Intracellular H2O2 Detection Research Articles

Gold nanoclusters stabilized with dopa-containing ligands: Catalyst-indicator integrated probe for tumor cell screening

Elevated hydrogen peroxide (H2O2) levels not only inflict cellular damage but also serve as a harbinger for various diseases. Tumor cells, in particular, often exhibit an abundance of H2O2. Hence, the detection of this pivotal molecule assumes paramount importance in monitoring physiological states and expediting cancer diagnosis. To this end, we have ingeniously devised an enzyme-free and monomeric system for intracellular H2O2 detection. Our astute selection of dopa-containing peptidomimetics, replete with ortho-bisphenol and amino acid moieties, has engendered the synthesis of distinctive fluorescent gold nanoclusters (AuNCs). These nanoclusters not only function as a peroxidase-like catalyst, catalyzing the decomposition of H2O2 into hydroxyl radicals (·OH), but also serve as an indicator, with their fluorescence quenched in response to varying H2O2 concentrations. Experimental results evince that our GDpE-AuNCs exhibit remarkable sensitivity, boasting a detection limit of 0.49 μM and a linear range of 5–1000 μM. Moreover, the amalgamation of catalyst and indicator within a single structure, facilitating efficient cellular uptake, engenders intracellular H2O2 detection and discernment of tumor cells. This pioneering approach bequeaths a valuable assay probe for monitoring physiological states and ushering in early disease diagnosis.

Novel mycophenolic acid precursor-based fluorescent probe for intracellular H2O2 detection in living cells and Daphnia magna and Zebrafish model systems.

Innovative for the scientific community and attracting attention in the extensive biomedical field are novel compact organic chemosensing systems built upon unique core molecular frameworks. These systems may demonstrate customized responses and may be adaptable to analytes, showing promise for potential in vivo applications. Our recent investigation focuses on a precursor of Mycophenolic acid, resulting in the development of LBM (LOD = 13 nM) - a specialized probe selective for H2O2. This paper details the synthesis, characterization, and thorough biological assessments of LBM. Notably, we conducted experiments involving living cells, daphnia, and zebrafish models, utilizing microscopy techniques to determine probe nontoxicity and discern distinct patterns of probe localization. Localization involved the distribution of the probe in the Zebrafish model within the gut, esophagus, and muscles of the antennae.

One-step fabrication of a self-driven point-of-care chip by femtosecond laser direct writing and its application in cancer cell H2O2 detection via semiconductor-based SERS

Self-driven microfluidic systems have attracted significant attention and demonstrated great potential in the field of point-of-care (POC) testing due to their device simplicity, low power consumption, increased portability, and reduced sample consumption. To develop POC detection chips with diverse characteristics that meet different requirements, there is a strong demand for feasible strategies that enable easy operation and reduce processing time. Here, a one-step processing approach using femtosecond laser direct writing technology was proposed to fabricate a capillary-actuated POC microfluidic chip. The driving force of the chip is highly dependent on its surface wettability, which can be easily adjusted by changing the laser processing parameters. This POC microfluidic chip allowed for the detection of intracellular H2O2 through a catalytic reaction system that incorporated 5-aminosalicylic acid -sensitized colloidal TiO2 nanoparticles and horse radish peroxidase, with integrating semiconductor-based surface-enhanced Raman scattering (SERS) quantitative technique. The concentration of H2O2 was determined by the SERS signal of the catalytic products in the microfluidic chip, resulting in rapid detection with minimal sample consumption. Our method provides a simple, feasible, and alternative strategy for POC testing of H2O2, with a linear range of 10−2∼10−6 M and a limit of detection of 0.55 μM. This approach was successfully applied to rapid detection of intracellular H2O2 in MCF-7 breast cancer cells with high sensitivity and minimal sample consumption. Additionally, this study not only demonstrates the exceptional advantages of femtosecond laser processing technology in fabricating diverse microfluidic chips for various applications, but also presents an efficient POC testing strategy for detecting cell signaling molecules.

Unraveling the Structure Transition and Peroxidase Mimic Activity of Copper Sites Over Atomically Dispersed Copper-Doped Carbonized Polymer Dots.

