Reversible bismuth reduction-driven dark photoelectrochemistry

Domain: neuroscience
Published: 2026-01-22
Editorial outcome: major_revision

Abstract
Current research in photoelectrochemical (PEC) systems focuses on designing photoelectrodes with light-enhanced charge transfer efficiency at the solid-liquid interface, which leads to the oxidation of various reductant molecules at the anode, posing a challenge in terms of selectivity. In this study, we introduce an original concept of dark-enhanced PEC system (dark-PEC) based on bismuth oxybromide (BiOBr)-photocathode. The reversible bismuth reduction causes the passivation and regeneration of oxygen reduction sites on the photocathode, thereby leading to an unconventional reverse photocurrent (|I_dark| > |I_light|) signal under air conditions. Significantly, owing to the selective binding and charge transfer between glutathione and BiOBr, the reversibility of bismuth reduction is increased, with a 10-fold enhancement of reverse photocurrent. Compared to traditional PEC systems, the dark-PEC system enables the selective recognition of reductant molecules by photocathodes, unveiling an original PEC reaction mechanism. This study will offer valuable insights into device design and redox processes in the PEC field.

Introduction
Photoelectrochemical (PEC) technology, which utilizes semiconductor materials to transform optical signals into electrical or chemical signals, has demonstrated significant potential for applications in sensing, batteries, catalysis, and other fields1–9. In particular, the redox reactions at the photoelectrode/electrolyte interface have garnered considerable attention as a critical process influencing the photoelectric conversion efficiency and catalytic activity in PEC applications10–16. A thorough understanding of the mechanisms and kinetics of these interfacial reactions is crucial for optimizing the performance of PEC devices17–20. Currently, to optimize PEC performance, researchers are concentrating on devising a range of strategies to enhance the charge transfer efficiency at the solid-liquid interface under illumination11–26. For a long time, the behavior of photoelectrodes in darkness was regarded as merely a weakened form of the light-driven reaction, assumed to have no impact on the photoelectric signal output process. However, within this light-enhanced PEC paradigm, as the light and dark reactions follow the same reaction pathway, different oxidant/reductant molecules can only be distinguished by the magnitude of the cathodic/anodic reaction current, making them highly susceptible to interference from similar oxidizing or reducing species (Fig. 1a). Although current approaches often employ enzymatic redox recognition or functionalized electrode surfaces with molecular capture units to improve selectivity, these methods are frequently constrained by limited stability, high operational costs, and challenges in achieving precise atomic-scale engineering between electrode and target27–32. Consequently, achieving high selectivity remains a critical challenge in the advancement of PEC sensing technologies33–35.

In this study, we report an original concept of dark-enhanced PEC system (dark-PEC) based on the bismuth oxybromide (BiOBr)-photocathode and demonstrate its distinctive photoelectron transfer pathway for facilitating the recognition of glutathione (GSH). The BiOBr-photocathode exhibits an unconventional reverse photocurrent (|I_dark| > |I_light|, defined as the photocurrent of the dark-enhanced type, contrasting with the light-enhanced type traditional photocurrent) in air condition, where the reversibility of bismuth reduction leads to the reconstruction and recovery of the oxygen reduction reaction (ORR) active site of the electrode during the alternation of light and dark. Upon illumination, BiOBr undergoes photoreduction to form low-valent Bi (Bi(3-&)+), thereby passivating the cathode surface for ORR. When the illumination ceases, dissolved oxygen in the electrolyte re-oxidizes Bi(3-&)+ to facilitate interfacial ORR. Based on the above dark-PEC mechanism, the selective binding and charge transfer between glutathione (GSH) and BiOBr facilitate the generation of high-valent bismuth (Bi(3+&)+), which greatly enhances the reversibility of ORR site reconstruction, thereby effectively increasing the reverse photocurrent (Fig. 1b). Finally, the selective responsiveness of this dark-PEC mechanism to GSH was validated through actual sample testing. This discovery represents a significant advancement in the solid-liquid interface mechanism of PEC devices.

Results
Reverse photocurrent characteristic of BiOBr
First, the morphology and structure of BiOBr were investigated. The transmission electron microscopy (TEM) image reveals that the as-synthesized BiOBr presents a nanosheet morphology with a size of ~50 nm (Fig. 2a). The diffraction peaks of BiOBr can be recognized in the XRD result (Fig. 2b), which are assigned to the standard lattice planes (PDF#78-0348) of BiOBr36,37. Further, high-resolution transmission electron microscopy (HRTEM) and selected-area electron diffraction (SAED) images verify the crystalline structure of BiOBr nanosheets, with the (101) crystal plane exposed (Supplementary Fig. 1). The corresponding energy-dispersive spectroscopy (EDS) element mapping images of BiOBr present that the Bi, O, and Br elements are uniformly distributed (Supplementary Fig. 2).

Then, the systematic research on photocurrent transients of the photocathodes modified by BiOBr in electrolytes was studied in different atmospheres. When the BiOBr-photocathode is measured in air-electrolyte, the photocurrent signal displays a positive direction (Fig. 2c), indicating a non-traditional reverse photocurrent (|I_dark| > |I_light|). We refer to this PEC process as Dark-PEC system. Afterward, nitrogen (N2) and oxygen (O2)-saturated electrolytes were used to examine the photocurrent response. Interestingly, when O2 is absent or saturated in the electrolyte, the photocurrents of the BiOBr photocathode show a negative direction (Fig. 2d), that is, the traditional cathode-photocurrent characteristic (|I_dark| < |I_light|). Moreover, we observed that the surface color of the BiOBr photocathode undergoes varying degrees of change during the conduct of the PEC test in electrolytes saturated with N2, O2, or air atmosphere (Fig. 2e). In an N2-saturated electrolyte, the color change of the BiOBr photocathode is most pronounced, shifting from white to brownish. However, during testing in the O2-saturated electrolyte, the BiOBr photocathode did not undergo any color change and remained white. In contrast, under the air condition, a slight color change can be observed at the edge of the tested photocathode, falling between the conditions of N2 saturation and O2 saturation. This indicates that the unconventional reverse photocurrent phenomenon is related to the O2 concentration in the electrolyte. Irrespective of whether the O2 concentration is excessively high or low (Fig. 2f), the dark-PEC process does not take place in the BiOBr photocathode. Therefore, to gain insight into this distinct dark-PEC characteristic in the BiOBr photocathode, we seek answers by exploring the color-changing mechanism and the ORR of the electrode surface.

