A Novel NIR Fluorescent Probe for Rapid Response to Hydrogen Sulfide

A Novel NIR Fluorescent Probe for Rapid Response to Hydrogen Sulfide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel NIR Fluorescent Probe for Rapid Response to Hydrogen Sulfide Xiaoci Lv, Yu Xie, Heping Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4598713/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jul, 2024 Read the published version in Journal of Fluorescence → Version 1 posted 7 You are reading this latest preprint version Abstract Hydrogen sulfide (H 2 S), as an important small molecule bioregulator, plays a key role in many physiological activities and signaling, and abnormal fluctuations in H 2 S concentration can lead to a variety of diseases. Therefore, it is of great significance to develop a near-infrared fluorescence probe to visualize fluctuations in H 2 S levels. This work is based on Sulfur-substituted dicyanomethylene-4H-chromene (DCM), A novel NIR fluorescent probe (E) -3 - (2 - (4 - (dicyanomethylene) -6-methyl-4H-Thiochromen-2-yl)vinyl-1-methylquinolin-1-ium (DMT) was synthesized successfully. Research has found that in weakly alkaline environments, the probe DMT reacts rapidly with H 2 S (only 10 s), the fluorescence intensity at 684 nm is enhanced by about 60 fold, the detection limit is as low as 0.1623 µM, the Stokes shift is large (94 nm), and strong selectivity as well as anti-interference ability towards H 2 S. This will provide a new method for the rapid detection and further application of H 2 S. NIR fluorescent probe Hydrogen sulfide Fluorescence intensity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Hydrogen sulfide (H 2 S) is known for its highly toxic and rotten egg smell, like nitric oxide (NO) and carbon monoxide (CO), is considered a neurotransmitter, also a gas signaling molecule that regulates physiological functions [ 1 – 3 ]. At present, the anthropogenic sources of H 2 S mainly come from industrial production, agricultural production, modern oil production and urban sewage system. These can all lead to the production, release and exposure of H 2 S, and large-scale acute exposure of H 2 S may cause poisoning [ 4 , 5 ]. H 2 S is known to be involved in many physiological activities, such as the balance of REDOX states within cells, regulation of vascular tone, protection of the heart, and regulation of cell growth in biological systems [ 6 – 8 ]. Hydrogen sulfide can stimulate vascular tumor formation at low concentrations, high concentration can inhibit biological energy and poison mitochondria [ 9 ]. In addition, H 2 S has been linked not only to the onset of Down syndrome, Alzheimer's disease, and diabetes, but also to Parkinson's disease (PD) and periodontitis [ 10 – 13 ]. At present, there are many methods reported to detect H 2 S signal molecules, mainly gas chromatography, colorimetry, electrochemical method and so on [ 14 – 17 ]. However, processing biological samples using these traditional methods requires complex procedures and has various limitations that make it challenging to quantitatively monitor H 2 S levels and dynamically detect H 2 S in real time [ 18 ]. Comparing with the traditional detection methods, the fluorescence detection method has the advantages of good selectivity, low background interference, high sensitivity and real-time detection [ 19 , 20 ]. There have been many reports on H 2 S probes. For example, Wang et al. [ 21 ] developed a fluorescence probe (EDPH) based on purine scaffold to detect H 2 S by inducing H 2 S to break ether bonds in EDPH, and successfully realized in-situ imaging in Hela cells. However, current fluorescent probes have the following limitations : (1) Weak anti-interference makes fluorescent probes susceptible to larger background signals from cytoplasm or other organelles [ 22 – 24 ]. (2) Many probes require a long response time to H 2 S, which is not conducive to the detection of H 2 S produced by biological tissues, because H 2 S is diffuse and unstable, as well as has a relatively short half-life [ 25 – 27 ]. (3) The detection limits of probes currently under development are usually higher than 100 µM, which limits their application in scenarios that produce lower concentrations of H 2 S [ 28 , 29 ]. Therefore, it is very important to develop a simple, efficient and sensitive fluorescent probe for rapid detection of hydrogen sulfide in vivo and in vitro. In this work, a probe DMT was prepared by methylation reaction of 3-quinoline formaldehyde and Sulfur-substituted dicyanomethylene-4H-chromene. Methylquinoline turned off the fluorescence, and the presence of H 2 S restored the intramolecular ICT effect and turned on the fluorescence. The detection of H 2 S was completed in EtOH/PBS solution, and the probe DMT showed excellent characteristics of fast response, high sensitivity, strong selectivity and low detection limit, and could distinguish between reaction and non-reaction from color changes before and after reaction. It provides a new method for visual detection of hydrogen sulfide in the environment and living cells. Materials All reagents are purchased from Shanghai Titan Technology Co., Ltd. without special instructions and do not require further purification. All solutions and buffers were prepared using deionized water. Dissolve the probe DMT in DMSO solution to prepare 1 mM of probe mother solution. Prepare leucine (Leu), valine (Val), phenylalanine (Phe), alanine (Ala), methionine (Met), arginine (Arg), proline (Pro), glycine (Gly), CaCl 2 , MgCl 2 , KCl, NaCl, NaBr, Na 2 CO 3 , NaNO 2 , NaF, Na 2 SO 4 , BaCl 2 ·2H 2 O, Na 2 S·9H 2 O, Na 2 S 2 O 4 , NaHSO 3 , glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) solutions using deionized water (10 mM). Prepare standard PBS (10 mM) solutions with different pH values using a PHS-3E pH meter. The probe