The lack of systematic structural resolution makes it difficult to build specific transition metal atoms-doped carbonized polymer dots(TMAs-doped-CPDs). Herein, the structure-activity relationship between Cu atoms and CPDs was evaluated by studying the peroxidase-like properties of Glu-Cu-CPDs prepared by using copper glutamate (Glu) with Cu-N2O2 initial structure. The results showed that the Cu atoms bound to Glu-Cu-CPDs in the form of Cu-N2C2, indicating that Cu-O bonds changed into Cu-C bonds under hydrothermal conditions. This phenomenon was also observed in other Cu-doped-CPDs. Moreover, the carboxyl and amino groups content decreased after Cu-atom doping. Theoretical calculations revealed a dual-site catalytic mechanism for catalyzing H2O2. Eventually, the detection of intracellular H2O2 suggested their application prospects. Our work provides an in-depth understanding on the formation and catalysis mechanism of TMAs-doped-CPDs, allowing for building specific TMAs-doped-CPDs.

Unraveling the Structure Transition and Peroxidase Mimic Activity of Copper Sites over Atomically Dispersed Copper‐Doped Carbonized Polymer Dots

AbstractThe lack of systematic structural resolution makes it difficult to build specific transition‐metal‐atom‐doped carbonized polymer dots (TMA‐doped CPDs). Herein, the structure‐activity relationship between Cu atoms and CPDs was evaluated by studying the peroxidase‐like properties of Glu−Cu−CPDs prepared by using copper glutamate (Glu) with a Cu−N2O2 initial structure. The results showed that the Cu atoms bound to Glu−Cu−CPDs in the form of Cu−N2C2, indicating that Cu−O bonds changed into Cu−C bonds under hydrothermal conditions. This phenomenon was also observed in other copper‐doped CPDs. Moreover, the carboxyl and amino groups content decreased after copper‐atom doping. Theoretical calculations revealed a dual‐site catalytic mechanism for catalyzing H2O2. The detection of intracellular H2O2 suggested their application prospects. Our study provides an in‐depth understanding of the formation and catalytic mechanism of TMA‐doped‐CPDs, allowing for the generation specific TMA‐doped‐CPDs.

Ultrasensitive multiplexed detection of small molecules and enzymes using stimuli-responsive nucleic acids

Small biomolecules and enzymes play essential roles in regulating various physiological functions and are highly correlated with many diseases, making them promising biomarkers for disease diagnosis. However, it remains challenging to achieve multiplexed detection of trace enzymes and small molecules due to the limited signal transformation and amplification capabilities in existing technologies. Here, we developed a signal transformation and amplification strategy based on stimuli-responsive nucleic acids (SR-NAs) by incorporating stimuli-responsive linker in single-stranded nucleic acid via click chemistry. The targets (e.g., enzymes, small molecules) could recognize the responsive linker and trigger the degradation of the SR-NAs, thus transforming the targets into DNA signals by generating new toehold nucleic acid strands, which were further augmented by nucleic acid amplification. We demonstrated that this strategy significantly improved the detection precision for small biomolecules (H2O2) and enzymes (esterase, β-galactosidase). Furthermore, the signal transformation function of SR-NA enables multiplexed detection via logical operation with multiple small molecules and enzymes as inputs, strand displacement reaction as Boolean logic-based algorithm, and fluorescence signal as output signal, as demonstrated by multiplexed detection of esterase and H2O2. More importantly, this method was capable of realizing ultrasensitive intracellular H2O2 detection. We envision that this SR-NA-based strategy could be expanded to detect other kinds of targets by designing self-immolative linkers, and thus provides a powerful toolbox for bioanalytical and biomedical applications.