Dark PEC mechanism of BiOBr photocathode
The BiOBr photocathode was directly exposed to light to investigate the dynamic changes in its surface coloration. As illustrated in Fig. 3a, the surface color of the electrode progressively darkens over time under continuous illumination. Notably, upon cessation of light exposure, the electrode’s surface color gradually diminishes, ultimately reverting to its initial white state. This phenomenon indicates that BiOBr possesses excellent photochromic reversibility. Based on the color changes of the electrode in different atmospheres as described above in Fig. 1e, it can be inferred that O2 in the air is capable of restoring the color state of BiOBr. Subsequently, to explore the influence of reversible color changes on the electronic structure of BiOBr, we utilized XPS technique to analyze the binding energies of elements before and after exposure to light. In the Bi 4f (Fig. 3b), O 1s (Supplementary Fig. 3a) and Br 3d (Supplementary Fig. 3b) spectra, noticeable negative shifts of binding energy were observed under light conditions, suggesting the accumulation of photogenerated electrons. It is worth noting that after illumination, an additional characteristic charge transfer (CT) satellite peak38 appears in the XPS of the Bi element at around 157.5 eV, between the characteristic peaks of Bi-metal and Bi3+. BiOBr nanosheets undergo photoreduction after illumination, leading to the formation of Bi(3-&)+ that store electrons39. Furthermore, electron paramagnetic resonance (EPR) measurements were employed to investigate the reversible behavior of BiOBr under alternating light and dark conditions. In comparison with the sample in the dark state, BiOBr after illumination exhibits a markedly enhanced EPR signal at g=2.01 (Supplementary Fig. 4). This enhancement can be attributed to an increased concentration of oxygen vacancy defects under photoexcitation, which is consistent with the appearance of the CT satellite peak in the Bi 4f XPS spectrum. To elucidate the nature of the dark-PEC phenomenon, cyclic voltammetry (CV) was employed to investigate the interfacial behavior between the BiOBr photoelectrode and the electrolyte. Obviously, under the saturated conditions of O2 and N2, the CV curves of BiOBr exhibit the same interfacial reaction before and after light exposure (Supplementary Fig. 5a, b). In an O2 atmosphere, the reduction of Bi3+ is unlikely to occur when the BiOBr photocathode is adequately surrounded by O2. Consequently, the photogenerated electrons facilitate the interfacial ORR, thereby enhancing the cathode current density. In a N2 atmosphere, an oxidation characteristic peak at approximately 0.3 V (vs. Ag/AgCl) is observed under both light and dark conditions. This peak can be ascribed to the oxidation behavior of Bi(3-&)+ on the BiOBr surface due to the anaerobic environment. The increased intensity of the oxidation peak after illumination suggests that the Bi3+ reduction on the BiOBr surface is substantially enhanced under light exposure. Meanwhile, the initial potential of the cathode reduction reaction exhibits a negative shift to approximately −0.3 V, and the current intensity under light exposure remains significantly higher than that in the dark. This phenomenon can be attributed to the transfer of photoelectrons to H2O in an O2-free environment. However, under air conditions, the CV curves of BiOBr show different interfacial behavior before and after light exposure. Specifically, the formation of Bi(3-&)+ still occurs during light exposure, but the oxidation peak associated with Bi(3-&)+ vanishes in the dark (Fig. 3c). Furthermore, upon illumination, the initial potential of ORR on the BiOBr photocathode exhibits a negative shift, and the reduction current diminishes. This behavior aligns with the reverse photocurrent observed in the above mentioned dark-PEC process.

Based on the above analysis, we further explore the underlying mechanism linking the formation of Bi(3-&)+, ORR and the reverse photocurrent under air conditions. The i-t curves of the BiOBr photocathode under varying light durations were measured by controlling the dark state duration to 40 s. Supplementary Fig. 6 shows that extending the illumination time from 5 s to 100 s increased reverse photocurrent intensity, with an enhancement of 70%. This positive correlation suggests that an increase in the content of Bi(3-&)+ leads to a greater difference in ORR current between light and dark. Next, the photocurrent behavior of the photocathode modified with varying concentrations of BiOBr under the stable air condition further elucidates how the generation of reverse photocurrent is influenced by the ratio of Bi(3-&)+ to O2 during alternating light/dark cycles. When the BiOBr concentration is low, the O2 in the air effectively inhibits the formation of Bi(3-&)+, thereby maintaining the conventional cathode-photocurrent. However, as the BiOBr concentration progressively increases, the conventional cathode-photocurrent diminishes and transitions to an unconventional reverse photocurrent when the BiOBr concentration ranges from 1 to 2 mg/mL, reaching a maximum at a BiOBr concentration of 6 mg/mL (Supplementary Fig. 7). Then, to elucidate the changes in the band structure of this dark-PEC mode, UV-visible diffuse reflectance spectra (Supplementary Fig. 8) were measured both before and after the illumination of BiOBr. The resultant Tauc plot indicates that the band gap of BiOBr decreases from 2.90 eV to 2.79 eV upon illumination. This suggests that the Bi(3-&)+ alters the position of the conduction band. The photoelectrode testing under air and O2-saturation conditions can be regarded as dark PEC and conventional PEC systems, respectively. Mott-Schottky curves for these systems were measured before and after light exposure. As illustrated in Fig. 3d, when the dark-PEC behavior occurs, the flat band potential (Efb) shifts from −0.27 V to −0.07 V (vs. Ag/AgCl) upon illumination, whereas Efb remains unchanged in the O2-electrolyte. This observation further corroborates the emergence of photo-induced new energy levels (corresponding to Bi(3-&)+) and the enhanced-dark reaction characteristics in the dark-PEC process of BiOBr. Meanwhile, the variations in reactive functional groups on the surface of the BiOBr photoelectrode were systematically characterized through in situ Fourier transform infrared spectroscopy (FTIR) under alternating light and dark conditions. As shown in Supplementary Fig. 9, after light irradiation, the intensities of the H-O-H bending vibration and O-H stretching vibration peaks associated with adsorbed water on the BiOBr surface were significantly reduced, indicating decreased ORR activity40,41. Therefore, the dark-PEC mechanism under air conditions occurring on the BiOBr-photocathode is summarized in Fig. 3e. The dark-PEC process is exclusively initiated under conditions where the O2 concentration enables the formation of reversible bismuth reduction on the BiOBr photocathode surface during light-dark alternations.

During illumination, Bi3+ in BiOBr undergoes photoreduction to form Bi(3-&)+ (a new energy band marked by the blue line), which induces surface passivation and decreases the interfacial ORR efficiency. Upon cessation of illumination, Bi(3-&)+ is re-oxidized by O2, facilitating surface recovery and enhancing the ORR performance at the photocathode/electrolyte interface. In contrast, under O2 and N2 conditions, since BiOBr is incapable of achieving reversible bismuth reduction reactions during the alternation between light and dark cycles, it exhibits a single bandgap characteristic (Fig. 3f). Specifically, under O2-saturated conditions, photoelectrons of pure Bi3+-O-Br facilitate the surface ORR, thereby increasing the current density. Under N2-saturated conditions, the photoelectron transfer from pure Bi(3-&)+-O-Br to H2O enhances the current density.

Generally, the majority of researchers focus on enhancing the ORR to boost the traditional cathodic photocurrent, as O2 serves as an efficient natural electron acceptor. However, in this dark-PEC system, the reverse photocurrent at the cathode is determined by reversible bismuth reduction occurring at the electrode-electrolyte interface. Introducing an excessive number of electron acceptors would only attenuate the intensity of the reverse photocurrent, a phenomenon that has been corroborated by the direction of the photocurrent under prior O2-saturated conditions. It was unexpected to observe that the reductant GSH could significantly enhance the reverse photocurrent signal, while ascorbic acid (AA) failed to exhibit a comparable enhancement effect (Fig. 4a). In addition, in the N2 atmosphere, neither induces an effective signal change compared to the blank group, which is consistent with the disappearance of the reverse photocurrent signal in the N2 atmosphere (Supplementary Fig. 10). Therefore, we selected the classical reductant AA as the control group for GSH to further investigate the specific mechanism by which GSH enhances the dark-PEC signal, despite traditionally being expected to oxidize at the anode.

Firstly, the CV curves of the two molecules were systematically evaluated before and after light exposure under varying atmospheric conditions. In the N2 atmosphere, the oxidation peak of Bi(3-&)+ in the GSH-electrolyte system was greatly enhanced following illumination (Supplementary Fig. 11a), with an amplification factor of 2.3 times compared to that observed in a blank-electrolyte system (Supplementary Fig. 5b). This suggests that GSH facilitates the formation of Bi(3-&)+. Conversely, in the AA system, the oxidation peak of Bi(3-&)+ remained largely unchanged before and after illumination (Supplementary Fig. 11b). Furthermore, under the air atmosphere, in comparison to the CV curve of pure BiOBr (Ipeak = 1.54 µA, Fig. 3c), the presence of GSH facilitates the formation of Bi(3-&)+ on the BiOBr surface under illumination (Ipeak = 2.34 µA, Fig. 4b). Conversely, the presence of AA hinders the formation of Bi(3-&)+ (Ipeak = 0.25 µA, Fig. 4c), as the oxidation peak current decreases. The photochromic (bismuth reduction) behavior of BiOBr in aqueous solution (Fig. 4d) also reveals that the presence of GSH results in the most significant color change, with the slowest recovery rate after illumination ceases.

