1 Introduction
Glutathione (GSH) is an active tripeptide with important physiological functions, and the sulfhydryl group (-SH) in its structure is easily oxidized and dehydrogenated. Therefore, GSH can not only scavenge oxygen free radicals and other free radicals in the body, but also combine with heavy metal ions, a variety of chemicals and their metabolites, and promote their excretion from the body to help maintain the normal function of the immune system in the body
[1⇓~3]. The content of GSH in human blood is 26 ~ 34 mg/100 G, and excessive GSH can cause diseases such as Alzheimer's disease, Warner syndrome, Parkinson's disease, cancer and heart disease. Deficiency can cause problems such as slow growth, hair loss, lethargy, psoriasis, liver damage, and loss of muscle and fat
[4,5]. Therefore, real-time and accurate detection of intracellular GSH content is of great significance for clinical diagnosis, disease treatment and pathogen detection.
At present, the detection methods of GSH include high performance liquid chromatography, electrochemical method, colorimetric method, fluorescence analysis, etc., among which the fluorescence analysis method based on fluorescent probes has high sensitivity, fast response, good selectivity and real-time monitoring, and has become a research hotspot in the field of GSH detection, and has been widely used for qualitative or quantitative detection in vivo
[6⇓⇓⇓~10][11].
Based on the unique properties of strong nucleophilicity, reducibility and high affinity for metal ions, GSH was detected by Michael addition reaction, nucleophilic substitution, reduction, thiol-induced cleavage of 2,4-dinitrobenzenesulfonyl (DNBS) and complexation
[12⇓⇓~15]. The detection mechanisms are mainly based on fluorescence resonance energy transfer (FRET), electron transfer (ET), intramolecular charge transfer (ICT), photoinduced electron transfer (PET), excited state intramolecular proton transfer (ESIPT) and inner filter fluorescence effect (IFE)
[16]. In order to further understand the molecular structure of GSH fluorescent probes, GSH fluorescent probes were summarized from the perspective of organic small molecule fluorescent probes and inorganic nano-fluorescent probes. The effects of 12 fluorophores on the fluorescence properties and biological applications of the probe were discussed, and the GSH response mechanism was analyzed
[16⇓~18].
2 Organic small molecule fluorescent probe for detecting GSH
Organic small molecule fluorescent probes that can be used to detect GSH can be divided into coumarins, BODIPY, rhodamines, cyanines, benzothiazoles, naphthalimides, metal-organic frameworks and other organic fluorescent probes.
2.1 Coumarin fluorescent probes for GSH detection
As a lactone compound, coumarin has the advantages of high fluorescence quantum yield, large Stokes shift, good optical properties, easy structure modification and the like, and is an ideal substrate for designing and synthesizing a fluorescence probe for specifically detecting GSH. Compared with the traditional fluorescence probe based on the supramolecular interaction of non-covalent bonds,Coumarin fluorescent probes are mostly reactive probes, and their recognition process is mainly to react with GSH in different types to produce fluorescent compounds, which change the optical signal of the fluorescent probe, and ultimately achieve the sensitive detection of GSH
[19].
2.1.1 Coumarin fluorescent probes based on Michael addition
Coumarin probes have electron-withdrawing groups and are easily attacked by the nucleophilic sulfur anion (GS-) in GSH. Eventually, the double bond between the α and β carbon atoms is broken, completing the Michael addition. Based on the above mechanism, a number of coumarin-based fluorescent probes based on Michael addition were designed (Fig. 1).
Reversible fluorescent probes facilitate quantitative, real-time monitoring of changes in GSH in living cells. Chen et al. Designed a reversible coumarin-based probe G-1
[20]. The — SH of GSH undergoes Michael addition to the maleimide of the probe (Fig. 2), resulting in an increase in fluorescence at 489 nm. The detection limit of G-1 was 760 nmol·L
-1, which was successfully applied to intracellular GSH imaging in L929 cells and HeLa cells.
Ratiometric probes improve the accuracy of GSH detection with built-in correction. Khatun et al. Synthesized a ratiometric fluorescent probe G-2 with coumarin as the main body
[21]. G-2 undergoes a Michael addition reaction with GSH, which blocks the expansion of π conjugation with the pyridine moiety, so that the fluorescence color changes from green to blue (Fig. 3). The fluorescence intensity ratio of F
460/F
510 increased linearly with the increase of GSH concentration, and the detection limit was 245 nmol·L
-1. G-2 can localize into GSH-depleted organelles (such as nucleoli, mitochondria, and lysosomes) and is used to track GSH during the cell cycle. Tian et al. Synthesized the ratiometric fluorescent probe G-3
[22]. G-3 undergoes a Michael addition reaction with GSH, which reduces the conjugation of GSH, resulting in a blue shift of the absorption peak and green fluorescence (Fig. 3). The G-3 fluorescence intensity ratio F
488/F
405 increased with the increase of GSH, and the detection limit for GSH was 50 mmol·L
-1. G-3 was successfully applied to ratiometric imaging of GSH levels in different living cells.
2.1.2 Coumarin fluorescent probes based on nucleophilic substitution
The nucleophilic substitution reaction between — SH in GSH molecule and electron-withdrawing group halogen occurs easily. Many researchers use this reaction to introduce halogen atoms into coumarin fluorophores to achieve fluorescence conversion through the substitution reaction between GSH and halogen atoms (Fig. 4).
