Fluorescent probe for the imaging of superoxide and peroxynitrite during drug-induced liver injury

Drug-induced liver injury (DILI) is an important cause of potentially fatal liver disease. Herein, we report the development of a molecular probe (LW-OTf) for the detection and imaging of two biomarkers involved in DILI. Initially, primary reactive oxygen species (ROS) superoxide (O₂˙⁻) selectively activates a near-infrared fluorescence (NIRF) output by generating fluorophore LW-OH. The C C linker of this hemicyanine fluorophore is subsequently oxidized by reactive nitrogen species (RNS) peroxynitrite (ONOO⁻), resulting in cleavage to release xanthene derivative LW-XTD, detected using two-photon excitation fluorescence (TPEF). An alternative fluorescence pathway can occur through cleavage of LW-OTf by ONOO⁻ to non-fluorescent LW-XTD-OTf, which can react further with the second analyte O₂˙⁻ to produce the same LW-XTD fluorescent species. By combining NIRF and TPEF, LW-OTf is capable of differential and simultaneous detection of ROS and RNS in DILI using two optically orthogonal channels. Probe LW-OTf could be used to detect O₂˙⁻ or O₂˙⁻ and ONOO⁻ in lysosomes stimulated by 2-methoxyestradiol (2-ME) or 2-ME and SIN-1 respectively. In addition, we were able to monitor the chemoprotective effects of tert-butylhydroxyanisole (BHA) against acetaminophen (APAP) toxicity in living HL-7702 cells. More importantly, TPEF and NIRF imaging confirmed an increase in levels of both O₂˙⁻ and ONOO⁻ in mouse livers during APAP-induced DILI (confirmed by hematoxylin and eosin (H&E) staining).

Drug-induced liver injury (DILI) is an important cause of potentially fatal liver disease.     

Pinkment: a synthetic platform for the development of fluorescent probes for diagnostic and theranostic applications

Reaction-based fluorescent-probes have proven successful for the visualisation of biological species in various cellular processes. Unfortunately, in order to tailor the design of a fluorescent probe to a specific application (i.e. organelle targeting, material and theranostic applications) often requires extensive synthetic efforts and the synthetic screening of a range of fluorophores to match the required synthetic needs. In this work, we have identified Pinkment-OH as a unique “plug-and-play” synthetic platform that can be used to develop a range of ONOO⁻ responsive fluorescent probes for a variety of applications. These include theranostic-based applications and potential material-based/bioconjugation applications. The as prepared probes displayed an excellent sensitivity and selectivity for ONOO⁻ over other ROS. In vitro studies using HeLa cells and RAW 264.7 macrophages demonstrated their ability to detect exogenously and endogenously produced ONOO⁻. Evaluation in an LPS-induced inflammation mouse model illustrated the ability to monitor ONOO⁻ production in acute inflammation. Lastly, theranostic-based probes enabled the simultaneous evaluation of indomethacin-based therapeutic effects combined with the visualisation of an inflammation biomarker in RAW 264.7 cells.

Pinkment, a resorufin based ONOO⁻ selective and sensitive ‘plug and play’ fluorescence-based platform for in vitro and in vivo use, enables facile functionalisation for various imaging and theranostic applications.     

Time-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe

Peroxynitrite (ONOO⁻) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO⁻ in cell biology, improved methods for the selective detection and real-time analysis of ONOO⁻ are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO⁻ in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO⁻ in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment.

Reactivity-based europium(iii) probe displays excellent selectivity for peroxynitrite (ONOO⁻), enabling its time-resolved luminescence detection in living cells.     

