Nicotinoproteins are a group of NAD+-dependent dehydrogenases that bind NAD+ tightly and catalyze reactions without using free NAD+. In this study, we investigated the role of the unique insertion loop in nicotinoproteins. Carveol dehydrogenase (CADh), a short-chain dehydrogenase/reductase (SDR) nicotinoprotein, and β-hydroxybutyrate dehydrogenase from Alcaligenes faecalis (AfBHBDh), a non-nicotinoprotein counterpart, were used as model enzymes. An insertion loop-deleted mutant, CADh Δ39-49, was constructed. An insertion loop from Mycobacterium paratuberculosis CADh (MpCADh) was introduced into AfBHBDh to generate the two mutants. The results showed that CADh Δ39-49 lost NAD+ tight binding capacity and could not utilize free NAD+. In contrast, the AfBHBDh mutants showed no dye-mediated dehydrogenase activity. Moreover, the KM and KD values for NAD+ were higher than those of the wild-type enzyme. Docking simulations revealed a stronger binding affinity between NAD+ and the mutants than with the wild-type AfBHBDh. Taken together, these results suggest that the insertion loop interferes with NAD+ entry into the active site of the enzyme while creating a more energetically favorable binding environment. This loop is necessary but alone is insufficient to achieve NAD+ tight binding. This study deepens understanding of NAD+ binding in SDR nicotinoproteins and provides insights for SDR enzyme engineering.

The growth of wearable electrophysiology is accelerating demand for gel-free biopotential electrodes that are skin-conformal, comfortable for extended use, and stable under typical human motion. Here we report sigma POMaC, a self-adhesive, conductive, skin-compatible elastomer based on poly(octamethylene maleate (anhydride) citrate) (POMaC), a citrate-derived polyester with mechanical characteristics that are easily tunable via changes to monomer ratios and curing. Although POMaC is readily processed into soft structures, achieving robust electronic conductivity that survives curing, drying, and handling remains challenging, hindering its use in bioelectronic interfaces. To address this, we co-formulate a soft conductor, poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS), with a surfactant, 4-dodecylbenzenesulfonic acid (DBSA), into the POMaC prepolymer to generate conductive sigma POMaC composites. Because these additives affect processing parameters and material properties, such as curing time and viscoelasticity, we optimized composition and processing to jointly achieve high conductivity (50 S/cm; similar to 0.02 Omega & centerdot;cm), skin-appropriate adhesion (0.013 +/- 0.004 N/mm on PDMS), and elastomeric compliance suitable for biopotential recording. Using optimized sigma POMaC, we fabricated a custom chest patch featuring conformal ECG electrodes and demonstrated clear, high-fidelity on-body ECG waveforms comparable in morphology and timing to simultaneous recordings made using commercial Ag/AgCl electrodes. Together, these results position sigma POMaC as a material platform for gel-free, self-adhesive, skin-interfaced bioelectronic electrodes, enabling simplified application and improved interface with the end-user and reducing disposable hydrogel waste in longitudinal monitoring.

We developed a rapid, convenient, and viable cell-selective sensing system for Escherichia coli (E. coli) based on a sequential enzymatic reaction on an intact cell surface. In food safety and infectious disease diagnostics, it is crucial to selectively detect viable cells that represent actual infection risk. However, conventional methods such as culture-based method and polymerase chain reaction are time-consuming and require specialized equipment, limiting their applicability for on-site testing. The proposed system is based on a cooperative reaction of antibody-enzyme complexes, in which chemiluminescent signals are generated only when two types of antibody-enzyme complexes localize in proximity on the bacterial surface. The system allows for homogeneous detection of E. coli simply by mixing reagents, eliminating washing and separation steps. The chemiluminescence intensity showed a concentration-dependent response and an approximately linear relationship with log (CFUmL-1) over the range of 1-104 CFU mL-1. Importantly, the signal was generated only in the presence of viable E. coli cells, indicating that the cooperative enzymatic reaction proceeds selectively on intact bacterial surface. This property allows clear discrimination between viable and dead cells, which has been difficult with conventional methods. Overall, the proposed approach provides a rapid, convenient, and viable-cell-selective platform for on-site microbial monitoring and risk assessment.

