Observation of glutathione intra- and extracellular membrane with probe modified by long alkyl chains

Observation of glutathione intra- and extracellular membrane with probe modified by long alkyl chains | 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 Observation of glutathione intra- and extracellular membrane with probe modified by long alkyl chains Lin Li, Shao-Bin Sun, Dan Qiao, Jing Zhou, Lin-qing Wang, Jian-Yong Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9217551/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract A naphthylimide dye-based fluorescent probe ( Nap-Mem-GSH ) was synthesized for the detection of Glutathione inside and outside cell membranes. This probe carried a 2-Hydroxyquinoline fragment as a recognition site on top of a naphthalimide dye, which exhibits better selective and fluorimetric response toward Glutathione in natural media. The long alkyl chain confers a cell membrane targeting role to Nap-Mem-GSH . Therefore, the detection of intra- and extracellular glutathione inside and outside the cell membrane can be realized by Nap-Mem-GSH . Meanwhile, it not only has a large Stokes shift (160 nm), but also has the advantages of low cytotoxicity and good membrane permeability to living cells, and has been successfully applied to effectively detect and image intracellular glutathione by confocal fluorescence imaging. Glutathione (GSH) large Stokes shift Cell membrane targeting Cell imaging Zebrafish imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Reduced glutathione (GSH), a biomarker of oxidative stress, is the most abundant intracellular non-protein thiol species [ 1 ] . GSH is a tripeptide composed of glutamate (Glu), cysteine (Cys) and glycine (Gly) [ 2 , 3 ] . Glutathione plays not only a role in maintaining the balance of cellular redox homeostasis through antioxidant protection and thiol-disulfide exchange of peptides and proteins, but also in the regulation of cell signaling and gene expression. Additionally, it is involved in detoxification of toxic compounds and the synthesis of eicosanoids [ 4 , 5 ] . Abnormal glutathione levels are associated with a variety of diseases such as AIDS, cancer, liver injury and neurodegenerative diseases [ 6 ] . Furthermore, high levels of GSH can lead to increased organismal resistance [ 7 – 11 ] . Therefore, the real-time detection of GSH is of great interest. Glutathione production and metabolism involves an intracellular and extracellular cycle, which is a cycle of six enzymatic reactions involving γ-glutamate (Fig. 1 ). The first stage is the formation of peptide bonds between cysteine and glutamate, catalyzed by γ-glutamate ligase (γ-GCL), to form a polypeptide substance of cysteine and glutamate, which plays a rate-limiting role in glutathione synthesis. The second stage is a reaction catalyzed by glutathione synthase (GS), which leads to the production of glutathione due to the binding of glycine to γ-glutamyltransferase [ 12 ] . GSH is transported from intracellular to extracellular via a transporter protein on the cytoplasmic membrane. Importantly, γ-glutamyltransferase (γ-GT) is an important enzymatic substance in the glutathione cycle that hydrolyzes some specific bonds between glutamate and cysteine residues in the glutathione molecule, which is mainly distributed in the outer cytoplasmic membrane of specific cell types, and its role is to facilitate the transfer of γ-glutamine residues to neutral amino acids for their transport into the cell. The GSH outside the cell membrane is catalyzed by γ-GT and re-entered into the cell to achieve the GSH cycle. Using the cellular GSH cycle, a number of modern intelligent drug delivery systems can be developed based on changes in intracellular and extracellular GSH -concentrations [ 13 , 14 ] . To date, there have been numerous analytical techniques for the determination of glutathione, including colorimetric methods [ 15 , 16 ] , high-performance liquid [ 17 – 19 ] chromatography [ 20 ] , electrochemistry, and spectroscopy [ 21 , 22 ] . However, these methods possess limitations. For instance, they are unable to accurately measure GSH levels in organisms and often come with a high cost. Compared with conventional detection methods, small-molecule fluorescent probes have attracted extensive research interest due to their low cost, fast response time, remarkable sensitivity, and simple operation [ 23 – 25 ] . The cell membrane is the boundary of the cell, separating the various substances and organelles within the cell from the external environment [ 26 ] . Cell membranes are responsible for signal transduction, transport of substances and maintaining the stability of the intracellular environment. Cell membranes are also associated with oxidative stress, which can cause peroxidative breakdown and property changes of membrane lipids. Therefore, probes for targeting cell membranes and detecting substances inside and outside cell membranes have been developed and widely used in biochemistry and medicine [ 27 ] . Hence, it