Protein encapsulation: a new approach for improving the capability of small-molecule fluorogenic probes

Herein, we report a protein-based hybridization strategy that exploits the host-guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO⁻ fluorescent probe Pinkment-OAc. Formation of a HSA/Pinkment-OAc supramolecular hybrid was confirmed by SAXS and solution-state analyses. This HSA/Pinkment-OAc hybrid provided an enhanced fluorescence response towards ONOO⁻versusPinkment-OAc alone, as determined by in vitro experiments. The HSA/Pinkment-OAc hybrid was also evaluated in RAW 264.7 macrophages and HeLa cancer cell lines, which displayed an enhanced cell permeability enabling the detection of SIN-1 and LPS generated ONOO⁻ and the in vivo imaging of acute inflammation in LPS-treated mice. A remarkable 5.6 fold (RAW 264.7), 8.7-fold (HeLa) and 2.7-fold increased response was seen relative to Pinkment-OAc alone at the cellular level and in vivo, respectively. We anticipate that HSA/fluorescent probe hybrids will soon become ubiquitous and routinely applied to overcome solubility issues associated with hydrophobic fluorescent imaging agents designed to detect disease related biomarkers.

Herein, we report a protein-based hybridization strategy that exploits the host–guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO⁻ fluorescent probe Pinkment-OAc.     

Novel Boronate Probe Based on 3-Benzothiazol-2-yl-7-hydroxy-chromen-2-one for the Detection of Peroxynitrite and Hypochlorite

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO^–), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H₂O₂ to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO⁻. The ability of the BC-BA probe to detect ONOO^– was measured using both authentic ONOO^– and the system co-generating steady-state fluxes of O₂^•– and ^•NO. BC-BA is oxidized by ONOO^– to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected.     

A novel method for determination of peroxynitrite based on hemoglobin catalyzed reaction

A novel spectrofluorimetric method for the determination of peroxynitrite is proposed. The method is based on a mimetic enzyme catalyzed reaction with hemoglobin as the catalyst and l-tyrosine as the substrate. A new fluorescent substance is produced that might probably be the coupled dimmer of tyrosine, which, instead of nitryl-tyrosine, is likely to be a new marking substance of ONOO⁻ injury in vivo. Kinetics of the reaction is studied and the possible reaction mechanism is also recommended. The proposed method is simple and highly sensitive with a detection limit of 5.00 × 10⁻⁸ mol L⁻¹ of peroxynitrite. A liner calibration graph is obtained over the peroxynitrite concentration range 5.60 × 10⁻⁷ to 2.10 × 10⁻⁵ mol L⁻¹, with a correlation coefficient of 0.9983. Interferences from some amino acids and metal ions normally seen in biological samples, and also some anions structurally similar to ONOO⁻ are studied.     

Study and application of perturbation of peroxynitrite on peroxidase-oxidase oscillation

Peroxynitrite was one of the important reactive oxygen species (ROS) which have been focused for many years. Peroxynitrite is an extremely strong and reactive oxidant which can cause many diseases linked to inflammatory processes and autoimmune diabetes, etc. ROS can significantly react with peroxidase and nicotinamide adanine dinucleotide (NADH) which is linked to numerous biological processes. NADH, NAD and horseradish peroxidase (HRP) were included in peroxidase-oxidase (PO) oscillation, dissolved oxygen was concerned with the oscillation and many ROS intermediates came into being in PO oscillation which was sensitive to the perturbation of ROS. The influence of peroxynitrite (ONOO⁻) on this oscillation system was investigated. It was found that the oscillation amplitude increased when the system was perturbed with peroxynitrite. There was a linear relationship between the increment ratio in the oscillation amplitude and perturbing peroxynitrite concentration in the range 2.50 × 10⁻⁸ to 1.56 × 10⁻⁶ mol/L. And further experimental results revealed that amplitude increasing may be caused by the effect of peroxynitrite on HRP. Based on this phenomenon, a highly sensitive method for the determination of peroxynitrite was developed.     