Subsequently, GSH and AA were individually mixed with BiOBr. The resultant samples were collected after centrifugation and further characterized using FTIR (Supplementary Fig. 12). The FTIR results reveal that the BiOBr treated with GSH exhibit prominent characteristic peaks at approximately 1640 cm−1 and 1500 cm−1, which can be attributed to the vibrational modes of the C=O and N-H functional groups in GSH. Furthermore, the characteristic peak positioned at approximately 417 cm−1 exhibits a shift towards the high-frequency region, which is indicative of the formation of the Bi-S bond. This observation was further corroborated by Raman spectroscopy, which verifies the binding behavior between BiOBr and GSH. As depicted in Fig. 4e, two distinct vibrational peaks were observed within the range of 300-400 cm−1, and these peaks can be ascribed to the vibrational characteristics associated with the Bi-S bond42–44. To further investigate the state of the photoelectrode after PEC processing, XPS characterization was conducted on different samples following a 1600 s photocurrent test (Supplementary Fig. 13). The findings reveal that the BiOBr photocathode treated with GSH exhibits Bi(3+&)+ and increased Bi(3-&)+ characteristic peaks in the high-resolution spectra of Bi elements (Fig. 4f). Due to the reversible redox reaction of GSH on the BiOBr photoelectrode surface, under illumination, GSH is oxidized by Bi3+ to form ox-GSH. When the light is turned off, ox-GSH is subsequently reduced back to GSH during the regeneration of high-valent bismuth from its low-valent state. Comparative colorimetric analysis of the electrolyte solution under light and dark conditions reveals a decrease in absorbance after light exposure (Supplementary Fig. 14), further confirming the reversibility of the redox interconversion between GSH and ox-GSH on the BiOBr photocathode. Therefore, it can be concluded that in the dark-PEC system of BiOBr photocathodes, Bi species serve as recognition sites for GSH molecules (forming Bi(3+&)+~GSH complexes). Under illumination, it functions as the pseudo anode to obtain electrons from GSH molecules to generate increased Bi(3-&)+. This process significantly reduces the surface ORR activity efficiency. Upon cessation of light exposure, increased Bi(3-&)+ was re-oxidized and facilitated the interfacial reduction reactions (ORR, ox-GSH reduction), thereby enhancing the reversibility of bismuth redox reactions and reverse photocurrent (Fig. 4g). Nevertheless, AA molecules possessing identical reducing properties are unable to be recognized by the pseudo anode and thus cannot enhance the reversibility of bismuth reduction (Fig. 4h). These two distinct redox pathways elucidate an original molecular recognition mechanism based on the dark-PEC pattern. The photocathode is also capable of detecting and quantifying reductant molecules with high sensitivity and accuracy, owing to the reversible nature of the valence state transformation of the Bi element.

Dark PEC sensing for GSH
Finally, we investigated the feasibility and practical application of the molecular recognition mechanism for GSH quantitative analysis in the dark-PEC system. By comparing the signal responses of various reductant molecules, including glucose (Glu), procyanidins (CY), uric acid (UA), mannitol (MA), dopamine (DA), and AA, our results demonstrate that GSH detection exhibits great selectivity. In addition, photocurrent response tests conducted on various thiol-containing compounds revealed that, apart from ox-GSH (which is capable of undergoing reversible redox reactions with GSH), the other thiol compounds failed to achieve reversible transformation during the light-dark cycles. As a result, they were unable to effectively facilitate the interconversion between high-valent and low-valent bismuth species, thereby further substantiating the high selectivity of this approach toward GSH molecules (Fig. 5a, b and Supplementary Fig. 15). Subsequently, leveraging this dark-PEC behavior, a linear relationship for GSH concentrations ranging from 80 to 500 μM was established (Fig. 5c). The linear regression equation is I_GSH = 4.792 C − 1.44 (R2 = 0.999), with a limit of detection of 0.02 μM (signal-to-noise ratio of 3) (Fig. 5d). Intra-day and inter-day recovery rate tests yielded results ranging from 98.8% to 107%, with relative standard deviations varying between 0.52% and 8.16% (Supplementary Table 1). The reliability and practicability of the dark-PEC sensor platform were assessed by evaluating GSH concentrations in real samples of onion, spinach, and broccoli. The analytical accuracy, in comparison with the colorimetric method (Supplementary Figs. 16, 17), ranged from 97.48% to 103.0%, with relative standard deviations between 2.33% and 5.06% (Fig. 5e and Supplementary Table 2). As presented in Supplementary Table 3, a comparative analysis was also conducted with the conventional PEC detection method for GSH. The dark-PEC signal proposed in this study demonstrated distinct advantages in terms of detection range, limit of detection, and selectivity. These findings confirm the high sensitivity and accuracy of the dark-PEC approach, highlighting its promising potential for practical sample analysis.

Methods
Apparatus
Transmission electron microscope (TEM) images were acquired from a Titan G2 60-300 (Thermo Fisher, United States). X-ray photoelectron spectroscopy (XPS) measurements were performed by Thermo ESCALAB QXi (Thermo Fisher, United States). The Ultraviolet-visible (UV-Vis) diffuse-reflectance absorption spectra were measured on a UV-Vis spectrophotometer (Agilent Technologies, Cary 100) using BaSO4 as reflectance standard reference. Mott-Schottky (M-S) curve, Electrochemical impedance spectroscopy (EIS) and photoelectrochemical (PEC) test were performed on a CHI660E electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a three-electrode system. PEC test was performed with 500 W Analog Daylight Xenon Light Source (PLS-FX300HU, Beijing Perfectlight Technology Co., Ltd.).

Reagents
Ascorbic acid (AA) and glutathione (GSH) were purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), Sodium sulfate (Na2SO4), mannitol, PVP (30K) and potassium bromide (KBr) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Indium-tin oxide coated glass (ITO, 10 Ω, 1.1 mm) was ordered from Luoyang Guluo Glass Co., Ltd. Unless stated, all the chemicals mentioned above were analytical reagents and used as received. The deionized water in all experiments was prepared in a three-stage Millipore Milli-Q plus 185 purification system.

Mott–Schottky tests
Electrodes for the Mott-Schottky analysis were drop-casted onto the surface of ITO. Ag/AgCl (3.5 M KCl) electrode was used as the reference electrode. Pt wire gauze electrode was applied as the counter electrode. Data points for open circuit potentials were recorded at the frequency of 1 kHz with the modulation signal amplitude of 5 mV in 0.2 M Na2SO4. The flat-band potential of the material was estimated to be the intercepts of the curve on the X-axis (vs. Ag/AgCl, pH 7). The measurements were carried out in the light and dark, respectively.

Synthesis of BiOBr
In a typical synthesis, Bi(NO3)3·5H2O (1 mmol), mannitol (3.3 mmol) and PVP (200 mg) were dissolved in 30 mL of deionized water under vigorous stirring for 10 min. Then, KBr (1.5 mmol) was added to the above solution, and the mixture was stirred for 1 h. The resulting solution was then transferred to a 50 mL Teflon-lined autoclave, sealed and heated at 160 °C for 3 h. Finally, the products were collected by centrifugation, washed multiple times with deionized water, and dried in a vacuum oven at 60 °C for 10 h. During the synthesis of BiOBr, PVP functions as a structure-directing agent and dispersant, guiding the formation of two-dimensional sheet-like morphologies and effectively preventing particle agglomeration. Mannitol acts as a reaction kinetics modulator and a mild coordinating and reducing agent, enabling controlled crystal growth by retarding the crystallization rate, thus facilitating the production of nanosheets with high crystallinity and well-defined morphology. The synergistic interaction between PVP and mannitol allows for precise control over the microstructural characteristics of the final product.