Diatomic active sites nanozymes: Enhanced peroxidase-like activity for dopamine and intracellular H2O2 detection

Diatomic active sites nanozymes: Enhanced peroxidase-like activity for dopamine and intracellular H2O2 detection

Multifunctional shape-dependent plasmonic nanoprobe by enzymatic etching of single gold triangular nanoplate

Hydrogen peroxide (H2O2), as a signaling molecule, plays a vital role in a wide variety of signaling transduction processes, aging, and diseases. However, the excessive production of H2O2 causes various diseases. Herein, we develop a novel method for H2O2 detection in live cells via dark-field scattering spectroscopy with gold triangular nanoprisms (AuTNPs) as probes. The corners of AuTNPs would be gradually oxidatively etched by the strong coordination of Br• which is generated by enzymatic reactions in the presence of horseradish peroxidase (HRP), bromide ion and trace hydrogen peroxide. Benefitting from the morphological change, the single AuTNP based plasmonic nanoprobe shows notable blueshifts and scattering color changes which could be real-time monitored under the dark-field microscopy. The peak position in the scattering spectra of individual AuTNP blueshifts linearly with the increase of H2O2 concentration, and exhibits high sensitivity to H2O2 in a large range from 2.5 to 100 µM with a low detection limit (LOD) of 0.74 µM. Moreover, the experimental results were supported by the simulated results via the finite-difference time-domain (FDTD) method. The nanoprobes have been further used for intracellular H2O2 detection in live cells. Besides, the etching of AuTNP also provides an alternative method to design novel plasmonic logic chips and write-once plasmonic memories.

Electrochemiluminescence cytosensing platform based on Ru(bpy)32+@silica-Au nanocomposite as luminophore and AuPd nanoparticles as coreaction accelerator for in situ evaluation of intracellular H2O2

An electrochemiluminescence (ECL) cytosensor was fabricated onto a microfluidic paper-based analytical device (μ-PAD) in order to detect hydrogen peroxide (H2O2) which was released from tumor cells. The ECL probe Ru(bpy)32+@silica-Au nanocomposite (Ru@SiO2-Au) was fabricated and served as ECL reagent. The ECL of Ru@SiO2-Au nanocomposite was quenched by the ferrocene (Fc). AuPd nanoparticles (AuPd NPs), which were modified on the paper working electrode (PWE), were served as the catalyst of H2O2 to produce hydroxyl radicals (•OH) for cleaving Fc-labelled DNA to achieve “signal-on”, and AuPd NPs also severed as coreaction accelerator. H2O2 was released from cells under the stimulation of phorbol myristate acetate. Fc-labelled DNA strand was cleaved via •OH. Fc molecule departed from the PWE surface, The ECL could be recovered. An ECL cytosensor on a 3D origami device was constructed. The ECL response of the Ru@SiO2-Au-Fc system was related to the number of cells. The ECL intensity was quantitatively related with the logarithm of MCF-7 cells number and H2O2 concentration, the detection limit was 30 cells mL−1. As a consequence, this work provided a really low-cost and disposable μ-PAD for sensitive detection of intracellular H2O2. What's more, this work had potential application value for studying cellular biology and pathophysiology.

Miniaturized electrochemiluminescent biochip prepared on gold nanoparticles-loaded mesoporous silica film for visual detection of hydrogen peroxide released from living cells

Au nanoparticles (NPs) has been widely used for the detection of intracellular H2O2 to enhance the electron transfer process. But AuNPs are easy to aggregate in the live cells environment. Herein we report a rapid, reliable and low-cost electrochemiluminescent (ECL) biochip integrated by AuNPs-loaded mesoporous silica film (MSF) to detect H2O2 released by macrophage cells. The MSF was employed as a template to load AuNPs within the nanochannels to avoid aggregation. H2O2 could be catalyzed by AuNPs to promote the ECL reaction of luminol molecules in solution. The ECL intensity was significantly enhanced, and the peak potential was negatively shifted by 400 mV due to the excellent electrocatalytic ability of AuNPs. The integrated biochip demonstrated good reproducibility, with a wide linear range of 0.1–200 μM and an LOD of 25.3 nM. The reliability was evaluated by applying for the assessment of antioxidant activity of resveratrol using RAW 264.7 macrophage model. The AuNPs-loaded MSF integrated biochip can be easily adapted to the development of improved devices in biosensing, lab-on-a-chip, and nanofluidic systems.

