A cyanobacteria-based living biophotovoltaic (LBPV) system was developed by integrating Leptolyngbya sp. with conductive polymer-gold nanoparticle-modified electrodes for simultaneous green energy generation and herbicide detection. The photoanode was fabricated through the electropolymerization of dithieno[3,2-b:2',3'-d] pyrrole derivatives, followed by the incorporation of aniline-functionalized AuNPs to enhance electron transfer. Optimization of the polymer thickness, AuNP loading, and cyanobacterial concentration revealed 60 electropolymerization cycles and 450 mg/mL cyanobacteria as the ideal parameters for photocurrent output. The biocathode, modified with bilirubin oxidase, enabled efficient oxygen reduction, ensuring stability and reproducibility. To extend the experimental findings, deep learning architectures (LSTM, BiLSTM, and GRU) were employed to model and forecast chronoamperometric photocurrent dynamics. Among all tested configurations, the BiLSTM-SGDM model exhibited the best predictive performance with R2 = 0.92, RMSE ≈ 48 μA, and MAE ≈ 38 μA on the test set, effectively capturing nonlinear variations and transient response behaviors of the LBPV system. The deep-learning-based predictions closely matched the experimental measurements, confirming the capability of AI-assisted models to reproduce complex photoelectrochemical kinetics. The optimized system produced stable photocurrents under visible light of ∼1 sun (1400 W/m2) and maintained 56% of its initial activity after 50 days. As a biosensor, the LBPV exhibited remarkable sensitivity with detection limits of 1.12 nM for diuron and 9.70 nM for linuron. The integration of AI-based photocurrent forecasting with biohybrid photovoltaic design offers a promising framework for next-generation sustainable energy and environmental monitoring systems. Interference studies further confirmed high selectivity against common environmental contaminants. These findings underscore the potential of LBPVs as dual-function devices, combining sustainable energy harvesting with highly sensitive photoelectrochemical biosensing of phenyl urea herbicides in aquatic environments.

Herein we report the self-powered biosensor for detection of dissolved oxygen (DO) detection using a paper-based enzymatic biofuel cell (BFC) employing screen-printed electrodes composed of MgO-templated mesoporous carbon (MgOC). The sensor used an anode modified by flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and a cathode modified by bilirubin oxidase (BOD) to enable selective oxygen reduction under glucose-rich conditions. Electrochemical analyses revealed a linear relationship between the cathodic current and DO concentration over the range of 0–22 mg L−1, with a maximum power output of 398 µW cm−2 at 20 mg L−1 DO. The biosensor system was successfully used to quantify DO in both pure water and a commercial soft drink, without requiring external power sources. These findings demonstrate the feasibility of low-cost, disposable, and scalable DO sensing by using cathode-targeting enzymatic BFCs, thereby opening new avenues for environmental and food quality monitoring.

Self-powered detection of T4 polynucleotide kinase activity based on DNA structure transformation-modulated direct electron transfer of bilirubin oxidase.

In high-risk neonates, such as very low birth weight infants or those undergoing abdominal surgery, elevated direct bilirubin (DB) levels are frequently observed. Under such conditions, unbound bilirubin (UB) measured using peroxidase-based analyzers may appear spuriously elevated, complicating clinical interpretation. Retrospective analysis was performed on laboratory datasets with complete measurements of total bilirubin (TB), DB, UB, and albumin from January 2021 to December 2023. DB was measured enzymatically using the Nescauto VL D-bil bilirubin oxidase method. Indirect bilirubin (iDB) was calculated as TB minus DB, and its molar ratio to albumin (iDB/albumin) was evaluated for correlation with UB across varying DB levels and DB/TB ratios. Outlier-high UB values were defined as those exceeding the 95% confidence interval of the iDB/albumin ratio within the physiological range (DB < 1 mg/dL and DB/TB < 10%). A total of 5970 datasets from 1386 neonates were analyzed. As DB levels and DB/TB ratios increased, the correlation between the iDB/albumin ratio and UB weakened, and the regression slope became steeper. The proportion of outlier-high UB values rose significantly: 4.9%, 10.8%, 32.5%, and 92.2% for DB <1, 1-2, 2-3, and ≥3 mg/dL, respectively; and 4.2%, 10.3%, 17.2%, and 51.7% for DB/TB <10%, 10%-20%, 20%-30%, and ≥30%. UB values tend to rise spuriously as DB increases, particularly when DB ≥2 mg/dL or DB/TB ≥20%. In such situations, estimating UB from the iDB/albumin ratio may provide a more reliable basis for risk assessment.

