1 Introduction
Small biological thiols mainly include three compounds: cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). They are widely distributed in cells and organisms and play an important role in physiological and pathological activities (Figure 1), and abnormal changes in their concentrations are often closely related to various diseases
[1⇓⇓⇓~5]. Cysteine plays an important role in protein synthesis and metabolism. A series of pathological symptoms such as slow growth, hair depigmentation, edema, lethargy, liver function damage, muscle relaxation, obesity and physical weakness are related to the change of its concentration
[6⇓~8]. The concentration level of homocysteine is called H value, which can accurately predict the risk index of heart disease and stroke, and high concentration level of Hcy can easily lead to cardiovascular disease and Alzheimer's disease
[9⇓~11]. Glutathione is the most abundant small molecular biological thiol (1-10 mM) in the body, which is divided into two forms: reduced glutathione and oxidized glutathione. In general, reduced glutathione is the main form. It is an important substance for regulating metabolism in cells, participating in a variety of biochemical reactions in organisms, maintaining normal immunity, regulating oxidation and chelating detoxification
[12⇓~14].
Thiol amino acids play an important role in maintaining the normal life activities of organisms, so it is particularly important to detect their concentration levels. The general methods for the detection of thiols include electrochemical method, high performance liquid chromatography, capillary electrophoresis, mass spectrometry, and immunoassay, etc. These methods are cumbersome, require a large number of samples, have high cost, and take a long time to detect
[15⇓⇓⇓~19]. Fluorescent probe method has been paid more and more attention in the detection of thiols because of its advantages of simple operation, high spatial and temporal resolution, visualization and less damage
[20⇓⇓~23].
At present, many fluorescent probe molecules have been reported, but their absorption and emission wavelengths are short (< 550 nm), which are easily interfered by biological background signals, and their tissue penetration ability is poor, so the imaging effect is limited. Fluorescent probes with long wavelength emission, that is, emitting red or near-infrared light signals (> 600 nm), can well circumvent these problems. Their signal penetration depth is deep, and the interference of self-background is small, and with the increase of wavelength, Raman scattering decreases, so that the scattering interference is greatly reduced, which has greater advantages in practical applications
[24⇓⇓~27]. Therefore, this paper mainly focuses on the fluorescent probes with red or near-infrared light signals, which are classified according to the types of fluorophores that constitute the probes, specifically rhodamine, dipyrrole boron fluoride, cyanine, natural pigments, general donor-acceptor conjugated compounds and aggregation-induced luminescent compounds.Molecular design, optical properties, recognition mechanism and applications in cells and in vivo are reviewed.
2 Rhodamine-Based Thiol Fluorescent Probe
Rhodamine is a catechol fluorescent dye (Figure 2), which has been widely used in biotechnology fields such as fluorescent labeling, small molecule detection and biological imaging since it was first discovered in 1887
[28]. It has the advantages of large molar absorptivity, high fluorescence quantum yield and long excitation wavelength. At present, researchers have developed a variety of thiol fluorescent probes based on this structure
[28,29].
According to the reported literature, we found that the structure of rhodamine can be modified, and the excitation and emission wavelengths can be significantly shifted by alkylating the nitrogen atom. As reported by Fan et al., a ratiometric fluorescent probe for the detection of Cys/Hcy targeted to mitochondria 1 (fig. 3)
[30]. The probe uses 4-methylthiophenol as a thiol recognition group, diethylrhodamine as a fluorescent group, and diethylamine cation as a mitochondrial localization group. After the addition of Cys/Hcy in PBS buffer solution (10 mM, pH 7. 4), the thiol group of thiol reacted with 4-methylthiophenol to form a nucleophilic aromatic substitution.Then the amino group attacked the thiol group to form an intramolecular rearrangement.After the reaction, the absorption peak of the molecule was blue-shifted from 590 nm to 460 nm.The fluorescence intensity at 620 nm decreased under 580 nm excitation, and a new emission peak at 540 nm appeared under 455 nm excitation. The ratiometric detection of Cys/Hcy was realized by the change of the fluorescence intensity ratio of the F
540 nm/F
620 nm. Probe 1 has high selectivity for Cys/Hcy, good sensitivity (detection limit: Cys 22 nM; Hcy 23 nM), fast response (Cys 5 min; Hcy 10 min), and large change in fluorescence signal ratio (Cys 163 times; Hcy 125 times).
