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.

Simple and rapid chemiluminescence aptasensor for Hg2+ in contaminated samples: A new signal amplification mechanism.

Detection of ultralow concentration of heavy metal ion Hg2+ is important for human health protection and environment monitoring because of the gradual accumulation in environmental and biological fields. Herein, we report a convenient chemiluminescence (CL) biosensing platform for ultrasensitive Hg2+ detection by signal amplification mechanism from positively charged gold nanoparticles ((+)AuNPs). It is based on (+)AuNPs charge effect and aptamer conformation change induced by target to stimulate the generation of CL in the presence of H2O2 and luminol without high salt medium. Notably particularly, the typical problem of the high salt medium from (-) AuNPs system, like influencing aptamers' bind with target and hindering CL reaction can be effectively addressed through the direct introduction of (+)AuNPs. Therefore, the proposed biosensing exhibits a high sensitivity toward target Hg2+ with a detection limit of 16 pM, which is far below the limit (10nM) defined by the U.S. Environmental Protection Agency in drinkable water, and is about 10-fold lower than the previously reported aptamer-based assays for Hg2+. This sensing platform provides a simple, rapid, and cost-effective approach for label-free sensitive detection of Hg2+. Moreover, it is universal for the detection of other targets. Undoubtedly, such a direct utilizing of (+)AuNPs' charge effect will provide a new signal amplification way for label-free aptamer-based CL analysis.

N-Hydroxysuccinimide as an effective chemiluminescence coreactant for highly selective and sensitive detection.

The development of novel coreactants for chemiluminescence is very important to improve performance and widen its applications without using any other catalyst. N-Hydroxysuccinimide (NHS), a highly popular amine-reactive, activating, or protecting reagent in biochemical applications and organic synthesis, has been explored as an efficient and stable chemiluminescence coreactant for the first time. The chemiluminescence intensity of the newly developed luminol-NHS system is about 22 times higher than that of the traditional luminol-H2O2 system. Chemiluminescence of this system is dramatically enhanced by Co2+. This new chemiluminescence system is then applied for the highly selective and ultrasensitive detection of Co2+ with limit of detection (0.01 nM) better than those of several conventional analytical methods. This system also enables the efficient detection of luminol (LOD = 7 pM) and NHS (LOD = 3.0 µM) with excellent sensitivity. This chemiluminescence method was then also utilized to detect Co2+ in tap water and blue silica gel with excellent recoveries in the range 99.20-103.07 %. This novel chemiluminescence system has several advantages, including simple, cost-effective, highly sensitive, selective, and wide linear range. We expect that this chemiluminescence system will be a promising candidate for chemical and biological sensing. Graphical Abstract Comparison of CL peak intensities of classical luminol-H2O2 CL system and newly developed luminol-NHS CL system.

An ultra-facile and label-free immunoassay strategy for detection of copper (II) utilizing chemiluminescence self-enhancement of Cu (II)-ethylenediaminetetraacetate chelate.

The establishment of facile, rapid, sensitive and cost-effective protocols for the detection of heavy metals is of great significance for human health and environmental monitoring. Hereby, an ultra-facile and label-free immunoassay strategy was designed for detecting heavy metal ion by using Cu (II) as the model analyte. Cu (II) reacted previously with ethylenediaminetetraacetate (EDTA) was captured by immobilized monoclonal antibody for Cu (II)-EDTA chelate. Then Cu (II) was detected based on the self-enhancing effect of Cu (II)-EDTA chelate to luminol-H2O2 chemiluminescence reaction. The CL intensity is linear relative with Cu (II) concentration in a very wide range of 1.0-1000ng/mL, with a detection limit of 0.33ng/mL (S/N=3). Since the specificity of this proposed strategy relied on both the specificity of monoclonal antibody and the specificity of luminol-H2O2 system, it could avoid interference from most common ions. The proposed method was used successfully to detect Cu (II) in traditional Chinese medicine and environmental water samples with acceptable recovery values of 82-113%. This proof-of-principle work demonstrated the feasibility of the label-free immunoassay for heavy metal ions, and opened a new avenue for rapid screening and field assay for drug safety, environmental monitoring and clinical diagnosis.

Iodophenol blue-enhanced luminol chemiluminescence and its application to hydrogen peroxide and glucose detection.

In this study, we found that iodophenol blue can enhance the weak chemiluminescence (CL) of luminol-H2O2 system. With the aid of CL spectral, electron spin resonance (ESR) spectral measurements and studies on the effects of various free radical scavengers on the iodophenol blue-enhanced luminol-H2O2 system, we speculated that iodophenol blue may react with H2O2 and oxygen to produce oxidizing radical species such as OH(•) and O2(•-) resulting the formation of (1)O2. The generated (1)O2 may react with luminol anion generating an unstable endoperoxide and subsequent 3-aminophthalate* (3-APA*). When the excited-state 3-APA returned to the ground-state, an enhanced CL was observed. Based on the H2O2 concentration dependence of the catalytic activity of iodophenol blue, a cheap, simple, sensitive CL assay for the determination of H2O2 was established. Under the optimum experimental conditions, a linear relationship between the relative CL intensity and H2O2 concentration in the range of 0.025-10 µM was obtained. As low as 14 nM H2O2 can be sensitively detected by using the proposed method. The relative standard deviation for 5, 1 and 0.25 µM H2O2 was 2.58%, 5.16% and 4.66%, respectively. By combining the glucose oxidase (GOx)-catalyzed oxidation reaction, CL detection of glucose was realized. The linear range of glucose detection was 0.1-30 µM with a detection limit of 0.06 µM. The proposed method has been applied to the detection of glucose in diluted serum.

