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The self-catalyzing chemiluminescence of luminoldiazonium ion and its application for catalyst-free hydrogen peroxide detection and rat arthritis imaging Chunxin Zhao, Hongbo Cui, Jing Duan, Shenghai Zhang, and Jiagen Lv Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04544 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on January 6, 2018

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Analytical Chemistry

The self-catalyzing chemiluminescence of luminol-diazonium ion and its application for catalyst-free hydrogen peroxide detection and rat arthritis imaging Chunxin Zhao, Hongbo Cui, Jing Duan, Shenghai Zhang, Jiagen Lv* Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Shaanxi Normal University, Xi'an, 710119, P. R. China *Corresponding author. Tel: +86-29-81530726, fax: +86-29-81530727, E-mail addresses: [email protected]

ABSTRACT: We report the unique self-catalyzing chemiluminescence (CL) of luminol-diazonium ion (N2+-luminol) and its analytical potential. Visual CL emission was initially observed when N2+-luminol was subjected to alkaline H2O2 aqueous without the aid of any catalysts. Further experimental investigations found the peroxidase-like activity of N2+-luminol on the cleavage of H2O2 into OH• radical. Together with other experimental evidence, the CL mechanism is suggested as the activation of N2+-luminol and its dediazotizaiton product 3-hydroxyl-luminol by OH• radical into corresponding intermediate radicals, and then further oxidation to excited state 3-N2+-phthalic acid and 3-hydroxyl-phthalic acid which finally produce 415 nm CL. The self-catalyzing CL of N2+luminol provides us an opportunity to achieve the attractive catalyst-free CL detection of H2O2. Experiments demonstrated the 10−8 M level detection sensitivity to H2O2 as well as to glucose or uric acid if pre-subjected to glucose oxidase or uricase. With the exampled determination of serum glucose and uric acid, N2+-luminol shows its analytical potential for other analytes linking the production or consumption of H2O2. Under physiological condition N2+-luminol exhibits the high selective and sensitive CL toward 1 O2 among the common reactive oxygen species. This capacity supports the significant application of N2+-luminol for detecting 1O2 in live animals. By imaging the arthritis in LEW rats, N2+-luminol CL is demonstrated a potential tool for mapping the inflammation relevant biological events in live-body.

Without the requirement of light source to excite the luminescent structure, chemiluminescence (CL) detection is performed in the dark circumstance that enables the high signalto-noise ratio optical signal readout.1−3 CL analysis has been established as a branch of analytical chemistry which offers a simple, low-cost, and sensitive analytical technique. For typical CL events, the reaction mechanism is known as a CL reagent (e.g., luminol or Schaap’s dioxetane) being oxidized by an oxidant, reactive oxygen species (ROS) particular, to yield a corresponding excited state product, along with its decay to ground state, the reaction energy being released as light emission directly or via energy transfer by a exogenous flourophor.1,3 While current analytical utilization of CL has covered a wide range of analytes far beyond ROSs,4−7 the limit in selectivity for direct CL detection has been realized.1,3 For example, when detecting a reductive analyte in complex matrix the coexisting reductant would also participate in the CL redox reaction, hampering the selective CL response to target analyte. Consequently, indirect CL methods are developed. These approaches usually employ the techniques for analyte separation or bio-molecule recognition including high performance liquid chromatography (HPLC),8,9 solid phase extraction (SPE),10,11 molecular imprinting (MI),12,13 capillary electrophoresis (CE),14,15 and bio-affinity based separation.16,17 Along with the greatly strengthened selectivity, these strategies involve sophisticated instruments, complex preparation of separation materials, or costly biological reagents that decrease the simpleness and cheapness of CL detection. In terms of either aligning with the referred unique selling point of

simple detection or meeting the new analytical demands as monitoring biological events in live bodies, direct CL methods are greatly welcome. Intensive efforts have been made by researchers to explore the selective and direct CL systems. Penetrating the interactions among analyte, CL reagents/catalysts, and functional nano-materials to achieve the analyte-responsive CL has been proved to be effective strategy. Hydroxylamine-O-sulfonic acid was demonstrated an effective oxidant for highly selective Co2+ ion detection against other transition metal ions.18 Via the resonance energy transfer between 1O2 and aggregation induced emission (AIE)-active fluorophores, 1O2 during photodynamic therapy was selectively monitored.19 A list of CL sensors for volatile analytes were developed by forcing the sample vapor to pass through a heater coated with specially appointed nano-particles.20–23 Organophosphorus pesticide in tea drink was directly measured with the aid of an Au–Fe3O4 nano-cascade.24 Free clorine in water was detected by using a carbon nitride quantum dots based CL system.25 Utilizing an orderly arranged nano-structure, CL recognition of peroxynitrite against common ROSs was achieved.26 More recently, CL imaging (CLI) is deemed as advantageous in sensitivity for its elimination of autofluorescence and light scattering.27−31 In terms of mapping biological events in live animals, targetresponsive/triggered CL probes are even more preferred owing to the difficulty in tissue to manage the separation or recognition of targets. With the synthesis of specific CL probes that can be in vivo turned-on by corresponding targets, native O2•−,

