Sensitive and specific detection of peroxynitrite and in vivo imaging of inflammation by a “simple” AIE bioprobe

Huilin Xie a, Jingtian Zhang b, Chao Chen ab, Feiyi Sun a, Haixiang Liu c, Xinyuan He a, Kristy W. K. Lam a, Zhao Li a, Jacky W. Y. Lam ac, Guo-Qiang Zhang *b, Dan Ding b, Ryan T. K. Kwok *ac and Ben Zhong Tang *acde
aDepartment of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute for Advanced Study, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, China. E-mail:
bKey Laboratory of Bioactive Materials, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, China
cHKUST Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area Hi-Tech Park, Nanshan, Shenzhen 518057, China
dSCUT-HKUST Joint Research Institute, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China
eAIE Institute, Guangzhou Development District, Huangpu, Guangzhou 510530, China

Received 30th November 2020 , Accepted 31st December 2020

First published on 5th January 2021


A luminogenic bioprobe TPE-DMAB for simple and specific detection of peroxynitrite (ONOO) has been developed. TPE-DMAB exhibits aggregation-induced emission (AIE) characteristic and shows fluorescence enhancement (up to 100-fold) upon cleavage of phenylboronic moieties by ONOO. TPE-DMAB can be facilely synthesized and displays excellent specificity and sensitivity towards ONOO (detection limit of 54 nM). Based on these novel properties of TPE-DMAB, we further produced its encapsulated nanoparticles and successfully used them to detect endogenous ONOO in macrophage cells and visualize the inflammation sites of mice, verifying the great potential of TPE-DMAB for biological applications.


Reactive oxygen and nitrogen species (RONS) are critically important oxidative stress mediators in various biological processes, such as immune activation and cell signaling.1–3 But excessive RONS release may also result in harmful effects, thereby causing cell death and biological structure damage.4–6 Peroxynitrite (ONOO), one of the RONS, is an biological oxidant that contributes to the regulation of redox homeostasis,7 activation of signal transductions,8 and induction of normal immune responses.9,10 ONOO is constantly being produced and consumed, maintaining a normal trace amount.11 However, an overwhelming level of ONOO is often accompanied by inflammatory,12,13 neurodegenerative disorders,14,15 rheumatoid arthritis,16 cancer,17 and other diseases.18 Therefore, developing a bioprobe that enables monitoring of the ONOO level on-site with high sensitivity is of extreme significance in the early diagnosis of diseases in the human body.

The primary methods for detecting RONS include chemiluminescence,19,20 fluorescence,21–25 and electrochemical-based approaches.26,27 Among them, fluorescence-based probes have drawn increasing attention from scientists because they offer highly sensitive, noninvasive, and real-time detection.28,29 However, only a few fluorescent systems could realize in vitro and in vivo detection of ONOO.30,31 In addition, conventional fluorescent dyes, such as rhodamine, fluorescein, and cresyl violet, are highly emissive in their dilute solutions but their light emission is quenched partially or even completely upon aggregate formation.32,33 As a result of the aggregation-caused quenching (ACQ) effect, these conventional fluorescent probes usually operate in a fluorescence turn-off mode and suffer from the insensitive and non-linearity response, which greatly limits their applications.34 In contrast, luminogens with aggregation-induced emission (AIE) features fluoresce weakly or not at all in dilute solutions but are brightly fluorescent when aggregated.35–40 The AIE effect permits the use of dye solutions with a high concentration for bioassays and enables the development of turn-on biosensors with superior sensitivity by taking the advantage of luminogenic aggregation.41–44 Based on these unique characteristics of the AIE effect, several probes have been designed to detect various RONS under distinct conditions,45 but only a few could specifically detect ONOO and even fewer could realize in vivo detection.46–48 We have previously developed a fluorescent light-up bioprobe by encapsulating an AIE luminogen (TPE-IPB) with a lipid-PEG matrix for selective detection of in vivo inflammation with increased ONOO concentrations.49 However, the synthesis of TPE-IPB involved complicated procedures and TPE-IPB was not chemically stable as the high reactivity of the Schiff base. Therefore, new AIE bioprobes with simple molecular structure, good chemical stability, and superior sensitivity towards ONOO would be more valuable for practical applications.

