Tentative identification of phytochelatins, their derivatives, and Cd-phytochelatin complexes in Ocimum basilicum L. roots by Paper Spray Mass Spectrometry.

An unprecedented and direct PS-MS (paper spray ionization mass spectrometry) method was proposed for the detection of native peptides, that is, glutathiones (GSHs), homoglutathiones (hGSHs), and phytochelatins (PCs), in basil (Ocimum basilicum L.) roots before and after cadmium exposure. The roots were submitted to cold maceration followed by sonication with formic acid as the extractor solvent for sample preparation. PS-MS was used to analyze such extracts in the positive mode, and the results allowed for the detection of several GSHs, hGSHs, and PCs. Some of these PCs were not distinguished in the control samples, that is, basil roots not exposed to cadmium. Other PCs were noticed in both types of roots, uncontaminated and cadmium-contaminated, but the intensities were higher in the former samples. Moreover, long-time exposure to cadmium stimulated the formation of some of these PCs and their cadmium complexes. The results, therefore, provided some crucial insights into the defense mechanism of plants against an external stress condition due to exposure to a toxic heavy metal. The present study represents a promising alternative to investigate other crucial physiological processes in plants submitted to assorted stress conditions.

Glutathione-activated biotin-targeted dual-modal imaging probe with improved PDT/PTT synergistic therapy.

BACKGROUND: Glutathione (GSH), a highly abundant thiol compound within cells, plays a critical role in physiological processes and exhibits close correlation with cancer. Among molecular imaging technologies, most probes have relatively short emission wavelengths and lack photoacoustic imaging (PA) capability, resulting in the inability to obtain tissue images with high penetration depth. The presence of GSH in the tumor microenvironment neutralizes ROS, diminishing the therapeutic effect of PDT, thus resulting in often unsatisfactory therapeutic efficacy. Therefore, it is imperative to develop a dual-modal probe for the detection of GSH and the diagnosis and treatment of cancer. RESULTS: In this study, we synthesized a novel dual-modal probe, Cy-Bio-GSH, utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging techniques for GSH detection. The probe integrates cyanine dye as the fluorophore, nitroazobenzene as the recognition moiety, and biotin as the tumor-targeting moiety. Upon reacting with GSH, the probe emits NIR fluorescence at 820 nm and generates a PA signal. Significantly, this reaction activates the photodynamic and photothermal properties of the probe. By depleting GSH and employing a synergistic photothermal therapy (PTT) treatment, the therapeutic efficacy of photodynamic therapy (PDT) is remarkably enhanced. In-vivo experiments confirm the capability of the probe to detect GSH via NIRF and PA imaging. Notably, the combined tumor-targeting ability and PDT/PTT synergistic therapy enhance therapeutic outcomes for tumors and facilitate their ablation. SIGNIFICANCE: A novel tumor-targeting and dual-modal imaging probe (Cy-Bio-GSH) is synthesized, exhibiting remarkable sensitivity and selectivity to GSH, enabling the visualization of GSH in cells and the differentiation between normal and cancer cells. Cy-Bio-GSH enhances PDT/PTT with effective killing of cancer cells and makes the ablation of tumors in mice. This work represents the first tumor-targeting probe for GSH detection, and provides crucial tool for cancer diagnosis and treatment by dual-modal imaging with improved PDT/PTT synergistic therapy.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Phase and Defect Engineering of MoSe2 Nanosheets for Enhanced NIR-II Photothermal Immunotherapy.

Inducing immunogenic cell death (ICD) during photothermal therapy (PTT) has the potential to effectively trigger photothermal immunotherapy (PTI). However, ICD induced by PTT alone is often limited by inefficient PTT, low immunogenicity of tumor cells, and a dysregulated redox microenvironment. Herein, we develop MoSe2 nanosheets with high-percentage metallic 1T phase and rich exposed active Mo centers through phase and defect engineering of MoSe2 as an effective nanoagent for PTI. The metallic 1T phase in MoSe2 nanosheets endows them with strong PTT performance, and the abundant exposed active Mo centers endow them with high activity for glutathione (GSH) depletion. The MoSe2-mediated high-performance PTT synergizing with efficient GSH depletion facilitates the release of tumor-associated antigens to induce robust ICD, thus significantly enhancing checkpoint blockade immunotherapy and activating systemic immune response in mouse models of colorectal cancer and triple-negative metastatic breast cancer.

Bioinspired Surface Ligand Engineering Regulates Electron Transfers in Gold Clusterzymes to Enhance the Catalytic Activity for Improving Sensing Performance.

