1 Introduction
Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are three common biological thiols in cells, which play a very important role in many physiological processes such as regulating human metabolism and cellular redox homeostasis
[1]. Although Cys shares similar structural units (Fig. 1) and reaction properties with Hcy and GSH, they play different roles in physiological processes
[2]. As an essential thiol-containing amino acid, cysteine is involved in important processes such as protein synthesis, metabolism, and detoxification. Cys is the metabolite of Hcy, the precursor of antioxidant GSH, and the source of sulfide in human metabolism. The normal concentration of Cys in human body is 30 ~ 200 μM
[3]. Abnormal levels of three biothiols in the human body can lead to various health problems. Lack of Cys levels in the body can lead to growth retardation, hair discoloration, liver and muscle damage, fat loss and other related problems. Too much Cys can cause diseases such as rheumatoid arthritis, Parkinson's disease and Alzheimer's disease
[4⇓⇓⇓⇓~9]. As far as the current research is concerned, the presence of Hcy and GSH will interfere with the detection of Cys, and the specific recognition and detection of Cys in vivo is still a great challenge. Therefore, it is urgent to develop an effective real-time specific detection technology for Cys in human cells and tissues, and it is of great clinical significance.
At present, the reported methods for the detection of Cys mainly include electrochemical method, capillary electrophoresis, high performance liquid chromatography, gas chromatography-mass spectrometry, surface enhanced Raman scattering (SERS) and spectrophotometry
[10⇓⇓~13]. However, these methods have some shortcomings, such as expensive instruments, complex detection and long detection time. In recent years, fluorescence analysis has been widely used for specific detection of Cys due to its high sensitivity, good selectivity, small sample size, low cost and good biocompatibility
[14]. In addition, the widespread use of optical microscopy has accelerated the real-time detection of cysteine in vitro and in vivo by fluorescence techniques, which allow visualization of the distribution of cysteine within the target due to its high temporal and spatial resolution
[9]. The fluorescent probe is mainly composed of three parts, namely a Fluorophore, a Relay and a Receptor. After the reaction between the substance to be detected and the recognition group, the signal is transmitted to the fluorophore through the connecting arm to cause the fluorescence change, and the fluorophore is the information source of the optical signal
[15,16]. Each fluorophore has its advantages and disadvantages. Different fluorophores have different fluorescence quantum yields, different emission wavelengths, and different Stokes shifts.
In this paper, the fluorescent probes used to detect cysteine in the past three years are reviewed. According to the preparation methods, they are mainly divided into two parts: chemical fluorescent probes and genetic fluorescent probes. Chemical fluorescent probes mainly include organic small molecule fluorescent probes and nano-fluorescent probes. These probes are again distinguished by the structure of the fluorophore, and the characteristics of these probes are carefully compared and comprehensively summarized.
2 Small Organic Molecule Fluorescent Probes for Cys Detection
Organic small molecule fluorescent probe is mainly through the specific interaction with the analyte, causing the chemical environment of the fluorophore to change, through the change of color, the shift of spectrum, the change of fluorescence intensity and other manifestations to quantitatively detect the analyte. These specific effects are due to Photoinduced electron transfer, intramolecular charge transfer, Twisted intramolecular charge transfer, Excited-state intramolecular proton transfer, Fluorescence resonance energy transfer, cross-bond
[17⇓~19]. The fluorophore of organic small molecule fluorescent probes is based on compounds containing conjugated structures. The common fluorophores used to detect cysteine are coumarin, benzothiazole, rhodamine, fluorescein, fluoroboropyrrole, etc.
2.1 Coumarin-based Cys fluorescent probe
Coumarin is found in many plants in nature and contains a 1-benzopyran-2-one group, which is a very large family of compounds and a natural product
[20]. Coumarin compounds can be used in fluorescent dyes, laser dyes, antifungal and antitumor agents, such as anticancer compounds, antiproliferative agents, antioxidants and antifungal agents, because of their excellent optical properties and life activities
[21,22][23][24]. The parent structure of coumarin (Figure 2) is a two-ring structure, including pyranone and benzene rings, and the molecular structure contains carbon-carbon double bond units and is fixed in the cis conformation, so it can effectively avoid the cis-trans isomerization of normal carbon-carbon double bonds in vinyl compounds, which is helpful for coumarin to emit strong fluorescence and show good photostability. Fluorescent probes based on coumarin derivatives have been widely used in the construction of fluorescent chemical sensors because of their good photostability, large Stokes shift, high molar extinction coefficient and fluorescence quantum yield, excellent biocompatibility, perfect cell permeability, many modification sites, and simple synthesis
[25,26][27][28][29]. Because of the strong affinity of Cys, it can react with α, β-unsaturated ketone group to add to coumarin derivatives.
By introducing certain substituents at the 3, 4, 7, or 8 position of the coumarin parent, a stable and strong fluorescence signal is generated. Cao et al. Synthesized a multi-signal fluorescent probe 1 by introducing three different substituents at the 3, 4, and 7 positions of coumarin (Fig. 3)
[30]. The different reaction mechanisms of the probe with the three biothiols lead to three products with different spectral responses. The detection limit of probe 1 for Cys (0 ∼ 20 μm) was 6 nM. In the live cell fluorescence imaging experiment, probe 1 produced obvious fluorescence signals in the green, blue and red emission channels of Cys, Hcy and GSH, respectively, indicating the application potential of probe 1 in the specific detection of Cys in live cells. Because multiple reaction sites have higher sensitivity and selectivity than single reaction sites, their group recently synthesized a probe 2 with two potential thiol recognition sites (Fig. 4)
[31]. When Cys was added, a significant fluorescence enhancement was observed when the probe first used the α, β-unsaturated acetyl moiety as the thiol Michael addition site, and then the fluorescence began to decrease slowly when the probe cleaved with the 2-thiophene carbonyl group as the fluorescence quenching group and the thiol nucleophilic substitution reaction site. Compared with Hcy/GSH, Cys has less steric hindrance and stronger affinity for sulfhydryl groups, so the probe can rapidly and specifically detect Cys with a detection limit of 0.24 μM. This is also the first probe reported so far that utilizes a two-level hierarchical reaction for specific single detection of Cys. This two-level hierarchical reaction makes it possible to dynamically monitor Cys in living cells, providing a design strategy for the development of new specific recognition probes in the future.
Cheng et al. Designed and synthesized a new fluorescent probe 3 to detect three biological thiols by introducing different substituents at the 3 and 7 positions of the coumarin parent, using Michael addition, and intramolecular rearrangement strategy (Fig. 5)
[32]. When Cys/Hcy was added to the probe, the conjugated alkyne on the substituent at the third position of the probe underwent Michael addition and intramolecular rearrangement, and the fluorescence enhancement was obvious at 495 nm under the excitation of 430 nm wavelength. When GSH was added to the probe, the intramolecular S, N rearrangement could not occur, so no fluorescence was produced. The detection limit of Cys (0 ~ 10 μM) was 49 nM. In the fluorescence imaging experiment of mouse liver tissue, the probe showed two-photon fluorescence characteristics, which could detect Cys in deep tissue, and the imaging depth could be up to 107 μm. However, because Hcy and Cys have similar structures, probe 2 cannot specifically detect Cys.
Chu et al. Designed and synthesized a colorimetric fluorescent probe 4 (Fig. 6) for the differential detection of Cys and Hcy by introducing acrylate and allyl groups at the 7 and 8 positions of the coumarin parent, respectively
[33]. Probe 4 itself is weakly fluorescent. After adding Cys, the acrylate group of the probe is easy to nucleophilic addition with Cys, followed by rapid intramolecular rearrangement and cyclization. The removal of a 7-membered ring can cause a 5-fold fluorescence enhancement, and the solution changes from colorless to yellow. The addition of Hcy and other amino acids did not change the color and fluorescence intensity of the solution. The detection limit of probe 3 for Cys was 2 μm. The introduction of an allyl group at the ortho position of the acrylate is of enlightening significance for the development of fluorescent probes for the specific detection of Cys.
Zou et al. Also synthesized probe 5 by introducing an acrylate group at the 7-position of the coumarin parent (fig. 7)
[34]. The Michael addition of Cys to the acrylate group on the probe initiated the ESIPT process, which induced a 35-fold fluorescence enhancement, which was more obvious than that of probe 4. The detection limit of the probe for Cys was 88. 2 nM, which was more sensitive than that of probe 4. Even the addition of Hcy/GSH, which is similar in molecular structure, did not cause interference, and the Stokes shift of the probe was 216 nm. Probe 5 also has good biocompatibility and low cytotoxicity, and has been successfully used for Cys fluorescence imaging in living cells.
Zhang et al. Designed and synthesized a fluorescent probe 6 for detecting Cys in lysosomes by introducing morpholine and acrylate groups at the 4 and 7 positions of the coumarin parent, which were used as lysosomal targeting groups and specific recognition units of Cys, respectively (Fig. 8)
[35]. Probe 6 itself does not fluoresce due to the PET process between the morpholine group and the acrylate group, but when Cys is added, the probe fluoresces strongly due to the conjugate addition of Cys and acrylate, with a detection limit of 0.28 μM. Confocal fluorescence microscopy imaging in HeLa cells showed that the probe could specifically detect lysosomal Cys in living cells, which provided an important reference for the future study of lysosomal Cys-related diseases.
Xiao et al. Designed and synthesized a fluorescent probe 7 (Fig. 9) for differential detection of Cys and GSH with different emission channels at different excitation wavelengths by coupling coumaryl aldehyde and naphthyl groups
[36]. The probe itself has weak fluorescence, and after adding Cys, it emits strong fluorescence at 470 nm under the excitation of 360 nm wavelength, and the fluorescence response of the probe to Hcy/GSH is weaker than that to Cys. In the HeLa cell fluorescence imaging experiment, Cys and GSH can have obvious fluorescence emission in the blue and green channels, respectively, indicating that the probe can efficiently detect Cys in living cells.
Hien et al. Synthetically designed a complex formed between Cu
2+ and a coumarin fluorescent ligand as a novel chemical sensor for detecting biological thiols based on a complex exchange reaction8 (Fig. 10)
[37]. The fluorescence of coumarin was quenched by Cu
2+, and the fluorescence intensity was almost completely recovered when Cys was gradually added to twice the concentration of the sensor, because the probe reacted with Cys to form a [CuX
2]
2+ complex, which led to the release of the fluorophore and the recovery of fluorescence. Hcy and GSH can also cause different degrees of fluorescence recovery, but the recovery rate is less than that of Cys, and the detection limit of the probe for Cys is 0. 3 μM. The probe provides a new research direction for the detection of biological thiols by using the complex formed between metal ions and small organic molecule fluorophores.