DMT reacts with Na 2 S in EtOH/PBS (v/v = 1:1, 10 mM, pH = 7.4) solution. Except for time measurement, the response time between the probe and Na 2 S was greater than 30 minutes. All fluorescence spectrum measurements were recorded at 590/684 nm, with excitation slit 10 nm, emission slit 10 nm, and photomultiplier tube voltage 800 V. All data were measured in parallel three times, all at room temperature. Instruments The UV visible absorption spectrum was collected using a UV-1800 spectrophotometer. Use the F-7000 fluorescence spectrophotometer to record fluorescence emissions. Using TMS as the internal reference, 1 H NMR and 13 C NMR spectra were recorded on the Bruker INOVA-400 NMR spectrometer wspectrometer. High-resolution spectral analysis (HRMS) was performed on the LCMS-9030 QT0F plus mass spectrometer.Prepare standard PBS (10 mM) solutions with different pH values using a PES-3E pH meter. Other instruments include vertical rotary steamer, SHB-III water circulation multi-purpose vacuum pump, Gas phase mass spectrometry (GC-MS), Organic Synthesis Monitoring Mass Spectrometer (OMS). Synthesis of Probe DMT Synthesis of compounds 1 and 2: Synthesis according to known literature [ 30 – 32 ], see supporting literature for details. Synthesis of compound 3: Compound 2 (71.4 mg, 0.3 mmol) and 3-quinoline formaldehyde (51.8 mg, 0.33 mmol) were dissolved in 10 mL ultra-dry acetonitrile solution in a 25 mL round-bottled flask, and 2 drops of piperidine were added as a catalyst. The reaction was carried out at 80°C for 8 h by reflux and monitored by TLC until the reaction was complete (development agent:Petroleum ether/ethyl acetate = 2:1). The obtained mixed solution was filtered by vacuum to obtain a deep red solid 101 mg with a yield of 89.7%. No further purification required. ESI-MS: calculated for C 24 H 15 N 3 S: [M + H] + 378.1; measured: 377.9. Synthesis of probe DMT: Compound 3 (75.4 mg, 0.2 mmol) and methyl trifluoromethanesulfonate (131 mg, 0.8 mmol) were added into a 50 mL round-bottom flask and dissolved in 30 mL anhydrous CH 2 Cl 2 solution. The reaction was stirring at room temperature for 24 h, and was monitored by TLC until the reaction was complete (developing agent:Dichloromethane/methanol = 20:1). The mixed solution was filtered by vacuum to obtain 106 mg orange solid with a yield of 97.7%. 1 H NMR (Fig. S5) (400 MHz, DMSO- d 6 ) δ 9.93 (s, 1H), 9.43 (s, 1H), 8.52 (s, 1H), 8.48 (d, J = 8.7 Hz, 1H), 8.30 (d, J = 7.9 Hz, 1H), 8.28–8.20 (m, 1H), 8.04 (t, J = 7.0 Hz, 1H), 7.95 (d, J = 16.4 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.57 (d, J = 12.1 Hz, 2H), 4.64 (s, 3H), 2.40 (s, 3H). 13 C NMR (Fig. S6) (100 MHz, DMSO- d 6 ) δ 155.07, 150.14, 146.26, 144.08, 139.29, 137.87, 136.33, 134.62, 131.54, 131.08, 131.03, 130.10, 129.61, 129.19, 128.52, 127.72, 124.58, 123.49, 119.71, 117.44, 116.14, 68.68, 46.25, 21.46. ESI-MS (Fig. S4): calculated for C 25 H 18 N 3 S + : [M] + 392.1216; measured: 392.1213. Results and discussion Study on UV-VIS absorption spectra of hydrogen sulfide by probe DMT In order to evaluate the spectral properties of probe DMT on hydrogen sulfide, the UV-visible absorption spectra after the interaction between probe DMT and S 2− were tested, as shown in Fig. 1 . As can be seen from Fig. 1 , the maximum absorption wavelength of probe DMT is at 480 nm. After the addition of S 2− , the maximum absorption peak at 480 nm gradually decreased, and the peak at 378 nm also gradually decreased, and a new strong absorption peak appeared at 600 nm, indicating that S 2− reacted with the probe DMT, changing the original conjugate structure of the probe molecules leading to these phenomena.The methylquinoline group has fluorescence quenching effect on the probe, and the reaction of S 2− with the probe causes the probe to restore the intramolecular ICT effect, and the intramolecular charge distribution occurs a new layout. When the concentration of S 2− reached 500 µM, the absorption peak intensity did not change. After the probe reacted with S 2− , the color of the solution changed from yellow to blue, and the change could be directly observed by the naked eyes. Study on fluorescence spectra of hydrogen sulfide by probe DMT The fluorescence spectra of the interaction between probe DMT and S 2− are shown in Fig. 2 a. Probe DMT shows almost no fluorescence in PBS buffer, while the probe showed an emission band centered at 680 nm (λ ex = 590 nm) after interacting with S 2− . With the increase of S 2− concentration, the fluorescence intensity at λ em = 684 nm gradually increased by about 60 fold, which was attributed to the recovery of intramolecular ICT effect. When the concentration of S 2− reached 500 µM, the fluorescence intensity hardly changed. As can be seen from the small figure in the upper right corner, the product solution after the reaction of probe DMT and S 2− was purple red fluorescence. The relationship between fluorescence intensity and concentration of S 2− is shown in Fig. 2 b, and the concentration of S 2− below 500 µM has a good linear relationship (R 2 = 0.9926, K = 2.1706). Stability of probe DMT for S 2- In order to explore the stability effect of the probe in buffer solution (EtOH:PBS = 1:1), the fluorescence intensity of DMT in different pH environments was measured, as shown in Fig. 3 . The probe was almost unaffected by the pH of the environment, and the fluorescence intensity of the solution was enhanced with the addition of S 2− . As can be seen from Fig. 3 , in a strongly acidic environment, the fluorescence intensity of the probe did not increase significantly after adding S 2− , but showed obvious fluorescence enhancement in an environment with pH = 6.5–10.5. This indicates that probe DMT can be used as a detection tool for in vivo cell imaging and biological imaging applications. Reactivity of probe DMT