A AIE probe for visual monitoring of endogenous and exogenous H2S and food freshness

A AIE probe for visual monitoring of endogenous and exogenous H2S and food freshness | 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 AIE probe for visual monitoring of endogenous and exogenous H 2 S and food freshness Rongqi Wang, Si Chen, Yamin Li, Tuotuo Fang, Zhanbin Jin, Tingting Wei, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8862141/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Hydrogen sulfide (H 2 S) is a crucial endogenous gas transmitter for the evaluation of food spoilage and disease diagnosis. Thus, we have effectively prepared a novel AIE probe EHAT , which displayed a obvious fluorescence color change after the addition of H 2 S (from orangered to green), suggesting that probe EHAT can identify H 2 S with the naked eye. The probe EHAT -based test strip was also developed to conveniently detect H 2 S gas generated during food spoilage. Moreover, the probe EHAT were successfully utilized to image endogenous and exogenous H 2 S in live cells. Aggregation-Induced Emission (AIE) Endogenous and exogenous H2S Visual detection Food freshness Cell imageing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Hydrogen sulfide (H 2 S), a well-known gas for its characteristic foul odour of rotten egg, is participated in lots of physiological and pathological activities [ 1 – 5 ]. Abnormal H 2 S level is associated with the progression of a variety of diseases, such as Parkinson's disease, diabetes and liver diseases [ 6 – 8 ]. In addition, H 2 S is one of the main volatiles generated in the process of protein food spoilage and has negative impacts on human health [ 9 – 12 ]. Therefore, for the protection of human health, it is particularly important to develop powerful tools for the rapid detection of H 2 S derivatives. Currently, several assays for detecting H 2 S have been developed, such as chromatography, spectroscopy, and electronic noses. Nevertheless, these sensing methods have limitations as they are destructive, expensive and relying on expensive instruments and skilled operators [ 13 – 16 ]. Fluorescent probes have attracted tremendous interest for monitoring the biological active molecules in living system owning to its good sensitivity, high selectivity, easy operation as well as good biocompatibility [ 17 – 20 ]. Compared with traditional fluorescent dyes with ACQ effect, the AIE molecules are nonemissive in the dispersed state, but in the aggregate state, they are intensively emissive, which enable high fluorescence in water solution [ 21 – 25 ]. The colormetric fluorescent probes with two independent fluorescence color could provide visual recognition [ 26 – 28 ]. It’s highly urgent to develop an ultrasensitive ratiometric AIE probe for selectively imaging of H 2 S in mitochondria. Therefore, it’s highly urgent to develop a colormetric AIE probes for multifunctional applications in food and biological system. In this content, we developed a AIE probe EHAT , which displayed strong yellow fluorescence in solid state. Moreover, EHAT displayed a fluorescence ratiometric response from dark red to green upon addition of H 2 S over other interfering species in 90% aqueous EtOH solution. The EHAT- based test paper was prepared by loading EHAT onto filter paper, are capable of real-time visual monitoring of freshness of protein food based on H 2 S production from microbial growth. Interestingly, EHAT can image exogenous and endogenous H 2 S in living cells. Moreover, the EHAT will emerge as a promising and valuable tool for food safety inspection and bioimaging. 2. Experimental 2.1. General comments Fluorescence spectra were carried out in PerkinElmer LS55. The 1 H NMR and 13 C NMR spectra were collected at 600 MHz and 500 MHz using a Bruck AV-600 spectrometer in DMSO- d 6 , respectively. The IR spectra was acquired with a Perkin-Elmer IR spectrophotometer using KBr pellet. Mass spectra were obtained on an Agilent 6220 Quadruple LC/MS (Agilent Co, USA). All the materials for synthesis and spectral analysis were commercial available. For fluorescence measurements, excitation wavelength was 400 nm and the excitation and emission wavelength band passes were both set at 5 nm and 5 nm. 2.2 synthesis In Scheme 1 , 2 -(3-Formyl-4-hydroxy-phenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester (291 mg, 1 mmol) and N-Aminorhodanine (148 mg, 1 mmol) were added to ethanol and added the two drops of glacial acetic acid and the mixture was refluxed with stirring for 8 h accompanied by stirring and cooled to room temperature [29] . The precipitate was filtrated to give the red powders of HNTE (265 mg, yield 64%). 1 H NMR spectrum of EHAT (Figure. S1) (DMSO- d 6 , 600 MHz) δ 11.45 (s, 1H), 9.06 (s, 1H), 8.53 (d, 1H), 8.06 (q, 1H), 7.15 (d, 1H), 4.35 (q, 1H), 4.29 (s, 2H), 2.69 (t, 3H), 1.31 (t, 3H). 13 C NMR spectrum of EHAT (Figure. S2) 13 C NMR (101 MHz, DMSO- d 6 ) δ 166.08, 161.86, 161.40, 160.73, 143.24, 133.26, 126.30, 133.26, 126.50, 124.45, 120.97, 118.96, 118.27, 61.61, 35.24, 18.72, 17.67, 14.61. HRMS (Figure. S3): calcd for C 17 H 16 N 3 O 4 S 3 : 422.0303 [ EHAT + H] + , found: 422.0301. 2.3 Preparation of test strips The filter paper was cut into circle pieces and dipped in probe EHAT ethanol solution (10 µM), and it was subsequently dried to get the EHAT -based test strips. Then these strips exposed to the different concentration of H 2 S. 2.4 Cell incubation and fluorescence image A549 cells were cultured with DMEM which supplemented with 10% (v/v) fetal bovine serum. The Hela cells were incubated with 10 µM of EHAT for 30 min at 37 ◦ C. Then the other analytes were added and incubated for another 30 min, respectively. All of the cell imagings were collected on a confocal scanning microscope. All the measure was performed at room temperature. 