Unique biomedical application of fluorescence derivatization based on palladium-catalyzed coupling reactions for HPLC analysis of pharmaceuticals and biomolecules.

Palladium-catalyzed coupling reactions are versatile and powerful tools for the construction of carbon-carbon bonds in organic synthesis. Although these reactions have favorable features that proceed selectively in mild reaction conditions using aqueous organic solvents, no attention has been given to their application in the field of biomedical analysis. Therefore, we focused on these reactions and evaluated the scope and limitations of their analytical performance. In this review, we describe the pros and cons and future trends of fluorescence derivatization of pharmaceuticals and biomolecules based on palladium-catalyzed coupling reactions such as Suzuki-Miyaura coupling, Mizoroki-Heck coupling, and Sonogashira coupling reactions for HPLC analysis.

LC-MS/MS analysis of components in smoke from e-cigarettes that use guarana extract as the caffeine source.

Currently, e-cigarette products to inhale caffeine (Caf) are commercially available and widely used. Guarana extract (GE) is used as the caffeine source in some e-cigarette products. In this study, an LC-MS/MS analysis of components in the smoke from e-cigarettes with GE was performed. The concentration ranges of Caf and the minor components theophylline (TP), theobromine (TB), and paraxanthine (PX) in e-liquid and cigarette smoke extract (CSE) of five e-cigarette products were determined. The concentration ranges of e-liquid and CSE were 2.17-8.62 mg/mL and 0.17-1.17 µg/puff for Caf, 0.09-37.58 µg/mL and 0.03-11.88 ng/puff for TB, 50.28-185.26 ng/mL and 0.00-0.05 ng/puff for TP, and 0.44-4.09 µg/mL and 0.03-0.20 ng/puff for PX, respectively. By comparing the peak area ratios of e-liquid and CSE, we clarified that the heat degradation of Caf to its related components in GE products was accelerated. Epicatechin, which is another typical component in GE, was determined for CSE, but not for e-liquid.

Biotinylated Quinone as a Chemiluminescence Sensor for Biotin-Avidin Interaction and Biotin Detection Application.

Biotin, or vitamin B7, is essential for metabolic reactions. It must be obtained from external sources such as food and biotin/vitamin supplements because it is not biosynthesized by mammals. Therefore, there is a need to monitor its levels in supplements. However, biotin detection methods, which include chromatographic, immune, enzymatic, and microbial assays, are tedious, time-consuming, and expensive. Thus, we synthesized a product called biotin-naphthoquinone, which produces chemiluminescence upon its redox cycle reaction with dithiothreitol and luminol; then it was used as a chemiluminescence sensor for biotin-avidin interaction. When a quinone biotinylated compound binds avidin, the chemiluminescence decreases noticeably due to the proximity between quinone and avidin, and when free biotin is added in a competitive assay, the chemiluminescence returns. The chemiluminescence is regained as the free biotin displaces biotinylated quinone in its complex with avidin, freeing biotin-naphthoquinone. Many experiments, including the use of a biotin-free quinone, proved the competitive nature of the assay. The competitive assay method used in this study was linear in the range of 1.0-100 µM with a detection limit of 0.58 µM. The competitive chemiluminescence assay could detect biotin in vitamin B7 tablets with good recovery of 91.3 to 110% and respectable precision (RSD < 8.7%).

A Turn-On Quinazolinone-Based Fluorescence Probe for Selective Detection of Carbon Monoxide.

Carbon monoxide (CO) is a toxic, hazardous gas that has a colorless and odorless nature. On the other hand, CO possesses some physiological roles as a signaling molecule that regulates neurotransmitters in addition to its hazardous effects. Because of the dual nature of CO, there is a need to develop a sensitive, selective, and rapid method for its detection. Herein, we designed and synthesized a turn-on fluorescence probe, 2-(2'-nitrophenyl)-4(3H)-quinazolinone (NPQ), for the detection of CO. NPQ provided a turn-on fluorescence response to CO and the fluorescence intensity at 500 nm was increased with increasing the concentration of CO. This fluorescence enhancement could be attributed to the conversion of the nitro group of NPQ to an amino group by the reducing ability of CO. The fluorescence assay for CO using NPQ as a reagent was confirmed to have a good linear relationship in the range of 1.0 to 50 µM with an excellent correlation coefficient (r) of 0.997 and good sensitivity down to a limit of detection at 0.73 µM (20 ppb) defined as mean blank+3SD. Finally, we successfully applied NPQ to the preparation of a test paper that can detect CO generated from charcoal combustion.