is critical for monitoring substances inside and outside cell membranes. In recent years, many fluorescent probes had been developed for imaging GSH [ 28 – 31 ] . However, conventional probes can only target certain organelles or subpopulations within cells and detect intracellular GSH levels. Although a number of fluorescent probes have been available to detect biothiols, there are fewer specific assays for GSH, Cys and Hcy [ 32 – 34 ] . There are no probes capable of detecting extracellular GSH. In this paper, a novel naphthylamine based fluorescent probe ( Nap-Mem-GSH ) was firstly developed for the detection of GSH inside and outside the cell membrane. The probe Nap-Mem-GSH could respond selectively with glutathione and insignificantly with other amino acids, common metal ions and other analytes. The long alkyl chain conferred Nap-Mem-GSH the ability of cell membrane targeting, and GSH detection and cell membrane targeting could be performed simultaneously, allowing verification of the intra- and extra-membrane recycling of GSH. This low toxicity probe had been successfully used to detect glutathione on cell membranes in living cells, as well as for imaging zebrafish. 2. Experimental Detailed methods are provided in the ESI. 3. Synthesis 3.1 Synthesis of Nap-Mem-GSH : 4-Bromo-1,8-naphthalic anhydride (2.0 g, 8.6 mmol) and 1-dodecylamine (3.2 g, 17.2 mmol) were dissolved in Ethanol (30.0 mL). After cooling to room temperature, the crude product was purified by filtration and column chromatography to give 1 ( 2.356 g, 62% yield). Then, 1 (443.0 mg, 1.0 mmol) and 2-Hydroxyquinoline (291.0 mg, 2.0 mmol) were dissolved in DMF (5.0 mL). Sodium tert -butoxide (48.0 mg, 0.5 mmol) was added, reacted under nitrogen atmosphere for 12 h and cooled to room temperature. The residue was obtained by filtration and purified by silica gel column to give a yellow solid ( 228.6 mg, 45% yield). 1 H NMR (400 MHz, DMSO) δ 8.57–8.43 (m, 2H), 8.37 (dd, J = 7.7, 4.4 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.77 (t, J = 7.3 Hz, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.50 (t, J = 7.0 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.17 (t, J = 6.5 Hz, 2H), 6.50 (d, J = 9.4 Hz, 1H), 4.00 (d, J = 8.5 Hz, 2H), 1.60 (s, 2H), 1.30 (s, 4H), 1.21 (s, 14H), 0.84 (t, J = 6.4 Hz, 3H). 13 C NMR (101 MHz, DMSO): δ = 164.30, 159.43, 133.70, 131.91, 129.82, 129.04, 127.72, 125.58, 123.62, 122.65, 122.31, 114.30, 110.05, 40.50, 31.92, 29.63, 28.22, 27.22, 22.70, 14.21. HRMS (ESI + ): m/z calcd for C 24 H 13 N 2 O 3 [M + H + ] + :509.2804, found: 509.2808 4. Results and discussion 4.1 Design strategy of probe Nap-Mem-GSH 2-Hydroxyquinoline and its derivatives are an important class of alkaloids, and many compounds containing the 2-hydroxyquinoline group have a variety of effects, and have a wide range of applications in anticancer [ 35 ] , antibiotic [ 36 ] , antiviral [ 37 ] , antibacterial [ 38 ] , and blood pressure lowering [ 39 ] , and so on, so 2-hydroxyquinoline derivatives have a broad application prospect [ 40 ] . Most cytoplasmic membrane probes such as DiI(1,1'-Di-n-octadecyl-3,3,3',3'-tetraMethylindocarbocyanine perchlorate) and DiO(3,3’-dioctadecyloxacarbocyanine) have long Long alkyl chains. Long alkyl chains are highly hydrophobic, and the introduction of long aliphatic chains allows the probe to interact hydrophobically with the phospholipids of the cytoplasmic membrane of the cell and immobilize in the cytoplasmic membrane of the cell [ 41 ] . Therefore, Nap-Mem-GSH can be used to detect the sensitivity of cell membranes. 4-Bromo-1,8-naphthalene anhydride was reacted with 1-dodecylamine to form compound 1 , and then with 2-Hydroxyquinoline to obtain the probe Nap-Mem-GSH. The probe Nap-Mem-GSH was structurally characterized comprehensively via standard 1 H NMR, 13 C NMR and HRMS. 4.2 Spectra response towards biothiols Response tests were conducted using various common biothiols, such as Cys, Hcy, GSH and H 2 S (Fig. 1 A). During the screening of the corresponding substances, It was found that Nap-Mem-GSH responded well to GSH, while other biothiols had less effect on Nap-Mem-GSH . The response of Nap-Mem-GSH to GSH led us to the next detailed study. DMSO/PBS buffer was configured at room temperature, then GSH was added and Nap-Mem-GSH (10 µM) was tested in UV, and it could find that the maximum absorption wavelength of the solution without the addition of GSH to Nap-Mem-GSH solution was 475 nm, After the addition of GSH, the absorption intensity at 390 nm increased and the UV absorption intensity at 475 nm decreased. Also, testing the fluorescence emission of Nap-Mem-GSH in the presence or absence of GSH, using 390 nm as the excitation wavelength, it could be found that Nap-Mem-GSH showed weak emission in the absence of GSH, while the fluorescence intensity of the solution increased significantly to 40 times the original one with the addition of 100 µM GSH and incubation. In DMSO/PBS buffer, the GSH concentration was increased from 1 µM to 140 µM and