When the probe was used to monitor the redox balance of thiols in mitochondria of MCF-7 cells under the induction of H2O2 (Fig. 4A),H2O2, thiols in the cells could be oxidized, resulting in a decrease in their content. It can be seen from the figure that after the cells are treated with H2O2, the fluorescence signal of the green channel is weakened and the signal of the red channel is enhanced, which indicates that the H2O2 does play a role in regulating the oxidation of thiols in cells. The probe was also successfully applied to the detection of Cys/Hcy ratio in kidney tissue and fluorescence imaging of Daphnia magna (Fig. 4B). After the Daphnia was incubated with Cys, the fluorescence signal of the green channel was significantly enhanced, which showed the selective response effect of the probe to Cys in vivo.
A new fluorophore can be obtained by replacing the oxygen atom of rhodamine with silicon. Guo et al. reported a fluorescent probe 2 constructed with silicon-rhodamine as the fluorophore and 4-methoxythiophenol as the recognition group (fig. 5)
[31]. After the addition of Cys in PBS buffer solution (10 mM, pH 7. 4), the fluorophore 2b was rapidly formed through successive nucleophilic aromatic substitution and intramolecular rearrangement.The maximum absorption peak of 2b was blue-shifted from 688 nm to 472 nm within 2 min, and the fluorescence emission intensity at 620 nm was significantly enhanced. The addition of GSH also reacted with the probe to form compound 2C within 1 min, which decreased the initial absorption peak at 688 nm, but did not change the fluorescence. In particular, when Cys was added to 2 C, the absorption peak at 688 nm further decreased to 0 within 4 min, while a new absorption peak appeared at 472 nm and the fluorescence emission at 620 nm was enhanced. This indicates that Cys can rapidly displace the GSH unit in compound 2C. Therefore, the presence of GSH does not interfere with the specific detection of Cys. They further studied the regulatory role of Cys in cancer cells and found that depleting Cys in cells could make cancer cells more sensitive to chemotherapy.
Urano et al. reported a reversible ratiometric fluorescent probe 3 for the detection of GSH based on fluorescence resonance energy transfer (FRET) (fig. 6)
[32]. The probe 3 is formed by connecting tetramethylrhodamine (TMR) as an energy donor and silicon-rhodamine (SiR) as an energy acceptor. It has good cell penetration and can selectively accumulate in mitochondria because it is a cationic compound. In PBS/DMSO (V ∶ V = 95 ∶ 5, 0.2 M, pH = 7.4) solution, the fluorescence of the probe mainly came from SiR (632 nm) due to the FRET effect. After the addition of GSH, the conjugated structure of SiR was destroyed by Michael addition reaction with the probe, and the fluorescence intensity at 632 nm decreased gradually, while the fluorescence intensity from TMR (593 nm) increased gradually. The K
d value (dissociation constant of GSH) of probe 3 was calculated to be 3. 0 mM by fitting the fluorescence ratio (F
593 nm/
) and GSH concentration, and the optimal K
d value was 1 to 10 mM according to the actual content of GSH in cells. Therefore, probe 3 is very suitable for the detection of GSH content in real cell environment. They used the probe to quantify GSH concentrations in a variety of cell lines.
In addition to the above two structural modifications, rhodamine can also be combined with other fluorophores to construct ratiometric fluorescent probes. In 2019, Liu et al. Also reported a ratiometric near-infrared fluorescence probe 4 based on FRET (fig. 7)
[33]. The probe 4 is connected with coumarin as an energy donor, rhodamine as an energy acceptor, piperazine as a spacer group, and a trifluoromethyl phenyl thioester part as a thiol recognition group. In the mixed solution of PBS/ethanol (V ∶ V = 7 ∶ 3, pH = 7. 4), the fluorescence emission of the probe was very weak at 467 nm (coumarin moiety) and very strong at 645 nm (rhodamine moiety) due to the FRET effect. However, the addition of Cys resulted in the substitution and intramolecular rearrangement of the thiol group with the phenylthioester moiety of the probe, followed by the formation of a non-fluorescent closed spirolactam structure in the rhodamine moiety, and the FRET process was blocked, resulting in a 45% decrease in the fluorescence intensity of the rhodamine moiety (645 nm) and an 18-fold increase in the fluorescence intensity of the coumarin moiety (467 nm). At the same time, the fluorescence intensity ratio of the
/F
645 nm was increased from 0.235 to 4.237, and the ratiometric detection of Cys was realized. The probe was successfully applied to monitor the concentration of Cys in HeLa cells and Drosophila larvae.
