Recent Progress in Organic Small-Molecule Fluorescent Probe Detection of Hydrogen Peroxide.

The fluorescent probe has become an important method for accurate detection of H2O2, with advantages of simple operation, high sensitivity, good selectivity, and real-time dynamic monitoring. This paper reviews the research progress in organic small-molecule fluorescent probe H2O2 detection methods that are based on different recognition reactions. In addition, the application prospect of fluorescent probes in the detection of trace H2O2 is anticipated.

Introduction

As one of the important reactive oxygen species (ROS), H2O2 is an important signal molecule for cell growth, proliferation, and differentiation.[1] Abnormal H2O2 can induce diabetes, inflammatory diseases, cancer, and so on. Therefore, accurate detection of trace H2O2 is necessary. In addition, H2O2 is widely used in medical sterilization, textile bleaching, agricultural pesticides, peroxide explosive raw materials, and other fields. Accurate detection of hydrogen peroxide is of great importance to industrial production and human health.

At present, the general detection of H2O2 uses redox titration, spectrophotometry, electrochemical methods, chromatography, and spectroscopy.[2] Because the sample preparation for these research methods is very complicated, they cannot dynamically reflect the change of H2O2, so it is impossible to effectively detect the concentration of H2O2 in living cells. The fluorescent probe method has many advantages including high sensitivity and selectivity, fast response, simple operation, good biocompatibility, real-time imaging, etc. With the ability to accurately detect H2O2, it has become an important tool in the field of chemical sensing and biological imaging.

Currently, organic small-molecule fluorescent probes used to detect H2O2 are mainly based on the boric acid reaction, the Baeyer–Villiger-type reaction, and the sulfonyl oxidation reaction. Therefore, this paper focuses on the research status of different recognition reactions for trace detection of H2O2.

Types of H2O2 Detection

Arylboronate-Based Fluorescent H2O2 Probes

As a recognition group, phenylborate is widely used to detect H2O2. In the presence of H2O2, arylboronic acids form an unstable tetrahedral borate complex that is negatively charged. After migration, rearrangement, and hydrolysis, phenolic compounds and boronic acids are produced. Ultimately, this causes a change in the fluorescence signal (Figure ).

Figure 1

Reaction mechanism of H2O2 recognition by arylboronate.

On the basis of the intramolecular charge-transfer mechanism (ICT), the fluorescent probe forms a strong push–pull conjugate system through electron-donating and electron-accepting groups. The fluorescence emission spectrum varies with the electron capacity of the fluorescent probe after binding with the object to be detected. Li’s group reported a novel near-infrared fluorescent probe (example 1); a quinolinium–xanthene dye was used as the fluorophore, and borate ester was chosen as the response group.[3] In the presence of H2O2, there was a strong near-infrared fluorescence signal at 772 nm (Figure ). In comparison to the emission wavelength of common hemicyanine dyes (about 700 nm), probe 1 showed a stronger fluorescence enhancement (10 times).

Figure 2

Sensing mechanism of probe 1 to H2O2.

Zhang et al. designed an off–on fluorescent probe (example 2) based on ICT for sensing H2O2.[4] A novel fused quinoxaline was used as the fluorescent group, and the boronate moiety was the reaction site. As a typical ‘‘push–pull” electron-conjugated structure, probe 2 possessed good fluorescence properties such as higher fluorescence quantum yields. The fluorescence intensity increased 11-fold after detecting H2O2 (Figure ). Probe 2 could be utilized as an efficient fluorescent tool to detect H2O2 in aqueous solutions or living systems.

Figure 3

Sensing mechanism of probe 2 to H2O2.

The mechanism of excited-state intramolecular proton transfer (ESIPT) refers to that, when the fluorescence spectrum of the donor molecule overlaps with the excitation spectrum of the receptor molecule, the excitation of the donor molecule can induce the receptor molecule to emit fluorescence. Zhou’s group reported a fluorescent probe (example 3) for detecting H2O2 based on the ESIPT mechanism.[5] Probe 3 made use of a modified 2-(20-hydroxyphenyl)benzothiazole as the fluorophore and a pinacol phenylborate ester as a reactive moiety (Figure ). The probe molecules showed almost no fluorescence signal when excited at 380 nm. However, in the presence of H2O2, the fluorescence was significantly enhanced at 542 nm, and the Stokes displacement was large (162 nm). It has been successfully used to monitor the exogenous and endogenous production of H2O2 and to identify the accumulation of H2O2 during iron poisoning.

Figure 4

Sensing mechanism of probe 3 to H2O2.