Metal nanoclusters feature a hierarchical structure, facilitating their ability to mimic enzyme-catalyzed reactions. However, the lack of true catalytic centers, compounded by tightly bound surface ligands hindering electron transfers to substrates, underscores the need for universal rational design methodologies to emulate the structure and mechanisms of natural enzymes. Motivated by the electron transfer in active centers with specific chemical structures, by integrating the peroxidase cofactor Fe-TCPP onto the surface of glutathione-stabilized gold nanoclusters (AuSG), we engineered AuSG-Fe-TCPP clusterzymes with a remarkable 39.6-fold enhancement in peroxidase-like activity compared to AuSG. Fe-TCPP not only mimics the active center structure, enhancing affinity to H2O2, but also facilitates the electron transfer process, enabling efficient H2O2 activation. By exemplifying the establishment of a detecting platform for trace H2O2 produced by ultrasonic cleaners, we substantiate that the bioinspired surface-ligand-engineered electron transfer can improve sensing performance with a wider linear range and lower detection limit.

Oxygen Vacancy Piezoelectric Nanosheets Constructed by a Photoetching Strategy for Ultrasound "Unlocked" Tumor Synergistic Therapy.

Piezoelectric dynamic therapy (PzDT) is an effective method of tumor treatment by using piezoelectric polarization to generate reactive oxygen species. In this paper, two-dimensional Cu-doped BiOCl nanosheets with surface vacancies are produced by the photoetching strategy. Under ultrasound, a built-in electric field is generated to promote the electron and hole separation. The separated carriers achieve O2 reduction and GSH oxidation, inducing oxidative stress. The bandgap of BiOCl is narrowed by introducing surface oxygen vacancies, which act as charge traps and facilitate the electron and hole separation. Meanwhile, Cu doping induces chemodynamic therapy and depletes GSH via the transformation from Cu(II) to Cu(I). Both in vivo and in vitro results confirmed that oxidative stress can be enhanced by exogenous ultrasound stimulation, which can cause severe damage to tumor cells. This work emphasizes the efficient strategy of doping engineering and defect engineering for US-activated PzDT under exogenous stimulation.

Scattering/Fluorescence Dual-Mode Imaging in MnO2-Coated Silicon Nanospheres for Cancer Cell Detection.

A tumor microenvironment (TME)-responsive nanoprobe composed of a fluorescent dye-decorated silicon (Si) nanosphere core and a thin MnO2 shell is proposed for simple and intelligent detection of cancer cells. The Si nanosphere core with diameters of 100-200 nm provides environment-independent Mie scattering imaging, while, simultaneously, the MnO2 shell provides the capability to switch the on/off state of the dye fluorescence reacted to the glutathione (GSH) and/or H2O2 levels in a cancer cell. Si-MnO2 core-shell nanosphere probes are fabricated in a solution-based process from crystalline Si nanosphere cores. The fluorescence switching under exposure to GSH is demonstrated, and the mechanism is discussed based on detailed optical characterizations including single-particle spectroscopy. Different types of human cells are incubated with the nanoprobes, and a proof of concept experiment is performed. From the combination of the robust scattering images and GSH- and H2O2-sensitive fluorescence images, the feasibility of cancer cell detection by the multimodal nanoprobes is demonstrated.

O-Phthalaldehyde Derivatization for the Paper-Based Fluorometric Determination of Glutathione in Nutritional Supplements.

Herein, a new, direct paper-based fluorimetric method is described for the quantitative determination of glutathione (GSH) molecules in nutritional supplements. Briefly, the proposed analytical method is based on the fluorescence emission resulting from the direct and selective chemical reaction of GSH molecules with the derivatization reagent that is o-phthalaldehyde (OPA) in acidic conditions at room temperature. The intensity of the emitted fluorescence on the surface of the analytical paper devices after irradiation with a lamp at 365 nm is proportional to the concentration of GSH and is measured using a smartphone as the detector. This methodology, which is suitable for measurements in laboratories with limited resources, does not require specialized instrumentation or trained personnel. The protocol governing the proposed method is simple and easily applicable. Essentially, the chemical analyst should adjust the value of pH on the surface of the paper by adding a minimal amount of buffer solution; then, after adding a few microliters of the derivatization reagent, wait for the surface of the paper to dry and, finally, add the analyte. Subsequently, the irradiation of the sensor and the measurement of the emitted fluorescence can be recorded with a mobile phone. In the present study, several parameters affecting the chemical reaction and the emitted fluorescence were optimized, the effect of interfering compounds that may be present in dietary supplements was examined, and the stability of these paper sensors under different storage conditions was evaluated. Additionally, the chemical stability of these paper devices in various maintenance conditions was studied, with satisfactory results. The detection limit calculated as 3.3 S/N was 20.5 µmol L-1, while the precision of the method was satisfactory, ranging from 3.1% (intra-day) to 7.3% (inter-day). Finally, the method was successfully applied to three different samples of dietary supplements.