to hydrogen sulfide Time is a key factor to evaluate the properties of fluorescent probes. In order to study the reaction performance of probe DMT to S 2− , the response time of probe and its reaction was measured, as shown in Fig. 4 . It can be seen from Fig. 4 that the probe can respond to S 2− in a very short time and reach an equilibrium state at 10 s. Selectivity of probe DMT to hydrogen sulfide Selectivity is one of the important parameters to evaluate the performance of fluorescent probes. In this paper, Leu, Val, Phe, Ala, Met, Arg, Pro, Gly, CaCl 2 , MgCl 2 , KCl, NaCl, NaBr, Na 2 CO 3 , NaNO 2 , NaF, Na 2 SO 4 , BaCl 2 ·2H 2 O, Na 2 S·9H 2 O, Na 2 S 2 O 4 , NaHSO 3 , GSH, Cys and Hcy were analyzed to explore the selectivity of probe DMT and its interaction, as shown in Fig. 5 . As can be seen from Fig. 5 , after adding S 2− , the probe DMT solution showed obvious fluorescence enhancement at 684 nm, and the fluorescence intensity increased slightly after adding GSH. Since the probe DMT solution added with GSH has no significant change under visible light and 365 nm UV lamp, and there is a significant difference in fluorescence enhancement ratio, it can be well distinguished between the two. The addition of other anions, cations, amino acids and other biological mercaptans had no obvious effect. The results show that probe DMT has good selectivity for S 2− . The anti-interference of probe DMT to S 2- As shown in the Fig. 6 , there was no obvious fluorescence change in probe DMT solution after adding other analytical species except S 2− . After adding 500 µM S 2− solution to different solutions, the fluorescence intensity of probe DMT at 684 nm showed obvious enhancement, and the fluorescence intensity was only slightly different from that of only 500 µM S 2− solution. Therefore, it can be shown that the probe DMT has excellent selectivity to S 2− and strong anti-interference to other analytical substances. Sensing mechanism study Through theoretical analysis and literature investigation, it is speculated that the nucleophilic addition reaction occurs between H 2 S and the double bond on the pyridine ring in the probe molecule DMT (Fig. 7 a), and the fluorescence is turned on [ 33 , 34 ]. The proposed mechanism was verified by measuring the molecular weight of the probe DMT reacting with H 2 S. As shown in the figure, after adding 250 eq H 2 S to fully combine it with the probe molecule, the mass spectrum peak of the reaction product [DMT + H 2 S + Na + ] was measured to be m/z = 448.0657, and its theoretical value was 448.0918, both values were basically consistent (Fig. 7 b). Conclusion In conclusion, a novel NIR fluorescence probe DMT for H 2 S recognition was designed and synthesized in this work. The structure of the probe was determined by high-resolution mass spectrometry and NMR. The sensing mechanism of DMT and H 2 S response was explained by HRMS. In vitro titration experimental data show that probe DMT has some excellent properties: The reaction with H 2 S can be complete within 10 s, the maximum fluorescence emission wavelength can reach the near infrared 684 nm and the fluorescence intensity is increased by about 60 fold, the large Snokes shift (94 nm), and the weak alkaline conditions show good selectivity and excellent anti-interference. It provides a new method for the rapid detection of H 2 S in the environment and living cells. Declarations Funding This study was supported by the Hunan Provincial Natural Science Foundation of China (2019JJ40295) . Acknowledgements This study was supported by the Hunan Provincial Natural Science Foundation of China (2019JJ40295) .The authors acknowledge Analyzing & Testing Center, School of chemistry and chemical engineering, Changsha University of Science and Technology for characterizing of samples. Authors’ contributions Xiaoci Lv was mainly responsible for probe DMT synthesis and article writing; Yu Xie was involved in performance testing; Heping Li was responsible for revising the article. All authors read and approved the final manuscript. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Conflicts of Interest The authors declare no conflicts of interest. 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Chem Eng J 427:131563. https://https://doi.org/10.1016/j.cej.2021.131563 Schemes Scheme 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx image1.png Scheme 1 The synthesis route of compound DMT Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2024 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 04 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviewers agreed at journal 03 Jul, 2024 Reviewers invited by journal 03 Jul, 2024 Editor assigned by journal 20 Jun, 2024 Submission checks completed at journal 20 Jun, 2024 First submitted to journal 18 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4598713","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322684198,"identity":"0d5e5f47-c0b5-4abc-b16b-66d37204555e","order_by":0,"name":"Xiaoci Lv","email":"","orcid":"","institution":"Changsha University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoci","middleName":"","lastName":"Lv","suffix":""},{"id":322684199,"identity":"83314bae-0a82-489a-a45e-17a594242808","order_by":1,"name":"Yu Xie","email":"","orcid":"","institution":"Changsha University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Xie","suffix":""},{"id":322684200,"identity":"b084c0f3-d7d7-44e6-b203-a7e9bb0b0918","order_by":2,"name":"Heping Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACPmYeIGkAQswHDnyoIEILG0ILW+LBGWeI0cLAA2EYMPAYH+ZtIUYLO+/BxwUFd+y2S/d8OMDbwCDPL3aAkMP4ko1nGDxL3jnn7IYDkjsYDGfOTiCkhcdMmsfgcLLBjdwNBwzPMCQY3Casxfw3REvOgwOJbcRpMWMGarEDamE4cJBILcbSMwwOJxjcSDM42HBGgrBf+PnPGH4u+HPY3uBG8uPPfyps5PmlCWgBAWYgTmyAsCUIK4dpsSdO6SgYBaNgFIxIAACVdENb/hRanAAAAABJRU5ErkJggg==","orcid":"","institution":"Changsha