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A novel strategy for colorimetric detection of hydroxyl radicals based on a modified Griess test
Author’s Accepted Manuscript A Novel Strategy for Colorimetric Detection of Hydroxyl Radicals Based on a Modified Griess Test Tao Deng, Shiyou Hu, Xin-an Huang, Jianping Song, Qin Xu, Yi Wang, Fang Liu www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)31194-9 https://doi.org/10.1016/j.talanta.2018.11.044 TAL19277
To appear in: Talanta Received date: 22 September 2018 Revised date: 4 November 2018 Accepted date: 13 November 2018 Cite this article as: Tao Deng, Shiyou Hu, Xin-an Huang, Jianping Song, Qin Xu, Yi Wang and Fang Liu, A Novel Strategy for Colorimetric Detection of Hydroxyl Radicals Based on a Modified Griess Test, Talanta, https://doi.org/10.1016/j.talanta.2018.11.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel Strategy for Colorimetric Detection of Hydroxyl Radicals Based on a Modified Griess Test Tao Denga, Shiyou Hua, Xin-an Huanga, Jianping Songa, Qin Xua, Yi Wangb, Fang Liua*
a
Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 501405,
China b
Center Laboratory in the First Affiliated Hospital, Guangdong Pharmaceutical University,
Guangzhou510080, China
Corresponding author:
Fang Liu, Email: [email protected]
Abstract: Hydroxyl radicals (•OH) is the most reactive oxygen species involved in many environmental and biological processes. The development of simple and reliable methods to quantitatively determine hydroxyl radicals is desired. Herein, a colorimetric strategy based on a modified Griess test has been presented. The detection started with the nitrite release from nitroimidazoles (nIm) upon its specific reaction with •OH radicals and subsequently nitrite quantification by Griess reagent. The result showed that this nitroimidazole modified Griess test (nIm-Griess) was successfully adapted for the measurement of •OH radicals generated by Fenton reaction, water radiolysis, as well as the activation of two antimalarials artemisinin (Art) and dihydroartemisinin (DHA). Graphical abstract:
1
Key words: Hydroxyl radicals, Colorimetric detection, Griess test, Nitrite release 1. Introduction: Reactive oxygen species (ROS), consist of radical and non-radical oxygen species formed by the partial reduction of oxygen, which play important roles in environmental and biological processes. Among all ROS, hydroxyl radical •OH has the greatest chemical reactivity, thus being considered as the most harmful species. It causes oxidative damage to lipids, proteins, nucleic acids and other biomolecules in cells. Excessive exposure to •OH radicals can cause cell death, which is often involved in the development of various diseases [1]. However, •OH radical is not always a harmful oxygen species, which is also an important oxidant for many advanced oxidation processes. For example •OH radical could be used for degradation of organic pollutants in electro/photocatalysis systems. Meanwhile, •OH radical is one of the major therapeutic ROS in ionizing radiation treatment of cancer [2, 3]. Detection of hydroxyl radicals was desired because of its importance. However •OH radical detection is highly challenged due to its extreme short life-time and uncontrollable reactivity [4]. Currently, several methods have been developed to detect •OH radicals, including electrochemical methods [5, 6], electron spin resonance (EPR) [7, 8], chemiluminescence [9-11], UV-Vis spectroscopy and fluorescent spectroscopy [12-17], and more recently the nanoparticle based fluorescent probes 2
[18-24]. Although, fluorescence strategies hold the advantages for intracellular measurements [25, 26], most of them have various limitations with respect to low cost effective and complexity. Moreover, •OH radical is highly reactive not only to the designed responsive moieties but also to the backbone of dye molecules (eg. fluorescein or rhodamine), thus leading to partial degradation of probes and the subsequent undesirable fluorescence quenching [27, 28]. Visual color detection is one of the popular methods for various analytes including reactive oxygen species. It is found that methylene blue could be oxidative degraded by •OH radicals from Fenton reaction to form colorless products, which make possibilities for visual color detection [15]. Similarly, the metal-dye complex fast blue BB salt (FBBs)has been used for visual turn-off detection of •OH radicals during Fenton reaction [14]. However, due to its complex reaction and intrinsic drawbacks of turn-off strategies, the detection sensitivity in both methods above is limited, thus making them only applicable for detection of •OH radicals over tens-of-micromolar concentrations. Therefore, more sensitive and reliable methods for visual color detection of •OH radicals are still desired. Herein, a simple and sensitive method has been presented on the basis of nitroimidazoles and Griess reagents. As illustrated in Scheme 1, reacting with hydroxyl radicals can release nitrite ions from nitroimidazoles, which could be subsequently detected by Griess method. The initial colorless Griess solution turns red upon the reaction with nitrite ions in a concentration dependent manner, thus reflecting the concentrations of •OH radicals. In our study, the proposed nIm-Griess strategy has been successfully used to determine •OH radicals generated from Fenton reaction and X-ray radiation. More interestingly, the nIm-Griess method has also been successfully applied for monitoring •OH radicals produced from the catalyzed activation of two classic antimalarial artemisinins.
3
2 Experimental 2.1 Materials Ferrous chloride tetrahydrate was purchased from Alfa Aesar (China Limited, Shanghai). Griess reagent R2 N-1-napthylethylenediamineaqueous solution (0.1%) was purchased from Cayman (Michigan, USA). Artemisinins, p-amino benzene sulfonamide, hydrogen peroxide (30%) and other chemicals were purchased from Adamas (Shanghai, China). 96-well plate was purchased from Thermo Fisher.
