In this work, we present (hemi)spherical atomic force microscopy (AFM) sensors for the detection of hydrogen peroxide. Platinum-black (Pt-B) was electrodeposited onto conductive colloidal AFM probes or directly at recessed microelectrodes located at the end of a tipless cantilever, resulting in electrocatalytically active cantilever-based sensors that have a small geometric area but, due to the porosity of the films, exhibit a large electroactive surface area. Focused ion beam-scanning electron microscopy tomography revealed the porous 3D structure of the deposited Pt-B. Given the accurate positioning capability of AFM, these probes are suitable for local in situ sensing of hydrogen peroxide and at the same time can be used for (electrochemical) force spectroscopy measurements. Detection limits for hydrogen peroxide in the nanomolar range (LOD = 68 ± 7 nM) were obtained. Stability test and first in situ proof-of-principle experiments to achieve the electrochemical imaging of hydrogen peroxide generated at a microelectrode and at photocatalytically active structured poly(heptazine imide) films are demonstrated. Force spectroscopic data of the photocatalyst films were recorded in ambient conditions, in solution, and by applying a potential, which demonstrates the versatility of these novel Pt-B-modified spherical AFM probes.

Producing clean energy is one of the world's greatest challenges today. The use of solar energy to split water into hydrogen and oxygen is therefore of great interest. In addition to semiconductor materials, heterogenized molecular catalysts for the hydrogen evolution reaction (HER) and water oxidation catalysts (WOC) have attracted considerable interest. The development and design of artificial molecular devices (i.e. artificial photosynthesis systems) using visible light requires the heterogenization of catalysts (CAT) and photosensitizers (PS). However, the correlation of structural heterogeneity, leaching of active components and degradation processes during illumination, as well as the continuous monitoring of reactivity have not been fully addressed.1,2 Scanning electrochemical probe microscopy such as atomic force microscopy (AFM), scanning electrochemical microscopy (SECM), scanning electrochemical cell microscopy (SECCM), and scanning photoelectrochemical microscopy (SPEM), have been used to map light-driven water splitting and local electron transfer kinetics under in situ and operando conditions.3-5 In this contribution, we present the deposition of mixed CAT/PS nanowires using earth-abundant BPh2-bridged, organometallic cobaloxime CATs and Ru-based ([Ru(tbbpy)2(mmip)]Cl3) PS on various substrates using SECCM. Based on different studies an cabolime microarrays6, the nanowires are characterized with respect to their stabilty, elemental composition as well as their light-driven H2 evolution activity. The H2 evolution of nanowire arrays is imaged via SECM using Pd- and Pt-Black modified microelectrodes. In addition, submicron-sized electrocatalytically active cantilever-based probes, recently developed by our group, are used for improved lateral resolution. These probes can be used for colloidal force spectroscopy7,8 providing nanomechanical properties of the studied materials. A thorough characterisation of the cantilever-based sensors will be presented together with nanomechanical and activity mapping of nanowires with different CAT/PS ratios. References: 1 C. Kranz, M. Wächtler, Chem. Soc. Rev. 50, 1407 , 2021 . 2 C. Santana Santos et al., Chem. Rev. 123, 4972, 2023. 3 J. Kund et al., Angew. Chem. Int. Ed. 22, e202217196, 2023. 4 G. Askarova et al., Anal. Chem. 94, 7169, 2022. 5 J.T. Mefford, et al., Nature, 593, 67, 2021. 6 E. Oswald et al, Chem. Eur. J. 27, 16896 , 2021. 7 H.-J. Butt, Biophys. J. 60, 1438, 1991. 8 S. Daboss, Anal. Chem. 92, 8404, 2020. This work is part of the TRR 234 “CataLight” project that has received funding from the Deutsche Forschungsgemeinschaft (German Science Foundation), DFG. Project number 36454990.

ATP is one of the most widely distributed extracellular signaling molecules, regulating many physiological and pathological processes via activation of purinergic receptors. Extracellular ATP may originate from cell-regulated release but also from cell membrane injury. To measure ATP release in-vitro or invivo for insight into the mechanisms of transmission requires its direct, sensitive, non-invasive detection, ideally with high spatial and temporal resolution. This current opinion article highlights recent research in ATP detection using electrochemical methods and presents examples how in-vitro electrochemical ATP measurements can contribute to insights in purinergic signaling for pivotal functions like alveolar homeostasis and how ATP acts as major excitatory neurotransmitter in the central nervous system.

