1 Introduction
H2S has been demonstrated to act as a gas transmitter to exert functionally modulatory roles in human biology, similar to NO and CO. H2S can render controllable regulation of cellular functions by affecting intracellular signaling processes. As an upstart among various vital biological gases, H2S has received significant research interest, which has resulted in unraveling its biological functions in various cellular processes, especially in the cardiovascular system [1], inflammation system [2], nervous system [3].
One of the first observations of H2S biology was its effect on the cardiovascular system. For instance, up-regulation of H2S in rat blood vessels resulted in the expansion of vessels through opening of the vascular smooth muscle KATP channels [4]. In addition, in cecal ligation and puncture-induced rats, H2S ameliorated cardiac dysfunction [5]. Up-regulation of cystathionine γ-lyase (CSE) in vivo, by means of increasing the local H2S concentration, helped to protect experimental mice from the development of atherosclerosis [6]. In another study related to the evaluation of morpholin-4-ium-4-methoxyphenyl(mopholino) phosphinodithiote, a H2S donor showed anti-atherosclerotic function [7].
Another most reported function of H2S is its effect on the inflammatory system. In a recent study on H2S, it was confirmed that levels of H2S in vivo increased under inflammatory or sepsis conditions [2]. The interaction between H2S and inflammatory attracts great attention. It was demonstrated that H2S has a benign influence on mitigation of vascular inflammation via up-regulation of glutathione (GSH) and glutamate-cysteine (Cys) ligase catalytic subunit and inhibition of interleukin-1β in U937 monocytes [8]. It was demonstrated that exogenous H2S can inhibit hyperplasia of the endothelial lining by partially restraining the interleukin-1β and selectively controlling mitogen-activated protein kinases and the phosphatidylinositol 3′-kinase/protein kinase B pathway on fibroblast-like synoviocytes, and can thus be used for the treatment of osteoarthritis [9].
The significant biological roles of H2S have been revealed broadly and its importance obtains unprecedented attention. In addition to its effects on the cardiovascular system and inflammatory system, H2S has also been closely related to Alzheimer’s disease [10], Parkinson’s disease [11], gastrointestinal disease [12], and cancer [13]. Although there is still more to be studied, the functional roles of H2S in human biology will be revealed clearly. Furthermore, depending on these developments, more effective and powerful H2S-based therapeutic methods will be identified.
2 Current engineering strategies for detecting H2S
H2S in vivo is mainly derived from Cys and 3-mercaptopyruvate via enzymatic reactions catalyzed by cystathionineβ-synthase (CBS), CSE, Cys aminotransferase, and 3-mercaptopyruvate sulfurtransferase [14]. However, due to the dynamic conversion between various H2S-existing forms, the accurate biological concentration of H2S in vivo is untraceable. For example, experiments conducted in the Whiteman lab revealed that the concentration of H2S in healthy volunteers was around 43.8 ± 5 μmol·L−1, and was markedly increased (up to 200 μmol·L−1 in one case) during septic shock [15]. Therefore, reliable analytical assays that measure the accurate concentration of H2S in vivo are warranted. In recent years, the technology of fluorescent sensing was employed and small-molecular fluorescent probes have been identified as promising tools to interrogate biological H2S.
Effective fluorescence sensors for bio-imaging must conquer stringent requirements. First, the probe must be exclusively selective to its corresponding target substrate without being interfered by other co-existing substrates. Moreover, since most biochemical analytes are at low concentrations in their native environment, the sensitivity of the probe has to be ensured for low-abundant analytes. In addition, for further in vivo applications, the probe should do no harm to human biology and its reactive product ought to be non-toxic. Furthermore, optical properties, solubility, membrane permeability, etc. should be taken into consideration.
The relative high concentrations of other biological sulfur species, such as GSH and Cys residues, are among the most challenging factors for probes to selectively detect H2S in vivo [16]. At present, based on the reductive and nucleophilic ability of H2S and its high binding affinity towards Cu2+, various reaction-based H2S probes have been developed, and are generally categorized into three types: reduction-based H2S probes, nucleophilicity-based probes, and copper sulfide (CuS)-based probes. Several review articles have comprehensively summarized these probes [17–20]. However, new probes have also been rapidly developed in the last few years. Furthermore, hydrogen polysulfides (H2Sn) is the redox partner of H2S in terms of chemical properties. They coexist in biological systems and collectively regulate the sulfur redox balance. To study the distribution and regulation mechanism of H2Sn in organisms, a number of probes have been developed. In this minireview, a brief review of these probes will be summarized and they can be divided into four groups: H2S reducibility-based probes, H2S nucleophilicity-based probes, metal sulfide precipitation-based probes and H2Sn probe. The detection performance of each probe and biological application are shown in Table 1.
Tab.1 The detection performance of the fluorescent probes summarized in this minireview |
| Probe | Selectivity | Ex/Em/nm | Reported change in fluorescence | Limit of detection (LOD) | Biological system |
|---|---|---|---|---|---|
| H2S reducibility-based probes | |||||
| 1 (DDP-1) | H2S/H2Sn | 360/452,542 | 100 nmol·L−1 24 nmol·L−1 | HeLa cells | |
| 2 (Flu-N3) | H2S | 470/538 | 50-fold | 0.031 μmol·L−1 | HepG-2 cells and nude mice |
| 3 (RPC-1) | H2S/HClO | 360/445 545/580 | 192.1 nmol·L−1 19.8 nmol·L−1 | HeLa cells, HepG-2 cells, RAW264.7 cells and drug-induced liver injury mice | |
| 4 (Lyso-HA-HS) | H2S/HOCl | 380/448 550/580 | 490-fold | 3.4 × 10–7 mol·L−1 7.3 × 10–8 mol·L−1 | HepG-2 cells and RAW264.7 cells |
| 5 (Mito-HS) | H2S | 345/540 | 21-fold | HeLa cells and BALB/C nude mice with xenograft breast cancer tumor | |
| 6 (Lyso-HS) | H2S | 345/540 | 15-fold | HeLa cells and BALB/C nude mice with xenograft breast cancer tumor | |
| 7 (MT-HS) | H2S | 440/540 | 40-fold | 1.65 µmol·L−1 | HeLa cells and fresh rat liver slices |
| 8 (Na-H2S-ER) | H2S | 440/545 | 45-fold | 7.77 × 10–6 mol·L−1 | HeLa cells, living liver tissue slices and zebrafish |
| 9 (ASNHN-N3) | H2S | 454/545 | 0.75 µmol·L−1 | HeLa cells, RAW264.7 cells, the fresh liver tissues and heart arteries | |
| 10 (BN-H2S) | H2S | 440/544 | 71 nmol·L−1 | HeLa cells and NIH 3T3 cells | |
| 11 (CouN3-BC) | H2S | 405/450 | 35-fold | HeLa cells | |
| 12 | H2S | 370/490 | 0.67 µmol·L−1 | HEK293A cells | |
| 13–14 | H2S | ||||
| 15 (Mito-HS) | H2S | 380/450 | ~43-fold | 24.3 nmol·L−1 | HeLa cells, MDA-MB-231 cells, DU145 cells and 3T3-L1 cells |
| 16 (Lyso-C) | H2S | 355/458 | >20-fold | 47 nmol·L−1 | HepG-2 cells |