The sensitivity of GSH detection can be improved by increasing the reactive sites of the probe with — SH and —NH
2 in GSH. Li et al. First introduced α, β-unsaturated malononitrile at the 3-position and a chlorine atom at the 4-position of coumarin to prepare the probe G-4. The fluorescence of the probe was quenched due to the presence of electron-withdrawing groups in the molecule
[23]. The — SH of GSH replaced the chlorine atom of the probe G-4, the —NH
2 cyclized with malononitrile, the ICT process was inhibited, G-4 fluorescence recovered and emitted yellow fluorescence (Fig. 5), and the detection limit for GSH was 0.43μmol·L
-1. The probe can be used for human cervical cancer cell level GSH imaging. Based on the same design strategy, Yin et al. Developed a new fluorescent probe G-5 by combining coumarin chloride and benzothiazole acetonitrile
[24]. The chlorine atom at position 4 in the coumarin moiety of the probe is halogen-substituted with — SH. Under the influence of the cyano group, the benzothiazole moiety with an unsaturated bond can serve as another reactive site for molecular cyclization with —NH
2 (Fig. 5). The detection limit of G-5 was 6.9 nmol·L
-1. G-5 was successfully applied to fluorescence imaging of exogenous living cell GSH.
Clinical studies have shown that the level of GSH in laryngeal cancer cells is significantly higher than that in normal or peritumoral tissues, but the application of fluorescent probes in clinical specimens is still rare. Based on this, Zou et al. Designed a two-photon fluorescence probe G-6 based on coumarin derivatives
[25]. The probe G-6 is non-fluorescent due to the heavy atom effect of the bromine atom. In the presence of GSH, the brominated moiety can be nucleophilically displaced by — SH with a strong fluorescence emission phenomenon (Fig. 6). The fluorescence intensity of the probe showed a good linear relationship with the concentration of GSH (0~15 mmol·L
-1), and the detection limit was 90μmol·L
-1. G-6 successfully distinguishes laryngeal cancer tissue from normal tissue and can be used to detect GSH in living cells, mouse models, and human clinical cancer tissue.
Yang et al. Synthesized a near-infrared fluorescent probe G-7 from coumarin and semi-cyanamide
[26]. Nucleophilic substitution and intramolecular cyclization occurred during the reaction between the probe and GSH (Fig. 7). With the addition of GSH, G-7 bioimages endogenous GSH in A375 cells by red emission.
2.1.3 Coumarin fluorescent probes based on demetalation reaction
The coumarin-like fluorescent probe is coordinated with a metal ion, and the fluorescence of the coumarin-like fluorescent probe is quenched by PET or ligand-metal charge transfer (LMCT). However, — SH in GSH molecule has stronger complexing ability with metal ions, and can demetalate with the complex to form GSH-metal complex, which restores the fluorescence (Fig. 8).
The preparation of the Schiff base is simple and convenient, and the imine/alkylimino group in the structure enhances the coordination ability of the probe and the metal ion. He et al. Selected coumarin hydrazide and 2,6-pyridinedicarboxaldehyde as raw materials to synthesize bis-Schiff base coumarin probe G-8 based on the easy coordination characteristics of Schiff base
[27]. G-8 itself has a strong fluorescence, and when coordinated with Cu
2+, the fluorescence of the probe is quenched due to paramagnetism and photoinduced electron transfer. Upon addition of GSH, a demetalation reaction occurs (Fig. 9), thereby allowing the fluorescence at 485 nm to recover. The probe can be applied to fluorescence imaging and specifically detect GSH in human cervical cancer cells. He et al. Designed and prepared a novel fluorescent probe G-9 from coumarin hydrazide and imidazole-2-carbaldehyde
[28]. Cu
2+ is complexed with G-9, so that the electron-withdrawing ability of G-9 is enhanced. The interaction between the two provides an additional fluorescence quenching pathway. The exciton energy is transferred from the ligand to the metal d orbital or LMCT occurs, resulting in the fluorescence quenching of G-9. Due to the strong ability of GSH to capture copper metal ions from G-9-Cu
2+, G-9-Cu
2+ was deetched, G-9 was released, and fluorescence was restored (Fig. 9). The detection limit of G-9-Cu
2+ for GSH was 0.12μmol·L
-1. The probe can be used for the detection of endogenous GSH under physiological conditions, and provides a new method for the biomolecular detection of GSH.
2.2 BODIPY fluorescent probe for detecting GSH
BODIPY is an important class of organic fluorescent dyes. Its molecular structure is easy to modify, and it has the characteristics of high quantum yield and high stability. The modified BODIPY fluorescent probe can achieve the purpose of detecting GSH by using the complex molecular structure of the probe.
2.2.1 BODIPY fluorescent probe based on aromatic ring substitution
Fluorophores containing labile substituents readily undergo nucleophilic substitution reactions with GSH to form thioethers (Fig. 10).
Liu et al. Designed fluorescent probe G-10 by introducing a chlorine atom to BODIPY
[29]. The chlorine atom can react with the — SH on GSH by aromatic nucleophilic substitution to give thioether (Fig. 11), which decreases the fluorescence intensity at 557 nm and increases the fluorescence intensity at 588 nm, and the detection limit for GSH is 1.1μmol·L
-1. By introducing a positively charged triphenylphosphine functional group at the 5 position of the probe, it can be used as a mitochondrial delivery carrier to deliver the probe to mitochondria by electrostatic attraction. In addition, the probe can be used for exogenous GSH imaging of human cervical cancer cells.
Aza-BODIPY has a longer emission wavelength, which is conducive to penetrating into the body and causing less damage to biological samples during molecular imaging. Xiang et al. Reported a BODIPY NIR probe G-11 containing two 7-nitrobenzo-2-oxa-1,3-diazole (NBD) groups
[30]. NBD is not only the leaving group of GSH-induced nucleophilic substitution reaction, but also can quench the fluorescent probe through the PET process. The NBD in G-11 is replaced by -SG of GSH, producing thio-NBD and aza-BODIPY and releasing red fluorescence at 730 nm (Fig. 12). G-11 can be used to detect GSH in human cervical cancer cells.
The electron-withdrawing ability of the substituent at position 3 of BODIPY can affect the activity of nucleophilic aromatic substitution reaction. Chen et al. Introduced 3,4-aminophenol into chlorinated BODIPY and developed fluorescent probe G-12
[31]. The phenol derivative attached to the BODIPY core reacts with GSH by aromatic nucleophilic substitution, resulting in red fluorescence (Fig. 13). There was a good linear relationship between the fluorescence intensity of the probe and the concentration of GSH(0~100μmol·L
-1), and the detection limit was 56 nmol·L
-1. The probe could obviously observe the increase of GSH in inflammatory mediators induced in RAW 264.7 cells, and successfully detected exogenous and endogenous GSH in macrophages.
