Hemin as a Molecular Probe for Nitric Oxide Detection in Physiological Solutions: Experimental and Theoretical Assessment.

Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (●NO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric ●NO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting ●NO in diverse solutions. We investigated how the luminescence kinetics was influenced by ●NO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and ●NO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with ●NO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the ●NO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the ●NO-donor. The examined reactions aid in comprehending the mechanism of ●NO/hemin/H2O2/luminol interactions and how these can be used for detecting ●NO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.

Antagonistic cooperativity between crystal growth modifiers.

Ubiquitous processes in nature and the industry exploit crystallization from multicomponent environments1-5; however, laboratory efforts have focused on the crystallization of pure solutes6,7 and the effects of single growth modifiers8,9. Here we examine the molecular mechanisms employed by pairs of inhibitors in blocking the crystallization of haematin, which is a model organic compound with relevance to the physiology of malaria parasites10,11. We use a combination of scanning probe microscopy and molecular modelling to demonstrate that inhibitor pairs, whose constituents adopt distinct mechanisms of haematin growth inhibition, kink blocking and step pinning12,13, exhibit both synergistic and antagonistic cooperativity depending on the inhibitor combination and applied concentrations. Synergism between two crystal growth modifiers is expected, but the antagonistic cooperativity of haematin inhibitors is not reflected in current crystal growth models. We demonstrate that kink blockers reduce the line tension of step edges, which facilitates both the nucleation of crystal layers and step propagation through the gates created by step pinners. The molecular viewpoint on cooperativity between crystallization modifiers provides guidance on the pairing of modifiers in the synthesis of crystalline materials. The proposed mechanisms indicate strategies to understand and control crystallization in both natural and engineered systems, which occurs in complex multicomponent media1-3,8,9. In a broader context, our results highlight the complexity of crystal-modifier interactions mediated by the structure and dynamics of the crystal interface.

A simple, fast, label-free colorimetric method for detection of telomerase activity in urine by using hemin-graphene conjugates.

Telomerase, a widely accepted cancer biomarker for early cancer diagnostics, is considered as an important therapeutic target. To now, it is still a challenging subject to develop a simple and sensitive strategy for telomerase activity detection. Herein, we reported a simple colorimetric strategy for label-free quantification of human telomerase activity in urine by using hemin-graphene nanomaterial (H-GNs). H-GNs possessed tailored dispersibility in the high salt concentration and highly active biomimetic oxidation catalyst property. In this strategy, H-GNs were adjusted to coagulate to appropriate degree by carefully selecting the contained NaCl amount in the presence of original TS primer. The supernatant of the solution contained few H-GNs and showed light blue color. Under the action of telomerase, TS primer was elongated with repeating sequences of (TTAGGG)n. These negatively charged DNA enhanced individual H-GNs electrostatic repulsion and resisted salt-induced H-GNs coagulation. As a result, the supernate of the corresponding solution containing more dispersed H-GNs and showed dark blue color after chromogenic reaction. Thus, telomerase activity could be quasi-quantified by naked eye and precise quantified by UV spectrometer. The proposed method has the linear range from 100 to 2300 HeLa cells/mL and the detection limit was 60 cells/mL. It has been successfully applied to detect telomerase activity in real urine samples. Obtained results were in good agreement with the clinical diagnosis. Therefore, this colorimetric approach affords simplicity, sensitivity and reliability in telomerase activity detection.

A facile label-free colorimetric aptasensor for acetamiprid based on the peroxidase-like activity of hemin-functionalized reduced graphene oxide.