Advanced scanning probe microscopy techniques such as atomic force – scanning electrochemical microscopy (AFM-SECM) und scanning electrochemical microscopy (SECM) are highly attractive hybrid techniques for studying biomedically relevant samples down to the single entity level. Our group introduced a new type of AFM-SECM probe having a conductive colloid instead of a sharp AFM tip. As this spherical microelectrode, which is located at the end of an electrically insulated AFM cantilever can be easily modified by electrodeposition with e.g., conductive polymers and electrocatalytic layers, single cell force spectroscopy under potential control allows studying adhesion properties [1,2]. Cell adhesion is a crucial parameter not only for developing new materials serving as substrates for neural scaffolds, electrodes, and biomedical devices but also in biofilm formation. Biofilms are well-organized aggregates of bacteria able to attach to and proliferate at almost every type of solid surface resulting in increased resistance to conventional antimicrobial and antibiofouling agents [3].The first attachment of bacterials is a process highly influenced by the nature of the surface, such as hydrophobicity, chemical structure, surface charge, and presence of antimicrobials [4]. In this contribution, we present the potential of various scanning probe microscopy techniques, as suitable tools to locally investigate the early stages of biofilm formation and the effects of various antibiofouling and antimicrobial systems against Escherichia coli and Pseudomonas fluorescens. For example, the properties of polydopamine, a bio-compatible polymer, are studied in respect to early stages of bacterial adhesion in dependence of surface charge density and applied potential [5]. Colloidal conductive AFM probes can also be modified electrocatalytic layers, suitable for detecting signaling molecules and at the same time allow electrochemical force spectroscopy at soft samples like biomedically relevant single entities. References [1] P. Knittel, H. Zhang, C. Kranz, G. Wallace and M. Higgins, Nanoscale 8, 4475 (2016). [2] S. Daboss, J. Lin M. Godejohan, C. Kranz, Anal. Chem. 92, 8404 (2020). [3] O. Ciofu, C. Moser, P. Østrup Jensen, N. Høiby Nat. Rev. Microbiol. 20, 621 (2022). [4] A. Viljoen, et al. J . Bacteriology, 202, e00125 (2020). [5] G. Caniglia, A. Teuber, B. Mizaikoff, C. Kranz Anal. Bioanal. Chem. 415, 2059 (2023).

Introduction Sodium thiosulfate (Na2S2O3), an H2S releasing agent, was shown to be organ-protective in experimental hemorrhage. Systemic inflammation activates immune cells, which in turn show cell type-specific metabolic plasticity with modifications of mitochondrial respiratory activity. Since H2S can dose-dependently stimulate or inhibit mitochondrial respiration, we investigated the effect of Na2S2O3 on immune cell metabolism in a blinded, randomized, controlled, long-term, porcine model of hemorrhage and resuscitation. For this purpose, we developed a Bayesian sampling-based model for 13C isotope metabolic flux analysis (MFA) utilizing 1,2-13C2-labeled glucose, 13C6-labeled glucose, and 13C5-labeled glutamine tracers. Methods After 3 h of hemorrhage, anesthetized and surgically instrumented swine underwent resuscitation up to a maximum of 68 h. At 2 h of shock, animals randomly received vehicle or Na2S2O3 (25 mg/kg/h for 2 h, thereafter 100 mg/kg/h until 24 h after shock). At three time points (prior to shock, 24 h post shock and 64 h post shock) peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from whole blood, and cells were investigated regarding mitochondrial oxygen consumption (high resolution respirometry), reactive oxygen species production (electron spin resonance) and fluxes within the metabolic network (stable isotope-based MFA). Results PBMCs showed significantly higher mitochondrial O2 uptake and lower O 2 • − production in comparison to granulocytes. We found that in response to Na2S2O3 administration, PBMCs but not granulocytes had an increased mitochondrial oxygen consumption combined with a transient reduction of the citrate synthase flux and an increase of acetyl-CoA channeled into other compartments, e.g., for lipid biogenesis. Conclusion In a porcine model of hemorrhage and resuscitation, Na2S2O3 administration led to increased mitochondrial oxygen consumption combined with stimulation of lipid biogenesis in PBMCs. In contrast, granulocytes remained unaffected. Granulocytes, on the other hand, remained unaffected. O 2 • − concentration in whole blood remained constant during shock and resuscitation, indicating a sufficient anti-oxidative capacity. Overall, our MFA model seems to be is a promising approach for investigating immunometabolism; especially when combined with complementary methods.