An ultra-stable bio-inspired bacteriochlorin analogue for hypoxia-tolerant photodynamic therapy

Photodynamic therapy (PDT) greatly suffers from the weak NIR-absorption, oxygen dependence and poor stability of photosensitizers (PSs). Herein, inspired by natural bacteriochlorin, we develop a bacteriochlorin analogue, tetrafluorophenyl bacteriochlorin (FBC), by one-step reduction of tetrafluorophenyl porphyrin (TFPP). FBC can realize deep tissue penetration, benefitting from the strong NIR absorption. The reactive oxygen species (ROS) generation capacity of FBC can retain around 60% with a 1.0 cm-thick pork skin as the barrier. Besides, FBC could not only produce oxygen-dependent ¹O₂, but also generate less oxygen-dependent O₂⁻˙ and ˙OH to achieve excellent PDT even in hypoxic tumors. Moreover, FBC exhibits an ultra-high stability and it is almost unchanged even under visible light at room temperature for 15 months. Interestingly, the high reactivity of the fluorophenyl group makes it easy for FBC to produce FBC derivatives. A biocompatible FBC nanogel could be directly formed by blending FBC with SH–PEG–SH. The FBC nanogel displays excellent photodynamic efficacy in vitro and in vivo. Thus, FBC would be a promising PS for the clinical PDT of deep tumors.

A hypoxia-tolerant photosensitizer FBC-based nanoplatform with strong NIR absorbance and ultra-high stability was facilely prepared for PDT of deep tumors.     

Electrochemical Nucleic Acid Biosensors for the Detection of Interaction Between Peroxynitrite and DNA

We studied the reactivity of peroxynitrite and different nucleic acid molecules using DNA electrochemical biosensors. SIN‐1 (3‐morpholinosydnonimine) has been used for the simultaneous generation of NO?and superoxide, i.e., as a peroxynitrite (ONOO^?) donor. Double strand DNA (dsDNA), single strand DNA (ssDNA) and 15 guanine bases oligonucleotide (Oligo(dG)₁₅) were immobilized on a carbon paste electrode to generate the biosensor and DPV was selected as the electroanalytical technique. Results showed that electrochemical biosensors were very sensitive for detecting interaction between ONOO^? and DNA. A down/up effect was observed, i.e., at low ONOO^? concentrations the guanine oxidation signal decreased while at high ONOO^? concentrations the guanine oxidation current increased. Oligo(dG)₁₅ exhibited greater interaction at low ONOO^? concentrations than the other DNA molecules. The reactivity between ONOO^? and DNA was also evaluated in solution phase, showing the same down/up effect. Finally, the capacity of DNA to hybridize was prevented after interaction with ONOO^?.     

Plasmonic O2 dissociation and spillover expedite selective oxidation of primary C–H bonds

Manipulating O₂ activation via nanosynthetic chemistry is critical in many oxidation reactions central to environmental remediation and chemical synthesis. Based on a carefully designed plasmonic Ru/TiO_2−x catalyst, we first report a room-temperature O₂ dissociation and spillover mechanism that expedites the “dream reaction” of selective primary C–H bond activation. Under visible light, surface plasmons excited in the negatively charged Ru nanoparticles decay into hot electrons, triggering spontaneous O₂ dissociation to reactive atomic ˙O. Acceptor-like oxygen vacancies confined at the Ru–TiO₂ interface free Ru from oxygen-poisoning by kinetically boosting the spillover of ˙O from Ru to TiO₂. Evidenced by an exclusive isotopic O-transfer from ¹⁸O₂ to oxygenated products, ˙O displays a synergistic action with native ˙O₂⁻ on TiO₂ that oxidizes toluene and related alkyl aromatics to aromatic acids with extremely high selectivity. We believe the intelligent catalyst design for desirable O₂ activation will contribute viable routes for synthesizing industrially important organic compounds.

Room-temperature O₂ dissociation and spillover, as driven by plasmonic Ru on oxygen-deficient TiO₂, expedite the selective oxidation of primary C–H bonds in alkyl aromatics for synthesizing industrially important organic compounds.     

Electrochemical approach to detect the presence of peroxynitrite in aerobic neutral solution

This work describes the conditions of use of bare gold electrode to detect electrochemically the presence of peroxynitrite ONOO^? in phosphate buffer solution at pH 7.1. As ONOO^? is extremely unstable in neutral solution, current–potential curve was reconstructed between ?0.5 and 0.7 V vs SCE by amperometry experiments at rotating disk electrode at different potentials. Comparison of this reconstructed curve with voltammograms of the common interfering species (dopamine, hydrogen peroxide, nitrite, ascorbic acid and glutamate) shows that the presence of ONOO^? can be selectively determined at ?0.1 V vs SCE. This detection occurs through the electrochemical reduction of peroxynitrous acid ONOOH, the conjugated acid of ONOO^?. Detection of ONOO^? produced in situ by the reaction of nitric oxide with superoxide was also achieved.     