Biosensors, utilizing glucose oxidoreductases as biological recognition elements (BREs), underpin current blood glucose monitoring (BGM) and continuous glucose monitoring (CGM) technologies. Glucose oxidase remains the gold standard due to its high selectivity and stability, yet its oxygen dependency limits it to first- and second-generation sensing. Fungal flavin adenine dinucletotide (FAD)-dependent glucose dehydrogenase (GDH) has emerged as the standard for mediator-based second-generation sensors due to its oxygen insensitivity, though it requires further engineering to match the stability and selectivity for CGM applications. Recent research increasingly prioritizes third-generation sensors capable of direct electron transfer (DET). Bacterial FADGDHs are premier candidates, possessing intrinsic DET capabilities via dedicated covalently bound heme subunits, while cellobiose dehydrogenase represents a rising star utilizing non-covalently bound heme b as a built-in mediator. Both require extensive protein engineering to overcome inherent limitations in substrate specificity and catalytic efficiency toward glucose. This review introduces the protein and enzyme sciences of glucose oxidoreductases, which have been and expected to be applied to blood, interstitial fluid, and other biological fluids. It reviews their reported and expected sensing modalities and engineering strategies, including rational design and chimera construction, to enhance enzyme robustness, selectivity, and the availability of electron acceptors, including DET ability. It also introduces perspectives on future engineering approaches to create ideal glucose-sensing oxidoreductases. This review article is an updated version of “Review of Glucose Oxidases and Glucose Dehydrogenases: A Bird’s Eye View of Glucose Sensing Enzymes,” which was published in 2011 in the Journal of Diabetes Science and Technology (JDST) ( https://doi.org/10.1177/193229681100500507 ).

We established a rapid and convenient electrochemical detection system for quantifying mouse fecal hemoglobin to evaluate colitis severity. A human myoglobin-binding aptamer was cross-reactive with mouse hemoglobin, and coupled with redox enzymes, enabled sensitive detection in feces (12.5-200 nM) within 20 min. This highlights the potential of cross-species hemoglobin detection.

Wash-free immunoassays with enzyme-linked immunosorbent assay (ELISA)-level performance remain challenging because peroxidase-based signal generation typically requires multiple washing steps and the handling of unstable H2O2. Here, we report a bead-based and glucose-fueled ELISA-like platform that co-localizes target capture and catalytic amplification on biogenic magnetic nanoparticles (MNPs) with peroxidase-like activity from magnetotactic bacteria. To generate biogenic antibody-displaying magnetic nanoparticle (bioMagbody), we used the SpyCatcher/SpyTag (SC/ST) system as a covalent protein coupling module. SC was genetically displayed via fusion to the abundant membrane protein Mms13 on the biogenic MNPs, enabling covalent ligation of ST-fused single-chain variable fragments (scFvs) to generate bioMagbody. SC expression and scFv display preserved the intrinsic peroxidase-like activity and target binding. Using glucose oxidase (GOx)-scFv complex as the H2O2 donor and bioMagbody as the acceptor, C-reactive protein (CRP) was quantified in buffer with a limit of detection of 4.5 nM, and specific detection was demonstrated in human serum by simple mixing without washing steps. The wash-free operation arises from target-localized H2O2 generation by GOx-scFv and proximity-enabled transfer to bioMagbody, which drives luminol chemiluminescence upon glucose addition as the reaction initiator. The platform was readily reconfigured to detect inactivated SARS-CoV-2 by exchanging scFvs. This work establishes a genetically programmable biogenic nanozyme interface for wash-free immunoassays.

L-glutamate (L-Glu) is the primary excitatory neurotransmitter in the mammalian central nervous system. Developing a real-time monitoring system is essential to understanding the onset and progression of related conditions. However, the absence of an L-Glu dehydrogenase that is insensitive to oxygen limits the development of oxygen-independent electrochemical enzymatic sensors. Additionally, the most commonly used L-Glu-specific oxidase requires site-specific proteolytic post-translational modifications in specific host microorganisms, which makes protein engineering difficult. To address these issues, L-Glu oxidase derived from Streptomyces mobaraensis (SmEOx), which does not require post-translational modifications, was engineered to function as a dehydrogenase. Residues crucial for the oxidative half reaction with oxygen in SmEOx were identified, and mutagenesis studies were conducted. Mutant SmEOx variants with suppressed oxidase activity and improved dye-mediated dehydrogenase activity compared to the wild-type enzyme were successfully obtained. The ratio of dehydrogenase activity to oxidase activity (Dh/Ox) increased ~2900-fold in mutant M117I and ~6700-fold in mutant M117F/K400N compared to wild-type recombinant SmEOx. The resulting virtually L-Glu dehydrogenases (vEDHs) were modified with a redox mediator and evaluated using transient open-circuit potential (OCP)-based L-Glu measurements. As a result, the vEDH (M117F/K400N mutant)-immobilized electrode enabled electrochemical L-Glu detection under ambient oxygen without the need for an external electron mediator, unlike the wild-type enzyme. The created vEDH, together with the OCP sensor developed using it, paves the way for future development of miniaturized, real-time L-Glu monitoring systems with high temporal and spatial resolution.

