“Dual-Lock-Dual-Key” Controlled Second Near-Infrared Molecular Probe for Specific Discrimination of Orthotopic Colon Cancer and Imaging-Guided Tumor Excision

Open AccessCCS ChemistryRESEARCH ARTICLE7 Nov 2022"Dual-Lock-Dual-Key" Controlled Second Near-Infrared Molecular Probe for Specific Discrimination of Orthotopic Colon Cancer and Imaging-Guided Tumor Excision Kun Dou, Chen Fan, Wenqi Feng, Yao Kong, Yunhui Xiang, Zijun Wang and Zhihong Liu Kun Dou College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 , Chen Fan College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 , Wenqi Feng College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 , Yao Kong Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules and College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062 , Yunhui Xiang College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 , Zijun Wang College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 and Zhihong Liu *Corresponding author: E-mail Address: [email protected] College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules and College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062 https://doi.org/10.31635/ccschem.021.202101444 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Fluorescence imaging in the second infrared window (1000–1700 nm) has emerged as a promising approach to tumor diagnosis. However, the currently available second near-infrared (NIR-II) imaging agents are based on the "always on" modality or single biomarker activation, which are subject to limited imaging contrast, nonspecific response, and even false-positive diagnosis. Here, we developed a H2S/H+ dual-stimuli responsive NIR-II fluorescent probe, WH-N3, for precise tumor delimitation and intraoperative fluorescence-guided surgical resection. WH-N3 itself is nonfluorescent, and it can only light up through synergistic activation by H2S and in the tumor acidic environment (TEM). Such a "dual-lock-dual-key" strategy-based activatable probe exhibited significantly higher tumor-to-normal tissue (T/N) ratios than the "always on" agent (ICG) and single parameter responsive counterpart probes in the imaging of colon tumors, which overexpresses H2S. WH-N3 was also able to visualize the tumor-derived endogenous H2S fluctuation and accurately differentiate tumor types based on H2S content discrepancy. More excitingly, under the guidance of the probe's highly specific NIR-II fluorescence, a tiny orthotopic colon tumor with diameter down to 0.8 mm was facilely resected. We expect our dual-stimuli responsive strategy will contribute more reliable tools for specific discrimination and imaging-guided excision of tumor. Download figure Download PowerPoint Introduction Malignancies involve uncontrolled cell growth, division, and metastasis, becoming one of the most serious life-threatening diseases in the world.1,2 Among various cancer therapy modalities, surgical resection of lesions is currently a primary clinical therapeutic approach and, in many cases, is the only effective treatment option.3,4 Delimiting the tumor profile and highly specific identification of a tumor of small size during the operation determine the therapeutic outcome.5 Unfortunately, surgeons that mainly rely on experience and visual inspection face a tremendous challenge in discriminating between the margins of cancerous lesions and normal tissues. Additionally, conventional diagnostic approaches for clinical prediagnosis and intraoperative determination of tumors, such as X-ray computed tomography (CT) or magnetic resonance imaging, usually suffer from unsatisfactory resolution and sensitivity.6–8 In contrast, fluorescence imaging has become a powerful tool for investigation of biological species and processes, benefiting from its unique advantages of high sensitivity, real-time detection, and fast feedback.9–12 Especially, fluorescence imaging in the second near-infrared (NIR-II) window (1000–1700 nm) has recently drawn increasing attention since it affords increased temporal–spatial resolution and deeper tissue penetration.13–17 Owing to the superior optical property of NIR-II fluorescence imaging, NIR-II fluorescent probes have been designed for accurate cancer diagnosis, treatment, and postsurgical evaluation in the past few years.15,18–21 However, most reported NIR-II tumor diagnostic agents are in an "always on" mode. They continuously emit fluorescent signal regardless of the environment, which inevitably causes poor tumor imaging contrast and insufficient specificity.22,23 As an alternative approach, a reaction-based responsive agent, that is, an activatable probe, is much more promising to