The fluorescence property of that coumarin can be better improve by selectively modifying the substituent on the coumarin matrix. Most of the probes based on coumarin fluorophore for Cys detection introduce recognition sites at the 3 and 7 positions of the parent. The introduction of an amino group at the 7 position of the coumarin parent can enhance the intramolecular charge transfer (probes 1, 2, 3, 7, 8), and the introduction of a Cys recognition group at the 3 position of the parent has a lower detection limit and a faster reaction rate (probes 1, 3).
2.2 Rhodamine-based Cys fluorescent probe
Rhodamine compound is a basic fluorescent dye with xanthene as the parent (Fig. 11). Its spirolactam has two structures of ring opening and ring closing. In the ring opening state, the whole molecule is in a conjugated state, emitting strong fluorescence, and the color changes significantly. In the closed loop state: colorless and non-fluorescent
[38,39]. The rhodamine dye has the advantages of large rigid planar structure, good photostability, high molar extinction coefficient, high fluorescence quantum yield, longer excitation wavelength, less background interference, good water solubility and sensitivity
[40,41][42][43,44][45][46,47][48,49]. Rhodamine compounds are usually used to design ratiometric fluorescent probes by combining with other fluorophores. The ratiometric fluorescent probe has self-calibration capability through dual-channel emission intensity variation, which can improve the sensitivity and accuracy of the sensor and can be used for better imaging applications. Near-infrared fluorophores have unique ratiometric detection and near-infrared imaging characteristics, such as good penetration deep into tissues, low cellular and tissue background fluorescence
[50,51].
Yu et al. designed and synthesized a novel near-infrared ratiometric fluorescent probe 9 (Fig. 12) by introducing a quinoline group into the molecular skeleton of rhodamine. Compared with the traditional rhodamine dye, the probe enlarges the π conjugation system, increases the Stokes shift, and has excellent fluorescence quantum yield
[52]. After treatment with Cys, the spirolactam of the probe was in a closed loop state, the near-infrared fluorescence emission at 712 nm decreased, and the visible fluorescence at 462 nm caused by the quinoline group increased. When 40 μm Cys was added, the fluorescence intensity ratio (I
462 nm/I
712 nm) of probe 7 increased from 0.063 to 7.206, and the fluorescence enhancement was 114-fold. When Hcy is added, there is a similar spectral response, but the reaction can reach saturation faster due to the larger pseudo-first-order rate constant of Cys. The detection limit of the probe for Cys was 0.12 μm. In Caov 3 cells imaging, probe 7 was found to have two different fluorescence emissions, which could visually and specifically detect Cys in cells.
In addition to detecting Cys in living cells, Liu et al. Also synthesized a ratiometric fluorescent probe 10 (Figure 13) to detect Cys in bovine serum, water, milk, apples, etc. By introducing acrylate as a recognition site on the rhodamine skeleton, the fluorescence emission of the probe itself at 592 nm decreased after adding Cys, and a new fluorescence emission peak appeared at 584 nm with a blue shift in emission.The fluorescence intensity did not change significantly when other analytes were added, which was due to the Michael addition of Cys to the probe, followed by the removal of a 7-membered ring by intramolecular cyclization, and the fluorescence intensity reached a plateau within 1. 5 min, which was faster than that of most previously reported fluorescent probes for Cys detection
[53]. The seven-membered ring is more favorable than the eight-membered ring induced by Hcy in kinetics, while GSH has a slow reaction rate due to its steric hindrance. The detection limit of probe 10 for Cys is 20 μM.
Xia et al. Designed and synthesized a near-infrared ratiometric fluorescent probe 11 based on the FRET mechanism using coumarin as an energy donor and rhodamine as an energy acceptor (Fig. 14)
[54]. When Cys was gradually added to the probe solution, the fluorescence of the coumarin donor increased significantly, and the fluorescence of the rhodamine receptor decreased gradually, because the ring-closed spirolactam structure was formed on the rhodamine receptor, and the reaction with Cys led to the fluorescence quenching of the rhodamin receptor. When Cys was added, the fluorescence intensity ratio finally increased from 0.235 to 4.237, and the fluorescence was enhanced by 18 times. The ratio of the probe to Hcy changed much less than that of Cys, and the probe was not sensitive to GSH. The detection limit of the probe for Cys was 1.5 μm. The probe can achieve dual-channel targeted fluorescence detection of Cys in mitochondria of living cells, and has been successfully applied to in vivo imaging of Drosophila melanogaster.
Based on the FRET mechanism, Liu et al. Designed and synthesized a two-photon fluorescent probe 12 (Fig. 15) by using rhodamine as the energy donor of FRET and nalimide as the donor
[55]. The probe uses 2,4-dinitrobenzenesulfonyl (DNBS) as the recognition unit, and the fluorescence at 590 nm increases with the increase of Cys concentration under the excitation of 395 nm ultraviolet lamp, and the fluorescence response of Hcy/GSH is weaker than that of Cys. The detection limit of the probe for Cys was 0.47 μm. Under the excitation of 800 nm (two-photon) wavelength, the probe can not only realize the fluorescence imaging of endogenous and exogenous Cys in the mitochondria of living cells, but also realize the tissue imaging with the imaging depth of 80 μm.
Based on the redox reaction between rhodamine B (RhB) and manganese dioxide nanosheets (MnO
2NFs) and biothiols, Xue et al. Constructed a ratiometric fluorescent probe 13 for rapid and selective detection of biothiols (Fig. 16)
[56]. Due to the strong oxidizing property of MnO
2NFs, RhB is oxidized into its derivatives, and the color of the solution changes from purple to light pink, and the fluorescence changes from red to green, forming a MnO
2NFs-RhB. When Cys was gradually added to the MnO
2NFs-RhB solution from 0 to 40 μm, the green fluorescence of the probe itself at 523 nm gradually decreased and the red fluorescence of the RhB derivative at 579 nm increased under the excitation of a UV lamp with a wavelength of 365 nm, which was due to the fact that Cys could reduce MnO
2NFs to Mn
2+ to inhibit the oxidation of RhB. The detection limit of the probe for Cys (0 – 15 μm) was 0.282 μm. The probe can be used to detect Cys in human serum.
Maiti et al. Designed and synthesized a colorimetric fluorescence sensor 14 for highly selective detection of L-Cys by using the interaction between RhB and gold nanoparticles (AuNPs) in aqueous medium (Fig. 17)
[57]. Due to the electrostatic interaction, the RhB dye is tightly packed on the surface of the AuNPs to initiate the FRET process, resulting in almost no fluorescence of the system. When L-Cys was added, there was obvious fluorescence, and the solution changed from wine red to blue-black, because L-Cys and Au NPs had a stronger chemical adsorption through the Au-S bond, which made RhB released and the fluorescence recovered. The detection limit of the probe for L-Cys was 0.01 μM. L-Cys in human urine can make the fluorescence recovery rate of the probe reach 35%, indicating that the probe can be used as an effective way to diagnose cysteinuria.
Rhodamine was combined with other fluorescent dyes or nanomaterials to design ratiometric fluorescent probes to detect Cys, and the fluorescence change was more obvious (probes 11, 12, 13, 14), and the detection limit was lower when combined with nanomaterials (probes 13, 14).
2.3 Cys fluorescent probe based on benzothiazole
Benzothiazole is widely used as a fluorescent probe due to its rigid planar structure (fig. 18) and large delocalized π bond, high quantum yield, large Stokes shift, good photostability and fast response rate
[58][59]. Benzothiazole fluorescent probes can detect metal ions, anions, small molecules and biological macromolecules through complexation reaction, hydrolysis reaction, nucleophilic addition reaction and the like, and can be used for biological cell imaging and pH sensing
[60][61]. Benzothiazole probes exhibit some special fluorescence properties, such as ESIPT, ICT, AIE, etc.
Ren et al. Designed and synthesized a near-infrared sensor 15 for Cys based on the ESPIPT mechanism (Fig. 19)
[62]. When Cys was added to the solution containing the probe, a strong emission peak appeared at 686 nm due to the ESIPT process. With the addition of Hcy or GSH, there was no significant fluorescence enhancement at 686 nm except a slight enhancement at 470 nm, because Hcy/GSH did not induce intramolecular cyclization and blocked the ESIPT process, which could distinguish Cys from Hcy/GSH. Probe 15 showed a detection limit of 0.20 μm for Cys in pH 8 solution and a Stokes shift of 263 nm in response to Cys. The probe realizes the fluorescence imaging of Cys in HeLa cells and mice, which indicates that the probe can effectively detect Cys in vivo.
Anusuyadevi et al. Also designed and synthesized a fluorescent probe 16 for highly selective detection of Cys based on the ESIPT mechanism with the acrylate group as the recognition site (Fig. 20)
[63]. Because the acrylate group hinders the ESIPT process of the probe, the probe itself does not fluoresce, and the fluorescence intensity at 478 nm increases with the increase of the concentration of Cys added, and the presence of other anions and amino acids does not interfere with the detection of Cys. The detection limit of the probe for Cys was 0.478 μM. In the fluorescence imaging experiment of HeLa cells, the probe can detect the concentration change of Cys through the difference of fluorescence intensity, thereby monitoring the intracellular redox state, which indicates that the probe can be used as an efficient probe to study the rapid determination of Cys in intracellular redox imbalance.
Zhang et al. Designed and synthesized a near-infrared ratiometric fluorescent probe 17 based on the ICT process (Fig. 21)
[64]. This probe uses methacrylate as a Cys recognition site and a trigger point for the ICT process. The probe itself emitted blue fluorescence when excited by a UV lamp with a wavelength of 365 nm, and the probe emitted red fluorescence at 710 nm when Cys was added, and the fluorescence intensity ratio (
/I
430 nm) of the probe was found to be linearly related to the concentration of Cys (1 – 40 μM), with a detection limit of 0.5 μM. Probe 17 was highly selective for Cys in the presence of other amino acids, anions, and cations. In addition, the probe can detect endogenous and exogenous Cys in HeLa cells.
Wei et al., also based on the ICT process, utilized acrylate as a Cys recognition site, mitochondria-targeting ratiometric fluorescent probe 18 (Fig. 22)
[65]. Different from probe 14, probe 18 uses naphthalene as an electron donor and benzothiazole as an electron acceptor. When Cys is added, nucleophilic addition occurs between probe 18 and acrylate on naphthalene, and the ICT process is started. The fluorescence emission of the probe decreases at 525 nm and increases at 595 nm, accompanied by a change in fluorescence color from green to orange, with a Stokes shift of 119 nm. The detection limit of probe for Cys was 74 nM. The fluorescence from the probe was found to overlap with that from MTDR (a mitochondria-targeting deep red dye) by co-fluorescence imaging of HepG2 cells, indicating that the probe can achieve targeted fluorescence imaging of Cys in mitochondria.