Potentiometric bioelectrochemical sensors offer advantages over amperometric ones due to their size-independent Nernstian response. However, their reliance on Ag/AgCl reference electrodes presents instabilities with environmental changes and prolonged use. Ag/AgCl instabilities are due to their reliance on a stable equilibrium between Ag+ and Cl- which may shift with fluctuations in chloride. To address this issue, we propose a reference electrode that incorporates an "inactivated" biological recognition element (inBRE) harboring identical redox species to the active BRE on the working electrode but lacking catalytic activity, thereby expecting a shift in its redox potential symmetrically with the working electrode, termed as a bioreference electrode. As proof of concept, we developed a potentiometric glucose bioelectrochemical sensor using a genetically and structurally inactivated direct electron transfer (DET)-type Burkholderia cepacia glucose dehydrogenase (inBcGDH) as the inBRE. Mutations (αHis476Ala/Asn519Ala) rendered BcGDH catalytically inactive while preserving its redox cofactors, creating a stable redox potential against which to measure. The inBcGDH was covalently immobilized on a 2 mm diameter gold electrode to form a bioreference electrode and integrated into an open-circuit potential (OCP)-based glucose sensor, which was evaluated for stability over 10 days and tested in various environmental factors (ionic strength, pH, and temperature) against traditional Ag/AgCl electrodes. Compared to Ag/AgCl, which experienced 67% signal loss over 10 days, the bioreference electrode exhibited only 23% loss. Additionally, after glucose addition (1 mM) in NaCl concentrations ranging from 50 to 500 mM, the bioreference electrode had only 6.2% signal bias, while Ag/AgCl showed up to 100% signal bias. Further, the bioreference electrode was able to be miniaturized using a 100 μm electrode, maintaining a linear signal response with glucose addition. These results demonstrate the bioreference electrode's ability to enhance potentiometric sensor stability and accuracy under varying conditions.

Levodopa, the primary treatment for Parkinson’s Disease, has a narrow therapeutic window further complicated by the lack of real-time feedback, primarily due to the absence of an enzyme specific to levodopa. We addressed this by developing a novel direct electron transfer type (DET) enzyme, copper dehydrogenase (CoDH), engineered from an extremophile derived multicopper oxidase (MCO), for use in a continuous levodopa sensor. By introducing mutations into the type 2 and type 3 copper ligand histidine residues, the enzyme drastically decreased its oxidase activity while enhancing DET activity with the electrode. Using this developed CoDH, a chronoamperometric levodopa sensor was constructed, which was minimally affected by environmental changes, or by interferents, including levodopa metabolites, adjunct medications, and common plasma and interstitial fluid components. A miniaturized levodopa sensor was constructed and was able to detect levodopa as low as 138 nM, suggesting its future application for in vivo subcutaneous measurement. The multicopper oxidase protein from Pyrobaculum aerophilum (McoP) was mutated, yielding copper dehydrogenase (CoDH). CoDH was then utilized to prepare a levodopa sensor across the entire physiologically relevant levodopa range.

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.

Anticancer bispecific antibody (BsAb) fragments are effective therapeutic agents because of their high tissue penetration and cytotoxic effects, which can be produced in cost-effective bacterial hosts. We used the cyanobacterium Synechocystis sp. PCC6803 (PCC6803) as an alternative host for sustainably producing BsAb fragments from CO2. Cyanobacteria are sustainable and independent of external carbon sources such as glucose, because of producing substances via photosynthesis. Cyanobacteria can be cultured using wastewater or seawater, reducing freshwater consumption and the environmental impact of the process, suggesting that cyanobacteria can be used as a sustainable BsAb production platform. We examined the production of various antibody fragments, namely, single-chain bispecific diabody (scBsDb), single-chain fragment variable (scFv), variable domains of the heavy-chain antibody VHH, and bispecific tandem VHH (BsVHH), using PCC6803. scBsDb showed partial antigen-binding ability and no cytotoxic effects. In contrast, all scFvs and VHH had binding abilities and we prepared BsVHH up to 2.4 mg/L culture. BsVHH showed binding activity with the targets and cytotoxic effects against carcinoma, unlike scBsDb. Our results highlight the suitability of VHH and VHH-based engineered antibody fragments for using PCC6803 and indicate their potential for producing other therapeutic proteins. We report producing functional BsAb fragments using cyanobacteria. Our findings contribute to developing cyanobacterial bioprocesses for producing therapeutic BsAb fragments, expanding the possibilities for sustainably producing valuable materials from CO2.