address these issues. A few well-designed activatable NIR-II probes that can light up under the stimulation of cancer biomarkers have been used for tumor imaging.24–27 Despite the evident improvement achieved, these activatable probes were only single-target responsive. Since most biological parameters that are overexpressed in tumor environments may also exist to a certain extent in normal tissues and plasma, using single-factor activation as a criterion may fail to provide enough contrast to effectively distinguish tumor margins from normal tissues. Moreover, single pathological parameter response may face the issue of nonspecific activation and even cause false-positive signal to influence the accuracy of diagnostic and therapeutic outcomes. In comparison, the strategy that relies on the cooperative activation of multibiomarkers should be a more reliable and effective approach for clinical application.28–31 With this multistimuli response strategy, the fluorescence signal can only be emitted in the co-existence of multibiomarkers upon their specific and synergistic reaction with the probe, maximally eliminating fake signal derived from normal tissues, thereby improving the accuracy and specificity of cancer imaging. However, due to the difficulty of constructing NIR-II fluorophore and the limitation of the reaction-based activation strategy, a multibiomarker co-activated NIR-II molecular fluorescent probe is still in its infancy. Inspired by the pH-dependent equilibrium of intramolecular spirocyclization, we herein report the H2S/acidic tumor environment (TEM) (pH 6.5) dual-stimuli-activated NIR-II fluorescent probe ( WH-N3) for high-specificity discrimination and imaging-guided resection of orthotopic colon tumor (Schemes 1a and 1b). The smart probe ( WH-N3) was composed of two functional "locks": (1) The rhodamine section, which follows the carboxylic acid-controlled spirocyclization mechanism, could reversibly switch fluorescence OFF–ON by changing the π-conjugation of the xanthene ring under different pH conditions. (2) The azide group, as the highly specific H2S recognition site and the fluorescence quencher, was introduced into the BODIPY fluorophore. As designed, WH-N3 is essentially nonfluorescent because the large dihedral angle of the rotatable bond destroys the rigid planar structure and blocks the intramolecular charge transfer (ICT) process from the BODIPY fluorophore to the rhodamine derivative. While in the presence of H2S and high acidity (the two "keys"), the azide group is initially reduced to the amine group by H2S. Then 1,6 intramolecular self-elimination occurs and finally liberates the thiol group as a strong electron donor. Subsequently, NIR-II fluorescence signal is lit up through triggering and enhancing the ICT process from the BODIPY to the rhodamine derivative ( Supporting Information Scheme S1). Upon the dual-stimuli activation, the probe exhibited an evident fluorescence enhancement at 1020 nm, along with excellent selectivity, sensitivity, and acceptable quantum yield (1.5% in aqueous solution). Owing to the smart "dual-lock-dual-key"—in other words, an "AND" logic gate design strategy—accurate differentiation and localization of the tumor edge was successfully achieved through real-time monitoring of endogenous H2S. The dual-stimuli synergistic activation endowed WH-N3 with significantly higher tumor-to-normal tissue (T/N) ratios than that of the "always on" agent (indocyanine green [ICG]) and single parameter responsive counterpart probes. Moreover, in the combination of its deep tissue penetration capability with highly specific dual-activation characteristics, WH-N3 was successfully applied for orthotopic colon tumor real-time imaging and intraoperative imaging-guided tumor resection. Large cancerous tissues and small metastatic lesions were precisely located by the probe. Under the guidance of NIR-II fluorescence imaging, very tiny tumor tissues with diameters down to about 0.8 mm were facilely resected. Scheme 1 | (a) Schematic illustration of the design strategy of "dual-lock-dual-key" NIR-II fluorescent probe WH-N3. (b) Schematic illustration of tumor-specific imaging and imaging-guided resection of orthotopic colon tumor. Download figure Download PowerPoint Experimental Methods Materials and apparatus Unless otherwise stated, all reagents and materials were purchased from commercial companies and used without further purification. Aminooxy acetic acid (AOAA) and IR-26 were obtained from Sigma-Aldrich (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ICG, S-adenosyl-l-methionine (SAM), 3-ethyl-2,4-dimethylpyrrole, and 4-Nitrobenzyl bromide were