University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Heping","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-18 09:00:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4598713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4598713/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-024-03857-9","type":"published","date":"2024-07-26T00:32:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60470141,"identity":"f1d12542-1e4f-4cde-9345-9d6bb9ddd6f8","added_by":"auto","created_at":"2024-07-17 06:26:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157530,"visible":true,"origin":"","legend":"\u003cp\u003eThe ultraviolet spectrum of probe DMT (20 μM) before and after adding S\u003csup\u003e2- \u003c/sup\u003e(0-500 μM) into EtOH/PBS solution (v/v=1:1, 10 mM, pH=7.4). Illustration: Color change of solution before and after reaction under natural light\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/b6d14dc4a7a87bb16e2963ff.png"},{"id":60469672,"identity":"1a4a5595-3c9b-4728-a2a5-a3697687fa18","added_by":"auto","created_at":"2024-07-17 06:18:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eFluorescence spectra (λ\u003csub\u003eex\u003c/sub\u003e=590 nm) of probe DMT (20 μM) before and after adding S\u003csup\u003e2-\u003c/sup\u003e (0-500 μM) into EtOH/PBS solution (v/v=1:1, 10 mM, pH=7.4). Illustration: Color change of solution under 365 nm UV lamp. \u003cstrong\u003eb\u003c/strong\u003e Linear relationship of fluorescence intensity at 590 nm with different concentration of S\u003csup\u003e2-\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/8fb9da74fe8f4c4b0e370195.png"},{"id":60470143,"identity":"d7fafd63-01d8-4520-ac59-1466aa63d0fb","added_by":"auto","created_at":"2024-07-17 06:26:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49953,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of probe DMT (20 μM) before and after interaction with S\u003csup\u003e2-\u003c/sup\u003e (500 μM) in EtOH/PBS (v/v=1:1, 10 mM) solution at different pH (λ\u003csub\u003eex\u003c/sub\u003e=590 nm,λ\u003csub\u003eem\u003c/sub\u003e=684 nm)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/adf0b88084443c53049365b9.png"},{"id":60469677,"identity":"94974ae6-2b16-47e3-a91b-e8da9413e30e","added_by":"auto","created_at":"2024-07-17 06:18:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80868,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of probe DMT (20 μM) interacting with S\u003csup\u003e2-\u003c/sup\u003e (500 μM) in EtOH/PBS solution (v/v= 1:1, 10 mM, pH=7.4), λ\u003csub\u003eex\u003c/sub\u003e=590 nm,λ\u003csub\u003eem\u003c/sub\u003e=684 nm\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/821c10e92679e3355c482da2.png"},{"id":60470638,"identity":"50cd0fd8-383a-461b-bd6b-0de635b9f844","added_by":"auto","created_at":"2024-07-17 06:34:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44498,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of EtOH/PBS (v/v= 1:1, 10 mM, pH=7.4) solution of probe DMT (20 μM) after interaction with various analytes (500 μM) at 590 nm. Analytes: 1, blank; 2, Ba\u003csup\u003e2+\u003c/sup\u003e; 3, Ca\u003csup\u003e2+\u003c/sup\u003e; 4, Mg\u003csup\u003e2+\u003c/sup\u003e; 5, K\u003csup\u003e+\u003c/sup\u003e; 6, Na\u003csup\u003e+\u003c/sup\u003e; 7, Cl\u003csup\u003e-\u003c/sup\u003e; 8. Br\u003csup\u003e-\u003c/sup\u003e; 9, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e; 10, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e; 11, F\u003csup\u003e-\u003c/sup\u003e; 12, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e; 13, S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e; 14, HSO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e; 15, Cys; 16, Hcy; 17, Leu; 18, Val; 19, Phe; 20, Ala; 21, Met; 22, Arg; 23, Pro; 24, Gly; 25, GSH; 26, S\u003csup\u003e2-\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/ed7ababbc0c02615c798a2fa.png"},{"id":60470145,"identity":"b5b30a4a-c831-44d1-99dd-b7f67a83dd67","added_by":"auto","created_at":"2024-07-17 06:26:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":117990,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of EtOH/PBS (v/v= 1:1, 10 mM, pH=7.4) solution of probe DMT (20 μM) after interaction with various analytes (500 μM) at 590 nm. Analytes:1, Ba\u003csup\u003e2+\u003c/sup\u003e; 2, Ca\u003csup\u003e2+\u003c/sup\u003e; 3, Mg\u003csup\u003e2+\u003c/sup\u003e; 4, K\u003csup\u003e+\u003c/sup\u003e; 5, Na\u003csup\u003e+\u003c/sup\u003e; 6, Cl\u003csup\u003e-\u003c/sup\u003e; 7, Br\u003csup\u003e-\u003c/sup\u003e; 8, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e; 9, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e; 10, F\u003csup\u003e-\u003c/sup\u003e; 11, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e; 12, S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e; 13, HSO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e; 14, Cys; 15, Hcy; 16, Leu; 17, Val; 18, Phe; 19, Ala; 20, Met; 21, Arg; 22, Pro; 23, Gly; 24, GSH; 25, S\u003csup\u003e2-\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/419b25ada7eb229b7a5a3e23.png"},{"id":60470144,"identity":"6aed08fa-59b7-4af8-b3ae-aa13625b438d","added_by":"auto","created_at":"2024-07-17 06:26:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Reaction mechanism diagram of probe DMT and H\u003csub\u003e2\u003c/sub\u003eS. \u003cstrong\u003eb\u003c/strong\u003e Ultrahigh resolution mass spectrometry after probe reaction\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/828beb1f2150107a82b2a57b.png"},{"id":61197477,"identity":"9d07ce7f-d027-4f53-a3c8-6bf34eb536c1","added_by":"auto","created_at":"2024-07-27 00:32:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1026935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/624550c0-30fc-4e05-921d-25a69e5c50c0.pdf"},{"id":60469680,"identity":"c7a34234-7097-4c65-b6bb-f53b9442894c","added_by":"auto","created_at":"2024-07-17 06:18:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":347223,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/a1dfbdcca5202a5322315873.docx"},{"id":60469674,"identity":"bc87d7f3-65f3-46d8-b126-9798d4e88b1a","added_by":"auto","created_at":"2024-07-17 06:18:32","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":51895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e The synthesis route of compound DMT\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4598713/v1/c30a477f50ce906a3042a6fa.