3. Results and discussion 3.1 The optical properties of AIE To determine whether the probe EHAT have AIE properties, their emission behaviors in EtOH-H 2 O mixtures and solid state were studied. As shown in Fig. 1 b, when probe EHAT was dissolved in ethanol, its dilute pure ethanol solution emitted orange-yellow fluorescence at 504 nm. With increasing water volume fraction ( f w ) values from 10 to 90%, the fluorescence intensity of the solution of probe EHAT (10 µM) decreased gradually and a new emission peak at 590 nm appeared. The probe EHAT in EtOH/H 2 O (1/9, v/v) solution displayed faint red fluorescence. Above result suggestted that the probe EHAT exhibited obvious AIE activity. Further, the absorption spectra of probe EHAT (10 µM) in the EtOH/water mixtures are shown in Fig. S4. There were two characteristic absorption peaks at 340 and 380 nm. Obvious absorption decrease was observed when water fraction reached a volume fraction ( f w ) over 60%. The result further suggested that water molecular induced aggregation of probe EHAT . 3.2 The fluorescence sensing behaviour studies of EHAT The fluorescent responses of probe EHAT (5 µM) to various analytes (H 2 S, K + , Na + , Ca 2+ , Cd 2+ , Co 2+ , Ni 2+ , Cysteine, Glutathione, Phenylalanine, Glycine, Glutamate, Tyrosine, Leucine, Valine, Lsoleucine, Tryptophan, Serine, Aspartic acid, Methionine, Fluconazole, Chloramphenicol, Penicillin G sodium, Cefalexin, Sulfadiazine, Metronidazole) was assessed in EtOH/H 2 O (1/9, v/v, pH = 7.4) solution. Upon addition of H 2 S (1.0 µM) into the solution, the maximum emission wavelength of probe EHAT displayed a blue shift from 590 nm to 515 nm, while no apparent fluorescence changes were observed in presence of other analytes under the same conditions. Also, the fluorescence of probe EHAT solution changed from red to green after addition of H 2 S. Therefore, the above results indicated that the probe EHAT could detect H 2 S by “naked eye” with high selectivity. Further, the studies for quantitative detection of H 2 S were performed through fluorescence spectra. The fluorescence intensity of probe EHAT increased remarkably along with blue-shift from 590 nm to 515 nm upon addition of H 2 S gradually. The pleasurable linear relationship between fluorescence intensity at 515 nm and the concentration of H 2 S were observed (Fig. S5). Furthermore, the detection limit was calculated to be 1.5×10 − 7 M based on the 3σ/k rules. Above results indicated that the probe could be used to detect H 2 S quantitatively by fluorescence spectra. The competition experiment was performed to study the possible analytes interference (Fig. S6). The fluorescence intensity at 515 nm decreased or changed slightly after added 50 µM of H 2 S to the 10 µM probe EHAT solution in the presence of other above analytes. The above results indicate that the response of probe EHAT to H 2 S is hardly interfered by other analytes. To confirm the effect of pH on probe EHAT fluorescence detection of H 2 S, the experiments were performed at a pH ranging from 2 to 12 (Fig. S7). After addition of H 2 S to the probe EHAT solution, the fluorescence tensity at 515 nm varied significantly in the pH range from 6.0 to 8.0, these results indicated that the probe EHAT can detect H 2 S under complex physiological conditions and is suitable for cell imaging. 3.3. Sensing mechanism of EHAT to H 2 S At the same time, 1 H NMR titration of probe EHAT with H 2 S was described in Fig. S8. The chemical shift at 9.06 ppm (Ha) was obviously weakened upon addition of 1 equiv. H 2 S. Meanwhile, the new characteristic NH absorption peak at 3430 cm − 1 appeared (Fig. S9). Taken together, above findings suggested that the C = N group was attacked by H 2 S based on nucleophilic addition mechanism. The peak at m/z 478.1588 of [ EHAT + H] + and a new peak at m/z 337.1700 of [ EHAT+ H 2 S + H] + (the product of EHAT reacted with H 2 S ) were all found in Fig. S10, indicating that the probe EHAT was interacted with H 2 S by 1:1 addition reaction. The proposed sensing mechanism of probe EHAT towards H 2 S was shown in scheme 2 . The presence of H 2 O molecular will help for the formation of new intermolecular hydrogen bond (O … H-O), which is benefit for the formation of aggregation state and showed the obvious red fluorescence. After the nucleophilic addition reaction with H 2 S, the intramolecular conjugation of EHAT was weaken, another O … S-H intramolecular hydrogen bond will be formed and resulted the the new aggregates was formed between EHAT and H 2 S, causing the blue-shift of emission wavelength. Furthermore, to better understand the optical properties of probe EHAT and EHAT- H 2 S, we calculated energy gap by using the Gaussian 09 program. The optimized geometries and electronic cloud distribution on highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were depicted in Fig. 2 . The electron distribution of probe EHAT is asymmetrical: The LUMO orbital locates on the benzene ring, C = N double bond and adjacent thiazole ring, while the HOMO orbital was primarily distributed over the benzene ring and adjacent thiazole ring. After reaction of probe EHAT with H 2 S, the LUMO (-2.10 eV) are mainly located at the benzene ring and adjacent thiazole group; However its HOMOs (-6.04 eV) were distributed over thiazole unit. The energy gaps of EHAT and EHAT- H 2 S were 3.70 ev and 3.94 ev, respectively. 3.4 Response of probe EHAT -based test strips to H 2 S To evaluate the practical application of the portable probe EHAT , we tentatively prepared EHAT -based strips to detect H 2 S conveniently. The filter paper was cut into various pieces and dipped in probe EHAT ethanol solution (10 µM), and it was subsequently dried to get the EHAT -based test strips. The fluorescence colors of EHAT -based test strips caused significant changes from orangered to green upon the addition of various concentration of H 2 S solutions (10 µM, 20 µM, 30 µM, 40 µM and 50 µM). Seeing from the fluorescence color change of the EHAT -based test strips, there is an obvious H 2 S concentration-dependent relationship (Fig. 3 ) by smartphone software (RGB analytical app). The H 2 S concentration can be correctly read out by the naked eye. Above results suggested that the smartphone sensing platform could quantitatively measure the H 2 S value. 