3 Thiol fluorescent probe based on dipyrrole fluoroborate
Dipyrrole boron fluoride (BODIPY) is a kind of fluorophore with excellent photophysical and chemical properties (Fig. 8). It is widely used in the field of fluorescent probes because of its large molar absorptivity, high fluorescence quantum yield, easy modification and good light stability
[34,35]. However, the absorption and emission of the parent compounds are generally in the short wavelength region, so researchers have modified the molecule to make its emission wavelength longer.
Extending the conjugation length of the molecule and enhancing the strength of the donor-acceptor in the system are common methods to make the emission wavelength of the molecule red-shift. In 2019, Gu et al. Reported a ratiometric fluorescent probe 5 for detecting GSH (fig. 9)
[36]. Probe 5 introduced trifluoromethyl substituted cyanostyrene at the 2nd position of BODIPY, which expanded the conjugation degree of the molecule and enhanced the strength of the receptor. At the same time, the introduction of the electron-withdrawing group can enhance the reactivity of the recognition group chlorine atom and accelerate the response rate of the probe. When GSH was added into the PBS/acetonitrile (V ∶ V = 1 ∶ 1, pH = 7. 4) mixed solution of probe 5, the thiol group attacked the chlorine atom and the nucleophilic substitution reaction occurred, and the reaction was completed within 15 min. The electron donating effect of the thiol group caused the red shift of its fluorescence spectrum. During this process, the absorption peak at 574 nm gradually decreases, and a new absorption peak appears at 609 nm. At the same time, under the excitation of 550 nm light, the fluorescence emission peak at 590 nm gradually decreased, and the emission at 621 nm increased. When Cys/Hcy was added, the chlorine atom in the probe was replaced by thiol, and the intramolecular rearrangement reaction occurred, and finally the amino substituted product was formed, which led to the decrease and blue shift of the absorption peak at 574 nm and the decrease of the fluorescence intensity at 590 nm, which was different from the signal after GSH was added. Probe 5 successfully distinguished GSH from Cys in HeLa cells by the difference in the ratio of fluorescence in the red and green channels.
Qian et al. reported a highly sensitive two-photon near-infrared fluorescence probe for the detection of GSH 6 (fig. 10)
[37]. The donor triphenylamine was introduced into the 3 and 5 positions of BODIPY to enhance the intramolecular charge transfer (ICT) effect, and the emission spectrum of probe 6 entered the near infrared region. 2,4-dinitrobenzenesulfonyl is introduced at the 8th position of BODIPY to serve as a fluorescence quenching group and a thiol recognition group. When GSH was added to the PBS/THF (V ∶ V = 1 ∶ 1, pH = 7. 4) solution of the probe, the 2,4-dinitrobenzenesulfonyl group was cleaved from the molecule due to the nucleophilic aromatic substitution reaction between GSH and the probe, and the photoinduced electron transfer (PET) effect was eliminated, thus the fluorescence emission of the probe was restored. During this process, the absorption peak was blue-shifted from 691 nm to 679 nm. Under the excitation of 670 nm light, the fluorescence emission intensity at 719 nm was significantly enhanced. The detection limit of the probe for GSH is 25.46 nM, and the fluorescence quantum yield of the fluorophore after recognizing GSH is 0.44. Due to its large two-photon absorption cross section (σ
2=410 GM), the probe has the potential to be used in two-photon imaging of GSH.