Chen et al. constructed an ESIPT-based near-infrared (NIR)-emitting ratiometric fluorescent probe (example 4) for monitoring H2O2.[6] The ESIPT process in the probe molecule is effectively blocked due to the protection of hydroxyl groups by borate esters (Figure ). In response to H2O2, the probe molecule undergoes oxidation and 1,6-elimination reactions, resulting in the release of the fluorophore, which in turn produces a bright red-shift emission (from 538 to 656 nm). In addition, probe 4 has been successfully used to detect intracellular H2O2 in living cells and zebrafish.

Figure 5

Sensing mechanism of probe 4 to H2O2.

Benzoyl-Based Fluorescent Probes for H2O2

The phenyl group is broken through oxidation by H2O2 and eventually hydrolyzed to benzoic acid. Benzil has the lowest empty orbital to accept electrons from the highest occupied orbital of the fluorophore. In the process of oxidative light-induced electron transfer, the fluorescence of the fluorophore is quenched. The fluorescence will be restored only after reacting with H2O2, resulting in strong fluorescence (Figure ).

Figure 6

Reaction mechanism of H2O2 recognition by benzoyl-based probes.

The mechanism of photoinduced electron transfer (PET) is that, after the probe is excited by light, electron transfer will occur between the excited receptor and the donor, leading to the switching on or off of the probe’s fluorescence. Hu’s group reported a probe (example 5) based on rhodamine derivatives as fluorophores.[7] With 4-nitro-α-ketoamide as the recognition group, the probe underwent a process of donor excitation light-induced electron transfer. After adding H2O2, the recognition group was cleaved to release a new fluorophore, which had a significant fluorescence at 730 nm (Figure ). The Stokes shift of the common rhodamine derivatives was less than 50 nm. In contrast, the Stokes shift of the new fluorophore was 140 nm; therefore, it could greatly reduce the influence of serious background interference in fluorescence detection. By virtue of the high selectivity and sensitivity for H2O2 detection, with a detection limit of 61 nM, probe 5 was suitable for targeting mitochondria and visually detecting endogenous H2O2 in living cells.

Figure 7

Sensing mechanism of probe 5 to H2O2.

A near-infrared fluorescent probe (example 6) based on the cysteine backbone to specifically detect H2O2 was constructed by Cao’s group.[8] By utilizing the cysteine’s characteristics of near-infrared fluorescence and absorption, probe 6 had a good ability to target mitochondria. The goal of minimizing unnecessary background noise in biological specimens was also achieved. The probe itself showed very weak fluorescence in the near-infrared region. After reacting with H2O2, the fluorescence intensity was significantly increased, and a maximum emission peak was detected at 702 nm, with a detection limit as low as 0.186 μM (Figure ). The probe had good biocompatibility and mitochondrial targeting ability. Besides, it had been successfully applied to the imaging of exogenous and endogenous H2O2 in cells and mice.

Figure 8

Sensing mechanism of probe 6 to H2O2.

Sulfonate-Based Fluorescent Probes for H2O2

Under the action of H2O2, the sulfonic acid group undergoes oxidative hydrolysis to produce fluorescence. In comparison to the borate group, the sulfonate group is more stable under hydrolysis conditions, and the pentafluorobenzene ring can improve the reactivity of the sulfonate to H2O2 (Figure ).

Figure 9

Reaction mechanism of H2O2 recognition by an arylsulfonyl ester group.

Yu’s group developed a near-infrared ratiometric fluorescent probe (example 7) for specific imaging of endogenous H2O2 in cells and in vivo.[9] The absorbance of probe 7 itself was the highest at 830 nm (Figure ). In the presence of H2O2, a new absorbance appeared at 560 nm. The fluorescence emission center of probe 7 was 836 nm. It could selectively and sensitively detect H2O2 within 200 s without interference from other ROS.

Figure 10

Sensing mechanism of probe 7 to H2O2.

Wang’s group designed a pentafluorobenzene-containing fluorescent probe (example 8) for monitoring H2O2.[10] The fluorescence of the probe was activated by designing a double-lock model system consisting of a spiro location and the object to be measured. The addition of H2O2 leads to a nucleophilic addition reaction, and the quenched groups are cleaved to form a strong conjugate system, which ultimately leads to fluorescence enhancement (Figure ). The fluorescence intensity increased significantly at 549 nm. The probe had the advantage of an emission 24 times that of open emission, indicating that the sensor could detect H2O2 in real time by the fluorescence signal under physiological conditions.

Figure 11

Sensing mechanism of probe 8 to H2O2.

Selenium Oxide Based Fluorescent Probes for H2O2

Xu and Qian reported a simple selenamorpholine-based fluorescent probe (example 9) by the combination of selenamorpholine and BODIPY fluorophore.[11] As PET inhibits protonation of selenofenomorphine, probe 9 can partially turn on fluorescence in lysosomes. Protonation of amino nitrogen atoms in selenium porphyrins changes the electronic state, leading to the quenching of the PET process and the recovery of fluorescence. The fluorescence intensity of probe 9 increased gradually with the decrease of pH from 7 to 3. Probe 9 had a higher pH response than other organelles in the lysosomal pH range of 4–6. It showed a weak fluorescence signal at 504 nm until H2O2 was gradually added, and its fluorescence intensity increased accordingly (Figure ). The probe was successfully used for fluorescence detection of endogenous/exogenous H2O2 in living cells and normal zebrafish.