In this work, a novel turn-on fluorescent bioprobe, namely TPE-DMAB, for specific detection of ONOO has been developed. A commercially available AIE luminogen (TPE-DMA) was coupled with 4-(bromomethyl)phenylboronic acid, which serving as the recognition site for ONOO to yield the probing molecule TPE-DMAB (Scheme 1). The cationic tertiary amino groups, and the phenylboronic acid moiety render TPE-DMAB exhibit hydrophilicity, making it display a weak emission in the aqueous medium. However, TPE-DMAB could be cleaved through oxidation in the presence of OONO, followed by the release of p-quinonemethide and eventually yield TPE-DMA. TPE-DMA reveals strong hydrophobicity and shows typical AIE properties. As a consequence, the aggregation forms and turns on the bright fluorescence due to the poor solubility of TPE-DMA in the aqueous solution. Notably, TPE-DMAB quickly responses to ONOO and converts into TPE-DMA, which triggers a dramatic fluorescence enhancement up to 100-fold. Also, a good linear response exists between fluorescence intensity and ONOO concentration in the range of 3–12 μM, and the detection limit is 54 nM. Furthermore, TPE-DMAB is highly selective for ONOO over various RONS. Encouraged by these properties, we further fabricated a fluorescent light-up nanoprobe by encapsulating TPE-DMAB with a lipid-PEG matrix for in vivo visualization of inflammation sites on a mouse model.

image file: d0qm01004a-s1.tif
Scheme 1 Synthetic route to TPE-DMAB and design rationale for ONOO detection.

Results and discussion

Materials and instruments

TPE-DMA was purchased from AIEgen Biotech Co., Ltd., and it was used as received without further purification. All other chemicals and solvents were purchased either from Sigma-Aldrich or VWR Chemicals and were used as received. UV-vis absorption spectra were recorded on a Varian Cary 50 UV-visible spectrophotometer. Fluorescence spectra were measured on the Edinburgh Instruments Spectrofluorometer FS5. The size and morphology of nanoparticles were investigated using transmission electron microscopy (JEM-2010F JEOL, Japan). The size distribution of nanoparticles was analysed by the dynamic light scattering with a particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90 degrees at room temperature. NMR spectra were measured on a Bruker AVIII 400 MHz NMR spectrometer. The mass spectrum was obtained on a Xevo G2-XS Top mass spectrometer by Water's at the ESI mode.

Synthesis and characterization of TPE-DMAB

The synthetic route of TPE-DMAB and its reaction with ONOO were depicted in Scheme 1. TPE-DMA (100 mg, 0.24 mmol) and 4-(bromomethyl)phenylboronic acid (120 mg, 0.56 mmol) were added in a round-bottom flask and then 20 mL of acetonitrile was added. The reaction was kept stirring at room temperature for 30 h under a nitrogen atmosphere. The solvent was removed under reduced pressure and then the crude product was washed with excessive ethyl acetate to afford TPE-DMAB as a white solid in a yield of 23%. 1H NMR (400 MHz, CD3OD) δ (ppm): 8.19 (s, 4H), 7.71 (d, J = 8.1 Hz, 4H), 7.57 (d, J = 8.2 Hz, 4H), 7.26 (t, J = 7.3 Hz, 4H), 7.20 (d, J = 7.1 Hz, 2H), 7.12 (d, J = 8.9 Hz, 4H), 7.02 (d, J = 7.0 Hz, 4H), 6.78 (d, J = 8.2 Hz, 4H), 4.90 (s, 4H), 3.49 (s, 12H). 13C NMR (400 MHz, CD3OD) δ (ppm): 145.1, 142.8, 142.4, 140.5, 136.9, 134.6, 132.2, 131.7, 131.0, 129.9, 128.7, 127.9, 122.0, 72.6, 53.1. HRMS: m/z calcd for [M-H]+ C44H45B2N2O4+ 687.3560; found 687.3579. The corresponding NMR spectra and mass spectra were depicted in Fig. S2–S4 (ESI).

Photophysical properties of TPE-DMA and TPE-DMAB

The absorption and fluorescence properties of TPE-DMA and TPE-DMAB were investigated. TPE-DMA can be dissolved in most organic solvents such as THF and chloroform. In a dilute THF solution, TPE-DMA shows a board absorption band with a maxima at 365 nm (Fig. S5, ESI). It is almost non-emissive in the dilute THF solution, indicating the active intramolecular motion of TPE-DMA dissipates most of the exciton energy. To further demonstrate its AIE characteristics, we recorded its photoluminescence (PL) spectra in THF/H2O mixtures with different water fractions (Fig. S6 and S7, ESI). When the water fraction is less than 70%, there is no emission. However, by further increasing the water fraction, the fluorescence intensity of TPE-DMA increases dramatically with an obvious emission peak (λem) at 530 nm, demonstrating a typical AIE property. On the other hand, TPE-DMAB exhibits poor solubility in THF but it can be well dissolved in polar solvents like DMSO. The absorption spectrum of TPE-DMAB was measured in DMSO and its absorption was found in the range of 280–440 nm with a maxima at 315 nm (Fig. S8, ESI). The blue-shifted absorption peak is attributed to the existence of electron-withdrawing cationic tertiary amino groups of TPE-DMAB.