Multistep energy and electron transfer processes in novel rotaxane donor–acceptor hybrids generating microsecond-lived charge separated states

A new set of [Cu(phen)₂]⁺ based rotaxanes, featuring [60]-fullerene as an electron acceptor and a variety of electron donating moieties, namely zinc porphyrin (ZnP), zinc phthalocyanine (ZnPc) and ferrocene (Fc), has been synthesized and fully characterized with respect to electrochemical and photophysical properties. The assembly of the rotaxanes has been achieved using a slight variation of our previously reported synthetic strategy that combines the Cu(i)-catalyzed azide–alkyne cycloaddition reaction (the “click” or CuAAC reaction) with Sauvage''s metal-template protocol. To underline our results, complementary model rotaxanes and catenanes have been prepared using the same strategy and their electrochemistry and photo-induced processes have been investigated. Insights into excited state interactions have been afforded from steady state and time resolved emission spectroscopy as well as transient absorption spectroscopy. It has been found that photo-excitation of the present rotaxanes triggers a cascade of multi-step energy and electron transfer events that ultimately leads to remarkably long-lived charge separated states featuring one-electron reduced C₆₀ radical anion (C₆₀˙^–) and either one-electron oxidized porphyrin (ZnP˙⁺) or one-electron oxidized ferrocene (Fc˙⁺) with lifetimes up to 61 microseconds. In addition, shorter-lived charge separated states involving one-electron oxidized copper complexes ([Cu(phen)₂]²⁺ (τ < 100 ns)), one-electron oxidized zinc phthalocyanine (ZnPc˙⁺; τ = 380–560 ns), or ZnP˙⁺ (τ = 2.3–8.4 μs), and C₆₀˙^– have been identified as intermediates during the sequence. Detailed energy diagrams illustrate the sequence and rate constants of the photophysical events occurring with the mechanically-linked chromophores. This work pioneers the exploration of mechanically-linked systems as platforms to position three distinct chromophores, which are able to absorb light over a very wide range of the visible region, triggering a cascade of short-range energy and electron transfer processes to afford long-lived charge separated states.     

Oxyresveratrol-Loaded PLGA Nanoparticles Inhibit Oxygen Free Radical Production by Human Monocytes: Role in Nanoparticle Biocompatibility

Oxyresveratrol, a polyphenol extracted from the plant Artocarpus lakoocha Roxb, has been reported to be an antioxidant and an oxygen-free radical scavenger. We investigated whether oxyresveratrol affects the generation of superoxide anion (O₂⁻) by human monocytes, which are powerful reactive oxygen species (ROS) producers. We found that oxyresveratrol inhibited the O₂⁻ production induced upon stimulation of monocytes with β-glucan, a well known fungal immune cell activator. We then investigated whether the inclusion of oxyresveratrol into nanoparticles could modulate its effects on O₂⁻ release. We synthesized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on monocytes. We found that empty PLGA nanoparticles induced O₂⁻ production by resting monocytes and enhanced the formation of this radical in β-glucan-stimulated monocytes. Interestingly, the insertion of oxyresveratrol into PLGA nanoparticles significantly inhibited the O₂⁻ production elicited by unloaded nanoparticles in resting monocytes as well as the synergistic effect of nanoparticles and β-glucan. Our results indicate that oxyresveratrol is able to inhibit ROS production by activated monocytes, and its inclusion into PLGA nanoparticles mitigates the oxidative effects due to the interaction between these nanoparticles and resting monocytes. Moreover, oxyresveratrol can contrast the synergistic effects of nanoparticles with fungal agents that could be present in the patient tissues. Therefore, oxyresveratrol is a natural compound able to make PLGA nanoparticles more biocompatible.     