They also reported a number of fluorescent probes with similar structures to Probe 6, which can adjust the emission wavelength by modifying the donor moiety. For example, the near infrared fluorescence probe 7 (Figure 11) introduced 4-methoxystyrene groups at the 3 and 5 positions of BODIPY as electron donors. After GSH was recognized, the fluorescence at 656 nm was turned on, and the emission intensity was increased by 45 times
[38]. Similarly, the donor 4-hydroxystyrene was introduced at the 3 and 5 positions of BODIPY, and 2,4-dinitrobenzenesulfonyl was introduced at both ends of BODIPY as recognition sites, respectively.After the reaction with GSH, the fluorescence at 675 nm was enhanced by 27 times, and the fluorescence was also excited by 800 nm laser, which indicated that the probe had potential application in two-photon biological imaging
[39]. In probe 9 reported by Liu et al., a pentafluorophenyl group was introduced at position 8 of BODIPY to increase the intensity of the fluorophore acceptor, and the fluorescence emission intensity at 610 nm was enhanced by 43 times after reaction with Cys
[40].
4 Cyanine based thiol fluorescent probe
The history of cyanine dyes can be traced back to a century and a half ago. Williams discovered cyanine dyes in 1856. These dyes have extremely sensitive photosensitive ability and are a typical class of near-infrared luminescent materials. In general, these dyes can be divided into five categories: cyanine dyes, hemicyanine dyes, oxanisole dyes, merocyanine dyes and squaraine dyes. Among these dyes, cyanine and hemicyanine are more widely used in the development of fluorescent probes
[41,42].
4.1 Cyanine dye-based thiol fluorescent probe
The cyanine dye structure contains two nitrogen-containing heterocycles, one of which is positively charged and conjugated to the other nitrogen-containing center by a chain of an odd number of carbon atoms (fig. 12). The absorption and emission spectra of cyanine dyes can be tuned with the length of the conjugated polymethine chain (i.e., n)
[43]. The larger the conjugated system of the polymethine cyanine dye molecule is, the smaller the energy difference between the ground state and the first excited state of the molecule is, and thus the longer the absorption wavelength and the emission wavelength of the molecule are. Among them, indole heptamethine cyanine dyes (Cy7, n = 3) are often used to construct fluorescent probes because of their near-infrared emission properties.
In 2019, Yuan et al. Reported a near-infrared fluorescent probe 10 with dual emission signals for the detection of GSH in mitochondria (fig. 13)
[44]. The probe 10 takes 2- (4-hydroxystyryl) -4H-1-benzopyran-4-one (DCM) and cyanine dye (Cy7) as double fluorophores, and takes aryl ether as a response site of GSH. In the mixed solution of HEPES/DMSO (V ∶ V = 9 ∶ 1, 0.1 M, pH = 7.4), the probe only has weak fluorescence in the visible region. After the addition of GSH, the sulfhydryl group reacted with the ether bond of the probe to release two fluorophores, 10-DCM and 10-Cy7. During this process, the absorption peaks at 358 nm and 700 nm are enhanced. At the same time, the fluorescence intensity at 520 nm in the visible region and 810 nm in the near-infrared region also increased significantly. Concentration titration experiments showed that the detection limits of GSH in two different optical channels were 24 nM (visible light) and 32 nM (near infrared), respectively. The experiment of H22 tumor-bearing mouse model showed that the tumor tissue had strong blue fluorescence in the visible light channel and strong red fluorescence in the near-infrared channel. However, in tissues with inflammation, there is only weak fluorescence in the visible light channel, and there is no fluorescence signal in the near-infrared channel. The multi-channel fluorescence imaging property of the probe makes it have the potential to distinguish tumors from inflammatory tissues.
The fluorescent probes 11 and 12 reported by Yin et al. Are constructed from cyanine and naphthalimide (fig. 14)
[45,46]. Cationic cyanine in probe 11 targeted mitochondria and morpholine-containing naphthalimide targeted lysosomes. After GSH was added to a mixed solution of PBS/DMSO (V: V = 99:1, 10 mM, pH = 7.4), two products with different fluorescence emission (11-Cy and 11-Na) were obtained. In this process, a new absorption peak appears at 785 nm. Under the excitation of 710 nm light, the fluorescence intensity at 812 nm increased significantly. They also found that GSH tends to preferentially attack the cyanine portion of the probe to form 11-Cy. In probe 12, the fluorescence of the probe is quenched due to the ICT process caused by the tertiary amine nitrogen in the piperazine ring. After reaction with GSH, the fluorescence intensity at 495 nm and 795 nm increased. Yang et al. reported that probe 13, with 2-mercaptopyridine as a leaving group, turned on fluorescence emission at 750 nm after the addition of Cys/Hcy
[47].