2.2.2 BODIPY fluorescent probe based on -SH cleavage strategy
It is also a good strategy to design GSH probes by eliminating the covalent connection in the probe molecule through — SH to achieve the effect of fluorescence signal conversion (Fig. 14).
Wang et al. Designed and synthesized a probe G-13 containing DNBS and BODIPY as the mother nucleus, based on the research idea that the fluorescence of the probe recovered after the fluorescence quenching group DNBS was cut off by -SH
[32]. DNBS in the probe was cleaved by — SH of GSH, resulting in orange fluorescence at 582 nm (Fig. 15). The probe can quickly enter human cervical cancer cells and highly selectively detect intracellular GSH.
Liu et al. Used coumarin as an internal standard fluorophore to synthesize fluorescent probe G-14 by covalent attachment of chlorinated BODIPY and coumarin using nucleophilic substitution reaction on the aromatic ring
[33]. G-14 has green fluorescence, and coumarin and the corresponding BODIPY derivative are obtained after the covalent connection between coumarin and BODIPY is cut off by -SH on GSH. The coumarin moiety gave blue fluorescence at 453 nm, and the GSH-substituted BODIPY derivative gave yellow fluorescence at 558 nm. Addition of excess GSH resulted in the formation of a disubstituted BODIPY derivative with pink fluorescence at 588 nm (fig. 16). The detection limit of the probe for GSH is 1.07μmol·L
-1, and the probe can be used for imaging study of exogenous GSH in human cervical cancer cells.
2.3 Rhodamine fluorescent probe for detecting GSH
Rhodamine is a xanthene fluorescent dye, and the opening of the spiro ring of its derivative causes a fluorescence "switch" phenomenon accompanied by a fluorescence color change (Figure 17). The specific detection of GSH has been a hot spot in the research of rhodamine probes.
The introduction of hydrophilic groups to the bottom ring can improve the poor water solubility of rhodamine fluorescent probes. Ouyang et al. Synthesized a carboxyl-containing rhodamine probe G-15 from trimellitic anhydride
[34]. When the — SH in GSH was added to the aldehyde group of rhodamine, the ring was opened and hydrolyzed into a fluorescent rhodamine derivative, the fluorescence intensity at 576 nm increased significantly, and the color of the solution changed from colorless to pink by naked eye observation (Fig. 18), and the detection limit for GSH was 27 nmol·L
-1. The probe G-15 can be used to detect GSH at the level of human cervical cancer cells.
Chen et al. Developed a rhodamine-based fluorescent probe G-16
[35]. The lactam ring of the spirosome in the structure disrupts the conjugation of the rhodamine fluorophore, leaving G-16 without fluorescence. The nucleophilic substitution of GSH with the cyclobromoamide group promotes the ring opening of the spirochetal lactone, releasing the rhodamine chromophore, which changes the fluorescence color from colorless to pink (Fig. 19). The fluorescence intensity of G-16 showed a good linear relationship with the concentration of GSH (5~50μmol·L
-1), and the detection limit was 0.17μmol·L
-1. G-16 can be used to monitor the cellular redox state of GSH induced by oxidants.
Near-infrared fluorescence emission probes have attracted more and more attention in recent years because of their strong tissue penetration and low autofluorescence interference. Tong et al. Designed and synthesized a novel near-infrared probe G-17 for GSH based on conjugate addition and intramolecular amino-induced ring opening of spirochetal lactam
[36]. The -SH in GSH undergoes an addition reaction with the aldehyde group in G-17 to form an unstable intermediate. Then, the carbonyl group of rhodamine reacted with the —NH
2 group of GSH, which induced the ring opening of rhodamine lactam, and the fluorescence color changed from yellow to green (Fig. 20), and the detection limit was 0.1μmol·L
-1. G-17 can be applied to both endogenous and exogenous GSH imaging in MCF-7 cells, and it is a rapid and effective method to detect excess GSH in living cells. Yang et al. Also designed and synthesized a near-infrared fluorescent probe G-18
[37]. G-18 is not fluorescent due to the spirolactam ring of the rhodamine moiety. Upon addition of GSH, the hydrogen ion interacts with the carboxyl group of GSH to form an unstable intermediate. As the distance between the rhodamine moiety and the negatively charged carboxylic acid group decreases, a highly acidic microenvironment is created, which causes the helix ring of rhodamine to partially open and the fluorescence to turn on, emitting bright red fluorescence (fig. 20). There was a good linear relationship between the probe and the concentration of GSH in the range of 1~3μmol·L
-1, and the detection limit was 70 nmol·L
-1. The probe has been successfully applied to the determination of GSH in serum and bioimaging of GSH in living cells with low cytotoxicity.
2.4 Cyanine fluorescent probes for the detection of GSH
The fluorescent nucleus of the cyanine fluorescent probe is a cyanine dye (cyanine dye, hemicyanine dye, Oxonol dye). These probes contain quaternary ammonium cations, have good affinity and can be effectively enriched in mitochondria. In addition, the synthesis procedure is simple, the background interference is small, and the quantum yield is high. Therefore, it is of practical significance to study and expand this kind of probe. The thioether bond of the probe is the GSH recognition site, which undergoes a nucleophilic reaction to produce a thioether compound with a fluorescence change (fig. 21).
Lee et al. Developed two fluorescence-enhanced probes G-19 and G-20 based on cyanine
[38]. The probe G-19 connects the two cyanine units via a phenyl thioether bond, resulting in probe self-quenching. The probe G-20 contains a 3,5-bis (trifluoromethyl) phenyl sulfide group, and the cyanine moiety undergoes PET with 3,5-bis (trifluoromethyl) phenyl sulfide, so that the probe is non-fluorescent. GSH cleaves the thioether bond of G-19 and G-20, resulting in a significant fluorescence enhancement at 805 nm for both NIR probes (Figure 22). The detection limits for GSH were 0.63 and 0.33μmol·L
-1 for probes G-19, G-20, respectively. They can be used for fluorescence detection and imaging of GSH in SCC7 tumor-bearing mice.