Integration of direct electron transfer-type (DET-type) Burkholderia cepacia glucose dehydrogenase (BcGDH) with an extended gate field effect transistor (EGFET) transducer to measure glucose in human plasma is a promising approach to overcome technology limitations in commercial continuous glucose monitors (CGM). Sensors were fabricated using microwire electrodes and were characterized for selectivity against interferents, reversibility, stability, and validated ex vivo. DET-type EGFET sensors showed low signal bias against a variety of interfering compounds and demonstrated acute reversibility and stability, while also successfully measuring glucose ex vivo in human plasma with a limit of detection of 0.94 mM. The DET-type EGFET glucose sensor was operated ex vivo over a physiological concentration range, demonstrating the feasibility of using EGFET-based transduction of DET-BcGDH for future use in CGM applications.

Glycated albumin (GA) provides unique advantages, offering a mid-term reflection of glycemic status over 2-3 weeks. GA is defined as the percentage ratio of glycated human serum albumin (GHSA) to total albumin, computed as the sum of GHSA and non-glycated human serum albumin (HSA). Current GA measurement methods rely on multi-step enzymatic assays requiring proteolytic digestion and separate quantification of GHSA and total albumin, limiting their applicability to point-of-care testing (POCT) platforms. In this study, we introduce an innovative electrochemical aptamer-based (E-AB) sensing method for GA monitoring. This approach enables single-step GA measurement using a single aptamer on a single electrode, without the need to quantify GHSA and HSA separately. This method leverages a novel analytical parameter, referred to as the "evolution", which is based on the observation of distinct behaviors of the aptamer, revealing a rapid response for HSA and a slower and more complex interaction for GHSA. Using the evolution parameter, we developed a robust method for determining glycation ratios (10 %, 20 %, and 40 %) irrespective of total albumin concentration fluctuations within clinically relevant ranges (5.26-7.52 μM). The developed E-AB sensor shows statistically significant differences in the evolution signal (p - value < 0.05) across all three glycation ratios, with a coefficient of determination greater than 0.94. The sensor has a limit of detection (LOD) of 10.6 % across 9 replicate sensors. This work represents an advancement in GA monitoring, offering a rapid, accessible, and reliable tool suitable for decentralized diagnostic applications, including POCT and personal healthcare devices.

The biosensors are generally described as analytical devices that transduce a signal derived from molecular recognition by biological recognition element (BRE) to quantify analytes or species in samples by combining varieties of transducers with different detection modalities. Therefore, the characteristics of biosensors are highly dependent on BREs. BREs are categorized into two types; a biocatalytic type BRE (BioCat-BREs) and a bioaffinity type BRE (BioAff-BREs). Following the currently most successful biosensors, the sensors for continuous glucose monitors (CGMs) for diabetes management, the development of biosensors for continuous sensing of biomarkers and drugs, such as small molecule drugs, peptides and proteins such as therapeutic antibodies is of increasing interest to both researchers and clinicians. However, unlike glucose oxidoreductases and the concentration of glucose in the mM range, the target molecules for the future continuous monitoring system require the development of innovative BioCat-BREs, which ideally are direct transfer type oxidoreductases, and BioAff-BREs which maintain their high affinity and specificity to the target while their binding site is regenerable under in vivo sensor operating condition, as well as the development of new modalities and devices to detect targets in the µM - pM range.

Electrochemical impedance spectroscopy (EIS) represents a promising and rapidly growing biosensing technique, enabling researchers and clinicians to perform label-free analyte detection; however, the scarcity of small, low-cost, and energy-efficient mobile devices for impedance measurement presents a major obstacle to further utilization of this method. Many EIS analog frontend (AFE) integrated circuits meeting these criteria are not commercially available; equivalent discrete circuits are frequently too expensive, large, or energy-inefficient for broad deployment. We present a discrete EIS-AFE which encodes impedance magnitude and phase as DC potentials; our AFE is optimized to minimize energy expenditure (<21 μJ per point at 10 kHz), size (<91 mm ^2 for the detector circuits), computational overhead (requiring only three ADC samples), and design complexity to target edge sensing applications (such as single-frequency EIS). We characterize the performance of the custom AFE, perform a comparative power analysis, and demonstrate successful EIS sensing using a series of dummy cells and a synthetic tissue analog saturated with artificial sweat. Our AFE enables accurate acquisition of impedance data with considerable power and cost savings relative to similar devices, while enabling modular expansion of the system to facilitate EIS sensing in a variety of mobile sensing applications.