Iminocoumarin-based fluorescence probe for intracellular H2O2 detection with a red emission and a large Stokes shift

A highly sensitive and selective fluorescence probe for H2O2 has been developed based on an iminocoumarin derivative using aromatic boronic ester as sensing unit. A significant fluorescence enhancement was observed with λmaxem = 619 nm when this probe was treated with H2O2. In the detection of H2O2, this probe exhibited a large Stokes shift (148 nm) and a low detection limit (6.0 × 10−8 mol/L). Furthermore, cell imaging studies had revealed that this probe had the potential to detect intracellular H2O2 in living cells.

Ultrasensitive electrochemiluminescence assay of tumor cells and evaluation of H2O2 on a paper-based closed-bipolar electrode by in-situ hybridization chain reaction amplification

In this manuscript, a disposable paper-based analytical device comprised of a closed bipolar electrode (BPE) was fabricated for the ultrasensitive electrochemiluminescence (ECL) detection of intracellular H2O2 and the number of cancer cells. In this approach, wax printing was used to fabricated reaction zone, and carbon ink-based BPE and driving electrodes were screen-printed into the paper. AuPd nanoparticles (NPs), which served as a carrier of the capture aptamer and as the catalyst for the ECL reaction of luminol and H2O2, were used to modify the BPE. Luminol/Au NPs were attached to the surface of the captured cells via hybridation chain reaction with two hairpin structure DNA labelled luminol/Au NPs. In the stimulation of phorbol myristate acetate, The coreactant H2O2 was released from the target cells. The ECL response of the luminol-H2O2 system was related to the number of cancer cells in the testing buffer, which served as a quantitative signal for the determination of cancer cells and the concentration of H2O2. In order to decrease the external voltage, K3[Fe(CN)6] was introduced in the cathode resevoir of BPE because it gained electrons at the cathode more easily than oxygen. The ECL intensity was quantitatively related to the concentration of MCF-7 in the range of 1.0 × 102–1.0 × 107 cells/mL. The detection limit was 40 cells/mL and it showed good specificity for cells with high overexpression of mucin-1 receptor, it was concluded that the developed protocol could be effectively utilized for the detection of MCF-7 cells.

RGO quantum dots/ZnO hybrid nanofibers fabricated using electrospun polymer templates and applications in drug screening involving an intracellular H2O2 sensor.

Throughout the years, reported intracellular H2O2 sensors just focused on unrelated measurements of intracellular H2O2 generated from the stimulus of Cd2+, ascorbic acid (AA) etc., leading to difficulty in data interpretation. Here, a novel reduced graphene oxide quantum dots (rGO QDs)/ZnO hybrid nanofibers-based electrochemical biosensor for the detection of intracellular H2O2 released from cancer and normal cells under the stimuli of the corresponding anticancer drugs permits a quantitative study of the interaction between the target drug compound and the cancer cell, which is suitable for candidate drug screening. Nylon 6/6 nanofibers are used as robust templates for the facile fabrication of novel rGO QDs/ZnO hybrid nanofibers via electrospinning followed by a step hydrothermal growth method. The as-made sensor was applied to determine H2O2 released from a prostate cancer cell (PC-3) versus a noncancerous cell (BPH-1) under the stimuli of the corresponding anticancer drugs (apigenin, antisense CK2αetc.). The amount of H2O2 released from the PC-3 cancer cell is about (320 ± 12) amol per cell and about (210 ± 6) amol per cell for the BPH-1 noncancerous cell under the stimuli of specific therapy drug antisense CK2α. These results demonstrate that the rGO QDs/ZnO hybrid nanofibers-based electrochemical biosensor can efficiently detect the distinct amounts of H2O2 released from cancer and noncancer cells.