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.

3D origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device.

A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.

Disposable luminol copolymer-based biosensor for uric acid in urine.

A new electrochemiluminescent (ECL) disposable biosensor for uric acid was manufactured by immobilization in a double-layer design of luminol as a copolymer with 3,3',5,5'-tetramethylbenzidine (TMB) and the enzyme uricase in chitosan on gold screen-printed cells. The good mechanical and improved electroluminescent characteristics of the new copolymer poly(luminol-TMB) make it possible to determine uric acid by measuring the growing ECL emission with the analyte concentration. The combination of enzymatic selectivity with ECL sensitivity results in a disposable analytical device with a linear range for uric acid from 1.5×10(-6) to 1.0×10(-4) M, a limit of detection of 4.4×10(-7) M and a precision of 13.1% (1.0×10(-5) M, n=10) as relative standard deviation. Satisfactory results were obtained for uric acid determination in 24h-urine samples compared to a reference procedure. This uric acid biosensor can be used as a low-cost alternative to conventional methods.

A novel electrochemiluminescence strategy for ultrasensitive DNA assay using luminol functionalized gold nanoparticles multi-labeling and amplification of gold nanoparticles and biotin-streptavidin system.

Luminol functionalized gold nanoparticles were used as labels for electrochemiluminescence signal amplification and an ultrasensitive, highly selective, convenient, low cost DNA detection strategy was developed.

The phytochemical analysis and antioxidant activity assessment of orange peel (Citrus sinensis) cultivated in Greece-Crete indicates a new commercial source of hesperidin.

The flavonoid content of several methanolic extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Crete (Greece) was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The chemical structures of the constituents fractionated were originally determined by comparing their retention times and the obtained UV spectral data with the available bibliographic data and further verified by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated flavanones and phenolic acids along with their ester derivatives. In addition, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. Interestingly enough, its quantity at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of hesperidin. The antioxidant activity of the orange peel methanolic extract fractions was evaluated by applying two complementary methodologies, DPPH(*) assay and the Co(II)/EDTA-induced luminol chemiluminescence approach. Overall, the results have shown that orange peel methanolic extracts possess moderate antioxidant activity as compared with the activity seen in tests where the corresponding aglycones, diosmetin and hesperetin were assessed in different ratios.

Real-time detection of dopamine released from a nerve model cell by an enzyme-catalyzed luminescence method and its application to drug assessment.

A real-time observation of neurotransmitter release from a nerve cell is a useful method for not only neuroscience research, but also assessing of the influence of chemicals, including drugs, on the human nervous system. In this study, a more simple and sensitive method for real-time monitoring of dopamine release from a nerve model cell was developed. Highly sensitive detection of dopamine was performed by using tyramine oxidase for dopamine oxidation, which was followed by a luminol luminescence reaction. This enzyme-catalyzed luminescence method was applied to observe dopamine release from the PC12 cell as a nerve model cell upon stimulation with acetylcholine and an acetylcholine receptor agonist. The results demonstrated that the real-time monitoring of the activation of the PC12 cell was easily performed by this method. This method possessed many advantages, such as high sensitivity, rapid measurement and no pretreatment for cells. It might be applied to drug screening and the assessment of harmful influences of food additives and pesticides on the nerves.

A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background.

We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other non-commercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested.

Assessment of training effects on activity levels of alveolar macrophage in matured rats using chemiluminescent technique.

Chemiluminescence responses have been used for the evaluation of phagocyte function. In this study, to evaluate effects of training started after maturation on pulmonary immunity, the activity levels of rat alveolar macrophages (AMs) were assessed as reactive oxygen species (ROS) generating capacity, measured by lucigenin- and luminol-dependent chemiluminescence, using a parallel luminometer. One group of male Wistar rats started training at 11 weeks old and another group at 17 weeks old. The experimental period was 12 weeks, and about half of the rats were sacrificed after 6 weeks. The forced and voluntary exercises affect the mean levels of body weights and cell populations in the bronchoalveolar lavage fluid in younger animals; however, the voluntary exercise group in younger animals seemed to adapt after 12 weeks. By contrast, chemiluminescence responses in older rats observed after 6 weeks suggest that AMs are primed, and the maximum releasing activities of ROS are reduced. These changes in AM activity may be caused by the exercise and separation stresses and the rats may adapt to those stressors after 12 weeks. The chemiluminescent technique is thought to be useful to evaluate the changes of AM activity.