All reagents were used without further purification. The distilled deionized water from a
Milli-Q Plus system is used throughout the experiments. 96-well plate reading was done on a Multi-detector microplate reader - VICTOR™ X3 (Thermo Fisher).
2.2 Griess reaction and standard curve establishment. As indicated from Fig. S1, the Griess reagent consists of two parts p-amino benzene sulfonamide aqueous solution (1%, R1) and N-1-napthylethylenediamine aqueous solution R2 (0.1%). R2 was purchased from Cayman and used directly. R1 was prepared by dissolving 500 mg p-amino benzene sulfonamide into 50 mL phosphoric acid solution (5%) according to the standard procedure. R1 and R2 were mixed freshly before testing. Briefly, for a test, 100 μL mixture of Griess reagent R1 and R2 was added into a 96-well plate followed by the addition of 50 μL nitrite containing samples. Under acidic conditions, the nitrite ions will react with R1to forma reactive diazonium salt, which can further react with R2 to form a red azo product with maximum absorption at 540 nm. The plate was kept under dark at room temperature for 10 minutes before data recording by a plate reader. Unless additional instructions, all Griess tests have been done at room temperature (25゜C). To establish the standard curve for nitrite, a series concentration of sodium nitrite solutions was freshly prepared, and then 50 μL 4
of each solution was added into the Griess reagent for colorimetric observation. Three repeats were set for each sample. The absorptions at 540 nm for each well after subtracting the values from reagent control were plotted versus the concentrations of nitrite. A linear relationship was found between the absorption and nitrite concentration (Fig.S2).
2.3 Detection of hydroxyl radical from Fenton reaction Fenton reaction was the most widely used model reaction to generate •OH radicals. The generation of •OH radicals from hydrogen peroxide in the presence of ferrous ions was estimated to be quantitative. The first step for detection is to generate nitrite ions from nitroimidazoles upon the reaction with •OH radicals. To do the test, nitroimidazole solution (500 μM, 2-nIm or 5-nIm) and FeCl2 solution (400 μM) were firstly mixed. To the mixture, different concentrations of hydrogen peroxide (H2O2) were added followed by the addition of pure water to make the final concentrations of nitroimidazoles and FeCl2 are 250 μM and 200 μM respectively. The reaction was kept at room temperature for 5 minutes. Then to 100 μL prepared Griess reagent mixture in a 96-well plate, 50 μL reaction solution was added. After 10 minutes, the data was recorded by a plate reader. Three repeats were done for each treatment. It’s well known that DMSO was the most widely used reagent to neutralize •OH radicals. Herein, DMSO was added into the reaction mixture in our control assay at the final concentration at 4% to examine the reaction specificity. The detection limit (Cmin) is estimated by a reported method Cmin=3*σ/B, where σ is the standard deviation obtained from three individual absorbance measurements (abs at 540 nm) without any analyte (H2O2). B is the slope obtained after linearly fitting the titration curves within the analyte concentrations. 5
2.4 Detection of hydroxyl radicals from water radiolysis To monitor•OH radicals generation during water radiolysis by X-ray. 5-nIm was dissolved in PBS buffer (pH 7.4) to prepare series of solution with different concentrations (0, 2, 5, 20, 50, 200, 500 μM). Meanwhile, the concentration of 5-nIm was fixed at 50 μM to observe X-ray dosage dependent nitrite release. The solution was prepared in 1.5 mL transparent eppendorf tube and sealed before irradiation. The dosage of X-ray (6 MeV) was set as 0,5, 25, 50 Gy, where Gy is a unit defined as the absorption of one joule of radiation energy per kilogram of matter. Generally, the clinical relevant dosage for a single therapy usually ranged from 0 to 20 Gy. An X-ray source with 6 MeV photon beams was used in this study. 2.5 Detection of •OH radicals production from the activation of artemisinins Artemisinin (Art) and Dihydroartemisinin (DHA) solutions were prepared in Milli-Q water. Hemin was prepared in 0.5M NaOH solution, since it has poor solubility in neutral pH conditions. To perform the detection, Art or DHA was firstly mixed with 5-nIm (250 μM), followed by the addition of hemin solution to reach the final concentration of hemin at 100 μM. The reaction solution was prepared in 1.5 mL eppendorf tubes, which were then incubated at 37゜C for 20 minutes before Griess test. The nitrite ions generated in the solution was quantified by the same method as described above. Hemin at 100μM can cause a little background absorption at 540 nm, which has been subtracted from all the tested groups before data processing. 3. Results and Discussion: It’s found in our previous study that nitroimidazole solutions are able to release nitrite ions upon water radiolysis probably by reaction with the hydrated electrons and the subsequent product •OH 6
radicals [29]. The phenomenon inspired us to consider new strategies for •OH radical detection based on nitrite release. Among all •OH radical generation system, Fenton reaction is the most widely used model reaction, which has been extensively used to develop •OH radical probes. In most cases, the generation of •OH radical from H2O2 by ferrous ions is often considered as chemical quantitatively (see Equation S1) [30]. To prove our design, the classic Fenton system was firstly used as the source of •OH radicals. In a typical detection, nitroimidazoles were mixed with ferrous ions firstly followed by the addition of H2O2. The in situ generated •OH radical reacted with nitroimidazoles, which led to the release of detectable nitrite ions by Griess test. As indicated in Fig.1A, both 2-nIm and 5-nIm can react with Fenton generated •OH radicals, thus resulting in the release of nitrite. When the concentrations of hydroxyl radicals below 10 µM, both 2-nIm and 5-nIm (both at 250 