4.2 Thiol fluorescent probes based on hemicyanine dy
Hemicyanine dyes are structurally characterized as donor-pi-acceptor (D-pi-a) (fig. 15). One end of the compound takes hydroxyl, alkoxy or amino as an electron donor, the other end takes a nitrogen heterocycle with positive charge as an electron acceptor, and the compound and the nitrogen heterocycle are connected through a conjugated system. Hemicyanine dyes generally have a large Stokes shift
[48].
Guo et al. reported a reversible ratiometric fluorescent probe for real-time monitoring of GSH 14 (fig. 16)
[49]. The probe 14 has a hemicyanine composed of rhodamine and benzothiazole as a fluorophore. When GSH was added to the probe in PBS buffer solution (10 mM, pH = 7. 4), the Michael addition reaction of GSH with the double bond occurred, which blocked the π conjugation of benzothiazole and led to the near-infrared fluorescence quenching of hemicyanine, thus releasing the fluorescence of rhodamine. During this process, the absorption peak at 580 nm decreases and a new absorption peak appears at 530 nm. At the same time, the fluorescence emission peak at 688 nm decreased, and a new fluorescence emission peak appeared at 560 nm. The fluorescence intensity of the probe was proportional to the concentration of GSH in the range of 0-7 mM with a detection limit of 9. 43 μM.
In 2019, Liu et al. Reported a fluorescent probe 15 with multiple response sites (fig. 17)
[50]. The probe 15 uses a hemicyanine composed of coumarin and indole salt as a fluorophore, and a sulfonate alkyl chain is introduced to improve the water solubility of the probe. When Cys/Hcy was added to the probe in PBS buffer solution (10 mM, pH = 7.4), nucleophilic substitution, intramolecular rearrangement and intramolecular cyclization occurred successively, resulting in 15-a3 (seven-membered ring)/15-b3 (eight-membered ring). However, the thiol group of GSH undergoes a nucleophilic substitution reaction with site 1 to form 15-c1, followed by an intramolecular cyclization to form 15-c2 (a 14-membered ring). However, due to the instability of the 10-membered ring, no intramolecular rearrangement occurs in 15-c1 to form 15-c3.
The three biothiols showed different fluorescence responses under different excitation light, indicating that the probe could effectively distinguish biothiols, and the A375 cell imaging experiment proved that the probe had the ability to monitor endogenous biothiols in living cells in real time. In 2010, Chen et al. Also reported a hemicyanine fluorescent probe 16 composed of 7,8-pyranocoumarin and an indole salt
[51]. After it reacts with thiol, the fluorescence at 751 nm is turned on, and the probe can also be used to detect the oxidative stress state of cells (fig. 17).
Tang et al. Reported a photoacoustic and fluorescence dual-mode imaging probe for detecting Cys17 (fig. 18)
[52]. The probe 17 has a hemicyanine as a fluorophore and an acrylate group as a fluorescence quenching group and a thiol recognition group. In the PBS/DMSO (V ∶ V = 99 ∶ 1, pH 7. 4) solution of the probe, the conjugated addition and intramolecular cyclization of Cys with the acrylate group of the probe occurred, and the lactam was cleaved from the molecule, thus restoring the emission of the hemicyanine fluorophore. During this process, the absorption peak was red-shifted from 585 nm to 680 nm, and the fluorescence emission intensity at 710 nm was enhanced by a factor of 26.7. The detection limit of the probe for Cys was 0.21 μM. GSH concentrations are low in the lungs of patients with pulmonary fibrosis (PF), but it is not known whether this is due to low levels of Cys. They found that the content of Cys in the lungs of mice with pulmonary fibrosis was higher than that of healthy mice, indicating that the limited synthesis of GSH was not caused by Cys deficiency. By comparing the fluorescence and photoacoustic signals of the probe in PF mice, pneumonia mice and healthy mice, it was found that only PF mice showed significant fluorescence and photoacoustic signals. This indicates that the probe can be used as an effective means to distinguish pulmonary fibrosis from pneumonia in clinic, and can also be used as a potential target for new drugs for pulmonary fibrosis.
