1 Introduction
One of the most important reactive oxygen species (ROS) is endogenous hypochlorous acid (HClO), which is catalyzed by myeloperoxidase (MPO) in leukocytes under certain physiological conditions and is a double-edged sword in the life process
[1][2]. On the one hand, HClO can protect human health by killing pathogens and bacteria that invade the immune system of organisms
[3]; On the other hand, excessive HClO can lead to the oxidation of biomolecules such as proteins, nucleic acids, lipids, and enzymes, causing tissue damage, inflammation, and a range of diseases such as liver ischemia-reperfusion injury and even cancer
[4⇓⇓~7]. In view of the special importance of hypochlorous acid in biology and medicine, it has become a hot topic in the field of biomedicine to explore methods with high selectivity, high sensitivity and easy detection of endogenous hypochlorous acid
[8].
There are many detection methods for hypochlorous acid, such as starch-potassium iodide method, N, N-diethyl-p-phenylenediamine colorimetry, chemiluminescence method, spectrophotometry and so on
[8][9]. However, the amount of hypochlorous acid produced in normal cells of organisms is very small, about 0.47 nmol of hypochlorous acid molecules per minute per 10 neutrophils. The method for detecting the hypochlorous acid is complex to operate and requires to be carried out in an organic medium in many cases, and the physiological environment in an organism is an aqueous medium environment, which is not favorable for detecting the concentration and distribution of the hypochlorous acid by using the method. Therefore, it is a key issue to find a suitable detection method for hypochlorous acid in general physiological environment.
Fluorescent probe technology has been widely concerned because of its good selectivity, high sensitivity, low detection limit, simple operation and low cytotoxicity. The luminescence mechanisms of the fluorescent probes mainly include photoinduced electron transfer (PET), intramolecular charge transfer (ICT), excited state intramolecular proton transfer (ESIPT), aggregate fluorescence enhancement (AIE), electron energy transfer (EET) and fluorescence resonance energy transfer (FRET). To date, a variety of fluorescent probes for HClO detection have been developed based on unique reaction mechanisms, such as oxidative cleavage of lactams or lactones, oxidative cleavage of double bonds, oxidation of thioethers or selenides, or other oxidation-based methods
[10⇓⇓⇓~14][7,15⇓⇓⇓⇓⇓ ~21][22⇓⇓⇓⇓⇓~28][29⇓⇓⇓~33].
In view of the fact that fluorescent probes can effectively detect hypochlorous acid in vivo, this paper mainly reviews the progress in the design, synthesis and biological application of fluorescent probes for hypochlorous acid in the past three years. These fluorescent probes have the advantages of high spatial and temporal resolution, high sensitivity and easy observation in cell imaging technology, and are widely used in biology and medicine.
2 Fluorescent probes for hypochlorite based on different fluorophores
Hypochlorous acid fluorescent probes are mainly hypochlorous acid recognition groups linked to different fluorophores, combined with different reaction mechanisms, thus releasing fluorescent signals. According to the different fluorescent groups, it can be divided into BODIPY, rhodamine, fluorescein, coumarin, cyanine, benzothiazole, naphthalimide and other fluorescent probes.
2.1 BODIPY-based fluorescent probe for HClO
Treibs et al. First synthesized BODIPY molecule in 1968. Its molecular configuration is that two pyrrole ring molecules are fixed in the same plane through boron bridge bond and methine bridge bond, thus improving the rigidity of the whole molecular structure. This structure enables the molecule to produce strong fluorescence under the action of laser
[34].
As the parent structure of the fluorescent probe (Figure 1), BODIPY has the advantages of stable chemical properties, high molar extinction coefficient and high fluorescence quantum yield, and can introduce modification groups on different reaction sites to make the molecule show different functional characteristics. Therefore, the design of fluorescent probes has been widely developed and studied, which promotes the development of fluorescent probe technology.
Gao et al. designed and synthesized BODIPY fluorescent probe 1 based on aldoxime recognition group, which can specifically detect HClO (fig. 2)
[35]. The probe 1 was oxidatively dehydrogenated by HClO, which eliminated the rotation and isomerization, thus producing a fluorescence signal with a detection limit of 2. 33 μM. The probe showed good biocompatibility, and the imaging in RAW264.7 cells showed that the probe had good permeability, and exogenous or endogenous HClO could cause bright red fluorescence in the cells, which showed its potential hypochlorite specific sensing ability in biological systems.