purchased from Aladdin Reagent, Ltd. (Shanghai, China). Other reagents, including common solvents, were analytical grade or higher and were used without further purification. All aqueous solutions were prepared using ultrapure water (Mill-Q, Millipore, Massachusetts, United States, 18.2 MΩ·cm resistivity). The pH measurements were conducted by the Mettler Toledo Delta 320 pH meter (Shanghai, China). Mass spectrometry analysis was performed on a system consisting of the OrbitrapExploris 480 mass spectrometer (Thermo Fisher, Massachusetts, United States) with a Ultimate3000 high performance liquid chromatography system (Thermo Fisher, Massachusetts, United States). The 1H NMR and 13C NMR spectra were acquired over a Bruker Avance III HD 400 spectrometer (Switzerland). Absorption spectra were recorded on a UV–vis–NIR spectrophotometer (Hitachi, Japan). NIR-II fluorescence spectra were excited by an 808 laser (Beijing Hi-Tech Optoelectronic Co., Ltd., Beijing, China) and recorded with a fluorometer (FLS 1000, Edinburgh, UK). In vivo NIR-II fluorescence images were acquired by an In-Vivo Master NIR-II fluorescence imaging system (Grand Imaging Technology Co. Ltd., Wuhan, China). Spectroscopic measurements and solution preparation For photophysical characterization, compounds WH-1, WH-Cl, and WH-N3 were dissolved in dimethylformamide (DMF) to prepare the stoke solution (1 mM), which were further diluted to 10 μM as the testing solutions with phosphate-buffered saline (PBS) (pH 7.4)/DMF (7/3, v/v) or PBS (pH 6.2)/DMF (7/3, V/V). The absorption spectroscopic study was performed on a Hitachi UV–vis–NIR spectrometer. Fluorescence emission spectra in the NIR-I region were obtained with a xenon lamp, and NIR-II spectra were obtained by an 808 nm laser. Theoretical calculations To shed light on the proposed molecular engineering strategy and verify fluorescence optical characteristics, molecular geometries of probe WH-N3 and its corresponding product WH-N3-HS/WH-N3-S −, WH-N3-HS-LH were initially optimized at the B3LYP/6-31+G(d,p) level with a solvent model called polarizable continuum model (PCM; water as the solvent). Then, the corresponding highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were also calculated at the same level of theory. Measurement of fluorescence quantum yield (Φ) The quantum yields were calculated by the equation: Φsam = Φref × (Ksam/Kref) × (nsam/nref)2, where subscripts sam and ref denote test sample and reference, respectively. Φ is the fluorescence quantum yield, n is the refractive index of the solvent, and K is the slope of the plot, where the slope was determined by plotting the integrated fluorescence intensity versus absorbance value. In brief, five samples with different absorbance at 808 nm were prepared, and then the fluorescence integrated intensity under the corresponding excitation wavelength was collected. Notably, to maximize illumination homogeneity and optical transparency, all absorbance in tests were kept below 0.1 at the excitation wavelength. In this work, the quantum yields of WH-N3-HS in PBS buffer were determined with IR 26 as the reference(Φ = 0.5% in 1,2-dichloroethane).14,18 Statistical analysis Data were normalized to control groups, and statistical analysis was performed with a two-tailed Student's t-test (n = 10 for cells imaging and n = 3 for mice imaging). *P < 0.05, **P < 0.01, and ***P < 0.005. Error bars represent the standard deviation (SD). All data were analyzed by GraphPad Prism7. Cell culture and imaging at NIR-II regions We initially performed the cell imaging experiment to evaluate the bioimaging capability of WH-N3. In this work, HCT-116 cells were obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), which were incubated in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 15% fetal bovine serum (FBS) and 1.5% penicillin-streptomycin (100 U/mL, 100 μg/mL, Invitrogen) at 37 °C under a humidified atmosphere containing 5% CO2. Cell-specific experiments were divided into four groups. Group 1: cells were only incubated with 10 μM probe for 90 min as the control. Group 2, to verify the capability of the probe to track H2S fluctuation, exogenous H2S (200 μM NaHS) was added into the cells and coincubated with probe for 90 min. Next, to clarify that probe was capable of monitoring endogenous H2S fluctuation, inhibitor and activator assays were performed as another two experiment groups. Specifically, group 3, AOAA (25 μM), a commonly used cystathionine-β-synthase (CBS) inhibitor, was added into the cells for 1 h, and then cells were incubated for another 90 min. Group 4 cells were preincubated with 75 μM SAM, the CBS activator, for 