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eA Novel NIR Fluorescent Probe for Rapid Response to Hydrogen Sulfide\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) is known for its highly toxic and rotten egg smell, like nitric oxide (NO) and carbon monoxide (CO), is considered a neurotransmitter, also a gas signaling molecule that regulates physiological functions [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. At present, the anthropogenic sources of H\u003csub\u003e2\u003c/sub\u003eS mainly come from industrial production, agricultural production, modern oil production and urban sewage system. These can all lead to the production, release and exposure of H\u003csub\u003e2\u003c/sub\u003eS, and large-scale acute exposure of H\u003csub\u003e2\u003c/sub\u003eS may cause poisoning [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. H\u003csub\u003e2\u003c/sub\u003eS is known to be involved in many physiological activities, such as the balance of REDOX states within cells, regulation of vascular tone, protection of the heart, and regulation of cell growth in biological systems [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Hydrogen sulfide can stimulate vascular tumor formation at low concentrations, high concentration can inhibit biological energy and poison mitochondria [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, H\u003csub\u003e2\u003c/sub\u003eS has been linked not only to the onset of Down syndrome, Alzheimer's disease, and diabetes, but also to Parkinson's disease (PD) and periodontitis [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt present, there are many methods reported to detect H\u003csub\u003e2\u003c/sub\u003eS signal molecules, mainly gas chromatography, colorimetry, electrochemical method and so on [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, processing biological samples using these traditional methods requires complex procedures and has various limitations that make it challenging to quantitatively monitor H\u003csub\u003e2\u003c/sub\u003eS levels and dynamically detect H\u003csub\u003e2\u003c/sub\u003eS in real time [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Comparing with the traditional detection methods, the fluorescence detection method has the advantages of good selectivity, low background interference, high sensitivity and real-time detection [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. There have been many reports on H\u003csub\u003e2\u003c/sub\u003eS probes. For example, Wang et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] developed a fluorescence probe (EDPH) based on purine scaffold to detect H\u003csub\u003e2\u003c/sub\u003eS by inducing H\u003csub\u003e2\u003c/sub\u003eS to break ether bonds in EDPH, and successfully realized in-situ imaging in Hela cells. However, current fluorescent probes have the following limitations : (1) Weak anti-interference makes fluorescent probes susceptible to larger background signals from cytoplasm or other organelles [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. (2) Many probes require a long response time to H\u003csub\u003e2\u003c/sub\u003eS, which is not conducive to the detection of H\u003csub\u003e2\u003c/sub\u003eS produced by biological tissues, because H\u003csub\u003e2\u003c/sub\u003eS is diffuse and unstable, as well as has a relatively short half-life [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. (3) The detection limits of probes currently under development are usually higher than 100 \u0026micro;M, which limits their application in scenarios that produce lower concentrations of H\u003csub\u003e2\u003c/sub\u003eS [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, it is very important to develop a simple, efficient and sensitive fluorescent probe for rapid detection of hydrogen sulfide in vivo and in vitro.\u003c/p\u003e \u003cp\u003eIn this work, a probe DMT was prepared by methylation reaction of 3-quinoline formaldehyde and Sulfur-substituted dicyanomethylene-4H-chromene. Methylquinoline turned off the fluorescence, and the presence of H\u003csub\u003e2\u003c/sub\u003eS restored the intramolecular ICT effect and turned on the fluorescence. The detection of H\u003csub\u003e2\u003c/sub\u003eS was completed in EtOH/PBS solution, and the probe DMT showed excellent characteristics of fast response, high sensitivity, strong selectivity and low detection limit, and could distinguish between reaction and non-reaction from color changes before and after reaction. It provides a new method for visual detection of hydrogen sulfide in the environment and living cells.