Figure 12

Sensing mechanism of probe 9 to H2O2.

A new type of probe (example 10) that quickly reacts with H2O2 in cells was constructed by Koide’s group.[12] The probe underwent Mislow–Evans rearrangement with H2O2, followed by acetal hydrolysis. In a few seconds, green fluorescent molecules were produced, which in turn led to a significant increase in the fluorescence signal of the detection system (Figure ).

Figure 13

Sensing mechanism of probe 10 to H2O2.

Acetyl-Based Fluorescent Probes for H2O2

H2O2 is a good nucleophile because of the α-effect of adjacent nonbonding orbitals. Acetate as an electrophile can react with HOO– to form a negatively charged tetrahedral complex. Finally, the reaction produces a phenolic compound (Figure ).

Figure 14

Reaction mechanism of H2O2 recognition by an acetyl group.

Wang’s group reported a near-infrared fluorescent probe (example 11) based on acetyl salt.[13] In view of the advantages of the near-infrared emission, large Stokes shift, and light stability, dicyanomethyl-4-H-pyridine was used as the fluorophore, and an acetyl group was the recognition group for detecting H2O2. When H2O2 was added, the fluorescence emission at 704 nm was significantly enhanced; the detection limit was 2.1 × 10–8 M (Figure ). Moreover, the solution color could be observed by naked eyes, changing from yellow to pink. Probe 11 was successfully used to monitor and image endogenous H2O2 in mouse axillary cells.

Figure 15

Sensing mechanism of probe 11 to H2O2.

A new colorimetric near-infrared fluorescent probe (example 12) with an acetyl heterocyclic aromatic amine was designed by Wang’s group.[14] Heterocyclic aromatic amines have the advantages of a novel and simple chemical structure, large Stokes shift, and near-infrared emission wavelength. The identification group, acetyl, had the ability to enhance the ICT effects. Therefore, when the probe reacted with H2O2, the deviation of the acetyl group would lead to the obstruction of the ICT process; the fluorescence intensity gradually decreased at 700 nm (Figure ). The detection limit of the probe, which could be used to specifically detect endogenous and exogenous H2O2 in living cells, was 0.85 μM.

Figure 16

Sensing mechanism of probe 12 to H2O2.

Payne/Dakin Reaction-Based Fluorescent Probes for H2O2

H2O2 mediates the conversion of aromatic aldehydes to phenol under strong alkaline conditions. H2O2 is activated by alkali catalytic addition of nitrile to produce peroxide acid in situ. The Payne/Dakin tandem reaction activates H2O2 and aldehyde, respectively, providing a new research idea for the specific detection of H2O2. In the tandem reaction, H2O2 is activated by electron-deficient nitrile to produce peroxide acid, which is oxidized under neutral conditions. Subsequently, the original hydroxybenzaldehyde is converted to phenol (Figure ).

Figure 17

Reaction process of Payne and Dakin.

Yang’s group synthesized a fluorescent probe (example 13) that detected H2O2 through the tandem Payne/Dakin reaction as a response method.[15] In this probe, rhodamine was the fluorophore, and the salicylaldehyde group was connected to the fluorophore by carbamate. In order to form hydrogen-bond-activated aldehydes, a hydroxyl group was added to the ortho position of the aromatic aldehydes, and the Payne reaction activity was increased with CCl3CN. The protected fluorophore is not fluorescent due to electron delocalization. After being treated with H2O2 and CCl3CN, the salicylaldehyde part was effectively converted into a catechol part, which finally triggered the cleavage of carbamate to release the fluorophore (Figure ). The fluorophore rhodamine had excellent photophysical properties, such as high quantum yield, excellent light stability, pH insensitivity, and good water solubility. After adding H2O2, the fluorescence was increased significantly within 30 min, and the detection limit could reach 0.53 nM.

Figure 18

Sensing mechanism of probe 13 to H2O2.

Yang’s group reported a coumarin-based fluorescent probe (example 14) for H2O2 based on the Payne/Dakin tandem reaction.[16] Probe 14 converted electron-withdrawing aldehyde groups into electron-donating hydroxyl groups (Figure ). This probe had fast response speed and high sensitivity for detecting H2O2. The detection limit was 31 nM. It can directly display the basal and endogenous production of H2O2 in living cells.

Figure 19

Sensing mechanism of probe 14 to H2O2.