Shi et al. designed and synthesized a BODIPY fluorescent probe 2 containing tellurium atoms (fig. 3)
[36]. X-ray diffraction analysis showed that divalent tellurium formed a relatively small angle (96.6 (8) °), so the tellurium atoms were easily oxidized, thus inhibiting the photoinduced electron transfer of tellurium atoms to BODIPY. The probe utilizes the rapid oxidation characteristic of tellurium atoms to detect low-concentration ClO
-, and green fluorescence can be clearly seen by naked eyes under fluorescent light. The detection limit is 0.12 μM and the probe is suitable for a wide pH range (4 ~ 12). Confocal fluorescence microscopy imaging in RAW264.7 and HepG2 cells showed that the probe could be used as an effective fluorescent probe for the detection of ClO
- in living cells.
Gao et al. designed and synthesized a new fluorescent probe 3 based on aza-BODIPY derivatives (fig. 4)
[37]. In the detection of HClO, due to the cleavage of the imine C = N bond, resulting in the formation of a fluorescent aza-BODIPY-CHO, it showed a rapid red fluorescence response with a detection limit of 0.27 μM. Fluorescence imaging experiments of the probe on living cells and RAW264.7 living cells showed red fluorescence in the cells, indicating the potential application of detecting exogenous and endogenous HClO in living cells.
Jin et al. designed and synthesized a water-soluble BODIPY derivative fluorescent probe 4 (fig. 5)
[38]. The probe can rapidly and selectively detect hypochlorous acid. Pyridinium salt was linked to BODIPY to improve water solubility, and the recognition group of HClO was 4-methylmercaptophenoxy. After the addition of HClO, the PET process was terminated due to the oxidation of thioether to sulfoxide, and the probe showed a unique response with a significant increase in fluorescence intensity (~ 100-fold) within 1 min. The probe has a linear response in the range of 0 μM to 10 μM with a detection limit of 53 nM. The solubility of the probe in PBS (10 mM, pH 7.4) is 83.9μg/mL
-1 and the fluorescence quantum yield is very low (Φ
f=0.0013). The probe has been successfully applied to HClO imaging in living cells.
Sun et al. designed and synthesized BODIPY fluorescent probe 5 (fig. 6)
[39]. The probe has ultra-sensitivity (LOD = 0.56 nM) and fast response (< 1 s). Unlike traditional monatomic electron donors, the electron-rich pyrrole ring in the probe was first used to construct a PET probe that suppresses fluorescence (Φ
f=0.006) through the process of "enhancing PET". When the probe is converted to the corresponding ketone by HClO oxidation, the fluorescence gradually increases (Φ
f=0.347). The probe shows extremely low background fluorescence and high signal-to-noise ratio when recognizing HClO, and detects HClO in cancer cells for the first time.
Xu Et al. designed and synthesized three BODIPY-based selenides 6, 7, 8 with different hydrocarbon groups (Et, PhCH
2, and Ph) (fig. 7)
[40]. All of the three non-fluorescent probes can induce the formation of fluorescent selenides by HClO/ClO
-. The response time and sensitivity were in the order of Probe 6 (2 s, 0.3 nM) < Probe 7 (5 s, 0.8 nM) < Probe 8 (> 100 s, 9.2 nM). The selectivity of probe 7 for HClO was higher than that of probe 6. Probe 7 can target both exogenous and endogenous HClO in live cell mitochondria.
Duan et al. designed and synthesized a high-brightness red BODIPY fluorescent probe 9 (fig. 8)
[41]. After the thioether group was oxidized to sulfoxide by HClO, the probe showed an obvious ratiometric fluorescence response to the ClO
- with a fast response (< 30 s) and a low detection limit (59 nM). The advantages of the probe are excellent photostability, high fluorescence quantum yield, stable fluorescence output (resistant to pH changes), low cytotoxicity and good membrane permeability, and the probe has been successfully applied to the imaging of endogenous and exogenous HClO/ClO
- in zebrafish and mice.
Liu et al. designed and synthesized a water-soluble BODIPY-P fluorescent probe 10 (fig. 9)
[42]. After the addition of NaClO, the ClO
- promotes the oxidative cleavage of the C = C double bond of the olefin, so that the probe responds quickly and shows a visible fluorescence change from red to orange, and has good selectivity, high sensitivity and low detection limit (31.6 nM). The probe is cell permeable, has low toxicity and is preferentially distributed in mitochondria, and can effectively observe exogenous and endogenous HClO in living cells.
2.2 Fluorescent probe for HClO based on fluorescein
Fluorescein is a molecule of the xanthene family that is often used to design fluorescent dyes. It was first prepared in 1871 from resorcinol and phthalic anhydride by Friedel-Crafts acylation. The structure is shown in Figure 10
[29]. In acid environment, it exists in the form of closed ring and has no fluorescence. In an alkaline environment, the spiro lactone opens and then fluoresces strongly. As a dye molecule, fluorescein has many advantages, such as good water solubility, high molar absorption coefficient, high fluorescence quantum yield, high emission peak and low cytotoxicity, and is widely used in biochemistry, medicine and other fields.