µM) show similar responsibility in nitrite release. However, more nitrites were released from 5-nIm solution than 2-nIm when the •OH radical was increased from 10 µM to 100 µM, which indicated that 5-nIm is probably more reactive towards •OH radical. The release curves become flat for both 2-nIm and 5-nIm when •OH radicals concentration is over 50 µM, probably because non-specific reactions happened due to the relative higher local concentration of •OH radicals during solution mixing. Plotting the nitrite release from 5-nIm versus •OH radical concentrations, a good linear relationship was observed with a high R-squared value (R2 =0.998)(Fig.1B). According to the linear fitting equation, the detection limit (Cmin) is estimated to be 0.398 μM, which is comparable to some recently reported fluorescence probes [16, 18]. Table 1 summarized the recent developments for •OH radicals determination. Although the nanoparticle based probes exhibited the greater sensitivity than small molecular probes, they were usually suffering from complex manipulation. Moreover, significant visual color changes were observed during detection (Fig.1C), thus making the naked-eye detection available that is not 7
achievable by most of the previous methods. Therefore, nIm-Griess offers an easier and technically achievable method for the detection of •OH radicals from Fenton reactions. Since •OH radical mediated nitrite release from nitroimidazole is the most critical process in the detection, we then looked into the reaction mechanisms. As previously proposed, nitrite release from 5-nIm in water radiolysis system might be initialized with the addition reaction between the nitro group substituted carbon atom (C5) and •OH radical. The addition intermediate bearing nitro group is unstable followed by the rapid elimination of nitrite ions (Fig.2A) [31]. Fortunately, a direct evidence to support proposed mechanism was obtained by analyzing the product using ESI-MS. As illustrated in Fig.S3, a typical mass signal (98.10) corresponding to the denitrated product was observed. The reaction specificity was further confirmed by introducing a •OH radical quencher DMSO to the reaction mixture. As shown in Fig. 2B, normally 25 µM •OH radical can cause the release of around 15.5 µM nitrite, whereas only background level release was observed in the presence of 4% DMSO. The result indicated that •OH radical quenching can indeed suppress nitrite release from 5-nIm, thus implying that nitrite release is specifically responding to •OH radicals. With the favorable performance in hydroxyl radical detection, we then examined the selectivity of nIm-Griess towards •OH radical over other ROS. As indicated from Fig.2C, only •OH radical is able to cause obvious nitrite release, all other tested ROS including hydrogen peroxide (H2O2), singlet oxygen (1O2 ), tert-Butyl hydroperoxide (TBHP), Superoxide (O2- ) and the neutrophil oxidant hypochlorous acid (HOCl) can just result in background level signals. Nitroimidazoles and their derivatives have been applied as chemical radiosensitizers in radiotherapy for decades, the mechanism may relate to the denitration and imidazole radical formation. •OH radicals was thought as the major radicals during water radiolysis, which could be generated 8
directly from water radiolysis or indirectly from the subsequent reaction between hydrated electrons and H2O (Fig.3A). Under 25 Gy irradiation, nitrite release from 5-nIm solution increased along with the raising of 5-nIm concentration. However, the release reached a saturated condition when concentration was further increased over 200 µM, mostly because 200 µM is sufficient to consume all the •OH radicals released upon 25 Gy irradiation (Fig.3B). After that, the dosage dependent nitrite release was studied while fixing the concentration of 5-nIm at 50 µM. As indicated in Fig. 3C, an increased nitrite release was observed with the X-ray dosage ranging from 0 to 50 Gy. Plotting the dosage versus nitrite concentration gives us a linear like relationship (R2=0.988). Thus actually, nIm-Griess based assay could be used to monitor the energy delivery by X-ray through the measurement of the radiolysis product •OH radicals. The pH effects on nitrite release upon irradiation were then evaluated by irradiating the 5-nIm (50 µM) PBS solution of various pH values. As illustrated from Fig. S4, •OH radical induced nitrite release did not prefer acidic conditions, mostly because the intermediate radical anions is not stable in acidic environments [32]. With the excellent performance on •OH radicals detection in Fenton reaction and X-ray irradiation system, we made further exploration to answer the questions involved in action mechanisms of classic antimalarials artemisinin (Art) and its derivatives. Although, Arts are most commonly used drugs against malaria, their action mechanism is not fully clear yet [33, 34]. There is a common sense that Arts can generate ROS and alkyl radicals during the activation by ferrous containing hemoglobin or free ferrous ions, which might be one of the mechanisms of their bioactivity [35]. However, what kinds of ROS were released during Arts activation is still unknown. To check if the highly toxic •OH radicals was involved in action of Arts, our nIm-Griess method has been applied (Fig.4A). Art and dihydroartemisinin (DHA) were mixed with 5-nIm firstly, then the metal catalyst hemin was added. 9