\u003c/p\u003e"},{"header":"Materials","content":"\u003cp\u003eAll reagents are purchased from Shanghai Titan Technology Co., Ltd. without special instructions and do not require further purification. All solutions and buffers were prepared using deionized water. Dissolve the probe DMT in DMSO solution to prepare 1 mM of probe mother solution. Prepare leucine (Leu), valine (Val), phenylalanine (Phe), alanine (Ala), methionine (Met), arginine (Arg), proline (Pro), glycine (Gly), CaCl\u003csub\u003e2\u003c/sub\u003e, MgCl\u003csub\u003e2\u003c/sub\u003e, KCl, NaCl, NaBr, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, NaNO\u003csub\u003e2\u003c/sub\u003e, NaF, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, BaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, Na\u003csub\u003e2\u003c/sub\u003eS\u0026middot;9H\u003csub\u003e2\u003c/sub\u003eO, Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, NaHSO\u003csub\u003e3\u003c/sub\u003e, glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) solutions using deionized water (10 mM). Prepare standard PBS (10 mM) solutions with different pH values using a PHS-3E pH meter. The probe DMT reacts with Na\u003csub\u003e2\u003c/sub\u003eS in EtOH/PBS (v/v\u0026thinsp;=\u0026thinsp;1:1, 10 mM, pH\u0026thinsp;=\u0026thinsp;7.4) solution. Except for time measurement, the response time between the probe and Na\u003csub\u003e2\u003c/sub\u003eS was greater than 30 minutes. All fluorescence spectrum measurements were recorded at 590/684 nm, with excitation slit 10 nm, emission slit 10 nm, and photomultiplier tube voltage 800 V. All data were measured in parallel three times, all at room temperature.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInstruments\u003c/h2\u003e \u003cp\u003eThe UV visible absorption spectrum was collected using a UV-1800 spectrophotometer. Use the F-7000 fluorescence spectrophotometer to record fluorescence emissions. Using TMS as the internal reference, \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded on the Bruker INOVA-400 NMR spectrometer wspectrometer. High-resolution spectral analysis (HRMS) was performed on the LCMS-9030 QT0F plus mass spectrometer.Prepare standard PBS (10 mM) solutions with different pH values using a PES-3E pH meter. Other instruments include vertical rotary steamer, SHB-III water circulation multi-purpose vacuum pump, Gas phase mass spectrometry (GC-MS), Organic Synthesis Monitoring Mass Spectrometer (OMS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Probe DMT\u003c/h2\u003e \u003cp\u003eSynthesis of compounds 1 and 2: Synthesis according to known literature [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], see supporting literature for details.\u003c/p\u003e \u003cp\u003eSynthesis of compound 3: Compound 2 (71.4 mg, 0.3 mmol) and 3-quinoline formaldehyde (51.8 mg, 0.33 mmol) were dissolved in 10 mL ultra-dry acetonitrile solution in a 25 mL round-bottled flask, and 2 drops of piperidine were added as a catalyst. The reaction was carried out at 80\u0026deg;C for 8 h by reflux and monitored by TLC until the reaction was complete (development agent:Petroleum ether/ethyl acetate\u0026thinsp;=\u0026thinsp;2:1). The obtained mixed solution was filtered by vacuum to obtain a deep red solid 101 mg with a yield of 89.7%. No further purification required. ESI-MS: calculated for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eS: [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e378.1; measured: 377.9.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSynthesis of probe DMT: Compound 3 (75.4 mg, 0.2 mmol) and methyl trifluoromethanesulfonate (131 mg, 0.8 mmol) were added into a 50 mL round-bottom flask and dissolved in 30 mL anhydrous CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e solution. The reaction was stirring at room temperature for 24 h, and was monitored by TLC until the reaction was complete (developing agent:Dichloromethane/methanol\u0026thinsp;=\u0026thinsp;20:1). The mixed solution was filtered by vacuum to obtain 106 mg orange solid with a yield of 97.7%. \u003csup\u003e1\u003c/sup\u003eH NMR (Fig. S5) (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 9.93 (s, 1H), 9.43 (s, 1H), 8.52 (s, 1H), 8.48 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 1H), 8.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 8.28\u0026ndash;8.20 (m, 1H), 8.04 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 1H), 7.95 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.4 Hz, 1H), 7.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.57 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.1 Hz, 2H), 4.64 (s, 3H), 2.40 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (Fig. S6) (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 155.07, 150.14, 146.26, 144.08, 139.29, 137.87, 136.33, 134.62, 131.54, 131.08, 131.03, 130.10, 129.61, 129.19, 128.52, 127.72, 124.58, 123.49, 119.71, 117.44, 116.14, 68.68, 46.25, 21.46. ESI-MS (Fig. S4): calculated for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e: [M]\u003csup\u003e+\u003c/sup\u003e392.1216; measured: 392.1213.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStudy on UV-VIS absorption spectra of hydrogen sulfide by probe DMT\u003c/h2\u003e \u003cp\u003eIn order to evaluate the spectral properties of probe DMT on hydrogen sulfide, the UV-visible absorption spectra after the interaction between probe DMT and S\u003csup\u003e2\u0026minus;\u003c/sup\u003e were tested, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAs can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the maximum absorption wavelength of probe DMT is at 480 nm. After the addition of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e, the maximum absorption peak at 480 nm gradually decreased, and the peak at 378 nm also gradually decreased, and a new strong absorption peak appeared at 600 nm, indicating that S\u003csup\u003e2\u0026minus;\u003c/sup\u003e reacted with the probe DMT, changing the original conjugate structure of the probe molecules leading to these phenomena.The methylquinoline group has fluorescence quenching effect on the probe, and the reaction of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e with the probe causes the probe to restore the intramolecular ICT effect, and the intramolecular charge distribution occurs a new layout. When the concentration of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e reached 500 \u0026micro;M, the absorption peak intensity did not change. After the probe reacted with S\u003csup\u003e2\u0026minus;\u003c/sup\u003e, the color of the solution changed from yellow to blue, and the change could be directly observed by the naked eyes.