Zhang et al. designed and synthesized a novel HClO fluorescent probe 11 based on the rapid oxidation of p-aminophenyl ether (fig. 11)
[43]. The fluorophore chosen to increase the fluorescence response is methoxyfluorescein. The helicized probe 11 is colorless and non-fluorescent, and upon reaction with HClO, the spiro structure is unlocked to the coupling conformation and green fluorescence (> 1000-fold) is released. The probe shows a fast response (t
1/2<30 s) to HClO and a very low detection limit (0.65 nM) with high sensitivity and selectivity. The probe has been successfully applied to the detection of exogenous and endogenous HClO in living cells.
Peng et al. successfully developed a light-controlled fluorescent probe 12 (fig. 12) capable of directly detecting ClO
- near the membrane by combining a light-controlled group (nitrobenzyl), a membrane localization unit (cholesterol), and a ClO
- recognition group (benzoyl) with fluorescein
[44]. The probe has low detection limit (260. 77 nM) and high selectivity for ClO
-. In the absence of ClO
-, the fluorescence intensity of probe 12 was very weak, and after reaction with ClO
-, the fluorescence intensity increased significantly (pH = 7.0 ~ 10.0). Cell-based experiments show that the probe has low cytotoxicity and the ability to directly detect the ClO
- in the near membrane region.
2.3 Rhodamine-Based HClO Fluorescent Probe
Rhodamine compounds belong to a class of basic fluorescent dyes. Its structure contains an oxanthene and a spiro ring, which can be designed and modified by introducing groups to control the opening or closing of the spiro ring structure so as to achieve the purpose of detecting substances. Based on this special structure, rhodamine is often used in probes designed as "off-on" probes. Rhodamine fluorescent dyes have many excellent characteristics, such as stable photochemical properties, long excitation wavelength, not affected by pH and so on. Rhodamine B and Rhodamine 6G compounds are the most widely used rhodamine compounds (Figure 13), in which the parent xanthene plays an important role in the fluorescence characteristics of rhodamine, so the introduction of different groups on the aromatic ring can change the fluorescence properties of probe molecules. In the case of high pH, rhodamine exists in the lactone structure, and the molecule will not emit fluorescence. When the pH is low, it exists in the quinoid structure, and the molecule will fluoresce.
Mao et al. designed and synthesized a near-infrared fluorescent probe 13 for lysosomal HClO imaging based on the silicon-containing rhodamine structure for the first time (fig. 14)
[45]. The probe uses morpholine as a lysosomal targeting group and a HClO-induced cyclization reaction site as a response group. The probe 13 has high selectivity and good sensitivity, the linear range is 5.0×10
-8~1.0×10
-5M, and the detection limit is 2.0×10
-8M. The probe was used to observe endogenous HClO in lysosome-involved inflammatory responses, including cells infected with bacteria and mouse models of inflammation. This probe has potential applications in the study of the biological function and pathology of HClO in lysosomes.
Gong et al. designed and synthesized a two-photon fluorescence probe 14 with long-wavelength emission (fig. 15)
[46]. The fluorescence of the probe was significantly enhanced (~ 106-fold) in the detection of HClO in vitro due to a specific HClO-induced cyclization reaction. The probe has a rapid response time, high sensitivity (LOD = 40 nM), high selectivity, and low toxicity. The two-photon fluorescence probe can be used for detecting HClO in living cells and tissues.
Wang et al. designed and synthesized a ratiometric two-photon rhodamine derivative fluorescent probe 15 (fig. 16)
[47]. When probe 15 detects HClO, the lactam is cleaved by HClO to open the rhodamine spiro ring and restore the conjugated system of rhodamine, thus releasing red emission light, and the fluorescence intensity is slightly reduced at 471 nm, while the fluorescence intensity is enhanced near 580 nm. The probe has high selectivity. The probe can image exogenous and endogenous HClO in living cells, and can also detect HClO in living tissues and zebrafish.
Hu et al. prepared a coumarin-rhodamine ratio fluorescent probe 16 with a thiosemicarbazide group as a linker based on the TBET (via bond energy transfer) effect (fig. 17)
[48]. The probe can display the characteristic fluorescence emission of coumarin, and after HClO is added, the connecting group is reacted to form oxadiazole, so that the rhodamine spiro ring is opened. Due to HClO triggering the intramolecular desulphurization-cyclization cascade of mercaptohydrazine, the double emission and absorption bands change significantly. The probe has the advantages of high selectivity, good sensitivity (140 nM), fast response time (< 2 min), and detection system pH range of 3 to 7. 6. The probe was applied to the determination of spiked HClO in water samples and endogenous HClO in living cells.