| 17 (1-H2S) | H2S | 580/635 | 7-fold | ~4.7 × 10–5 mol·L−1 | HeLa cells, zebrafish and fresh liver tissue slices of Kunming mice |
| 18 (AC-N3) | H2S | 360/500 | 18 nmol·L−1 | HeLa cells | |
| 19 (QME-N3) | RSH/H2S | 350/– | MCF-7 cells | ||
| 20 (TPC-N3) | H2S | –/498 | HepG-2 cells and fresh liver and muscle slices of liver cirrhosis induction mice | ||
| 21 (Lyso-HS) | H2S | 365/505 | 95-fold | 214.5 nmol·L−1 | A549 cells, HepG-2 cells and rat renal tubular epithelial cells |
| 22 (QL-Gal-N3) | H2S | 365/521 | 102-fold | 126 nmol·L−1 | HeLa, A549 cells and HepG-2 cells |
| 23 (Gol-H2S) | H2S | 405/515 | 0.11 µmol·L−1 | HeLa, EK293A cells and SMMC-7721 cells | |
| 24 (Diketopyrrolopyrrole, DPP-NO2) | H2S | 506/550 | 800-fold | 5.2 nmol·L−1 | HeLa cells |
| 25 | H2S | 485/522 | 2.55 µmol·L−1 | HepG-2 cells | |
| 26 (azo1) | |||||
| 27 (azo2) | H2S | 468/517 | 103-fold | 5 µmol·L−1 | Fresh male Sprague-Dawley (SD) rat blood plasma and tissues |
| 28 (azo3) | H2S | 468/517 | 148-fold | 500 nmol·L−1 | Fresh male SD rat blood plasma and tissues |
| 29–32 | |||||
| 33 (PHS1) | H2S | 393/486 | 8.87 nmol·L−1 | HeLa cells | |
| H2S nucleophilicity-based probes | |||||
| 34 | 190 nmol·L−1 (buffer) 380 nmol·L−1 (serum) | ||||
| 35 (Cy-Cl) | H2S | 760/795 | HeLa cells | ||
| 36 (CyCl-1) | HS− | 0.16 µmol·L−1 | Living mice | ||
| 37 (CyCl-2) | HS− | 0.37 µmol·L−1 | Living mice | ||
| 38 (BH-HS) | H2S | 450/535 | 57-fold | 1.7 × 10–6 mol·L−1 | HeLa cells |
| 39 (CPC) | H2S | 410/474, 582 | 56-fold | 40 nmol·L−1 | HeLa cells |
| 40 (TP-PMVC) | H2S | 405/550 | 3.2 µmol·L−1 | A549 cells | |
| 41 (CP-H2S) | H2S | 355/454, 573 | 252.7-fold | 2.2 × 10–7 mol·L−1 | SMMC-7721 hepatoma cells |
| 42 (Mi) | H2S | 520/596 | 15 nmol·L−1 | HeLa cells | |
| 43 (CyT) | H2S | 575/595, 655 | 7.33 nmol·L−1 | HeLa cells | |
| 44 (Indo-TPE-Indo) | H2S | 488/560, 710 | 0.19 µmol·L−1 | HeLa, MCF-7 and HUVEC cells | |
| 45 (TP-MIVC) | RNA/H2S | 488/625 405/550 | 12-fold | 1.0 × 10–6 mol·L−1 3.2 × 10–6 mol·L−1 | HeLa cells, zebrafish, normal mice and tumor mice |
| 46 (CTN) | H2S | 370/424 | 200-fold | 90 nmol·L−1 | HeLa cells |
| 47 (Near-infrared (NIR)-HS) | H2S | 670/723 | 50-fold | 38 nmol·L−1 | MCF-7 cells and living mice |
| 48 (TPE-3) | H2S | 452/550 | 0.09 µmol·L−1 | HeLa cells, zebrafish | |
| 49 (TP-NIR-HS) | H2S | 800/675 | 83 nmol·L−1 | A375 cells and nude rat liver frozen slices | |
| 50 (2-CHO-OH) | H2S | 550/655 | 32-fold | 8.3 × 10–8 mol·L−1 | HeLa cells |
| 51 (NDCM-2) | H2S | 490/655 | 160-fold | 58.797 nmol·L−1 | MCF-7 cells, the kidney tissue slices and living Kunming mice |
| 52 (NIPY-DNP, 2,4-dinitrophenyl) | H2S | 340/505 | 273-fold | 0.36 µmol·L−1 | A549 cells |
| 53 (TMSDNPOB) | H2S | 574/592 | 30-fold | 1.27 µmol·L−1 | HeLa cells and raw 264.7 macrophage cells |
| 54 (LC-H2S) | H2S | 571/664 | 27-fold | 4.05 µmol·L−1 | HeLa cells |
| 55 (A) | H2S | 440/537 | 49 nmol·L−1 | LoVo cells and SW480 cells | |
| 56 (DMC) | H2S | 384/547 | 0.069 µmol·L−1 | HeLa cells | |
| 57 (Cda-DNP) | H2S | 405/450 | 120-fold | 0.18 µmol·L−1 | HeLa cells, A549 cells, HFL1 cells, and zebrafish |
| 58 (NR-NO2) | H2S | 675/710 | L929 cells, HeLa cells, HCT-116 cells and BALB/c nude mice | ||
| 59 (Mito-NIR-SH) | H2S | 670/720 | 14-fold | 89.3 nmol·L−1 | HeLa cells |
| 60 (DMOEPB) | H2S | ||||
| 61 (DMONPB) | H2S | 543/625 | 1.3 µmol·L−1 | RAW264.7 macrophages and HeLa cells and liver tissues of Kunming mice | |
| 62 | H2S | 570/623 | HCT-116 and CT-26 cells | ||
| 63 | H2S | 590/680 | 115-fold | 11 nmol·L−1 | HCT16, HT29, A549, H1944, MCF-7, MDA-MB-468, MDA-MB-231, PANC1, HeLa, HepG-2 cells and Kunming living mice |
| 64 (QCy7-HS) | H2S | 580/715 | 25-fold | 1 µmol·L−1 | HeLa, HepG-2 cells and female BALB/c mice |
| 65 (Z1) | H2S | 480/537 | 0.15 µmol·L−1 | Ec1 cells | |
| 66 (L) | H2S | 496/607 | 1.05 × 10–5 mol·L−1 | MCF-7 cells | |
| 67 | H2S, Cys/homocysteine (Hcy), GSH | 415/465 415/465 415/465 | 0.10 µmol·L−1 0.08 µmol·L−1 0.06 µmol·L−1 | HeLa cells | |
| 68 | H2S | 382/550 | HeLa cells | ||
| 69 | H2S | 382/455 | 150 nmol·L−1 | HeLa cells | |
| 70 | H2S | 502/530 | 65-fold | 0.057 µmol·L−1 | HEK293A cells |
| 71 | H2S | 530/589 | 4.5-fold | 0.58 µmol·L−1 | HEK293A cells |
| 72 | H2S | 565/585 | 19-fold | 0.36 µmol·L−1 | HEK293A cells and zebrafish |
| 73 | H2S | 405/480 | 45-fold | 9 µmol·L−1 | HEK293 cells and HeLa cells |
| 74 | H2S | 405/496 | 200-fold | 0.9 µmol·L−1 | HEK293A cells, A549 cells and zebrafish |
| 75 | H2S | 394/486 | 45-fold | 56 nmol·L−1 | HEK293 cells |
| 76 (Endoplasmic reticulum (ER)-CN) | H2S | 383/490 | 6.5-fold | 4.9 µmol·L−1 | HeLa cells |
| 77 | H2S | 395/532 | 68-fold | 2.46 µmol·L−1 | HeLa cells |
| 78 | H2S | 346/516 | 25-fold | 20 nmol·L−1 | A431 cells |
| 79 (BDP-N1) | H2S | 540/587 | 150-fold | 0.06 µmol·L−1 | A549 cells and zebrafish |
| 80 (BDP-N2) | H2S | 625/587 | 170-fold | 0.08 µmol·L−1 | A549 cells and zebrafish |
| 81 | H2S/GSH Cys/Hcy | 620/685 460/540 | 253-fold/448-fold | 70 µmol·L−1/0.38 µmol·L−1 52 nmol·L−1/38 nmol·L−1 | HeLa cells and living mice |
| 82 | H2S | 539/565 | 160-fold | 4.80 × 10–8 mol·L–1 | HeLa cells |
| 83 (NIR-H2S) | H2S | 730/830 | 68-fold | 2.7 × 10–7 mol·L−1 | MCF-7 cells, athymic nude mice and Kunming mice |
| 84 (L) | H2S | 780/468 | 29-fold | 24 nmol·L−1 | HeLa cells |
| 85 (RHP) | H2S | 410/550, 475 | 4-fold | A549 cells | |
| 86 (RHP-2) | H2S | 415/467, 532 | 27-fold | 270 nmol·L−1 | MCF-7 cells and mouse hippocampus |
| 87 | H2S | 465/520 | 80-fold | 0.15 µmol·L−1 | HeLa cells |
| 88 (NS1) | H2S | 365/539, 444 | 1.7 × 10–6 mol·L−1 | MCF-7 cells | |
| 89 (MeRho-TCA) | H2S | 476/520 | 65-fold | ||
| 90 (LR-H2S) | H2S | 410/541, 475 (one-photon) 840/541,475 (two-photo) | 80-fold | 0.70 µmol·L−1 | SGC-7901 cells |
| 91 (PyN3) | H2S | 410/455 | 158 nmol·L−1 | MCF-7 cells, HeLa cells, zebrafish | |
| 92 (NIR-Az) | H2S | 680/720 | 200-fold | 0.26 µmol·L−1 | HeLa cells, RAW 264.7 murine macrophages and BALB/c nude mice |
| 93 (Mito-VS) | H2S | 370/510 | 7-fold | 0.17 µmol·L−1 | HeLa cells |
| 94 (BDP-N3) | H2S | 475/515 | 10-fold | 2.05 µmol·L−1 | HepG-2 cells |
| 95 (Mito-N3) | H2S | 680/736 | 20 nmol·L−1 | MCF-7 cells and BALB/c(nu/nu) mice | |
| 96 | H2S | 485/610 | 5.7 nmol·L−1 | HeLa cells | |
| 97 (MF-N3) | H2S | 530/560 | 16-fold | 0.09 µmol·L−1 | HepG-2 cells |
| 98 (HF-PBA) | H2S/biothiols | 345/520, 400 | 75 nmol·L−1 | HeLa cells | |
| 99 (HS-1) | H2S | 350/403, 519 | 0.020±0.001 mmol·L−1 | A549 cells | |
| 100 (DCM-PBA) | H2S | 560/680 | 1.1 nmol·L−1 | HeLa cells | |
| 101 (Cy-PBA) | H2S | 675/725 | 21 nmol·L−1 | A549 cells and nude mice | |
| 102 | H2S | 380/495, 525 | 91 nmol·L−1 | HeLa cells | |
| 103 | H2S | 560/633 | 25-fold | 8.37 µmol·L−1 | Hi5 insect cells and Caenorhabditis elegans |
| 104 (DCN-S) | H2S | 420/550, 580 | 5.7-fold | 88 nmol·L−1 | HeLa cells |
| 105 (HBTSeSe) | H2S | 380/460 | 47-fold | 0.19 µmol·L−1 | RAW264.7 cells |
| 106 (SFP-1) | H2S | 300/391 | HeLa cells | ||
| 107 (SFP-2) | H2S | 465/510 | 16-fold | HeLa cells | |
| 108 (ZS1) | H2S | 520/561 | 62-fold | 2.5 µmol·L−1 | RAW 264.7 macrophage cells |
| 109 (P1) | H2S | 378/524 | HeLa cells | ||
| 110 (P2) | H2S | 370/450 | |||
| 111 (P3) | H2S | 375/500 | 50 nmol·L−1 | HeLa cells | |
| 112 (P5) | H2S | 485/638 | 0.9 µmol·L−1 | HeLa cells | |
| 113 (RB-PE-1) | H2S | 560/590 | HeLa cells | ||