3.5. Application of probe EHAT in the foodstuffs The H 2 S are generated during storage and processing of protein-rich foods. To estimate the practical application of the probe EHAT , the EHAT- based strips was used to detect the freshness of shrimp, chicken, pork and fish. When EHAT- based strips were treated with protein-rich foods (shrimp, chicken, pork and fish) for 0 h, 6 h, 12 h, 18 h, 24 h and 36 h, obvious fluorescence colour changes of EHAT- based strips from orangered to green were obviously seen by the naked eye under 365 nm UV lamp (Fig. 4 ). Above results illustrated that the probe EHAT could be employed as a simple tool of on-site detection of protein-rich freshness. 3.6. Imaging of endogenous and exogenous H 2 S generation in living cells The cytotoxicity of probe EHAT was studied. In Fig. S11, the viability of A549 cells was all over 90% even the concentration of probe EHAT increased from 0 to 50 µM, which indicated probe EHAT featured with low cytotoxicity. Encouraged by the prominent optical performances of probe EHAT , its ability to visualize H 2 S levels in living cells was then investigated. The fluorescence images of living A549 cells were obtained by fluorescence confocal microscope and red channel (570–670 nm) and green channel (500–540 nm) was selected for observations. When A549 cells were nurtured with 10 µM EHAT , a weak red fluorescence and green fluorescence signal was observed. When the cells were pretreated with 10 µM EHAT for 30 min, and then stained with varied concentrations of H 2 S (5 µM, 10 µM, 20 µM, 30 µM, 40 µM and 50 µM) for 30 min. With the gradual addition of exogenous H 2 S, the significantly enhanced red fluorescence and attenuated green fluorescence were presented (Fig. 5 ), suggesting that the probe EHAT performed well for supervising of the exogenous H 2 S generation in living cells. Next, that the probe EHAT was used to detect the endogenous H 2 S generation in living cells was studied (Fig. 6 ). The A549 cells was incubated with 10 µM EHAT and NEM (a hydrogen sulfide remover) for 30 min, which displayed red fluorescence signals and green fluorescence signals. Then on the addition and subsequent incubation with an endogenous hydrogen sulfide inducer (L-Cys) for 30 min, the obvious green fluorescence was observed. The above results indicated that the probe EHAT could as a ideal tool for ratiometric monitoring endogenous H 2 S in living cells via ratiometric image way. we expected that the probe EHAT could be employed to the ratiometric imaging of exogenous and endogenous H 2 S in living cells. Declarations Acknowledgements Funding Declaration This work was supported by the Scientic Research and Technology Development Plan of Baise (Grant No. 20250315), the Fundamental Research Funds for the Guangxi Natural Science Foundation Program (Grant No. 2025GXNSFHA069268), the Young academic leaders of Xinzhou Normal University (No. X20240013), Scientific and Technological Inno-vation Programs of Higher Education Institutions in Shanxi (No. 2022L465 and No. 2021L454), Fundamental Research Program of Shanxi Province (No. 202203021222307 and 202303021222235), Natural Science Foundation of China (22277104) for financial assistance. Author Contributions Rongqi Wang, Si Chen, Yamin Li, Tuotuo Fang, Zhan-Bin Jin, Ting-Ting Wei, Jia-Ping Li and Hai-Xian Ren wrote the main manuscript text. Zi-Ao Zong and Na-Na Li prepared Figs.1, 2, 3, 4, 5, 6, scheme 1 and scheme 2. All authors reviewed the manuscript. 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Zong, Fluorescence probe with aggregation induced emission effect and application for colormetric detection of endogenous and exogenous hypochlorite, Dyes Pigments 210 (2023) 110965. Scheme Scheme 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Scheme1.png Scheme2.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 15 Apr, 2026 Reviews received at journal 26 Mar, 2026 Reviews received at journal 22 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers invited by journal 16 Mar, 2026 Editor assigned by journal 26 Feb, 2026 Submission checks completed at journal 26 Feb, 2026 First submitted to journal 12 Feb, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-8862141","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608023262,"identity":"db175778-b851-403a-a538-7e2b3980b87b","order_by":0,"name":"Rongqi Wang","email":"","orcid":"","institution":"Affiliated Hospital of Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Rongqi","middleName":"","lastName":"Wang","suffix":""},{"id":608023267,"identity":"6d84d361-f0a0-49fc-a686-2005bd98eb9a","order_by":1,"name":"Si Chen","email":"","orcid":"","institution":"Youjiang Medical College for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Si","middleName":"","lastName":"Chen","suffix":""},{"id":608023270,"identity":"5f966b6d-5861-4a62-ae87-e8ad81ca1223","order_by":2,"name":"Yamin Li","email":"","orcid":"","institution":"Guangdong Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Yamin","middleName":"","lastName":"Li","suffix":""},{"id":608023273,"identity":"8bdfae0d-399c-458c-88fa-f78497126bc5","order_by":3,"name":"Tuotuo Fang","email":"","orcid":"","institution":"ZhongKe (Guangdong) Refinery \u0026 Petrochemical Company Limited","correspondingAuthor":false,"prefix":"","firstName":"Tuotuo","middleName":"","lastName":"Fang","suffix":""},{"id":608023275,"identity":"2638ea60-6d61-4add-b75e-1f81bf3c716a","order_by":4,"name":"Zhanbin Jin","email":"","orcid":"","institution":"Xinzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhanbin","middleName":"","lastName":"Jin","suffix":""},{"id":608023282,"identity":"3270ec18-1967-4b20-9ffe-e062df8d26f6","order_by":5,"name":"Tingting Wei","email":"","orcid":"","institution":"Xinzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Wei","suffix":""},{"id":608023285,"identity":"8278a90c-48c6-463e-8548-a34e06969181","order_by":6,"name":"Jiaping Li","email":"","orcid":"","institution":"Xinzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jiaping","middleName":"","lastName":"Li","suffix":""},{"id":608023291,"identity":"d2654620-5ea3-4486-8df9-1a5f79b2198e","order_by":7,"name":"Haixian Ren","email":"","orcid":"","institution":"Xinzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Haixian","middleName":"","lastName":"Ren","suffix":""},{"id":608023292,"identity":"326592c3-a728-42e8-8da8-e73f03a2c046","order_by":8,"name":"Ziao