PEC measurements system
The obtained BiOBr sample was dispersed in a mixture of Nafion/H2O (v/v: 1/100) to form a uniform slurry suspension with a concentration of 6 mg/mL under the action of ultrasound. Then, 40 µL of BiOBr dispersion was successively modified with the ITO electrode surface with a circular area of ~0.28 cm2 (diameter = 6 mm) and dried at 50 °C to obtain the working photoelectrode. All photocurrent measurement was carried out in 0.01 M Na2SO4 (pH 7.0) as the electrolyte with a classical three-electrode system, using a modified-ITO electrode, Pt wire, and Ag/AgCl as working electrode, counter electrode and reference electrode, respectively. Under oxygen and nitrogen conditions, the corresponding gases were passed through for 3 min before PEC testing. All the reported potential/voltage values are not IR corrected for electrochemical data.

Photochromic test of BiOBr
In a 96-well plate, BiOBr (6 mg/mL 40 µL) was mixed with GSH (10 µL, 0.01 M) solution or AA (10 µL, 0.01 mM) in the deionized water (100 µL, pH = 7.0) at room temperature (25 °C). The mixture was exposed to alternating light/dark cycles for 3 min each. During the photochromic experiment conducted on the BiOBr photoelectrode (6 mg/mL, 40 μL), the electrode was pre-wetted before illumination. Subsequently, it was exposed to light for three minutes to observe and document the color changes before and after illumination through video recording.