| 114 (RB-PE-2) | H2S | 560/590 | HeLa cells | ||
| 115 (RB-PE-3) | H2S | 560/590 | HeLa cells | ||
| 116 (FEPO-1) | H2S | 455/522 | 14 µmol·L−1 | HeLa cells and zebrafish | |
| 117 (FLVN-OCN) | H2S | 415/525 | 0.25 µmol·L−1 | A-549 cells | |
| 118 (ZX-NIR) | H2S | 520/600 650/700 | 37 nmol·L−1 | HCT116 cells, HepG-2 tumor-bearing mouse model and HCT116 tumor-bearing mice | |
| 119 (Coumarin-tetrazine (Tz)) | H2S | 375/456 | 16-fold | ||
| 120 (boradiazaindacene (BODIPY)-Tz-I) | H2S | 580/660 | 22.7-fold | 0.68 µmol·L−1 | 3T3 fibroblast cells |
| 121 (BODIPY-Tz-II) | H2S | 580/660 | 31-fold | 0.66 µmol·L−1 | 3T3 fibroblast cells |
| 122 | H2S | 18.2 µmol·L−1 | |||
| 123 (PTZ-P1) | H2S | –/488 | 25-fold | ||
| 124 (PTZ-P2) | H2S | ||||
| 125 (PTZ-P3) | H2S | 330/480, 540 | |||
| 126 (PTZ-P4) | H2S | 580/638 | HeLa cells and Caenorhabditis elegans | ||
| H2S metal sulfide-based fluorescent probes | |||||
| 127 | H2S | 470/517 | 420 nmol·L−1 | ||
| 128 | Cu2+/H2S | 410/505 | 1.3 × 10–7 mol·L−1 | HeLa cells | |
| 129 | H2S | 456/612 | 0.25 µmol·L−1 | ||
| 130 (CuHCD) | S2– and HNO | 484/595 484/595 | 0.7 µmol·L−1 23 µmol·L−1 | ||
| 131 (TACN) | H2S | ||||
| 132 (Cyclam) | H2S | ||||
| 133 (Hsip-1) | H2S | 491/516 | 50-fold | HeLa cells | |
| 134 (TMCyclen) | H2S | ||||
| 135 | H2S | 680/765 | 80 nmol·L−1 | RAW264.7 cells and HEK 293 cells | |
| 136 | H2S | 600/680 | MCF-7 cells | ||
| 137 | H2S | 446/605 | ~130-fold | 21.6 nmol·L−1 | |
| 138 [Cu(MaT-cyclen)2] | H2S | 375/430 | 205 nmol·L−1 | HeLa cells and zebrafish | |
| 139 | H2S | –/794 | 27-fold | 280 nmol·L−1 | |
| 140 (L1Cu) | H2S | 495/534 | HeLa cells and L929 mouse fibroblast cell lines | ||
| 141 (L1) | H2S | 494/523 | 25-30-fold | 1.7 µmol·L−1 | HepG-2 cells |
| 142 (L1-Cu) | H2S | 495/557 | HeLa cells | ||
| 143 (L2) | |||||
| 144 (L) | Cu2+/H2S | 310/373,495 | 9.12 × 10–7 mol·L−1 | ||
| 145 (NJ1) | Cu2+/H2S | 360/492 | HeLa cells | ||
| 146 (NL) | Cu2+/H2S | 430/519 | 0.17 µmol·L−1 | MDA-MB-231 cells | |
| 147 | Cu2+/HS− | 340/480 | 25-fold | 2.24 µmol·L−1 | HepG-2 cells |
| 148 | Cu2+/H2S | 345/540 | |||
| 149 | Cu2+/H2S | 405/540 | 47 nmol·L−1 | NIH/3T3 cells | |
| 150 (TAB-3) | |||||
| 151 (CAH-Cu2+) | H2S | 350/425 | 31-fold | 65 nmol·L−1 | |
| 152 (L-Cu) | H2S | 495/525 | 31 nmol·L−1 | HeLa cells and zebrafish | |
| 153 (DPD-Cu2+) | Cu2+/S2– | 440/510 440/510 | 0.73 nmol·L−1 0.87 nmol·L−1 | A549 cells | |
| 154 (Cu-1) | HS− | 543/600 | 14-fold | HeLa cells | |
| 155 (Cu(BB)2) | H2S | 384/590 | 0.11 µmol·L−1 | ||
| 156 (aggregation-induced emission (AIE)-S) | Cu2+/H2S | 350/533 | HeLa cells | ||
| 157 (6-CdII) | H2S | 550/599, 619 | HeLa cells | ||
| 158 | H2S | 365/500 | ~13-fold | 30 nmol·L−1 | |
| Fluorescent probes for H2Sn | |||||
| 159 (Cy-Sn) | H2Sn | 680/720 | 2.2 × 10–8 mol·L−1 | RAW264.7 cells and living mice | |
| 160 (KB1) | H2Sn | 535/682 | >30-fold | 8.2 nmol·L−1 | MCF-7 cells |
| 161 (RPHS1) | H2Sn | 395/482, 655 | 5.8-fold | 43 nmol·L−1 | HeLa cells |
| 162 | H2Sn | 397/534 | 328-fold | 26 nmol·L−1 | A549 cells and zebrafish |
| 163 (PZC-Sn) | H2Sn | 480/620 | 1 nmol·L−1 | RAW 264.7 cells and zebrafish | |
| 164 (Re-SS) | H2Sn | 550/589 | 24 nmol·L−1 | RAW 246.7 cells | |
| 165 (BDP-PHS) | H2Sn | 525/574, 618 | 57 nmol·L−1 | HeLa cells | |
| 166 (JCCF) | H2Sn | 480/543 | 52-fold | 98.3 nmol·L−1 | MCF-7 cells and zebrafish |
| 167 | H2Sn | 360/502 | 18-fold | 5.0 × 10–7 mol·L−1 | HepG-2 cells |
| 168 | H2Sn H2S | 410/468, 606 410/519, 606 | 194-fold 37-fold | 21 nmol·L−1 34 nmol·L−1 | RAW264.7 cells, living mice liver tissue and zebrafish |
| 169 (NIPY-NF) | H2Sn | 340/520 | 69-fold | 84 nmol·L−1 | A549 cells |
| 170 (Lyso-NRT-HP) | H2Sn | 405/548 | 10 nmol·L−1 | HeLa cells and the freezing kidney slices | |
| 171 (BCy-FN) | H2Sn | 653/727 | 46 nmol·L−1 | RAW264.7 cells, ZF4 cells, zebrafish larvae and BALB/c mice | |
| 172 | H2Sn | 680/708 | 44-fold | 35 nmol·L−1 | HeLa cells, RAW264.7 cells and BALB/c mice |
| 173 (τ-Probe) | H2Sn | 2 nmol·L−1 | HeLa cells and zebrafish | ||
| 174 (MB-Sn) | H2Sn | 530/584 | 26.01 nmol·L−1 | RAW 264.7 cells | |
| 175 (HQO-PSP) | H2Sn | 520/633 | 86-fold | 95.2 nmol·L−1 | A549 cells and mouse lung tissues |
| 176 (SPS-M1) | H2Sn | 372/430, 506 | 0.1 µmol·L−1 | HeLa cells, transgenic mice expressing human A53 T α-syn, SH-SY5Y cells and fresh mice brain slices | |
| 177 (PP-PS) | H2Sn | 300/478 | 20.3-fold | 1 nmol·L−1 | A549 cells, mouse tumor tissues and inflamed mouse models |
3 Reducibility-based probes for H2S
Since the 19th century, H2S, sodium sulfide, and sodium hydrosulfide have been used as synthetic reducing agents. Utilizing the reducibility of H2S, reduction-based probes, which are designed based on the nitrogen atom redox states, have been developed. Four categories of nitrogen-containing groups have been reported, including the azide group, the nitro group, the hydroxamino group as well as the azo group, thereby realizing pragmatic availability of reaction mechanisms and integrating diversity into reduction-based probes.
3.1 Probes based on reduction of azide group for sensing
Since the first successful attempt by the Chang group in 2011, the azide group has become the most widely used functionality to design reduction-based H2S probes [21]. The detection occurs via the chemoselective reduction of azide to amine. To date, almost all fluorophores, including rhodamine and its derivatives, coumarin, 1,8-naphthalimide, have been utilized to carry the azide group to generate structurally diverse probes for H2S [19]. As a H2S reporter, the biocompatibility and the versatility of the azide group make it the most promising engineering strategy for sensing H2S in human biology.
In 2016, Chen et al. reported probe 1 (DDP-1) that could distinguish H2S and H2Sn [22]. The probe utilized two specific groups for sensing H2S and H2Sn. H2Sn are oxidative products of H2S that have a weaker reducing ability; the azide group can differentiate H2S from H2Sn. Biological experiments also proved that probe 1 (DDP-1) has cell membrane permeability and can recognize H2S and H2Sn from a different emission spectrum in cells. In 2018, Zhao et al. generated fluorescent probe 2 (Flu-N3) by removing the H2Sn reactive site in probe 1 (DDP-1) [23]. Probe 2 (Flu-N3) performed good sensitivity and selectivity for H2S compared to other biothiols and triumphantly imaged H2S in vivo. Moreover, Jiao et al. chose rhodamine B instead of fluorescein and introduced HClO-specific thiolactone to create a two-photon fluorescent probe 3 (RPC-1) [24]. Probe 3 (RPC-1) exhibits two emission bands corresponding to H2S and HClO, respectively. Upon excitation with a two-photon laser, the presence of HClO/H2S results in significant fluorescence enhancement within their own channels. Therefore, through simultaneously imaging H2S and HClO, probe 3 (RPC-1) can evaluate drug-induced liver injury caused by antidepressants duloxetine and fluoxetine, and explore the molecular mechanism associated with H2S protection. In 2019, another multi-responsive probe 4 (Lyso-HA-HS) was developed utilizing diformylhydrazine as the HOCl reaction site, which can simultaneously detect H2S and HOCl in lysosomes [25]. This probe was the first to track endogenous H2S and HOCl in lysosomes in living cells.
