Bioelectrosynthesis of Signaling Molecules for Selective Modulation of Cell Signaling

Abstract

Bioelectrosynthesis holds great potential for studying and regulating biological systems through the in situ synthesis and delivery of cell signaling molecules with high spatiotemporal precision. Despite recent advancements, precise control over multiple signaling molecules within a single platform remains challenging. Here, we introduce a bioelectrosynthesis approach capable of selectively producing two types of signaling molecules from a single precursor. This system leverages multi-metal sulfide electrocatalysts inspired by denitrifying enzymes, which generate signaling molecules, nitric oxide (NO), and ammonia (NH₃) from nitrite ions. By controlling catalytic active sites, NO or NH₃ can be selectively produced under mild electric fields in physiologically relevant conditions. In situ product analyses and first-principles calculations reveal that NO intermediate binding affinity determines product selectivity. These electrocatalysts integrate seamlessly with biological systems, allowing precise, on-demand modulation of NO- or NH₃-mediated signaling pathways in human cell lines. By combining electrochemical precision with selective cell control, this strategy may advance the study and regulation of biological systems.

Keywords: Biocatalysis; Bioelectrosynthesis; Computational chemistry; Electrochemistry; Signal transduction.

Figure 1

A schematic of a bioelectrosynthesis approach for the independent and selective production of signaling molecules from the NO₂ ⁻ ion. The control of active sites in the electrocatalysts determines the selectivity toward NO and NH₃, offering an independent modulation of corresponding cell signaling.

Figure 2

a) Schematic representation of the crystal structure of Cu₂MoS₄. Mo, Cu, and S atoms are marked in purple, orange, and yellow, respectively. b) XRD patterns for synthesized Cu₂MoS₄ and FeCuMoS₄. c) HRTEM, d) STEM, and e) STEM‐EDS mapping images of synthesized Cu₂MoS₄. f) A schematic of the Fe doping process for Cu₂MoS₄ to synthesize FeCuMoS₄. Doped Fe ions are depicted in dotted circles. g) ICP‐OES results of Cu₂MoS₄ and FeCuMoS₄ indicated that doped Fe ions preferentially substituted the Cu ions. h) HRTEM, i) STEM, and j) STEM‐EDS mapping images of FeCuMoS₄. The STEM‐EDS mapping image indicated uniform distributions of Cu (red), Mo (blue), and Fe (green) ions inside the crystal.

Figure 3

CV curves of the a) bare carbon, b) Cu₂MoS₄‐loaded, and c) FeCuMoS₄‐loaded electrodes in the presence or absence of 0.1 M NO₂ ⁻ containing Tyrode's solution at a scan rate of 50 mV s⁻¹. Chronoamperometry profiles of d) Cu₂MoS₄‐loaded and e) FeCuMoS₄‐loaded electrodes at the applied voltage from −1.0 to −2.0 V versus Ag/AgCl. FE_NH4+ (mean ± SD) with the f) Cu₂MoS₄ or h) FeCuMoS₄ electrocatalysts and FE_NO (mean ± SD) with the g) Cu₂MoS₄ or i) FeCuMoS₄ electrocatalysts at various applied voltage conditions, respectively (n = 3 independent experiments per group). A potential range from −1.0 to −2.0 V versus Ag/AgCl was utilized for the analysis.

Figure 4

Configurations illustrating the binding of NO₂ ⁻ to a) Cu₂MoS₄ and b) FeCuMoS₄ surfaces. Cu, Mo, Fe, S, N, and O atoms were marked in orange, purple, cyan, yellow, blue, and red circles, respectively. c) Gibbs free energy diagrams for the NO₂ ⁻ reduction reaction catalyzed by Cu₂MoS₄ at an applied potential (U) of 0 V (black) and 0.38 V versus the RHE (blue). d) Gibbs free energy diagrams for the NO₂ ⁻ reduction reaction catalyzed by FeCuMoS₄ at U = 0 V (black) and 0.77 V versus RHE (blue). Atomic Bader charge analyses for e) Cu in Cu₂MoS₄ and f) Fe in FeCuMoS₄ before (gray) and after (black) binding to NO. g) Changes in Bader charge of NO after the binding to Cu₂MoS₄ and FeCuMoS₄. h) A schematic illustrating the strong back‐pi bonding between the Fe active site and NO intermediates in FeCuMoS₄.

Figure 5

TRPV1 activation with electrochemically produced NO. a) Schematic representation of two plasmids used to generate genetically engineered cells co‐expressing TRPV1 and GCaMP6s (left) and the mechanism of NO‐mediated activation of TRPV1 and subsequent Ca²⁺ influx (right). b) Schematic illustrating selective activation of TRPV1 by electrochemically produced NO, but not by NH₃. Here, cells were positioned near the electrode where localized electrosynthesis of NO or NH₃ occurred. Individual GCaMP6s fluorescence traces for 150 TRPV1⁺ cells upon application of a voltage of −1.2 V versus Ag/AgCl to c) Cu₂MoS₄ and d) FeCuMoS₄ electrocatalysts. The voltages were turned on at 30 s (dashed lines), and continuous electrochemical synthesis was performed until 900 s. Here, HEK ID represents individual cells analyzed, and each horizontal line displays the GCaMP6s fluorescence changes of the individual cell over time. A total of 150 cells were analyzed for each condition (n = 3 independent experiments per group). e) Schematic depiction of time‐lapse imaging showing sequential activation of TRPV1⁺ cells during electrosynthesis of NO. Time points t₁, t₂, and t₃ represent increasing durations of electrolysis, with TRPV1⁺ cells located farther from the electrode being activated at later time points due to diffusion of NO. f) Representative time‐lapse images of Ca²⁺ responses in TRPV1⁺ cells in the presence of applied voltages of −1.2 V versus Ag/AgCl and FeCuMoS₄ electrocatalysts. Electrodes loaded with FeCuMoS₄ electrocatalysts were located at the left in all four images (scale bar: 100 µm). The vertical axis corresponds to GCaMP6s fluorescence intensity, and the color scale was utilized to visualize the GCaMP6s fluorescence changes.

Figure 6

OTOP1 activation mediated by electrosynthesized NH₃. a) Schematic of plasmids used to prepare cells co‐expressing OTOP1 and SEpHluorin (left) and the mechanism of NH₃‐mediated intracellular alkalinization and consequent activation of OTOP1 (right). b) Schematic illustrating selective intracellular alkalinization by electrochemically produced NH₃, but not by NO. Here, cells were positioned near the electrode where NO or NH₃ signaling molecules were locally produced. Individual SEpHluorin fluorescence traces for 150 OTOP1⁺ cells following the electrosynthesis mediated by c) Cu₂MoS₄ and d) FeCuMoS₄ electrocatalysts. −1.2 V versus Ag/AgCl was applied from 30 s (dashed lines) to 900 s. Here, HEK ID refers to individual cells, with each horizontal line representing the SEpHluorin fluorescence changes of the individual cell over time. For each condition, fluorescence responses were recorded from a total of 150 cells (n = 3 independent experiments per group). Time‐lapse images of OTOP1⁺ cells, which were placed in the vicinity of the electrode, upon applying voltages of −1.2 V versus Ag/AgCl to e) Cu₂MoS₄ or f) FeCuMoS₄ electrocatalysts (scale bar: 100 µm). The vertical axis represents SEpHluorin fluorescence intensity, and the color scale was employed to visualize the SEpHluorin fluorescence changes.