β-hydroxybutyrate binding proteins (BHBBPs) are a newly identified group of periplasmic solute-binding proteins (SBPs) that interact with β-hydroxybutyrate (BHB), a key physiological metabolite. In this study, we systematically characterized the interaction properties of both previously reported and newly identified BHBBPs, including "NovoS" and "EDC10" from Gram-negative bacteria. Following recombinant production, we assessed the specificity and affinity of these proteins against a library of 23 different metabolites using a label-free derivative of differential scanning fluorimetry (nanoDSF). Positive interactions were further evaluated for their binding affinity via tryptophan fluorescence spectroscopy, which confirmed D/L-BHB as the preferred ligand for all proteins, with slight enantioselectivity. BHBBPs also exhibited binding to other compounds such as acetoacetate, D/L-α-hydroxybutyrate, L-lactate, and pyruvate, albeit with reduced affinity. These findings expand the classification of BHBBPs, suggesting that similar proteins and associated transporters may be widespread in prokaryotes involved in the carbon cycle of polyhydroxybutyrate. Guided by the crystal structure of the homologous BMA2936 protein, we introduced targeted point mutations in conserved polar residues of the BHBBPs EDC24 and NovoS. It was determined through this experimental pipeline that their affinity towards BHB was reduced by a factor between 25 and 750, shifting their binding constants towards the millimolar range. Collectively, the affinities of both wild-type and mutant proteins span 4 orders of magnitude, from nanomolar to millimolar recognition of BHB. Leveraging the versatility of SBP-based biosensing, these receptors and their wide affinity range could facilitate the development of effective bioanalytical tools for BHB detection in diverse physiological environments.

Therapeutic monoclonal antibodies (mAbs) exhibit significant interindividual pharmacokinetic (PK) variability, necessitating therapeutic drug monitoring (TDM). However, current monitoring methods require complex and time-consuming laboratory analysis, making rapid dose adjustments challenging. This review discusses emerging technologies for on-site mAb monitoring, with a focus on recent advances in recognition elements and detection strategies. Novel recognition elements, such as anti-idiotype molecules and meditopes, enable specific detection, whereas direct detection methods that eliminate washing steps facilitate rapid bedside testing. We examine the prospects for continuous mAb monitoring based on current technological advances and regulatory considerations. Although challenges regarding sensor regeneration and stability remain, the technologies discussed hold promise to enable real-time therapeutic monitoring at the bedside.

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

Conventional genetic engineering approaches for bacterial metabolic pathway manipulation, although highly applicable, still face limitations including metabolic burden, irreversibility, and dependency on host cellular machinery. Cell-penetrating peptide-peptide nucleic acid conjugates (CPP-PNAs), known for their applicability as antibacterial tools and in the elucidation of protein function, offer a promising alternative to overcome such limitations. Since the application of CPP-PNA in metabolic engineering and pathway elucidation remains largely unexplored, we developed and validated a CPP-PNA platform using Synechocystis sp. PCC 6803 as a model system to demonstrate targeted metabolic pathway evaluation. High compatibility and dose-dependent permeation efficiency in strain PCC 6803 was first observed when the amphipathic CPP (KFF)₃K was employed, achieving clear cell growth inhibition at 10 µM and above. Specific targeting of D-lactate dehydrogenase (Ddh) using CPP-Syn6803ddh conjugates achieved near-complete protein translation knockdown within 24 h, as confirmed by Western blot analysis. Metabolomics analysis using LC-MS on predetermined metabolites revealed that CPP–PNA treatment produced metabolic effects comparable to stable genetic knockout strains, with both approaches showing a significant 2.5-fold increase in pyruvate accumulation compared to wild-type controls. Further elucidating the reason for pyruvate accumulation, we observed compensatory activation of the glyoxalase pathway at 48 h post-treatment, resulting in 3-fold increased D-lactate production presumably through methylglyoxal detoxification. Validating this observation, RT-qPCR analysis confirmed 2-3-fold upregulation of the glyII gene, encoding for the glyoxalase II (GlyII) enzyme, in both CPP-PNA treated and knockout strains, while double CPP-PNA inhibition experiments targeting both Ddh and glyoxalase pathways suppressed D-lactate accumulation. This study establishes CPP-PNAs as efficient tools for rapid, and simple metabolic pathway investigation. The approach produces results comparable to conventional genetic knockouts while offering dose-dependent control and avoiding permanent genomic alterations. Our findings reveal unexpected metabolic complexity in Synechocystis sp. PCC 6803 D-lactate synthesis under light conditions and demonstrate the utility of CPP-PNA for uncovering compensatory pathway activation. This platform represents a valuable addition to bacterial genetic engineering, addressing some of the critical limitations faced by conventional approaches, while showing potential for further understanding the biochemistry of metabolite-producing bacteria.