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β-galactosidase activity, and tumor-related nitroreductase and hypoxia in rodents were imaged, respectively.31−33 All aforementioned approaches shed light on developing the direct CL systems, as indicated, exploring the intrinsic selectivity of chemical reaction/interaction and looking for the new selective CL reagents.7 Luminol-diazonium ion (N2+-luminol) had been used labeling antibody of IgG and glutathione with CL moiety for the subsequent detection of IgG and glutathione.34,35 The CL behavior of N2+-luminol has yet been studied in detail. Here, we investigated the CL of N2+-luminol to explore its potential for direct CL analysis. We found the bright CL of N2+-luminol to alkaline H2O2 in absence of any transient metal ions regardless of the cations of BF4− or Cl− in contrast to the really poor CL to ClO− (Figure 1). After a series of experiments, it is found that N2+-luminol possesses the peroxidase-like activity to catalyze the cleavage of H2O2 into OH• radical. Together with more experiment evidence, the CL mechanism of N2+-luminol to H2O2 in alkaline aqueous is suggested as the activation of N2+-luminol and its dediazotizaiton product 3-hydroxylluminol by OH• radical into corresponding intermediate radicals, which are further oxidized to excited state 3-N2+-phthalic acid and 3-hydroxyl-phthalic acid to produce 415 nm CL during their decay to ground state. In physiological aqueous N2+luminol exhibits the very weak CL response to H2O2 due to the restriction of poor ionization of the cyclic-hydrazide structure. Meanwhile, N2+-luminol contributes the highly sensitive CL toward 1O2 among common ROSs. Based on above first findings, we propose the catalyst-free CL detection of H2O2 and serum glucose and uric acid. This has been proved by the 10−8 M level detection sensitivity to H2O2, glucose, and uric acid, respectively, as well as the determination of serum samples. The highly selective CL toward 1O2 under physiological condition implies the potential of N2+-luminol for detecting inflammation in live animals. Such potential has also been demonstrated by imaging the arthritis in LEW rats.

Experimental section Chemicals and Reagents. Ultrapure water from a Millipore water purification system (18.2 MΩ·cm, Direct-Q 3UV, Millipore, FR) was used in all experiments. Analytical grade chemicals, including HCl, NaOH, Na2HPO4·12H2O, NaH2PO4·2H2O, Na2CO3, NaHCO3, HAc, NaAc, uric acid, and all of metal slats were obtained from Sinopharm Chemical Reagent Co., Ltd. NaNO2, NaBF4, NaClO, Na2S2O4 were purchased from Tianjin chemical reagent company. Luminol, superoxide dismutase (SOD), uricase, ascorbate oxidase, and glucose oxidase were obtained from Sigma-Aldrich (St. Louis, USA). Xanthine and xanthine oxidase were purchased from Solarbio Life Sciences (Beijing, China). Pristane was purchased from Accelerating Scientific and Industrial Development thereby Serving Humanity (Beijing, China). Stock solution of 0.1 M H2O2 was prepared by diluting 30% (v/v) H2O2 (Tianjin Tianli Chemical Reagents Ltd.) and placed for 8 hours before the use for experiments. ClO− was freshly prepared from NaClO solution (Tianjin Fuchen Reagents Ltd.) with ultrapure water. Both H2O2 and ClO− stock solutions were standardized by KMnO4 and iodometric titration, respectively. Working solution of 3,3',5,5'-tetramethylbenzidine (TMB, Shanghai Macklin Biochemical Co., Ltd) was prepared by dissolving solid TMB in DMSO. SOD, xanthine and xanthine oxidase were freshly prepared by dissolving in 0.1 M pH 7.4 PBS.