Zong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYNACAwYGfmbmgw9I0yLZzpZsQKJF53nMBIhSKd/ee/jljwI7OePDDGYMDDU20QS1MPacS7OQMEg2NjvMkPaA4VhabgMhLcwSOWYGBgbMidsOMxw3YGw4TFgLG0hLgkF94uZmxjYJorTwSOQYPzhgcDhxAzMzG3FaJHjOmDE2GBw3ljjMxmyQQIxf5Nt7jD/++FMtx99//uODDzU2hLWAvQNnJhChHASYPxCpcBSMglEwCkYqAADcPjirwIRWVQAAAABJRU5ErkJggg==","orcid":"","institution":"Youjiang Medical College for Nationalities","correspondingAuthor":true,"prefix":"","firstName":"Ziao","middleName":"","lastName":"Zong","suffix":""},{"id":608023293,"identity":"5d18142b-499e-4d03-9f2e-9dc749e79f8a","order_by":9,"name":"Nana Li","email":"","orcid":"","institution":"Xinzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-02-12 12:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8862141/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8862141/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105039048,"identity":"ad2df2f8-1b14-4c88-8669-c93c16ad7ee5","added_by":"auto","created_at":"2026-03-20 07:45:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402021,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of \u003cstrong\u003eEHAT \u003c/strong\u003e(10 µM) in EtOH/H\u003csub\u003e2\u003c/sub\u003eO with different water fractions (a) and solid state (b). (c) Fluorescence spectra of \u003cstrong\u003eEHAT\u003c/strong\u003e (10 µM) in the presence of various analytes (50 μM) in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1/9, v/v, pH = 7.4) medium. Inset: The color change of \u003cstrong\u003eEHAT\u003c/strong\u003e (10 μM) before and after addition of H\u003csub\u003e2\u003c/sub\u003eS (50 μM) in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (9/1, v/v, pH = 7.4) medium under UV light of 365 nm. (d) The Emission spectral changes of \u003cstrong\u003eEHAT\u003c/strong\u003e (10 μM) upon gradual addition of increasing amounts of H\u003csub\u003e2\u003c/sub\u003eS in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1/9, v/v, pH = 7.4) solution.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/4236d7e70c0a1bbcfb79f1e4.png"},{"id":105038099,"identity":"ed9a0bb1-2efa-4e41-bbef-a13ff2cc92e4","added_by":"auto","created_at":"2026-03-20 07:42:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":259319,"visible":true,"origin":"","legend":"\u003cp\u003eThe calculated molecular orbitals and the HOMO-LUMO gaps of \u003cstrong\u003eEHAT \u003c/strong\u003eand\u003cstrong\u003e EHAT -H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/1ba48d781730cea11373c124.png"},{"id":105038040,"identity":"23c456b7-d664-4a1c-8800-14a6a0cee06e","added_by":"auto","created_at":"2026-03-20 07:41:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116422,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The images of \u003cstrong\u003eEHAT\u003c/strong\u003e-based test strips for the detection of H\u003csub\u003e2\u003c/sub\u003eS under 365 nm UV lamp. (b) RGB value of the fluorescent photo analyzed by the color recognizer of the smartphone.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/4aa99cef1453e1ee0625204c.png"},{"id":105039963,"identity":"75123a13-60fb-429f-b8c5-1927aaf72b43","added_by":"auto","created_at":"2026-03-20 07:47:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":972510,"visible":true,"origin":"","legend":"\u003cp\u003eImages of chicken, shrimp, fish and pork under sunlight and 365 nm UV light.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/b43452bb6d4a25e3e1f642c6.png"},{"id":105039009,"identity":"a0627882-94eb-4417-b25d-956555c35e8e","added_by":"auto","created_at":"2026-03-20 07:44:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":477347,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence images of probe \u003cstrong\u003eEHAT\u003c/strong\u003e exogenous H\u003csub\u003e2\u003c/sub\u003eS in A549 cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/364753146f2a1ba099ca819d.png"},{"id":105038144,"identity":"88eadfdc-0a46-4417-a869-a65196645130","added_by":"auto","created_at":"2026-03-20 07:42:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":347969,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence images of probe \u003cstrong\u003eEHAT\u003c/strong\u003e for endogenous H\u003csub\u003e2\u003c/sub\u003eS in A549 cells.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/09bba0998e0603e9aa9d164b.png"},{"id":107479706,"identity":"ec3a8774-59ae-4c5a-8580-8f9562bc5733","added_by":"auto","created_at":"2026-04-22 01:45:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2907193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/0dcde054-dec1-44ea-89d0-cbdf1ca0693f.pdf"},{"id":105039761,"identity":"a0d2d07c-9dd7-4e5a-81f0-f095436ceb6c","added_by":"auto","created_at":"2026-03-20 07:47:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1242365,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/b9f3eacfa91c71fda8641b51.docx"},{"id":105038844,"identity":"4bea3777-a5a6-42cc-b4ef-1104ec0733ba","added_by":"auto","created_at":"2026-03-20 07:44:41","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28517,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/2259dedb37207d43bd722db1.png"},{"id":105039061,"identity":"a18e9924-4df3-46a9-bf81-5d6b81882282","added_by":"auto","created_at":"2026-03-20 07:45:04","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":134121,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-8862141/v1/641559cfc8b9b8b859968aa0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eA AIE probe for visual monitoring of endogenous and exogenous H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS and food freshness\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS), a well-known gas for its characteristic foul odour of rotten egg, is participated in lots of physiological and pathological activities [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Abnormal H\u003csub\u003e2\u003c/sub\u003eS level is associated with the progression of a variety of diseases, such as Parkinson's disease, diabetes and liver diseases [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, H\u003csub\u003e2\u003c/sub\u003eS is one of the main volatiles generated in the process of protein food spoilage and has negative impacts on human health [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, for the protection of human health, it is particularly important to develop powerful tools for the rapid detection of H\u003csub\u003e2\u003c/sub\u003eS derivatives.