Li et al. designed and synthesized a fluorescent probe 17 based on rhodamine (Fig. 18). The probe 17 was designed and synthesized by functionalization with phenyl isothiocyanate. Due to the cyclization of rhodamine-thiosemicarbazide, rhodamine-oxadiazole was formed in the presence of HClO, resulting in enhanced fluorescence
[49]. The probe 17 has excellent selectivity and high sensitivity to HClO, and functions well under physiological pH conditions. Confocal microscopy images of HeLa cells showed that the fluorescence intensity of probe 17 was significantly increased after reaction with HClO.
Ren et al. designed and synthesized a single rhodamine-based fluorescent probe 18 (fig. 19)
[50]. The probe can detect oxidized (HClO) and reduced (H
2S) substances in organelles (lysosomes). Reactions of the probe with H
2S and HClO result in blue and red channel emissions, respectively, providing different signal responses to oxidizing and reducing species in the lysosome. The probe realizes dual-channel imaging of endogenous hypochlorous acid and hydrogen sulfide in lysosomes of living cells, respectively.
2.4 Coumarin-Based Fluorescent Probe for HClO
Coumarin is a kind of benzofuranone compound which is widely distributed in natural species. Because of its unique photophysical properties, coumarin is used as the parent material of synthetic probes. Coumarin fluorescent probes have a wide range of applications and excellent photochemical properties. The structure of coumarin is shown in fig. 20. Derivatives with different functions can be synthesized by modifying the eight different sites of the coumarin, so as to further improve the defects of the coumarin fluorescent probe and play a greater role in the application of biological imaging.
Wang et al. designed and synthesized a fluorescent probe 19 with excellent selectivity (fig. 21)
[51]. The fluorophore of the probe is 4-methylcoumarin, and the HClO recognition group is dimethylthiocarbamoyl. After the probe reacts with hypochlorous acid, a coumarin derivative is formed, so that the fluorescence is remarkably enhanced. In addition, the probe can respond to HClO in solution within 60 s with a detection limit of 34. 75 nM. The probe can be used to detect HClO in tap water, and the confocal images confirm that the probe can also be used to identify HClO in living cells.
Wu et al. Designed and synthesized a near-infrared coumarin fluorescent probe 20 targeting liposomes (fig. 22)
[52]. Based on the FRET mechanism, a ratiometric fluorescence probe of coumarin-malononitrile derivatives was constructed for the detection of HClO/ClO
- with high selectivity and sensitivity. Two well-resolved emission bands (470/672 nm) ensure accurate detection of HClO/ClO
- both in vivo and in vitro. For the first time, the ratiometric fluorescent probe was successfully used to detect exogenous and endogenous HClO/ClO
- in RAW264.7 cells by targeting liposomes.
Yan et al. constructed a ratiometric fluorescent probe for rapid detection of HClO/ClO
- based on coumarin (donor) and pyridinium (acceptor) 21 (fig. 23)
[53]. The probe has red emission signal (610 nM), large Stokes shift (190 nM), high energy transfer efficiency (95. 3%), high selectivity and sensitivity, low detection limit (25 nm), wider detection range (25 nm ~ 30 mM), rapid reaction (< 13 s) and good biocompatibility. The probe has been successfully used for real-time detection of endogenous HClO/ClO
- in RAW264.7 cells.
Chen et al. Developed a novel ratiometric fluorescent probe 22 using a phenothiazine coumarin backbone (fig. 24)
[54]. The introduction of the phenothiazine moiety leads to an increase in the red shift and the Stokes shift of the emission, thereby effectively detecting HClO. After HClO oxidation of the sulfur atom on phenothiazine to sulfoxide, probe 22 showed a significant ratio fluorescence response (more than a 150-fold change in F
534/F
626), a fast response time (< 30 s), and an ultrasensitivity (LOD = 15 nM). The probe is capable of detecting exogenous and endogenous HClO in live RAW 264.7 cells by a ratio fluorescence imaging technique.
Han et al. designed and synthesized a coumarin fluorescent probe 23 (fig. 25) capable of detecting HClO and H
2O
2 simultaneously for the first time, in which phenothiazine coumarin was used as the fluorophore and recognition group for detecting HClO, and coumarin containing boric acid ester was used as the recognition group for detecting H
2O
2[55]. The probe can selectively detect HClO and H
2O
2, and generate a distinct fluorescence signal pattern in living cells. The probe can be used as a potential tool for biomedical scientists to study and dissect the involvement of HClO and H
2O
2 in biological processes under physiological and pathological conditions.
2.5 HClO fluorescent probe based on cyanine dy
Cyanine dyes generally contain two structures centered on the N atom, forming a "push-pull" electronic structure. The initial cyanine dye was synthesized by Willimas with quinoline and alkyl iodide. It is a common parent for the synthesis of probes and has the characteristics of strong affinity and wide spectral range. The structure of the cyanine dye is shown in Figure 26. However, there are still many limitations in the application of cyanine dyes, such as the need to modify the fluorophore molecules with good photochemical stability and high fluorescence efficiency. In order to develop more cyanine dye probe molecules, different groups should be introduced into their structure to change the structure of the molecule, so as to achieve the goal.