Determination of Anthraquinone-Tagged Amines Using High-Performance Liquid Chromatography with Online UV Irradiation and Luminol Chemiluminescence Detection.

Quinones are frequently used as derivatization reagents in HPLC analysis to improve detection sensitivity. In the present study, a simple, sensitive, and selective chemiluminescence (CL) derivatization strategy for biogenic amines, prior to their HPLC-CL analysis, was developed. The novel CL derivatization strategy was established based on using anthraquinone-2-carbonyl chloride as derivatizing agent for amines and then using the unique property of the quinones' moiety to generate reactive oxygen species (ROS) in response to UV irradiation. Typical amines such as tryptamine and phenethylamine were derivatized with anthraquinone-2-carbonyl chloride and then injected into an HPLC system equipped with an online photoreactor. The anthraquinone-tagged amines are separated and then UV-irradiated when they pass through a photoreactor to generate ROS from the quinone moiety of the derivative. Tryptamine and phenethylamine can be determined by measuring the chemiluminescence intensity produced by the reaction of the generated ROS with luminol. The chemiluminescence disappears when the photoreactor is turned off, suggesting that ROS are no longer generated from the quinone moiety in the absence of UV irradiation. This result indicates that the generation of ROS could be controlled by turning the photoreactor on and off. Under the optimized conditions, the limits of detection for tryptamine and phenethylamine were 124 and 84 nM, respectively. The developed method is successfully applied to determine the concentrations of tryptamine and phenethylamine in wine samples.

Anthracycline-Functionalized Dextran as a New Signal Multiplication Tagging Approach for Immunoassay.

The most used kind of immunoassay is enzyme-linked immunosorbent assay (ELISA); however, enzymes suffer from steric effects, low stability, and high cost. Our research group has been developing quinone-linked immunosorbent assay (QuLISA) as a new promising approach for stable and cost-efficient immunoassay. However, the developed QuLISA suffered from low water-solubility of synthesized quinone labels and their moderate sensitivity. Herein, we developed a new approach for signal multiplication of QuLISA utilizing the water-soluble quinone anthracycline, doxorubicin, coupled with dextran for signal multiplication. A new compound, Biotin-DexDox, was prepared in which doxorubicin was assembled on oxidized dextran 40, and then it was biotinylated. The redox-cycle-based chemiluminescence and the colorimetric reaction of Biotin-DexDox were optimized and evaluated, and they showed very good sensitivity down to 0.25 and 0.23 nM, respectively. Then, Biotin-DexDox was employed for the detection of biotinylated antibodies utilizing avidin as a binder and a colorimetric assay of the formed complex through its contained doxorubicin redox reaction with NaBH4 and imidazolium salt yielding strong absorbance at 510 nm. The method could detect the plate-fixed antibody down to 0.55 nM. Hence, the application of Biotin-DexDox in QuLISA was successfully demonstrated and showed a significant improvement in its sensitivity and applicability to aqueous assays.

Analysis of vaporized caffeine in smoke from e-cigarettes using liquid chromatography-tandem mass spectrometry and clarification of minor components.

PURPOSE: Electronic cigarettes (e-cigarettes) are used widely, and e-cigarettes containing caffeine (Caf) have recently become commercially available. However, no risk evaluation of these Caf-containing products has been performed to date. Such an evaluation requires a sensitive analytical method for quantifying Caf in smoke from e-cigarettes. The aim of this study was to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying vaporized Caf from commercially available e-cigarettes, and to determine minor components related to Caf in cigarette smoke extract (CSE). METHODS: A sampling system for Caf using a suction pump was designed and sampling conditions were optimized. RESULTS: The optimized LC-MS/MS conditions allowed the sensitive determination of Caf in smoke with a limit of detection of 0.03 ng/mL at a signal-to-noise ratio of 3. The method was applied to CSEs from five e-cigarette products and the concentration of Caf ranged from 0.894 ± 0.090 to 3.32 ± 0.14 µg/mL smoke (n = 3). Additionally, minor components related to Caf, such as theobromine, theophylline, and paraxanthine, were detected in CSE and in e-liquid at very low concentrations, indicating that they were impurities in e-liquid and vaporized along with Caf. CONCLUSION: This is the first report to determine the concentration of vaporized Caf using an LC-MS/MS method and to clarify several minor components in smoke from e-cigarettes.

Development of signal multiplication system for quinone linked immunosorbent assay (Multi-QuLISA) by using poly-l-lysine dendrigraft and 1,2-naphthoquinone-4-sulfonate as enzyme-free tag.