Enzymatic biofuel cell on flexible nanoporous gold electrodes.

Bilirubin oxidase nanoparticles’ (BOxNPs) were prepared from commercial bilirubin oxidase by using the ‘desolvation method’. An amperometric bilirubin biosensor was developed by immobilizing BO

Understanding the redox properties and catalytic behavior of proteins is critical for harnessing their functions in biocatalysis and to promote efficient bio-inspired catalysts design. Enzymatic X-ray absorption spectroelectrochemistry (XA-SEC) combines the insights of X-ray absorption spectroscopy with the precision of electrochemical methods to elucidate enzymes' redox properties and catalytic behavior. Here we describe how to perform enzymatic XA-SEC experiments. The procedure begins with the preparation of the carbon-based working electrode to enhance enzyme immobilization. We exemplify with the efficient immobilization of bilirubin oxidase from Myrothecium verrucaria on the electrode surface, utilizing nanomaterials to enhance biomaterial loading and electron-transfer at the enzyme-electrode interface. Next, we guide researchers through setting up a standard three-electrode electrochemical cell, ensuring proper electrical connections and electrolyte preparation. Our Protocol details the Cu K-edge X-ray absorption spectroscopy measurement procedure at the synchrotron light sources, with in situ electrochemical control. Real-time redox processes are monitored through direct electron transfer analysis, providing valuable thermodynamic and kinetic information. It is important to determine the stability and activity of the analyzed protein under X-ray beam exposure; our approach typically results in stable electrochemical and spectroscopic signals for long experimental runs, showcasing the enzyme's robust performance and efficient protein immobilization. The method's ability to correlate XA-SEC data with direct electron transfer and substrate-biding analysis provides a powerful tool for advancing our understanding of enzymatic electrocatalysis and opens new avenues for developing sustainable bioelectrochemical technologies.

Enzymatic biofuel cell (EBFC)-based self-powered sensors (SPSs) have emerged as a promising class of portable sensing devices; therefore, it is crucial to develop a novel and efficient strategy for the fabrication of EBFC-SPSs. Herein, we present a novel strategy for self-powered sensing by regulating the direct electron transfer (DET) of bilirubin oxidase (BOD) in a glucose-oxygen biofuel cell. The cathode is fabricated by immobilizing BOD on a gold nanoparticle (AuNP)-multiwalled carbon nanotube (MWCNT) nanocomposite by using an aptamer-complementary DNA (cDNA) duplex as a bridge. In the absence of aflatoxin B1 (AFB1), BOD remains distant from the AuNP-MWCNT nanocomposite, so the DET of BOD is hindered, preventing the catalytic reduction of oxygen at the cathode. In the presence of AFB1, the specific binding of the aptamer to AFB1 triggers its dissociation from the cathode, while the cDNA forms a hairpin structure due to the self-complementary sequences at both ends. The DNA structure switching brings BOD into close proximity with the AuNP-MWCNT nanocomposite, so BOD can effectively catalyze the reduction of oxygen at the cathode through DET. As a result, the biofuel cell transitions from the initial "open-circuit" to the "closed-circuit" state, enabling self-powered sensing of AFB1. The linear range for AFB1 detection is from 10-2 to 105 pg mL-1, with an ultralow detection limit of 3 × 10-3 pg mL-1. This work not only offers a novel strategy for self-powered sensing but also develops a portable device for fungal toxin detection.

This contribution studies enzymatically driven graphene monolayer swimmers for the direct transformation of biochemical energy into motion. The swimmers are elaborated via bipolar electrodeposition of gold and asymmetric immobilization of glucose oxidase and bilirubin oxidase on small pieces of a graphene monolayer. Most importantly, we demonstrate that the speed of the free-standing hybrid objects can be enhanced by an external magnetic field, allowing also controlled rotation, despite the absence of ferromagnetic construction elements. These results illustrate the potential of 2D materials for designing, in synergy with magnetic fields, original enzyme-based energy conversion devices.