Pan et al. designed and synthesized a bifunctional ratio probe 24 (fig. 27) that linked an electron-donating carbazole to the central site of the heptamethylamine cyanine
[56]. The probe 24 may exhibit high sensitivity and selectivity to CN
- and HClO, respectively, with rapid response. The strong oxidation of C = C bond by NaClO leads to a gradual decrease in the absorbance and fluorescence of the probe, and a good linear correlation between the emission intensity and the concentration of NaClO is observed. The detection limit was reduced to 14 nM for HClO in PBS buffer. The probe shows good recovery and analytical precision in the detection of CN
- and HClO in actual water samples. In addition, probe-loaded test strips have become portable tools that can effectively detect CN
- and HClO with the naked eye.
Zhang et al. designed and synthesized a cyanine derivative ratiometric fluorescent probe 25 (fig. 28) to detect endogenous HClO in lung cancer cells
[21]. The probe contains one n-octadecyl chain and two caproic acid 2- [ [2- (dimethylamino) ethyl] methylamino] ethyl ester chains, and the lipid chain and the amino group serve as targeting groups and can specifically recognize lung cancer A-549 cells. The detection limit of the probe was 0.1 μM. Probes 25 were demonstrated to be able to capture LPS-induced HClO in mouse lungs based on in vivo results. These findings suggest the potential for noninvasive and real-time monitoring of HClO in vivo.
Ma et al. designed and synthesized a near-infrared fluorescent probe 26 for selective detection of HClO (fig. 29)
[57]. The probe can selectively recognize HClO based on the formation mechanism of chlorine oxygen (Cl — O) bond for the first time. The probe showed excellent selectivity to the ClO
- with fast response time (< 0.2 s) and high sensitivity (LOD = 39.44 nM). The probe was successfully applied to the imaging of exogenous and endogenous HClO in RAW 264.7 macrophages.
2.6 HClO fluorescent probe based on benzothiazole
Benzothiazole (HBT) has a narrow band gap and good photothermal and chemical stability due to the large π-conjugated system formed by the condensation of thiazole and benzene rings (Fig. 30). Benzothiazole is a very active fluorophore, and its derivatives are well known for their ESIPT effect in the change of fluorescence spectrum and are widely used in the framework of new fluorescent probes
[58][59⇓~61].
Wang et al. designed and synthesized a benzothiazole-based fluorescent probe 27 (fig. 31)
[62]. After the addition of HClO to probe 27, an intramolecular cyclization reaction occurred, the unbridged C = N was converted to bridged C = N, and the fluorescence color changed from orange to cyan. The quantum yield of probe 27 increased from 0.26 to 0.34 after the addition of HClO (10 eq). The probe has high selectivity and sensitivity (24 nM). The probe provides an effective method for analyzing HClO residues in tap water and can image HClO in HeLa cells.
Yang et al. designed and synthesized a benzothiazole-based "off-on" fluorescent probe 28 (fig. 32)
[63]. The detection limit of probe 28 in 100% aqueous solution is in the 8.2×10
-9M,pH range of 5 to 11. Confocal fluorescence imaging and MTT assay demonstrated the excellent membrane permeability and low toxicity of the probe, demonstrating its potential application in living cells.
He et al. designed and synthesized an "on-off" fluorescent probe 29 (fig. 33) by using the inhibition of azobenzene group on benzothiazole derivative fluorophore
[64]. The fluorophore of the probe is 2- (2 '-hydroxyphenyl) -benzothiazole, an aromatic azo group is used for inhibiting the fluorescence emission of the probe, and after the probe reacts with HClO, the azo group is removed, and the fluorophore makes a rapid and sensitive fluorescence response. The probe has high fluorescence quantum yield (Φ
f≈0.75), low detection limit (20.3 nM) and low cytotoxicity. In addition, the probe has been successfully used to detect HClO in tap water, exogenous HClO in HeLa cells, and endogenous HClO in MCF-7 cells.
Wu et al. designed and synthesized a fluorescent probe 30 based on ESIPT (fig. 34)
[18]. The fluorophore of the probe is methoxy hydroxy benzothiazole. Probe 30 has high sensitivity (LOD = 0.16 nm), good selectivity and fast response time (< 10 s). The probe can detect endogenous and exogenous HClO/ClO
- in HeLa cells. Test strips containing the probe have been developed to selectively detect HClO/ClO
- in water.