Artificial Evolution of Graphene Oxide Chemzyme with Enantioselectivity and Near‐Infrared Photothermal Effect for Cascade Biocatalysis Reactions

It is highly desirable and challenging when the chemzyme can be not only simply duplicating and imitating the properties of natural enzymes, but also introducing additional new features for practical applications. Herein, we report a zinc-finger-protein like α-helical chiral metallo-supramolecular complex ([Fe2L3](4+)) functionalized graphene oxide (GO-COOH) as a peroxidase mimic. This artificial enzyme integrates the characteristics of both chiral metallo-supramolecular complex and GO-COOH, and shows excellent catalytic activity. More intriguingly, the novel chemzyme turn out to have enantioselectivity and near-infrared photothermal effect. To the best of our knowledge, this is the first example that the chemzyme has such new features. Based on these properties, we have demonstrated three examples for the applications of our designed enzyme: 1) Intracellular H2O2 detection in PC12 cells against Alzheimer's disease; 2) Discrimination between the chiral drug, Levodopa (L-dopa), the gold standard for treating Parkinson's disease and its enantiomer, D-dopa. This is important because L-dopa is the most effective drug at present used to combat Parkinson's disease while D-dopa is inactive and can even cause side effects, thus for drug efficacy it must be free of D-dopa in the formulation; 3) Remote control of enzyme cascade biocatalysis reactions using high transparent, bio-friendly near-infrared (NIR) light. NIR allows remote activation with relatively high spatial and temporal precision. Our work will provide new insights into design and construction of novel chemzyme with more advanced features beyond intrinsic enzyme property.

Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H₂O₂) production in brain cells. For selective imaging of H₂O₂ over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H₂O₂ and mitochondria peroxy yellow 1 detects mitochondrial H₂O₂. Two-photon absorption cross sections for these H₂O₂ probes are measured with a mode-locked Ti:sapphire laser in the wavelength range of 720 to 1040 nm. TPF imaging is demonstrated in the HT22 cell line to monitor both cytoplasmic H₂O₂ and localized H₂O₂ production in mitochondria. Endogenous cytoplasmic H₂O₂ production is detected with TPF imaging in rat astrocytes modified with d-amino acid oxidase. The TPF H₂O₂ imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H₂O₂.

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H2O2 in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H2O2 source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H2O2 is sensitive to intracellular H2O2 competitors: it decreases when H2O2 is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H2O2. Oxidation of the fluorescent probes following reaction of H2O2 with endogenous HRP overcomes most criticisms in their use for intracellular H2O2 detection. The method can be applied for direct determination in plate reader and is proposed to detect H2O2 generation in physio-pathological cell models.

Effect of dihydrotestosterone on hydrogen peroxide‐induced apoptosis of mouse embryonic stem cells

Steroid hormones have been reported to activate various signal transducers that trigger a variety of cellular responses. Among these hormones, testosterone has been identified as an antioxidant that protects against cellular damage. Therefore, using mouse embryonic stem (ES) cells as a model system, this study evaluated the effects of dihydrotestosterone (DHT), a biologically active testosterone metabolite, on H2O2-induced apoptosis. H2O2 increased the release of lactate dehydrogenase (LDH) and DNA fragmentation but reduced the cell viability in a time-dependent manner (> or =8 h). Moreover, H2O2 decreased the level of DNA synthesis and the levels of the cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H2O2 were inhibited by a pretreatment with DHT. However, a treatment with flutamide (androgen receptor inhibitor, 10(-3) M) abolished the protective effects of DHT. This result was supported by the presence of the androgen receptor in mouse ES cells. The activity of the antioxidant enzyme, catalase, was increased by the DHT treatment but not by a co-treatment with DHT and flutamide. Using CM-H(2)DCFDA (DCF-DA) for the detection of intracellular H2O2, DHT decreased the intracellular H2O2 levels but flutamide blocked this effect. H2O2 also increased the level of p38 MAPK, JNK/SAPK, and NF-kappaB phosphorylation, which were inhibited by the DHT pretreatment. Catalase inhibited the effect of H2O2 on MAPKs and NF-kappaB. However, the flutamide treatment abolished the inhibitory effects of DHT on the H2O2-induced increase in the levels of p38 MAPK, JNK/SAPK, and NF-kappaB phosphorylation. DHT inhibited the H2O2-induced increase in caspase-3 expression and decreased the level of Bcl-2 and the cellular inhibitor of apoptosis protein (cIAP)-2. These effects were abolished by the flutamide treatment. In conclusion, DHT prevents the H2O2-induced apoptotic cell death of mouse ES cells through the activation of catalase and the downregulation of p38 MAPK, JNK/SAPK, and NF-kappaB via the androgen receptor.