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Apparatus. The fluorescence spectra were measured using a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). The CL detection was performed with a MCDR-A Chemiluminescence Analyzer (Xi’an Ruimai Electronic Sci. Tech. Co. Ltd., Xi’an, China). The flow-injection system was consisted of a BT100-02 peristaltic pump (Baoding Qili Precision Pump Co., Ltd, Baoding, China) and a six-way valve. The CL spectrum was measured by placing the CL cell at the window of the photomultiplier tube (PMT) of Hitachi F-7000 fluorescence spectrophotometer. The UV-vis absorption spectra were obtained from a TU-1901 double beam UV-vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd, Beijing, China). Rodent CL imaging were taken using a Bruker In-Vivo Xtreme II Imaging System (Bruker Corporation, USA) which equipped with a sensitive Charge Coupled Device (CCD) camera and a VMR anesthesia machine (Matrx, USA). Preparation of diazonium salts. All arenediazonium salts were synthesized according to the reported method.36 Briefly for N2+-luminol-BF4−, in a 25 mL beaker, the luminol (0.18 g, 1 mmol) was dissolved in 10 mL ultrapure water and a solution of HCl (3 mL, 3 mmol) was added dropwise into the suspension of luminol under stirring at 0-5 °C. Then, NaBF4 (0.11 g, 1 mmol) and NaNO2 (0.072 g, 1.05 mmol) were successively added into the solution above. After responding for 30 min, the precipitated N2+-luminol-BF4− was filtered off. After addition of diethyl ether to the filtrate, the precipitation of N2+luminol-BF4− was filtered off. Above precipitation together was washed with 10 mL diethyl ether for three times. The obtained brown N2+-luminol-BF4− was dried overnight at room temperature and stored at 4-30 °C in glass vials with airtight and light-free. The ESI-MS characterization of N2+-luminolBF4− is shown in Figure S1. Other diazonium salts were prepared with corresponding chemicals instead of luminol or NaBF4 as detailed in S1. Safety Considerations. We prepared N2+-luminol-BF4− and the N2+-luminol-Cl− in this work. Solid diazonium halides are known explosive while the tetrafluoroborate salts can be stable at room temperature.37 Here, besides the experiments to demonstrate that N2+-luminol responds H2O2 with regardless of anion species, N2+-luminol-Cl− was crashed out in all other experiments despite the lack of direct experimental evidence on its explosive. This principle was also applied to the preparation of N2+-phthalate acid and N2+-benzene salts. Solid N2+luminol-BF4− has been proved stable for more than one year in air tight glass vial with light shield at 4-30 °C. Here after, N2+luminol refers to the water solution prepared from solid N2+luminol-BF4−. CL dynamic profiles and CL spectrum measurements. To determine the CL reaction kinetics, dynamic profiles with respect to all mentioned CL reactions were tested with static method. ROSs were generated according to the reported procedures (S2).38,39 The CL spectrum of N2+-luminol was measured with a continuous flow manifold as shown in Figure S2A. The procedures and flow manifolds for ROSs generation and dynamic profiles measurement are detailed in S2 and Figure S2B-C, respectively. Peroxidase-like activity investigation on arenediazonium salts. TMB can be oxidized to a blue color product by H2O2 upon the presence of arenediazonium salts. One diazonium salt was added into 0.1 M NaAc-HAc buffer solution (pH 4.6), followed by adding TMB solution (0.04 mol in 10 mL


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DMSO). After that, H2O2 was added into the mixture. The peak absorbance at 650 nm was measured by UV-vis spectrophotometer after reaction for a determined time at room temperature. CL detection of H2O2, serum glucose and uric acid. The detection of H2O2 and serum glucose and uric acid was performed with a flow-injection manifold as shown in Figure S2D. Glucose standards were prepared by adding 50 µL glucose peroxides (5 u/mL) into each of 10 mL glucose and keeping at 37 °C for 30 min. The serum samples were 10000 folds diluted and treated as that for glucose standards. Uric acid standards were prepared by adding 50 µL ascorbate oxidase (5 u/mL) into each of 10 mL uric acid and keeping at 37 °C for 5 min, and then, adding 50 µL uricase (5 u/mL) into each solution and keeping at 37 °C for another 5 min. The serum samples were 1000 folds diluted and treated as that for uric acid standards. The carrier (NaOH solution) and H2O2 solution were pumped through at a flow rate of 1.5 mL/min. 100 µL of N2+-luminol-BF4− solution was injected into the carrier stream at a flow rate of 1.8 mL/min, which then reacted with the H2O2 stream. When the reaction solution was passing through the CL flow cell, the light emission was measured by the PMT. Pristane Induced Arthritis (PIA) rat model and imaging. LEW rats (female) were housed in groups of 2 individuals per cage in climate-controlled (24 °C/50-60% humidity) circumstance. Rats were fed ordinary solid diet and distilled water, and were subjected to 14 h light/10 h dark cycles. Health status of the rats was monitored daily and weight was controlled at least once a week. After two weeks of acclimatisation, 150 µL pristane were injected intradermally into the tail of the rats under anesthesia induced by an oxygen/isoflurane mixture in 0.7 L/min.40 Above rats were cultured under the normal condition for 16 days until the red swelling at the ankle of hind paw was observed. The imaging experiment was carried out on the subsequent day. After fasting and water deprivation for 3 hours, the test rat was given an intraperitoneal (i.p.) administration of 200 µL N2+-luminol (1.0×10−3 M in 0.1 M PBS buffer, pH 7.4), under anaesthesia with a 0.7 L/min oxygen/isoflurane stream. Imaging was performed by the Bruker In-Vivo Xtreme II Imaging System with the working conditions as exposure time, 5 min; binning, 4×4; field of view, 19 cm; CCD working temperature, −86 °C; anesthesia maintaining oxygen/isoflurane stream, 350 mL/min. X-ray images were taken with the same imaging system. Fusion of CL image and X-ray image was completed with the MI software equipped by Bruker Xtreme II system. At the end of the study, the animals were sacrificed by diethyl ether inhalation.