Dual-Stimuli Regulation of DNAzyme Cleavage Reaction by Coordination-Driven Nanoprobes for Cancer Cell Imaging.

Endowing current artificial chemical reactions (ACRs) with high specificity and intricate activation capabilities is crucial for expanding their applications in accurate bioimaging within living cells. However, most of the reported ACR-based evaluations relied on either single biomarker stimuli or dual activators without obvious biological relevance, still limiting their accuracy and fidelity. Herein, taking the metal-ion-dependent DNAzyme cleavage reaction as a model ACR, two regulators, glutathione (GSH) and telomerase (TE) activated DNAzyme cleavage reactions, were exploited for precise discrimination of cancerous cells from normal cells. DNA probe was self-assembled into the ZIF-90 nanoparticle framework to construct coordination-driven nanoprobes. This approach enhances the stability and specificity of tumor imaging by utilizing biomarkers associated with rapid tumor proliferation and those commonly overexpressed in tumors. In conclusion, the research not only paves the way for new perspectives in cell biology and pathology studies but also lays a solid foundation for the advancement of biomedical imaging and disease diagnostic technologies.

S-scheme heterojunction phthalocyanine/TiO2 photoelectrochemical sensor for innovative glutathione detection.

A novel photoelectrochemical sensor, employing an S-scheme heterojunction of phthalocyanine and TiO2 nanoparticles, has been developed to enable highly sensitive determination of glutathione. By integrating the favorable stability, environmental benignity, and electronic properties of the TiO2 matrix with the unique photoactivity of phthalocyanine species, the designed sensor presents a substantial linear dynamic range and a low detection limit for the quantification of glutathione. The sensitivity is attributed to efficient charge transfer and separation across the staggered heterojunction energy levels, which generates measurable photocurrent signals. Systematic variation of phthalocyanine content reveals an optimal composition that balances light harvesting capacity and electron-hole recombination rates. The incorporation of phosphotungstic acid (PTA) in sample preparation effectively minimizes interference from compounds like L-cysteine and others. Consequently, this leads to an improvement in accuracy through the reduction of impurity levels. Appreciable photocurrent enhancements are observed upon introduction of both oxidized and reduced glutathione at the optimized composite photoanode. Coupled with advantageous features of photoelectrochemical transduction such as simplicity, cost-effectiveness, and resistance to fouling, this sensor holds great promise for practical applications in complex biological media.

Design of Ratio-Fluorescence Nanohybrid Based on Radix Hedysari Green-Synthesized CDs and GSH-AuNCs for Sensitive Detection of Cefodizime Sodium in Urine Sample.

A dual-emission ratio-fluorescent sensing nanohybrid based on Radix Hedysari green-synthesized carbon quantum dots (CDs) and glutathione-functionalized gold nanoclusters (GSH-AuNCs) had been developed for the determination of cefodizime sodium (CDZM). The designed fluorescence nanohybrid had two significant fluorescence emission peaks at 458 nm and 569 nm when excited at 360 nm, which was attributed to the CDs and GSH-AuNCs. With the addition of CDZM, the fluorescence at 458 nm was slightly weakened while the fluorescence at 569 nm was enhanced obviously. Based on the relationship between the I569/I458 fluorescence intensity ratio and the concentration of CDZM, the designed nanohybrid exhibited a good linearity range of 1.0-1000.0 µM and the limit of detection (LOD) was 0.19 µM. The method was finally applied in the detection of CDZM in urine, showing the potential applications in complicated biological samples.

Colorimetric array sensor based on bimetallic nitrogen-doped carbon-based nanozyme material to detect multiple antioxidants.

Copper-cobalt bimetallic nitrogen-doped carbon-based nanoenzymatic materials (CuCo@NC) were synthesized using a one-step pyrolysis process. A three-channel colorimetric sensor array was constructed for the detection of seven antioxidants, including cysteine (Cys), uric acid (UA), tea polyphenols (TP), lysine (Lys), ascorbic acid (AA), glutathione (GSH), and dopamine (DA). CuCo@NC with peroxidase activity was used to catalyze the oxidation of TMB by H2O2 at three different ratios of metal sites. The ability of various antioxidants to reduce the oxidation products of TMB (ox TMB) varied, leading to distinct absorbance changes. Linear discriminant analysis (LDA) results showed that the sensor array was capable of detecting seven antioxidants in buffer and serum samples. It could successfully discriminate antioxidants with a minimum concentration of 10 nM. Thus, multifunctional sensor arrays based on CuCo@NC bimetallic nanoenzymes not only offer a promising strategy for identifying various antioxidants but also expand their applications in medical diagnostics and environmental analysis of food.

