydrazone linkage, 4.

Figure 2

Real-time monitoring of the shift in resonance frequency for ten microrings within the same sample flow chamber during organic modification via reaction with HyNic silane. The chamber was initially filled with 100% ethanol and a solution of 1 mg/mL HyNic silane in 95% EtOH and 5% DMF introduced under flow (5 μL/min) at t = 8 min. After 30 min, the HyNic silane was flushed from the chamber and microrings returned to 100% ethanol. The residual wavelength shift corresponds to immobilized HyNic silane.

Figure 3

Real-time shifts in resonance wavelength from representative microrings upon covalent immobilization of anti-(human thrombin) antibody onto the sensor surfaces at three different pHs and in the presence (a) and absence (b) of 100 mM aniline. In each trace the sensors were initially incubated in buffer (with or without aniline, respectively) at a flow rate of 30 μL/min and a 5 μg/mL solution of 4FB-modified antibody with or without 100 mM aniline was flowed for 30 min. After switching back to the original buffer for 20 minutes, non-covalently attached antibody was removed with a low pH (pH 2.2) glycine buffer rinse. The sensors were then returned to the respective buffer at t = 57 min to determine the residual net shift corresponding to the amount of covalently immobilized antibody.

Figure 4

Time-resolved response from human α-thrombin binding to microrings modified with anti-(human thrombin) antibody at pH 7.4 in the presence (a) and absence (b) of 100 mM aniline. The microrings are initially in BSA-PBS buffer and thrombin is introduced at t = 5 min. Thrombin is then introduced and incubated for 10 min, after which BSA-PBS buffer is flowed to observe antigen dissociation. The remaining antigen-antibody interactions on the surface are then disrupted via exposure to a low pH (pH 2.2) glycine buffer for 2 min (not shown) to regenerate the sensor for subsequent detection experiments.