Results and discussion

Figure 1. (A) The CL responses of luminol-diazonium salts to alkaline H2O2 and ClO−. N2+-luminol-BF4− and N2+-luminol-Cl−, 3.3×10−4 M; NaOH, 0.033 M; H2O2 and ClO−, 0.033 M. (B) CL comparison to H2O2 with respect to luminol, N2+-luminol-Cl−, and N2+-luminol-BF4− obtained from a flow-injection setup (Figure S2D). H2O2, 4.0×10−3 M; PMT voltage, −700 V. (C) CL dynamic curves of N2+-luminol-BF4− with respect to H2O2 and ClO−. H2O2, 1.0×10−3 M; ClO−, 1.0×10−3 M; PMT voltage, −300 V. In (B) and (C), luminol, N2+-luminol-Cl−, and N2+-luminol-BF4−, 1.0×10−4 M; NaOH, 1.0×10−3 M.

Investigation on N2+-luminol CL. We occasionally observed the bright CL when alkaline H2O2 was added into the luminol diazotization solution in absence of any transient metal ions (e.g., Co2+, Cu2+, Fe2+, Cr3+). We initially attributed such phenomenon to the production of peroxynitrite via reaction between NO2− and H2O2 in the acidic aqueous which was subsequently oxidizing N2+-luminol.41,42 However, when using the synthesized N2+-luminol-BF4− and N2+-luminol-Cl− solids to prepare the solutions, where the production of peroxynitrite had been eliminated, bright CL by H2O2 were visualized and recorded with a mobile phone camera (Figure 1A). Further evidence shown in Figure 1B demonstrates the great difference in CL between N2+-luminol salts and luminol when they were subjected to H2O2. Also in Figure 1B, it can be found that N2+-luminol ion is intrinsically CL sensitive to H2O2 regardless of the anion species. Observing the CL dynamic curves from static experiment (Figure 1C), the CL of N2+luminol reaches its maximum within 4 s. This shows N2+luminol kinetically reacts with H2O2 fast. It was supposed that N2+-luminol would vigorously react with ClO− to produce strong CL. To our surprise, N2+-luminol acts really poor CL response to ClO− as depicted in Figure 1A and 1C.



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It seems that N2+-luminol can be directly oxidized by H2O2 without catalysts. To explore this, we firstly examined 14 metal ions to look for their effects on N2+-luminol CL. Among them, Co2+, Cr3+, Cu2+, Cd2+, Fe2+, Fe3+, Mn2+, Ni2+, and Ag+ are “catalytic” for Feton-like reaction while Al3+, Ba2+, Mg2+, Pb2+, and Zn2+ are “non-catalytic”. Our results show that there is no enhancement effect on CL for all above metal ions (Figure 2). The “non-catalytic” ions perform the similar CL signals as that of metal free blank. For “catalytic” ions, in particular those “highly catalytic” ones including Cu2+, Co2+, Cr3+, Ni2+, Mn2+, and Fe2+, even the decreased CL has been observed. This phenomenon is estimated for the dual roles played by “catalytic” ions. On one hand, their presence increases the hydroxyl radical production and hence enhances the CL. On the other hand, since partial of the short-lived hydroxyl radicals quickly decay to O2 before they encounter and react with N2+-luminol, their presence causes the loss of H2O2. As a whole, “catalytic” ions result in the CL decrease. Based upon above findings, N2+-luminol is confirmed to possess the CL behavior very different from that of luminol, implying the new potential for CL analysis.