A facile aptasensor has been developed for the colorimetric detection of acetamiprid by using the hemin-functionalized reduced graphene oxide (hemin-rGO) composites. The as-prepared hemin-rGO composites possessed both the ability of rGO to physically adsorb the aptamers and the peroxidase-like activity of hemin that could catalyse 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2, to produce a solution with blue color. The well-dispersed hemin-rGO composites coagulated completely at the proper salt concentration; however, the coagulation of hemin-rGO was vanished when abundant aptamers were adsorbed on its surface because the attached negatively charged DNA backbone increased individual hemin-rGO electrostatic repulsion. In the detection scheme, acetamiprid with different concentrations was firstly incubated with the same amount of aptamer. The more acetamiprid in the tested solution, the less free aptamers were absorbed on the hemin-rGO surface, making the composites coagulate to a higher degree in the presence of the optimum NaCl concentration. As a consequence, the content of hemin-rGO in the supernatant was decreased after centrifugation, which catalysed oxidation of TMB in the presence of H2O2 to produce light blue color with a low absorbance. The color variation relavant to the acetamiprid concentration can be judged by the naked eyes and easily monitored by the inexpensive UV-vis spectrometer. Such designed aptasensor displayed a linear response for acetamiprid in the range from 100nM to 10µM with a detection limit of 40nM (S/N=3). This colorimetric aptasensing platform offers great advantages including the simple operation process, low-cost portable instrument, and user-friendly applications.

Chemiluminescence assay for the sensitive detection of iodide based on extracting Hg2+ from a T-Hg(2+)-T complex.

A simple and sensitive chemiluminescence assay for iodide (I(-)) detection was reported, which was based on iodide extracting Hg(2+) from DNA featuring a stem-loop structure containing T-Hg(2+)-T. Specifically, Hg(2+) induced random coiled G-rich single-strand DNA to form a stem-loop structure containing T-Hg(2+)-T. Because the binding of Hg(2+) and I(-) is much stronger than that of Hg(2+) and thymine (T), I(-) could extract Hg(2+) from the stem-loop structure, releasing the DNA, which then bound with K(+) and transformed into a K(+)-stabilized G-quadruplex (with hemin as a cofactor), which catalyzed the H(2)O(2)-mediated oxidation of luminol. The produced chemiluminescence as a sensing signal was applied to sensitively and selectively detect iodide with a detection limit of 12 nM. This system exhibited the first DNAzyme-based iodide sensor. Finally, the sensor was successfully applied for iodide detection in real lake water samples.

Colorimetric detection of DNA damage by using hemin-graphene nanocomposites.

A colorimetric method for detection of DNA damage was developed by using hemin-graphene nanosheets (H-GNs). H-GNs were skillfully synthesized by adsorping of hemin on graphene through π-π interactions. The as-prepared H-GNs possessed both the ability of graphene to differentiate the damage DNA from intact DNA and the catalytic action of hemin. The damaged DNA made H-GNs coagulated to different degrees from the intact DNA because there were different amount of negative charge exposed on their surface, which made a great impact on the solubility of H-GNs. As a result, the corresponding centrifugal supernatant of H-GNs solution showed different color in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, which could be discriminated by naked eyes or by ultraviolet (UV)-visible spectrometer. Based on this, the damaged effects of styrene oxide (SO), NaAsO2 and UV radiation on DNA were studied. Results showed that SO exerted most serious damage effect on DNA although all of them damaged DNA seriously. The new method for detection of DNA damage showed good prospect in the evaluation of genotoxicity of new compounds, the maximum limit of pesticide residue, food additives, and so on, which is important in the fields of food science, pharmaceutical science and pesticide science.

Hemin-graphene hybrid nanosheets with intrinsic peroxidase-like activity for label-free colorimetric detection of single-nucleotide polymorphism.