Nguyen et al. designed and synthesized an open fluorescent probe 31 for the detection of HClO (fig. 35)
[65]. The fluorophore of the probe is a benzothiazole derivative and the recognition group is a hydroxylamine group. Aft reaction with HClO, that ICT process is enhance due to the suppression of the decay process involved in the C = N isomerization, because the resulting nitrile oxide is a strong electron-withdrawing group compared to unreacted hydroxylamine, resulting in a significant fluorescence enhancement at 540 nm. HPLC-MS technique confirmed that the oxime group of the probe was specifically converted to nitrous oxide under the induction of HClO, with high selectivity and rapid response, and the detection limit was 0. 08 μM. Additionally, that probe show excellent cell membrane permeability and low cytotoxicity and has been successfully applied to HClO image in living cells.
2.7 Naphthalimide-based HClO fluorescent probe
Naphthalene-1,8-dicarboximide derivatives are a unique class of environmentally sensitive fluorophores with strong donor-acceptor (D-a) conjugated systems and co-planar naphthalene rings (fig. 36). Naphthalimide derivatives have a wide range of applications in materials, biology and medicine because of their bright yellow-green fluorescence, high fluorescence quantum yield, large Stokes shift, good photostability and thermal stability, and easy structural modification.
Ma et al. designed and synthesized a novel ratiometric fluorescent probe 32 based on naphthalene-1,8-dicarboximide derivatives (fig. 37)
[66]. The recognition group is (2-amino-ethyl) -thiourea group, and the lysosome localization group is morpholine. In the absence of HClO, the probe emits green fluorescence. In the presence of excess HClO, the ICT process was interrupted, resulting in a 57 nm blue shift in the fluorescence emission from 533 to 476 nm and a decrease in the green fluorescence. The linear response of the I
476nm/I
533nm to HClO was obtained over a 1.0×10
-6~1.0×10
-4mol·L
-1 range of HClO concentration. The probe has the advantages of short response time, wide pH range, high sensitivity (LOD=8.0×10
-7M), high selectivity and specificity for hypochlorous acid, and little cytotoxicity. In addition, the probe shows ideal lysosome targeting ability and has been successfully used for confocal imaging of HClO in the lysosomes of HepG2 cells.
Xia et al. designed and synthesized a two-photon fluorescence probe 33 based on naphthalene-1,8-dicarboximide derivatives (Figure 38)
[67]. The probe has a fast response (~ 150 s) to HClO, a high sensitivity (LOD = 0.12 μM), a detection range of 1.0 ~ 160 μM, low cytotoxicity and endoplasmic reticulum (ER) targeting ability. These outstanding properties make the probe useful for monitoring exogenous and endogenous HClO in living cells and zebrafish. The probe is the first to detect the HClO produced by zebrafish induced by sleep deprivation, which can be used to further study the metabolism of HClO during sleep deprivation and its role in biological activities in the future.
Lu et al. designed and synthesized a fluorescent probe 34 based on ICT targeting ER (fig. 39)
[68]. The ER targeting group of the probe is toluenesulfonamide, the recognition group of HClO is N, N-dimethylthiocarbamate, and the fluorophore is 4-hydroxy-naphthalene-1,8-dicarboximide. After reaction with HClO, the probe provided a significant turn-on fluorescence signal (60-fold enhancement) within 2 min based on the cleavage of N, N-dimethylthiocarbamate to a hydroxyl group. The detection limit of the probe was 0. 13 μM for HClO with high selectivity and fast response (< 2 min). Biological imaging experiments show that the probe can specifically monitor the endogenous HClO level in the ER of living cells, and can be successfully used for imaging HClO in zebrafish.
Song et al. designed and synthesized a novel two-photon fluorescence probe 35 targeting ER (fig. 40)
[69]. The fluorophore of the probe is naphthalimide, the ER targeting group is toluenesulfonamide, the recognition group of HClO is methylthio, and the probe has strong green fluorescence. After responding to HClO, the green fluorescence of the probe at 502 nm was greatly reduced due to the inhibitory effect of ICT, and the probe showed high selectivity for HClO. The detection limit is 0.76 μM and the pH range is 4.0 ~ 9.0. Imaging experiments show that the probe can be used to detect HClO in living cells.
Zhang et al. designed and synthesized a ratio fluorescent probe 36 based on the combination of ICT mechanism and FRET principle (Fig. 41)
[70]. After the probe reacted with HClO, the electron-donating thiourea moiety was oxidized and converted to an imidazoline moiety (electron-withdrawing group), resulting in a significant blue shift (about 100 nm) in the fluorescence emission. The prepared probe shows good water dispersibility (100% aqueous medium), fast response (< 40 s), high sensitivity (LOD = 1.75 nM) and high selectivity. Furthermore, in vivo imaging experiments demonstrated that the probe is useful for the visual detection of endogenous HClO production in LPS-induced acute liver injury in zebrafish models.