Chen et al. reported a far-red to near-infrared fluorescent probe 18 for the specific detection of Cys (fig. 19)
[53]. Nucleophilic substitution and intramolecular rearrangement occurred when Cys was added to the probe in PBS/ethanol (V ∶ V = 99 ∶ 1, 10 mM, pH = 7.4). During this process, the maximum absorption of probe 18 was blue-shifted from 660 nm to 540 nm. Under the excitation of 550 nm light, with the increase of Cys concentration, the emission peak at 710 nm gradually decreased, and a new fluorescence emission peak at 626 nm appeared and gradually increased. The fluorescence intensity ratio (
/
) was increased from 0.23 to 9.5, a 41-fold enhancement, achieving ratio detection for Cys.
The common disadvantage of cyanine dye-based fluorescent probes is poor photostability, and their conjugated chains are easily attacked by other active substances in vivo, which makes their selectivity also face problems. Therefore, in order to give full play to the advantages of long wavelength emission, the subsequent development of cyanine dyes should start with improving the light stability and structural stability.
5 Thiol fluorescent probe based on natural pigment
5.1 Thiol fluorescent probe based on anthocyanin
Anthocyanins are a class of natural pigments widely found in plants in nature (Fig. 20)
[54]. Based on the parent structure of anthocyanins, the fluorescence quantum yield and two-photon absorption cross section of anthocyanins can be increased by fixing the rotatable carbon-carbon single bond through ring formation.
In 2019, Cheng et al. reported a two-photon fluorescence probe 19 and Song et al. reported a red light probe 20 (fig. 21), both of which were constructed with an anthocyanin derivative as the fluorophore and acrylate as the recognition group of Cys
[55][56]. When Cys was added to the probe 19 in PBS/acetonitrile (V ∶ V = 4 ∶ 1, 25 mM, pH 7. 4) for 15 min, the fluorescence emission intensity at 611 nM was enhanced by a factor of 51 under the excitation of 550 nm light, and the detection limit of probe 19 was 24. 1 nm. After the probe 20 reacted with Cys, the maximum absorption peak was red-shifted from 540 nm to 580 nm, and the fluorescence emission at 615 nm was turned on.
5.2 Curcumin-based thiol fluorescent probe
Curcumin is a diketone compound extracted from the rhizomes of some plants in Zingiberaceae and Araceae (fig. 22)
[57,58]. Hydroxyl groups at the two ends can be easily functionalized by chemical modification, and the beta-diketone structure in the middle can be coordinated with boron fluoride to enhance the electron-withdrawing ability of boron fluoride, so that the emission wavelength of boron fluoride is red-shifted.
The fluorescent probes 21 and 22 (fig. 23) reported by Chen et al. Are D-A-D symmetric fluorescent probes constructed with curcumin as the fluorophore
[59,60].
2,4-dinitrobenzenesulfonyl was used as the response group of thiol in probe 21. When thiol was added to the mixed solution of ethanol/HEPES (V ∶ V = 4 ∶ 1, pH 7.4), the fluorescence at 610 nm was turned on and the fluorescence intensity was significantly enhanced (Cys: 17.8 times; Hcy: 14.2 times; The detection limits were 0.59 μM for Cys, 2.25 μM for Hcy, and 3.38 μM for GSH. The maximum absorption peak of probe 22 was red-shifted from 461 nm to 510 nm, and the fluorescence intensity at 615 nm was increased by 11 times after it reacted with Cys. Probe 22 was successfully applied to the selective imaging of endogenous Cys in MCF-7 cells.
6 Thiol fluorescent probe based on donor-acceptor conjugated compound
Organic fluorophores generally have large π-electron conjugated systems, and the introduction of donor-acceptor (D-a) structure into the system can make the emission spectrum red-shift by enhancing the ICT effect, and then be used to construct red or near-infrared fluorescent probes. Common electron donors are triphenylamine, alkylamine, hydroxyl and other groups; Common electron acceptors include cyano, 2- (3-cyano-4,5,5-trimethylfuran-2 (5H) -methylene) malononitrile (TCF), and isophorone (fig. 24)
[61⇓~63].