A sensitive and stable signal multiplied quinone-linked immunosorbent assay (Multi-QuLISA) was developed. In Multi-QuLISA, an oligomerized quinone linked to biotin, namely biotin-8mer-naphthoquinone (Bio8mer-NQ), is used as a signal-generating label. Bio8mer-NQ is formed from a dendrigraft poly-l-lysine generation 1 (DPLL G1), a controlled branched oligomer composed of eight lysine moieties with nine free amino groups as a backbone. One of the nine amino groups of DPLL G1 is attached to biotin moiety, while the other eight are attached to 1,2-naphthoquinone-4-sulfonate (NQS). Bio8mer-NQ labels a biotinylated detection antibody using avidin as a co-binder. Then, multi-quinones in Bio8mer-NQ undergo a redox cycle with dithiothreitol and luminol, generating strong chemiluminescence. Standard ELISA uses a label enzyme that suffers from vulnerability in different conditions and poor stability. Bio8mer-NQ showed better stability than the enzyme (biotin-HRP) under different drastic pH and temperature conditions, hydrolytic enzymes, etc. Furthermore, Bio8mer-NQ was used as both chemiluminescence and colorimetric label based on the redox cycle of quinone, and it had LODs of 1.5 and 6.5 nM, respectively. The method could detect biotinylated immunocomplex in an in-house designed immunoassay down to 0.2 nM, which is about 25 times more sensitive than biotin HRP. Eventually, Bio8mer-NQ was applied successfully in Multi-QuLISA for detecting ß-casein with a sensitivity of 3.2 ng/mL, while the conventional ELISA had an LOD of 35 ng/mL. Overall, Bio8mer-NQ is a stable compound that could be used as an excellent replacement for the enzyme in immunoassay and can be used in both colorimetric and chemiluminescence assays with good sensitivity.

Selective fluorescence labeling of myristicin using Mizoroki-Heck coupling reaction. Application to nutmeg powder, oil, and human plasma samples.

Myristicin [5-allyl-1­methoxy-2,3-(methylenedioxy)benzene] is the major constituent of the seasoning nutmeg oil and powder. Sometimes myristicin is abused via its ingestion at high doses to cause hallucination. In these high doses, myristicin could cause severe adverse health effects, including convulsion, delirium, and palpitation. Hence there is a strong need for a sensitive method for its analysis, such as fluorescence determination. Myristicin has a very weak fluorescence and also lacks derivatizable groups like the carboxylic, hydroxyl, or amino group in its structure, which makes its fluorescence derivatization challenging. In this research, we developed a fluorescence labeling method for myristicin based on the Mizoroki-Heck coupling reaction of its terminal alkene with a fluorescent aryl iodide derivative, 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIB-I). Then, we developed an HPLC fluorescence detection method for the determination of myristicin utilizing this labeling reaction. The developed method showed a good linear response for myristicin (r = 0.995) in the range of 0.01-10 µmol/L with excellent sensitivity down to the detection limit of 2.9 nmol/L (9.6 fmol/injection). Finally, the developed method could be successfully applied to determine myristicin content in nutmeg powder, oil samples, and human plasma with simple extraction methods and good recoveries ranging from 89.3 to 106%.

Development of a Selective Assay of Tyrosine and Its Producing and Metabolizing Enzymes Utilizing Pulse-UV Irradiation-Induced Chemiluminescence.

A new pulse UV irradiation-induced chemiluminescence (CL) determination method was developed for l-tyrosine using the luminol derivative L-012. The proposed method depends on the formation of reactive oxygen species (ROS) upon pulse UV irradiation of l-tyrosine; then, these ROS react with L-012 producing strong CL. The proposed method showed excellent sensitivity and ultraselectivity toward l-tyrosine. The mechanism of the developed CL method was studied using ROS scavengers, HPLC, and mass spectrometry. The method was linear for l-tyrosine in the range of 0.03-50 µM. Minor changes in the l-tyrosine structure, including hydroxylation, dehydroxylation, phosphorylation, or decarboxylation, were found to lead to a strong decrease in CL. Using the excellent selectivity of the proposed method for l-tyrosine, we have developed a CL assay for measuring alkaline phosphatase activity in the range of 0.02-15 U/L with the limit of detection (LOD) of 4 mU/L using the nonchemiluminescent O-phospho-l-tyrosine as a substrate. Furthermore, the CL reaction was applied for tyrosinase activity assay as this enzyme can convert l-tyrosine to the nonchemiluminescent l-dopa. The decrease in CL is correlated with the tyrosinase activity in the range of 0.025-0.75 U/mL with an LOD of 1.5 mU/mL. Moreover, the tyrosinase activity assay was successfully applied for the determination of IC50 of the tyrosinase inhibitors kojic acid and benzoic acid. Therefore, our novel pulse UV irradiation CL method for the determination of l-tyrosine was not only suitable for the determination of this vital amino acid but also extended to the successful determination of its producing and metabolizing enzymes and their inhibitors.