Zhao et al. Designed and synthesized a novel near-infrared fluorescent probe 19 (Fig. 23) based on the interaction of ESIPT and ICT. The fluorophore was synthesized from benzothiazole derivatives and methylquinoline, and acrylate was used as the recognition site. The positively charged methylquinoline group hindered the ICT process, so the probe fluorescence was quenched
[66]. After the addition of Cys, the hydroxyl bond formed between the acrylate recognition group and the fluorophore is easily cleaved, and ICT and ESIPT processes are initiated between the phenolic hydroxyl group and the benzothiazole group, resulting in a large Stokes shift (310 nm) and an enhanced fluorescence signal. Cys can be detected with high selectivity even in the presence of other amino acids and cations, with a detection limit of 0.062 μM for Cys. The probe can also detect endogenous Cys produced by A549 cells.
Long et al. Also designed and synthesized probe 20 based on the ESIPT and ICT mechanisms (fig. 24)
[67]. By expanding the π conjugated system of benzothiazole, the ICT process is initiated and the maximum emission peak wavelength is shifted to the near-infrared region. The probe itself did not show fluorescence under the excitation of 411 nm ultraviolet lamp, but showed a gradual increase in fluorescence at 713 nm after the addition of Cys, and the selectivity of the probe to Cys was still high in the presence of other analytes. The detection limit of the probe for Cys was 102 nM. The Stokes shift in response to Cys was 302 nm. The probe can detect endogenous and exogenous Cys in HeLa cells under one/two-photon excitation. In addition to this, the probe can also be applied to fluorescence imaging of Cys in zebrafish and mouse brain and abdominal cavity.
Tang et al. Designed and synthesized an efficient benzothiazole-based fluorescent probe 21 based on the far-infrared fluorescence "turn-on" response strategy (Fig. 25)
[68]. After Cys was gradually added into the probe solution, the fluorescence emission at 607 nm was observed under the excitation of a 365 nm UV lamp, which was due to the conjugate addition cyclization reaction between the probe and Cys, and the fluorophore was released. The detection limit of the probe for Cys (0 ~ 8 μM) was 0. 12 μM, and the Stokes shift of the response was 210 nm. The probe can distinguish Cys from other amino acids and human macroelements through different colors of fluorescence. The probe has good water solubility, low cytotoxicity and high cell membrane permeability, and has been successfully applied to the targeted fluorescence imaging detection of Cys in aqueous solution and mitochondria of MCF-7 cells.
Based on the white light emission strategy, Yang et al. Designed and synthesized a novel fluorescent sensor 22 (Fig. 26) by combining benzothiazole and nitrobenzofurazan (NBD)
[69]. In the presence of other amino acids, anions and metal ions, only Cys can cleave the two fluorophores of the probe to produce two products, resulting in white light. The detection limit of the probe for Cys was 37 nM. In the fluorescence imaging experiment of HeLa cells, the white light emission system is also applicable, indicating that the probe has good biocompatibility, which makes its application in the medical field possible.
Gholami et al. Took advantage of the strong affinity of mercury for sulfur-containing amino acids to design and synthesize a new benzothiazole-based dual chemical sensor23 (Fig. 27), which can detect both Hg
2+ and Cys in aqueous solution
[70]. After the addition of Hg
2+ to the ACN:H
2O(V∶V=1∶1) solution, the solution changed from red to blue due to the formation of coordination complex between the sensor and Hg
2+ ions. After the addition of Cys to it, the high affinity of Hg
2+ for amino acid sulfhydryl groups led to the release of the dye, and the solution turned pink again. The reaction was completed 20 s after the addition of Cys. The detection limit of the sensor -Hg
2+ complex for Cys was 0.1 μM. It was also found that the strip coated with the sensor -Hg
2+ complex could also cause color change when exposed to purified thiol-containing amino acids, indicating that the complex could be used as a simple colorimetric sensor to screen purified biothiols, but the complex sensor could not distinguish three biothiols.
Benzothiazole-based fluorescent probes for Cys detection are mostly modified by introducing related substituents at their first position, and most of these probes have large Stokes shifts, which can effectively reduce the interference of background fluorescence (probes 15, 19, 20, 21), and have high detection sensitivity. In addition, they can also form complexes with metal ions to improve the response rate to Cys (probe 23).
2.4 Fluorescein-based Cys fluorescent probe
Fluorescein, also known as fluorescent yellow, fluorescent orange, fluorescent red and so on, belongs to xanthene fluorescent dyes. According to the molecular structure (fig. 28), the fluorescein molecule contains two parts of a hydroxyl-modified xanthene ring and a spirolactone. Under acidic conditions, fluorescein is in a closed loop state and no fluorescence is produced; Under alkaline conditions, the spironolactone ring is opened, and the xanthene ring forms a conjugated quinone structure with strong fluorescence. Fluorescein has large molar extinction coefficient, high fluorescence quantum yield, stable luminescence signal, good water solubility, excitation wavelength and emission wavelength in the visible range
[71][72,73][74][75].
Ji et al. Designed and synthesized a new fluorescent probe 24 (Figure 29) based on the addition cyclization reaction of Cys and acrylate. The probe uses fluorescein and julolidine as fluorophores, and the probe exhibits very low background fluorescence in PBS buffer solution (pH 7.4).The addition of Cys showed a fluorescence enhancement of nearly 30 times at 567 nm under the excitation of 538 nm ultraviolet lamp, the addition of GSH showed only a weak fluorescence enhancement, and the addition of other amino acids showed almost no fluorescence
[76]. The detection limit of the probe for Cys was 39.2 nM. Due to the low cytotoxicity and good cell permeability of the probe, they successfully applied it to live HepG2 cell fluorescence imaging. Shi et al. Also designed and synthesized a ratiometric fluorescent probe 25 for quantitative detection of Cys in living cells using acrylate as a recognition site (fig. 30)
[77]. Under physiological pH conditions, the signal intensity of the probe at 520 nm increased with the increase of Cys concentration under 480 nm excitation, and the fluorescence emission intensity at 700 nm was almost unchanged. The concentration of Cys can be determined by calculating the ratio of fluorescence intensity, so as to realize the quantitative detection of Cys.
Karaku et al. designed and synthesized an open-type fluorescent response probe 26 to detect Cys by using alkynyl ester as a recognition site (Fig. 31)
[78]. Similar to acrylate, alkynyl ester acts as the acceptor of Michael addition reaction, and the probe itself has no fluorescence. With the increase of the concentration of Cys, the fluorescence intensity at 515 nm increases gradually, which is due to the addition of — SH on Cys to the alkynyl group at the end of the probe and the subsequent cyclization to promote the cleavage of the ester, resulting in the release of the fluorophore. The detection limit of the probe for Cys was 182 nM. The addition of other amino acids had no significant change in fluorescence intensity, indicating that the probe was highly selective for Cys detection. In addition, the probe has high selectivity for Cys in the physiological pH range, and realizes fluorescence imaging of Cys in A549 cells.
Wang et al. Designed and synthesized a colorimetric vision sensor 27 (fig. 32) using DNBS as a recognition site
[79]. The fluorescence of the sensor itself is very weak, and the color of the solution changes from light yellow to yellow-green when Cys is added, and the presence of other amino acids and metal ions does not interfere with the detection of Cys. The detection limit of the probe for Cys was 6.5 μm. It was found that the recovery rates of the probe for Cys in water and milk were as high as 106.7% and 109.2%, respectively, indicating that the sensor made it possible to detect Cys in food.
Similarly, using DNBS as a Cys recognition site, Du et al. Modified the fluorescein skeleton with a carbon-nitrogen triple bond, and introduced two DNBS to design and synthesize an "on" response fluorescent probe 28 (fig. 33)
[80]. When Cys, Hcy and GSH were added to the aqueous solution of the probe, obvious green fluorescence emission at 519 nm was observed, and the fluorescence intensity caused by Cys was the largest, which was due to the reaction of Cys with DNBS, followed by intramolecular cyclization to remove the five-membered ring and form the quinoid structure. The detection limit of the probe for Cys was 0. 021 μM, which was more sensitive than probe 27, and could be applied to endogenous and exogenous Cys fluorescence imaging in HeLa cells.
Liu et al. Designed a reversible fluorescent probe 29 to monitor Cys and HClO in living cells by reacting phenylselenenyl chloride with fluorescein (fig. 34)
[81]. The probe itself shows very weak fluorescence due to the PET process before the Se with lone pair electrons and the fluorescein parent, but after the oxidizing HClO is added, the electron-rich Se in the probe is oxidized into electron-deficient SeO, which blocks the PET process and causes fluorescence emission. When the reducing Cys was added, SeO was reduced to electron-rich Se, and the fluorescence decreased with the increase of Cys concentration.The response of the probe to HClO and Cys was highly reversible, and the detection limit was low, 1.1 and 3 nM, respectively. According to the cell imaging experiment, the probe can monitor the endogenous HClO and Cys by monitoring the fluorescence intensity in the green channel, and this highly reversible fluorescent probe provides a strategy for the study of redox homeostasis in living systems.
Qin et al. designed and synthesized an "open-type" fluorescent probe 30 based on the combination of fluorescence Inner filter effect (IFE) and nanoparticle polymerization mechanism by using the strategy that biological thiols can be combined with AuNPs through sulfur groups (Fig. 35)
[82]. Due to the IFE process between fluorescein and AuNPs, the fluorescein fluorescence was quenched and the probe was almost non-fluorescent. After the addition of Cys/Hcy/GSH, respectively, the three biothiols caused different degrees of fluorescence recovery due to their induced aggregation of AuNPs. The detection limit of the probe for Cys was 0.6 μ. In addition, the recovery rate of Cys in human serum was 95. 6% ~ 103.9%, which indicated that the probe could be used for the specific detection of Cys in human serum.
Most fluorescein-based fluorescent probes have a maximum emission wavelength between 500 and 520 nm (probes 25, 27, 28), which is shorter than that of structurally similar rhodamine dyes. In addition, the sensitivity of detection can be improved by introducing a Cys recognition site at the 3-position (or 6-position) of the lactone structure of fluorescein, in which two recognition groups are introduced at the 3-position and 6-position of the probe.