Reaction-based Indicator displacement Assay (RIA) for the selective colorimetric and fluorometric detection of peroxynitrite

Using the self-assembly of aromatic boronic acids with Alizarin Red S (ARS), we developed a new chemosensor for the selective detection of peroxynitrite. Phenylboronic acid (PBA), benzoboroxole (BBA) and 2-(N,N-dimethylaminomethyl)phenylboronic acid (NBA) were employed to bind with ARS to form the complex probes. In particular, the ARS–NBA system with a high binding affinity can preferably react with peroxynitrite over hydrogen peroxide and other ROS/RNS due to the protection of the boron via the solvent-insertion B–N interaction. Our simple system produces a visible colorimetric change and on–off fluorescence response towards peroxynitrite. By coupling a chemical reaction that leads to an indicator displacement, we have developed a new sensing strategy, referred to herein as RIA (Reaction-based Indicator displacement Assay).     

On the Synergism of Biogenic Gold Nanoparticles and Hydroxyaluminum Phthalocyanines in the Photoeradication of Staphylococcus aureus

Due to the unusual properties of gold nanoparticles, these structures are widely used in medicine and biology. This paper describes for the first time the synthesis of colloidal gold nanoparticles by the cell-free filtrate obtained from the Coriolus versicolor biomass and the use of these biogenic nanostructures to increase the photosensitizing efficiency of di- (AlPcS₂) and tetrasulfonated (AlPcS₄) hydroxyaluminum phthalocyanines in antibacterial photodynamic therapy. The obtained monodisperse particles were extremely stable, and this remarkable stability was due to the presence of phosphoprotein as a capping agent. The studied gold nanoparticles had a spherical shape, were uniformly distributed, and were characterized by a single plasmon band at wavelength of 514–517 nm. Almost 60% of the gold particles were found to be in the range of 13 to 15 nm. In accordance with the regulations of the American Microbiological Society, indicating that any antimicrobial technique must kill at least 3 log CFU (99.9%) to be accepted as “antimicrobial”, this mortality of Staphylococcus aureus was shown to be achieved in the presence of AlPcS₄ + AuNPs mixture and 4.8 J cm⁻² light dose compared to AlPcS₄ alone, which required a light dose of 24 J cm⁻². The best effect of increasing the effectiveness of combating this pathogen was observed in the case of AlPcS₂ + AuNPs as a photosensitizing mixture. The light dose of 24 J cm⁻² caused a lethal effect of the studied coccus in the planktonic culture.     

Realizing high hydrogen evolution activity under visible light using narrow band gap organic photocatalysts

The design and synthesis of conjugated semiconducting polymers for photocatalytic hydrogen evolution have engendered intense recent interest. However, most reported organic polymer photocatalysts show a relatively broad band gap with weak light absorption ability in the visible light region, which commonly leads to a low photocatalytic activity under visible light. Herein, we synthesize three novel dithieno[3,2-b:2′,3′-d]thiophene-S,S-dioxide (DTDO) containing conjugated polymer photocatalysts by a facile C–H arylation coupling polymerization reaction. The resulting polymers show a broad visible light absorption range up to 700 nm and a narrow band gap down to 1.81 eV due to the introduction of the DTDO unit. Benefiting from the donor–acceptor polymer structure and the high content of the DTDO unit, the three-dimensional polymer PyDTDO-3 without the addition of a Pt co-catalyst shows an attractive photocatalytic hydrogen evolution rate of 16.32 mmol h⁻¹ g⁻¹ under visible light irradiation, which is much higher than that of most reported organic polymer photocatalysts under visible light.

Narrow band gap conjugated polymer photocatalysts containing dithieno[3,2-b:2′,3′-d]thiophene-S,S-dioxide show an attractive photocatalytic hydrogen evolution rate of 16.32 mmol h⁻¹ g⁻¹ under visible light irradiation.     