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Figure 2. Effect of metal ions on CL of N2+-luminol with H2O2. Blank, metal ions free; N2+-luminol, 1.0×10−4 M; H2O2, 1.0×10−3 M; NaOH, 0.01 M; metal ions, 4.0×10−6 M; Flow manifold, Figure S2E, PMT voltage, -300 V.

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Figure 3. (A) CL spectrum of N2+-luminol + H2O2. N2+-luminol, 1.0×10−3 M; H2O2, 0.01 M; NaOH, 0.01 M. (B) Fluorescence spectra of 3-hydroxyphthalic acid, 3-N2+-phthalic acid, and phthalic acid. 3-hydroxyphthalic acid, 3-N2+-phthalic acid, and phthalic acid, 1.0×10−4 M; NaOH, 0.01 M. (C) N2+-luminol dynamic profiles with respect to H2O2, ClO−, 1O2, OH•, and O2•− in alkaline aqueous. O2•−, 5.0×10−3 M Na2S2O4 + 0.01 M NaOH; N2+-luminol for 1O2, 1.0×10-5 M. (D) N2+-luminol dynamic profiles with respect to H2O2, ClO−, 1O2, OH•, and O2•− in physiological aqueous. O2•−, 3.0×10−4 M xanthine + 0.05 u/mL xanthine oxidase for 1 min. In (C) and (D), N2+-luminol, 1.0×10−4 M； H2O2, 1.0×10−3 M; OH•, 2.0×10−3 M H2O2 + 2.0×10−4 M Fe2+; 1 O2, 2.0×10-3 M H2O2 + 2.0×10-3 M ClO−. All of CL dynamic


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profiles were measured with the flow manifolds as shown in Figure S2B and Figure S2C.

More experiments were conducted to gain further insight into the CL of N2+-luminol. The CL dependence of N2+-luminol + H2O2 at different pH was firstly investigated. Experimental results show N2+-luminol gives the best CL in strong alkaline environment. (Figure S3). Then, the CL spectrum of N2+luminol was measured with a fluorospectrometer as shown in Figure 3A. It can be seen that the CL peaks at 415 nm. This provides us the evidence to look for the CL fluorophor of N2+luminol oxidation. In consideration of the possible oxidation products, 3-N2+-phthalic acid, 3-hydroxyl-phthalic acid, and phthalic acid were suggested as the candidate fluorophors. After the synthesis of 3-N2+-phthalic acid, we measured the fluorescence spectra of above three candidates. As shown in Figure 3B, 3-N2+-phthalic acid and 3-hydroxyl-phthalic acid present the peak fluorescence at 415 nm that just in coincidence with the peak CL of N2+-luminol. Phthalic acid is ignored for both its 429 nm peak emission and poor fluorescence efficiency. MS characterization on N2+-luminol + NaOH solution finds the production of hydroxyl-luminol (Figure S4A). This indicates the decay of N2+-luminol to hydroxyl-luminol in strong alkaline aqueous. Followed MS characterization on N2+-luminol + NaOH + H2O2 solution finds the appearance of N2+-phthalic acid and hydroxyl-phthalic acid (Figure S4B). Above MS evidence indicates that in strong alkaline aqueous while portion of N2+-luminol being directly oxidized to N2+phthalic acid, another portion of N2+-luminol was firstly dediazotized to hydroxyl-luminol and subsequently oxidized to hydroxyl-phthalic acid. In combination of the MS findings with the fluorescence spectra, 3-hydroxyl-phthalic acid and 3N2+-phthalic acid are suggested as the CL fluorophors. It is intensely curious about the CL performance of N2+luminol toward common ROSs. Here, we examined the CL dynamic profiles of N2+-luminol with respect to H2O2, ClO−, 1 O2, OH•, and O2•− in the pH=11.2 alkaline aqueous. The obtained results shown in Figure 3C demonstrate that N2+luminol kinetically reacts fast with H2O2, 1O2, and OH• in contrast to with ClO− and O2•−. For easy comparison, we collected the data on CL profile area and peak height in Table S1. Where, the integral area under a CL dynamic profile may represent the CL efficiency while the CL peak height may represent the sensitivity for flash-type measurement. It can be seen that N2+-luminol behaves the best CL to 1O2, and the sensitive CL to H2O2 and OH•. When comparing the case to H2O2 with that to OH• radical, their CL performance are found close to each other in all aspects of CL efficiency, CL peak height, and even the shape of CL profile. It is clear that OH• radical does not take the priority over H2O2 to active N2+-luminol. One step further, we assume the cleavage of H2O2 into OH• radical by N2+-luminol itself in alkaline aqueous. We also investigated the CL behavior of N2+-luminol under biological condition to explore its analytical potential. Observed from the results shown in Figure 3D and in Table S1, N2+-luminol responds very weak CL to H2O2, O2•− radical, OH• radical, and ClO−. It exhibits the selective CL to 1O2 against common ROSs. Noting the CL cases to OH• radical in Table S1, the concentration increase of Fe2+ brings about the decrease of CL. This result reminds us of the estimation on “catalytic” metal ions, the more the presence of Fe2+ ion the more the decomposition of H2O2. From above experimental evidence, we conclude N2+-luminol is a potential 1O2 selective