PEC analysis of GSH samples
First, the linear relationship of GSH with different standard concentrations was established by the dark PEC method. Then, 1 g of onion, spinach and broccoli samples were weighed and extracted by adding a small amount of 5% metaphosphoric acid buffer, and the samples were stored in deionized water for 40 mL, centrifuged at 8000 rpm for 10 min, and the supernatant was taken for use. The real samples were then tested for GSH by using the colorimetric method. (1) 100 µL of various sample solutions were mixed with 100 µL of 5,5’-Dithiobis-(2-nitrobenzoic acid) solution (4 mM), and the GSH content was quantified based on the extent of color development. Subsequently, the quantified samples were diluted to achieve different concentrations of GSH, and the recovery rates were determined. (2) The diluted real sample was detected by using the current dark-PEC method based on the calibration curve.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript entitled “Reversible bismuth reduction-driven dark photoelectrochemistry” reports a so‑called “dark‑PEC” system based on a BiOBr photocathode, which exhibits reverse photocurrent behavior (Idark| > Ilight) under air-saturated conditions and is claimed to enable selective glutathione (GSH) detection. While the topic is within the scope of the journal, the study suffers from major concerns in terms of novelty, mechanistic rigor, and data sufficiency.\nThe core phenomenon – coupling of PEC activity with reversible valence changes of the electrode material leading to modulation of photocurrent – has already been extensively documented in PEC–electrochromic systems (e.g., WO3, Bi-based oxides) over the past decade. The present work does not introduce a fundamentally new physical effect but rather labels an existing mechanism with new terminology. Furthermore, the dataset presented is not sufficiently robust to support the proposed mechanism or the claimed high selectivity toward GSH. The reported effect only occurs under narrowly defined oxygen concentrations, lacks comprehensive parametric study, and is supported mainly by indirect evidence. Without stronger mechanistic validation and broader demonstration of robustness, the manuscript does not meet the novelty and rigor standards of the journal. Based on the above concerns, I recommend rejection of this manuscript in its current form.\n1.Experimental scope is too narrow, with inherently weak photocurrent signals leading to poor robustness for sensing applications. The effect occurs only within a narrow oxygen concentration window, raising concerns about reproducibility in practical environments.\n2.The reverse photocurrent is only tested under air-, O2-, and N2-saturated electrolytes. Other critical parameters (pH, applied bias, ionic strength, light intensity) are not systematically varied, limiting understanding of universality.\n3.No long-term cycling data (> hours) or batch-to-batch reproducibility tests are provided to assess electrode stability.\n4.Given that Bi(3-&)+ is chemically unstable, potential degradation pathways during repeated light–dark cycling are not addressed.\n5.Application relevance is overstated. The GSH sensing range and detection limit are comparable to or weaker than existing PEC sensors, while the operational constraints (air-saturation requirement) reduce practical applicability.", "reviewer_2": "The authors report a novel “dark-enhanced” photoelectrochemical (PEC) system based on a BiOBr photocathode, where reversible bismuth reduction drives an unconventional reverse photocurrent (|Idark| > |Ilight|) under air conditions. The work demonstrates that glutathione (GSH) selectively interacts with BiOBr, enhancing the reversibility of the bismuth redox cycle and amplifying the reverse photocurrent. This mechanism enables selective detection of reductant molecules at the photocathode interface, addressing a key challenge of selectivity in PEC sensing systems. The study combines electrochemical, spectroscopic, and surface characterization analyses to validate the mechanism and demonstrate practical applications in GSH quantification in real samples.\nThe manuscript introduces a compelling conceptual advancement in photoelectrochemical systems by shifting the paradigm from light-enhanced to dark-enhanced PEC processes. The mechanistic insights into reversible bismuth redox cycles, coupled with selective molecular recognition at the cathode, represent an original approach with potential implications for selective PEC sensing.\nThe experimental validation is thorough, with adequate characterization (XPS, TEM, EDS, Raman, Mott-Schottky) supporting the proposed mechanism. However, there are points requiring further clarification, especially regarding the reproducibility, scope, and broader applicability of the dark-PEC concept beyond the GSH model system.\nI believe the manuscript has high novelty and significance, but several substantial issues need to be addressed before it can be considered for publication in Nature Communications.\n1. Mechanistic depth – Role of oxygen concentration\nThe proposed dark-PEC mechanism relies on a delicate balance between O₂ availability and Bi reduction/oxidation dynamics. However, the authors should quantitatively assess the oxygen concentration threshold where the transition from traditional PEC to dark-PEC behavior occurs. Could this be correlated with electrochemical O₂ reduction kinetics?\n2. Generality of the mechanism\nWhile the work demonstrates GSH selectivity, it remains unclear if this mechanism could extend to other thiol-containing molecules or reductants capable of interacting with Bi species. Testing a broader set of analytes with similar functional groups would help position this platform as a more general sensing strategy.\n3. Photocurrent reversibility and stability over time\nThe manuscript lacks a detailed stability assessment. How does the reverse photocurrent behavior evolve over multiple cycles or prolonged operation? Could there be degradation of the BiOBr surface or accumulation of irreversible Bi(0)?\n4. Signal amplification and sensitivity comparison\nThe LOD reported (20 nM GSH) is excellent, but a benchmarking against state-of-the-art PEC sensors for GSH (or similar reductants) should be included to contextualize the advancement in sensitivity and selectivity.\n5. Origin of the unconventional energy band (Bi(3-&)+)\nThe description of Bi(3-&)+ as an intermediate oxidation state is central but speculative. The notation is non-standard and chemically ambiguous. The XPS data (binding energy shift, new peak at ~157.5 eV) lacks deconvolution or comparison with Bi⁰/Bi(I) standards, making the assignment uncertain. The authors should clarify the nature of this species and support its existence with additional spectroscopic evidence (e.g., XANES, in-situ XPS) or, at minimum, a rigorous XPS fitting.\nMinor Comments\n1. Figure Quality\nFigures (e.g., Fig. 1, Fig. 4) would benefit from clearer labels and a more schematic visualization of the electron flow directions.\n2. Terminology Consistency\nThroughout the text, the term “reverse photocurrent” is used. It might be helpful to explicitly define this term early on (i.e., reverse with respect to light-on condition).\n3. Supporting Information – Missing Spectroscopic Data\nIn SI, including high-resolution XPS spectra for GSH-interacted BiOBr after long-term cycling (post-mortem analysis) would strengthen the stability claims.", "reviewer_3": "This paper proposes a “dark-enhanced” photocatalytic system based on a BiOBr photoanode, achieving a reverse photocurrent (|Idark| > |Ilight|) through the reversible reduction of bismuth, and applies it to the selective detection of glutathione, demonstrating a certain degree of innovation. However, the paper still has several shortcomings and requires major revisions and improvements. The following are the revision suggestions:\n1. The discussion on selectivity in traditional PEC systems is overly brief. The manuscript lacks a systematic review of recent advances in enhancing PEC selectivity, resulting in an unclear benchmark for evaluating the innovation of the proposed system. Please supplement the introduction with representative studies from the past five years that focus on PEC selectivity control.\n2. The functions of mannitol and PVP in the synthesis of BiOBr nanosheets are not clearly explained. Please elaborate on their roles during the preparation process (line 265).\n3. The characterization of BiOBr's structural and optoelectronic properties is based on a single synthesis batch, without replicates. To confirm reproducibility, results from at least three independent syntheses should be included, particularly regarding the consistency of morphology, crystallinity, and photoelectrochemical performance (refer to line 61).\n4. In line 83, it is claimed that the reverse photocurrent is influenced by a “moderate O2 concentration,” yet no quantitative data or correlation is provided. Please conduct a gradient experiment (0-100% O2) and plot the relationship between O2 partial pressure and photocurrent response to define the “moderate” range more precisely.\n5. The valence state change of bismuth is currently inferred only from XPS binding energy shifts and electrochemical behavior, which is indirect. To convincingly demonstrate the reversible redox process and presence of intermediate oxidation states (line 98), additional direct evidence such as in situ X-ray absorption fine structure and electron paramagnetic resonance data is recommended.\n6. Line 151 states that Bi(3–δ)+ species may passivate ORR sites and reactivate O₂ oxidation in the dark. However, this claim lacks in situ spectroscopic support. Please consider including data from in situ Raman spectroscopy or in situ AFM to support this hypothesis.\n7. The manuscript does not discuss the impact of light parameters (e.g., intensity, wavelength) on the reduction degree of Bi species, which may directly influence the generation of Bi(3–δ)+ and hence affect the reverse photocurrent. Please address this in the discussion.\n8. The validation system is not rigorous enough. Only interference from five substances, including CY and UA, was compared, but interference from other structurally similar reducing agents (such as cysteine and glutathione disulfide bonds) was not ruled out (line 237).\n9. How does this detection system perform in terms of interference resistance in complex matrices such as serum?\n10. The recovery rate experiment for actual sample testing (243 lines) was not compared with other standard methods, making it difficult to prove the accuracy of the method. At the same time, intra-day and inter-day comparison data need to be supplemented.\n11. This study did not compare the dark-PEC system with the traditional PEC (GSH detection based on anodic oxidation) in terms of sensitivity (detection limit), selectivity, response time, and other quantitative aspects, making it difficult to highlight its technical advantages. A comparison table should be added to highlight the technical advantages of this work."}, "author_response": "Response to Reviewers' Comments (manuscript NCOMMS-25-56414)\nReply to reviewer 1: Thank you very much for your valuable comments.\nReviewer #1: The manuscript entitled “Reversible bismuth reduction-driven dark photoelectrochemistry” reports a so-called “dark-PEC” system based on a BiOBr photocathode, which exhibits reverse photocurrent behavior (|I | > |I |) under air-saturated conditions and is dark light\nclaimed to enable selective glutathione (GSH) detection. While the topic is within the scope of the journal, the study suffers from major concerns in terms of novelty, mechanistic rigor, and data sufficiency.