The mixture was incubated at 37 °C for 20 minutes before the measurement by Griess reagent. As indicated in Fig. 4B, both Art and DHA can cause the release of nitrite with catalyzing reaction, thus implying the generation of •OH radicals during artemisinins’ activation. In contrast, there is no nitrite in 5-nIm solutions when only with Arts or hemin. The result indicated that both peroxide bridge and hemin are required for hydroxyl radical generation. The process may start with the catalyzed decomposition of the peroxide bridge, which subsequently generates a peroxide intermediate. •OH radical was presumably released from the peroxide intermediate through a Fenton-like reaction. Incubation with 500 μg mL-1 DHA can cause 24.1 µM nitrite release from 5-nIm, whereas only 10.6 µM nitrite was released from Art incubation, thus indicating different efficiency in •OH radical formation between Art and DHA. The differences on •OH radical release may attribute to the different reactivity of their peroxide bridges. Furthermore, concentration dependent •OH radical release from DHA was observed, where the amount of •OH radicals increases as the concentration of DHA increasing (Fig. 4C). 4. Conclusion A novel strategy has been developed for colorimetric detection of hydroxyl radicals on the basis of a modified Griess test. Before a standard Griess test, nitroimidazoles were used as the moieties to release nitrite upon the reaction with •OH radicals. The final azo product from Griess reaction exhibits red color with maximum absorption at 540 nm, making the •OH radical detection available not only by UV-Vis spectrometer but also by naked eyes. It should be noticed that Griess test is initially developed for nitrite, thus the intrinsic nitrite in tested samples will cause interferences. This method can sensitively and selectively detect •OH radical from Fenton reaction with a detection limit down to nanomolar level. The successful detection of •OH radical from water radiolysis has also been achieved, 10
thus making the possibilities to monitor the energy delivery during ionizing irradiation. More interestingly, we successfully detected the •OH radical release from two classic antimalarials Art and DHA catalyzed by hemin. This is the first evidence that hydroxyl radicals may be one of the major reactive oxygen species during artemisinins’ activation. Furthermore, different efficiencies in •OH radical generation between Art and DHA have been observed, which properly indicates different reactivities between these two drugs. In a word, the presented method is a simple and easier strategy for •OH radical detection. Acknowledgements We gratefully acknowledge the National Natural Science Foundation of China (21807018), Dongguan Talents Program for Innovation and Entrepreneurship（No. 2017-16）and the start-up fund from Guangzhou University of Chinese Medicine. We thank Prof. Hristov, Dimitre from the Department of Radiation Oncology-Radiation Physics of Stanford University for his nice help on X-ray radiation study. We thank Ms. Xin Liu, the CEO of Dongguan Bioshine Biotechnology Ltd. Co. for her nice assistant in measurement. We also acknowledge the Lingnan Medical Research Center of Guangzhou University of Chinese Medicine for the support on facilities.
Competing interests There are no conflicts to declare. Appendix A. Supplementary data Supplementary data related to this article can be found at xxxx
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Scheme 1. Schematic illustration of •OH radical detection based on nIm-Griess method
Table 1: Comparison of the analytical performances with different methods Materials Rhodamine based small molecular Fluorescent small molecular Fluorecein based small molecule BODIPY based small molecule
Selectivity over
Naked-eye
Detection Limits/Linear
other ROS
detectable
range
•OH
yes
no data/few to tens μM
[26]
38 nM/few to tens μM
[17]
390 nM/few to tens μM
[16]
no data/no data
[13]
•OH
•OH
•OH
no description no description
no
14
Ref.
BODIPY based fluorescent probe Lysozyme-silver nanoclusters Coumarin-activated silica NPs
•OH
no
11 nM/0.1-120 μM
[12]
ClO−, 1O2,
no
200 nM/0.8-200 μM
[18]
•OH
no
1.56 μM/0.5-220 μM
[23]
slightly response to
Fluorescent
both •OH
nanoprobe
and O2•−
no
0.2 nM/0-20 μM
[24]
•OH
no
2 nM/4 nM-16 μM
[20]
•OH
no
12 nM/0.018-6 μM
[21]
Methylene Blue
no data
Yes
no data/no data
[15]
Fast blue BB salt
no data
Yes
no data/25-150 μM
[14]
•OH
Yes
398 nM/0-25 μM
Upconversion nanoprobe Graphene quantum dots
Modified-Griess based
This work
Fig.1 A) Nitrite release from 5-nIm (250 μM) and 2-nIm (250 μM) upon the reaction with hydroxyl radicals generation from Fenton reaction; B) Linear relationship of nitrite release from 5-nIm (250μM) as a function of hydroxyl radical concentrations (0 to 25μM); C) Reaction in the eppendorf tubes show the color changes upon the reaction with ·OH (0, 5, 10, 25, 50 μM corresponding to tube 1 to tube 5 15
respectively).
Fig. 2 A) Proposed reaction mechanism of hydroxyl radical induced nitrite release from 5-nIm; B) nitrite release specifically responds to ·OH and the process can be suppressed by ·OH selective quencher DMSO; C) nIm-Griess can selectively detect hydroxyl radicals against other ROS. The
concentrations of analytes were set as ·OH (25 μM), H2O2, TBHP and HOCl (100 μM), 1O2 and O2were generated according to the reported methods [16].
Fig.3 A) Hydroxyl radical generation during water radiolysis; B) Nitrite release from different concentrations of 5-nIm (0, 2, 5, 20, 50, 200, 500 μM) under 25 Gy irradiation; C) A good linear 16
relationship was observed when plotting nitrite release versus X-ray doses.
Fig. 4 A) Schematically show hydroxyl radical release from artemisinin (Art) and dihydroartemisinin (DHA) through hemin catalyzed activation; B) the quantification of nitrite release from 5-nIm (250 μM) upon the reaction with hydroxyl radicals release from artemisinins (500 μg mL-1)/hemin (100 μM) system; C) DHA concentration dependent nitrite release from 5-nIm/DHA/hemin system. The concentrations for the components were 5-nIm (250 µM), hemin (100 µM), the reaction was incubated at 37 °C for 20 min before Griess test at rt.
17
Highlights:
A simple method nIm-Griess has been developed for visual colorimetric detection of hydroxyl radicals.
This method is based on the modified Griess test, where all the chemicals are commercial available, thus making it a low cost and easily achievable way for hydroxyl radical determination.
This method shows excellent responsibility and selectivity in detection of hydroxyl radicals produced from Fenton reaction and water radiolysis.