The fluorescent response of TPE-DMAB to ONOO

Fig. 1a shows the PL spectra of TPE-DMAB after incubation with different concentrations of ONOO. In the absence of analyte, TPE-DMAB in the mixture of DMSO/PBS (1[thin space (1/6-em)]:[thin space (1/6-em)]99, v/v) is almost non-emissive. However, an emission peak at 530 nm appears and the intensity rises gradually along with the increasing of ONOO concentration. A 100-fold fluorescence enhancement is observed when the ONOO concentration reaches 20 μM. Furthermore, a good linear relationship exists between fluorescence intensity and ONOO concentration in the range of 3–12 μM (inset of Fig. 1b). The detection limit of ONOO is estimated to be 54 nM based on 3σ/slope. The proposed reaction mechanism of TPE-DMAB and ONOO is depicted in Fig. S1 (ESI).
image file: d0qm01004a-f1.tif
Fig. 1 (a) Photoluminescence (PL) spectra of TPE-DMAB incubated with different ONOO concentrations. (b) Plot of I/I0vs. ONOO concentrations. I0 and I are the PL intensities of TPE-DMAB (10 μM) before and after treatment with ONOO at 530 nm, respectively. Inset: Linear range for ONOO detection. (c) Kinetic studies of fluorescence activation of TPE-DMAB (10 μM) by ONOO in PBS buffer (pH 7.4). (d) Plot of I/I0vs. various reactive oxygen and nitrogen species (RONS) in PBS buffer (pH 7.4). I and I0 are the PL intensities of TPE-DMAB (10 μM) in the presence and absence of RONS (10 μM), respectively.

Then we studied the reaction kinetics of TPE-DMAB towards ONOO. Upon addition of different concentrations of ONOO to the solution of TPE-DMAB, the normalized PL intensity versus the reaction time was plotted in Fig. 1c. The PL intensity of the probe grow dramatically at the beginning and reach a maximum intensity within 8 min. Moreover, the higher concentration of ONOO leads to a more rapid and larger PL enhancement. While the PL intensity of the control group remains unchanged after 8 min, indicating the good chemical stability of TPE-DMAB.

The pH effect on the detection is further evaluated (Fig. S9, ESI). The results show that the optimum pH for the probe to detect ONOO is pH = 7.4, with the largest PL enhancement. The probe also works well in basic buffers, but it cannot operate in acidic conditions as low pH is not favourable for the cleavage of phenylboronates.

The selectivity of the fluorescent probe was investigated. A series of RONS including ClO, H2O2, ˙OH, tert-butyl hydroperoxide (TBHP), ˙OBu, O2, 1O2, NO2, NO3 and ONOO were selected to treat with TPE-DMAB. Remarkably, only ONOO could significantly trigger the fluorescence enhancement, while other RONS show negligible fluorescent response (Fig. 1d). The results indicate TPE-DMAB is highly selective to ONOO.

Fabrication of TPE-DMAB nanoparticles as a nanoprobe for ONOO detection

To increase the solubility of TPE-DMAB and reduce the usage of cosolvent, we then fabricated it into nanoparticles (NPs) by encapsulating TPE-DMAB with the DSPE-PEG2000.50 The detailed procedure of nanoparticle fabrication can be found in ESI. The hydrodynamic diameter of the TPE-DMAB NPs was found to be around 150 nm by dynamic light scattering (DLS) (Fig. 2b). The transmission electron microscopy image indicates that the TPE-DMAB NPs are in a uniform spherical shape and the sizes are consistent with the DLS results. In the nanoparticles, because TPE-DMAB has two charges, the molecules are not well arranged and show very loose arrangement. ONOO is a small molecule, it can enter the nanoparticles easily. When the ONOO molecules react with the probe, the fluorophore, TPE-DMA, could be generated, which can form tight aggregates and show largely enhanced emission (Fig. 2a).
image file: d0qm01004a-f2.tif
Fig. 2 (a) Schematic illustration of fluorescence response of TPE-DMAB nanoparticles (NPs) to ONOO. (b) DLS profile of TPE-DMAB NPs. Inset: TEM image of TPE-DMAB NPs. (c) PL spectra of TPE-DMAB NPs treated with different ONOO concentrations. Inset: Fluorescent images of TPE-DMAB NPs in PBS buffer (pH 7.4) before and after treatment with 200 μM ONOO.

The fluorescent response of TPE-DMAB NPs to ONOO

We subsequently determined the performance of TPE-DMAB NPs in sensing ONOO. The fluorescent response of TPE-DMAB NPs to ONOO behaves similarly to the TPE-DMAB molecule. As shown in Fig. 2c, the fluorescence intensity gradually enhanced upon increasing the concentration of ONOO from 30 to 200 μM. Meanwhile, the TPE-DMAB NPs also show very good selectivity for ONOO (Fig. S10, ESI), and it could be activated within the pH ranging from 6.8 to 7.4 (Fig. S11, ESI). Notably, the ONOO production rate in the inflammation-related regions could reach 50–100 μM min−1,14,18,20 Therefore, TPE-DMAB NPs are very suitable to be used as a nanoprobe for visualizing inflammation areas by taking advantages of the rapid and sensitive fluorescent response of TPE-DMAB to ONOO.