Thionated organic compounds as emerging heavy-atom-free photodynamic therapy agents

This minireview focuses on recent progress in developing heavy-atom-free photosensitizers based on the thionation of nucleic acid derivatives and other biocompatible organic compounds for prospective applications in photodynamic therapy. Particular attention is given to the use of thionated nucleobase derivatives as “one-two punch” photodynamic agents. These versatile photosensitizers can act as “Trojan horses” upon metabolization into DNA and exposure to activating light. Their incorporation into cellular DNA increases their selectivity and photodynamic efficacy against highly proliferating skin cancer tumor cells, while simultaneously enabling the use of low irradiation doses both in the presence and in the absence of molecular oxygen. Also reviewed are their primary photochemical reactions, modes of action, and photosensitization mechanisms. New developments of emerging thionated organic photosensitizers absorbing visible and near-infrared radiation are highlighted. Future research directions, as well as, other prospective applications of heavy-atom-free, thionated photosensitizers are discussed.

This minireview focuses on recent progress in developing heavy-atom-free photosensitizers based on the thionation of nucleic acid derivatives and other biocompatible organic compounds for prospective applications in photodynamic therapy.     

A Simple Method for Evaluating the Bioactive Phenolic Compounds’ Presence in Brazilian Craft Beers

There are a significant number of analytical methodologies employing different techniques to determine phenolic compounds in beverages. However, these methods employ long sample preparation processes and great time consumption. The aim of this paper was the development of a simple method for evaluating the phenolic compounds’ presence in Brazilian craft beers without a previous extraction step. Catechin, caffeic acid, epicatechin, p-coumaric acid, hydrated rutin, trans-ferulic acid, quercetin, kaempferol, and formononetin were analyzed in fifteen different craft beers. The method showed good linearity (R² ≥ 0.9966). The limit of detection ranged from 0.08 to 0.83 mg L⁻¹, and limits of quantification were between 0.27 and 2.78 mg L⁻¹. The method showed a satisfactory precision (RSD ≤ 16.2%). A good accuracy was obtained by the proposed method for all phenolic compounds in craft beer (68.6% ˂ accuracy ˂ 112%). Catechin showed higher concentrations (up to 124.8 mg L⁻¹) in the samples, followed by epicatechin (up to 51.1 mg L⁻¹) and caffeic acid (up to 8.13 mg L⁻¹). Rutin and formononetin were observed in all analyzed samples (0.52 mg L⁻¹ to 2.40 mg L⁻¹), and kaempferol was less present in the samples. The presence of plant origin products was determinant for the occurrence of the highest concentrations of phenolic compounds in Brazilian craft beers.     

A spontaneous solid-state NO donor to fight antibiotic resistant bacteria

This study substantiates the chemical origin of a free-radical-driven antibacterial effect at the surface of biomedical silicon nitride (Si₃N₄) in comparison with the long-known effect of oxygen reduction by oxidized TiO₂ at the surface of biomedical titanium alloys. Similar to the antibacterial effect exerted by reactive oxygen species (ROS; i.e., superoxide anions, hydroxyl radicals, singlet oxygen, and hydrogen peroxide) from TiO₂, reactive nitrogen species (RNS), such as nitrous oxide (N₂O), nitric oxide (NO), and peroxynitrite (^?OONO) in Si₃N₄, severely affect bacterial metabolism and lead to their lysis. However, in vitro experiment with gram-positive Staphylococcus epidermidis (S. epidermidis, henceforth) revealed that ROS and RNS promoted different mechanisms of lysis. Fluorescence microscopy of NO radicals and in situ time-lapse Raman spectroscopy revealed different metabolic responses of living bacteria in contact with different substrates. After 48 h, the DNA of bacteria showed complete destruction on Si₃N₄, while carbohydrates of the peptidoglycan membrane induced bacterial degradation on Ti-alloy substrates. Different spectroscopic fingerprints for bacterial lysis documented the distinct effects of RNS and ROS. Spontaneously activated in aqueous environment, the RNS chemistry of Si₃N₄ proved much more effective in counteracting bacterial proliferation as compared to ROS formed on TiO₂, which requires external energy (photocatalytic activation) to enhance effectiveness. Independent of surface topography, the antibacterial effect observed on Si₃N₄ substrates is due to its unique kinetics ultimately producing NO and represents a new intriguing avenue to fight bacterial resistance to conventional antibiotics.