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStudy on fluorescence spectra of hydrogen sulfide by probe DMT\u003c/h2\u003e \u003cp\u003eThe fluorescence spectra of the interaction between probe DMT and S\u003csup\u003e2\u0026minus;\u003c/sup\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Probe DMT shows almost no fluorescence in PBS buffer, while the probe showed an emission band centered at 680 nm (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;590 nm) after interacting with S\u003csup\u003e2\u0026minus;\u003c/sup\u003e. With the increase of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e concentration, the fluorescence intensity at λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;684 nm gradually increased by about 60 fold, which was attributed to the recovery of intramolecular ICT effect. When the concentration of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e reached 500 \u0026micro;M, the fluorescence intensity hardly changed. As can be seen from the small figure in the upper right corner, the product solution after the reaction of probe DMT and S\u003csup\u003e2\u0026minus;\u003c/sup\u003e was purple red fluorescence. The relationship between fluorescence intensity and concentration of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, and the concentration of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e below 500 \u0026micro;M has a good linear relationship (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9926, K\u0026thinsp;=\u0026thinsp;2.1706).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStability of probe DMT for S\u003csup\u003e2-\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eIn order to explore the stability effect of the probe in buffer solution (EtOH:PBS\u0026thinsp;=\u0026thinsp;1:1), the fluorescence intensity of DMT in different pH environments was measured, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The probe was almost unaffected by the pH of the environment, and the fluorescence intensity of the solution was enhanced with the addition of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, in a strongly acidic environment, the fluorescence intensity of the probe did not increase significantly after adding S\u003csup\u003e2\u0026minus;\u003c/sup\u003e, but showed obvious fluorescence enhancement in an environment with pH\u0026thinsp;=\u0026thinsp;6.5\u0026ndash;10.5. This indicates that probe DMT can be used as a detection tool for in vivo cell imaging and biological imaging applications.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReactivity of probe DMT to hydrogen sulfide\u003c/h3\u003e\n\u003cp\u003eTime is a key factor to evaluate the properties of fluorescent probes. In order to study the reaction performance of probe DMT to S\u003csup\u003e2\u0026minus;\u003c/sup\u003e, the response time of probe and its reaction was measured, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e that the probe can respond to S\u003csup\u003e2\u0026minus;\u003c/sup\u003e in a very short time and reach an equilibrium state at 10 s.\u003c/p\u003e \n\u003ch3\u003eSelectivity of probe DMT to hydrogen sulfide\u003c/h3\u003e\n\u003cp\u003eSelectivity is one of the important parameters to evaluate the performance of fluorescent probes. In this paper, Leu, Val, Phe, Ala, Met, Arg, Pro, Gly, CaCl\u003csub\u003e2\u003c/sub\u003e, MgCl\u003csub\u003e2\u003c/sub\u003e, KCl, NaCl, NaBr, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, NaNO\u003csub\u003e2\u003c/sub\u003e, NaF, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, BaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, Na\u003csub\u003e2\u003c/sub\u003eS\u0026middot;9H\u003csub\u003e2\u003c/sub\u003eO, Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, NaHSO\u003csub\u003e3\u003c/sub\u003e, GSH, Cys and Hcy were analyzed to explore the selectivity of probe DMT and its interaction, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAs can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, after adding S\u003csup\u003e2\u0026minus;\u003c/sup\u003e, the probe DMT solution showed obvious fluorescence enhancement at 684 nm, and the fluorescence intensity increased slightly after adding GSH. Since the probe DMT solution added with GSH has no significant change under visible light and 365 nm UV lamp, and there is a significant difference in fluorescence enhancement ratio, it can be well distinguished between the two. The addition of other anions, cations, amino acids and other biological mercaptans had no obvious effect. The results show that probe DMT has good selectivity for S\u003csup\u003e2\u0026minus;\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe anti-interference of probe DMT to S\u003csup\u003e2-\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, there was no obvious fluorescence change in probe DMT solution after adding other analytical species except S\u003csup\u003e2\u0026minus;\u003c/sup\u003e. After adding 500 \u0026micro;M S\u003csup\u003e2\u0026minus;\u003c/sup\u003e solution to different solutions, the fluorescence intensity of probe DMT at 684 nm showed obvious enhancement, and the fluorescence intensity was only slightly different from that of only 500 \u0026micro;M S\u003csup\u003e2\u0026minus;\u003c/sup\u003e solution. Therefore, it can be shown that the probe DMT has excellent selectivity to S\u003csup\u003e2\u0026minus;\u003c/sup\u003e and strong anti-interference to other analytical substances.