Dong et al. reported a fluorescent probe based on excited state intramolecular proton transfer (ESIPT) 23 (fig. 25)
[64]. When Cys was added to HEPES buffer (H
2O∶THF=99∶1,50 mM,pH 7.4) for 8 min, the fluorescence intensity at 640 nm increased to the maximum under 450 nm excitation. In this process, the probe reacts with Cys to release the proton donor (-OH), and the intramolecular hydrogen bond is formed between the proton donor (-OH) and the adjacent acceptor (-C = O), which excites the ESIPT effect and produces a strong red fluorescence emission. The probe has a detection limit of 5 nM for Cys and has been successfully applied to the fluorescence imaging of endogenous and exogenous Cys in HepG2 cells.
In 2019, Chen et al. Reported a red-emitting fluorescent probe 24 with a large Stokes shift (fig. 26)
[65]. Probe 24 is a fluorophore composed of hydroxynaphthalene as an electron donor and TCF as an electron acceptor. When thiol was added to the HEPES/ethanol (V ∶ V = 1 ∶ 1, pH = 7. 4) mixture solution, the maximum absorption peak of the probe was red-shifted from 420 nm to 480 nm, and the fluorescence intensity at 640 nm was enhanced by 32 times under the excitation of 480 nm light, and its Stokes shift reached 160 nm, which effectively avoided the influence of fluorescence self-absorption. The detection limits were 0.11 μM for Cys, 0.47 μM for Hcy and 0.35 μM for GSH. In the same year, Qi et al. Also reported a D-a type fluorescent probe 25 containing TCF
[66]. The probe 25 is constructed using xanthene as a donor, TCF as an acceptor, and acrylate as a recognition group. The fluorescence at 770 nm turned on after the reaction with Cys. In addition, the probe solution changed from light purple to green (Cys), light green (GSH) and colorless (Hcy) after the addition of thiols, indicating that probe 25 can also be used as a colorimetric indicator to distinguish thiols.
In 2018, Jiang et al. reported a fluorophore composed of naphthol and the acceptor dicyanoisophorone derivative, and a fluorescent probe 26 constructed with acrylate as the recognition group of Cys (fig. 27)
[67]. When Cys was added into the PBS/DMSO (V ∶ V = 1 ∶ 1, pH = 7.4) solution, the absorption peak at 418 nm decreased, and a new absorption peak appeared at 441 nm with an isosbestic point at 433 nm. At the same time, the fluorescence intensity at 631 nm increased significantly. In the same year, Zhu et al. Also reported a fluorescent probe 27 with dicyanoisophorone as an acceptor
[68]. The probe has 3,5-dinitropyridine as a recognition site.After the reaction with thiol, 3,5-dinitropyridine leaves, and the fluorescence at 697 nm is turned on, and the intensity is enhanced by 71 times.
7 Thiol fluorescent probes based on AIE-type compound
Tang et al first proposed the concept of aggregation-induced luminescence (AIE) in 2001
[69]. Common molecular fluorescent probes are prone to aggregation in aqueous solution, resulting in fluorescence quenching (ACQ), which affects the performance of the probe, while AIE probes can avoid this problem
[70⇓~72]. Tetraphenylethylene (TPE) is one of the most classical and widely used structures in the construction of AIE probe molecules (fig. 28). The four benzene rings in TPE are connected to the central olefin structure. In the dissolved state, the rotation of the benzene rings dissipates energy by non-radiative transition, resulting in almost no fluorescence of TPE. However, in the molecular aggregation state, the rotation of the benzene ring is physically limited, that is, intramolecular motion is limited (RIM), which turns on the fluorescence emission of TPE. Moreover, the twisted structure of AIE molecules can hinder the π-π stacking interaction, avoid fluorescence quenching, and further contribute to the enhancement of fluorescence
[73,74].
