A chemical timer approach to delayed chemiluminescence
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Although the onset time of chemical reactions can be manipulated by mechanical, electrical, and optical methods, its chemical control remains highly challenging. Herein, we report a chemical timer approach for manipulating the emission onset time of chemiluminescence (CL) reactions. A mixture of Mn 2+ , NaHCO 3 , and a luminol analog with H 2 O 2 produced reactive oxygen species (ROS) radicals and other superoxo species (superoxide containing complex) with high efficiency, accompanied by strong and immediate CL emission. Surprisingly, the addition of thiourea postponed CL emission in a concentration-dependent manner. The delay was attributed to a slow-generation-scavenging mechanism, which was found to be generally applicable not only to various types of CL reagents and ROS radical scavengers but also to popular chromogenic reactions. The precise regulation of CL kinetics was further utilized in dynamic chemical coding with improved coding density and security. This approach provides a powerful platform for engineering chemical reaction kinetics using chemical timers, which is of application potential in bioassays, biosensors, CL microscopic imaging, microchips, array chips, and informatics.Keywords:
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Correspondence should be addressed to Yunbo Li, yli@vcom.vt.edu and Ge Wang, wangg@vt.eduReceived 6 October 2008; Accepted 19 December 2008Recommended by Guowei WeiWe have applied the highly sensitive chemiluminescence (CL) imaging technique to investigate the in situ ROS formation incultured monolayers of rat H9c2 cardiomyocytes. Photon emission was detected via an innovative imaging system after incubationof H9c2 cells in culture with luminol and horseradish peroxidase (HRP), suggesting constitutive formation of ROS by thecardiomyocytes. Addition of benzo(a)pyrene-1,6-quinone (BPQ) to cultured H9c2 cells resulted in a 4-5-fold increase in theformation of ROS, as detected by the CL imaging. Both constitutive and BPQ-stimulated CL responses in cultured H9c2 cells weresustained for up to 1 hour. The CL responses were completely abolished in the presence of superoxide dismutase and catalase,suggesting the primary involvement of superoxide and hydrogen peroxide (H
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Reactive oxygen species (ROS) are produced by numerous biological systems and by several phagocytes such as neutrophils and macrophages. ROS include mostly superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radical, which are involved in a variety of biological processes such as immunity, inflammation, apoptosis and cell signaling. Thus, there is a need for a sensitive and reliable method to measure ROS. The luminol-amplified chemiluminescence technique is widely used to measure ROS production by neutrophils; however, it is unclear which ROS species are detected by this technique. In this study, we show that Xanthine/Xanthine oxidase (XXO), a known superoxide-producing system, stimulated a luminol-amplified chemiluminescence in the absence of horseradish peroxidase (HRPO), while the presence of HRPO enhanced the response. Both reactions were inhibited by superoxide dismutase (SOD), but not by catalase, confirming that superoxide anion, and not hydrogen peroxide, is the species oxidizing luminol to produce chemiluminescence. Glucose/Glucose oxidase (GGO), a known hydrogen peroxide-producing system, did not induce luminol-amplified chemiluminescence in the absence of HRPO; however, addition of HRPO resulted in a chemiluminescence response, which was inhibited by catalase, but not by SOD. Myeloperoxidase (MPO), isolated from human neutrophils, was also able to enhance the superoxide- and hydrogen peroxide-dependent luminol-amplified chemiluminescence. The production of ROS by stimulated human neutrophils was detected by luminol-amplified chemiluminescence, which was only partially inhibited by SOD and catalase. Interestingly, adding HRPO to stimulated neutrophils increased the luminol-amplified chemiluminescence, which was strongly inhibited by SOD, but not by catalase. These results show that (a) luminol-amplified chemiluminescence is able to detect superoxide anion in the absence of peroxidases, but not hydrogen peroxide; (b) in the presence of peroxidases, luminol-amplified chemiluminescence is able to detect both superoxide anion and hydrogen peroxide; and
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Studies of functional activity of neutrophilic granulocytes from patients with chronic rhinosinusitis was performed using luminol- or lucigenin-dependent chemiluminescence. We have revealed increased production of reactive oxygen species (ROS) both under basal conditions, and in zymozan-induced chemiluminescent reaction, employing luminol as an activator, and determining total functional activity of neutrophils. Meanwhile, in lucigenin-induced chemiluminescence associated with production of superoxide anion radicals, a delayed ROS production was revealed, both in spontaneous and zymozan-induced response. One may suggest that, when studying these pathological events in neutrophils, ROS production in the cells proceeds mostly by myeloperoxidase pathway.
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Although the onset time of chemical reactions can be manipulated by mechanical, electrical, and optical methods, its chemical control remains highly challenging. Herein, we report a chemical timer approach for manipulating the emission onset time of chemiluminescence (CL) reactions. A mixture of Mn 2+ , NaHCO 3 , and a luminol analog with H 2 O 2 produced reactive oxygen species (ROS) radicals and other superoxo species (superoxide containing complex) with high efficiency, accompanied by strong and immediate CL emission. Surprisingly, the addition of thiourea postponed CL emission in a concentration-dependent manner. The delay was attributed to a slow-generation-scavenging mechanism, which was found to be generally applicable not only to various types of CL reagents and ROS radical scavengers but also to popular chromogenic reactions. The precise regulation of CL kinetics was further utilized in dynamic chemical coding with improved coding density and security. This approach provides a powerful platform for engineering chemical reaction kinetics using chemical timers, which is of application potential in bioassays, biosensors, CL microscopic imaging, microchips, array chips, and informatics.
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The generation and transformation of radicals on the cathode of indirect electrochemical oxidation were studied by chemilumines- cence(CL)and UV-Visible spectra in the reactor with a salt bridge that connected the separated chambers.The CL intensity of 4×10~(-9)mol/L luminol on the cathode with bubbling oxygen was about seven times that of the intensity without it,which was because of the generation of reactive oxygen species(ROS).The existence of ROS,especially the generation of the superoxide radical,c...
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Reactive oxygen species (ROS) play important roles in the pathogenesis of vascular disease states. In particular, superoxide anion participates in endothelial dysfunction mainly owing to its rapid interaction with NO, but also as it causes direct biological effects and serves as a progenitor for many other ROS. Detection of ROS in intact tissues and cells is much more difficult than in chemical systems. We describe advantages and potential pitfalls of chemiluminescent methods of vascular ROS detection. Lucigenin and luminol-enhanced chemiluminescent methods are described in the detection of vascular superoxide and peroxynitrite production and NAD(P)H oxidase activity. We also describe the use of new chemiluminescent probes, including cypridina luciferin analogs (coelenterazine; CLA and MCLA) and pholasin. The validity of some of these chemiluminescent methods (in particular lucigenin-enhanced chemiluminescence) recently has been questioned. It has been suggested that lucigenin itself, especially at high concentrations (>50 micromol/L), may produce superoxide via redox cycling. Using intact human vascular rings and vascular homogenates, we show that lucigenin, particularly at lower concentrations (5 micromol/L), provides an accurate assessment of the rate of superoxide production as assessed by close correlations with the SOD inhibitable ferricytochrome c reduction assay. Chemiluminescent techniques provide a useful approach for vascular ROS measurements, but should be always interpreted in the context of measurements obtained using other complementary techniques.
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