60 min, then washed, and 10 μM WH-N3 was further added and incubated for 90 min. For NIR-II cell imaging, the above cells were collected in a centrifuge tube to prepare the cell suspension, and then the cell image was collected in the 1000–1700 nm region under the 808 nm laser excitation (50 mW/cm2, exposure time: 200 ms, 1000 nm LP). Examination of the ability of WH-N3 in differentiating colon tumor based on H2S contents To demonstrate the feasibility of WH-N3 to identify and differentiate types of cancers based on H2S contents, the H2S overexpressed HCT-116 cells and other tumor cell lines, including 4T1, U87, and MCF-7 cells were selected for cell imaging experiments. Before imaging, cells were placed in 25 Petri dishes and allowed to adhere for 24 h. Four cell lines were incubated with 10 μM probe for 90 min, and then collected for NIR-II imaging (808 nm excitation with 1000 nm long-pass filter, 60 mW/cm2, exposure time: 300 ms). Fluorescence imaging of H2S in vivo All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Wuhan University and approved by the Animal Ethics Committee of Wuhan University. Briefly, HCT-116 cells (1 × 107 cells) suspended in 100 μL of PBS were transplanted around the armpit or hind legs of female nude BALB/c mice to establish the animal experiment models. First, to verify the high specificity response of WH-N3 to H2S in tumor region, our tailored probe WH-N3 and the control probe WH-Cl were loaded into the mice through subcutaneous injection (20 μL, 1 mM in physiological saline containing 1% Tween-80 and 4% dimethyl sulfoxide (DMSO) for increasing water solubility), respectively. Mice were anesthetized with isoflurane, and time-dependent NIR-II fluorescence images were recorded under 808 nm excitation with 1000 nm long-pass filter 50 mW/cm2, exposure time: 50 ms). Then, to further validate that WH-N3 was capable of tracking tumor-derived endogenous H2S in vivo, an inhibitor and an activator of enzyme were introduced. To this end, tumor sites were pretreated with SAM, a CBS activator (3 mM, 25 μL), and AOAA (CBS inhibitor, 1 mM, 25 μL) to establish two experimental groups. The control group (loaded with probe only) was injected with 25 μL saline. Four hours after reagent administration, three groups of mice were subject to subcutaneous injection of WH-N3 in the tumor regions. Then, NIR-II images were collected at various time points (0–180 min) using an InGaAs camera equipped with an 808 nm laser and a 1000 nm long-pass filter (50 mW/cm2; exposure time, 30 ms). Next, WH-N3 (20 μL, 1 mM in physiological saline, containing 1% Tween-80 and 4% DMSO) was further applied to identify and differentiate types of cancers at the living-body level through subcutaneous injection into H2S-rich HCT116 tumor-bearing mice and H2S-deficient MCF-7 tumor-bearing mice. Briefly, HCT-116/MCF-7 tumor cells were harvested by centrifugation and resuspended in sterile PBS. Cells (1 × 107 cells) were then implanted subcutaneously into the armpits of female mice. When the tumors reached around 0.7 mm in diameter (15 days after implant), the tumor-bearing mice were subjected to imaging studies. Then, time-dependent (0–180 min) NIR-II images were collected from an InGaAs camera equipped with an 808 nm laser and a 1000 nm long-pass filter (50 mW/cm2; exposure time, 50 ms). For the comparative study on the dual-stimuli responsive probe, the "always on" agent and single-parameter activated counterpart, WH-N3, WH-1, and commercial tumor contrast agent ICG (100 μL, 1 mM in physiological saline containing 1% Tween-80 and 4% DMSO) were intravenously injected into nude mice bearing subcutaneous HCT-116 cancer xenografts separately. Then, time-dependent (0–24 h) NIR-II images were collected using an InGaAs camera equipped with the 808 nm laser and a 1000 nm long-pass filter (50 mW/cm2; exposure time, 200 ms). Intraoperative fluorescence-guided surgical resection of subcutaneous tumor The tumors were observed under NIR-II fluorescence imaging in the optimal surgical time window [16–24 h post-injection (PI)]. Under the guidance of NIR-II imaging, the tumor was precisely resected and further analyzed by hematoxylin-eosin (H&E) staining. NIR-II images were collected using an InGaAs camera equipped with the 808 nm laser and a 1000 nm long-pass filter (50 mW/cm2; exposure time, 200 ms). NIR-II imaging-guided orthotopic colon tumor lesions surgery The orthotopic colon cancer model was prepared as follows: HCT-116 cells (5 × 105 dish−1) were seeded in cell culture flash in 15 mL of DMEM medium supplemented with 15% FBS and 1.5% antibiotics and incubated in CO2 at 37 °C for 24 h. The cells were harvested