Methylenonitrile-Based Fluorescent Probes for H2O2

Gao’s group reported a novel methylenemalononitrile–BODIPY-based fluorescent probe (example 15) for highly selective detection of hydrogen peroxide in living cells.[17] Methylenonitrile has a strong electron-absorbing capacity through the double bond. Nucleophilic addition and oxidation reactions occurred with the addition of H2O2 that split the double bond, resulting in fluorescence opening and a ratio effect at 540 nm. Probe 15 had a detection limit of 2–10 μM and was suitable for use in the pH range from 6 to 10. It could be used as a highly sensitive fluorescent probe for the detection of endogenous and endogenous H2O2 in living cells (Figure ).

Figure 20

Sensing mechanism of probe 15 to H2O2.

Dual-Function Fluorescent Probes

Most of the current fluorescent probes are only suitable for the detection of one analyte and cannot fully meet the actual detection needs. Therefore, the detection of two or more target analytes by one fluorescent probe has become a research hot spot, and it is also full of challenges.

Yang et al. a constructed fluorescent probe (example 16) for detecting H2O2/H2S redox events in living cells and organisms.[18] The probe was selective and sensitive to H2O2 and H2S and generated an obvious fluorescence signal and fluorescence pattern. It exhibited a blue fluorescence with a maximum at 413 nm. When H2O2 was present, the emission maximum shifted to 486 nm. The ratio between the fluorescence intensities at 486 and 413 nm increased with the accumulation of H2O2. However, in the presence of H2S, a bright red fluorescence signal was observed, and the original blue fluorescence was intensified in a concentration-dependent manner (Figure ). The detection limits of probe 16 for H2O2 and H2S were estimated to be 0.044 and 0.058 μM, respectively. The probe’s excellent multicolor imaging capabilities were ideal for monitoring dynamic H2O2 and H2S redox processes in living cells and organisms.

Figure 21

Sensing mechanism of probe 16 to H2O2/H2S.

Yang’s group designed a novel dual-function fluorescent probe (example 17) for the rapid detection of HSO3– and H2O2 in aqueous solution and living cells.[19] Rapid detection of HSO3– in the aqueous phase was based on a 1,4-addition reaction, with a minimum detection limit of 120 nM (Figure ). The detection of H2O2 was achieved by oxidation of phenylboric acid, and the minimum detection limit was 70 nM.

Figure 22

Sensing mechanism of probe 17 to H2O2/H2S.

Wang’s group constructed a dual-ratiometric fluorescent probe (example 18) that can simultaneously detect HClO and H2O2 by two channels (Figure ).[20] In probe 18, coumarin was the fluorescent parent, and the recognition group of HClO and H2O2 was skillfully combined. The probe had high selectivity and sensitivity to H2O2 and HClO and meanwhile could be used to monitor the generation of endogenous H2O2 and HClO.

Figure 23

Sensing mechanism of probe 18 to H2O2/HClO.

Conclusions and Outlook

At present, the research of reactive organic small-molecule fluorescent probes that recognize H2O2 is mainly based on the oxidation mechanism of phenylborate, the Baeyer–Villiger oxidation rearrangement reaction, and the sulfonate cleavage mechanism. Although the large number of fluorescent probe examples mentioned above can achieve the goal of detecting trace amounts of H2O2, there are still certain shortcomings in detection. For example, phenylborate can also react with ONOO– and ClO–, and the reaction speed is much faster than that with H2O2. Therefore, it is of great significance to explore new reaction mechanisms to detect H2O2. In the past 2 years, the tandem Payne/Dakin reaction has provided a new option for preparing H2O2-selective probes. This reaction mechanism affects the photophysical state of the fluorophore through significant changes in electron-withdrawing aldehydes and electron-donating hydroxyl groups, which in turn leads to visible changes in its optical properties.

The detection of trace H2O2 by organic small-molecule fluorescent probes is mainly applied in the biomedical field. The researchers modified the dye matrix structure and multiple active sites with excellent fluorescence properties to recognize H2O2. The reaction mechanism affects the change of the fluorescence signal and finally achieves the purpose of detection.

The development of excellent fluorescent probes with dual detection functions has received much attention. Being able to meet the needs of identifying two target analytes means that this fluorescent probe needs to be accurately designed with two response modes and have excellent fluorescence imaging performance. The ratio fluorescent probe can measure the fluorescence of two different emission wavelengths, realize the built-in correction of interference factors, and is more suitable for qualitative and quantitative detection. In addition, near-infrared fluorescence with emission and excitation wavelengths of 600–900 nm can penetrate deep tissue, prevent the interference of background fluorescence, reduce tissue damage, and facilitate in vivo imaging applications.

In conclusion, it is a future research goal to explore multifunctional fluorescent probes with excellent synthetic performance, low cost, high sensitivity, good specificity, short reaction time, and real-time detection.