\nThe core phenomenon-coupling of PEC activity with reversible valence changes of the electrode material leading to modulation of photocurrent has already been extensively documented in PEC-electrochromic systems (e.g., WO , Bi-based oxides) over the past decade. The present work does 3\nnot introduce a fundamentally new physical effect but rather labels an existing mechanism with new terminology. Furthermore, the dataset presented is not sufficiently robust to support the proposed mechanism or the claimed high selectivity toward GSH. The reported effect only occurs under narrowly defined oxygen concentrations, lacks comprehensive parametric study, and is supported mainly by indirect evidence. Without stronger mechanistic validation and broader demonstration of robustness, the manuscript does not meet the novelty and rigor standards of the journal. Based on the above concerns, I recommend rejection of this manuscript in its current form.\n(1) Experimental scope is too narrow, with inherently weak photocurrent signals leading to poor robustness for sensing applications. The effect occurs only within a narrow oxygen concentration window, raising concerns about reproducibility in practical environments.\nResponse: Weak photocurrent signals and sensing robustness are not synonymous. In photoelectrochemical systems, the robustness of “signal-enhanced” PEC sensing fundamentally relies on the inherently low blank photocurrent signal. As the concentration of the target analyte increases, the photocurrent response correspondingly intensifies. The research under different atmosphere conditions aims to explore the essential mechanism of reverse photocurrent generation, which does not mean that our detection application is limited to a narrow oxygen concentration window. This phenomenon is observed under ambient air conditions—a stable and well-controlled environment—that ensures high reproducibility. To validate this consistency, we synthesized three independent batches of BiOBr material and performed PEC measurements, which collectively demonstrated excellent reproducibility of the inverted photocurrent effect (Figure R1). In addition, intra-day and inter-day recovery tests were conducted to evaluate method reliability. The recovery rates varied between 98.8% and 107%, with relative standard deviations ranging from 0.52% to 8.16%, indicating excellent repeatability and robustness in sample analysis (Table R1).\n(2) The reverse photocurrent is only tested under air-, O -, and N -saturated electrolytes. Other 2 2\ncritical parameters (pH, applied bias, ionic strength, light intensity) are not systematically varied, limiting understanding of universality.\nResponse: According to the reviewer’s suggestion, we have tested reverse photocurrent under other parameters (pH, applied bias, ionic strength, light intensity). As pH increases, the reverse photocurrent signal gradually diminishes, attributable to the slowed kinetics of the ORR under alkaline conditions (Figure R2a). With increasingly negative applied potentials, the reverse photocurrent intensifies as a result of enhanced electron injection and increased photocathode current driven by the external potential (Figure R2b). Regarding ion concentration, the mechanism of the reverse photocurrent primarily depends on the reversible equilibrium between oxygen concentration and the valence state of bismuth. Consequently, the signal intensity does not exhibit significant enhancement with rising ion concentration (Figure R2c). Finally, an increase in light intensity leads to a stronger reverse photocurrent signal, attributable to improved light absorption efficiency at the photoelectrode (Figure R2d). In this study, the final experimental conditions were determined to be a pH of 7.0, an applied potential of -0.5 V, a 0.01 M Na SO 2 4\nelectrolyte solution, and a light intensity of 100 mW/cm², which were subsequently employed for the identification and quantitative analysis of reductant molecules.\n(3) No long-term cycling data (> hours) or batch-to-batch reproducibility tests are provided to assess electrode stability.\nResponse: According to the reviewer’s suggestion, we tested the long-term cycling and batch-to-batch reproducibility of the reverse photocurrent. For the pristine BiOBr photoelectrode under prolonged on-off light cycling, the reverse photocurrent exhibited instability (Figure R3a). In contrast, upon the addition of glutathione, both the intensity and stability of the photocurrent response were markedly improved (Figure R3b). This behavior can be attributed to the underlying mechanism of reverse photocurrent generation. Under illumination, Bi3+ is reduced, leading to decreased ORR activity. In the dark, the intermediate state Bi(3-&)+ is oxidized, resulting in enhanced ORR activity. Therefore, during the long-term cycling process, the pure BiOBr photoelectrode is difficult to maintain a reversible Bi redox reaction at equal time intervals (10 seconds of light and 10 seconds of dark), resulting in reaction imbalance and photocurrent attenuation. However, after adding GSH, due to its reversible reaction characteristics, it can alleviate the above imbalance to a certain extent, thereby maintaining a more stable photocurrent for a longer time. Notably, since quantitative analysis of the target analyte is conducted within a relatively short timeframe, maintaining photocurrent stability for approximately 100 seconds is sufficient to meet the experimental requirements of this study. Furthermore, comprehensive morphological (Figure R4), crystallographic (Figure R5), and photocurrent (Figure R6) characterizations were performed on three independently synthesized batches of BiOBr, confirming consistent batch-to-batch reproducibility. In conclusion, the dark-PEC signal in this study exhibited excellent reliability with respect to stability and repeatability, thereby providing robust support for subsequent research endeavors.\n(4) Given that Bi(3-&)+ is chemically unstable, potential degradation pathways during repeated light-dark cycling are not addressed.\nResponse: As highlighted by the reviewer, Bi(3-&)+ exhibits chemical instability. To validate the presence of this intermediate species, we conducted comprehensive XPS measurements and in-depth analyses (Figure R7). The results indicate that the charge transfer (CT) satellite peak observed in the low binding energy region of the Bi 4f XPS spectrum following light exposure arises from the effective shielding effect of O 2p electrons on 4f holes, without inducing a significant alteration in the Bi3+ chemical state. During this process, Bi exhibits two distinct final state configurations: the primary peak corresponds to the [Xe] 4f13 5d10 6s2 configuration, while the CT satellite peak is associated with the [Xe] 4f13 5d10 6s2 6p1 configuration. In addition, the stable oxidation states of bismuth in reality are only three: 0, +3, and +5. It is difficult to correspond to the standard XPS characteristic peaks of +1 and +2 oxidation states. Accordingly, we designate the bismuth species with oxidation states between 0 and +3 as Bi(3-&)+. Furthermore, electron paramagnetic resonance (EPR) measurements were employed to investigate the reversible behavior of BiOBr under alternating light and dark conditions. In comparison with the sample in the dark state, BiOBr after illumination exhibits a markedly enhanced EPR signal at g = 2.01 (Figure R8). This enhancement can be attributed to an increased concentration of oxygen vacancy defects under photoexcitation, which is consistent with the appearance of the CT satellite peak in the Bi 4f XPS spectrum.\nThe observation of the “dark-enhanced” reverse photocurrent during the light-dark cycle can be attributed precisely to the high instability of this intermediate state. During the illumination process, Bi3+ in the BiOBr photocathode undergoes a self-reduction reaction, generating Bi(3-&)+, which leads to a decrease in the interfacial ORR activity. When the light is turned off, this metastable Bi(3-&)+ is gradually oxidized by molecular oxygen and returns to the stable Bi3+ state, significantly enhancing the interfacial ORR activity. Therefore, the possible degradation path of Bi(3-&)+ mentioned by the reviewer is actually the process of being oxidized by oxygen in the absence of light and reverting to the original Bi3+ state (Figure R8).\n(5) Application relevance is overstated. The GSH sensing range and detection limit are comparable to or weaker than existing PEC sensors, while the operational constraints (air-saturation requirement) reduce practical applicability.\nResponse: Based on the reviewer’s comment, we conducted a comparative analysis of this work with existing studies on GSH detection using traditional PEC signals. The results indicate that the proposed method exhibits significant advantages in terms of both sensing range and detection limit. Notably, no prior research has thoroughly investigated the distinctive characteristics of the “dark-enhanced” PEC signaling mechanism in molecular detection. To the best of our knowledge, this study is the first to propose and systematically explore this mechanism, revealing that its cathodic reverse photocurrent enables selective recognition of GSH molecules. By regulating reaction sites under alternating light and dark conditions, this study establishes a new-type strategy for small molecule detection. As summarized in Table R2, the approach demonstrates superior performance with respect to signal type, sensitivity, selectivity, sensing range, and detection limit. In particular, the high selectivity allows for effective discrimination of GSH from other thiol-containing compounds, representing a notable advancement in PEC-based sensing applications. Furthermore, all PEC sensing platforms were evaluated under air-saturated conditions. This environment offers a relatively stable detection setting without requiring additional gas introduction procedures, thereby ensuring strong practical applicability.\nReply to reviewer 2: Thank you very much for your valuable comments.\nReviewer #2: The authors report a novel “dark-enhanced” photoelectrochemical (PEC) system based on a BiOBr photocathode, where reversible bismuth reduction drives an unconventional reverse photocurrent (|I | > |I |) under air conditions. The work demonstrates that glutathione dark light\n(GSH) selectively interacts with BiOBr, enhancing the reversibility of the bismuth redox cycle and amplifying the reverse photocurrent. This mechanism enables selective detection of reductant molecules at the photocathode interface, addressing a key challenge of selectivity in PEC sensing systems. The study combines electrochemical, spectroscopic, and surface characterization analyses to validate the mechanism and demonstrate practical applications in GSH quantification in real samples.\nThe manuscript introduces a compelling conceptual advancement in photoelectrochemical systems by shifting the paradigm from light-enhanced to dark-enhanced PEC processes. The mechanistic insights into reversible bismuth redox cycles, coupled with selective molecular recognition at the cathode, represent an original approach with potential implications for selective PEC sensing.