Boosting the enzymatic activity of CxxC motif-containing PDI family members.

Compounds harboring high acidity and oxidizability of thiol groups permit tuning the redox equilibrium constants of CxxC sites of members of the protein disulphide isomerase (PDI) family and thus can be used to accelerate folding processes and increase the production of native proteins by minimal loading in comparison to glutathione.

Chemodynamic PtMn Nanocubes for Effective Photothermal ROS Storm a Key Anti-Tumor Therapy in-vivo.

Background: Chemodynamic therapy (CDT) is a new treatment approach that is triggered by endogenous stimuli in specific intracellular conditions for generating hydroxyl radicals. However, the efficiency of CDT is severely limited by Fenton reaction agents and harsh reaction conditions. Methods: Bimetallic PtMn nanocubes were rationally designed and simply synthesized through a one-step high-temperature pyrolysis process by controlling both the nucleation process and the subsequent crystal growth stage. The polyethylene glycol was modified to enhance biocompatibility. Results: Benefiting from the alloying of Pt nanocubes with Mn doping, the structure of the electron cloud has changed, resulting in different degrees of the shift in electron binding energy, resulting in the increasing of Fenton reaction activity. The PtMn nanocubes could catalyze endogenous hydrogen peroxide to toxic hydroxyl radicals in mild acid. Meanwhile, the intrinsic glutathione (GSH) depletion activity of PtMn nanocubes consumed GSH with the assistance of Mn3+/Mn2+. Upon 808 nm laser irradiation, mild temperature due to the surface plasmon resonance effect of Pt metal can also enhance the Fenton reaction. Conclusion: PtMn nanocubes can not only destroy the antioxidant system via efficient reactive oxygen species generation and continuous GSH consumption but also propose the photothermal effect of noble metal for enhanced Fenton reaction activity.

A ratiometric fluorescence and colorimetry dual-signal sensing strategy based on o-phenylenediamine and AuNCs for determination of Cu2+ and glyphosate.

A ratiometric fluorescence sensing strategy has been developed for the determination of Cu2+ and glyphosate with high sensitivity and specificity based on OPD (o-phenylenediamine) and glutathione-stabilized gold nanoclusters (GSH-AuNCs). Water-soluble 1.75-nm size GSH-AuNCs with strong red fluorescence and maximum emission wavelength at 682 nm were synthesized using GSH as the template. OPD was oxidized by Cu2+, which produced the bright yellow fluorescence oxidation product 2,3-diaminophenazine (DAP) with a maximum fluorescence emission peak at 570 nm. When glyphosate existed in the system, the chelation between glyphosate and Cu2+ hindered the formation of DAP and reduced the fluorescence intensity of the system at the wavelength of 570 nm. Meanwhile, the fluorescence intensity at the wavelength of 682 nm remained basically stable. It exhibited a good linear relationship towards Cu2+ and glyphosate in water in the range 1.0-10 µM and 0.050-3.0 µg/mL with a detection limit of 0.547 µM and 0.0028 µg/mL, respectively. The method was also used for the semi-quantitative determination of Cu2+ and glyphosate in water by fluorescence color changes visually detected by the naked eyes in the range 1.0-10 µM and 0.30-3.0 µg/mL, respectively. The sensing strategy showed higher sensitivity, more obvious color changes, and better disturbance performance, satisfying with the detection demands of Cu2+ and glyphosate in environmental water samples. The study provides a reliable detection strategy in the environment safety fields.

Construction of logic gate computation for the assay of the nerve agent sarin based on an AChE-based dual-channel sensing system.

Nerve agents have posed a huge threat to national and human security, and their sensitive detection is crucial. Herein, based on the oxidation of Ce4+ and the aggregation-induced emission (AIE) of glutathione-protected gold nanoclusters (GSH-Au NCs), a cascade reaction was designed to prepare oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) and GSH-Au NCs crosslinked by Ce3+ (Ce3+-GSH-Au NCs). oxTMB had a broad UV-visible absorption range (500-700 nm) and was capable of quenching the fluorescence of Ce3+-GSH-Au NCs at 590 nm through the internal filtration effect (IFE). Thiocholine (TCh), the hydrolysis product of acetylthiocholine chloride (ATCl) catalyzed by acetylcholinesterase (AChE), reduced oxTMB completely, resulting in a decrease in the absorption of oxTMB and the recovery of IFE-quenched fluorescence of Ce3+-GSH-Au NCs. Nerve agent sarin (GB) hindered the production of TCh and the reduction of oxTMB by inhibiting the AChE activity, leading to the fluorescence of Ce3+-GSH-Au NCs being quenched again. The dual-output sensing system (AChE + ATCl + oxTMB + Ce3+-GSH-Au NCs) exhibited a low limit of detection to GB (2.46 nM for colorimetry and 1.18 nM for fluorimetry) and excellent selectivity toward common interferences being unable to inhibit AChE. Moreover, the intelligent logic gate constructed based on the sensing system showed promising applications in the field of smart sensing of nerve agents.