probe. Imaging biological events/processes with the production or consumption of 1O2 is further expected.

Figure 4. Investigation on the catalytic effect of N2+-luminol on TMB oxidation. (A) Typical Uv-absorption curves of TMB with respect to the presence of (a) TMB, (b) H2O2 + TMB, (c) H2O2 + TMB + N2+-luminol. N2+-luminol, 8.3×10−5 M; (B) Timedependent Uv-absorbance changes with respect to N2+-luminol concentrations from 0 to 8.3×10−5 M. TMB, 1.7×10−3 M; H2O2, 1.3×10−3 M; buffer, pH 4.6 (HAc-NaAc); incubation, 25 °C for 10 min.

As aforementioned, N2+-luminol is assumed to promote the cleavage of H2O2 into OH• radical. If it is true, N2+-luminol should possess the peroxidase-like property. To prove this assumption, common experiments to demonstrate the peroxidase-like activity for specified mimetics were conducted.43–45 As depicted in Figure 4A, with the addition of N2+-luminol into TMB + H2O2 mixture, the solution color immediately turned to blue while the blank control remained clear. The dependence of the N2+-luminol amount on TMB oxidation was also investigated as shown in Figure 4B. Further demonstration on such peroxidase-like activity of N2+-luminol was conducted by adding trace amount of N2+-luminol into luminol + H2O2 mixture. As shown in Figure S5, deducting the CL contribution from N2+-luminol itself, the CL of luminol was enhanced more than 1000 folds (the green region in Figure S5). To look for the reactive species produced during N2+-luminol catalyzed oxidation process of TMB by H2O2, we tested several scavengers that can quench the relevant reactive species of OH• radical, 1O2, and O2•− radical, respectively. As shown in Figure S6, ascorbic acid and thiourea effectively quench the TMB oxidation while SOD and NaN3 do not. These results indicate that OH• radical is the main reactive species in the catalytic reaction rather than O2•− radical and 1O2.46 These



results suggest N2+-luminol catalyze the cleavage of H2O2 into OH• radical. To further evaluate the catalytic activity of N2+luminol, we measured its steady-state kinetic performance as shown in Figure S7. The apparent kinetic data of Michaelis– Menten constant (Km) and maximum reaction rate (Vmax) were also measured as listed in Table S2. The obtained results also support the peroxidase-like activity of N2+-luminol. It is interesting to investigate whether other diazonium salts also possess the peroxidase-like activity. As shown in Figure S8, similar to N2+-luminol, 3-N2+-phthalic acid and N2+benzene exhibit the catalytic activity on TMB oxidation while phenol and 3-hydroxyl-phthalic acid do not. Such result suggests diazo moiety to be the catalytically active site. Learning from the recent study46, the diazo moiety is supposed to act as the stabilizer for OH• radical. Despite the mechanism of the N2+-luminol peroxidase-like activity has yet been explicitly revealed, to the best of our knowledge, this is the first finding of diazonium compounds that possess the peroxidase-like activity. This property of N2+-luminol suggests the attractive analytical potential for catalyst-free detection of H2O2.

Scheme 1. The proposed mechanism on N2+-luminol + H2O2 CL oxidation. Together with the well acknowledged CL mechanism of luminol and its derivatives,2,3,47–50 researches on dediazoniation reaction of aryldiazonium ions,51–53 relevant report on small molecule catalyzed oxidation with H2O2,46 and our experiment