Hydroxyl radical releasing from antimalarial artemisinins was observed by nIm-Griess, thus offering direct evidences for the action mechanisms of artemisinins.
18
PII: DOI: Reference:
S0039-9140(18)31194-9 https://doi.org/10.1016/j.talanta.2018.11.044 TAL19277
To appear in: Talanta Received date: 22 September 2018 Revised date: 4 November 2018 Accepted date: 13 November 2018 Cite this article as: Tao Deng, Shiyou Hu, Xin-an Huang, Jianping Song, Qin Xu, Yi Wang and Fang Liu, A Novel Strategy for Colorimetric Detection of Hydroxyl Radicals Based on a Modified Griess Test, Talanta, https://doi.org/10.1016/j.talanta.2018.11.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel Strategy for Colorimetric Detection of Hydroxyl Radicals Based on a Modified Griess Test Tao Denga, Shiyou Hua, Xin-an Huanga, Jianping Songa, Qin Xua, Yi Wangb, Fang Liua*
a
Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 501405,
China b
Center Laboratory in the First Affiliated Hospital, Guangdong Pharmaceutical University,
Guangzhou510080, China
Corresponding author:
Fang Liu, Email: [email protected]
Abstract: Hydroxyl radicals (•OH) is the most reactive oxygen species involved in many environmental and biological processes. The development of simple and reliable methods to quantitatively determine hydroxyl radicals is desired. Herein, a colorimetric strategy based on a modified Griess test has been presented. The detection started with the nitrite release from nitroimidazoles (nIm) upon its specific reaction with •OH radicals and subsequently nitrite quantification by Griess reagent. The result showed that this nitroimidazole modified Griess test (nIm-Griess) was successfully adapted for the measurement of •OH radicals generated by Fenton reaction, water radiolysis, as well as the activation of two antimalarials artemisinin (Art) and dihydroartemisinin (DHA). Graphical abstract:
1
Key words: Hydroxyl radicals, Colorimetric detection, Griess test, Nitrite release 1. Introduction: Reactive oxygen species (ROS), consist of radical and non-radical oxygen species formed by the partial reduction of oxygen, which play important roles in environmental and biological processes. Among all ROS, hydroxyl radical •OH has the greatest chemical reactivity, thus being considered as the most harmful species. It causes oxidative damage to lipids, proteins, nucleic acids and other biomolecules in cells. Excessive exposure to •OH radicals can cause cell death, which is often involved in the development of various diseases [1]. However, •OH radical is not always a harmful oxygen species, which is also an important oxidant for many advanced oxidation processes. For example •OH radical could be used for degradation of organic pollutants in electro/photocatalysis systems. Meanwhile, •OH radical is one of the major therapeutic ROS in ionizing radiation treatment of cancer [2, 3]. Detection of hydroxyl radicals was desired because of its importance. However •OH radical detection is highly challenged due to its extreme short life-time and uncontrollable reactivity [4]. Currently, several methods have been developed to detect •OH radicals, including electrochemical methods [5, 6], electron spin resonance (EPR) [7, 8], chemiluminescence [9-11], UV-Vis spectroscopy and fluorescent spectroscopy [12-17], and more recently the nanoparticle based fluorescent probes 2
[18-24]. Although, fluorescence strategies hold the advantages for intracellular measurements [25, 26], most of them have various limitations with respect to low cost effective and complexity. Moreover, •OH radical is highly reactive not only to the designed responsive moieties but also to the backbone of dye molecules (eg. fluorescein or rhodamine), thus leading to partial degradation of probes and the subsequent undesirable fluorescence quenching [27, 28]. Visual color detection is one of the popular methods for various analytes including reactive oxygen species. It is found that methylene blue could be oxidative degraded by •OH radicals from Fenton reaction to form colorless products, which make possibilities for visual color detection [15]. Similarly, the metal-dye complex fast blue BB salt (FBBs)has been used for visual turn-off detection of •OH radicals during Fenton reaction [14]. However, due to its complex reaction and intrinsic drawbacks of turn-off strategies, the detection sensitivity in both methods above is limited, thus making them only applicable for detection of •OH radicals over tens-of-micromolar concentrations. Therefore, more sensitive and reliable methods for visual color detection of •OH radicals are still desired. Herein, a simple and sensitive method has been presented on the basis of nitroimidazoles and Griess reagents. As illustrated in Scheme 1, reacting with hydroxyl radicals can release nitrite ions from nitroimidazoles, which could be subsequently detected by Griess method. The initial colorless Griess solution turns red upon the reaction with nitrite ions in a concentration dependent manner, thus reflecting the concentrations of •OH radicals. In our study, the proposed nIm-Griess strategy has been successfully used to determine •OH radicals generated from Fenton reaction and X-ray radiation. More interestingly, the nIm-Griess method has also been successfully applied for monitoring •OH radicals produced from the catalyzed activation of two classic antimalarial artemisinins.
3
2 Experimental 2.1 Materials Ferrous chloride tetrahydrate was purchased from Alfa Aesar (China Limited, Shanghai). Griess reagent R2 N-1-napthylethylenediamineaqueous solution (0.1%) was purchased from Cayman (Michigan, USA). Artemisinins, p-amino benzene sulfonamide, hydrogen peroxide (30%) and other chemicals were purchased from Adamas (Shanghai, China). 96-well plate was purchased from Thermo Fisher.
All reagents were used without further purification. The distilled deionized water from a
Milli-Q Plus system is used throughout the experiments. 96-well plate reading was done on a Multi-detector microplate reader - VICTOR™ X3 (Thermo Fisher).