Polymeric carbon nitrides (PCN) are sustainable, tunable, non-toxic and chemically stable materials that represent highly promising heterogeneous photocatalysts for light-driven hydrogen peroxide production via selective reduction of dioxygen. However, most of the studies on photocatalytic H2O2 production using PCN-based photocatalysts reported so far have used PCN powder suspensions and have been carried out in the presence of additional (sacrificial) electron donors, such as aliphatic or aromatic alcohols. Herein, we report the first multicomponent hybrid photocathode based on PCN that is capable of selective reduction of dioxygen to H2O2 under visible light irradiation (420 nm LED). A comparative analysis of various photocathode architectures is carried out using electronic absorption spectroscopy, surface photovoltage spectroscopy, open-circuit photopotential spectroscopy, and photocurrent measurements, including in-situ detection of formed H2O2 using microelectrodes. Notably, the ability of PCN-based photocathodes to catalyze the light-driven reduction of O2 to H2O2 in the absence of any additional electron donor is unambiguously demonstrated. Our study thus highlights the intrinsic nature of the photocatalytic activity of PCN in H2O2 production, and paves the way for the development of further PCN-based photocathodes in which PCN could be coupled with more effective light absorbers to increase the overall performance.

Microelectrochemistry has a long history in the life sciences for in vivo and in vitro measurements of e.g., pH, oxygen, and signaling molecules such as nitric oxide (NO), adenosine-5`-triphosphate (ATP), hydrogen peroxide (H2O2) and neurotransmitters. In particular, gaining information on signaling molecules at live cells, e.g., during and after stimulation is of significance for understanding cell signaling. Within recent years, our research team has focused on the development of micro-sized sensing approaches for the detection of ATP [1,2], which is considered among the most important autocrine and paracrine signaling molecules [3]. H2O2 belongs to the group of reactive oxygen species (ROS), which is in contrast to radical ROS rather stable. H2O2 is involved in many physiological processes such as hypoxic signal transduction, cell differentiation and proliferation but is also involved in mediating immune responses [4]. The effect is, among other parameters, dependent on its local concentration and its exposure time. Platinum black is a highly suitable electrocatalytic nanomaterial for the electrochemical detection of H2O2 [5]. We recently demonstrated that Pt black modified microelectrodes can be used for H2O2 at granulocytes and peripheral blood mononuclear cells from pigs [6]. Within this presentation, we will introduce dual functional miniaturized probes that allow further miniaturization of electrochemical sensors for biomedical applications. We will demonstrate that these probes have great potential for cell measurements, i.e., at macrophages that produce and release ROS in response to phagocytosis or stimulation. References [1] E. Hecht, A. Liedert, A. Ignatius, B. Mizaikoff, C. Kranz, Biosens. Bioelectron. 2013, 44, 27. [2] C. Ziller, et al., ChemElectroChem 2017, 4, 864. [3] R. Corriden, P. A. Insel, Sci. Signal. 2010, 3, 104. [4] C. Lennicke et al., Cell Commun. Signal. 2015, 13, 39. [5] Y. Li, C. Sella, F. Lemaitre, M.G. Collignon, L. Thouin, C. Amatore, Electroanalysis 2013, 25, 895. [6] A. Hellmann, S. Daboss, F. Zink, C. Hartmann, P. Radermacher, C. Kranz, Electrochim. Acta 2020, 353, 30851

Electrochemical biosensors combine the inherent selectivity of biorecognition elements with the superior sensitivity of electroanalytical methods. The progress in the field directly benefits from the application of nanomaterials and nanostructured/nanosized electrochemical transducers that enhance the sensitivity, enable measurements in exceedingly small volumes and facilitate in vivo studies. Furthermore, miniaturized biosensors as scanning probe microscopy probes provide spatially resolved information. Considering the extensive body of literature in this field, we focus herein on an introduction of nanosized transducers along with selected applications in the field of biomedical science.