Lin et al. Prepared the fluorescent probe G-21 by connecting hydroxybenzothiazocyclocyanine (HBTMC) and NBD through an ether bond
[39]. G-21 itself is not fluorescent. GSH undergoes nucleophilic substitution reaction with the probe, and the red fluorescence at 609 nm is enhanced (Fig. 23). The probe can be used for fluorescence detection of GSH at the cellular level.
2.5 Benzothiazole fluorescent probes for the detection of GSH
Compared with the classical fluorescence mechanisms (PET, ICT and FRET), ESIPT has a large Stokes shift and high environmental sensitivity, which is more attractive. The 2- (benzothiazol-2-yl) phenol derivative has excellent photostability, high fluorescence quantum yield, and good cell membrane permeability, so the ESIPT process of its conversion from the phenol form to the ketone form can be used as a design strategy to develop a fluorescent probe for the detection of GSH (Figure 24).
Chen et al. Synthesized a biphenylbenzothiazole derivative probe G-22 with a large Stokes shift (202 nm)
[40]. DNBS was used as a recognition group and electron acceptor, and its interaction with hydroxybenzothiazole (HBT) inhibited the ESIPT of HBT, resulting in the fluorescence quenching of the probe. When GSH reacted with the probe, the PET between DNBS and benzothiazole derivatives was blocked, and the generated 4-hydroxy-3- (benzothiazol-2-yl) -4-biphenyl nitrile produced intramolecular ESIPT, and the fluorescence turned on to emit strong cyan fluorescence (Fig. 25), with a detection limit of 0.17μmol·L
-1 for GSH. Zheng et al. Developed the fluorescent probe G-23 for tracking GSH
[41]. G-23 was not fluorescent. After the addition of GSH, G-23-OH was formed and ESIPT occurred, showing a large Stokes shift and green fluorescence of the probe (fig. 25). The detection limit of the probe for GSH(0~1 mmol·L
-1) was 330 nmol·L
-1. G-23 can be applied to image endogenous GSH in living cells and zebrafish.
Zhang et al. Synthesized a novel single-excitation dual-emission fluorescent probe G-24 based on HBT
[42]. GSH reacts with the probe via Michael addition and ester cleavage followed by ESIPT, and the color of the probe changes from colorless to light blue (fig. 26). The detection limit of the probe for GSH was 0.24μmol·L
-1. G-24 provides a new way to prepare multi-selective probes by using chemical reactions and weak intermolecular interactions.
2.6 Naphthalimide fluorescent probe for detecting GSH
Naphthalimide derivatives have the characteristics of large conjugated system and strong electron-donating groups. An electron-donating group is introduced to the 4-position of the naphthalimide, and its conjugated system is changed to form a compound with intramolecular donor-acceptor charge transfer, which often produces strong fluorescence. This type of probe has a sulfoxide group, which is often used by GSH as a recognition site for aromatic nucleophilic reaction to produce thioether compounds (Figure 27).
Liu et al. Developed two GSH fluorescent probes, G-25 and G-26
[43]. The fluorophore of G-25 is connected with a non-oxidized morpholine group, which is a switch-type fluorescent probe with no fluorescence in the initial state. G-26 has two morpholine oxides (thiomorpholine-S-dioxide and morpholine-N-oxide) as functional groups, and the probe has blue fluorescence. The substitution reaction of GSH with the sulfonamide of the two probes gave the same product, which emitted green fluorescence at 495 nm (fig. 28). The probe can be used to detect GSH in lysosomes.
Two-photon probes usually use two fluorophores and recognition groups to design the probe structure, which has the advantages of lower phototoxicity, better three-dimensional localization, deeper penetration depth and lower self-absorption for cell imaging. Xu et al. Designed the fluorescent probe G-27
[44]. GSH attacks G-27 by an aromatic nucleophilic substitution mechanism, with the nucleophilic substitution product producing green emission and the cleavage product producing orange emission (Fig. 29). After the addition of GSH(2~150μmol·L
-1), the fluorescence intensity of the probe at 495 nm gradually increased, showing a good linear relationship, and the detection limit was 0.10μmol·L
-1. The probe was successfully applied to two-color fluorescence imaging to detect GSH in living cells.
In order to explore the key structural factors determining the sensitivity of the probe in living cells, Zong et al. Synthesized a GSH ultrasensitive probe G-28 with a sulfoxide group as the recognition group
[45]. Upon addition of GSH, — SH attacks the sulfinyl group in the probe, and nucleophilic substitution occurs (Fig. 30). The fluorescence intensity of G-28 showed a single exponential increase in response to GSH with a detection limit of 0.11 mmol·L
-1 for GSH(0~500 mmol·L
-1). G-28 tracks subtle fluctuations in GSH levels in developing neurons, bioimaging GSH flux in developing neurons.
2.7 Metal organic framework fluorescent probe for detecting GSH
Metal-organic frameworks (MOFs) are usually a series of porous framework materials composed of metal ions or metal clusters and organic ligands (usually containing carboxylic acid, nitrogen, sulfur) arranged in order and connected by coordination bonds. Unlike organic molecules, the rigid framework formed by metal ions or metal clusters and organic ligands in MOFs can separate the ligand from the metal-centered node, enhance fluorescence, and reduce self-quenching. In addition, MOFs-based sensors are more sensitive than other fluorescent sensors due to their tunable porous structure. At present, MOFs have been deeply developed to construct fluorescent chemical sensors, and this new material also has certain advantages for GSH detection.