\nThe experimental validation is thorough, with adequate characterization (XPS, TEM, EDS, Raman, Mott-Schottky) supporting the proposed mechanism. However, there are points requiring further clarification, especially regarding the reproducibility, scope, and broader applicability of the dark-PEC concept beyond the GSH model system.\nI believe the manuscript has high novelty and significance, but several substantial issues need to be addressed before it can be considered for publication in Nature Communications.\n(1) Mechanistic depth-Role of oxygen concentration\nThe proposed dark-PEC mechanism relies on a delicate balance between O availability and Bi 2\nreduction/oxidation dynamics. However, the authors should quantitatively assess the oxygen concentration threshold where the transition from traditional PEC to dark-PEC behavior occurs. Could this be correlated with electrochemical O reduction kinetics? 2\nResponse: Given that the dark-PEC mechanism depends on a delicate balance between oxygen availability and the Bi redox kinetics, and considering the challenges in precisely controlling and maintaining a constant dissolved oxygen concentration in the electrolyte during experiments, we leveraged the relatively stable dissolved oxygen levels in ambient air while investigating the transition from conventional PEC to dark-PEC behavior. To address this issue, quantitative analysis of the photocurrent direction reversal was achieved by systematically varying the amount of BiOBr deposited on the photoelectrode surface. As illustrated in Figure R10, under daytime air conditions, the dissolved oxygen concentration in the electrolyte solution remains stably maintained at approximately 5 mg/L. Photocurrent response measurements of photoelectrodes modified with varying concentrations of BiOBr reveal that when the C /C (10-3) ratio oxygen BiOBr\nranges from 5 to 2.5, the photocurrent behavior progressively transitions from the conventional PEC mode to the dark-PEC mode (Figure R11). This transformation is primarily governed by the threshold proportionality between the dissolved oxygen concentration and the BiOBr modification layer concentration.\n(2) Generality of the mechanism.\nWhile the work demonstrates GSH selectivity, it remains unclear if this mechanism could extend to other thiol-containing molecules or reductants capable of interacting with Bi species. Testing a broader set of analytes with similar functional groups would help position this platform as a more general sensing strategy.\nResponse: Based on the reviewer’s comment, we systematically evaluated the effects of various thiol-containing molecules on the photocurrent signal following their addition to the electrolyte solution. As illustrated in Figure R12, both reduced glutathione and oxidized glutathione significantly enhanced the reversed photocurrent signal, whereas the other tested molecules exhibited only a minimal effect in inducing this response. We hypothesize that this phenomenon is closely associated with the reversible redox mechanism of GSH. Since other molecules lack reversible redox properties, they may interfere with the reversible interconversion of bismuth in different valence states, consequently resulting in a reduction of the reverse photocurrent. The concentration of all compounds in the electrolyte was maintained at 600 μM.\n(3) Photocurrent reversibility and stability over time.\nThe manuscript lacks a detailed stability assessment. How does the reverse photocurrent behavior evolve over multiple cycles or prolonged operation? Could there be degradation of the BiOBr surface or accumulation of irreversible Bi(0)?\nResponse: According to the reviewer’s suggestion, we tested the long-term cycling of the reverse photocurrent. For the pristine BiOBr photoelectrode under prolonged on-off light cycling, the reverse photocurrent exhibited instability (Figure R13a). In contrast, upon the addition of glutathione, both the intensity and stability of the photocurrent response were markedly improved (Figure R13b). This behavior can be attributed to the underlying mechanism of reverse photocurrent generation: under illumination, Bi3+ is reduced, leading to decreased ORR activity; in the dark, the intermediate state Bi(3-&)+ is oxidized, resulting in enhanced ORR activity. Therefore, during the long-term cycling process, the pure BiOBr photoelectrode is difficult to maintain a reversible Bi redox reaction at equal time intervals (10 seconds of light and 10 seconds of dark), resulting in reaction imbalance and photocurrent attenuation. After adding glutathione, due to its reversible reaction characteristics, it can alleviate the above imbalance to a certain extent, thereby maintaining a more stable photocurrent for a longer time. Notably, since quantitative analysis of the target analyte is conducted within a relatively short timeframe, maintaining photocurrent stability for approximately 100 seconds is sufficient to meet the experimental requirements of this study.\nIn addition, XPS analysis was performed on the photoelectrodes following PEC testing (Figure R14). The results revealed no detectable accumulation or reduction of Bi(0) on the surface of the pristine BiOBr photoelectrodes. Moreover, upon exposure to ambient air after testing, the bismuth species were fully re-oxidized to the Bi3+ state.\n(4) Signal amplification and sensitivity comparison.\nThe LOD reported (20 nM GSH) is excellent, but a benchmarking against state-of-the-art PEC sensors for GSH (or similar reductants) should be included to contextualize the advancement in sensitivity and selectivity.\nResponse: According to the reviewer’s suggestion, we conducted a comparative analysis of this work with existing studies on GSH detection using traditional PEC signals. The results indicate that the proposed method exhibits significant advantages in terms of both sensing range and detection limit. Notably, no prior research has thoroughly investigated the distinctive characteristics of the “dark-enhanced” PEC signaling mechanism in molecular detection. To the best of our knowledge, this study is the first to propose and systematically explore this mechanism, revealing that its cathodic reverse photocurrent enables selective recognition of GSH molecules. As summarized in Table R3, the approach demonstrates superior performance with respect to signal type, sensitivity, selectivity, sensing range, and detection limit. In particular, the high selectivity allows for effective discrimination of GSH from other thiol-containing compounds, representing a notable advancement in PEC-based sensing applications.\n(5) Origin of the unconventional energy band (Bi(3-&)+).\nThe description of Bi(3-&)+ as an intermediate oxidation state is central but speculative. The notation is non-standard and chemically ambiguous. The XPS data (binding energy shift, new peak at ~157.5 eV) lacks deconvolution or comparison with Bi⁰/Bi(I) standards, making the assignment uncertain. The authors should clarify the nature of this species and support its existence with additional spectroscopic evidence (e.g., XANES, in-situ XPS) or, at minimum, a rigorous XPS fitting.\nResponse: According to the reviewer’s suggestion, we performed a systematic and rigorous analysis and validation of the valence state variations of bismuth under light and dark conditions. The results indicate that the charge transfer (CT) satellite peak observed in the low binding energy region of the Bi 4f XPS spectrum following light exposure arises from the effective shielding effect of O 2p electrons on 4f holes, without inducing a significant alteration in the Bi3+ chemical state. During this process, Bi exhibits two distinct final state configurations: the primary peak corresponds to the [Xe] 4f13 5d10 6s2 configuration, while the CT satellite peak is associated with the [Xe] 4f13 5d10 6s2 6p1 configuration. In addition, the stable oxidation states of bismuth in reality are only three: 0, +3, and +5. It is difficult to correspond to the standard XPS characteristic peaks of +1 and +2 oxidation states. Accordingly, we designate the bismuth species with oxidation states between 0 and +3 as Bi(3-&)+ (Figure R15). Furthermore, electron paramagnetic resonance (EPR) measurements were employed to investigate the reversible behavior of BiOBr under alternating light and dark conditions. In comparison with the sample in the dark state, BiOBr after illumination exhibits a markedly enhanced EPR signal at g = 2.01 (Figure R16). This enhancement can be attributed to an increased concentration of oxygen vacancy defects under photoexcitation, which is consistent with the appearance of the CT satellite peak in the Bi 4f XPS spectrum.\n(6) Figure Quality.\nFigures (e.g., Fig. 1, Fig. 4) would benefit from clearer labels and a more schematic visualization of the electron flow directions.\nResponse: According to the reviewer’s suggestion, we have further refined the annotations of the electron flow directions in Fig. 1 and Fig. 4 to enhance their clarity and readability (Figures R17 and R18).\n(7) Terminology Consistency.\nThroughout the text, the term “reverse photocurrent” is used. It might be helpful to explicitly define this term early on (i.e., reverse with respect to light-on condition).\nResponse: According to the reviewer’s suggestion, we have clearly defined the term “reverse photocurrent” in the early part of the article to enhance the clarity and readability of the expression.\n(8) Supporting Information – Missing Spectroscopic Data.\nIn SI, including high-resolution XPS spectra for GSH-interacted BiOBr after long-term cycling (post-mortem analysis) would strengthen the stability claims.\nResponse: According to the reviewer’s suggestion, we have incorporated high-resolution XPS spectral data of the GSH-BiOBr photoelectrode obtained after prolonged PEC testing. The results demonstrate that, during the testing period, the photoelectrode exhibits an enhanced and more stable reverse photocurrent response under light-dark cycling conditions, attributable to the significant and reversible transformation between high-valent Bi(3+&)+/Bi3+ states and low-valent Bi(3-&)+ species (Figure R19).\nReply to reviewer 3: Thank you very much for your valuable comments.\nReviewer #3: This paper proposes a “dark-enhanced” photocatalytic system based on a BiOBr photoanode, achieving a reverse photocurrent (|I | > |I |) through the reversible reduction of dark light\nbismuth, and applies it to the selective detection of glutathione, demonstrating a certain degree of innovation. However, the paper still has several shortcomings and requires major revisions and improvements. The following are the revision suggestions:\n(1) The discussion on selectivity in traditional PEC systems is overly brief. The manuscript lacks a systematic review of recent advances in enhancing PEC selectivity, resulting in an unclear benchmark for evaluating the innovation of the proposed system. Please supplement the introduction with representative studies from the past five years that focus on PEC selectivity control.\nResponse: According to the reviewer’s suggestion, we have supplemented the discussion in the introduction with representative studies on the selective control of PEC over the past five years.\n(2) The functions of mannitol and PVP in the synthesis of BiOBr nanosheets are not clearly explained. Please elaborate on their roles during the preparation process (line 265).\nResponse: According to the reviewer’s suggestion, we have provided a detailed explanation of the roles of PVP and mannitol in the synthesis process within the Methods section.