Development of a selective fluorescence derivatization strategy for thyroid hormones based on the Sonogashira coupling reaction.

A new fluorescence derivatization technique for the determination of the thyroid hormones, 3,3',5-triiodo-L-thyronine (T3, triiodothyronine) and 3,3',5,5'-tetraiodo-L-thyronine (T4, L-thyroxine), in human serum was developed based on the Sonogashira coupling reaction. This derivatization reaction was recently utilized by our research group as a promising solution for the derivatization of ortho-substituted aryl halides that suffer from steric hindrance. T3 and T4 possess amino groups that could be derivatized by many reagents; however, these reagents are not useful in the case of biological analysis as they could non-selectively react with many biogenic amines and amino acids. Thus, herein we aimed at labeling the iodo-phenyl group of T3 and T4 as a selective fluorescence labeling approach suitable for biological analysis. The fluorescent alkyne, 2-(4-ethynylphenyl)-4,5-diphenyl-1H-imidazole (DIB-ET), can label the ortho-substituted aryl halides T3 and T4 in the presence of palladium and copper as catalysts, overcoming the steric hindrance of ortho-substitution. Furthermore, the application of the proposed method for the selective analysis of T3 and T4 in biological samples was successfully performed even in the presence of numerous biological components. The formed fluorescent derivatives produced from the reaction of DIB-ET and T3 and T4 could be determined by an HPLC system with fluorescence detection. The proposed method was successfully applied for the selective and sensitive determination of T3 and T4 in human serum with detection limits (S/N = 3) of 4.0 and 6.1 ng/mL and the recovery rate in the ranges of 84.3-92.1% and 81.3-84.9%, respectively. Therefore, the proposed method could be used as a new simple tool for the simultaneous determination of T3 and T4 in biological samples.

A turn-on hydrazide oxidative decomposition-based fluorescence probe for highly selective detection of Cu2+ in tap water as well as cell imaging.

Copper (II) is one of the most important metal ions for the human body that act as a catalytic cofactor for many metalloenzymes and proteins, and its homeostasis disruption could lead to many neurological diseases. The reported probes for Cu (II) determination are mostly based on fluorescence quenching mechanism, which provides low precision and reliability. In the present work, a turn-on fluorescence probe, (Z)-1-[2-oxo-2-[2-[1-oxoaceanthrylen-2 (1H)-ylidene]hydrazinyl]ethyl]-pyridinium (OAHP), for highly selective detection of Cu2+ was developed. Hydrazide moiety of OAHP quenches probe fluorescence; however, upon its reaction with Cu, oxidative cleavage of the hydrazide moiety and intramolecular cyclization occurs, forming oxadiazole derivative with strong fluorescent properties. In this context, OAHP displayed significant fluorescence enhancement with increasing levels of Cu2+. OAHP could detect Cu2+ selectively with a detection limit of 18 nM (1.1 ppb). This is the first report for a probe that uses the ability of Cu2+ to induce oxidative decomposition of hydrazide with intramolecular cyclization, and it showed exceptional selective performance and exquisite sensitivity. Next, the method was applied successfully for monitoring Cu2+ in tap water samples with good accuracy (found% of 95.8-101.5%) and precisions (RSD<10%). Finally, OAHP was successfully applied for imaging Cu2+ in living cells, and this result indicates the potential of OAHP for selective detection of Cu2+ in complicated matrices.

Determination Method for Pyrroloquinoline Quinone in Food Products by HPLC-UV Detection Using a Redox-Based Colorimetric Reaction.

We have developed an HPLC-UV method for the determination of pyrroloquinoline quinone (PQQ), which utilizes a redox-based colorimetric reaction. In the proposed colorimetric reaction, the redox reaction between PQQ and dithiothreitol generates superoxide anion radicals that can convert 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to formazan dye. After PQQ separation on an octadecyl silica column, it was mixed online with dithiothreitol and INT, and the formed formazan dye was monitored by absorbance at 490 nm. The detection limit (S/N = 3) of the proposed method was 7.6 nM (152 fmol/injection). The proposed method could selectively detect PQQ in food products without any clean-up procedures.