\u003c/p\u003e \u003cp\u003eCurrently, several assays for detecting H\u003csub\u003e2\u003c/sub\u003eS have been developed, such as chromatography, spectroscopy, and electronic noses. Nevertheless, these sensing methods have limitations as they are destructive, expensive and relying on expensive instruments and skilled operators [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Fluorescent probes have attracted tremendous interest for monitoring the biological active molecules in living system owning to its good sensitivity, high selectivity, easy operation as well as good biocompatibility [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Compared with traditional fluorescent dyes with ACQ effect, the AIE molecules are nonemissive in the dispersed state, but in the aggregate state, they are intensively emissive, which enable high fluorescence in water solution [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The colormetric fluorescent probes with two independent fluorescence color could provide visual recognition [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It\u0026rsquo;s highly urgent to develop an ultrasensitive ratiometric AIE probe for selectively imaging of H\u003csub\u003e2\u003c/sub\u003eS in mitochondria. Therefore, it\u0026rsquo;s highly urgent to develop a colormetric AIE probes for multifunctional applications in food and biological system.\u003c/p\u003e \u003cp\u003eIn this content, we developed a AIE probe \u003cb\u003eEHAT\u003c/b\u003e, which displayed strong yellow fluorescence in solid state. Moreover, \u003cb\u003eEHAT\u003c/b\u003e displayed a fluorescence ratiometric response from dark red to green upon addition of H\u003csub\u003e2\u003c/sub\u003eS over other interfering species in 90% aqueous EtOH solution. The \u003cb\u003eEHAT-\u003c/b\u003ebased test paper was prepared by loading \u003cb\u003eEHAT\u003c/b\u003e onto filter paper, are capable of real-time visual monitoring of freshness of protein food based on H\u003csub\u003e2\u003c/sub\u003eS production from microbial growth. Interestingly, \u003cb\u003eEHAT\u003c/b\u003e can image exogenous and endogenous H\u003csub\u003e2\u003c/sub\u003eS in living cells. Moreover, the \u003cb\u003eEHAT\u003c/b\u003e will emerge as a promising and valuable tool for food safety inspection and bioimaging.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. General comments\u003c/h2\u003e \u003cp\u003eFluorescence spectra were carried out in PerkinElmer LS55. The \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were collected at 600 MHz and 500 MHz using a Bruck AV-600 spectrometer in DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, respectively. The IR spectra was acquired with a Perkin-Elmer IR spectrophotometer using KBr pellet. Mass spectra were obtained on an Agilent 6220 Quadruple LC/MS (Agilent Co, USA). All the materials for synthesis and spectral analysis were commercial available. For fluorescence measurements, excitation wavelength was 400 nm and the excitation and emission wavelength band passes were both set at 5 nm and 5 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 synthesis\u003c/h2\u003e \u003cp\u003eIn Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-(3-Formyl-4-hydroxy-phenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester (291 mg, 1 mmol) and N-Aminorhodanine (148 mg, 1 mmol) were added to ethanol and added the two drops of glacial acetic acid and the mixture was refluxed with stirring for 8 h accompanied by stirring and cooled to room temperature\u003csup\u003e[29]\u003c/sup\u003e. The precipitate was filtrated to give the red powders of \u003cb\u003eHNTE\u003c/b\u003e (265 mg, yield 64%). \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of \u003cb\u003eEHAT\u003c/b\u003e (Figure. S1) (DMSO- \u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, 600 MHz) δ 11.45 (s, 1H), 9.06 (s, 1H), 8.53 (d, 1H), 8.06 (q, 1H), 7.15 (d, 1H), 4.35 (q, 1H), 4.29 (s, 2H), 2.69 (t, 3H), 1.31 (t, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR spectrum of \u003cb\u003eEHAT\u003c/b\u003e (Figure. S2) \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 166.08, 161.86, 161.40, 160.73, 143.24, 133.26, 126.30, 133.26, 126.50, 124.45, 120.97, 118.96, 118.27, 61.61, 35.24, 18.72, 17.67, 14.61. HRMS (Figure. S3): calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e: 422.0303 [\u003cb\u003eEHAT\u003c/b\u003e\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found: 422.0301.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of test strips\u003c/h2\u003e \u003cp\u003eThe filter paper was cut into circle pieces and dipped in probe \u003cb\u003eEHAT\u003c/b\u003e ethanol solution (10 \u0026micro;M), and it was subsequently dried to get the \u003cb\u003eEHAT\u003c/b\u003e-based test strips. Then these strips exposed to the different concentration of H\u003csub\u003e2\u003c/sub\u003eS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell incubation and fluorescence image\u003c/h2\u003e \u003cp\u003eA549 cells were cultured with DMEM which supplemented with 10% (v/v) fetal bovine serum. The Hela cells were incubated with 10 \u0026micro;M of \u003cb\u003eEHAT\u003c/b\u003e for 30 min at 37 \u003csup\u003e◦\u003c/sup\u003eC. Then the other analytes were added and incubated for another 30 min, respectively. All of the cell imagings were collected on a confocal scanning microscope.\u003c/p\u003e \u003cp\u003eAll the measure was performed at room temperature.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The optical properties of AIE\u003c/h2\u003e \u003cp\u003eTo determine whether the probe \u003cb\u003eEHAT\u003c/b\u003e have AIE properties, their emission behaviors in EtOH-H\u003csub\u003e2\u003c/sub\u003eO mixtures and solid state were studied. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, when probe \u003cb\u003eEHAT\u003c/b\u003e was dissolved in ethanol, its dilute pure ethanol solution emitted orange-yellow fluorescence at 504 nm. With increasing water volume fraction (\u003cem\u003ef\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e) values from 10 to 90%, the fluorescence intensity of the solution of probe \u003cb\u003eEHAT\u003c/b\u003e (10 \u0026micro;M) decreased gradually and a new emission peak at 590 nm appeared. The probe \u003cb\u003eEHAT\u003c/b\u003e in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1/9, v/v) solution displayed faint red fluorescence. Above result suggestted that the probe \u003cb\u003eEHAT\u003c/b\u003e exhibited obvious AIE activity. Further, the absorption spectra of probe \u003cb\u003eEHAT\u003c/b\u003e (10 \u0026micro;M) in the EtOH/water mixtures are shown in Fig. S4. There were two characteristic absorption peaks at 340 and 380 nm. Obvious absorption decrease was observed when water fraction reached a volume fraction (\u003cem\u003ef\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e) over 60%. The result further suggested that water molecular induced aggregation of probe \u003cb\u003eEHAT\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.2 The fluorescence sensing behaviour studies of\u003c/em\u003e \u003cb\u003eEHAT\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe fluorescent responses of probe \u003cb\u003eEHAT\u003c/b\u003e (5 \u0026micro;M) to various analytes (H\u003csub\u003e2\u003c/sub\u003eS, K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Cysteine, Glutathione, Phenylalanine, Glycine, Glutamate, Tyrosine, Leucine, Valine, Lsoleucine, Tryptophan, Serine, Aspartic acid, Methionine, Fluconazole, Chloramphenicol, Penicillin G sodium, Cefalexin, Sulfadiazine, Metronidazole) was assessed in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1/9, v/v, pH\u0026thinsp;=\u0026thinsp;7.4) solution. Upon addition of H\u003csub\u003e2\u003c/sub\u003eS (1.0 \u0026micro;M) into the solution, the maximum emission wavelength of probe \u003cb\u003eEHAT\u003c/b\u003e displayed a blue shift from 590 nm to 515 nm, while no apparent fluorescence changes were observed in presence of other analytes under the same conditions. Also, the fluorescence of probe \u003cb\u003eEHAT\u003c/b\u003e solution changed from red to green after addition of H\u003csub\u003e2\u003c/sub\u003eS. Therefore, the above results indicated that the probe \u003cb\u003eEHAT\u003c/b\u003e could detect H\u003csub\u003e2\u003c/sub\u003eS by \u0026ldquo;naked eye\u0026rdquo; with high selectivity.\u003c/p\u003e \u003cp\u003eFurther, the studies for quantitative detection of H\u003csub\u003e2\u003c/sub\u003eS were performed through fluorescence spectra.\u003c/p\u003e \u003cp\u003eThe fluorescence intensity of probe \u003cb\u003eEHAT\u003c/b\u003e increased remarkably along with blue-shift from 590 nm to 515 nm upon addition of H\u003csub\u003e2\u003c/sub\u003eS gradually. The pleasurable linear relationship between fluorescence intensity at 515 nm and the concentration of H\u003csub\u003e2\u003c/sub\u003eS were observed (Fig. S5). Furthermore, the detection limit was calculated to be 1.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M based on the 3σ/k rules. Above results indicated that the probe could be used to detect H\u003csub\u003e2\u003c/sub\u003eS quantitatively by fluorescence spectra.\u003c/p\u003e \u003cp\u003eThe competition experiment was performed to study the possible analytes interference (Fig. S6). The fluorescence intensity at 515 nm decreased or changed slightly after added 50 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eS to the 10 \u0026micro;M probe \u003cb\u003eEHAT\u003c/b\u003e solution in the presence of other above analytes. The above results indicate that the response of probe \u003cb\u003eEHAT\u003c/b\u003e to H\u003csub\u003e2\u003c/sub\u003eS is hardly interfered by other analytes.\u003c/p\u003e \u003cp\u003eTo confirm the effect of pH on probe \u003cb\u003eEHAT\u003c/b\u003e fluorescence detection of H\u003csub\u003e2\u003c/sub\u003eS, the experiments were performed at a pH ranging from 2 to 12 (Fig. S7). After addition of H\u003csub\u003e2\u003c/sub\u003eS to the probe \u003cb\u003eEHAT\u003c/b\u003e solution, the fluorescence tensity at 515 nm varied significantly in the pH range from 6.0 to 8.0, these results indicated that the probe \u003cb\u003eEHAT\u003c/b\u003e can detect H\u003csub\u003e2\u003c/sub\u003eS under complex physiological conditions and is suitable for cell imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Sensing mechanism of \u003cb\u003eEHAT\u003c/b\u003e to H\u003csub\u003e2\u003c/sub\u003eS\u003c/h2\u003e \u003cp\u003eAt the same time, \u003csup\u003e1\u003c/sup\u003eH NMR titration of probe \u003cb\u003eEHAT\u003c/b\u003e with H\u003csub\u003e2\u003c/sub\u003eS was described in Fig. S8. The chemical shift at 9.06 ppm (Ha) was obviously weakened upon addition of 1 equiv. H\u003csub\u003e2\u003c/sub\u003eS. Meanwhile, the new characteristic NH absorption peak at 3430 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e appeared (Fig. S9). Taken together, above findings suggested that the C\u0026thinsp;=\u0026thinsp;N group was attacked by H\u003csub\u003e2\u003c/sub\u003eS based on nucleophilic addition mechanism. The peak at \u003cem\u003em/z\u003c/em\u003e 478.1588 of [\u003cb\u003eEHAT\u003c/b\u003e\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e and a new peak at \u003cem\u003em/z\u003c/em\u003e 337.1700 of [\u003cb\u003eEHAT+\u003c/b\u003e H\u003csub\u003e2\u003c/sub\u003eS\u0026thinsp;\u003cb\u003e+\u0026thinsp;H]\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e (the product of \u003cb\u003eEHAT\u003c/b\u003e reacted with H\u003csub\u003e2\u003c/sub\u003eS\u003cb\u003e)\u003c/b\u003e were all found in Fig. S10, indicating that the probe \u003cb\u003eEHAT\u003c/b\u003e was interacted with H\u003csub\u003e2\u003c/sub\u003eS by 1:1 addition reaction. The proposed sensing mechanism of probe \u003cb\u003eEHAT\u003c/b\u003e towards H\u003csub\u003e2\u003c/sub\u003eS was shown in scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The presence of H\u003csub\u003e2\u003c/sub\u003eO molecular will help for the formation of new intermolecular hydrogen bond (O\u003csup\u003e\u0026hellip;\u003c/sup\u003eH-O), which is benefit for the formation of aggregation state and showed the obvious red fluorescence. After the nucleophilic addition reaction with H\u003csub\u003e2\u003c/sub\u003eS, the intramolecular conjugation of \u003cb\u003eEHAT\u003c/b\u003e was weaken, another O\u003csup\u003e\u0026hellip;\u003c/sup\u003eS-H intramolecular hydrogen bond will be formed and resulted the the new aggregates was formed between \u003cb\u003eEHAT\u003c/b\u003e and H\u003csub\u003e2\u003c/sub\u003eS, causing the blue-shift of emission wavelength.