UiO-67 has excellent optical properties, and GSH can effectively enhance the fluorescence of UiO-67 through the electronic effect between GSH and the ligand, thus realizing GSH recognition. Based on the above principle, Zhu et al. Prepared a fluorescent probe G-29 with UiO-67 as the skeleton, because the ligand (4,4 '-diphenylethylenecarboxylic acid) has a conjugated structure, which leads to the fluorescence of G-29
[46].
When GSH is close to the ligand, the electron-donating groups of GSH (peptide bond and — SH) will increase the electron density of the conjugated structure, thereby enhancing the conjugation degree of the ligand and enhancing the solution fluorescence (Fig. 31). There was a good linear relationship between the fluorescence intensity of G-29 and the concentration of GSH (0.5~10 mmol·L-1), and the detection limit was 97.5μmol·L-1. G-29 is expected to serve as a diagnostic tool in clinical medicine.
Wang et al. Established a ratiometric fluorescent probe G-30 by using UiO-66-NH
2[47]. Dopamine is autoxidized in polyethyleneimine (PEI) solution to form copolymer (PDA-PEI). The fluorescence of G-30 was quenched by FRET with PDA-PEI. Due to the reducibility of GSH, the formation of PDA-PEI was prevented and the fluorescence of G-30 could be restored (Fig. 32). The detection limit of the probe for GSH(1~70μmol·L
-1) was 0.57μmol·L
-1. The probe was applied to the detection of GSH in human serum samples.
Jalili et al. Prepared a dual-emission metal-organic framework G-31 by encapsulating yellow and blue carbon dots with an imidazolium zeolite framework (ZIF-8)
[48]. In the presence of Cu
2+, the fluorescence of the yellow carbon dots in G-31 was enhanced, while the fluorescence of the blue carbon dots was quenched. After the addition of GSH to the Cu
2+-G-31 system, the fluorescence of the two carbon dots gradually recovered due to the strong interaction of GSH with Cu
2+ (Fig. 33). Under ultraviolet irradiation, the purpose of detection can be achieved through the change of fluorescence intensity. The detection range of G-31 for GSH was 3~25 nmol·L
-1, and the detection limit was 0.90 nmol·L
-1.
2.8 Other organic fluorescent probes for the detection of GSH
Aggregation-induced quenching (ACQ) can limit the use of probes in vivo, so it is of great significance to develop GSH-type fluorescent probes against ACQ effect. Cui et al. Designed a triphenylethane-containing quinoxaline pyridine compound G-32 with twisted intramolecular charge transfer (TICT) and aggregation-enhanced emission (AIE) properties
[49]. GSH cleaves the dinitrophenyl ether in the G-32 structure, and the product has AIE activity and low water solubility, thus turning on the fluorescence at about 516 nm, and the color changes from orange to green (Figure 34). The fluorescence intensity of the probe showed a good linear relationship with the concentration of GSH (0~10μmol·L
-1), and the detection limit was 434 nmol·L
-1. G-32 successfully detected GSH in bovine serum samples. In addition, GSH in HeLa cells can also be detected by confocal laser scanning microscopy.
Imidazole [1,5-α] pyridine has outstanding optical properties, such as strong photostability, high quantum yield, and large Stokes shift. Hou et al. Extended the conjugated system of imidazole [1,5] pyridine and synthesized a new fluorescent probe G-33
[50]. Due to ICT, the charge is transferred from the imidazole [1,5] pyridine moiety to the furan moiety, and G-33 produces red fluorescence. The Michael addition of GSH to the carbon-carbon double bond in G-33 partially inhibits ICT, and the fluorescence color of G-33 changes from red to cyan (fig. 35). The detection limit of G-33 for GSH(0~30μmol·L
-1) was 97 nmol·L
-1. The probe was successfully used to image endogenous GSH in MCF-7 cells and zebrafish, and is a powerful molecular tool to study endogenous GSH.
Porphyrins are a class of naturally occurring macrocyclic compounds that play an important role in the metabolism of living organisms. Porphyrins have been widely used in UV-vis sensors, Photoelectrochemical sensors (PEC), and electrochemiluminescence due to their special photoelectric properties, such as strong absorbance, high emissivity, and rich chemical properties. Chen et al. Prepared a naked-eye visible "switch" fluorescent probe G-34 based on 5,10,15,20- (4-sulfophenyl) porphyrin (TPPS4)-Hg
2+[51]. In the presence of Hg
2+, the lone pair of the electron donor nitrogen atom is transferred to the unoccupied orbital of Hg
2+ to form a complex, which leads to the fluorescence quenching of G-34. After the addition of GSH, the affinity of GSH to the Hg
2+ is stronger than that of the N atom of the porphyrin ring, which makes the probe turn to the "on" state and the fluorescence color change to pink. The naked-eye detection limit of the probe pair GSH(0~28.5μmol·L
-1, 38.15~61.87μmol·L
-1) was 0.43 nmol·L
-1 at 640 nm and 700 nm.
— SH in GSH has reducibility, which can break the disulfide bond in the probe molecule, resulting in fluorescence recovery/enhancement (fig. 36). Based on the disulfide-thiol exchange reaction, the following two probes were designed (figs. 37, 38).
Hu et al. Synthesized a Schiff base fluorescent probe G-35 based on AMC
[52]. The autofluorescence of the probe comes from the transmission of two carbon-nitrogen bonds in the Schiff base induced by n-π
*. In response to the disulfide bond, GSH is cleaved to — SH by a thiol-disulfide exchange reaction, resulting in an increase in fluorescence intensity (fig. 37). The detection limit of the probe for GSH was 36μmol·L
-1. G-35 can be used for GSH imaging in HeLa cells.
SQ is a typical NIR dye, and Zheng et al. Established a novel NIR self-quenching probe G-36 based on SQ
[53]. G-36 is composed of two SQ fluorophores connected by a disulfide bond, and the background fluorescence intensity is very weak due to ACQ enhancement and FRET-mediated self-quenching effect. GSH selectively cleaves the disulfide linker of G-36 through intermolecular hydrogen bonds and specific electrostatic interactions, interrupting the fluorescence self-quenching effect between the two SQ fluorophores (Fig. 38). The detection limit of this probe was 0.15μmol·L
-1. G-36 can monitor endogenous and exogenous GSH in tumor or normal cells.