Fluorescent probes based on 1,8-naphthalimide have been widely utilized in detecting H2S. However, the ineffectiveness of the organelle targeting ability has not been resolved. Wu et al. developed two fluorescence probes 5 (Mito-HS) and 6 (Lyso-HS) for sensing H2S in mitochondria and lysosomes by introducing triphenylphosphonium and dimethylamino moieties for organelle targeting [26]. These two probes showed a better response to H2S than structurally similar probes that are not charged. In addition, probe 5 (Mito-HS) was applied to image H2S in tumors in living mice. Furthermore, to explain the complexity of physiological H2S in subcellular organelles, Deng et al. developed two-photon fluorescent H2S probe 7 (MT-HS) with mitochondrial-targeting ability, which can be utilized for imaging H2S in deep penetrating living tissue [27]. Furthermore, they designed an ER-targeted fluorescent H2S probe 8 (NaH2S-ER) by using a sulfanilamide group as the target group [28]. Since none of probes can be used to monitor intercellular transmission of H2S, Fu et al. developed H2S probe 9 (ASNHN-N3), which showed good specificity on the cell surface with a long hydrophobic alkyl chain [29]. As expected, probe 9 (ASNHN-N3) visualized H2S in the cell membrane of living cells. Moreover, since H2S is closely related to tumor growth, it is of importance to monitor the H2S level in real-time for understanding its effects in tumor diagnosis and cancer cell proliferation. Therefore, Lin’s group developed a novel fluorescent probe 10 (BN-H2S) with a biotin group for detecting H2S, which has a special selectivity for cell cancer [30]. Because of the selectivity of the biotin group, probe 10 (BN-H2S) is capable of sensing H2S in cancer cells by two-photon imaging.
High quantum yield, as well as the small molecular and favorable permeability make coumarin a widely-applied fluorophore. Exploiting the protein labeling technologies, Chen et al. obtained a coumarin-based H2S probe 11 (CouN3BC), which connected a CLIP-tag substrate and had specifically targeting ability for the nucleus and mitochondria [31]. Utilizing coumarin as the fluorophore core, Zhu et al. modified the 4 and 6 positions of the coumarin ring and synthesized a series of multi-fluorinated fluorescent probes 12–14 to achieve a fast response for real-time H2S detection [32]. The data showed that increasing fluorine-substitution accelerated the H2S-mediated reduction reaction, and the tetra-fluorinated coumarin probe 14 showed a very fast response and outstanding selectivity to H2S both in vitro and in vivo. In addition, there are coumarin-based organelle targeting probes, of which Velusamy et al. developed chemodosimeter “off-on” fluorescent probe 15 (Mito-HS) [33]. Probe 15 (Mito-HS) quickly detected the formation of endogenous H2S in cancer cells with no external stimulations. Furthermore, specific fluorescence imaging in cancer cells showed that probe 15 (Mito-HS) can make a distinction between normal cells and cancer cells according to the level of H2S formation in vivo. Moreover, a commercially available fluorescent H2S probe was rendered to target lysosome (16) (Lyso-C) [34]. When it was employed in cell imaging studies, probe 16 (Lyso-C) can distinguish diverse levels of H2S in live cells and sensitively respond to exogenous H2S in lysosomes.
Other fluorophores were exploited to carry the azide group. In 2017, Liu et al. developed a biphoton fluorescent probe 17 (1-H2S), which was constructed through expanding the conjugation system of naphthalene and a coumarin analogue [35]. In biological experiments, probe 17 (1-H2S) was found to aggregate in the nucleolus region where H2S can be detected. By introducing azide at the 6-position of the chroman dye, Qiao et al. obtained a fluorescent probe 18 (AC-N3) [36]. Probe 18 (AC-N3) exhibited a better selectivity without interference from analytes, high sensitivity, and little cytotoxicity. In addition, the quinoline skeleton was employed to construct H2S probes. Based on the quinoline scaffold, Dai et al. prepared a two-input fluorescent probe 19 (QME-N3) [37]. This probe could generate intensive fluorescence sense RSH via the Michael addition and independently detected H2S through reduction of the azide group. In 2018, Ren et al. reasonable designed a novel series of electron donoracceptor type green fluorescent protein fluorophore scaffolds, which will improve the two-photon efficiency after forming a hydrogen-bond net in water [38]. For its excellent properties, Ren et al. developed a H2S selective probe 20 (TPC-N3) based on new scaffolds. Probe 20 (TPC-N3) had a good deep-tissue penetration for imaging H2S in the liver tissue. Accordingly, utilizing the click reaction, Dou et al. obtained fluorescent probe 21 (Lyso-HS), which introduced a tertiary amine as a lysosome-targeted moiety [39]. Probe 21 (Lyso-HS) showed enhanced fluorescence by 95-fold after sensing H2S and was applied to detect lysosomal H2S in living cells. In addition, probes 22 (QL-Gal-N3) and 23 (Gol-H2S) were successfully employed for respectively sensing endogenous H2S in hepatocyte and the Golgi apparatus by incorporating different organelle targeting groups [40,41]. Probe 22 (QL-Gal-N3) was developed through the introduction of a glycosyl moiety (as a hepatocyte targeting agent) to a quinoline fluorophore. In the presence of a glycosyl moiety, the water solubility and hepatocyte-targeting ability were obviously enhanced. Notably, probe 22 (QL-Gal-N3) was utilized to detect H2S in water samples and hepatocytes. Furthermore, the Golgi apparatus also played a significant role in eukaryotic organelles, which revealed a cytoprotective role of H2S in various physiological activities. Furthermore, Zhu and Sheng et al. developed a simple Golgi targeting fluorescent probe 23 (Gol-H2S) for accurate and sensitive detection of H2S. Considering the high cholesterol content of the Golgi membrane, a trifluoromethyl moiety was introduced into the quinoline structure to improve its fat solubility. Thus, probe 23 (Gol-H2S) could easily enter the Golgi apparatus through the barrier of the Golgi membrane because of its lipophilicity. Additionally, probe 23 (Gol-H2S) could monitor basal H2S and changes in the Golgi apparatus of cells and zebrafish. More importantly, real-time visualization of H2S production in the stress-induced Golgi apparatus was achieved by probe 23 (Gol-H2S). Chemical structures of azide-based probes are shown in Fig. 1.
Taken together, the azide group is an ideal H2S trigger, and these probes have good compatibility with biological systems. Moreover, they are not difficult to synthesize. Attention must be paid to the use of sodium azide because of its explosive nature. The potential flaw of such probes is its photoactivation, after continuous exposure to an optical microscope [42].
3.2 Probes based on reduction of nitro group for sensing
In addition to the azide group, other functional groups can be reduced by H2S. One is nitro, which can be reduced to an amino group by H2S. In 2017, a nitroolefin functionalized probe 24 (DPP-NO2) was generated by Wang’s group [43]. Taking advantage of its easy synthesis and modification, light resistance, and solvent resistance performances, DPP was chosen as the fluorophore, which has a red and high-performance pigment. Probe 24 (DPP-NO2) showed almost 800-fold enhanced fluorescence after the nitro reaction. Furthermore, probe 24 (DPP-NO2) was successfully used for imaging the fluorescence toward H2S in HeLa cells. In 2019, Zhou et al. developed a BODIPY-based probe 25 that introduced isoxazole to strengthen the oxidizability speed of the nitro group, and resulted in a significantly reduced response time within 55 s [44]. Due to its low toxicity and good cell membrane permeability, probe 25 can identify endogenous and exogenous H2S in living cells. Chemical structures of nitro-based probes are shown in Fig. 2.
3.3 Probes based on reduction of azo and hydroxamino group for sensing
In previous studies, probes utilizing the redox property of hydroxamino group and the azo group have been developed. In 2014, Li et al. reported probes 26–32 (azo1–azo7), which realized “off-on” fluorescence via reduction of the azo group for sensing H2S [45]. Probe 28 (azo3) carrying a pentafluorobenzyl group, had a 22-fold increasing selectivity towards H2S over other species and had a detection limit of 500 nmol·L−1. The data described above illustrated that utilization of a nitrogen atom redox state is an ideal and available method for designing fluorescent probes. In 2017, Chen et al. developed an excited-state intramolecular proton transfer (ESIPT)-based fluorescent probe 33 (PHS1) to sense H2S [46]. A hydroxylamine group was utilized as the H2S reporter, which was based on the fact that hydroxylamine can be reduced by H2S to form the corresponding amine group. Hydroxylamine as a protecting group of 3-amine nitrogen caused no emission of the fluorophore via preventing the ESIPT process until it was specifically reduced by H2S. Chemical structures of azo-based and hydroxamino-based probes are shown in Fig. 2.
4 Nucleophilicity-based fluorescent probes
In addition to the reductive property, H2S itself also possesses much stronger nucleophilicity than other thiols, which is due to its smaller size and lower pKa [47]. Such properties provide the possibility and availability to design fluorescent probes via a specific reaction between probes and H2S. Based on these properties, a lot type of specific reactions have been exploited for the development of probes, such as the cleavage of DNP group after nucleophilic reaction, H2S-induced S–S cleavage followed by intramolecular nucleophilic reaction, bringing great diversity and practicality to the family of fluorescent probes for H2S detection.
4.1 Probes based on substitution of chloride atom for sensing
Chloride is a latent site, which can be nucleophilically substituted by H2S, leading to changes in fluorescence intensity. Based on this, Montoya et al. developed an nitrobenzofurazan (NBD)-based colorimetric probe 34 [48]. However, the selectivity against biothiols, such as GSH and Cys was not as expected. In 2014, Han’s group reported an “on-off” cyanine-based a NIR probe 35 that could be utilized to selectively detect H2S in serum with a low detection limit in living cells [49]. Unfortunately, they did not screen for the selectivity of the probe. Inspired by Han’s work, Li et al. reported on probes 36 (CyCl-1) and 37 (CyCl-2), which could perform photoacoustic imaging of H2S in living mice [50]. The chemical structures of probes 34–37 are presented in Fig. 2.