2.2 Griess reaction and standard curve establishment. As indicated from Fig. S1, the Griess reagent consists of two parts p-amino benzene sulfonamide aqueous solution (1%, R1) and N-1-napthylethylenediamine aqueous solution R2 (0.1%). R2 was purchased from Cayman and used directly. R1 was prepared by dissolving 500 mg p-amino benzene sulfonamide into 50 mL phosphoric acid solution (5%) according to the standard procedure. R1 and R2 were mixed freshly before testing. Briefly, for a test, 100 μL mixture of Griess reagent R1 and R2 was added into a 96-well plate followed by the addition of 50 μL nitrite containing samples. Under acidic conditions, the nitrite ions will react with R1to forma reactive diazonium salt, which can further react with R2 to form a red azo product with maximum absorption at 540 nm. The plate was kept under dark at room temperature for 10 minutes before data recording by a plate reader. Unless additional instructions, all Griess tests have been done at room temperature (25゜C). To establish the standard curve for nitrite, a series concentration of sodium nitrite solutions was freshly prepared, and then 50 μL 4
of each solution was added into the Griess reagent for colorimetric observation. Three repeats were set for each sample. The absorptions at 540 nm for each well after subtracting the values from reagent control were plotted versus the concentrations of nitrite. A linear relationship was found between the absorption and nitrite concentration (Fig.S2).
2.3 Detection of hydroxyl radical from Fenton reaction Fenton reaction was the most widely used model reaction to generate •OH radicals. The generation of •OH radicals from hydrogen peroxide in the presence of ferrous ions was estimated to be quantitative. The first step for detection is to generate nitrite ions from nitroimidazoles upon the reaction with •OH radicals. To do the test, nitroimidazole solution (500 μM, 2-nIm or 5-nIm) and FeCl2 solution (400 μM) were firstly mixed. To the mixture, different concentrations of hydrogen peroxide (H2O2) were added followed by the addition of pure water to make the final concentrations of nitroimidazoles and FeCl2 are 250 μM and 200 μM respectively. The reaction was kept at room temperature for 5 minutes. Then to 100 μL prepared Griess reagent mixture in a 96-well plate, 50 μL reaction solution was added. After 10 minutes, the data was recorded by a plate reader. Three repeats were done for each treatment. It’s well known that DMSO was the most widely used reagent to neutralize •OH radicals. Herein, DMSO was added into the reaction mixture in our control assay at the final concentration at 4% to examine the reaction specificity. The detection limit (Cmin) is estimated by a reported method Cmin=3*σ/B, where σ is the standard deviation obtained from three individual absorbance measurements (abs at 540 nm) without any analyte (H2O2). B is the slope obtained after linearly fitting the titration curves within the analyte concentrations. 5
2.4 Detection of hydroxyl radicals from water radiolysis To monitor•OH radicals generation during water radiolysis by X-ray. 5-nIm was dissolved in PBS buffer (pH 7.4) to prepare series of solution with different concentrations (0, 2, 5, 20, 50, 200, 500 μM). Meanwhile, the concentration of 5-nIm was fixed at 50 μM to observe X-ray dosage dependent nitrite release. The solution was prepared in 1.5 mL transparent eppendorf tube and sealed before irradiation. The dosage of X-ray (6 MeV) was set as 0,5, 25, 50 Gy, where Gy is a unit defined as the absorption of one joule of radiation energy per kilogram of matter. Generally, the clinical relevant dosage for a single therapy usually ranged from 0 to 20 Gy. An X-ray source with 6 MeV photon beams was used in this study. 2.5 Detection of •OH radicals production from the activation of artemisinins Artemisinin (Art) and Dihydroartemisinin (DHA) solutions were prepared in Milli-Q water. Hemin was prepared in 0.5M NaOH solution, since it has poor solubility in neutral pH conditions. To perform the detection, Art or DHA was firstly mixed with 5-nIm (250 μM), followed by the addition of hemin solution to reach the final concentration of hemin at 100 μM. The reaction solution was prepared in 1.5 mL eppendorf tubes, which were then incubated at 37゜C for 20 minutes before Griess test. The nitrite ions generated in the solution was quantified by the same method as described above. Hemin at 100μM can cause a little background absorption at 540 nm, which has been subtracted from all the tested groups before data processing. 3. Results and Discussion: It’s found in our previous study that nitroimidazole solutions are able to release nitrite ions upon water radiolysis probably by reaction with the hydrated electrons and the subsequent product •OH 6
radicals [29]. The phenomenon inspired us to consider new strategies for •OH radical detection based on nitrite release. Among all •OH radical generation system, Fenton reaction is the most widely used model reaction, which has been extensively used to develop •OH radical probes. In most cases, the generation of •OH radical from H2O2 by ferrous ions is often considered as chemical quantitatively (see Equation S1) [30]. To prove our design, the classic Fenton system was firstly used as the source of •OH radicals. In a typical detection, nitroimidazoles were mixed with ferrous ions firstly followed by the addition of H2O2. The in situ generated •OH radical reacted with nitroimidazoles, which led to the release of detectable nitrite ions by Griess test. As indicated in Fig.1A, both 2-nIm and 5-nIm can react with Fenton generated •OH radicals, thus resulting in the release of nitrite. When the concentrations of hydroxyl radicals below 10 µM, both 2-nIm and 5-nIm (both at 250 µM) show similar responsibility in nitrite release. However, more nitrites were released from 5-nIm solution than 2-nIm when the •OH radical was increased from 10 µM to 100 µM, which indicated that 5-nIm is probably more reactive towards •OH radical. The