by centrifugation and resuspended in sterile PBS. Then, HCT-116 cells (5 × 107 cells per mouse) were intraperitoneally injected into the colon of 5-week-old female mice. Model confirmation and tumor size were observed on scarified mice 4 weeks after the injection of cells. Six weeks after intradermal injection, WH-N3 (100 μL, 1 mM in physiological saline containing 1% Tween-80 and 4% DMSO) were intravenously injected into nude mice and imaged under the NIR-II imaging system (808 nm, 1000 nm long-pass filter, 50 mW/cm2; exposure time, 400 ms). For the unguided surgery, tumors were removed at the optimal surgical time window (24 h PI) using the naked eye. Those eye-invisible tiny lesions were removed under an InGaAs camera with 808 nm laser and a 1000 nm long-pass filter (50 mW/cm2; exposure time, 300 ms). Finally, all the excised tumor tissues were analyzed by H&E staining. Results and Discussion Design and synthesis of the H2S/H+ dual-stimuli responsive probe WH-N3 To make a clinically applicable fluorescent probe with a "dual-lock-dual-key" characteristic, a fluorophore skeleton with NIR-II emission and readily modifiable structure is essential. To this end, among currently available NIR-II fluorophores, including donor–acceptor–donor framework dyes, cyanine dyes, and difluoroboron dipyrromethene (BODIPY) derivatives, we chose a BODIPY dye as the scaffold of our probe considering its facile modification and favorable photophysical properties.32 As H2S is overexpressed in colon cancer,33,34 an azide group as the highly specific recognition site for H2S and a fluorescence quencher, namely the first "lock", was introduced into BODIPY fluorophore. Because H2S is also found in normal tissues and blood plasma, though with relatively lower contents than in the tumor, it is still necessary to add a further "lock" so as to guarantee the probe is not lit up in advance before reaching the lesion region. We thus decided to further introduce an H+-responsive site in consideration of the higher acidity of tumor tissue (pH 6.2–6.9). Our previous work revealed that the ICT process tuned by the electronic push–pull effect of the molecule before and after response to the target could be a universal activation strategy for the NIR-II probe.35 Thus, as the electron-withdrawing group as well as the H+ responsive site, a rhodamine derivative with a carboxylic-acid controlled spirocyclization reaction was connected to the BODIPY fluorophore through a vinyl bond to prepare the second "lock". The H2S/H+ dual-stimuli responsive NIR-II probe WH-N3 was thus obtained. In addition, to highlight the advantages of our proposed "dual-lock-dual-key" probe in tumor high-contrast imaging, two analogues WH-1 and WH-Cl were also synthesized as the control probes. The structural and synthetic routes for all three compounds are shown in Supporting Information Scheme S2, and the intermediates and all the probes were fully characterized using 1H NMR and electrospray ionization high-resolution mass spectrometry (ESI-HRMS), as presented in Supporting Information Figures S16–S27. Spectroscopic properties and optical response of WH-N3 to H2S/H+ To verify the proposed "dual-lock-dual-key" strategy, the fluorescence response of WH-N3 to both H2S and H+ was first investigated by spectroscopic analysis. As expected, WH-N3 exhibited a pH-dependent equilibrium of intramolecular spirocyclization. It existed in its open-ring form with strong absorption at 625 nm under acidic conditions, whereas it gradually turned into its spirocyclic form accompanied with decreased and blue-shifted absorption in neutral to alkaline environments ( Supporting Information Figure S1a). The pKa value of WH-N3 was calculated to be 7.5 ( Supporting Information Figure S1b), indicating that WH-N3 mostly exists in its open-ring form in the tumor region with pH 6.2–6.9. Notably, in the absence of H2S, no NIR-II fluorescence signal was observed regardless of the acidity of the medium ( Supporting Information Figure S1c), which could be ascribed to the suppression of ICT process. The H2S/H+ coactivation of WH-N3 was then studied by absorption and fluorescence titration of the probe with H2S and H+. As shown in Figure 1a, WH-N3 itself had an absorption peak at 625 nm. After reacting with H2S at physiological acidity (pH 7.4), two new absorption peaks at 525 and 810 nm were generated, which were ascribed to the two forms of the reaction product, that is, WH-N3-HS-LH and WH-N3-HS, respectively (Scheme 1a), accompanied by the obviously decreased absorption at 625 nm. When the probe was incubated with NaHS in an acidic environment (pH 6.2), a strong absorption at 810 nm