Tang et al. Reported a ratiometric two-photon fluorescence probe based on TPE 28 (fig. 29)
[75]. The probe uses m-dinitrobenzene-substituted benzylphenol as a recognition group, and the molecule is positively charged and can target mitochondria. When GSH was added into the PBS/DMSO (V ∶ V = 1 ∶ 1, pH = 7. 4) solution, the thiol group attacked the dinitrobenzene and nucleophilic aromatic substitution reaction occurred, the recognition group was removed from the probe molecule, and the fluorescence emission at 631 nm decreased, while the fluorescence emission at 500 nm increased. The blue shift of the emission is attributed to the weaker ICT effect of tetraphenylvinylpyridine than that of the probe. At the same time, due to the large two-photon absorption cross section (σ
2=1790 GM) of the probe, it has been successfully applied to the two-photon fish larvae cell imaging experiment. In fig. 30, it can be seen that the fluorescence emission intensity of the fish larvae incubated with the probe increases in the green channel and decreases in the red channel. At the same time, they found that GSH may be mainly located in the eyes, liver and intestine. Together with GSH, it can be effectively absorbed into the whole intestinal epithelium through a specific absorption system and accumulated in liver and muscle, which provides a possibility for screening potential anti-tumor drugs in vivo. This is because GSH plays an important role in liver-dependent tissue flow in the treatment of cancer metastasis.
In 2019, Ma et al. Reported an AIE type fluorescent probe 29 for detecting Cys/Hcy (fig. 31)
[76]. The probe 29 combines the electron donor triphenylamine with the acceptor dicyanostyrene and the aldehyde group through the benzene ring and the carbon-carbon double bond to form a non-planar D-π-A-π-a type molecule. The aldehyde group is used as a recognition group of Cys/Hcy to form the thiazolidine/thiazidine through a cyclization reaction. When Cys/Hcy was added to the DMSO/H
2O(V∶V=3∶1) solution of the probe for 2 min, the weak fluorescence emission at 618 nm was blue-shifted to 596 nm, accompanied by an increase in fluorescence intensity. The blue shift of fluorescence was attributed to the conversion of aldehyde group to thiazolidine/thiazidine, which changed the structure of D-π-A-π-a to D-π-a. Moreover, compared with the probe 29, the solubility of the newly formed product is reduced, and the formed aggregates further restrict the intramolecular movement due to the intermolecular hydrogen bonding, so that the AIE effect is enhanced, resulting in enhanced orange-red fluorescence emission. At the same time, the yellow solution of probe 29 turned colorless during the detection process, indicating that the probe can also detect biothiols by colorimetry.
8 Conclusion and prospect
In view of the advantages of long-wavelength emission signal, such as low background interference, deep tissue penetration and low photodamage, this review focuses on the research of some representative red/near-infrared thiol fluorescent probes in the past three years. From the point of view of structure, on the one hand, the three small molecular biological thiols have something in common, that is, they are all amino acids containing sulfhydryl groups, and the nucleophilicity of sulfhydryl groups is strong in physiological environment; On the other hand, the differences are also prominent, that is, there are differences in nucleophilic ability and molecular size. Using these characteristics, researchers have designed and synthesized different fluorescent groups and recognition groups, and obtained fluorescent probes with selective response to thiols or specific thiols by nucleophilic aromatic substitution, Michael addition, intramolecular rearrangement and other reaction mechanisms. In the construction of fluorophores, the emission wavelength is red-shifted mainly by expanding the degree of molecular conjugation and introducing strong donor-acceptor groups. We classify the fluorophores into different types, and systematically discuss the research progress in this field. So far, not only the selective detection of endogenous thiols in living cells has been realized, but also the concentration of thiols in cells has been quantitatively detected by the design of ratiometric probes, and the change of concentration has been monitored in real time. In addition, the targeted detection of specific thiols in organelles can also be achieved by skillfully introducing positioning groups into the probe. The introduction of two-photon fluorescence imaging technology has realized the selective imaging of thiols in deeper tissues and even in vivo. Meanwhile, the design of the AIE type probe can avoid the fluorescence quenching effect of aggregation and increase the photobleaching resistance of the probe. Finally, the role of thiols in physiological activities was discussed.
In the field of red/near-infrared thiol fluorescent probes, great progress has been made, but there are still some obvious problems.Such as aggregation-induced fluorescence quenching, photobleaching and poor intracellular retention of the probe, as well as the selectivity problems caused by the large differences in the actual concentrations of the three thiols in cells, are worthy of further study. Here, we hope that the summary of the existing work can provide more useful ideas for researchers, and also hope that the progress in this field will eventually contribute to the diagnosis and treatment of related diseases.
National Natural Science Foundation of China(51703171)
Graduate Innovative Fund of Wuhan Institute of Technology(CX2019003)
PDF