HPLC Fluorescence Method for Eugenols in Basil Products Derivatized with DIBI.

Eugenols (Eugs) such as eugenol (Eug), methyleugenol (MeEug), and linalool (Lin) in basil product are the main bioactive components of basil products and have a terminal double-bond. A sensitive HPLC-fluorescence method for Eugs derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIBI) was developed. Good separation of DIB-Eugs was achieved within 20 min on an Atlantis T3 column (50 × 2.1 mm i.d., 3 µm) with a mobile phase of methanol-water. The calibration curves obtained with Eug standards showed good linearities in the range of 0.1-50 µM (r ≥ 0.999). The limits of detection at a signal-to-noise ratio (S/N) = 3 for Eug, MeEug, and Lin were 1.0, 6.0, and 4.8 nM, respectively. The limits of quantitation (S/N = 10) of the Eugs were lower than 19.9 nM. The accuracies for the Eugs were within 96.8-104.6%. The intra- and inter-day precisions as relative standard deviations for the Eugs were less than 1.2 and 9.6% (n = 3). The recoveries of Eug, MeEug, and Lin were 99.0 ± 0.1, 98.0 ± 0.2, and 96.0 ± 0.4% (n = 3), respectively. The DIB-Eugs were confirmed to be stable for 2 h (>90%) at room temperature and 24 h (>95%) at 4 °C. These parameters of the proposed method were useful for the simultaneous determination of Eugs in basil products. Therefore, the developed method may be a powerful tool for the quality evaluation of dried commercially available basil products.

A Comparative Study on the Reduction Modes for Quinone to Determine Ubiquinone by HPLC with Luminol Chemiluminescence Detection Based on the Redox Reaction.

Ubiquinone (UQ) is considered one of the important biologically active molecules in the human body. Ubiquinone determination in human plasma is important for the investigation of its bioavailability, and also its plasma level is considered an indicator of many illnesses. We have previously developed sensitive and selective chemiluminescence (CL) method for the determination of UQ in human plasma based on its redox cycle with dithiothreitol (DTT) and luminol. However, this method requires an additional pump to deliver DTT as a post-column reagent and has the problems of high DTT consumption and broadening of the UQ peak due to online mixing with DTT. Herein, an HPLC (high-performance liquid chromatography) system equipped with two types of online reduction systems (electrolytic flow cell or platinum catalyst-packed reduction column) that play the role of DTT was constructed to reduce reagent consumption and simplify the system. The newly proposed two methods were carefully optimized and validated, and the analytical performance for UQ determination was compared with that of the conventional DTT method. Among the tested systems, the electrolytic reduction system showed ten times higher sensitivity than the DTT method, with a limit of detection of 3.1 nM. In addition, it showed a better chromatographic performance and the best peak shape with a number of theoretical plates exceeding 6500. Consequently, it was applied to the determination of UQ in healthy human plasma, and it showed good recovery (≥97.9%) and reliable precision (≤6.8%) without any interference from plasma components.

A sensitive chemiluminescence detection approach for determination of 2,4-dinitrophenylhydrazine derivatized aldehydes using online UV irradiation - luminol CL reaction. Application to the HPLC analysis of aldehydes in oil samples.

Aldehydes are toxic carbonyl compounds that are identified in various matrices surrounding us. For instance, aldehydes could be formed during the cooking and frying of foods which affects the food quality and safety. Derivatization is a must for the determination of aldehydes as they lack intrinsic chromophoric groups. 2,4-Dinitrophenyl hydrazine (DNPH) is the most used derivatizing reagent for aldehydes and the formed hydrazones could be determined by either HPLC-UV or LC-MS. However, UV detection is non-sensitive, and the MS equipment is expensive and not widely available. Thus, herein we report a smart chemiluminescence (CL) detection method for the DNPH aldehydes derivatives. These derivatives are supposed to possess photosensitization ability due to the presence of strong chromophoric structures; nitrobenzene and phenyl hydrazone. Upon their UV irradiation, singlet oxygen is found to be produced which then converts the DNPH-aldehyde derivative into hydroperoxide. Next, the hydroperoxide reacts with luminol in an alkaline medium producing a strong CL. An HPLC system with online UV irradiation and online reaction with luminol followed by CL detection was constructed and used for the determination of aldehydes after their derivatization with DNPH. The developed method showed excellent sensitivity with detection limits down to 1.5-18.5 nM. The achieved sensitivity is superior to that obtained by HPLC-UV and LC-MS detection methods for DNPH-aldehydes derivatives. Additionally, our approach is an chemiluminogenic where the DNPH reagent itself does not produce CL which is an excellent advantage. The method was applied successfully for the determination of aldehydes in canola oil samples using simple liquid-liquid extraction showing good recovery (87.0-106.0%), accuracy (87.2-106.6), and precision (RSD≤10.2%). After analysis of fresh and heated oil samples, it was demonstrated that heating of oil, even for short time, strongly elevated the level of their aldehydes' content. At last, it was found that the results of the analysis of aldehydes in oil samples using the proposed method perfectly matched those obtained by a reference LC-MS method.