4.2 Probes based on breakage of conjugated systems for sensing
As universally recognized, H2S, a good nucleophile, can attack the electrophilic center of fluorescent molecules and break the conjugate system. Probe 38 (BH-HS) was developed based on this mechanism, performing great fluorescent imaging ability towards both intracellular and exogenous H2S [51]. In brief, BODIPY was selected as the fluorescence reporting group, with dimethyl amine as the electron donor and hemicyanine as recognition site for H2S. Thereafter, Feng et al. developed a ratiometric Förster resonance energy transfer (FRET)-based probe 39 (CPC) that integrated coumarin with hemicyanine [52]. Due to the nature of hemicyanine, probes 38 (BH-HS) and 39 (CPC) both demonstrated preferential distribution in mitochondria with excellent reaction kinetics with H2S, which enabled reaching a maximum fluorescence within several minutes. Taking hemicyanine as a H2S trigger, Liu et al. established an internal charge transfer (ICT)-based probe 40 (TP-PMVC) for tracking the H2S inside the lysosomes [53]. Two active sites (pyridine and hemicine) were included in the structure to react with H+ and H2S, respectively. The pyridine part with its appreciable pKa (≈5.0) was selected as the H+ site and lysosomal targeting unit. These innovations allowed for simultaneous screening of lysosomes and lysosomal H2S with double-color imaging. Moreover, based on the FRET mechanism, they introduced a H2S probe 41 (CP-H2S) with favorable colorimetric and ratiometric fluorescence, which selected the pyronine dye and coumarin chromophore as the energy acceptor and the energy donor, respectively [54]. In an aqueous solution, through the FRET process, probe 41 (CP-H2S) showed intrinsic red emission of the pyronine unit, while the presence of H2S inhibited the FRET process and resulted in blue emission from the coumarin part, while the red emission was reduced. Therefore, probe 41 (CP-H2S) was promising in living cell imaging.
In 2017, based on ICT, a concise and efficient fluorescent probe 42 (Mi) was synthesized [55]. Probe 42 (Mi) can be used to detect H2S with the naked-eye. Li et al. successfully applied probe 42 (Mi) to determine H2S on agar gels with satisfactory results. These results indicated that probe 42 (Mi) performed a promising application of H2S sensing in environmental samples. Compared with probe 42 (Mi), Ma et al. focused on improving reaction sensitivity. In 2018, they reported a ratiometric fluorescent probe 43 (CyT) of which its hemicyanine part selectively reacted with H2S in the mitochondria of living cells [56]. Moreover, this probe showed low toxicity to HeLa cells and had a good imaging effect in living cells and zebrafish. Except for traditional fluorophores with aggregation-caused quenching property, AIE luminogens (AIEgen) also received extensive attention. Its outstanding optical properties facilitated its application on biological imaging and sensing. Taking these factors into account, Ma et al. reported an AIEgen probe that was positively charged [57]. Probe 44 (Indo-TPE-Indo) with two indolium groups provided more opportunities to target mitochondria and had better responses to H2S in cells. Thus, as well as being applied for the detection of H2S in vivo, such as H2S in cancer cells and tumors, probe 44 (Indo-TPE-Indo) could function to visualize the H2S diversity in mitochondria of living cells. As mentioned above, the generation of H2S is related to enzyme CSE and CBS. In previous studies, it was confirmed that the CBS gene in humans is located on chromosome 21. Thus, enzyme CSE could produce H2S, and CBS is in close connection with RNA. To better understand the function of RNA, Liu et al. developed probe 45 (TP-MIVC), which was considered the first paradigm of probes capable for simultaneously reporting RNA and H2S with clear fluorescence signals [58]. Two different sites (carbazole and indolenium) respectively reacting with RNA and H2S of this probe created versatile imaging properties. In cancer cells, zebrafish, and living animals, probe 45 (TP-MIVC) performed clear fluorescence imaging of RNA and H2S. It is worth noting that by observing fluorescence intensity, it was found that probe 45 (TP-MIVC) distinguished tumor mice from normal mice. Chemical structures of probes 38–45 are shown in Fig. 3.
This type of probe was often used for developing the ratiometric H2S probes, which change the fluorescence emission by destroying the conjugate system. However, this strategy may be affected by other nucleophilic biothiols.
4.3 Probes based on removal of DNP for sensing
DNP is an extremely strong electron-withdrawing group and can lead to fluorescence quenching after being attached to fluorophores due to its strong electron absorption performance. The C–O bond between DNP and fluorophores is easy to be cleaved after nucleophilically attacking by H2S, with fluorescence turned on afterwards. The characteristics mentioned above make DNP an ideal group for engineering reaction-based probes for H2S. Obviously, incorporation of the DNP functional group onto different fluorophore scaffolds could yield in fruitful innovations of probes for H2S. For example, by uniting coumarin with benzothiazole, Cui et al. synthesized fluorescent probe 46 (CTN) for H2S based on thiolysis of the dinitrophenyl ether moiety [59]. By taking hemicyanine dye as the NIR skeleton, Zhang et al. developed NIR probe 47 (NIR-HS), which allowed for imaging and tracking H2S in vivo [60]. In 2017, Tang’s group successfully developed the first self-assembled fluorescent nanoprobe 48 (TPE-3) with both AIE and ESIPT characteristics to detect H2S through a modified nanoprecipitation [61]. Moreover, nanoprobe 48 (TPE-3) could be used for H2S imaging in live cells and in vivo due to its excellent water dispersibility and good biocompatibility. It is well known that two-photon-excited bioimaging has been widely utilized because of its deeper tissue penetration. Herein, Zhou et al. designed an efficient two-photon mitochondria-targeting dye 49 (TP-NIR-HS) with a H2S recognition moiety [62]. As expected, probe 49 (TP-NIR-HS) was essentially non-fluorescent, which might be ascribed to the ICT effect by the strong electron-withdrawing DNB group. Release of the fluorophore was the result of the addition of H2S, which caused the cracked portion of DNB to be left behind and turned on the fluorescent signals. Subsequently, probe 49 (TP-NIR-HS) was applied to imaging living cells and tissues, resulting in high imaging resolution and a deep-tissue imaging. The effect of pH on the sensitivity of probe 49 (TP-NIR-HS) is unknown.
In 2018, Gu et al. synthetized fluorescent probe 50 (2-CHO-OH) with DNP as the H2S reporter and an adjacent aldehyde group to improve sensing performances [63]. The strategy was also employed by Qian et al., who designed a NIR fluorescent probe 51 (NDCM-2) [64]. The mechanism was as follows. H2S nucleophilically added to the aldehyde group, resulting in a hemiactal, which promoted the intramolecular thiolysis process of the 2,4-dinitrophenyl ether. Based on the photoinduced electron transfer (PET) theory, Chen et al. developed a fluorescence probe 52 (NIPY-DNP) for H2S [65]. Later, Ji et al. reported a long wavelength fluorescent probe 53 (TMSDNPOB) based on the BODIPY structure to detect H2S [66]. The fluorescence signal of the probe was significantly enhanced after the sensing reaction. Starting with probe 49 and with the intention to overcome the shortcomings of a complex synthesis process and short emission wavelength, Li et al. obtained a long-wavelength probe 54 (LC-H2S) after only two synthesis steps [67]. Unfortunately, compared to probe 49, probe 54 (LC-H2S) was not a two-photon fluorescent probe. Chromone derivatives have also been used as fluorophores. Liu et al. designed a turn-on fluorescent 55 (A) for H2S detection, which was based on an ESIPT process [68]. Bearing the classical morpholine as a lysosome targetable marker, Wu et al. obtained a novel lysosome-targeting probe 56 (DMC) [69]. Moreover, coumarin had been adopted to load the DNP. Yang et al. reported a molecular probe 57 (Cda-DNP), which could access all compartments in the cell that detect H2S in cells and living animals [70]. Probe 57 (Cda-DNP) consisted of three functional domains: a H2S sensing domain, a fluorescence domain, and a biomembrane penetration domain. Moreover, the lateral chain N,N-dimethylethylenediamine played a significant role in enabling probe 57 (Cda-DNP) to enter cells and penetrate into different organelles.
Sun et al. developed a new molecular probe 58 (NR-NO2) taking benzothiazole-xanthene dyad as the fluorophore unit [71]. This probe not only tracked and analyzed H2S in mitochondria, but could also observe a mouse liver injury model caused by overdose of metformin via detecting hepatic H2S. The tracking and analysis of H2S in mitochondria was of great significance. Thus, Zhao et al. developed a mitochondria-targeting fluorescent probe 59 (Mito-NIR-SH) by introducing DNP into a Changsha NIR fluorophore, which was used to detect intracellular H2S [72]. Together, the experimental results demonstrated that 59 (Mito-NIR-SH) could selectively target mitochondria and image exogenously and endogenously H2S in the cellular environment. It is often effective to improve probe properties by modifying fluorophores. Using this rationale, Zhu et al. synthesized two different probes, by introducing methoxy groups on the BODIPY 3,5-positions, and designed and synthesized probes with large Stokes shift for detecting H2S [73]. Probe 60 (DMOEPB) had a dinitrophenyl ether as the reactive moiety and probe 61 (DMONPB) had a nitro group as a reactive group for H2S. In addition, based on BODIPY, Fang et al. obtained a naked-eye and “on-off” fluorescent probe 62 for detecting H2S [74]. The colorimetric sensing ability of this probe facilitated naked eye detection, thereby overcoming some drawbacks, such as probe concentration, sample environment and light scattering. Utilizing NIR dye cyanine as the fluorophore, Su et al. and Lin et al. developed probes 63 and 64 (QCy7-HS), respectively [75,76]. Due to blockage of the twisting of the N,N-diethylamino group at the fluorophore, Zhang et al. synthesized H2S probe 65 (Z1) with the functions of naked-eye colorimetry and efficient ER localization [77]. Moreover, Zhong et al. used 4-diethylaminosalicylaldehyde and 1,4-dimethylpyridinium iodide as synthetic raw materials and synthesized probe 66 (L) by a two-step reaction via eliminating d-PET and recovering ICT processes to identify H2S [78]. Chemical structures of DNP-based probes 46–66 are shown in Fig. 4.