3 Inorganic nano-fluorescent probe for detecting GSH
At present, the organic fluorescent probes used to detect GSH generally have the disadvantage of poor water solubility, while the designed inorganic fluorescent probes, such as semiconductor quantum dots (QDs), carbon dots (CDs), metal nanoclusters (NCs), etc., have good water solubility. Most of these fluorescent probes are based on the principle of fluorescence quenching and recovery to detect GSH.
3.1 Semiconductor quantum dots for the detection of GSH
QDs are small particles with a size of 0 ~ 100 nm synthesized from semiconductor materials, which are mainly composed of Ⅱ-Ⅵ or Ⅲ-Ⅴ group elements. Because of its excellent optical properties, it can be used for the specific detection of GSH.
Functionalized QDs based on crystal violet (CV) can be used to detect GSH conveniently, rapidly and at low cost. Sheng et al. Prepared a nanofluorescent probe G-37, which was a mixture of water-soluble mercaptopropionic acid (MPA) -capped QDs and CV. The fluorescence of the probe was quenched by FRET generated by the electrostatic interaction between the N
+ of CV and the —COO
- of MPA
[54]. Because that bin ability of GSH to Cd
2+ is stronger than that of MPA, MPA carry CV dissociated from the surface of QDs, and the fluorescence of QDs recovered (fig. 39). The fluorescence intensity of G-37 showed a good linear relationship with the concentration of GSH (0.02~50μmol·L
-1) at 550 nm, and the detection limit of GSH was 8.2 nmol·L
-1. The probe G-37 provides a new idea for the rapid determination of GSH in water and urine samples.
Among the metal-containing QDs, the chemical toxicity of ZnS is relatively small. As an eco-friendly fluorescent probe with high chemical stability, ZnS QDs have great potential in the field of fluorescence chemistry. Amouzegar et al. Synthesized an MPA-encapsulated probe G-38 based on this design strategy
[55]. After the addition of Zn
2+, Zn (oh)
was formed, and a passivation layer was formed on the surface of the quantum dots, resulting in the enhancement of the fluorescence of the quantum dots. After adding GSH to the Zn
2+-G-38, GSH can form a complex with Zn
2+, resulting in the cleavage of Zn (oh)
and the destruction of the QD passivation layer, thus reducing the fluorescence intensity (Fig. 40). The detection range of GSH was 1.95~104.0μmol·L
-1, and the detection limit was 0.92μmol·L
-1.
The surface modification of QDs can be used to develop sensitive and biocompatible fluorescent probes for the detection of GSH in cells. Sun et al. Added MnO
2 nanosheets on silica-coated semiconductor quantum dots (QD-SiO
2) to prepare a stable biocompatible fluorescent nanoprobe G-39
[56]. MnO
2 quenches the fluorescence of QD-SiO
2 by FRET. The addition of GSH led to the reductive degradation of MnO
2 nanosheets, and the fluorescence of G-39 was recovered. The fluorescence intensity of the probe increased gradually with the increase of GSH concentration, and showed a good linear relationship in the 0.01~120μmol·L
-1 range. G-39 has been successfully used for real-time imaging of intracellular GSH because of its good biocompatibility and strong cellular uptake ability.
3.2 Carbon dots for detection of GSH
CDs are a new type of fluorescent carbon nanoparticles in recent years, which can be divided into graphene quantum dots, carbon quantum dots, carbon nanodots and carbonized polymer dots. Because of its excellent optical properties, good biocompatibility, low toxicity and other advantages, it plays an important role in material detection and cell imaging. The principle of detecting GSH by CDs is generally indirect detection. First, the combination of CDs and substances leads to fluorescence quenching. The fluorescence of CDs can be restored by adding GSH, so as to achieve the purpose of detecting GSH.
Because of the strong complexing ability of — SH to Cu
2+, the detection of GSH can be realized based on the metal ion replacement method. Yang et al. Modified the surface of CDs with triethyltetramine to obtain probe G-40
[57]. Because the Cu
2+ is easy to be selectively captured by the amino and amide groups on the surface of G-40, the aggregate of G-40/Cu
2+ is formed, and the fluorescence is quenched. When GSH is added to the G-40/Cu
2+ aggregation system, the strong interaction between GSH and Cu
2+ disrupts the aggregate structure, releasing blue fluorescence at 470 nm (Figure 41). The fluorescence intensity of the probe has a good linear relationship with the concentration of GSH (0.2~175μmol·L
-1), and the detection limit is 0.11μmol·L
-1. G-40 can be used to detect Cu
2+ and GSH in living yeast cells.
The fluorescence of CDs obtained by direct pyrolysis of citric acid was greatly improved after surface passivation with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC). Pan et al. Designed the fluorescent probe G-41 by this method
[58]. GSH is mixed with excess EDC solution, and part of EDC forms a stable complex with GSH. When CDs is added to the above mixed solution, the surface of CDs is passivated by the remaining EDC, which is equivalent to that GSH indirectly controls the degree of surface passivation of CDs (Fig. 42). The detection limit of G-41 for GSH(1~50μmol·L
-1) was 0.943μmol·L
-1. The probe G-41 can be used for GSH monitoring in human serum.
When the halogen atom is introduced into the conjugated system of the fluorescent probe, the intersystem crossing rate increases and the fluorescence of the probe decreases, resulting in the "heavy atom effect", while the -SH compound can replace the halogen atom to restore the fluorescence of the probe
[59]. Based on this idea, our group designed and synthesized a fluorescent probe G-42 with CDs as the parent and bromoacetyl bromide as the functionalized group
[60]. The autofluorescence of G-42 is quenched due to the heavy atom effect. When the bromine atom is replaced by — SH of GSH, the fluorescence is restored. The fluorescence intensity of G-42 at 470 nm was linear with GSH(0~36μmol·L
-1), and the detection limit was 140 nmol·L
-1. G-42 can be used to detect GSH at the level of human umbilical vein endothelial cells because of its low toxicity and good cell permeability.