2.5 Cys fluorescent probe base on naphthalimide
The structure of the naphthalimide derivative (Figure 36) contains an "imide" structure with an electron-withdrawing effect and a group at the R
2 site that is easily modified and prepared, and when the R
2 site is an electron-withdrawing group, the fluorescence of the naphthalimide derivative is very weak; Naphthalimide derivatives emit strong fluorescence when the R
2 site is an electron-donating group, which is a classical dye based on the ICT mechanism
[83]. Naphthalimide derivatives are widely used in fluorescent dyes, light-emitting devices, biomedicine and other fields because of their rigid plane, large π-electron conjugated system, simple structure, easy modification, good photostability, high fluorescence quantum yield, large Stokes shift, good selectivity, high sensitivity and other excellent characteristics
[84,85][86][87][88,89].
Hou et al. Designed and synthesized a near-infrared fluorescent probe 31 (fig. 37) by introducing a polycyanofuran group into the 1,8-naphthalimide skeleton and using acrylate as a recognition site
[90]. The probe showed almost no fluorescence emission at an excitation wavelength of 620 nm, and a 40-fold fluorescence enhancement at 665 nm was observed after the addition of 10 EQ of Cys due to the recovery of the ICT process of the probe, and the detection limit of the probe for Cys (0 – 70 μm) was 0.093 μm. When Cys and Hcy/GSH coexist, there is little interference in the detection of Cys, indicating that the probe can distinguish Cys from other biological thiols with high selectivity, rapidity and sensitivity. In addition, the probe can also be applied to fluorescence imaging of endogenous and exogenous Cys in HeLa cells and zebrafish.
Aydin et al. Designed an "off-on" fluorescent probe 32 to recognize Cys with high selectivity by using methyl propiolate as the recognition site of Cys (fig. 38)
[91]. The probe itself has a strong blue fluorescence emission at 416 nM, and the Michael addition cyclization occurs when Cys is gradually added, so the fluorescence host structure of the probe is destroyed, the fluorescence emission blue shifts to 413 nm and the intensity decreases, while the addition of other amino acids has no obvious fluorescence change, and the detection limit of the probe for Cys is 9. 02 nm. Because of the different concentration of Cys in normal cells and cancer cells, normal cells and cancer cells can be distinguished by the color change of the probe fluorescence, which shows the potential application of the probe in medical diagnosis.
Zhu et al. Designed and synthesized fluorescent probe 33 (Fig. 39) using 4-chloro-7nitrobenzofurazan (NBD-Cl) as a Cys recognition site
[92]. The probe does not produce fluorescence due to the PET process of the reactive group and the fluorophore moiety. When the biological thiol is added, the nucleophilic extraction and subsequent molecular rearrangement with the probe occur, resulting in an increase in the fluorescence intensity at 550 nm, a red shift in the absorption spectrum, and a change from colorless to yellow in the solution. The detection limit of the probe for Cys was 0.31 nM, indicating that the probe was highly sensitive to Cys. However, it is unable to detect the three Cys/Hcy/GSH differently.
By introducing 7-chlorobenzofurazan-4-sulfonyl (SBD-Cl) on the 1,8-naphthalimide skeleton, Wang et al. designed a probe 34 based on the FRET mechanism and capable of differentially detecting three Cys/Hcy/GSH (fig. 40)
[93]. SBD-Cl served as the energy acceptor for FRET, and the naphthalimide moiety served as the energy donor. The probe itself showed a very weak fluorescence emission at 533 nm, and after the addition of Cys, the fluorescence emission at 559 nm gradually increased, and the yellow fluorescence turned on. The addition of Hcy/GSH did not induce the intramolecular rearrangement of the probe, so the fluorescence did not change significantly. The detection limit of the probe for Cys was 0.87 μm. Through the confocal fluorescence imaging experiment of the probe on HeLa cells, it was found that when exogenous Cys was added, the fluorescence of the probe in the original blue channel decreased, while the fluorescence appeared in the green channel, indicating that the probe could detect exogenous Cys in living cells. Similarly, using SBD-Cl as a recognition site, Li et al. Designed and synthesized a fluorescent probe 35 (fig. 41), which can distinguish and detect Cys and H
2S in different color channels
[94]. The triethylene glycol attached to the 1,8-naphthalimide can improve the water solubility of the probe and prevent fluorescence quenching caused by intermolecular aggregation. The detection limit of the probe for Cys was 16.7 nM.
Using DNBS as a recognition group, Rong et al. Designed and synthesized a fluorescent probe 36 with red emission and large Stokes shift for detecting biological thiols through switch response (fig. 42)
[95]. Due to the PET process between the recognition group DNBS and the fluorophore, the probe hardly fluoresces. With the increase of the concentration of the three biological thiols added, the recognition group is separated from the fluorophore due to the nucleophilic reaction between the probe and the biological thiol, which blocks the PET process and restores the fluorescence. The detection limit of Cys was 9. 87 nM. The probe can also detect endogenous and exogenous biological thiols in MCF-7 cells. Because the three biological thiols have similar structures, the probe can not detect them differently.
Cao et al. Designed and synthesized a fluorescent probe 37 for specific detection of Cys using 2-thiophene carbonyl as a recognition moiety (Fig. 43)
[96]. When 400 μM Cys, Hcy and GSH were added to the PBS buffer solution of the probe, it was found that the fluorescence emission intensity reached the maximum first when Cys was added, which was due to the largest pseudo-first-order rate constant and second-order rate constant, followed by GSH and Hcy. Therefore, the probe can realize the differential detection of the three biological thiols. The detection limit of the probe for Cys (0 – 30 μM) was 0.065 μM. The probe also allows imaging of biothiols in zebrafish.
The 3 and 4 positions and the R1 position in the parent structure of naphthalimide are reactive sites, which can be modified to improve its water/lipid solubility (probe 35), fluorescence emission wavelength (probe 36), response sensitivity (probe 33) and other characteristics, especially when an electron-donating group is introduced at the 3 or 4 position to form a D-A structure with the parent and induce the ICT process (probe 31).
2.6 Fluoroboropyrrole-Based Cys Fluorescent Probe
The BODIPY structure (Figure 44) is composed of two pyrrole units connected by methines and two pyrrole nitrogen coordinated BF
2 groups. Fluoroboropyrrole dyes have the advantages of high molar extinction coefficient, high fluorescence quantum yield, emission wavelength in the visible region, good optical and chemical stability, and high modifiability of the central skeleton
[97][98][99][100⇓~102]. However, it also has some disadvantages, such as poor water solubility, short emission wavelength and small Stokes shift, which limit its application. It can modify the parent structure, design and synthesize a variety of fluorescent probes with excellent properties, and has been widely used in biomedical, environmental detection and optical fields
[103][104][105,106].
Wang et al. Designed three fluorescent probes by introducing three different substituents, electron-withdrawing trifluoromethyl, neutral hydrogen and electron-donating methoxy, to the para-position of the 1-aryl group of BODIPY
[107]. The three probes were non-fluorescent, and the time response to Cys could be completed within 10 min. It was found that only the probe 38 with an electron-withdrawing trifluoromethyl group introduced responded the fastest (Figure 45), indicating that the probe with an electron-withdrawing substituent was more favorable for nucleophilic attack and the reaction was faster. The detection limit of probe 38 for Cys was 52 nM. Their group also designed and synthesized probe 39 by introducing methyl groups at positions 1 and 7, two aromatic groups at positions 3 and 5, and an electron-withdrawing thiazole thioether group in the middle of BODIPY (Figure 46)
[108]. Due to the aromatic substitution-rearrangement reaction between the probe and Cys, an 80-fold fluorescence enhancement was observed at 567 nM, and the fluorescence intensity reached three quarters of the maximum within 1 min, indicating that the probe 39 responded quickly to Cys, with a detection limit of 51 nm. When other amino acids and thiols were added to the two probes, there was no significant fluorescence change except for a weak fluorescence enhancement caused by Hcy, indicating that the two probes have good selectivity for Cys and can detect Cys in living cells in cell imaging experiments.
Pham et al. Designed near-infrared ratiometric fluorescent probes by introducing three different substituents containing 2,4-dinitrobenzene, toluene or (dimethylamino) naphthalene at the para-position of the 3-aryl group of BODIPY
[109]. It was found that the probe 40 introduced with 2,4-dinitrobenzene had stronger electron-withdrawing ability (Figure 47) and stronger nucleophilic substitution ability than the other two probes, and the detection limit of probe 40 for Cys was 5.23 μM. This probe has a longer fluorescence emission than probe 38 and reacts with Cys with a concomitant color change.
Ji et al. Designed and synthesized a mitochondria-targeting probe 41 for Cys-specific detection by introducing aromatic rings at positions 1 and 7 of BODIPY (Fig. 48)
[110]. Due to the PET process, the probe has almost no fluorescence, and the fluorescence at 617 nM is significantly enhanced after reaction with Cys, and the detection limit of the probe for Cys is 70 nm. There was only a weak fluorescence enhancement for Hcy and almost no fluorescence change for GSH. Shows that other analytes do not interfere with the detection of Cys. In the confocal fluorescence microscopy imaging experiment of HeLa cells, the probe has good mitochondrial targeting ability, which can realize the imaging of Cys in mice.
Tao et al. introduced a methoxyphenylthio group at the mesoposition of BODIPY, which made the background fluorescence of the probe extremely low, and introduced methyl groups at the 1 and 7 positions to provide steric hindrance, and designed and synthesized the near-infrared fluorescent probe 42 (Figure 49)
[111]. The fluorescence of the probe itself is very weak. After the addition of Cys, a 45-fold fluorescence enhancement was observed at 685 nm due to the aromatic substitution rearrangement of the probe and Cys under the excitation of a 580 nm ultraviolet lamp. When Hcy/GSH was added, there was no significant change in fluorescence. The detection limit of the probe for Cys was 118 nM. The probe was successfully applied to the fluorescence imaging of endogenous Cys in HeLa cells and deep tissue in mice.
Mei et al. Also synthesized probe 43 by inserting phenylselenium as an active group at the mesoposition and introducing two methyl groups at the 3- and 5-positions (fig. 50)
[112]. After the addition of Cys, the PET process between phenylselenium and BODIPY host was blocked, aromatic substitution rearrangement with phenylselenium occurred, and fluorescence was released. The detection limit for Cys was 4.1 nM. Compared with the probe 43 substituted by thioether at the meso position, the probe has a higher fluorescence quantum yield for Cys, and the response rate is faster, which can be stabilized within 2 min. Through the confocal fluorescence imaging experiment of HepG2 cells, it was found that the cells treated with Cys/Hcy only showed fluorescence in the blue channel, while the cells treated with GSH only showed fluorescence in the green channel, indicating that the probe could distinguish Cys/Hcy from GSH.