Photosystem II (PSII) is a vital photosynthetic enzyme with the potential for sustainable bioelectricity and fuel generation. However, interfacing PSII with intricate, small‐scale electrodes for practical applications has been challenging. This study addresses this by creating protonated macroporous carbon nitride (MCN) as support and developing a scalable spray‐freeze method to wire PSII with MCN. This facilitates the production of large‐area MCN‐PSII photoanodes up to 33 cm2 for biophotoelectrochemical water oxidation to O2, achieving efficient interfacial charge transfer and initial photocurrents in the mA range with Faradaic yield of 93.5 ± 8.5% over 5 h. A bias‐free biophotoelectrochemical (BPEC) device is designed by connecting the MCN‐PSII photoanode to a carbon nanotube cathode loaded with bilirubin oxidase. An array of eight tandem BPEC cells with a photoactive area of 72 cm2 successfully powers low‐power electronics, such as LEDs. This work paves an efficient way for bioelectrode fabrication, showcasing the potential of PSII‐based semi‐artificial systems for BPEC and biophotovoltaic applications.

Programming catalytic behavior at the microbial genome level is a frontier in synthetic biology with direct impact on bioelectrocatalysis. A key challenge is the coordinated control of gene expression, localization, folding, and cofactor maturation required to achieve proper bioelectrocatalytic activity. Here, a synthetic operon in Escherichia coli is engineered to reprogram its surfaceome for selective water oxidation. Using orthogonal IPTG‐inducible control and codon‐optimized expression, a fungal bilirubin oxidase (BOD) displayed at the cell surface is produced by ice nucleation protein anchoring (BOD‐E. coli). Post‐overexpression copper catalytic site reconstitution provides an active holoenzyme. The developed engineered living material performs water oxidation at near‐zero overpotential (27 mV at pH 9.1), with complete suppression of the oxygen reduction reaction. These results show how regenerable microbial platforms can be designed for selective catalysis and artificial photosynthesis.

Directed evolution relies on iterative cycles of variant generation, screening, and selection to identify enzyme variants with improved activities. Droplet‐based microfluidics accelerates this process by enabling rapid screening of enzyme variants in water‐in‐oil emulsions acting as picoliter‐scale microcompartments. In fluorescence‐activated droplet sorting (FADS), single E.coli cells are screened using a fluorogenic substrate at high throughput (≈2 kHz). However, fluorogenic assays for enzymatic systems are limited, while absorbance‐based detection assays represent a larger spectrum. At the micron scale, light absorption is weak, and scattering induced by droplet interfacial curvature further decreases detection sensitivity. Measurements are therefore performed at a cost of increasing droplet sizes or acquisition times, which limits throughput to <1 kHz. Here, this challenge is addressed with a confocal Absorbance‐Activated Droplet Sorting (cAADS) system. The platform achieves sensitive absorbance measurements at ultrahigh throughput (5.4 kHz) from droplets as small as 10 pL, and sorting of 50 pL droplets at frequencies up to 2.6 kHz. The cAADS methodology is demonstrated by enrichment of active Bilirubin Oxidase (BOD) variants, with a sorting efficiency of 99%. Its versatility and potential for absorbance‐based microfluidic screening in enzyme engineering are also demonstrated using a different enzyme: Glucose Oxidase.

Design of a thermostable bilirubin oxidase from Myrotheciumverrucaria.

BackgroundBilirubin photoisomers, generated during phototherapy or incidental light exposure, may interfere with direct bilirubin (DB) measurement using the bilirubin oxidase method. This interference is particularly relevant in neonates, who physiologically exhibit elevated levels of unconjugated bilirubin.MethodsResidual serum samples from 30 neonates were irradiated under controlled conditions to selectively produce bilirubin configurational isomers (BCIs) and structural isomers (BSIs). DB and total bilirubin (TB) were measured pre- and post- irradiation using the bilirubin oxidase method. BCI and BSI concentrations were quantified using high-performance liquid chromatography (HPLC), and their contributions to DB values were evaluated using linear and multiple regression analyses.ResultsPost-irradiation, DB levels increased significantly in correlation with BCI and BSI concentrations. Approximately 11% of BCI and 32% of BSI were quantified as DB using the bilirubin oxidase method. These findings were consistent across both individual and multiple regression models.ConclusionsBilirubin photoisomers significantly influence DB values measured by the bilirubin oxidase method, potentially leading to overestimation of conjugated bilirubin. In neonatal care, accurate interpretation of DB values requires attention to sample handling and awareness of photoisomer interference, particularly under light-expose conditions.