release curves become flat for both 2-nIm and 5-nIm when •OH radicals concentration is over 50 µM, probably because non-specific reactions happened due to the relative higher local concentration of •OH radicals during solution mixing. Plotting the nitrite release from 5-nIm versus •OH radical concentrations, a good linear relationship was observed with a high R-squared value (R2 =0.998)(Fig.1B). According to the linear fitting equation, the detection limit (Cmin) is estimated to be 0.398 μM, which is comparable to some recently reported fluorescence probes [16, 18]. Table 1 summarized the recent developments for •OH radicals determination. Although the nanoparticle based probes exhibited the greater sensitivity than small molecular probes, they were usually suffering from complex manipulation. Moreover, significant visual color changes were observed during detection (Fig.1C), thus making the naked-eye detection available that is not 7
achievable by most of the previous methods. Therefore, nIm-Griess offers an easier and technically achievable method for the detection of •OH radicals from Fenton reactions. Since •OH radical mediated nitrite release from nitroimidazole is the most critical process in the detection, we then looked into the reaction mechanisms. As previously proposed, nitrite release from 5-nIm in water radiolysis system might be initialized with the addition reaction between the nitro group substituted carbon atom (C5) and •OH radical. The addition intermediate bearing nitro group is unstable followed by the rapid elimination of nitrite ions (Fig.2A) [31]. Fortunately, a direct evidence to support proposed mechanism was obtained by analyzing the product using ESI-MS. As illustrated in Fig.S3, a typical mass signal (98.10) corresponding to the denitrated product was observed. The reaction specificity was further confirmed by introducing a •OH radical quencher DMSO to the reaction mixture. As shown in Fig. 2B, normally 25 µM •OH radical can cause the release of around 15.5 µM nitrite, whereas only background level release was observed in the presence of 4% DMSO. The result indicated that •OH radical quenching can indeed suppress nitrite release from 5-nIm, thus implying that nitrite release is specifically responding to •OH radicals. With the favorable performance in hydroxyl radical detection, we then examined the selectivity of nIm-Griess towards •OH radical over other ROS. As indicated from Fig.2C, only •OH radical is able to cause obvious nitrite release, all other tested ROS including hydrogen peroxide (H2O2), singlet oxygen (1O2 ), tert-Butyl hydroperoxide (TBHP), Superoxide (O2- ) and the neutrophil oxidant hypochlorous acid (HOCl) can just result in background level signals. Nitroimidazoles and their derivatives have been applied as chemical radiosensitizers in radiotherapy for decades, the mechanism may relate to the denitration and imidazole radical formation. •OH radicals was thought as the major radicals during water radiolysis, which could be generated 8
directly from water radiolysis or indirectly from the subsequent reaction between hydrated electrons and H2O (Fig.3A). Under 25 Gy irradiation, nitrite release from 5-nIm solution increased along with the raising of 5-nIm concentration. However, the release reached a saturated condition when concentration was further increased over 200 µM, mostly because 200 µM is sufficient to consume all the •OH radicals released upon 25 Gy irradiation (Fig.3B). After that, the dosage dependent nitrite release was studied while fixing the concentration of 5-nIm at 50 µM. As indicated in Fig. 3C, an increased nitrite release was observed with the X-ray dosage ranging from 0 to 50 Gy. Plotting the dosage versus nitrite concentration gives us a linear like relationship (R2=0.988). Thus actually, nIm-Griess based assay could be used to monitor the energy delivery by X-ray through the measurement of the radiolysis product •OH radicals. The pH effects on nitrite release upon irradiation were then evaluated by irradiating the 5-nIm (50 µM) PBS solution of various pH values. As illustrated from Fig. S4, •OH radical induced nitrite release did not prefer acidic conditions, mostly because the intermediate radical anions is not stable in acidic environments [32]. With the excellent performance on •OH radicals detection in Fenton reaction and X-ray irradiation system, we made further exploration to answer the questions involved in action mechanisms of classic antimalarials artemisinin (Art) and its derivatives. Although, Arts are most commonly used drugs against malaria, their action mechanism is not fully clear yet [33, 34]. There is a common sense that Arts can generate ROS and alkyl radicals during the activation by ferrous containing hemoglobin or free ferrous ions, which might be one of the mechanisms of their bioactivity [35]. However, what kinds of ROS were released during Arts activation is still unknown. To check if the highly toxic •OH radicals was involved in action of Arts, our nIm-Griess method has been applied (Fig.4A). Art and dihydroartemisinin (DHA) were mixed with 5-nIm firstly, then the metal catalyst hemin was added. 9