Secretion of pulmonary surfactant in the alveoli of the lungs is essential to maintain lung function. Stretching of alveoli during lung inflation is the main trigger for surfactant secretion. Yet, the molecular mechanisms how mechanical distension of alveoli results in surfactant secretion are still elusive. The alveolar epithelium consists of alveolar epithelial type I (ATI) and surfactant secreting type II (ATII) cells. ATI, but not ATII cells, express caveolae, small plasma membrane invaginations that can respond to plasma membrane stresses and serve mechanotransductive roles. Within this study, we investigated the role of caveolae as mechanosensors in the alveolus. We generated a human caveolin-1 knockout ATI cell (hAELVi(cav-/-)) using CRISPR/Cas9. Wildtype (hAELVi(wt)) and hAELVi(cav-/-)cells grown on flexible membranes responded to increasing stretch amplitudes with rises in intracellular Ca2+. The response was less frequent and started at higher stretch amplitudes in hAELVi(cav-/-)cells. Stretch-induced Ca2+-signals depended on Ca2+-entry via piezo1 channels, localized within caveolae in hAELVi(wt)and primary ATI cells. Ca2+-entry via piezo1 activated pannexin-1 hemichannels resulting in ATP release from ATI cells. ATP release was reduced in hAELVi(cav-/-)cells. In co-cultures resembling the alveolar epithelium, released ATP stimulated Ca(2+)signals and surfactant secretion from neighboring ATII cells when co-cultured with hAELVi(wt)but not hAELVi(cav-/-)cells. In summary, we propose that caveolae in ATI cells are mechanosensors within alveoli regulating stretch-induced surfactant secretion from ATII cells.

Immune cell activation leads to the acquisition of new functions, such as proliferation, chemotaxis, and cytokine production. These functional changes require continuous metabolic adaption in order to sustain ATP homeostasis for sufficient host defense. The bioenergetic demands are usually met by the interconnected metabolic pathways glycolysis, TCA cycle, and oxidative phosphorylation. Apart from glucose, other sources, such as fatty acids and glutamine, are able to fuel the TCA cycle. Rising evidence has shown that cellular metabolism has a direct effect on the regulation of immune cell functions. Thus, quiescent immune cells maintain a basal metabolic state, which shifts to an accelerated metabolic level upon immune cell activation in order to promote key effector functions. This review article summarizes distinct metabolic signatures of key immune cell subsets from quiescence to activation and demonstrates a methodical concept of how to assess cellular metabolic pathways. It further discusses why metabolic functions are of rising interest for translational research and how they can be affected by the underlying pathophysiological condition and/or therapeutic interventions.

It has been long known that hydrogen peroxide (H2O2) as an endogenous reactive oxygen species (ROS) belongs to a group of destructive molecules that can lead to protein oxidation, lipid peroxidation, and DNA damage. Whereas elevated ROS production leads to oxidative stress, it was also recently shown that at lower physiological levels, H2O2 acts as an intracellular signalling molecule regulating kinase-driven pathways1. As the involvement and concentration levels of H2O2 at the cellular level are not fully understood, its localized detection at a cellular level is of particular importance. Fluorescent redox-sensitive dyes are frequently used for the detection of H2O2 at the cellular level. H2O2 can also be detected electrochemically, e.g. at anodic potentials (0.6 V vs. Ag/AgCl) at Platinum (Pt) electrodes. However, such high oxidation potential can be affected by co-oxidizable substances such as ascorbic acid, catecholamine etc., which may be present in biological samples2. In this contribution, we present strategies of electrochemical H2O2 detection avoiding such interference problems based on electrocatalytically or enzyme-modified electrodes. Modification with Platinum black3 or Prussian Blue (PB)4 enables the detection at lower potential such as 0.3 V vs. Ag/AgCl at Pt-black modified electrodes and 0.0 V vs Ag/AgCl at PB modified electrodes, respectively. In particular, Prussian Blue (PB) modified electrodes show higher activity and significant higher electrochemical rate constants along with enhanced sensitivity. Hyperoxia treatment is controversial particularly in traumatic brain injury (TBI), due to increased radical and catecholamine production. To study increased release of ROS, measurements at porcine granulocytes and peripheral blood mononuclear cells will be presented using such modified microelectrodes. In addition, the co-detection of catecholamines such as epinephrine using dual sensors will be addressed. In order to study release at the single cell level, we recently introduced conductive colloidal AFM-SECM probes5, which serve as electrochemical transducers for the modification with Platinum black or Prussian Blue (PB) allowing the accurate provisioning of the biosensor at the cell surface. References: [1] D. R. Gough, T. G. Cotter, Cell Death and Disease 2, e213, (2011) [2] Y. Zang, et al., Anal. Chem., 66, 1183-1188 (1994). [3] S. Ben-Amor et al., Electrochim. Acta 126, 171–178 (2014) [4] A. A. Karyakin, Electroanalysis, 13, 813−819 (2001) [5] P. Knittel, H. Zhang, C. Kranz, G. G. Wallace and M. J. Higgins, Nanoscale,8, 4475–4481 (2016)