Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1–100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy.

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.

Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with K_D = 2.1 μM or K_D = 1.6 μM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

This study reports on the direct electron transfer (DET) ability of the enzyme spermidine dehydrogenase (SpDH) and its use in a DET-type enzymatic sensor for detecting spermine. SpDH was found to exhibit internal electron transfer from its cofactor, flavin adenine dinucleotide (FAD), to heme b. This was confirmed by observing the heme b-derived reduction peak at 560 nm in the presence of spermine, the substrate. SpDH was immobilized on a gold electrode via a dithiobis (succinimidyl hexanoate) self-assembled monolayer. The cyclic voltammetry analysis of the SpDH-immobilized gold electrode revealed an increased oxidation current in the presence of 0.1 mM spermine with an onset potential of −0.14 V vs. Ag/AgCl in the absence of an additional external electron acceptor. This result confirmed that SpDH is capable of DET. Chronoamperometric analyses were conducted using an SpDH-immobilized gold electrode with spermine as the substrate under a 0 V oxidation potential vs. Ag/AgCl using an artificial saliva matrix containing 10 µM ascorbic acid and 100 µM uric acid. The sensor exhibited good linear correlation between the current increase and spermine concentration from 0.2 to 2.0 µM, with a limit of detection of 0.084 µM, which encompasses the physiologically relevant spermine concentration found in the saliva. Primary structure alignments and 3D structure predictions revealed that all SpDH homologs possess two conserved histidine residues in the same location on the surface as the heme b ligand of SpDH. This indicates their potential for DET-ability with an electrode.

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

Therapeutic humanized monoclonal antibodies (mAbs) are focused on as effective medicine owing to their high specificity and efficacy compared with conventional small-molecule pharmaceuticals. To avoid side effects and improve the treatment efficacy, controlling the dose of therapeutic mAbs for the patient is the most crucial. However, the monitoring of their concentrations demands highly specific discrimination from human immunoglobulin G (IgG), since most therapeutic mAbs are designed based on IgG, which is naturally present in large quantities in human blood and differs only in residues at the complementarity determining region (CDR). Previously, we developed the therapeutic mAbs aptamer sensor utilizing an anti-idiotype aptamer, that recognizes the complementarity determining region, CDR, of a target therapeutic antibody, Bevacizumab [1,2]. This sensor can distinguish the target antibody and other human natural antibodies and succeed in detecting the bevacizumab from artificial serum that contains a high concentration of human natural IgG. Since this sensor does not require a chemical reaction nor a free redox probe in the solution, they are very suitable for in vivo continuous monitoring. However, due to the high binding constant (~nM-pM) of aptamer, binding site regeneration is only possible by denaturing both the target protein and aptamer structures with guanidine hydrochloride. Currently, the most challenging task for future therapeutic mAb sensors employing aptamers with a high binding constant to realize in vivo continuous monitoring is the development of versatile methods and technologies suitable for the in situ regeneration of aptamer binding sites. To tackle this challenging task, we have been engaged in the designing of engineered aptamers which can be modulated in their binding constant by external signals. Azobenzene is an ideal photoresponsive molecule, which reversibly changes its cis/trans forms, by UV or visible light irradiation and this structure change drives aptamer structure change and releases the target [3,4]. In this study, we designed an azobenzene-inserted anti-idiotype bevacizumab aptamer and developed an electrochemical sensor that can regenerate its binding site by light irradiation. To design the photo-regenerated anti-idiotype bevacizumab aptamer, we investigated the appropriate position to insert azobenzene without affecting the binding ability. After the confirmation of binding ability, we applied these aptamers to electrochemical measurement. Azobenzene-inserted anti-bevacizumab idiotype aptamers were immobilized on the planar electrode. Electrochemical measurements were carried out using a constructed aptamer electrode as a working electrode. After the confirmation of target binding on the electrode, we tried to regenerate the aptamer-immobilized electrode by UV light irradiation. The release of the target antibody was confirmed electrochemically. We will present the characterization of the photo-regenerated anti-idiotype aptamer, focusing on the change in binding toward the target therapeutic mAb, bevacizumab in UV and visible light irradiated conditions. Finally, we will show repeated detection of bevacizumab using this novel-engineered aptamer combined with electrochemical detection such as an extended gate FET-based sensor toward the continuous monitoring of a therapeutic mAb, bevacizumab. [1] Saito et al., Biosensors and Bioelectronics, 2022,203,114027 [2] Nagata et al., Int. J. Mol. Sci., 2023, 24(6) 5277 [3] Liang et al., JACS, 2003, 125, 16408 [4] Liang et al., Small, 2009, 5(5) 1761]