4.4 Probes based on removal of NBD group for sensing
Similar to the hydrolysis of DNP ether by H2S, a NBD group has also been widely used as a H2S-probe trigger for its fluorescence-quenching nature and easily-leaving property after nucleophilic reaction with H2S. Based on this, in 2016, Ding et al. reported a remarkably simple probe 67, which had fast fluorescence responses for all mercaptans [79]. Nevertheless, probe 67, like many other NBD ether-based probes, could not selectively detect H2S in the presence of other biological thiols. To solve this problem, their research team reported that the introduction of an aldehyde group on probe 67 resulted in highly selective H2S probes 68 and 69, both of which detected H2S in the presence of other biological thiols [80]. By attaching NBD to fluorescein and rhodamine dyes through a piperazine linker, Wang et al. obtained probe 70 and a NIR probe 71 [81]. Probe 71 showed a higher reaction rate toward H2S, which might be attributable to the positively-charged nitrogen in rhodamine and mitochondrial targeting. Enlightened by Grimm et al. [82], Ismail et al. rationally designed and synthesized a novel azetidinyl-rhodamine-NBD dyad 72 that quickly detected H2S in the range of infrared [83]. Compared to 71, obstructing the twisting amino side chain dramatically enhanced the performance of probe 72. Furthermore, Wei et al. developed the first H2S-specific fluorescence probe 73 based on the cleavage of NBD [84]. Later, their group designed a julolidine-fused coumarin-NBD probe 74 that allowed for the detection of H2S with improved performance [85]. Probe 74 showed excellent sensing performances with green-light emitting and was successfully used for biological imaging in cells and in zebrafish. In addition, Huang et al. developed an NBD-based fluorescent probe 75 based on a click reaction of alkyne-containing NBD derivative and azidocoumarin [86]. By choosing classical coumarin dye as the fluorophore, Zhang et al. prepared a fluorescent probe 76 (ER-CN) for sensing H2S by bearing a methyl sulfonamide group as an ER targetable marker [87].
Triphenylphosphonium can be used as the anchoring part for mitochondria, allowing it to enter mitochondria for selective monitoring and imaging. Therefore, Pak et al. engineered a mitochondria-target probe 77 with triphenylphosphonium as a mitochondria-oriented marker [88]. Utilizing 3-hydroxyflavone as fluorophore instead, Hou et al. developed a colorimetric and fluorescent dual probe 78 for H2S due to the color and fluorescence induced by the interaction of probe with H2S [89]. In 2017, two BODIPY-NBD based fluorescent probes namely 79 (BDP-N1) and 80 (BDP-N2) were prepared by integrating the styryl-BODIPY fluorophore with the NBD moiety using a one-pot reaction [90]. They were both fluorescence-off due to the quenching effect from the NBD group. Thiolysis was induced after introducing HS–, which made it exhibit off-on fluorescent signal. Zhang et al. obtained a long wavelength NIR fluorescent probe 81 based on BODIPY and carrying an NBD moiety, which linked with the benzyl pyridinium moiety through ether linkage at the meso position [91]. It simultaneously displayed distinct responses to H2S/GSH and Cys/Hcy from visible/NIR dual emission channels. Inspired by the pioneer’s work, through integration of an NBD amine reaction group into rhodamine fluorophore, Wang et al. presented an “off-on” fluorescent probe 82 for H2S [92]. In 2018, taking cyanine dye as NIR skeleton, Xiong et al. developed a NIR fluorescent probe 83 (NIR-H2S) based on a thiolysis reaction for H2S detection, which successfully monitored H2S in cells and mice [93]. So far, a large number of fluorescent probes for sensing H2S have been developed. However, all H2S probes based on NBD exhibited single photon excitation responses. Because of this kind of probe short excitation light, it sometimes had problems for biological imaging, especially for tissue imaging. On the contrary, two-photon probes can solve the above-mentioned problem. Tang and Jiang reported a two-photon fluorescent probe 84 (L) utilizing the FRET strategy [94]. Probe 84 (L) was employed to image exogenous and endogenous H2S in living cells. Chemical structures of NBD-based probes 67–84 are shown in Fig. 5.
4.5 Probes based on nitro or azide reduction-triggered self-immolation
Reduction of the azide or nitro group to amino followed by a self-immolative reaction to liberate free fluorophores, has been utilized to design reaction-based H2S fluorescent probes. Inspired by hypoxia pro-drug moiety the p-nitrobenzyl group, Qian’group developed ratiometric probe 85 (RHP) for hypoxia in 2011 [95]. Inspired by Qian’ work, Zhang et al. developed probe 86 (RHP-2) in 2014 [96]. Simultaneously, the tandem reaction mechanism was successfully confirmed by Cui et al. Based on this mechanism, Zhang et al. developed probe 87, which could be reduced by H2S, and proceeded intramolecular cyclization after removal of the p-aminobenzyl group to construct a renascent fluorophore [97]. Most reported probes of this type only responded to H2S with fluorescence intensity (based on the “turn on” or “turn off” mode) which was significantly affected by complex factors, such as the detection environment and probe location. Wang et al. developed a naphthalimide-based colorimetric and ratiometric fluorescent probe 88 (NS1) [98]. The azide portion of the probe was specifically reduced by H2S, and underwent a spontaneous 1,6-elimination reaction to form naphthimide compounds, which in turn exhibited strong fluorescence. This process resulted in a larger shift of the emission spectrum to achieve the colorimetric and ratiometric fluorescence response to H2S. Later, a novel strategy was developed by Steiger et al. for H2S based on the self-immolation of benzyl thiocarbamates to release carbonyl sulfide, which was quickly converted into H2S by carbonic anhydrase. Importantly, this strategy provided solutions to key challenges associated with both H2S delivery and detection. Finally, they designed and synthesized fluorescent probe 89 (MeRho-TCA) and confirmed triggering the release of H2S [99].
In 2017, by introducing the classical morpholine as a lysosome targetable marker, Feng et al. prepared a ratiometric double-photon fluorescent probe 90 (LR-H2S) for imaging lysosomal H2S [100]. Furthermore, Thirumalaivasan et al. obtained probe 91 (PyN3) based on pyrene [101]. By reducing the azide to an amine and self-immolative cleavage of the p-aminobenzyl group in the molecule, the fluorophore was released. What’s more, Park et al. obtained a new type of NIR probe 92 (NIR-Az) for H2S determination [102]. Probe 92 (NIR-Az) had a high selectivity for H2S among the 16 analytes tested including common reducing agents. Utilizing the excellent biocompatibility and rapid cell internalization of probe 92 (NIR-Az), Park et al. successfully proved its useful ability to monitor the concentration and time-dependent changes of H2S in living cells and animal aspect.
In 2018, a dual-response fluorescent probe 93 (Mito-VS) was designed and synthesized by Li’s group to monitor the level of viscosity and H2S, respectively [103]. Probe 93 (Mito-VS) was non-fluorescent due to a free intramolecular rotation between dimethylaniline and pyridine. After an increase in viscosity, rotation was prohibited and an intense red fluorescence was released. Upon the addition of H2S, probes reacted with H2S and a strong green fluorescence was observed. Utilizing the same principle, Yin’s group synthesized another BODIPY fluorescent probe 94 (BDP-N3), which had viscosity sensitivity and detected H2S with high selectivity [104]. Those probes allowed for the detection of both H2S and viscosity in a biological system. In 2019, due to the PET between fluorophore and azido moiety by a carbonate linker, Zhou et al. presented an “off-on” mitochondria-targeted NIR probe 95 (Mito-N3) [105]. Simultaneously, Yang et al. developed an interesting red-emitting fluorescent probe 96 [106]. Upon the addition of H2S, the reduction of the azido group generated an amino derivative, which rapidly released an imine intermediate and subsequently went through an intramolecular cyclization to release fluorescence. In addition, by introducing a self-immolative group to achieve a lower detection limit, Zhu et al. obtained a rhodamine-based probe 97 (MF-N3) that selectively accumulated in lysosomes and presented turn-on fluorescence when H2S and protons were present at the same time [107]. Chemical structures of probes 85–97 are shown in Fig. 6.
4.6 Probes based on electrophilic cleavage-triggered intramolecular tandem reactions
Due to the dual-nucleophilicity of H2S, a new tandem reaction between disulfide and H2S was discovered. This tandem reaction started with the nucleophilic substitution between disulfide and H2S. Successively, intramolecular cyclization between the freshly produced thiol and ester occurred, with simultaneous fluorophore release. Inspired by this tandem reaction, Li et al. selected 3-hydroxyflavone as the fluorophore and 2-(pyridine-2-yl-disulfanyl)benzoic acid as the H2S reporter, yielding fluorescent probe 98 (HF-PBA). Probe 98 (HF-PBA) became a potential multifunctional fluorescent probe, because it could also distinguish H2S and biothiol through different fluorescence bands [108]. In 2017, a water-soluble fluorescent probe 99 (HS-1) was prepared by integrating the 4-hydroxycoumarin fluorophore with a disulfide moiety by Yin’s group [109]. The reaction between probe 99 (HS-1) and H2S triggered the cleavage of the disulfide bond and subsequent intramolecular cyclization, thereby releasing 4-hydroxycoumarin, resulting in a ratio fluorescence response. Other relevant thiols induced no observable fluorescent response. In 2018, utilizing excellent properties of dicyanomethylene-4H-pyran as fluorophore, Men et al. rationally prepared a specific fluorescent probe 100 (DCM-PBA) by integrating PBA fragment to dicyanomethylene-4H-pyran via ester bridge [110]. Moreover, based on a semiheptamethine derivative, a classic NIR dye scaffold, Zhang et al. developed a fluorescent probe 101 (Cy-PBA) with NIR fluorescence emission [111]. In 2019, using the same strategy, a ratiometric-visualized fluorescent probe 102 was reported [112].
Similar to the above-mentioned mechanisms, Wang et al. introduced a colorimetric fluorescence probe 103 based on cyanine, allowing for the detection of H2S sensitively and selectively [113]. Nucleophilic attack of the disulfide bond by H2S led to cleavage of the disulfide bond and an intramolecular cyclization to promote release of the fluorophore, as detected through the enhancement of the fluorescence signal and color change of the reaction mixture. In addition, utilizing the same strategy, Wang et al. designed and synthesized fluorescence probe 104 (DCN-S) for H2S detection [114]. The disulfide bond of probe 104 (DCN-S) was broken under the nucleophilic substitution of H2S followed by the generation of dicyanoisophorone derivative, which emitted an orange fluorescence. Furthermore, the diselenide bond is about 5 orders of magnitude faster to be cleaved by H2S than the disulfide bond. Obviously, the diselenide group represents an ideal candidate for designing probes that respond quickly to H2S. Based on this rationale, Guan et al. reported a double-switch mechanism for fluorescence probe 105 (HBTSeSe) of sensing H2S employing a diselenide bond [115]. Chemical structures of probes 98–105 are shown in Fig. 7.