Wang et al. designed and synthesized fluorescent probe 44 by inserting an 8-hydroxyquinoline group at the meso position of BODIPY, and then introducing DNBS, which is the recognition group of Cys and the quenching site of fluorescence, into the hydroxyl position of quinoline (Figure 51)
[113]. The basic nitrogen atom on the quinoline provides a hydrogen bond in the molecule, and when Cys is added, the sulfur atom on the -SH of the quinoline is connected with the benzene sulfonate to form an unstable intermediate with a proton, and then the hydrogen proton on the sulfhydryl group is rapidly combined with the oxygen atom on the carboxyl group and the nitrogen atom of the quinoline structure through the formation of a double hydrogen bond,This deprotonation process can effectively accelerate the rate of the sulfonate cleavage reaction, and the three biological thiols can be differentially detected according to the reaction rate, and the reaction rate of the probe with Cys is the fastest, with a detection limit of 33 nM. The probe can also be used for Cys imaging in HepG2 cells and zebrafish in vivo.
Wang et al. designed and synthesized a ratiometric fluorescent probe 45 (Fig. 52) by connecting BODIPY as a FRET donor and near-infrared rhodamine as a FRET acceptor through a disulfide bond
[114]. The probe itself showed near-infrared rhodamine fluorescence at 656 nm under excitation by a 480 nm wavelength ultraviolet lamp, while BODIPY provided very weak fluorescence. When biothiol was added, the donor and acceptor were separated due to the destruction of the disulfide bond in the probe, and the FRET was interrupted, resulting in the decrease of rhodamine fluorescence intensity in the near-infrared region and the increase of BODIPY fluorescence intensity at 512 nm. After 60 min, the fluorescence intensity ratio (F
512 nm/F
656 nm) showed a 60-fold fluorescence enhancement. The probe was successfully used to image biothiols in mitochondria of HeLa cells, but the probe could not detect the three biothiols differently.
Jia et al. designed and synthesized a fluorescent probe 46 that can simultaneously recognize Cys, GSH and sulfite by connecting hemicyanine and BODIPY through ether bond (Fig. 53)
[115]. After adding Cys and GSH respectively, because the emission spectra of the probes have a certain overlap, in order to distinguish them, the probes were encapsulated into the micelle to prepare nano-fluorescent probes, and Cys, GSH and sulfite were added respectively to the micelle.Two strong emission peaks caused by sulfite and GSH were observed at 529 and 588 nm, and the fluorescence emission peaks of Cys and GSH were observed at 710 nm when excited at 670 nm, and the emission peak was more obvious when Cys was added. The detection limit of probe for Cys was 2.29 μM. The probe can also be used for bioimaging of Cys, GSH, and sulfite in living cells.
The fluorescent probes based on BODIPY have long emission wavelengths (probes 40, 41, 42, 46), and can be functionally modified by introducing corresponding substituents to the 1, 3, 5, 7 and meso positions of the parent. For example, the introduction of a strong electron-withdrawing group at its meso site can accelerate the reaction rate with Cys (probes 38, 40).
2.7 Cyanine-based Cys fluorescent probe
Cyanine structures (fig. 54) generally include two nitrogen-containing heterocycles linked together by a methine chain. The nitrogen at one end has a positive charge as an electron acceptor, and the nitrogen at the other end has no charge as an electron donor, so that a conjugated system with a push-pull electron effect is formed, and the central carbon of the conjugated system has an electrophilic property, so that various substituents can be introduced to the central carbon to modify the spectral properties of the conjugated system
[116]. The cyanine fluorescent molecular probe has the advantages of high molar coefficient in the visible region, good quantum yield, wide spectral range, strong anti-interference capability and good sensitivity
[117,118].
Yang et al. synthesized and designed a probe 47 showing turn-on fluorescence response to Cys/Hcy by introducing a thioaryl substituent on the central carbon of heptamethine cyanine and using the coupling reaction of biological thiol and fluorophore (Figure 55)
[119]. Under the excitation of 660 nm wavelength, the probe itself has no fluorescence, but after adding Cys/Hcy, a significant fluorescence emission at 750 nm is observed, accompanied by the solution changing from light green to blue, and the detection limit of the probe for Cys is 0. 39 μm. When GSH and other amino acids were added, no obvious fluorescence response was observed, indicating that the probe can be used as a colorimetric fluorescence sensor to selectively recognize Cys/Hcy, but it can not distinguish the two. The probe can also be used for fluorescence imaging of Cys in HeLa cells.
Zhu et al. Designed a fluorescent probe 48 that rapidly responds to biological thiols by introducing DNBS as a fluorescence quenching and Cys recognition site and a lipophilic cationic unit as a mitochondrial targeting site (Fig. 56)
[120]. Due to the PET process between DNBS and fluorophore, the probe itself has no fluorescence. After adding three biological thiols, the PET process is blocked, and the fluorescence at 604 nm is enhanced by different times. The detection limit of Cys is 13. 4 μM. Because the probe is positively charged and has a negative potential in the mitochondrial membrane, it can be well clustered in the mitochondria through electrostatic interaction. Mitochondrial targeted fluorescence imaging experiments on HeLa cells and other cancer cells showed that the red fluorescence of probe 48 in response to thiol was completely overlapped with the green fluorescence emitted by mitochondrial tracking dye.It is proved that the probe has a good ability to target the fluorescence imaging of biological thiols in mitochondria, and can be used as an effective tool to monitor the fluctuation level of biological thiols in cells induced by oxidative stress.
Zhang et al. Designed and synthesized a ratiometric near-infrared fluorescent probe 49 (Fig. 57) by adjusting the conjugated π-electron system of the cyanine fluorophore and using the acrylate group as the recognition site of the biological thiol
[121]. When Cys was gradually added to the buffer solution of the probe, the solution changed from green to red, the fluorescence emission of the probe at 794 nm decreased sharply, and a new fluorescence emission appeared at 635 nm, while the addition of Hcy and GSH caused very little fluorescence change, indicating that the probe could show a high level of ratiometric fluorescence emission for Cys, and the detection limit was 0. 09 μM. Experiments show that the probe can also detect the level of Cys in mitochondria of human lung cancer cells and realize fluorescence imaging, which indicates that the probe has great application potential in the field of medical diagnosis.
Men et al. Designed and synthesized a near-infrared open-type water-soluble fluorescent probe 50 (fig. 58) using pyrimidine as the recognition unit of Cys
[122]. With the addition of Cys/Hcy, the probe showed a significant fluorescence enhancement at 743 nm under 650 nm excitation, which was due to the reaction of Cys/Hcy with the probe to form sulfur substitution products, and the subsequent intramolecular rearrangement to form five-membered and six-membered ring transition States, respectively, resulting in stable amino substitution, but the reaction with Cys was faster. The detection limit of probe for Cys was 0.17 μM. GSH cannot undergo intramolecular rearrangement because it has no adjacent amino groups. Therefore, the probe can be used to distinguish and detect three biological thiols, and has been successfully applied to endogenous Cys/Hcy fluorescence imaging in living cells and mice.
Fang et al. Synthesized and designed a dual-mode fluorescent probe 51 based on near-infrared fluorescence imaging and ratiometric photoacoustic imaging of p-aminophenyl sulfide substituted cyanine dyes (fig. 59)
[123]. In PBS/DMSO solution (10 mM, pH = 7.4, V: V = 1:1), the probe produced a weak photoacoustic (PA) signal at 695 nm and a strong PA signal at 840 nm, but no fluorescence. When Cys/Hcy was added, the PA intensity at 840 nm decreased significantly, and the PA intensity at 695 nm increased with the increase of fluorescence intensity. The detection limit of the probe for Cys was 0.47 μm. Even in the presence of GSH, there is no interference, indicating that the probe can achieve fluorescence and photoacoustic dual mode to detect Cys/Hcy in solution, but can not distinguish the two. In addition, the probe also enabled the detection of endogenous Cys/Hcy in mice and living cells.
Li et al. designed and synthesized an enhanced fluorescent probe 52 with low background fluorescence (fig. 60) by using the NBD moiety as a recognition site and IR780 as a fluorophore, and connecting them through an ether bond
[124]. Because of the NBD-induced PET process, the probe itself did not produce fluorescence, and after the addition of Cys/Hcy and GSH, significant fluorescence responses were observed in the green and red channels, respectively. The probe can realize the targeted fluorescence imaging of mitochondrial biological thiols in living cells and the in vivo fluorescence imaging of acute liver injury in mice, which indicates that it is expected to have certain applications in medical diagnosis, but the probe can not distinguish between Cys and Hcy.
Niu et al. Constructed three probes by introducing the chlorobenzoate group into the cyanine skeleton according to the three different positions of the chlorine atom at the ortho, meta and para positions of the ester group, and their absorption spectra had no significant difference, indicating that the chlorine atom at the ortho, meta and para positions had no significant effect on the absorption spectra of the probes
[125]. When Cys was added to them respectively, only the absorption peak at 780 nM of probe 53 (Fig. 61) with the chlorine atom in the ortho position decreased significantly, and its fluorescence was induced to increase by 35 times. The detection limit of probe 53 for Cys (0 ~ 250 μM) was 228 nm. The absorption peak of probe 53 did not change significantly with the addition of Hcy and GSH. Shows that the probe can recognize Cys with high selectivity. The probe has been successfully applied to fluorescence imaging of Cys in HeLa cells and mice, as well as targeted fluorescence imaging in mitochondria.
Zhang et al. Also designed and synthesized three fluorescent probes by introducing different electron-withdrawing and electron-donating groups into the cyanine skeleton
[126]. They use electron-withdrawing 4- (trifluoromethyl) phenyl sulfide and 3,5-bis (trifluoromethyl) phenyl sulfide and electron-donating 3,4-methoxyphenyl sulfide as recognition units, respectively, and the reaction rate is regulated by adjusting the electron density of the recognition unit in the probe. The three probes showed very weak fluorescence due to the PET process initiated by the phenyl sulfide unit. After the addition of Cys, the probe 54 (fig. 62) with a strong electron-donating recognition unit had the fastest reaction rate, with a detection limit of 7.7 μM for Cys. The results showed that the strong electron donating ability was beneficial to the substitution rearrangement reaction between the probe and Cys, which provided a new strategy for the synthesis of probes by adjusting the electron density to control the reaction rate. The probe can be applied to endogenous Cys-targeted fluorescence imaging in HeLa cell mitochondria.
By introducing different Cys recognition sites on the central carbon of the cyanine dye parent, with different response rates. When introduced to the electron recognition unit, the response rate is the fastest (probe 54). Fluorescent probes based on cyanine dyes have long emission wavelength (probes 47, 50, 51, 54), good response to Cys in the near-infrared region, and the nitrogen at the end of the parent structure is positively charged, which is easy to combine with the negative charge in the mitochondrial membrane, thus realizing Cys-targeted fluorescence imaging in mitochondria (probes 48, 53, 54).