Wearable electronics, such as biosensors and e-skin, have been gaining attention for health monitoring applications. Nanomaterials such as metal and carbon nanotubes have been used for antiviral and antibacterial coatings, face masks, and immunosensors. Recent studies have explored electricity-based disinfection for face masks; however, these approaches often rely on metal catalysts, which lack biocompatibility, or require external power sources. Enzymatic biofuel cells (BFCs) have been proven to produce electric current through the catalysis of biofuels using enzymes and microorganisms. In this study, biocompatible and biosafety BFCs were employed to develop an electric face mask capable of bacteria sterilization via conductive surfaces.Figure 1 shows the schematic diagram of the BFC system and its illustration on the face mask. Bilirubin oxidase (BOD) and alcohol dehydrogenase (ADH) were used as biocatalysts for the cathodic and anodic reactions, respectively, enabling ion and electron flow through redox reactions. A polyester cloth face mask was modified by dip coating with a PEDOT:PSS mixture, and creating hydrogel-infused regions to manufacture an electrically conductive face mask. In utilizing carbon fiber electrodes, its surface structures were modified by carbon nanotubes and mediator to improve current performance of the electrodes as displayed in Figure 1.To evaluate the application, the response of bacteria (S. oneidensis and E. coli) to varying microampere electric current was determined (Figure 2a). Significant bacterial deaths were observed at currents of 20 µA and above. To prove the effect of current applied to the physical integrity of bacteria, staining with voltage-dependent dye was done, and fluorescence intensity was measured as shown in Figure 2b. Considering that increase in fluorescence intensity would indicate affecting the bacteria membrane potential, increase in fluorescence was observed from 20 µA and up. This proves that the applied microampere current was the cause of the increase in bacteria deaths as microampere current was increased.The biofuel cell was then fabricated and evaluated (Figure 3). The BOD biocathode had a peak current of around 380 µA at -0.2 V under stirring conditions, as compared to without stirring, thus confirming the enzymatic activity from BOD-immobilized biocathode fiber. For the methylene green (MG)-mediated bioanode, on the other hand, cyclic voltammogram (CV) in NADH was evaluated since the MG is a well-established NADH mediator. The significant increase in oxidation peak with NADH signifies the successful MG coating. To confirm the detection of ethanol, NAD+ cofactor and ADH enzyme solution was compared in the presence and absence of ethanol. Increase in oxidation peak to around 900 µA at 0.1 V was shown, thus indicating the successful detection of the ethanol oxidation.Since the performances of the electrodes have been evaluated, the cloth face mask was modified and examined. Figure 4a shows the conductivity of the PEDOT:PSS-modified face mask, which determines that significantly high conductivity was obtained at 1-2 hrs. of dip coating in the modified PEDOT:PSS mixture. To evaluate the overall performance of the BFC, the system in Figure 4b was fabricated, and resulting to the data shown in Figure 4c. The variable current signifies the current that passed through the variable resistance in the external circuit. However, upon calculation of the current that passes through the constant resistance of the PEDOT:PSS region of the face mask, modulation of the passing microampere current was obtained. With the previously determined minimum microampere current for bacteria death, with just a minimum cell potential of 0.1 V necessary current was obtained.In conclusion, this study shows that microampere electric currents of over 20 µA disrupt bacterial membrane potential, effectively killing S. oneidensis and E. coli. The BFC-powered face successfully produced sufficient current for bacterial sterilization, establishing its potential as a potable and biocompatible disinfection solution. Figure 1