4.7 Employing tandem reaction with proximal aldehyde and α,β-unsaturated carbonyl group for sensing H2S
Utilization of a proximal aldehyde group and α,β-unsaturated carbonyl group as a trapping moiety, another novel tandem reaction was developed. It was due to the dual-nucleophilicity of H2S that can lead to a sequential Michael-addition reaction with an aldehyde group and an α,β-unsaturated carbonyl successively. Qian et al. first designed two probes 106 (SFP-1) and 107 (SFP-2), which showed 50 to 100-fold selectivity against other biothiols, including GSH, Cys and the like [116]. In 2013, Li et al. developed an ICT-based probe 108 (ZS1) [117]. This probe possessed an incredibly over 5000-fold selectivity against biothiols. Moreover, inspired by this mechanism, Singha et al. developed three probes 109 (P1), 110 (P2), and 111 (P3) [118]. To enrich the electronic density of benzene by adding methoxy groups, high selectivity towards H2S over Cys was achieved. At higher pH values, a new HO–-condensation between an enolate and an aldehyde has been revealed. Later, to develop a H2S probe emitting in the longer wavelength region, preferably in the red or above (>625 nm), Ryu et al. exploited an “acetyl-benzocoumarin” as the dye platform and developed probe 112 (P5) [119]. The reactivity of 112 (P5) toward H2S was even faster than the previously-reported H2S probe 111 (P3) that showed signal saturation after 8 min. Chemical structures of probes 106–112 are shown in Fig. 8.
4.8 Other types of fluorescent probes for H2S
Other fluorescent probes not limited to the above-mentioned mechanisms for detecting H2S have also been reported. Chen et al. developed three rhodamine-propargylic ester-based probes 113 (RB-PE-1), 114 (RB-PE-2), and 115 (RB-PE-3) [120]. Tandem reactions between H2S and propargylic esters of rhodamine B(RB-Fes) led to spirocyclization of probes, rendering fluorescence off. Furthermore, an epoxide-based fluorescent probe 116 (FEPO-1) was developed by Chen’s group [121]. After being attacked by H2S at the C–O bond of epoxide, the epoxide ring opens and leads to alteration of the conjugation system of the probe. Karakus et al. was the first to use electrophilic cyanate as the H2S recognition group, and developed fluorescent probe 117 (FLVN-OCN) for H2S by modifying the fluorescent dye based on ESIPT [122]. In the presence of reactive sulfur species, they expected that the oxygen-nitrile bond of the pre-fluorescent dye 117 (FLVN-OCN) would result in selective splitting and then release the free hydroxyl derivative of the fluorophore. As we all know, the second NIR window (NIR-II) probes had a greatly improved in spatial resolution and tissue penetration depth. Xu et al. rationally designed a dye 118 (ZX-NIR), which can generate the NIR-II emission after reacting with H2S [123]. With the nanocomposites packaged, the designed nanoprobes had an excellent biocompatibility and good water solubility. This nanoprobe had a specific responsiveness to H2S, so it can identify and image H2S-rich colon cancer cells.
The well-known tetrazine structure was also used in fluorescence probes for detecting H2S. Zhao et al. reported a novel reactive fluorescent probe for the selective detection of H2S that adopted the Tz group and worked by the reduction of tetrazine to dihydio-tetrazine by H2S. They next designed and synthesized three fluorescence probes with a tetrazine group, 119 (Coumarin-Tz), 120 (BODIPY-Tz-I), and 121 (BODIPY-Tz-II) [124]. In addition, a H2S-induced deprotonation method would be another ideal strategy to design a probe because it has the advantage of non-interference with other thiols and a fast response time in physiological conditions. Utilizing this mechanism, Kaushik et al. reported a ratiometric colorimetric sensor 122 for the recognition of H2S [125]. The reason for the change in ratio between spectrum and color was that H2S induced the deprotonation of one of the –OH protons followed by changes of the resonance of probe 122. As is well known, many fluorescent probes are well-designed and can quickly and specifically respond to H2S through addition reactions to break the conjugated π-system of C=C bonds. Based on this, Wang et al. reported a novel strategy using H2S-mediated reduction of the C=C bond, which can effectively detect H2S with turn-on dual-color fluorescence [126]. Here, they designed and synthesized probes 123 (PTZ-P1), 124 (PTZ-P2), 125 (PTZ-P3), and 126 (PTZ-P4), in which phenothiazine ethylidene malononitrile derivatives reacted with H2S to form thiophene rings based on intramolecular cyclization reactions through reductive cleavage of C=C bonds. Of these probes, 126 (PTZ-P4) exhibited dual-color fluorescence after reductive cleavage.
Based on various types of reactions between H2S and other chemical species, a large number of reaction-based probes were obtained. Due to the feasibility and diversity, reaction-based strategy has become the most widely applied strategy. However, emphasis is still needed regarding the discovery and elucidation of novel reaction mechanisms, which are of better reaction kinetics and have a higher specificity. In addition, further modifications and improvements on available fluorescent probes should be promoted, in order to gain probes that are more compatible with the living system. Chemical structures of probes 113–126 are shown in Fig. 9.
5 Metal sulfide precipitation-based fluorescent probes for labeling H2S
The reaction between Cu(II) and S2–, generating CuS precipitate with Ksp about 10–45 (25 °C in water), has obtained broad utilization upon the design of fluorescent probes for detecting H2S. Underlying this principle, Choi et al. reported the first CuS-based probe 127 in 2009, utilizing dipicolylamine (DPA) as the Cu(II) bonding moiety [127]. Similarly, with DPA as a Cu(II) bonding moiety, Hou et al. developed probe 128 [128]. The Cu(II) complex of the probe can be used to detect sulfide anions. In 2015, Yue et al. reported dinuclear Ru(II)-Cu(II) complex-based fluorescent probe 129, using DPA as the Cu(II) bonding moiety, which could detect H2S in the rat brain [129]. In 2016, Lv et al. reported a fluorescent sensor 130 (CuHCD) that used hemicyanine-carbazole as the fluorophore and bipyridine-triazole-Cu2+ complex as the receptor, which selectively recognized HNO and H2S respectively through the non-covalent modulation of surfactant assemblies [130].
Taking advantage of the stability of Cu(II) complexes formed with azamacrocyclic rings, Sasakura et al. developed four azamacrocyclic probes 131 (TACN), 132 (Cyclam), 133 (Hsip-1), and 134 (TMCyclen) [131]. Among those four probes, 133 (Hsip-1) revealed the best photophysical properties and detected sulfides in living cells. Wu et al. took 1,4,7,10-tetraazacyclododecane (cyclen) as the optimal fragment of 133 (Hsip-1) to develop two BODIPY-based NIR probes 135 and 136 in 2014, and realized H2S imaging in living rats [132]. Also, in 2014, cyclen had been employed by Yuan’s group to develop a dinuclear Ru(II)-Cu(II) complex-based fluorescent probe 137 [133]. This dinuclear complex possessed a large Stokes shift (159 nm) and had a sensitive response to H2S (detection limit of 21.6 nmol·L−1). In 2015, Palanisamy et al. reported a mono anthracene functionalized cyclen fluorescent sensor MaT-cyclen forming complex 138 [Cu(MaT-cyclen)2] with Cu(II) ions, which caused fluorescence quenching [134]. Complex 138 [Cu(MaT-cyclen)2] acted as a fluorescent turn-on for H2S by utilizing the displacement method.
Other single-ligand Cu(II) complex-based fluorescent probes were also developed. With 8-aminoquinoline as a Cu(II) bonding moiety via the piperazine ring linked to fluorophore, Cao et al. developed a cyanine-based NIR probe 139 [135]. Later, in 2012, Hou et al. reported a fluorescein-based probe 140 (L1Cu), utilizing 2-(8-hydroquinoline) acetohydrazide as the Cu(II) bonding moiety [136]. Shortly after the design of 140 (L1Cu), Hou et al. reported another fluorescein-based probe 141 (L1), utilizing 2-benzyl-acetohydrazide as the Cu(II) bonding moiety [137]. When 141 (L1) is compared with 140 (L1Cu), the former showed better fluorescent properties both in sensitivity and selectivity. Kar et al. reported FRET-based probes 142 (L-1) and 143 (L-2), utilizing indole as the energy donor and a xanthene-Cu(II) complex as the acceptor [138]. This probe could be used to detect both Cu(II) and sulfides, depending on the off-on response of the xanthene fluorophore corresponding to the occurrence of FRET by binding with Cu(II). In 2014, Tang et al. developed a benzimidazole-based probe 144 (L), carrying N-(2-hydroxyethyl)-piperazinegrcup as the Cu(II) bonding moiety [139]. Moreover, in 2015, Qian et al. obtained an N,N-dimethyl naphthalene-based probe 145 (NJ1), bearing a 2-hydrazinylpyridine group as the Cu(II) bonding moiety [140]. Meng et al. furthered an NBD-based probe 146 (NL), utilizing salicyloylhydrazone as the Cu(II) binding moiety [141]. In 2015, Hai et al. reported a multifunctional pyridine-biquinoline-derivative probe 147, which was used to detect pyrophosphate and H2S in aqueous buffer and cells [142]. By selectively coordinating with metal ions (Cu2+ and Zn2+ mentioned in article), fluorescence quenching occurred via PET. This metal complex reacted with both pyrophosphate and H2S, resulting in the recovery of fluorescence. Through combining the triarylboron-fluorophore with cyclen and diphenylamine, Yang and coworkers developed three probes 148 (TAB-1), 149 (TAB-2), and 150 (TAB-3) [143]. Among these three probes, 148 (TAB-1) and 149 (TAB-2), bearing three and two cyclens, respectively, embodied a more suitable water solubility and cell permeability, and 149 (TAB-2) specifically had a mitochondria-target character. In 2016, a carbazole-based fluorescence probe was developed that presented an “on-off-on” type fluorescence response mode for sequential detection of Cu2+ and S2–. High selectivity and sensitivity of probe responses to Cu2+ were barely affected by the coexistence of other interfering analytes. The subsequent addition of S2– could effectively remove Cu2+ from the complex 151 (CAH-Cu2+) to immediately recover the quenched fluorescence by releasing the free probe [144]. In 2017, Wang et al. reported a peptide ligand L (FITC-Ahx-Ser-Pro-Gly-His-NH2) using fluorescein isothiocyanate as the fluorophore, Pro-Gly as the spacer, and histidine and serine as ionophores, which was designed and synthesized by solid phase peptide synthesis to chelate with Cu2+ to obtain the fluorescence chemosensor 152 (L-Cu) for H2S detection [145]. In 2019, based on pyrene and benzothiozole hydrazide, Rajasekaran et al. reported a “on–off–on” fluorescence chemosensor. Despite the interference of other ions, the chemosensor had a high affinity for Cu2+ ions, and the 153 (DPD-Cu2+) chelate still had a high sensitivity to S2– ions through the displacement method [146].