This paper demonstrated for the first time a simple wet-chemical strategy for synthesizing hemin-graphene hybrid nanosheets (H-GNs) through the π-π interactions. Significantly, this new material possesses the advantages of both hemin and graphene and exhibits three interesting properties. First, H-GNs have intrinsic peroxidase-like activity, which can catalyze the reaction of peroxidase substrate, due to the existence of hemin on the graphene surface. Second, their dispersion follow the 2D Schulze-Hardy rule, that is to say, the coagulation of H-GNs in electrolyte solution results from the interplay between van der Waals attraction and electric double-layer repulsion. Third, H-GNs exhibit the ability to differentiate ss- and ds-DNA in optimum electrolyte concentration, owing to the different affinities of ss- and ds-DNA to the H-GNs. On the basis of these unique properties of the as-prepared H-GNs, we have developed a label-free colorimetric detection system for single-nucleotide polymorphisms (SNPs) in disease-associated DNA. To our knowledge, this is the first report concerning on SNPs detection using functionalized graphene nanosheets. Owing to its easy operation and high specificity, it was expected that the proposed procedure might hold great promise in the pathogenic diagnosis and genetic diseases.

Non-stochastic quadratic fingerprints and LDA-based QSAR models in hit and lead generation through virtual screening: theoretical and experimental assessment of a promising method for the discovery of new antimalarial compounds.

In order to explore the ability of non-stochastic quadratic indices to encode chemical information in antimalarials, four quantitative models for the discrimination of compounds having this property were generated and statistically compared. Accuracies of 90.2% and 83.3% for the training and test sets, respectively, were observed for the best of all the models, which included non-stochastic quadratic fingerprints weighted with Pauling electronegativities. With a comparative purpose and as a second validation experiment, an exercise of virtual screening of 65 already-reported antimalarials was carried out. Finally, 17 new compounds were classified as either active/inactive ones and experimentally evaluated for their potential antimalarial properties on the ferriprotoporphyrin (FP) IX biocrystallization inhibition test (FBIT). The theoretical predictions were in agreement with the experimental results. In the assayed test compound C5 resulted more active than chloroquine. The current result illustrates the usefulness of the TOMOCOMD-CARDD strategy in rational antimalarial-drug design, at the time that it introduces a new family of organic compounds as starting point for the development of promising antimalarials.

Models and mechanisms of O-O bond activation by cytochrome P450. A critical assessment of the potential role of multiple active intermediates in oxidative catalysis.

Cytochrome P450 enzymes promote a number of oxidative biotransformations including the hydroxylation of unactivated hydrocarbons. Whereas the long-standing consensus view of the P450 mechanism implicates a high-valent iron-oxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound pathway, more recent studies on isotope partitioning, product rearrangements with 'radical clocks', and the impact of threonine mutagenesis in P450s on hydroxylation rates support the notion of the nucleophilic and/or electrophilic (hydro)peroxo-iron intermediate(s) to be operative in P450 catalysis in addition to the electrophilic oxenoid-iron entity; this may contribute to the remarkable versatility of P450s in substrate modification. Precedent to this mechanistic concept is given by studies with natural and synthetic P450 biomimics. While the concept of an alternative electrophilic oxidant necessitates C-H hydroxylation to be brought about by a cationic insertion process, recent calculations employing density functional theory favour a 'two-state reactivity' scenario, implicating the usual ferryl-dependent oxygen rebound pathway to proceed via two spin states (doublet and quartet); state crossing is thought to be associated with either an insertion or a radicalar mechanism. Hence, challenge to future strategies should be to fold the disparate and sometimes contradictory data into a harmonized overall picture.

[Comparative assessment of carboxymethylcellulose sorption properties with respect to hemin and globin]. / Sravnitel'naia otsenka sorbtsionnykh svoistv karboksimetiltselliulozy po otnosheniiu k geminu i globinu.

Adsorption of hemin and globin onto fibrous carboxymethylcellulose (CMC) that is commonly used in food industry was studied. The process of CMC saturation with hemin (and globin) was shown to depend on time and temperature. Constants for adsorption and the activation energy for the adsorption of globin were determined. A Langmuir-type hyperbolic adsorption was observed for globin, whereas the hemin adsorption on CMC was linear. Optimum conditions for globin isolation from hemoglobin solutions were found.