Copper metalloenzymes have been investigated for their ability to oxidize or reduce substrates at low overpotentials and high catalytic activities, owing to unique active site structures and catalytic mechanisms. In addition to their exceptional substrate diversity and selectivity, these enzymes have been explored at electrodes for energy conversion, environmental remediation and biosensing. The use of these enzymes in biofuel cells and third-generation electrochemical biosensors (in which enzymes are in direct electrical contact with the electrode) relies on establishing an optimal link between the enzyme active site or internal electron relays and the electrode surface. In this respect, the use of carbon nanomaterials such as carbon nanotubes (CNTs) has shown an ideal combination of enzyme compatibility and direct access to the copper active sites, achieving efficient electron tunnelling. Furthermore, their combination with enzymes has provided a straightforward way to integrate metalloenzymes into operational devices such as sensors and biofuel cells. Additionally, we have developed chemical or electrochemical approaches for the functionalization of carbon nanomaterials. In particular, the electrografting of aryldiazonium salts is aimed at introducing a variety of functional groups which can promote electron transfer through enzyme orientation, provide site-specific attachment groups or enzyme substrate mimics in order to boost enzymatic electrocatalysis.[1–3]. These approaches have been employed to study CNT-supported copper-containing nitrite reductase[4], bilirubin oxidases, laccases and their engineered variants with enhanced properties for oxygen reduction and pollutant detection such as polycyclic aromatic hydrocarbons.[5,6]References[1] S. Gentil, C. Pifferi, P. Rousselot-Pailley, T. Tron, O. Renaudet, A. Le Goff, Langmuir 2021, 37, 1001–1011.[2] S. Gentil, P. Rousselot-Pailley, F. Sancho, V. Robert, Y. Mekmouche, V. Guallar, T. Tron, A. Le Goff, Chem. Eur. J. 2020, 26, 4798–4804.[3] N. Lalaoui, M. Holzinger, A. Le Goff, S. Cosnier, Chem. Eur. J. 2016, 22, 10494–10500.[4] U. Contaldo, D. R. Padrosa, H. Jamet, M. Albrecht, F. Paradisi, A. Le Goff, Chem. Eur. J. 2023, 29, e202301351.[5] I. Sorrentino, I. Stanzione, A. Piscitelli, P. Giardina, A. Le Goff, Biosens. Bioelectron.: X 2021, 100074.[6] I. Sorrentino, M. Carrière, H. Jamet, I. Stanzione, A. Piscitelli, P. Giardina, A. L. Goff, Analyst 2022, 147, 897–904.

Abstract The coronavirus disease 2019 (COVID‐19) pandemic has raised awareness regarding the proper wearing and functionality of face masks to prevent viral and bacterial infections through the nasal and oral pathways. However, large‐scale daily use of disposable face masks introduces large volumes of waste. Reusable face masks will significantly reduce waste, but the lack of rapid sterilization prevents their use. To realize on‐the‐go sterilization, wearable biofuel cells are integrated into reusable conductive cloth face masks that generate electric current. To ensure softness and functionality, the face mask is modified with poly(3,4‐ethylenedioxythiophene) polystyrene sulfonate, a well‐known biocompatible conducting polymer. Meanwhile, fiber‐based bioanode and biocathode utilizing alcohol dehydrogenase for ethanol oxidation and bilirubin oxidase for oxygen reduction, respectively, are sewn on the conductive face mask. After assembling the “electric face mask”, modulation of current flow through its conductive region is demonstrated. In tests with two bacteria, Shewanella oneidensis MR‐1 and Escherichia coli, a minimum current flow of 20 µA significantly increases the bacteria's death rates. Therefore, this enzymatically powered mask provides necessary electric currents to kill bacteria on a conductive surface. Importantly, bacterial sterilization using the fabricated electric face masks is successfully demonstrated, adding a step to utilizing soft bioelectrical technology toward a more sustainable society.

Measurement of bilirubin in cerebrospinal fluid using the oxidase method on automated chemistry system advia XPT.

A novel biofuel cell based on galactose oxidase and bilirubin oxidase for efficient glycerol conversion and electricity generation

Self-powered bio-chip for ultra-sensitive dual MiRNAs detection: A portable platform for cancer biomarker analysis.