Zhang et al. designed and synthesized a two-photon fluorescent probe 37 targeting lysosomes (fig. 42)
[71]. The probe reacts with HClO, and the thiourea is partially oxidized to urea, which restores the fluorescence of naphthalene-1,8-dicarboximide (at 538 nm) due to the inhibition of the action of PET. The probe has short response time (< 30 s) to HClO, good selectivity, high photostability (Ф
f=0.1) and low detection limit (5.7 nM). OPM and TPM microscopy cell imaging experiments show that the probe has good membrane permeability and can successfully target lysosomes by taking advantage of the localization group, which proves its application prospect in the detection of lysosomal HClO in biological systems.
Zhang et al. design and synthesized "off" type fluorescent probe 38 and ratiometric fluorescent probe 39 based on naphthalene-1, 8-dicarboximide derivatives (fig. 43)
[72]. Reaction with HClO promotes the cleavage of the C = N bond. Both the "off" probe 38 and the ratiometric probe 39 have the advantages of fast response time, high sensitivity, and good selectivity. The detection limits of probe 38 and probe 39 for ClO
- are 0.12 μM and 26 nM, respectively. In addition, confocal fluorescence microscopy images using HepG2 cells showed that both probes could be used for live cell detection.
Xiong et al. designed and synthesized a ratiometric fluorescent probe 40 based on naphthalene-1,8-dicarboximide derivatives (fig. 44)
[73]. The double bond between naphthalimide and picoline in the probe can be oxidized by hypochlorous acid, causing a blue shift in emission, thus showing a change in ratio fluorescence. The probe shows red emission (λ
max=615 nm) and the solution fluoresces strongly green in the presence of HClO (λ
em,max=520 nm). The probe has fast response to HClO, low detection limit (19 nM) and good selectivity. The probe was successfully applied to the imaging of exogenous and endogenous HClO in living cells and zebrafish.
2.8 Other HClO fluorescent probe
Lan et al. designed a new ratiometric near-infrared fluorescent probe 41 (fig. 45) of dicyanoisophorone-phenylboronic acid based on ICT for rapid and specific detection of ClO
-[74]. The probe can be synthesized in one step, has long-wavelength emission in the NIR region (λ
em=652 nm) which can be detected by naked eyes, has fast response (< 30 s), excellent water solubility, high sensitivity (LOD = 15.7 nM) and good selectivity. Due to its good sensing properties and low cytotoxicity, the probe can be used to conveniently observe exogenous ClO
- in HepG2 cells and endogenous ClO
- in RAW264.7 macrophages. In addition, the probe has been successfully used for biological imaging of zebrafish with acute inflammation. Thus, the probe is a promising vector for identifying HClO levels in animals with related diseases.
Deng et al. designed and synthesized a new colorimetric open-type near-infrared (NIR) fluorescent probe 42 (fig. 46)
[75]. The recognition group for detecting HClO is dimethylthiocarbamate, and the probe exhibits a unique and highly selective colorimetric and NIR fluorescence response to HClO, showing a significant change in color (from yellow to blue) and fluorescence (off) in the NIR region. Short response time (< 3 s) and high sensitivity (LOD = 7.3 nM) in vitro. Probes were imaged for HClO in live HeLa cells, MCF-7 cells, zebrafish, and mice. It was further found that more endogenous HClO was produced in the liver tissue of injured mice, which indicated that the probe was not only effective for the detection of HClO in vivo, but also had broad application prospects.
Han et al. designed and synthesized two fluorescent probes 43, 44 based on aggregation-induced emission (AIE) (fig. 47)
[76]. The fluorophore of the probe is tetraphenylethylene (TPE). The chemical structures of probe 43 and probe 44 are slightly different, resulting in different responses to HClO. Probes 43 and 44 detect HClO by cleaving the Py
+-N
+, and the hydrophobicity of the probes changes as the aggregation-induced emission increases. The fluorescence response times of probe 43 and probe 44 were 10 s and 30 s, respectively, and probe 44 had excellent response (> 1000-fold) and low detection limit (0.108 μM). Probe 44 has been used to image inflammation and hepatocellular carcinoma in mice.
Wang et al. designed and synthesized a novel hydrazine-based naked-eye colorimetric fluorescence turn-on probe 45 (fig. 48)
[77]. In the presence of NaClO (0 ~ 3.0 eq), the fluorescence intensity of the probe at the 475 nm(λ
ex=360 nm) increases immediately due to the oxidation of the double bond between the hydrazone group and the pyrimidine group to an aldehyde group induced by ClO
-, accompanied by a significant color change from non-fluorescent (Φ = 0.062) to blue fluorescent (Φ = 0.494). The probe has high selectivity and low detection limit (53 nM). The probe was imaged in live RAW 264.7 cells, an arterial vascular nude mouse model, and human serum, indicating that the probe can be used as a potential tool for early diagnosis and monitoring of inflammation.