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facts as aforementioned, a possible self-catalyzing mechanism on N2+-luminol + H2O2 CL is proposed in Scheme 1. In brief, N2+-luminol cleaves H2O2 into OH• radical (Eq. 1). In alkaline aqueous, dediazoniation of N2+-luminol takes place along a homolytic pathway to produce the arene radical and subsequently to yield the hydroxyl-luminol (Eq. 2).51,54,55 OH• radical activates the N2+-luminol and hydroxyl-luminol into corresponding diformylhydrazine radicals (Eq. 3-4), respectively. Subsequently, these two diformylhydrazine radicals are further oxidized by O2 or O2•− radical into even more unstable peroxide intermediates (Figure S9). Then, the two peroxide intermediates decompose to the excited state N2+-phthalic anion and hydroxyl-phthalic anion, respectively. Finally, coupled with the decay of the excited anions to their ground state, 415 nm photon emission is released (Eq. 5-6). Under physiological condition, the low pH restricts the ionization of the cyclichydrazide in N2+-luminol, resulting in the poor N2+-luminol activation as well as the poor CL. The fact of the strongest CL response to 1O2 suggests the kinetically faster oxidation of N2+-luminol by 1O2 than by other ROSs (Eq. 7). Catalyst-free CL detection of H2O2, serum glucose and uric acid. Aforementioned experiments suggest the selfcatalyzing detection of H2O2 with N2+-luminol. We experimentally characterized such analytical potential. With a flowinjection setup (Figure S2D), the N2+-luminol and NaOH concentrations were firstly optimized (Figure S10). As expected, N2+-luminol shows the sensitive and quantitative response to H2O2. Figure S11 shows the linear relationships of net CL intensity (∆ICL) with H2O2 concentration in the 1.0×10−7 to 2.0×10−6 M range by following the equation of ∆ICL = 87.3C−1.94 (C, 10−6 M; n = 5, r2 = 0.999), and in the 2.0×10−6 to 8.0×10−6 M range by following the equation of ∆ICL = 145.7C−131.2 (C, 10−6 M; n = 7, r2 = 0.992), respectively. The detection reproducibility was tested by repeatedly measuring a 5.0×10−7 M standard for seven times. The obtained 1.3 % relative standard error (RSD) supports the precision for quantitative detection. According to the IUPAC principle, the detection limit is estimated to be 3×10−8 M. Above high sensitivity to H2O2 encourages us to apply N2+luminol to enzyme linked bioassays. Serum glucose and uric acid were selected as the model analytes for their best popularity in clinical diagnosis and bio-fluid research. The proved high sensitivity to H2O2 allows us to dilute the serum samples for 1000 to 10000 folds. This brings about either the save of serum sample or the sufficient solution volume to support the automatic analysis. Utilizing the same flow-injection setup and working conditions as those for H2O2 detection, linear relationships between the ∆ICL and glucose/uric acid concentration were investigated, respectively. For glucose, the linearity was found in the range of 1.0×10-7 to 3.0×10-5 M by following the equation of ∆ICL = 802.7C−8.7 (C, 10-6 M; n = 7, r2 = 0.990, Figure S12), with a detection limit of 3×10-8 M glucose. The detection repeatability was measured to be 2.8% (n = 7) for a 5.0×10-7 M glucose. For uric acid, the linearity was found in the range of 5.0×10-8 to 3.0×10-6 M by following the equation of ∆ICL = 1013C+8.8 (C, 10-6 M; n = 5, r2 = 0.998, Figure S13), with a detection limit of 2×10-8 M uric acid and 1.2% relative standard error (n = 7) for 5.0×10-7 M uric acid. We also evaluated the possible interference from common components in serum including inorganic ions, amino acids, and ascorbic acid (Table S3). It is found that all the normal levels of those components in serum are well below the tolerance concentrations


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given in Table S3. Considering of the possible abnormal high ascorbic acid cases, for uric acid detection ascorbate oxidase pretreatment was applied to serum samples to remove the possible interference. Thus, N2+-luminol CL can afford the selective determination of serum glucose and uric acid. At last, real serum samples obtained from a local hospital were determined. Our results agree well with those from the hospital method (Table S4 and S5). While featuring the convenience of catalyst-free, our N2+-luminol method exhibits the high sensitivity matching those reported CL systems.56,57 Above success to serum analytes demonstrate the use of N2+-luminol for sensitive detection of bio-analytes linking the yielding of H2O2.

for PIA model construction. (C) The T/NT value from (A) with respect to right and left paws. (D) The N2+-luminol accumulation in organs.