2.8 Cys fluorescent probe based on nitrobenzofurazan
Nitrobenzofurazan (NBD) is formed by replacing the 7-position of benzofurazan (BD) with nitro group. In the NBD structure (Fig. 63), the nitro group at the 7-position is an electron-withdrawing group. If an electron-withdrawing group is attached at the 4-position, the ICT process is blocked, and the compound is self-generated without fluorescence; If an electron-donating group is connected to the 4-position, a push-pull effect is formed, and fluorescence is generated. NBD derivatives are widely used in the field of fluorescence sensing because of their simple synthesis and good selectivity. NBD fluorophores have the advantages of high sensitivity, large Stokes shift, high quantum yield, good optical properties and cell permeability, as well as long wave emission and good modification
[127,128][129⇓~131][132].
An et al. Designed and synthesized An open-response probe 55 by introducing electron-deficient pentafluorophenol at the 4-position of NBD (fig. 64)
[133]. The probe itself has no fluorescence due to the electron-deficient nature of the substituent at the 4-position, while the probe solution after Cys treatment has fluorescence turned on again due to substitution and Smiles rearrangement. Similar fluorescence changes were not observed with the addition of other thiol species and H
2S. The probe has also been applied to the fluorescence imaging of Cys in GBM (glioblastoma) cells for the first time, indicating that the probe 55 can detect Cys in GBM cells with high selectivity and sensitivity, thus realizing the screening of GBM in the medical field.
Lu et al. Designed and synthesized a simple dual-emission fluorescent probe 56 (Figure 65) by connecting two fluorophores, NBD and phenanthroimidazole, with an ether bond at the 4-position of NBD
[134]. Under the excitation of 365 and 480 nM ultraviolet lamps, the probe did not produce fluorescence. After the addition of Cys, the ether bond was cleaved by aromatic substitution reaction with the probe, and the fluorophore was released, which showed blue and green fluorescence at 470 and 550 nm, respectively. The detection limit of the probe for Cys (0 ~ 100 μM) was 22. 6 nm. When Hcy/GSH was added, strong blue fluorescence was observed at 470 nm under 365 nm excitation. Under the excitation of 480 nm, the probe showed a very weak green fluorescence at 550 nm due to the steric hindrance after the addition of Hcy/GSH. The probe was successfully used to visually detect Cys in HeLa cells and zebrafish cells.
Zhou et al. Also introduced an ether bond at the 4-position of NBD to connect it with coumarin derivatives containing α, β-unsaturated ketones, and designed and synthesized a fluorescent probe 57 with large molar absorptivity and quantum yield (Fig. 66)
[135]. Due to the PET effect, the probe itself does not produce fluorescence. Upon addition of Cys, two fluorophores with the same emission peak were released with a detection limit of 0.44 μm. The reaction rate of adding Hcy is slower than that of adding Cys, and the volume of adding GSH is too large to induce the rearrangement of NBD derivatives, so there is no fluorescence. In the fluorescence imaging experiment of HeLa cells, probe 57 could image the fluorescence of Cys in oxidative stress induced by Cu
2+. In addition, the probe was successfully used for fluorescence imaging of endogenous and exogenous Cys in vivo.
Jing et al. Also designed a two-color mode fluorescent probe 58 (fig. 67) by combining coumarin and NBD with an ether bond
[136]. The probe itself has no fluorescence, but when Cys is added, the ether bond is cut off, resulting in a 10.7-fold and 11.2-fold fluorescence enhancement in the blue and green channels at 465 and 565 nM, respectively, and the detection limit of the probe for Cys is 8 nm. A weaker fluorescence enhancement was also shown in the blue and green channels when Hcy was added. With the addition of GSH or H
2S, fluorescence was observed only in the blue channel. The probe was successfully applied to detect three biological thiols and H
2S in different color channels in HeLa cells and zebrafish.
Yang et al. Also designed and synthesized a two-color mode probe 59 (fig. 68) by combining NBD with another fluorophore through an ether bond
[137]. The probe can simultaneously detect Cys/Hcy and GSH in green and blue color channels at two independent excitation wavelengths. The detection limit of the probe for Cys was 0.015 μM. However, unlike probe 58, this probe does not provide a differential detection of Cys/Hcy. They successfully used the probe to visually detect endogenous Cys/Hcy and GSH in Daphnia magna.
An electron-withdrawing group can be introduce into that 4-position of the NBD to design an 'off-on' type fluorescent response probe to realize the detection of Cys, and other fluorophores can also be introduce into the 4-position,Fluorescent probes with dual emission channels can be designed, which can better distinguish and detect three thiols in different color channels (probes 58, 59) in cell imaging experiments, and improve the response sensitivity of Cys.
2.9 Cys fluorescent probes based on other structures
Compared with traditional visible fluorescence imaging, near-infrared fluorescence imaging has a wide range of applications in the biomedical field because of its high spatial and temporal resolution, deeper tissue penetration, and deeper imaging.
Dicyanoisophorone derivatives are typical near-infrared fluorophores based on the ICT mechanism, with long wavelength fluorescence emission, large Stokes shift, and good photostability. Qian et al. Used a fluorescent probe 60 based on dicyanoisophorone derivatives with DNBS as the recognition group (fig. 69)
[138]. The probe itself does not fluoresce due to the strong fluorescence quenching effect of DNBS, and the fluorescence enhancement can be observed by adding Cys/Hcy/GSH respectively, which is most obvious caused by Cys, and the detection limit of Cys is 36.93 nM. Qian et al. Again introduced F —, Cl —, Br — and NO
2—4 different electron-withdrawing substituents to modify the structure of dicyanoisophorone fluorophore, and designed four probes with acrylate as the recognition site
[139]. Because the NO
2- is a strong electron-withdrawing group, which destroys the ICT process of the fluorophore, the probe substituted by the NO
2- has almost no fluorescence emission. The probe substituted by three halogens can make fluorescence response to Cys. The Br-substituted probe 61 (fig. 70) exhibited a maximum fluorescence enhancement of 214-fold and a minimum detection limit of 86.9 nM. The probe can be used for fluorescence imaging of endogenous and exogenous Cys in HeLa cells. Ge et al. Synthetically designed a novel red-emitting fluorescent probe 62 (fig. 71) using the thioester moiety as the Cys acceptor
[140]. After Cys treatment, the probe showed a Stokes shift of 210 nM, a 70-fold fluorescence enhancement, and a change from yellow to red in solution, which was due to the nucleophilic addition of Cys to the carbon-sulfur double bond of the thioester moiety of the probe to form a stable five-membered ring, and the detection limit of the probe for Cys was 79 nm. The addition of other analytes did not change the fluorescence significantly, indicating that the probe could specifically recognize Cys. The probe has also been successfully used for fluorescence imaging of endogenous and exogenous Cys in HepG2 cells and in vitro and in vivo in mice.
Based on the ICT mechanism, Zhao et al. Synthesized a fluorescent probe 63 with a large Stokes shift using chalcone derivatives as fluorophores and acrylates as recognition sites (Fig. 72)
[141]. Because of its central ketone structure, it has the property of accepting electrons, and as a recognition site, it is an electron-withdrawing group, which weakens the ICT effect of the probe. The probe produced almost no fluorescence, and the addition of Cys induced a 30-fold fluorescence enhancement with a detection limit of 80 nM for Cys. When Hcy/GSH was added, the fluorescence was negligible. , indicating that the probe can recognize Cys with high selectivity. In addition, the probe can detect endogenous Cys in HeLa cells.
Based on the ESIPT mechanism, Guo et al. Synthesized probe 64 (Figure 73) by using acrylate as the ESIPT blocking group and recognition site and the Schiff base synthesized from salicylaldehyde as the fluorophore host structure
[142]. After the addition of Cys, the ESIPT process was switched on due to the restoration of the intramolecular hydrogen bond of the probe, resulting in a 25-fold fluorescence enhancement with a detection limit of 36.4 nM. Through the fluorescence imaging of zebrafish, it was found that there was weak fluorescence in the zebrafish incubated with the probe, which indicated that the probe had good biological permeability and had important use value in monitoring the level of Cys in vivo. Sheng et al. Synthesized probe 65 with imidazopyridine derivatives as chromophores (fig. 74)
[143]. This kind of chromophore has large Stokes shift, low background fluorescence and high detection sensitivity. The three biological thiols were added because Cys induced a more pronounced fluorescence enhancement due to its relatively lower pK
a, and the detection limit of probe 65 for Cys was 0.07 μM. Li et al. Synthesized a novel fluorescence ratio probe 66 based on a flavonoid derivative as a fluorophore (Fig. 75)
[144]. The acrylate group blocks the ESIPT process of the probe, resulting in quenching of the fluorescence at 530 nm and normal emission of the fluorescence at 420 nm. The fluorescence ratio between the two emission peaks increased linearly only when Cys was added. Highly sensitive detection of Cys is achieved based on the fluorescence ratio relationship between these two different emission peaks. The detection limit of Cys was 42.3 nM. Probes 65, 66 were successfully applied to endogenous and exogenous Cys fluorescence imaging in HeLa cells.
Long et al. Synthetically designed a two-photon "turn-on" fluorescent probe 67 based on a naphthalene derivative using an α, β-unsaturated ketone as the Cys reaction site and a morpholine moiety as the lysosomal targeting moiety (fig. 76)
[145]. The addition of Cys to the α, β-unsaturated ketone resulted in a 136-fold fluorescence enhancement, and the detection limit of the probe for Cys was 11 nM. However, the addition of Hcy/GSH only increased the fluorescence intensity by 4/12 times, because of the low steric hindrance of Cys, which can quickly and selectively detect lysosomal Cys in living cells. In addition, the probe enables two-photon fluorescence imaging of endogenous Cys in HeLa cells and up to 184 μm deep in tissue.
Organic small molecule fluorescent probe is a reasonable modification of the structure of organic small molecule fluorophore by combining organic synthetic dyes with recognition groups. Many small organic molecule fluorescent probes have been developed to detect Cys. Compared with macromolecular markers, small organic molecules are easier to penetrate the cell membrane, which can achieve specific detection of Cys deep in the cell tissue. Therefore, small organic molecule fluorescent probes have attracted wide attention in the field of biosensing due to their simple synthesis, easy operation and excellent selectivity.
