Continuous Monitoring of Therapeutic Monoclonal Antibodies Using Photo-Regenerable Aptamer Based Electrochemical Sensors

Abstract

Introduction Therapeutic monoclonal antibodies (mAbs) have become central to oncology, providing targeted treatment with less toxicity compared to chemotherapy. Bevacizumab, a humanized mAb against vascular endothelial growth factor A (VEGF-A), is widely prescribed, yet patient outcomes vary greatly due to substantial interpatient pharmacokinetic variability. Serum half-lives have been reported from less than two to more than seven weeks, meaning that fixed dosing can leave some patients underexposed and others at risk of toxicity. Blood concentration of bevacizumab has been linked to treatment efficacy; thus, monitoring circulating bevacizumab could allow individualized dose adjustments to maintain concentrations within an optimal therapeutic window. Our group previously developed a single-use type electrochemical aptamer-based sensor for point-of-care (POC) testing using an anti-idiotype aptamer (A14#1) that selectively binds the complementarity-determining region of bevacizumab [1]. Integrating this aptamer into our sensor and utilizing square wave voltammetry (SWV) to measure signal changes upon binding enabled rapid detection over the physiologically relevant range. While effective for single-timepoint measurement, the problem with the POC format was that it can only capture a snapshot of the concentration upon binding of the drug to the aptamer and requires repeated assays to track mAb levels [2]. This limits the ability to detect concentration changes over an extended period or adjust dosing in real time. In the present work, we are adapting the A14#1 platform for continuous bevacizumab monitoring by incorporating photo-responsive azobenzene into the aptamer sequence. Azobenzene’s reversible trans-cis photoisomerization allows light-controlled switching between high-affinity (binding) and low-affinity (release) states [3]. This enables the aptamer to repeatedly bind and release bevacizumab, allowing multiple measurements from the same sensor surface without replacement, ideally for prolonged use during a patient’s treatment period. Materials and Methods BLI Evaluation Biolayer interferometry (BLI) was used to characterize binding between aptamers and bevacizumab. Biotinylated aptamers were immobilized on streptavidin-coated biosensors, and association/dissociation was monitored upon exposure to the bevacizumab. UV light was applied to the aptamer solution to induce trans–cis isomerization, and changes in binding/release of bevacizumab were recorded. Electrode Preparation with Aptamer Immobilization For preparations of aptamer-immobilized electrodes for testing concentration-dependent response with SWV and regeneration ability of the sensor surface, gold electrodes were mechanically polished and electrochemically cleaned. Thiolated A14#1 aptamers were immobilized onto the electrode surface overnight, followed by blocking with 6-mercaptohexanol (MCH) to minimize nonspecific adsorption. Prepared electrodes were stored in buffer until use for electrochemical experiments. Electrochemical Sensing with Ferricyanide Electrochemical measurements were performed using square wave voltammetry (SWV) with ferricyanide as a free redox probe. To assess photo-regeneration, we first record a baseline SWV signal from azobenzene-modified, aptamer-immobilized gold electrodes. Following this, the bevacizumab target is added to the solution at a specific concentration. Next, we apply UV light to induce azobenzene trans to cis isomerization, which disrupts the aptamer/drug complex and enables target release. Immediately afterward, we re-measure SWV; recovery of the redox current indicates photo-induced regeneration of the sensing surface. Multiple cycles of UV application and restoration of the peak current will determine the limits of the aptamer’s regeneration capability. Results and Discussion Preliminary BLI results suggest that azobenzene-modified A14#1 aptamers show a reduced binding affinity to bevacizumab after the application of UV light (Figure 1). Following these results, we will further analyze the effect of UV light on the azobenzene-modified bivalent A14#1 aptamer and investigate whether the peak current can be restored after UV application, to assess the regeneration capability of the sensor surface for continuous monitoring purposes. Compared to our previous point-of-care design, we aim to develop this continuous format to capture complete pharmacokinetic profiles from a single wearable sensor, detect unexpected fluctuations in concentration, and allow timely dose adjustments. We also aim to eliminate the need for repeated electrode preparation. By combining anti-idiotype aptamer specificity and azobenzene-controlled reversibility, this system is designed to enable patient-specific bevacizumab monitoring. The same approach could be adapted for continuous tracking of other therapeutic antibodies with variable pharmacokinetics. References: [1] M. Nagata, J. Lee, T. Saito, K. Ikebukuro, and K. Sode, “Development of an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody monitoring,” International Journal of Molecular Sciences , vol. 24, no. 6, p. 5277, 2023, doi: 10.3390/ijms24065277. [2] M. Nagata, E. D. Wilson, K. Ikebukuro, and K. Sode, “Challenges in realizing therapeutic antibody biosensing,” Trends in Biotechnology , advance online publication, 2025, doi: 10.1016/j.tibtech.2025.07.003. [3] X. Liang, T. Mochizuki, and H. Asanuma, “A supra-photoswitch involving sandwiched DNA base pairs and azobenzenes for light-driven nanostructures and nanodevices,” Small , vol. 5, no. 15, pp. 1761–1768, 2009, doi: 10.1002/smll.200900223. Figure 1