was found, indicating that the reaction product mainly exists in its open-ring form ( WH-N3-HS) in acidic condition. Meanwhile, bright fluorescence signal with an emission maximum at 1020 nm under 808 nm excitation was also observed at pH 6.2, indicating the good response of WH-N3 to H2S under acidic condition. Conversely, the fluorescence signal was very weak (the "off" state) after reaction with H2S in physiological acidity (pH 7.4) (Figure 1b and Supporting Information Figures S1d and S1e). These results can be attributed to the fact that the spirocyclic form at the physiological pH damages the π-conjugation of the molecular probe as well as its reaction product with H2S, reducing the electronic push–pull effect of the molecule, ultimately resulting in a dramatic decrease in the fluorescence intensity. Thus, the above results indicate that the bright NIR-II fluorescence signal of WH-N3 could be efficiently activated only in the coexistence of both an acidic environment and H2S. Furthermore, the reversible response of probe to H+ was verified. The fluorescence intensity of the probe reversibly switched between OFF and ON states upon its continuous response to pH 6.2 and 7.4 in the presence of H2S, indicating that the probe has a good reversibility response to H+ (Figures 1c and 1d). Such a reversible response to H+ can guarantee that the probe is not emissive in normal tissues in the body, even when some probe molecules diffuse from the tumor site to adjacent normal tissues, which is meaningful to improve the accuracy of tumor recognition by preventing undesired activation in normal tissue. Therefore, our tailored dual-stimuli responsive probe has shown great potential in cancer imaging. Figure 1 | (a) The absorption and photograph of WH-N3 (10 μM) before and after treatment with H2S at pH 6.2 and 7.4, respectively. (b) NIR fluorescence spectra and photograph of WH-N3 (10 μM) after responding to 200 μM NaHS at pH 6.2 and 7.4 under 808 nm excitation, respectively. (c and d) The reversible fluorescence response of probe to H+. 200 μM NaHS was added to the solution of WH-N3 (10 μM) in 10 mM PBS and incubated for 90 min. NaOH (2 M) and HCl (2 M) were used to adjust pH value between 7.4 and 6.2. Download figure Download PowerPoint Sensitivity and specificity of WH-N3 to H2S in acidic environment Because of the high complexity of in vivo environments, especially in disease regions, it is essential for activatable probes to maintain high sensitivity and specific recognition toward desired targets in order to improve accuracy of diagnosis and treatment. We then evaluated the performance of WH-N3with the azide group as a recognition site towards H2S under acidic conditions. As a comparison, we also synthesized a control probe, WH-Cl, which was designed by using a commonly reported monochlorinated BODIPY core as the H2S recognition site.36 First, H2S-dependent spectral profiles of WH-N3 (10 μM) were investigated in an acidic environment (PBS/DMF, 7:3, v/v, 10 mM, pH 6.2, 37 °C). As shown in Figure 2a, the absorption of WH-N3 at 625 nm gradually declined with the addition of NaHS (a commonly used H2S donor), along with the rise of a new absorption peak at 810 nm. Similarly, the fluorescence emission of WH-N3 in the NIR-I region (695 nm, under 600 nm excitation) was reduced by the addition of NaHS (Figure 2b). Meanwhile, a NaHS concentration-dependent fluorescence "turn-on" response in the NIR-II region (1020 nm) was observed under 808 nm excitation (Figures 2c and 2i), and a linear calibration was obtained for NaHS concentration ranging from 10 to 250 μM (R2 = 0.9905) with a detection limit of 249 nM (Figure 2d). Furthermore, with the characteristics of dual-excitation-dual-emission, the fluorescence ratio signal between the NIR-II/NIR-I also exhibited good linear response (10–200 μM, with a detection limit of 90 nM, Supporting Information Figure S2). Kinetic studies revealed that the fluorescence maximum can be reached within 90 min incubation ( Supporting Information Figure S3). Additionally, WH-N3 displayed a maximum fluorescence enhancement factor of 20-fold after H2S activation, and the reaction product had a relatively high fluorescence quantum yield of 1.5% in aqueous solution, with IR-26 as the reference ( Supporting Information Figure S4). The optical response of WH-Cl (10 μM) to H2S in acidic conditions was also examined (PBS/DMF, 7:3, v/v, 20 mM, pH 6.2, 37 °C). As shown in Figures 2e and 2f, with the addition of NaHS, the elevated absorption/emission signals at 795/1018 nm were also observed, and a linear correlation between the fluorescence intensity and NaHS concentration in the range