In order to avoid the complex process of covalent connection between fluorophore or acceptor and fluorophore, Wu et al. Developed a fluorescent nanoprobe G-43 based on IFE, with nitrogen-doped CDs as the fluorophore and DTNB as the recognition molecule
[61]. GSH reacts with DTNB in the probe to form 5-thio-2-nitrobenzoic acid (TNB), which has a high molar extinction coefficient and overlaps with the excitation spectrum of nitrogen-doped CDs to produce IFE, resulting in a gradual decrease in the fluorescence intensity of G-43 at 510 nm. The detection limit of G-43 for GSH was 30 nmol·L
-1. The probe can be used for the study of mouse oxidative stress model and the imaging of human liver cancer cells.
Jia et al. Designed a red carbon dot G-44 with high pyridine N content, which can detect GSH in both colorimetric and fluorescent modes
[62]. The color of G-44 changed from red to purple after the addition of GSH. The absorbance ratio of G-44 from 576 to 486 nm was linearly proportional to the logarithmic concentration of GSH(12.5~800μmol·L
-1), and the detection limit was 0.7μmol·L
-1. In addition, they also prepared cellulose composite hydrogel and test strip based on this probe, which provided a great opportunity for portable, accurate and sensitive detection of GSH.
On the basis of CDs synthesized by citric acid (CA) and polyetherimide (PEI), Wang et al. Developed a dual-wavelength emission probe G-45 using CA, PEI and rhodamine B as precursors
[63]. Excited at 360 nm, the fluorescence spectrum exhibited two emission bands of CDs (447 nm) and rhodamine B (581 nm), which were blue and orange, respectively. The fluorescence of G-45 at 447 nm was quenched by the addition of Hg
2+. While GSH can restore the fluorescence quenched by Hg
2+ (Fig. 43). The fluorescence intensity ratio (F
447/F
581) of this probe was proportional to the concentration of GSH (1~10μmol·L
-1), and the detection limit was 0.27μmol·L
-1. G-45 can be used for the determination of GSH in water, human blood, urine and other aqueous solutions.
Li et al. Prepared the dual-emission carbon dot G-46
[64]. GSH and the oxygen-containing functional groups on the surface of G-46 are easy to form intermolecular hydrogen bonds, which induce the orderly aggregation of the probe and the formation of large nanoparticles with uneven shapes, resulting in an upward trend of the fluorescence peak of G-46 at 430 nm and a proportional decrease of the fluorescence peak at 642 nm (Figure 44). The detection limit of the probe for GSH was 0.26μmol·L
-1. G-46 can be used as an effective tool for ratiometric GSH sensing and differentiating cancer cells from normal cells.
Most long-wavelength emission CDs are not affected by the blue autofluorescence of biological substrates and have less photodamage to biological tissues, but they have the disadvantages of low quantum yield and small Stokes shift. To solve this problem, our research group designed a yellow carbon dot G-47 with a large Stokes shift
[65]. Ag
+ binds to the carboxyl group of G-47, and a static quenching mechanism results in fluorescence quenching. GSH has a high affinity for Ag
+ and can restore the fluorescence of G-47. The detection of GSH was achieved by this fluorescence "off-on" process with a detection limit of 76 nmol·L
-1. G-47 can be used to detect GSH in tomato, purple grape and H1299 cells.
Graphene quantum dots (GQDs) can be used as a green alternative to toxic transition metal quantum dots, and can also be well applied to the detection of GSH. Wang et al. Synthesized a square MnO
2 nanocomposite G-48 coated with GQDs
[66]. GQDs fluorescence was suppressed by efficient FRET and IFE. GSH can decompose MnO
2 and restore the fluorescence of GQDs. The fluorescence intensity of G-48 at 456 nm was linear with the concentration of GSH (0.07~70μmol·L
-1), and the detection limit was 48 nmol·L
-1. Based on the same design strategy, Guo et al. Synthesized graphene oxide -MnO
2- fluorescein (GO-MnO
2-FL) nanocomposite G-49
[67]. ET occurred between FL and GO-MnO
2 nanocomposite, resulting in fluorescence quenching. GSH reduced MnO
2 to Mn
2+, which blocked the energy transfer between FL and GO-MnO
2, so that the fluorescence of the composite was significantly enhanced (Fig. 45), and the detection limit for GSH(10μmol·L
-1~2 mmol·L
-1) was 1.53μmol·L
-1. G-49 has excellent tumor-targeting ability and great potential for GSH-mediated cancer diagnosis.
3.3 Metal Nanoparticle Fluorescent Probes for Detection of GSH
NCs consist of a few to tens of atoms and are protected by sulfuric acid, sometimes even containing hundreds of metal atoms. Its size (< 2 nm) is between that of an atom and a nanoparticle. The diameter of NCs is comparable to the Fermi wavelength of electrons in metals, resulting in a continuous density of States split into discrete energy levels and thus unique optical, electrical, and chemical characteristics. In recent years, there has been an explosive growth in the research of NCs, which can be used as fluorescent probes in biosensing and bioimaging.
Li et al. Used polysaccharide (alginate dialdehyde, ADA), diphenylalanine (FF), Au nanoparticles (AuNPs) and 5-aminofluorescein (FI-NH
2) as raw materials to prepare fluorescent probe G-50
[68]. The fluorescence of (NSET),FI-NH
2 was quenched due to the nanometal energy transfer between FI-NH
2 and AuNPs. After the introduction of GSH, a stable Au — S bond was formed and the NSET process was blocked, the fluorescence of FI-NH
2 at 518 nm was recovered, and the fluorescence of Au nanospheres at 468 nm remained unchanged, which could be used as a built-in reference signal. The fluorescence ratio signal was positively correlated with GSH concentration within normal cells (0~200μmol·L
-1) and human cervical cancer cells (1~15 mmol·L
-1). G-50 provides important ideas for the diagnosis and treatment of cancer.