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSensing mechanism study\u003c/h2\u003e \u003cp\u003eThrough theoretical analysis and literature investigation, it is speculated that the nucleophilic addition reaction occurs between H\u003csub\u003e2\u003c/sub\u003eS and the double bond on the pyridine ring in the probe molecule DMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), and the fluorescence is turned on [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The proposed mechanism was verified by measuring the molecular weight of the probe DMT reacting with H\u003csub\u003e2\u003c/sub\u003eS. As shown in the figure, after adding 250 eq H\u003csub\u003e2\u003c/sub\u003eS to fully combine it with the probe molecule, the mass spectrum peak of the reaction product [DMT\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eS\u0026thinsp;+\u0026thinsp;Na\u003csup\u003e+\u003c/sup\u003e] was measured to be m/z\u0026thinsp;=\u0026thinsp;448.0657, and its theoretical value was 448.0918, both values were basically consistent (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, a novel NIR fluorescence probe DMT for H\u003csub\u003e2\u003c/sub\u003eS recognition was designed and synthesized in this work. The structure of the probe was determined by high-resolution mass spectrometry and NMR. The sensing mechanism of DMT and H\u003csub\u003e2\u003c/sub\u003eS response was explained by HRMS. In vitro titration experimental data show that probe DMT has some excellent properties: The reaction with H\u003csub\u003e2\u003c/sub\u003eS can be complete within 10 s, the maximum fluorescence emission wavelength can reach the near infrared 684 nm and the fluorescence intensity is increased by about 60 fold, the large Snokes shift (94 nm), and the weak alkaline conditions show good selectivity and excellent anti-interference. It provides a new method for the rapid detection of H\u003csub\u003e2\u003c/sub\u003eS in the environment and living cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Hunan Provincial Natural Science Foundation of China (2019JJ40295)\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Hunan Provincial Natural Science Foundation of China (2019JJ40295)\u0026nbsp;.The authors acknowledge Analyzing \u0026amp; Testing Center, School of chemistry and chemical engineering, Changsha University of Science and Technology for characterizing of samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoci Lv\u0026nbsp;was mainly responsible for probe DMT synthesis and article writing;\u0026nbsp;Yu Xie was\u0026nbsp;involved in performance testing;\u0026nbsp;Heping Li was responsible for revising the article. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDoes not apply to this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang K, Meng J, Bao W, Liu M, Wang X, Tian Z (2021) Mitochondrion-targeting near-infrared fluorescent probe for detecting intracellular nanomolar level hydrogen sulfide with high recognition rate. 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Chem Eng J 427:131563. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://https://doi.org/10.1016/j.cej.2021.131563\u003c/span\u003e\u003cspan address=\"https://10.1016/j.cej.2021.131563\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NIR fluorescent probe, Hydrogen sulfide, Fluorescence intensity","lastPublishedDoi":"10.21203/rs.3.rs-4598713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4598713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS), as an important small molecule bioregulator, plays a key role in many physiological activities and signaling, and abnormal fluctuations in H\u003csub\u003e2\u003c/sub\u003eS concentration can lead to a variety of diseases. Therefore, it is of great significance to develop a near-infrared fluorescence probe to visualize fluctuations in H\u003csub\u003e2\u003c/sub\u003eS levels. This work is based on Sulfur-substituted dicyanomethylene-4H-chromene (DCM), A novel NIR fluorescent probe (E) -3 - (2 - (4 - (dicyanomethylene) -6-methyl-4H-Thiochromen-2-yl)vinyl-1-methylquinolin-1-ium (DMT) was synthesized successfully. Research has found that in weakly alkaline environments, the probe DMT reacts rapidly with H\u003csub\u003e2\u003c/sub\u003eS (only 10 s), the fluorescence intensity at 684 nm is enhanced by about 60 fold, the detection limit is as low as 0.1623 \u0026micro;M, the Stokes shift is large (94 nm), and strong selectivity as well as anti-interference ability towards H\u003csub\u003e2\u003c/sub\u003eS. This will provide a new method for the rapid detection and further application of H\u003csub\u003e2\u003c/sub\u003eS.\u003c/p\u003e","manuscriptTitle":"A Novel NIR Fluorescent Probe for Rapid Response to Hydrogen Sulfide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 06:18:27","doi":"10.21203/rs.3.rs-4598713/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-04T11:23:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T08:24:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197018435991275167672628601308148350205","date":"2024-07-04T00:59:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T12:53:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-20T18:01:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-20T18:00:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2024-06-18T08:59:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3a7e76e3-9e26-4015-9528-e0b44c7db2e2","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-27T00:32:22+00:00","versionOfRecord":{"articleIdentity":"rs-4598713","link":"https://doi.org/10.1007/s10895-024-03857-9","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2024-07-26 00:32:22","publishedOnDateReadable":"July 26th, 2024"},"versionCreatedAt":"2024-07-17 06:18:27","video":"","vorDoi":"10.1007/s10895-024-03857-9","vorDoiUrl":"https://doi.org/10.1007/s10895-024-03857-9","workflowStages":[]},"version":"v1","identity":"rs-4598713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4598713","identity":"rs-4598713","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}