Dissociation of Phospholipids “Flip-Flop” and CD16 Downregulation in Stimulated Neutrophils with FMLP and TNFα in Relationship with ROS Production.

There are 3 kinds of PMN apoptosis: spontaneous, Fas or TNFα mediated system and ROS induced. The purpose of this work is to investigate the mechanisms of modulation of the CD16 decrease and the phospholipids “flip-flop” which are classically described as simultaneous events in apoptosis in relationship with the intracellular production of H2O2. Incubations during different times of PMN suspensions from healthy controls were performed in presence of Phorbol Myristate Acetate (PMA) at priming (10−9 M) and stimulating (2 10−6 M) concentrations, of fMLP 5 10−6M, of TNFα at 200ng/ml and finally of agonistic anti-Fas antibodies at 1 μg/ml. Inhibitors of PKC (staurosporin 10−5 M) and of the oxidase (DPI 400 μM) were tested with stimulating PMA. Standard two-colour flow-cytometry was performed for detection on one side of FITC-Annexin-V fixation and membrane impermeability to Propidium Iodide and on the other side of DCFH-DA for the intracellular H2O2 detection and CD16-PECy5.Results after 3 hours incubation are given in Table 1.Table 1.MEAN +/− SDCD16 %ANNEXIN V %DCFH-DA %nSPONTANEOUS85 +/−7.17 +/− 4.71 +/−0.933PMA 10-9 M75 +/−9.09 +/− 5.21 +/− 0.910PMA 2 10-6 M21 +/−27.1 ***51 +/− 21.4 ***83 +/− 11.8 ***33PMA & STAURO51 +/−23.3 °°°23 +/− 16.0 °°1 +/− 2.015PMA & DPI20 +/− 33.420 +/− 17.1 °°3 +/− 6.220fMLP 5 10-6M41 +/− 19.3 ***11 +/− 6.98 +/− 9.7 *23α TNF 200 ng/ml42 +/− 19.2 *5 +/− 3.5 *8 +/− 9.5 *7Anti-Fas μg/ml 140 +/− 25.2 *17 +/− 8.2 **1 +/− 0.97Wilcoxon test*: between spontaneous and the stimulant;°: between PMA and the inhibitorStimulating PMA induced both intracellular H2O2 production and apoptosis with increase of Annexin V fixation and CD16 down-regulation. Partial prevention of both features was observed when the ROS production was completely inhibited in presence of staurosporin. DPI had an effect on Annexin V fixation but no effect on CD16 decrease dissociating the 2 apoptotic features. Neutrophils activated with fMLP generated mainly extra-cellular H2O2 and did not undergo apoptosis measured thanks to Annexin V fixation, but CD16 down-regulation was observed. In presence of TNFα, a very slight stimulation of intracellular H2O2 production was observed with an inhibition of the Annexin V fixation and a CD16 down-regulation.The Fas mediated signalling system induced moderately both Annexin fixation and CD16 down-regulation without ROS production.Discussion: CD16 down-regulation seems to be present with fMLP and TNFα without other apoptotic features. Moreover, TNFα at 200ng/ml offers a protection for the phospholipids “flip-flop”. Dissociation of the classical PMN apoptotic features CD16 down-regulation and Annexin-V fixation with TNFα and fMLP demonstrates the complexity of the transduction signals used by these stimulants, and for the first time the independence of CD16 down-regulation from the phospholipids “flip-flop” which appears to be strongly dependent of intracellular ROS or more moderately of Fas stimulation and is never present without CD16 decrease. Only the presence of both events demonstrates apoptosis with certitude. On the opposite, it appears that the CD16 decrease alone cannot reflect the presence of the cell apoptosis. This decrease in absence of phospholipids “flip-flop”could be induced by extra-cellular ROS.