Li et al. Designed the probe G-51 based on the conversion strategy between the aggregated and dispersed States of gold nanocrystal clusters (AuNCs)
[69]. The aggregation agent 2-SH-1-methylimidazole (MMI) was added to adjust the state of AuNCs, so that the fluorescence color of AuNCs in the dispersed state and aggregated state was blue and red, respectively. In PBS solution, if GSH and MII were added at the same time, the AuNCs were dispersed, the fluorescence color of the probe did not change (Fig. 46), and the detection limit for GSH was 12 nmol·L
-1.
G-quadruplex is a special form of nucleic acid, which is easy to synthesize and modify, and can specifically bind to metal ions and porphyrins. Using N-methylporphyrin IX (NMM) combined with G-quadruplex as a signal unit for GSH detection, Ji et al. Developed a fluorescent probe G-52 based on Ag
+ and NMM-mediated G-quadruplex structure
[70]. Starting from a DNA strand with G-rich sequences, the initial intrastrand molecular hybridization forms a "hairpin" structure, and the G-rich sequence is partially blocked. After the addition of Ag
+, the "hairpin" structure is disrupted by the formation of a C-Ag
+-C base pair, and the G-enriched sequence is released to form a G-quadruplex structure, which can bind NMM and produce strong fluorescence at 612 nm. However, in the presence of GSH, the C-Ag
+-C base pair could not be formed due to the strong binding ability of GSH for Ag
+, and G-52 returned to its original structure (Fig. 47) with fluorescence quenching and a detection limit of 3.5 nmol·L
1- for GSH.
3.4 Manganese Dioxide Nanosheet-based Fluorescent Probe for the Detection of GSH
Manganese dioxide (MnO2) nanomaterials are widely used in fluorescent sensors because of their large molar extinction coefficient, good biocompatibility, and decomposition into non-toxic Mn2+ in vivo, which can be excreted by the kidney. MnO2 nanomaterials can achieve the detection of GSH by inducing fluorescence quenching or a unique redox reaction. At present, researchers have developed a variety of fluorescent probes based on MnO2 nanosheets for GSH detection.
Based on the MnO
2 nanosheet redox reaction, Yao et al. Reported a case of fluorescent probe G-53
[71]. MnO
2 nanosheets can oxidize OPDA to 2,3-diaminophenazine (OPDAox) with the best emission at 568 nm. The addition of GSH could reduce the MnO
2 nanosheets to Mn
2+, which reduced the effective oxidation activity of the nanosheets and inhibited the formation of OPDAox (Fig. 48). At the same time, the fluorescence at 568 nm was quenched. The detection limit of G-53 was 10 nmol·L
-1.
Zhang et al. Adsorbed the AIE molecule SiO
2-NPs on the MnO
2 nanosheet by electrostatic interaction to make probe G-54
[72]. Due to the steric hindrance between the negatively charged MnO
2 nanosheets and the anionic tetraphenylethylene derivative (TPE3), TPE3 could not aggregate on the surface of SiO
2 nanoparticles, and G-54 did not produce fluorescence. With the addition of GSH, the reduction of MnO
2 to Mn
2+ leads to the decomposition of nanosheets, releasing amino-functionalized SiO
2-NPs,TPE3 to aggregate on the surface of positively charged SiO
2-NP to form TPE3-SiO
2-NPs. TPE3 has restricted intramolecular rotation and emits intense fluorescence at 490 nm, the fluorescence intensity is positively correlated with the GSH concentration (0.5~100μmol·L
-1), and the detection limit is 200 nmol·L
-1. G-54 can be applied to complex probe fluorescent labeling processes.
MnO
2 is easily reduced under acidic conditions, and the color of the solution fades to colorless. Fu et al. Synthesized a multifunctional nanoprobe G-55 by combining the pH-responsive fluorescent dye AHC (3-acetyl-7-hydroxy-2H-chromium-2-group) and MnO
2 nanosheets
[73]. AHC is quenched by MnO
2 nanosheets with high efficiency due to IFE. After the addition of GSH, the MnO
2 nanosheets were reductively decomposed, and the blue fluorescence of AHC was recovered. Meanwhile, with the increase of acidic pH, not only the MnO
2 nanosheets were decomposed, but also the phenol group in AHC was deprotonated, which enhanced the ICT, resulting in the enhancement of AHC blue fluorescence (Fig. 49). The fluorescence of G-55 at 456 nm gradually increased with the addition of GSH(0.5~20μmol·L
-1), and the detection limit was 0.22μmol·L
-1.
4 Conclusion and prospect
In this paper, we reviewed the GSH fluorescent probes reported in the past five years from the perspectives of small organic molecules and inorganic nanoprobes, based on the classification of fluorophores. The response modes of GSH to different fluorophores were introduced, and the design strategy, fluorescence change and practical application of the probe were discussed. The comparison of the sensing performance and application of glutathione probes based on different fluorophores is summarized in Table 1.
At present, the selectivity, sensitivity, biocompatibility and intracellular distribution of GSH fluorescent probes have been improved. Among them: (1) Ratio probe and dual-emission probe can be effectively self-calibrated to improve the precision and accuracy of GSH detection; (2) The design of near-infrared and two-photon fluorophores reduces the fluorescence background interference and tissue damage ability of biological systems and improves the penetration depth, which is conducive to biomedical applications; (3) The combination of AIE molecule and probe not only avoids the ACQ effect of traditional fluorescent probes, but also effectively improves the fluorescent labeling process of the probe. The above three points provide new ideas for the development of GSH fluorescent probes.
In addition, the change of GSH content in human cells can indirectly reflect some diseases, but there are few probes that can track GSH in cells in real time and quantify it accurately. Therefore, future research should focus more on the clinical application of probes, which requires interdisciplinary cooperation among scientists in different fields. It is hoped that the review of relevant studies can provide reference and information for researchers.
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