Simple Fluorescence Assay for Triethylamine Based on the Palladium Catalytic Dimerization of Benzofuran-2-boronic Acid.

Although benzofuran-2-boronic acid hardly emits fluorescence, it can be rapidly converted to a highly fluorescent benzofuran dimer after mixing with a palladium catalyst and amine. We found that a fluorescence enhancement accompanying dimerization was quantitatively promoted upon increasing the concentration of amine. In the present study, we developed a simple fluorescence assay for amines based on the promotive effect. As the result of a fluorescence measurement of the reaction mixture of 19 kinds of typical amines, it was found that tertiary amines including triethylamine (TEA) provided a significant fluorescence enhancement. Finally, the fluorogenic reaction could be applied to develop a high-throughput fluorescent microplate assay for TEA with the limit of detection (blank + 3SD) of 0.091 µM.

A selective and highly sensitive high performance liquid chromatography with fluorescence derivatization approach based on Sonogashira coupling reaction for determination of ethinyl estradiol in river water samples.

A selective and highly sensitive high performance liquid chromatography (HPLC) with fluorescence derivatization method was developed for determination of ethinyl estradiol (EE); one of endocrine-disrupting compounds (EDCs). The fluorescence derivatization procedure was based on Sonogashira coupling reaction using 4-(4, 5-diphenyl-1H-imidazole-2-yl) iodobenzene (DIB-I), a fluorescence labeling reagent, to derivatize EE in presence of copper and palladium ions. The formed fluorescent product was separated on Cosmosil 5C18 MS-II by an isocratic elution with a mobile phase composed of acetonitrile: 5.0 mM Tris-HNO3 buffer, pH 7.4 (60:40, v/v %). The detection wavelengths were set at 310 and 400 nm as excitation and emission wavelengths, respectively. Various parameters affecting derivatization reaction were optimized. Further, the proposed method was validated and a good linearity with low detection limit (S/N=3) 7.4 ng L-1 was obtained in water sample after a simple solid-phase disk extraction (C18 SPE disk) method. The proposed method was successfully applied for detection of EE in river water samples in order to monitor EE concentration and to distinguish its effect on the ecosystem and human health.

A Smart Advanced Chemiluminescence-Sensing Platform for Determination and Imaging of the Tissue Distribution of Natural Antioxidants.

Antioxidants have gained marked attention owing to their ability to prevent the oxidation of biological components and to protect the body from reactive oxygen species, thereby maintaining human health. Thus, antioxidant-rich dietary supplements and natural foods can be effective against oxidative stress and can even act as chemopreventive agents. Therefore, a simple and rapid assay for evaluation of antioxidant capacity and assessment of their distribution profile in natural sources is vital. Herein, we report a rapid, innovative chemiluminescence (CL) platform for evaluation and visualization of antioxidant capacity. We found that intense and long-lasting CL was formed upon the redox reaction of quinones, e.g., menadione, with antioxidants, e.g., l-ascorbic acid, in the presence of luminol. The produced CL intensities were proportional to the antioxidants' concentrations with a detection limit of 0.18 µM for the model antioxidant, l-ascorbic acid. As the formed CL was long-lasting, it could be easily captured and detected with a charge-coupled device (CCD) camera. To evaluate the quantification ability of the CCD camera, we developed a smart and fast microplate-based assay based on photographing the generated CL with a cooled CCD camera. The photographed CL intensities were linearly proportional with the antioxidant concentrations, and then the method was applied for photographing multiple food sample extracts. Ultimately, we utilized our method for the distribution profiling of antioxidant capacity in food cut sections. Samples were dipped in luminol and then in quinone, followed by CCD camera photography, without the need for any pulverization/extraction procedure, giving precise antioxidant distribution information.