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, to better understand the optical properties of probe \u003cb\u003eEHAT\u003c/b\u003e and \u003cb\u003eEHAT-\u003c/b\u003eH\u003csub\u003e2\u003c/sub\u003eS, we calculated energy gap by using the Gaussian 09 program. The optimized geometries and electronic\u003c/p\u003e \u003cp\u003ecloud distribution on highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The electron distribution of probe \u003cb\u003eEHAT\u003c/b\u003e is asymmetrical: The LUMO orbital locates on the benzene ring, C\u0026thinsp;=\u0026thinsp;N double bond and adjacent thiazole ring, while the HOMO orbital was primarily distributed over the benzene ring and adjacent thiazole ring. After reaction of probe \u003cb\u003eEHAT\u003c/b\u003e with H\u003csub\u003e2\u003c/sub\u003eS, the LUMO (-2.10 eV) are mainly located at the benzene ring and adjacent thiazole group; However its HOMOs (-6.04 eV) were distributed over thiazole unit. The energy gaps of \u003cb\u003eEHAT\u003c/b\u003e and \u003cb\u003eEHAT-\u003c/b\u003eH\u003csub\u003e2\u003c/sub\u003eS were 3.70 ev and 3.94 ev, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.4 Response of probe\u003c/em\u003e \u003cb\u003eEHAT\u003c/b\u003e-based test strips \u003cem\u003eto H\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo evaluate the practical application of the portable probe \u003cb\u003eEHAT\u003c/b\u003e, we tentatively prepared \u003cb\u003eEHAT\u003c/b\u003e -based strips to detect H\u003csub\u003e2\u003c/sub\u003eS conveniently. The filter paper was cut into various pieces and dipped in probe \u003cb\u003eEHAT\u003c/b\u003e ethanol solution (10 \u0026micro;M), and it was subsequently dried to get the \u003cb\u003eEHAT\u003c/b\u003e-based test strips. The fluorescence colors of \u003cb\u003eEHAT\u003c/b\u003e-based test strips caused significant changes from orangered to green upon the addition of various concentration of H\u003csub\u003e2\u003c/sub\u003eS solutions (10 \u0026micro;M, 20 \u0026micro;M, 30 \u0026micro;M, 40 \u0026micro;M and 50 \u0026micro;M). Seeing from the fluorescence color change of the \u003cb\u003eEHAT\u003c/b\u003e-based test strips, there is an obvious H\u003csub\u003e2\u003c/sub\u003eS concentration-dependent relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) by smartphone software (RGB analytical app). The H\u003csub\u003e2\u003c/sub\u003eS concentration can be correctly read out by the naked eye. Above results suggested that the smartphone sensing platform could quantitatively measure the H\u003csub\u003e2\u003c/sub\u003eS value.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Application of probe \u003cb\u003eEHAT\u003c/b\u003e in the foodstuffs\u003c/h2\u003e \u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eS are generated during storage and processing of protein-rich foods. To estimate the practical application of the probe \u003cb\u003eEHAT\u003c/b\u003e, the \u003cb\u003eEHAT-\u003c/b\u003ebased strips was used to detect the freshness of shrimp, chicken, pork and fish. When \u003cb\u003eEHAT-\u003c/b\u003ebased strips were treated with protein-rich foods (shrimp, chicken, pork and fish) for 0 h, 6 h, 12 h, 18 h, 24 h and 36 h, obvious fluorescence colour changes of \u003cb\u003eEHAT-\u003c/b\u003ebased strips from orangered to green were obviously seen by the naked eye under 365 nm UV lamp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Above results illustrated that the probe \u003cb\u003eEHAT\u003c/b\u003e could be employed as a simple tool of on-site detection of protein-rich freshness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Imaging of endogenous and exogenous H\u003csub\u003e2\u003c/sub\u003eS generation in living cells\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of probe \u003cb\u003eEHAT\u003c/b\u003e was studied. In Fig. S11, the viability of A549 cells was all over 90% even the concentration of probe \u003cb\u003eEHAT\u003c/b\u003e increased from 0 to 50 \u0026micro;M, which indicated probe \u003cb\u003eEHAT\u003c/b\u003e featured with low cytotoxicity. Encouraged by the prominent optical performances of probe \u003cb\u003eEHAT\u003c/b\u003e, its ability to visualize H\u003csub\u003e2\u003c/sub\u003eS levels in living cells was then investigated. The fluorescence images of living A549 cells were obtained by fluorescence confocal microscope and red channel (570\u0026ndash;670 nm) and green channel (500\u0026ndash;540 nm) was selected for observations. When A549 cells were nurtured with 10 \u0026micro;M \u003cb\u003eEHAT\u003c/b\u003e, a weak red fluorescence and green fluorescence signal was observed. When the cells were pretreated with 10 \u0026micro;M \u003cb\u003eEHAT\u003c/b\u003e for 30 min, and then stained with varied concentrations of H\u003csub\u003e2\u003c/sub\u003eS (5 \u0026micro;M, 10 \u0026micro;M, 20 \u0026micro;M, 30 \u0026micro;M, 40 \u0026micro;M and 50 \u0026micro;M) for 30 min. With the gradual addition of exogenous H\u003csub\u003e2\u003c/sub\u003eS, the significantly enhanced red fluorescence and attenuated green fluorescence were presented (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that the probe \u003cb\u003eEHAT\u003c/b\u003e performed well for supervising of the exogenous H\u003csub\u003e2\u003c/sub\u003eS generation in living cells.