Chen et al. designed and synthesized a novel red ratio fluorescent probe 46 (fig. 49)
[78]. After reaction with the ClO
-, the probe showed a ratiometric fluorescence response to the ClO
- when the emission intensity ratio (I
480/I
612) increased from 0.28 to 27.46 due to the oxidative cleavage of the C = C double bond by the ClO
-, with a detection limit of 0.47 μM. The probe can respond quickly and has high selectivity and good emission of two kinds of separation. In addition, this biocompatible red-emitting ratiometric fluorescent probe has been used for fluorescence imaging of ClO
- in living cells and zebrafish.
He et al. Designed and synthesized a flavonoid-based fluorescent probe 47 (fig. 50)
[79]. The oxidation of the phenothiazine moiety in the probe by HClO significantly modulates the ICT process, causing a sharp change in fluorescence, thus enabling ratiometric detection. The addition of HClO to the probe causes a significant fluorescence color change from orange (λ
max,Em=586 nm) to green (λ
max,Em=524 nm). The probe has good selectivity, high sensitivity (LOD = 6.6 nm), long wavelength emission and large Stokes shift (140 nm). The probe has been successfully used to image HClO in living zebrafish cells.
Mao et al. designed and synthesized a new ratiometric two-photon fluorescence probe 48 (Figure 51)
[33]. The two-photon fluorophore of the probe is a quinolone skeleton, and the HClO recognition group is an aldehyde protected by a mercapto group. After reaction with HClO, the oxidized thioether group of the probe was deprotected to form a typical "push-pull" structure (6- (dimethylamino) quinoline-2-carboxaldehyde), which favored the two-photon absorption properties and showed a red shift in fluorescence emission due to the enhanced "ICT" effect. The probe response time was short (< 1 min), showing a decrease in blue emission and an increase in green emission. The probe can detect HClO quantitatively in vitro with a linear range of 0. 8-12 μM and a detection limit of 89 nM. The probe is the first ratiometric two-photon fluorescence probe to track HClO production during biopsy wound healing.
Lin et al. designed and synthesized a two-photon excited near-infrared fluorescence probe 49 based on Nile red (DyeA) derivatives (Fig. 52)
[80]. After the fluorescent probe reacts with HClO, the aniline group which quenches the fluorescence of the probe is oxidized by the ClO
- and cleaved from the DyeA fluorophore, resulting in an increase in fluorescence. The probe has two-photon excitation fluorescence, high light stability, high selectivity and low cytotoxicity, and the detection limit is about 4.37×10
-6M. The probe can rapidly respond to the HClO/ClO
- in HeLa cells and RAW cells.
Shao et al. designed and synthesized a highly efficient two-photon fluorescence probe 50 (Fig. 53) for real-time detection of endogenous HClO produced by MPO catalysis in the brain of kainic acid (KA) -induced epileptic mice
[81]. The probe itself shows a weak yellow fluorescence and a bright green fluorescence (~ 138-fold) in response to HClO (< 5 s). The probe exhibits efficient membrane permeability, excellent selectivity, high sensitivity (LOD = 104 nM), and high photostability. Using this probe, it was demonstrated in vivo and in vitro that MPO catalyzes the production of HClO in epileptic brains. Fluorescence imaging of epileptic brain showed that apigenin alleviated MPO-mediated oxidative stress and inhibited cellular iron death.
3 Conclusion and prospect
Based on different fluorophores (BODIPY, rhodamine, fluorescein, coumarin, cyanine, benzothiazole, naphthalimide, etc.), the design, synthesis and biological application of hypochlorous acid fluorescent probes in recent three years are reviewed in this paper. Compared with the traditional methods for detecting hypochlorous acid, the fluorescent probe has the advantages of high spatial and temporal resolution, high sensitivity and easy observation, and has important medical application prospects for real-time monitoring of HClO in organisms and effectively diagnosing diseases related to HClO. Despite the rapid development of fluorescent probes, there is still room for improvement in the research of fluorescent probes: (1) the currently developed hypochlorous acid fluorescent probes are one-way reaction type, and the design of reversible reaction probes is more conducive to improving their application ability; (2) Near-infrared ratiometric fluorescent probes for hypochlorous acid have many advantages over most small molecular fluorescent probes, and will be the focus of future research; (3) For the future clinical application of hypochlorous acid fluorescent probes, the design strategy should focus on improving biocompatibility and reducing cytotoxicity. In view of the importance of hypochlorous acid in organisms, the fluorescent probe of hypochlorous acid will have a broader application prospect.
Natural Science Foundation of Shandong Province(ZR2019QB017)
Scientific and Technological Innovation Fund for Postgraduates of Yantai University in 2020(YDYB2010)
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