The mixture was incubated at 37 °C for 20 minutes before the measurement by Griess reagent. As indicated in Fig. 4B, both Art and DHA can cause the release of nitrite with catalyzing reaction, thus implying the generation of •OH radicals during artemisinins’ activation. In contrast, there is no nitrite in 5-nIm solutions when only with Arts or hemin. The result indicated that both peroxide bridge and hemin are required for hydroxyl radical generation. The process may start with the catalyzed decomposition of the peroxide bridge, which subsequently generates a peroxide intermediate. •OH radical was presumably released from the peroxide intermediate through a Fenton-like reaction. Incubation with 500 μg mL-1 DHA can cause 24.1 µM nitrite release from 5-nIm, whereas only 10.6 µM nitrite was released from Art incubation, thus indicating different efficiency in •OH radical formation between Art and DHA. The differences on •OH radical release may attribute to the different reactivity of their peroxide bridges. Furthermore, concentration dependent •OH radical release from DHA was observed, where the amount of •OH radicals increases as the concentration of DHA increasing (Fig. 4C). 4. Conclusion A novel strategy has been developed for colorimetric detection of hydroxyl radicals on the basis of a modified Griess test. Before a standard Griess test, nitroimidazoles were used as the moieties to release nitrite upon the reaction with •OH radicals. The final azo product from Griess reaction exhibits red color with maximum absorption at 540 nm, making the •OH radical detection available not only by UV-Vis spectrometer but also by naked eyes. It should be noticed that Griess test is initially developed for nitrite, thus the intrinsic nitrite in tested samples will cause interferences. This method can sensitively and selectively detect •OH radical from Fenton reaction with a detection limit down to nanomolar level. The successful detection of •OH radical from water radiolysis has also been achieved, 10
thus making the possibilities to monitor the energy delivery during ionizing irradiation. More interestingly, we successfully detected the •OH radical release from two classic antimalarials Art and DHA catalyzed by hemin. This is the first evidence that hydroxyl radicals may be one of the major reactive oxygen species during artemisinins’ activation. Furthermore, different efficiencies in •OH radical generation between Art and DHA have been observed, which properly indicates different reactivities between these two drugs. In a word, the presented method is a simple and easier strategy for •OH radical detection. Acknowledgements We gratefully acknowledge the National Natural Science Foundation of China (21807018), Dongguan Talents Program for Innovation and Entrepreneurship（No. 2017-16）and the start-up fund from Guangzhou University of Chinese Medicine. We thank Prof. Hristov, Dimitre from the Department of Radiation Oncology-Radiation Physics of Stanford University for his nice help on X-ray radiation study. We thank Ms. Xin Liu, the CEO of Dongguan Bioshine Biotechnology Ltd. Co. for her nice assistant in measurement. We also acknowledge the Lingnan Medical Research Center of Guangzhou University of Chinese Medicine for the support on facilities.
Competing interests There are no conflicts to declare. Appendix A. Supplementary data Supplementary data related to this article can be found at xxxx
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Scheme 1. Schematic illustration of •OH radical detection based on nIm-Griess method
Table 1: Comparison of the analytical performances with different methods Materials Rhodamine based small molecular Fluorescent small molecular Fluorecein based small molecule BODIPY based small molecule
Selectivity over
Naked-eye
Detection Limits/Linear
other ROS
detectable
range
•OH
yes
no data/few to tens μM
[26]
38 nM/few to tens μM
[17]
390 nM/few to tens μM
[16]
no data/no data
[13]
•OH
•OH
•OH
no description no description
no
14
Ref.
BODIPY based fluorescent probe Lysozyme-silver nanoclusters Coumarin-activated silica NPs
•OH
no
11 nM/0.1-120 μM
[12]
ClO−, 1O2,
no
200 nM/0.8-200 μM
[18]
•OH
no
1.56 μM/0.5-220 μM
[23]
slightly response to
Fluorescent
both •OH
nanoprobe
and O2•−
no
0.2 nM/0-20 μM
[24]
•OH
no
2 nM/4 nM-16 μM
[20]
•OH
no
12 nM/0.018-6 μM
[21]
Methylene Blue
no data
Yes
no data/no data
[15]
Fast blue BB salt
no data
Yes
no data/25-150 μM
[14]
•OH
Yes
398 nM/0-25 μM
Upconversion nanoprobe Graphene quantum dots
Modified-Griess based
This work
Fig.1 A) Nitrite release from 5-nIm (250 μM) and 2-nIm (250 μM) upon the reaction with hydroxyl radicals generation from Fenton reaction; B) Linear relationship of nitrite release from 5-nIm (250μM) as a function of hydroxyl radical concentrations (0 to 25μM); C) Reaction in the eppendorf tubes show the color changes upon the reaction with ·OH (0, 5, 10, 25, 50 μM corresponding to tube 1 to tube 5 15
respectively).
Fig. 2 A) Proposed reaction mechanism of hydroxyl radical induced nitrite release from 5-nIm; B) nitrite release specifically responds to ·OH and the process can be suppressed by ·OH selective quencher DMSO; C) nIm-Griess can selectively detect hydroxyl radicals against other ROS. The
concentrations of analytes were set as ·OH (25 μM), H2O2, TBHP and HOCl (100 μM), 1O2 and O2were generated according to the reported methods [16].
Fig.3 A) Hydroxyl radical generation during water radiolysis; B) Nitrite release from different concentrations of 5-nIm (0, 2, 5, 20, 50, 200, 500 μM) under 25 Gy irradiation; C) A good linear 16
relationship was observed when plotting nitrite release versus X-ray doses.
Fig. 4 A) Schematically show hydroxyl radical release from artemisinin (Art) and dihydroartemisinin (DHA) through hemin catalyzed activation; B) the quantification of nitrite release from 5-nIm (250 μM) upon the reaction with hydroxyl radicals release from artemisinins (500 μg mL-1)/hemin (100 μM) system; C) DHA concentration dependent nitrite release from 5-nIm/DHA/hemin system. The concentrations for the components were 5-nIm (250 µM), hemin (100 µM), the reaction was incubated at 37 °C for 20 min before Griess test at rt.
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Highlights:
A simple method nIm-Griess has been developed for visual colorimetric detection of hydroxyl radicals.
This method is based on the modified Griess test, where all the chemicals are commercial available, thus making it a low cost and easily achievable way for hydroxyl radical determination.
This method shows excellent responsibility and selectivity in detection of hydroxyl radicals produced from Fenton reaction and water radiolysis.
Hydroxyl radical releasing from antimalarial artemisinins was observed by nIm-Griess, thus offering direct evidences for the action mechanisms of artemisinins.
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