Moreover, two-ligand and multi-ligand Cu(II) complex-based probes were developed. In 2013, Qu et al. developed a dipyrromethene-analogous NIR probe 154 (Cu-1) [147]. Later, in 2015, a BINOL-Benzimidazole-based probe 155 (Cu(BB)2) was developed by Wang’s group [148]. By applying the response of Cu(BB)2 to H2S, gaseous H2S in air can be detected using a simple test strip. Another two-ligand Cu(II) complex-based probe 156 (AIE-S) was developed by our group [149]. The AIE-S-Cu complex ameliorates the poor water solubility of AIE-S, and the reaction product of H2S and AIE-S will be fluorescent after rapid aggregation within seconds.
Other than taking Cu(II) complexation as a H2S capturer, Kawagoe et al. obtained a Cd(II)-based probe 157 (6-CdII), after optimizing the coordination fragmentations and fluorophores [150]. Probe 157 (6-CdII) showed high stability against highly oxidative species (H2O2, HClO, ONOO–, etc.) and had the best resistance towards GSH. Next, a mercury(II)-based probe 158 was developed by Wang’s group [151]. By means of fluorescent indicating paper, probe 158 was capable of imaging H2S gas even when coexisted with other gases, such as CO, NH3, NO, NO2, SO2, etc. Chemical structures of probes 127–158 are shown in Figs. 10 and 11.
Compared with a variety of other probes for detecting biological H2S, a key feature of metal sulfide-based probes is their prompt responses to H2S with complete fluorescence within seconds. However, attributing to the existences of endogenous complex ions (including Zn2+ and Mg2+), the proposed functions of designed probes may be abolished and the selectivity as well as the sensitivity would suffer from significant interference. In addition, the metabolism of the precipitate metal sulfides in human biology should be taken into consideration.
6 Fluorescent probes for H2Sn
In recent studies, it was revealed that H2Sn may play a practical mediator role in certain diseases related to H2S. Therefore, the importance of H2Sn in physiology and pathology is no less than that of H2S. Although significant progress has been made in H2Sn research in recent years, the distribution and regulatory mechanism of H2Sn in the organism still need to be elucidated. Therefore, using fluorescence spectroscopy to analyze and explore the role of H2Sn in a living system is a very effective method. At present, there are three types of probes for H2Sn according to recognition units: 1) 2-fluoro-5-nitrobenzoic ester; 2) phenyl 2-(benzoylthio)benzoate; 3) H2Sn mediated aziridine ring opening. In 2017, Gupta et al. had summarized the fluorescent probes for H2Sn [152].
In 2014, Liu et al. developed the first H2Sn-specific probes, employing a 2-fluoro-5-nitrobenzoic ester as H2Sn recognition units [153]. The probes undergo nucleophilic aromatic substitution with H2S2 to form the intermediate persulfide, which promotes intramolecular cyclization to release the fluorophore. Although, 2-fluoro-5-nitrobenzoic esters can be consumed by biothiols, cyclization does not occur and thus affects the fluorescent signal of the probe. Subsequently, a large number of H2Sn probes were developed, in which 2-fluoro-5-nitrobenzoic ester as H2Sn recognition units was connected to different fluorophores skeleton. Reporters for H2Sn based on semiheptamethine (159 (Cy-Sn)) [154], dicyanomethylene-benzopyran (160 (KB1)) [155], dicyanoisophorone (161 (RPHS1)) [156], phenothiazine (162–163 (PZC-Sn)) [157,158], resorufin (164 (Re-SS)) [159], BODIPY (165 (BDP-PHS)) [160], julolidine-coumarinocoumarins (166 (JCCF)) [161], naphthalene (167) [162] and so on have been described. Moreover, all those probes can be successfully applied in imaging of H2Sn in living cells. In 2020, Zhao et al. reported a fluorescent probe 168 (MC-Sn) [163], which have two specific reaction sites for sulfide and can distinguish H2S and H2Sn according different fluorescence signals after reaction. It is helpful for us to study the interaction of H2S and H2Sn in biological systems. Besides, due to the superior performance of two-photon probe 168 (MC-Sn), it was applied to detect biothiols in liver tissues. In order to explore endogenous lysosome-targetable H2Sn, Ren et al. developed a simple fluorescent probe 169 (NIPY-NF) [164]. In addition, utilizing morpholine group as the lysosomal targeting group, Han et al. designed probe 170 (Lyso-NRT-HP) for imaging of H2Sn [165]. The results showed that probe 169 (NIPY-NF) and probe 170 (Lyso-NRT-HP) was effective for detecting endogenous H2Sn, which produced in lysosome after lipopolysaccharides stimulates the cells. These probes are mainly used for imaging H2Sn. None of probes can research H2Sn formation via thionitrous acid (HSNO)-mediated. Zhang et al. developed an NIR fluorescent probe 171 (BCy-FN) for detection H2Sn, which had used for observed the generation of H2Sn in biological pathways for the first time [166]. The study reveals that this process was mediated by HSNO in living cells and in vivo under hypoxia stress. Chemical structures of probes 159–171 are shown in Fig. 12.
Utilizing the nucleophilicity and electrophilicity of H2Sn, phenyl 2-(benzoylthio) benzoate unit is an excellent responsive group for H2Sn. As depicted in Fig. 13, once the probe is in contact with H2Sn, the benzothioester will react with H2Sn to generate intermediate A; then H2Sn keep to react with A to form intermediate B, which further cyclizes to release the fluorophore. In 2017, Fang et al. reported a NIR probe 172 based on the combination of a hemicyanine skeleton and phenyl 2-(benzoylthio) benzoate unit [167]. This probe can enable to capture H2Sn effectively, which not only contribute to great sensitivity and selectivity, but also the potential functioning as living cells and mice imaging. Another probe 173 (τ-probe) was obtained to characterize H2Sn by the changes of fluorescence lifetime rather than the change of fluorescence intensity by Yang et al., which can be widely utilized as an ultrasensitive implement for detecting H2Sn in living systems [168]. Hoskere et al. designed a red-emitting probe 174 (MB-Sn) based on BODIPY scaffold, which performed conceivable amelioration of H2Sn visualization in ER [169]. Coordinately, phenyl 2-(benzoylthio) benzoate group and heptamethine cyanine (HQO) are two main structures of probe 175 (HQO-PSP). This probe valid in mitochondrial H2Sn sensing is versatile in tracking dynamic changes in living cells and tissues. Another two-photon fluorescent probe 176 (SPS-M1) was prepared by Kim’group [170]. Probe 176 (SPS-M1) was consisted of three functional parts: H2Sn receptor part, fluorophore scaffold and mitochondria-targeting unit, which was explored for distinguishing mitochondrial H2Sn in living cells with high sensitivity and selectivity. In 2020, Liang et al. reported probe 177 (PP-PS) for imaging endogenous H2Sn lysosomal localization, which can apply to further visualize endogenous H2Sn in the animal inflammation model [171]. Chemical structures of probes 172–177 are shown in Fig. 13.
In recent years, people have paid more attention to H2Sn, various probes emerge in endlessly. On the sensitivity and selectivity of the probes already have a lot of immense progress. However, combined current optical imaging technology, if the probe of H2Sn needs further development, its background fluorescence, toxicity and stability of the probe in complex systems should be worthy of consideration.
7 Conclusions
In the last decade, significant efforts have been made to develop fluorescent probes for detecting H2S. Various engineering strategies have been utilized to optimize the optical properties as well as the biological compatibility. Reduction-based, nucleophilicity-based, and metal sulfide-based strategies are the most widely applied strategies. According to these strategies, a considerable number of probes were gained, some of which have already been utilized to image H2S in cells, tissues, even within the living organism. At the same time, the advancement of modern imaging apparatuses also allows for distinct and high-quality detection for the low concentration of H2S in organisms. Moreover, due to the significant of H2Sn in physiology and pathology, we also introduced some probes for H2Sn. We believe that this review will enable more researchers to understand the design strategies of H2S and H2Sn, and lead to further research in this field.
The goal of small-molecular chemical probes for benignant is to realize in real-time, precise and convenient detection of the limited concentration of H2S and H2Sn in vivo, which will allow for the development of further applications upon this crucial biological matter. Also, the advances of H2S and H2Sn chemical probes will be beneficial to reveal the complexity and intricacy of its biological processes. However, there is still a gap between the current research status and practical applications. Except for improving the selectivity and sensitivity of probes, the biological compatibility and biological stability should also be taken into consideration. Although, in recent years, the research on H2S probes has slowed down, after we understand the complex pathological and physiological functions of H2S and H2Sn, the ultimate goal of our continuing research will be to achieve disease treatment. The future decade is going to witness an exciting and tremendous leap of fluorescent probes for H2S and H2Sn.