3 Nano fluorescent probe for detecting cystine
Most of the organic small molecule fluorescent probes have some disadvantages, such as raw materials pollution, fluorescence emission spectra and excitation spectra affected by the environment, and the instability of the probe, which limit its development in biological, medical and other fields to a certain extent. Therefore, nanomaterials as fluorescent sensors for the detection of Cys have attracted much attention. Nanoparticle fluorescent probes exhibit different optical properties from many bulk objects or homogeneous macromolecules due to their unique small size effect, quantum size effect and surface effect
[146]. Compared with organic small molecule fluorescent probes, nano-fluorescent probes have higher detection sensitivity and faster response rate for target analytes. These excellent properties make nano-fluorescent probes widely used in biosensing, medical engineering, environmental detection and other fields. At present, quantum dots, gold nanomaterials, carbon dots and other nano-fluorescent probe materials are commonly used to detect Cys.
3.1 Cys fluorescent probe based on quantum dot
Quantum dots (QDs) are nanoscale particles with semiconductor properties composed of very few atoms or molecules. Compared with traditional organic dyes, quantum dots have the following advantages: (1) narrow fluorescence emission spectrum makes them have higher purity of light absorption, broad excitation spectrum, and large Stokes shift can effectively avoid spectral overlap
[147]; (2) good photochemical stability and difficult decomposition; (3) The color is adjustable and the multi-color labeling can be realized. These excellent properties enable quantum to be used in the detection of various small molecules and ions.
Carbon quantum dots (CQDs) have strong photostability and resistance to photobleaching, as well as fluorescence modulation in the ultraviolet to near-infrared wavelength range
[148]. The working mechanisms of CQDs-based sensors and biosensors include fluorescence quenching, static quenching, dynamic quenching, energy transfer, internal filtering effect (IFE), photoinduced electron transfer (PET), and fluorescence resonance energy transfer (FRET)
[149]. However, due to the low fluorescence yield and single fluorescence color of CQDs, its application is limited, and doping elements has become a common method to improve its fluorescence yield. Using nitrogen-doped CQDs, Jiang et al. Synthesized a hemicellulose-based carbon quantum dot fluorescence sensor 68 to detect Ag
+ and L-Cys (Fig. 77)
[150]. The fluorescence of the sensor 68 is quenched due to the complex formation of the Ag
+ with the groups on the sensor surface. With the addition of L-Cys, the complexing ability of L-Cys to Ag
+ is stronger, which leads to its release from the sensor surface and the fluorescence recovery.It is found that the fluorescence intensity of the sensor can still maintain at about 85% of the initial intensity after 30 days, which indicates that the photoluminescence stability of the sensor is good, and the fluorescence intensity remains relatively stable in pH 3 ~ 9. The detection limit for l-Cys was 242 nM. The sensor was successfully applied to detect Ag
+ in river water.
Based on the FRET mechanism, Wang et al. Synthesized an "on-off-on" type sensor 69(NPCl-CQD
S) using Cl, N, and P elements co-doped CQDs, which can detect riboflavin, Ag
+, and Cys in sequence (Figure 78)
[151]. The sensor itself has a strong emission peak at 453 nm, showing blue fluorescence, and when riboflavin is added to the sensor, a new emission peak at 530 nm will be produced, because the emission spectrum of NPCl-CQD
S as an energy donor overlaps with the absorption spectrum of riboflavin as an energy acceptor.The FRET process was initiated by the addition of Ag
+, which destroyed the FRET process between riboflavin and the sensor. The fluorescence at 530 nm was turned on again by the addition of Cys, and the fluorescence intensity was basically restored after 2. 5 min. The detection limit for Cys was 0.96 nM, indicating high sensitivity. The sensor also exhibited low cytotoxicity, enabling fluorescence imaging of Cys in HeLa cells.
The absorption spectrum of Graphene quantum dots (GQDs) covers most of the visible region, and its surface groups and size will affect the shift of its ultraviolet absorption peak wavelength
[152]. Compared with organic small molecule fluorescent dyes, GQDs can emit light with higher energy and shorter wavelength when excited by light with lower energy and longer wavelength
[153]. GQDs have stable photoluminescence, biocompatibility and low cytotoxicity, which make them promising in the field of biosensing
[154][155][156]. The fluorescence of GODs can be changed by doping heteroatoms into GODs, which can change their catalytic ability and electronic band gap structure
[157]. Deng et al. Synthesized sensor 70 (Fe, N-GQDs) using Fe and N co-doped GQD to detect Cys (Fig. 79)
[158]. Due to the quantum confinement effect, Fe, N-GQDs emitted bright blue-green fluorescence at 480 nm when excited at 420 nm. They found that Fe, N-GQDs had peroxidase activity and could catalyze H
2O
2 to oxidize TMB (a peroxidase substrate) into blue oxTMB. When L-Cys was added to the Fe,N-GQDs+H
2O
2+TMB solution, the solution changed from blue to colorless, indicating that L-Cys could reduce the production of oxTMB, and L-Cys was easily oxidized to cystine by H
2O
2, which consumed the concentration of H
2O
2 in the solution and reduced the oxidation of TMB. The detection limit of the sensor for Cys was 140 nM.
Liu et al. Utilized N-doped GQDs (N-GQDs) synthetic sensor 71 to identify Hg
2+ and Cys (Figure 80)
[159]. The attachment of Hg
2+ to N-GQDs was shown to cause fluorescence quenching; When Cys is added, the fluorescence is restored again, and the formed N-GQDs/Hg
2+/cysteine can also be used for quantitative detection of the H
2O
2; When H
2O
2 was added, cysteine was oxidized to cystine, the fluorescence decreased, and the fluorescence intensity was negatively correlated with the concentration of H
2O
2.
Xue et al. designed and synthesized two novel fluorescent probes 72 and 73 (Fig. 81) by modifying GQDs with maleimide
[160]. In PBS buffer (pH 7.4), maleimide-functionalized GQDs (M-GQDs) had no significant fluorescence due to the quenching effect of maleimide. The sensing mechanism of probe 72 to biothiol is that the double bond of maleimide group reacts with — SH in biothiol to form a saturated maleimide ring, which blocks the ICT and PET processes, and the fluorescence intensity of M-GQDs increases gradually with the addition of biothiol. Probe 73 is based on the FRET process between M-GQDs and (4-aminophenyl) porphyrin (TAPP). — SH in biological thiols destroys the "donor-acceptor" relationship between M-GQDs and TAPP, which weakens the fluorescence intensity of TAPP and strengthens the fluorescence of M-GQPs, forming a ratio relationship. Probe 73 can detect biological thiols more rapidly and sensitively, with a detection limit of 0.234 nM for Cys.
3.2 Cys fluorescent probe based on gold nanomaterials
Gold nanoparticles (AuNPs) refer to gold particles with a diameter of 1 ~ 100 nm, which are generally divided into gold nanoparticles (AuNPs) and gold nanoclusters (AuNCs). The AuNPs have good chemical stability and strong surface modifiability. AuNCs are composed of several to hundreds of atoms and have good water solubility and biocompatibility. Compared with other metal nanomaterials, nanogold materials have a wide absorption range in the ultraviolet-visible region, and have better safety, so they have a wide range of applications in the field of biosensors.
Zhang et al. Synthesized a stable AuNCs enhanced fluorescence sensor 74 based on the AIE effect using GSH as a stabilizer (fig. 82)
[161]. Under the excitation of ultraviolet light, the probe emitted a weak orange fluorescence. The emission wavelength of AuNCs is stable at 600 nm even if the excitation wavelength is different. When 1. 5 mM Cys and 1. 0 mM GSH were added to the aqueous solution of AuNCs, obvious aggregation was observed in the transmission electron microscope, and the fluorescence intensity increased by 3 times, even under continuous UV irradiation for 10 min, the fluorescence intensity did not change. The detection limit of the probe for Cys (2.49 μM ~ 0.80 mM) was 0.42 μM, and the probe was successfully applied to the detection of biological thiols in human serum.
Niu et al. Designed a fluorescent sensor 75 (fig. 83) based on AuNCs using bovine serum albumin (BSA) as a stabilizer to react with AuCL
4- by using a "switch" strategy, which can be used to detect and identify Cys and Cu
2+[162]. Cys can react with the sensor by filling the surface defects of gold nanoclusters, resulting in fluorescence enhancement. When Cu
2+ is added, cysteine is oxidized to cystine, resulting in the destruction of the S — Au bond formed between Cys and the sensor, and the aggregation state of the nanoclusters can not be maintained, thus the fluorescence is quenched. The detection limit of the probe for Cys (0 – 15 μM) was 30.4 nM. When the sensor images Cys in the liver and heart of mice with Alzheimer's disease, the fluorescence intensity is significantly lower than that of normal mice, indicating that it can be used as an auxiliary medical method for the diagnosis of related diseases.
Li et al. Also designed and synthesized a probe 76 based on AuNCs and AuNPs based on the "switch" strategy (fig. 84)
[163]. Firstly, based on the principle of electron transfer, a reducing agent is added to synthesize the AuNCs composite material. The higher the concentration of this reducing agent, the stronger the fluorescence of the AuNCs composite. After readdition of AuNPs, probe 76 was synthesized. The fluorescence was quenched due to the obvious overlap of the absorption spectrum of AuNPs and the fluorescence emission spectrum of AuNCs composite, which induced the fluorescence inner filter effect. Upon addition of l-Cys, fluorescence was turned back on with a detection limit of 1.4 μm. The fluorescence recovery rate was as high as 100.7%. The probe has almost no fluorescence response to other common metal ions and amino acids, which indicates that the probe 76 has strong anti-interference ability and good selectivity for Cys, and can be used for the analysis and detection of Cys in human serum.
Nguyend et al. Wrapped AuNPs with a positively charged cationic polymer (PDADMAC) to improve its stability, and synthesized a colorimetric sensor 77 to detect Cys (Fig. 85)
[164]. The AuNPs solution itself is red, when Cys is added, due to the strong affinity between — SH in Cys and gold particles, Cys and AuNPs are closely bound together through Au — S bonds, and the strongest absorption peak appears at 520 nm after 13 min, accompanied by the change of solution color from red to blue (or purple).Moreover, when Cys was added to the AuNPs solution in the presence of Cu
2+, the time for the appearance of the strongest absorption peak at 520 nM was shortened to 7 min, which was due to the formation of a stable complex between the amino and carboxyl groups of Cys and Cu
2+, which aggregated more closely and improved the sensitivity of Cys detection, with a detection limit of 88 nm. Based on this phenomenon, the effect of AuNPs particle size on Cys detection was studied, and the results showed that AuNPs with smaller particle size showed higher sensitivity than those with larger particle size
[165].
Zeolite imidazolate frameworks (ZIFs) are metal-organic frameworks defined as ZIF-67 when cobalt (II) is the central atom. Liu et al. Designed probe 78 by modifying ZIF-67 with gold nanoparticles (Fig. 86)
[166]. Compared with ZIF-67, the color change of ZIF-67 modified by gold nanoparticles was more obvious when Cys was added to the solution. The results showed that the probe could be used as a colorimetric probe for visual detection of Cys with a detection limit of 1 μm.