In vitro detection of ONOO in macrophage cells

TPE-DMAB NPs were first applied to detect endogenously generated ONOO in macrophage cells. RAW 264.7 macrophage cells, which are known to induce the elevated generation of ONOO upon stimulation with bacterial endotoxin lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). After incubation with TPE-DMAB NPs for 3 h at 37 °C, negligible emission could be observed from the origin RAW 264.7 cells (Fig. 3a). While those cells treated by LPS and PMA exhibited strong yellow fluorescence (Fig. 3b), indicating the formation of TPE-DMA aggregates from the reaction of the TPE-DMAB NPs with ONOO. To further prove that the fluorescence enhancement is induced by the endogenously generated ONOO, RONS scavenger acetylcysteine (NAC) was added to the macrophage cells before the cells were treated with LPS and PMA. After pre-incubation with NAC for 2 h, only weak fluorescence could be observed in the macrophage cells (Fig. 3c). These results further indicate that the fluorescence is activated by the endogenously generated ONOO, and demonstrate that TPE-DMAB NPs are suitable for detecting ONOO in stressed cells. Moreover, the cytotoxicity of the presented NPs was evaluated via MTT assays (Fig. S12, ESI), and the results indicate that the nanoprobe possesses good biocompatibility.
image file: d0qm01004a-f3.tif
Fig. 3 CLSM images of live murine macrophages (RAW 264.7) incubated 3 h with TPE-DMAB NPs (3 μg mL−1). (a) Control: cells without chemical treatments. (b) Cells treated with LPS (2 h) and PMA (0.5 h). (c) Cells treated with NAC before treating LPS (2 h) and PMA (0.5 h), then treated with NAC (2 h) again. [LPS] = 1 mg mL−1; [PMA] = 5 mg mL−1; [NAC] = 1 mM. Scale bars: 20 μm.

In vivo imaging of inflammation in a mouse model

After verifying the detection capacity of TPE-DMAB NPs for the endogenous ONOO in stressed macrophage cells, the TPE-DMAB NPs were applied to specifically imaging of inflammation sites on mice. It has been reported that during the inflammation process, immune cells such as neutrophils and macrophages are recruited to the inflammation sites and stimulated to release a mass of ONOO.51,52 To verify whether TPE-DMAB NPs could be used to image ONOOin vivo, LPS-induced inflammation-bearing mice were chosen and incubated with TPE-DMAB NPs. As demonstrated in Fig. 4, intense fluorescence signals were observed in the inflammation sites after post injection of TPE-DMAB NPs for 30 min, while no detectable fluorescence signal was found on the mouse that without injection of the fluorescent probe or the mouse injected by uric acid (a natural RONS scavenger). This experiment indicates that TPE-DMAB NPs are highly desirable for in vivo detection of ONOO and imaging of inflammation.
image file: d0qm01004a-f4.tif
Fig. 4 In vivo fluorescence images of LPS-induced inflammation in nude mice. (a) Before and (b) after 30 min in situ injection of TPE-DMAB NPs. (c) Image was taken after pretreatment with uric acid in PBS buffer 1 h before injecting the TPE-DMAB NPs. The blue circles indicate the inflammatory region at the mice's left-back.


In summary, we have developed an AIE-active fluorescent bioprobe, namely TPE-DMAB, via a one-step simple modification of a commercially available AIE luminogen TPE-DMA. TPE-DMAB could specifically detect peroxynitrite with minimal interference from other reactive oxygen and nitrogen species. Meanwhile, the fluorescence intensity had a good linear relationship with the ONOO concentration in the range of 3–12 μM, and the detection limit was 54 nM. We also demonstrated the TPE-DMAB NPs can realize in vitro ONOO detection and in vivo visualization of inflammation.

Conflicts of interest

There are no conflicts to declare.


The authors acknowledge funding from the National Natural Science Foundation of China (21788102, 51873092 and 51961160730), the Research Grants Council of Hong Kong (N-HKUST609/19, 1606620 and C6009-17G), the Innovation and Technology Commission (ITC-CNERC14SC01), the National Key Research and Development Program of China (2018YFE0190200), the National Key R&D Program of China (Intergovernmental Cooperation Project, 2017YFE0132200), the Science and Technology Plan of Shenzhen (JCYJ20180507183832744), the Tianjin Science Fund for Distinguished Young Scholars (19JCJQJC61200), and the Fundamental Research Funds for the Central Universities, Nankai University.

Notes and references

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Electronic supplementary information (ESI) available. See DOI: 10.1039/d0qm01004a
These authors equally contributed to this work.

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