\n(3) The characterization of BiOBr's structural and optoelectronic properties is based on a single synthesis batch, without replicates. To confirm reproducibility, results from at least three independent syntheses should be included, particularly regarding the consistency of morphology, crystallinity, and photoelectrochemical performance (refer to line 61).\nResponse: According to the reviewer’s suggestion, comprehensive morphological (Figure R20), crystallinity (Figure R21), and photoelectrochemical performance (Figure R22) characterizations were performed on three independently synthesized batches of BiOBr, confirming consistent batch-to-batch reproducibility.\n(4) In line 83, it is claimed that the reverse photocurrent is influenced by a “moderate O 2 concentration,” yet no quantitative data or correlation is provided. Please conduct a gradient experiment (0-100% O ) and plot the relationship between O partial pressure and 2 2\nphotocurrent response to define the “moderate” range more precisely.\nResponse: Given that the dark-PEC mechanism depends on a delicate balance between oxygen availability and the Bi redox kinetics, and considering the challenges in precisely controlling and maintaining a constant dissolved oxygen concentration in the electrolyte during experiments, we leveraged the relatively stable dissolved oxygen levels in ambient air while investigating the transition from conventional PEC to dark-PEC behavior. To address this issue, quantitative analysis of the photocurrent direction reversal was achieved by systematically varying the amount of BiOBr deposited on the photoelectrode surface. As illustrated in Figure R23, under daytime air conditions, the dissolved oxygen concentration in the electrolyte solution remains stably maintained at approximately 5 mg/L. Photocurrent response measurements of photoelectrodes modified with varying concentrations of BiOBr reveal that when the C /C (10-3) ratio oxygen BiOBr\nranges from 5 to 2.5, the photocurrent behavior progressively transitions from the conventional PEC mode to the dark-PEC mode (Figure R24). This transformation is primarily governed by the threshold proportionality between the dissolved oxygen concentration and the BiOBr modification layer concentration.\n(5) The valence state change of bismuth is currently inferred only from XPS binding energy shifts and electrochemical behavior, which is indirect. To convincingly demonstrate the reversible redox process and presence of intermediate oxidation states (line 98), additional direct evidence such as in situ X-ray absorption fine structure and electron paramagnetic resonance data is recommended.\nResponse: According to the reviewer’s suggestion, we performed a systematic and rigorous analysis and validation of the valence state variations of bismuth under light and dark conditions. The results indicate that the charge transfer (CT) satellite peak observed in the low binding energy region of the Bi 4f XPS spectrum following light exposure arises from the effective shielding effect of O 2p electrons on 4f holes, without inducing a significant alteration in the Bi3+ chemical state. During this process, Bi exhibits two distinct final state configurations: the primary peak corresponds to the [Xe] 4f13 5d10 6s2 configuration, while the CT satellite peak is associated with the [Xe] 4f13 5d10 6s2 6p1 configuration. In addition, the stable oxidation states of bismuth in reality are only three: 0, +3, and +5. It is difficult to correspond to the standard XPS characteristic peaks of +1 and +2 oxidation states. Accordingly, we designate the bismuth species with oxidation states between 0 and +3 as Bi(3-&)+ (Figure R25). Furthermore, electron paramagnetic resonance (EPR) measurements were employed to investigate the reversible behavior of BiOBr under alternating light and dark conditions. In comparison with the sample in the dark state, BiOBr after illumination exhibits a markedly enhanced EPR signal at g = 2.01 (Figure R26). This enhancement can be attributed to an increased concentration of oxygen vacancy defects under photoexcitation, which is consistent with the appearance of the CT satellite peak in the Bi 4f XPS spectrum.\n(6) Line 151 states that Bi(3-δ)+ species may passivate ORR sites and reactivate O₂ oxidation in the dark. However, this claim lacks in situ spectroscopic support. Please consider including data from in situ Raman spectroscopy or in situ AFM to support this hypothesis.\nResponse: According to the reviewer’s suggestion, we experimentally verified the inference that the intermediate state Bi(3-&)+ was generated after light exposure and thereby inhibited the ORR through in situ Fourier transform infrared spectroscopy (FTIR). As shown in Figure R27, after light irradiation, the intensities of the H-O-H bending vibration and O-H stretching vibration peaks associated with adsorbed water on the BiOBr surface were significantly reduced, indicating decreased ORR activity. Meanwhile, a comparison of the CV curves before and after illumination reveals that the initial reduction potential for the ORR on the BiOBr photoelectrode shifts to more negative values upon illumination, accompanied by a decrease in reduction current density (Figure R28). This observation further suggests that light irradiation retards the electron transfer kinetics at the solid-liquid interface, leading to passivation of the ORR active sites.\n(7) The manuscript does not discuss the impact of light parameters (e.g., intensity, wavelength) on the reduction degree of Bi species, which may directly influence the generation of Bi(3-δ)+ and hence affect the reverse photocurrent. Please address this in the discussion.\nResponse: According to the reviewer’s suggestion, we conducted reverse photocurrent tests under different light intensities and wavelengths (Figure R29). The results indicated that the reverse photocurrent significantly increased with the rise of light intensity. This was attributed to the fact that the enhanced light intensity promoted the generation of the intermediate state Bi(3-&)+, thereby increasing the extent of reversible electron transfer. Additionally, the tests under different wavelength conditions revealed that the reversible conversion between the intermediate state Bi(3-&)+ and Bi3+ was more efficient under 380 nm light, resulting in the largest reverse photocurrent response. In this study, the final experimental conditions were were established using simulated solar irradiation with a wavelength range corresponding to AM 1.5G and a light intensity of 1000 mW/cm², which were subsequently employed for the identification and quantitative analysis of reductant molecules.\n(8) The validation system is not rigorous enough. Only interference from five substances, including CY and UA, was compared, but interference from other structurally similar reducing agents (such as cysteine and glutathione disulfide bonds) was not ruled out (line 237).\nResponse: According to the reviewer’s suggestion, we systematically evaluated the effects of various thiol-containing molecules on the photocurrent signal following their addition to the electrolyte solution. As illustrated in Figure R30, both reduced glutathione and oxidized glutathione significantly enhanced the reversed photocurrent signal, whereas the other tested molecules exhibited only a minimal effect in inducing this response. We hypothesize that this phenomenon is closely associated with the reversible redox mechanism of GSH. Since other molecules lack reversible redox properties, they may interfere with the reversible interconversion of bismuth in different valence states, consequently resulting in a reduction of the reverse photocurrent. The concentration of all compounds in the electrolyte was maintained at 600 μM.\n(9) How does this detection system perform in terms of interference resistance in complex matrices such as serum?\nResponse: In this work, we have collected the experimental data on the detection of GSH in actual plant samples, thereby further validating the reliability of the dark-PEC method for analyzing GSH in spinach, onion, and broccoli (Fig. 5e and Supplementary Table 2). Furthermore, we have assessed the analytical performance of GSH in 10-fold diluted saliva samples, and the results demonstrate its excellent anti-interference capability and practical applicability in biological matrices (Figure R31).\n(10) The recovery rate experiment for actual sample testing (243 lines) was not compared with other standard methods, making it difficult to prove the accuracy of the method. At the same time, intra-day and inter-day comparison data need to be supplemented.\nResponse: According to the reviewer’s suggestion, the proposed dark-PEC method was compared with the colorimetric method in the detection of actual samples in this study. The results demonstrated a high degree of consistency between the two methods in terms of concentration determination. Digital photographs, photocurrent response signals, and absorbance response data for the three actual samples are presented in Figure R32, while the corresponding quantitative analysis results are summarized in Table R4. The analytical accuracy ranged from 97.48% to 103.0%, further confirming the high precision of the proposed method. In addition, intra-day and inter-day recovery tests were conducted to evaluate method reliability (Table R5). The recovery rates varied between 98.8% and 107%, with relative standard deviations ranging from 0.52% to 8.16%, indicating excellent repeatability and robustness in sample analysis.\n(11) This study did not compare the dark-PEC system with the traditional PEC (GSH detection based on anodic oxidation) in terms of sensitivity (detection limit), selectivity, response time, and other quantitative aspects, making it difficult to highlight its technical advantages. A comparison table should be added to highlight the technical advantages of this work.\nResponse: According to the reviewer’s suggestion, we conducted a comparative analysis of this work with existing studies on GSH detection using traditional PEC signals. The results indicate that the proposed method exhibits significant advantages in terms of both sensing range and detection limit. Notably, no prior research has thoroughly investigated the distinctive characteristics of the “dark-enhanced” PEC signaling mechanism in molecular detection. To the best of our knowledge, this study is the first to propose and systematically explore this mechanism, revealing that its cathodic reverse photocurrent enables selective recognition of GSH molecules. As summarized in Table R6, the approach demonstrates superior performance with respect to signal type, sensitivity, selectivity, sensing range, and detection limit. In particular, the high selectivity allows for effective discrimination of GSH from other thiol-containing compounds, representing a notable advancement in PEC-based sensing applications."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all the queries in the revised manuscript. I suggest its publication in its current version.", "reviewer_2": "The authors fully respond to my concerns, especially the mechanism understanding with valuable new data. The paper can published with no changes.", "reviewer_3": "The authors have addressed all the points satisfactorily I raised in my previous review. I have no further concerns and support the acceptance of the manuscript."}, "author_response": ""}]

Decision letter