Chaicham et al. Designed and synthesized a colorimetric fluorescence sensor 79 based on AuNPs and GQDs to detect Cys (Fig. 87)
[167]. GQDs can effectively promote the formation and stabilization of AuNPs, forming the AuNPs/GQDs structure. In PBS buffer solution (pH 7. 4), AuNPs/GQDs showed a broad absorption band at 525 nm, and the maximum absorption peak at 525 nm decreased significantly after the addition of Cys, accompanied by the appearance of a new absorption band at 645 nm, and the solution changed from red to blue, with a detection limit of 5. 88 μM for Cys. The addition of other amino acids resulted in no significant change in the UV-Vis spectrum. The probe can realize the specific detection of Cys in milk and human urine.
3.3 Cys fluorescent probe based on carbon dots
Carbon dots (CDs) are Carbon nanoparticles with size less than 10 nm and good fluorescence emission characteristics. There are many functional groups on the surface of CDs, which can be modified. For example, carboxyl groups can make CDs have good water solubility. The existence of these functional groups makes CDs better combined with other materials or biological reagents. In addition, the synthetic raw materials of CDs have the advantages of wide sources, simple synthesis and low toxicity, which make them receive more and more attention and widely used.
Mohandoss et al. Designed and synthesized a sensor 80 with high quantum yield to detect Cys using N, S co-doped CD (N, S-CDs) (Fig. 88)
[168]. The solution of N, S-CDs is colorless under sunlight and emits green fluorescence under excitation at 450 nm, indicating that N, S-CDs has good photoluminescence properties, and this property depends on its particle size and surface morphology. The addition of Cys enhanced the photoluminescence obviously due to the nucleophilic interaction between -SH and the surface —NH
2 of N, S-CDs, and the green fluorescence changed into yellow fluorescence, while the addition of other molecules or metal ions had no significant change, and the detection limit of Cys was 23 nM. The sensor also penetrates the cell membrane well, allowing fluorescence imaging of intracellular Cys in HCT 116 cells.
Xiang et al. Combined (N, S-CDs) with silver nanoparticles (AgNPs) to design and synthesize a pH-dependent "on-off-on" fluorescence sensor 81 (Fig. 89)
[169]. Due to the presence of —NH
2 and — SH groups with strong affinity on the surface of N, S-CDs, the AgNPs were adsorbed on its surface and the fluorescence was quenched. When the three biothiols were added, the AgNPs were dissociated from the surface of the sensor 81 due to the stronger affinity of the thiols, and the fluorescence was restored. At pH 3, all three biothiols caused a similar degree of fluorescence recovery, and at pH 7, only Cys/Hcy caused fluorescence recovery. The sensor has a detection limit of 68.5 nM for Cys and has been successfully used to determine the concentration of biological thiols in human serum. Natesan et al. Also designed a CDs sensor 82 (fig. 90) based on an "on-off-on" strategy to detect Ag
+ and Cys
[170]. CDs has a strong fluorescence emission peak at 530 nm, and the fluorescence of CDs is quenched after the addition of Ag
+ due to the binding of Ag
+ with CDs, while the fluorescence of CDs is recovered after the addition of Cys due to the dissociation of Cys from the surface of CDs due to its strong binding with Ag
+. The sensor has a detection limit of 0.34 μM for Cys, enabling fluorescence imaging of Cys in living cells. In addition, the sensor is also used as a fluorescent dye for fingerprint staining.
Zhao et al. designed a dual-emission ratiometric fluorescent probe 83 based on N-doped yellow fluorescent carbon dots (y-CDs) and copper nanoparticles with blue fluorescence (CuNCs) to detect biological thiols (Fig. 91)
[171]. This is due to the fact that Cu
2+ can coordinate with the nitrogen-containing groups on the surface of Y-CDs, blocking the PET process, resulting in the enhancement of the yellow fluorescence of Y-CDs at 567 nm, and the quenching of the blue fluorescence of CuNCs at 450 nm through the FRET process. The addition of biothiol can prevent the chelation of Y-CDs with Cu
2+, resulting in the quenching of the yellow fluorescence of Y-CDs and the recovery of the blue fluorescence of CuNCs. The quantitative detection of Cys was achieved by calculating the fluorescence intensity ratio, and the detection limit was 0. 21 μM. The sensor also allows ratiometric fluorescence detection of Cys in serum.
Lu et al. Designed and synthesized a ratiometric fluorescent probe 84 based on nanoenzyme for the first time (Fig. 92)
[172]. Because of the strong oxidizing property of iron ions, CDs doped with iron ions has excellent peroxidase activity, and then combined with o-phenylenediamine (OPD) as the enzyme substrate, it can be oxidized to oxidized OPD with photoluminescence characteristics, so that probe 78 has two emission peaks at 460 and 560 nm under the excitation of 360 nm wavelength ultraviolet lamp. When L-Cys was added, the production of oxidized OPD was inhibited, resulting in a decrease in the fluorescence intensity at 560 nm and an unchanged fluorescence intensity at 460 nm, with a detection limit of 0.047 μM. The probe can still detect L-Cys with high selectivity in the presence of other amino acids and cations. In addition, the probe has also been successfully used to detect L-Cys in water and serum, which provides a reference for the development of more fluorescent probes based on nanozyme in the future.
Based on CDs, Liu et al. Reported a fluorescence sensor 85 (Scy-CDs) for quantitatively detecting the concentration of Cys/Hcy and pH in lysosomes according to the change of fluorescence intensity (fig. 93)
[173]. The sensor has a strong fluorescence emission at 556 nm at pH 7, the fluorescence intensity gradually decreases with the addition of H
+,pH, and the fluorescence intensity is linearly related to pH at pH 5.0 to 5.6, which indicates that the sensor can be used to determine the pH value in lysosome (pH 4.4 to 6.5), and the PET process is blocked and the fluorescence can be quickly recovered in the presence of Cys or Hcy, which indicates that the sensor can be used for targeted fluorescence imaging of Cys in lysosome.
The detection of Cys by nano-fluorescent probes can mostly be carried out in aqueous solution, and has good stability, simple operation, lower detection limit and higher sensitivity than organic small molecule fluorescent probes, but the selectivity for Cys is low, and it is difficult to achieve specific detection of Cys, so there is still a lot of research space to develop nano-fluorescent probes with high selectivity for Cys detection.
4 Fluorescent proteins
Fluorescent proteins (FPs) are proteins produced in cells by transfection of plasmid DNA, with a molecular weight of 25-30 KDa. Fluorescent proteins have good targeting, low cytotoxicity, strong tissue penetration, and can provide accurate labeling in cells or organisms. However, due to the poor photostability and small Stokes shift of fluorescent proteins, it has a certain negative impact on bioluminescence imaging. In recent years, green fluorescent protein (GFP) has been widely used in various biosensors
[174,175].
Using GFP as a chromophore, Shen et al. Designed and synthesized a fluorescent response probe 86 with a red emission wavelength (fig. 94) by using acrylate as both a fluorescence quenching group and a Cys recognition group
[176]. When the probe was excited by a 493 nm UV lamp, the fluorescence intensity at 620 nm increased with the increase of Cys concentration, and the fluorescence intensity remained stable in the pH range of 4. 2-8. 5. The detection limit of Cys (0-200 μM) was 18. 7 μM. When Hcy/GSH was added, the probe reacted with Cys most rapidly due to the different kinetic rates of the reaction, indicating that the probe could detect Cys with high selectivity in the physiological pH range. The probe enables the detection of endogenous and exogenous Cys in living cells by confocal fluorescence microscopy imaging in Bel-7402 cells. They also designed a lysosome-targeting fluorescent probe 87 (Fig. 95)
[177]. The probe has DNBS as a fluorescence quenching and Cys detection group, and a morpholino group is introduced as a lysosomal targeting group. Probe 87 showed no fluorescence at 470 nm. After incubation with 50 μM of different biological thiols and amino acids for 30 min, only Cys and GSH caused a significant fluorescence turn-on change, but GSH responded faster than Cys due to the higher activity of — SH on GSH than Cys. The detection limit of the probe for Cys (0 ~ 14 μM) was 4.98 nM. It was successfully applied to lysosomal endogenous and exogenous Cys-targeted fluorescence imaging in Bel-7402 cells.
5 Conclusion and outlook
In this paper, the latest research progress of 87 fluorescent probes for cysteine detection in the past three years is reviewed. The design strategy of these fluorescent probes and the sensing mechanism of Cys were summarized. According to the different chromophores, these fluorescent probes are divided into three categories: organic small molecule fluorescent probes, nano-fluorescent probes and gene fluorescent probes, and then in each category, the similar detection mechanisms and the types of recognition groups are compared and summarized. It is found that acrylate is the most common Cys recognition group in small organic molecule fluorescent probes, which can detect Cys by conjugate addition reaction with Cys, followed by intramolecular cyclization, release of fluorophore and fluorescence switching on. Organic small molecule fluorescent probes have good biocompatibility and can specifically detect Cys in deep tissues of cells, with imaging depth greater than 100 μm (probe 3, probe 67), and can also achieve single/two-photon imaging (probe 20). However, due to the relatively simple structure of the fluorophore of organic small molecule probes, the luminescence stability is easily affected by environmental factors. Because of its small size, the nano-fluorescent probe has a lower detection limit than the organic fluorescent probe, as low as 0.234 nM (probe 73), which can achieve ultra-trace detection of Cys. However, because most of the nano-fluorescent probes need third-party reagents to detect Cys, such as metal ions, and it is difficult to distinguish three biological thiols with similar structures, the selectivity is low. Gene fluorescent probes can provide precise labeling in cells or organisms because of their low cytotoxicity and strong tissue penetration, such as targeted fluorescence imaging of lysosomes (probe 87).
Based on these findings, our future research on Cys detection can be carried out in the following three aspects: (1) by modifying the structure of fluorophores or developing new fluorophores and sensing mechanisms, we can prepare fluorescent probes with more stable luminescence, more sensitive detection and faster response on the basis of ensuring the fluorescence characteristics; (2) develope a dual-photon (multi-photon) fluorescent probe with specific target site recognition to realize functional fluorescent imaging of cell; (3) Develop a visual fluorescent sensor that can detect Cys in vivo in real time and dynamically.
In conclusion, the development of fluorescent probes that can specifically recognize and detect Cys in cells and tissues is of great significance to the field of clinical medical diagnosis.
Scientific Research Fund of Hunan Provincial Education Department(20C0593)
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