Mice arthritis imaging. During inflammation, a granule enzyme myeloperoxidase (MPO) is activated and released into the extracellular space which mediates the production of ClO− from H2O2 and Cl− ion. For the importance of ClO− in nonspecific immunity, the native MPO level has been regarded as an independent biomarker to predict the risk of coronary heart disease and to evaluate the therapeutic effect on rheumatic arthritis.58−60 Since ClO− can react with H2O2 to yield 1O2 in situ,61 the level of 1O2 indicates the activity of MPO. Thus, imaging 1O2 level provides us an opportunity to locate and evaluate the location and status of inflammation in live-body. We have demonstrated the selective CL of N2+-luminol to 1O2 against other ROSs. The detection sensitivity to 1O2 is confirmed by the finding of remarkable CL enhancement to sub10−7 M level 1O2 (Figure S14). PIA rat model was prepared in this work for its many similarities to human rheumatic arthritis.62 Imaging results are shown in Figure 5A, where clear CL evidence indicates the severe inflammation at ankle join, metatarsal joint, and interphalangeal joint of the right hind limb rather than the entire paw swelling by macroscopic observation (Figure 5B). Notably, CL evidence is also found at the left hind paw (at the metatarsal joint in 15-20 min image) while no red swelling was observed (Figure 5A and B left) until 5 days later. This shows at the imaging moment the inflammation was initiating at the left hind paw where the fourth digital joint displayed the clear CL signal. That is, N2+-luminol imaging can sensitively detect the early inflammation. The signal ratio of target/non-target (T/NT) represents the capacity of distinguishing the interest region from the normal tissue. As depicted in Figure 5C, in the initial 20 min N2+-luminol shows the high T/NT. And 40 min after N2+-luminol almost decayed as the CL is too weak to be detected (result has not shown). This result shows that N2+-luminol is suitable for detecting the inflammation location and status. In case of monitoring the inflammation, repeated N2+-luminol administration is necessary. To have a preliminary understanding of the possible N2+luminol accumulation in live animal, a dissection experiment was performed. As shown in Figure 5D, 30 min after the N2+luminol administration N2+-luminol is found accumulating in spleen, intestine, and stomach. Above results support the potential of N2+-luminol for mapping inflammation status in live animals.

Conclusion

Figure 5. (A) CL imaging results of PIA rat with N2+-luminol (in upward view). Red numbers at the middle of images represent the imaging duration. The moment after N2+-luminol administration was set as the 0 min for time counting. Yellow circle labeled at the fore limb represents the assumed non-target region. (B) Photos of the hind limbs of test rat before imaging (in vertical view). Obvious redness and swelling at right paw indicates the success

We explored the CL behavior and analytical potential of N2+-luminol. N2+-luminol is found to possess the unique catalytic property on the CL oxidation of itself by catalyzing the cleavage of H2O2 into OH• radical. The novel catalyst-free and sensitive CL detection of H2O2 has been experimentally demonstrated as well as the further application to serum glucose and uric acid. The common peroxidase-like property of arenediazonium ions is also firstly reported. The highly selective and sensitive CL of N2+-luminol to 1O2 under physiological condition enables the attractive mapping of inflammation in live animals that has been demonstrated by imaging the arthritis model rats. Despite the self-catalyzing mechanism of N2+-luminol has yet been fully revealed, our results support the capacity of N2+-luminol for direct and simple CL detection of analytes which link the production or consumption of H2O2, so as to a useful live-body tool for investigation of inflammation



relevant biological events or diseases by detecting the yielding of 1O2.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Details of N2+-luminol preparation and experimental procedures S1-S2 and Figures S1−S14 and Table S1-S5, including the synthesis method for diazonium compounds other than N2+luminol, procedures for ROSs generation CL measurement; MS

characterization on N2+-luminol salts, collection of the flow manifolds, N2+-luminol CL pH dependence, MS identification of N2+-luminol decay and oxidation products, N2+-luminol catalytic activity on luminol CL, reactive ROS species investigation during N2+-luminol catalyzed TMB oxidation, steadystate kinetic assay of N2+-luminol, catalytic effects of N2+phthalic acid, N2+-benzene, hydroxyl-phthalic acid, and phenol on TMB oxidation, reaction equations of two diformylhydrazine radicals during N2+-luminol oxidation, NaOH and N2+luminol optimization, plots of CL vs. H2O2 concentration,. plots of CL vs. glucose concentration, plot of CL vs uric acid concentration, N2+-luminol CL response to sub-10-7 M 1O2; table collections of N2+-luminol CL response to each ROS, the apparent Michaelis–Menten constant and maximum reaction rate of N2+-luminol, interference study on N2+-luminol CL, serum glucose determination results, and serum uric acid determination results. (docx)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ORCID Jiagen Lv: 0000-0003-2761-9789

Author Contributions J.L. devised the experiments, C.Z, H.C., and J.D. completed the experimental work. Manuscript was written by J.L. with contributions from all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 21175090).

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