A Combination of Biocatalysis and Fenton-Like Reaction Induced OH• Burst for Cascade Amplification of Cancer Chemodynamic Therapy.

Chemodynamic therapy (CDT) is a novel antitumor strategy that employs Fenton or Fenton-like reactions to generate highly toxic hydroxyl radical (OH•) from hydrogen peroxide (H2O2) for inducing tumor cell death. However, the antitumor efficacy of the CDT strategy is harshly limited by the redox homeostasis of tumor cells; especially the OH • is easily scavenged by glutathione (GSH) and the intracellular H2O2 level is insufficient in the tumor cells. Herein, we propose the Mn2+-menadione (also known as vitamin K3, MK3) cascade biocatalysis strategy to disrupt the redox homeostasis of tumor cells and induce a OH• storm, resulting in enhanced CDT effect. A nanoliposome encapsulating Mn-MK3 (Mn-MK3@LP) was prepared for the treatment of hepatic tumors in this study. After Mn-MK3@LPs were taken up by tumor cells, menadione could facilitate the production of intracellular H2O2 via redox cycling, and further the cytotoxic OH • burst was induced by Mn2+-mediated Fenton-like reaction. Moreover, high-valent manganese ions were reduced by GSH and the depletion of GSH further disrupted the redox homeostasis of tumor cells, thus achieving synergistically enhanced CDT. Overall, both cellular and animal experiments confirmed that the Mn-MK3@LP cascade biocatalysis nanoliposome exhibited excellent biosafety and tumor suppression efficacy. This study may provide deep insights for developing novel CDT-based strategies for tumor therapy.

A detection system using sensing motif-tethered oligodeoxynucleotides for multiplex biomolecular analysis.

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H2O2. After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.

Cryo-EM structure of cadmium-bound human ABCB6.

ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, we present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.1 Å, respectively. The overall folding of the two structures resembles the inward-facing apo state but with less separation between the two halves of the transporter. Two GSH molecules are symmetrically bound to the Cd(II) ion in a bent conformation, with the central cysteine protruding towards the metal. The N-terminal glutamate and C-terminal glycine of GSH do not directly interact with Cd(II) but contribute to neutralizing positive charges of the binding cavity by forming hydrogen bonds and van der Waals interactions with nearby residues. In the presence of PC2, Cd(II) binding to ABCB6 is similar to that observed with GSH, except that two cysteine residues of each PC2 molecule participate in Cd(II) coordination to form a tetrathiolate. Structural comparison of human ABCB6 and its homologous Atm-type transporters indicate that their distinct substrate specificity might be attributed to variations in the capping residues situated at the top of the substrate-binding cavity.

Stealth and Biocompatible Gold Nanoparticles through Surface Coating with a Zwitterionic Derivative of Glutathione.

Gold nanoparticles (AuNPs) hold promise in biomedicine, but challenges like aggregation, protein corona formation, and insufficient biocompatibility must be thoroughly addressed before advancing their clinical applications. Designing AuNPs with specific protein corona compositions is challenging, and strategies for corona elimination, such as coating with polyethylene glycol (PEG), have limitations. In this study, we introduce a commercially available zwitterionic derivative of glutathione, glutathione monoethyl ester (GSHzwt), for the surface coating of colloidal AuNPs. Particles coated with GSHzwt were investigated alongside four other AuNPs coated with various ligands, including citrate ions, tiopronin, glutathione, cysteine, and PEG. We then undertook a head-to-head comparison of these AuNPs to assess their behavior in biological fluid. GSHzwt-coated AuNPs exhibited exceptional resistance to aggregation and protein adsorption. The particles could also be readily functionalized with biotin and interact with streptavidin receptors in human plasma. Additionally, they exhibited significant blood compatibility and noncytotoxicity. In conclusion, GSHzwt provides a practical and easy method for the surface passivation of AuNPs, creating "stealth" particles for potential clinical applications.