\u003c/p\u003e \u003cp\u003eNext, that the probe \u003cb\u003eEHAT\u003c/b\u003e was used to detect the endogenous H\u003csub\u003e2\u003c/sub\u003eS generation in living cells was studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The A549 cells was incubated with 10 \u0026micro;M \u003cb\u003eEHAT\u003c/b\u003e and NEM (a hydrogen sulfide remover) for 30 min, which displayed red fluorescence signals and green fluorescence signals. Then on the addition and subsequent incubation with an endogenous hydrogen sulfide inducer (L-Cys) for 30 min, the obvious green fluorescence was observed. The above results indicated that the probe \u003cb\u003eEHAT\u003c/b\u003e could as a ideal tool for ratiometric monitoring endogenous H\u003csub\u003e2\u003c/sub\u003eS in living cells via ratiometric image way. we expected that the probe \u003cb\u003eEHAT\u003c/b\u003e could be employed to the ratiometric imaging of exogenous and endogenous H\u003csub\u003e2\u003c/sub\u003eS in living cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Scientic Research and Technology Development Plan of Baise (Grant No. 20250315), the Fundamental Research Funds for the Guangxi Natural Science Foundation Program (Grant No. 2025GXNSFHA069268), the Young academic leaders of Xinzhou Normal University (No. X20240013), Scientific and Technological Inno-vation Programs of Higher Education Institutions in Shanxi (No. 2022L465 and No. 2021L454), Fundamental Research Program of Shanxi Province (No. 202203021222307 and 202303021222235), Natural Science Foundation of China (22277104) for financial assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRongqi Wang, Si Chen, Yamin Li, Tuotuo Fang, Zhan-Bin Jin, Ting-Ting Wei, Jia-Ping Li and Hai-Xian Ren\u0026nbsp;wrote the main manuscript text.\u0026nbsp;Zi-Ao Zong and Na-Na Li\u0026nbsp;prepared Figs.1, 2, 3, 4, 5, 6, scheme 1 and scheme 2. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. Wang, F. Tang, T. Zang, X. Liu, G. Cheng, W. Chen, W. Shu, D. Li, Y. Tang, X. Sun, H. Xiao, Ingenious fluorescent probes for biogenic amine and their applications in bioimaging and food spoilage detection, Food Chem 454 (2024) 139714.\u003c/li\u003e\n\u003cli\u003eX. Yang, S. Zhang, F. Luo, Y. Zhang, D. Yan, M. Lai, Y. Ye, K. Sun, X. Ji, An AIE-based ratiometric fluorescent probe for highly selective detection of H\u003csub\u003e2\u003c/sub\u003eS in plant stress responses, Biosens Bioelectron 267 (2025) 116798.\u003c/li\u003e\n\u003cli\u003eM. Chen, R. Chen, Y. Shi, J. Wang, Y. Cheng, Y. Li,. 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Zhou, X. Liu, X. Peng, Rational design of AIE-based fluorescent probes for hypochlorite detection in real water samples and live cell imaging, J. Hazard. Mater. 418 (2021) 126243\u003c/li\u003e\n\u003cli\u003eR. Jia, W. Tian, H. Bai, J. Zhang, S. Wang, J. Zhang, Amine-responsive cellulose-based ratiometric fluorescent materials for real-time and visual detection of shrimp and crab freshness, Nat. Commun. 10 (2024) 795\u0026ndash;798.\u003c/li\u003e\n\u003cli\u003eH.F. Xie, X.H. Jiang, F. Zeng, C.M. Yu, S.Z. Wu, A novel ratiometric fluorescent probe through aggregation-induced emission and analyte-induced excimer dissociation. Sensor Actuator B Chem 203 (2014) 504\u0026ndash;510.\u003c/li\u003e\n\u003cli\u003eN. Li, N. Shi, D. Yang, R. Wu, C. Xu, B. Zhu, F. Shao, X. Zhang, S. Bi, Y. Fan, Solid-state fluorescent switch based on the intercoversion of J-aggregation and dimer and aggregation pattern-dependent fluorescence colorimetric sensing of GSH/Zn\u003csup\u003e2+\u003c/sup\u003e/Cd\u003csup\u003e2+\u003c/sup\u003e, J Mol Liq 342 (2021) 116946.\u003c/li\u003e\n\u003cli\u003eN. Li, Y. Gao, X. Xu, P. Qiu, Y. Gao, M. Yan, Q. Zhang, W. Lin, Z. Xing, Z. Zong, Fluorescence probe with aggregation induced emission effect and application for colormetric detection of endogenous and exogenous hypochlorite, Dyes Pigments 210 (2023) 110965.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Aggregation-Induced Emission (AIE), Endogenous and exogenous H2S, Visual detection, Food freshness, Cell imageing","lastPublishedDoi":"10.21203/rs.3.rs-8862141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8862141/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) is a crucial endogenous gas transmitter for the evaluation of food spoilage and disease diagnosis. Thus, we have effectively prepared a novel AIE probe \u003cb\u003eEHAT\u003c/b\u003e, which displayed a obvious fluorescence color change after the addition of H\u003csub\u003e2\u003c/sub\u003eS (from orangered to green), suggesting that probe \u003cb\u003eEHAT\u003c/b\u003e can identify H\u003csub\u003e2\u003c/sub\u003eS with the naked eye. The probe \u003cb\u003eEHAT\u003c/b\u003e-based test strip was also developed to conveniently detect H\u003csub\u003e2\u003c/sub\u003eS gas generated during food spoilage. Moreover, the probe \u003cb\u003eEHAT\u003c/b\u003e were successfully utilized to image endogenous and exogenous H\u003csub\u003e2\u003c/sub\u003eS in live cells.\u003c/p\u003e","manuscriptTitle":"A AIE probe for visual monitoring of endogenous and exogenous H2S and food freshness","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 06:56:54","doi":"10.21203/rs.3.rs-8862141/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-16T03:08:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-26T04:28:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T00:18:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T03:37:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133668221890085986225938045942373171259","date":"2026-03-18T06:14:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216720892016439108038888078456353364273","date":"2026-03-17T16:35:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112336453223259532849713740151272042280","date":"2026-03-16T23:51:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T16:15:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-27T03:21:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-27T03:20:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2026-02-12T12:28:48+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":"8cbc4598-9b26-499b-887b-e0cc3fd97690","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-01T02:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 06:56:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8862141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8862141","identity":"rs-8862141","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}