reacted with Nap-Mem-GSH (10 µM). The fluorescence spectra were recorded and the fluorescence intensity at 545 nm was plotted against the concentration (Fig. 1 C.). The fluorescence intensity was linearly proportional to the GSH concentration in the range of 1 µM ~ 130 µM (Fig. 1 D.). The strong emission signal corresponding to GSH compared to other biological thiols indicated that Nap-Mem-GSH is capable of detecting GSH, which provides the possibility of cellular imaging of intracellular concentrations of GSH at 1–10 mM. This GSH cycle was simultaneously present outside the cell membrane. To investigate the temporal stability changes of Nap-Mem-GSH and also to investigate whether Nap-Mem-GSH can respond rapidly to GSH detection. The probe Nap-Mem-GSH was tested for temporal stability, and over time the fluorescence intensity was relatively stable before the addition of GSH, while it rose to a maximum within 2 minutes after the addition of GSH, allowing rapid adaptation to intracellular conditions. As shown in Fig. S2, the response of Nap-Mem-GSH to intracellular GSH concentration resulted in a rapid increase in fluorescence and good fluorescence stability over 100 min. A further possibility for validating cell imaging was provided. 4.3 The effect of pH Responsiveness of Nap-Mem-GSH to GSH was tested as a function of pH, as shown in the Fig. 2 . It was found that Nap-Mem-GSH itself has the strongest and most stable fluorescence intensity in the pH range of 7.0–8.0. Because the reactivity of GSH increases with increasing pH, Meanwhile, Nap-Mem-GSH was affected under acidic conditions, leading to a decrease in the activity of Nap-Mem-GSH in reaction with glutathione, which resulted in a weak fluorescence intensity. Nap-Mem-GSH reacted best with GSH in the pH range of 7.0–8.0. T More assuredly, Nap-Mem-GSH was able to adapt to the cellular environment and enable cellular imaging. 4.4 Selective response of Nap-Mem-GSH to GSH Nap-Mem-GSH was then tested for its selectivity for other analytes, including other amino acids and common biometallic ions. It was found that only the addition of GSH to the Nap-Mem-GSH solution significantly enhanced the fluorescence intensity. Other substances did not trigger fluorescence enhancement. Thus Nap-Mem-GSH had high selectivity for GSH. To further demonstrate the selective recognition of GSH by Nap-Mem-GSH . Competition experiments between GSH and other substances were further tested by adding 20 µM aliquots of Nap-Mem-GSH to a 200 µM solution of other analytes for testing, followed by 20 µM of GSH for testing (Fig. 3 ). As shown in Fig. 3 , it was found that the other analytes did not affect the detection of GSH by Nap-Mem-GSH . GSH can also be easily detected in the presence of other analytes at the same time. 4.5 The cell imaging of Nap-Mem-GSH To demonstrate the feasibility of the designed probe for glutathione imaging in intracellular medium using cell cultures. We performed MTT experiments to test the cytotoxicity of Nap-Mem-GSH (Fig. S3). The results showed that Nap-Mem-GSH had good cell viability, even when the probe concentration reached more than 20 µM. And Nap-Mem-GSH had no effect on cell growth, indicating that the probe should be used in biological cell experiments. According to the practical application requirements, HeLa cells were selected as a cell model to analyze the performance of Nap-Mem-GSH for the detection of intracellular GSH and GSH on cell membrane. Then, confocal laser scanning microfluorescence imaging studies were performed. As shown in the Fig. 4 , HeLa cells could see brighter yellow fluorescence after the addition of Nap-Mem-GSH (10 µM) and incubation for 20 minutes. When HeLa cells were pretreated with 5 mM of n -ethylmaleimide (NEM, a well-known biological thiol scavenger) [ 42 ] and incubated with Nap-Mem-GSH , only a small amount of yellow fluorescence was seen. To further investigate the cell membrane targeting of Nap-Mem-GSH , In co-localization studies, imaging was performed using a widely used commercial cell membrane targeting dye, perchlorate DiD (Fig. 4 ). the signal generated by Nap-Mem-GSH overlapped very well with the fluorescence of DiD perchlorate. the Pearson's co-localization coefficient (describing the correlation of the intensity distribution between the two channels) was calculated to be 0.95, confirming that Nap-Mem-GSH was specifically located in the cell membrane of live HeLa cells. 4.6 The zebrafish imaging of Nap-Mem-GSH Further, Nap-Mem-GSH was investigated in zebrafish. The same part of the same zebrafish was imaged by fluorescence confocal microscopy test Nap-Mem-GSH . Yellow fluorescence was seen to appear in zebrafish, and then, by adding exogenous GSH, it was found enhanced yellow fluorescence in zebrafish, demonstrating the ability of Nap-Mem-GSH to detect exogenous GSH in zebrafish. Then added NEM exogenous to the solution while detecting the change in fluorescence intensity, and it was found that the fluorescence intensity in zebrafish diminished in vivo, indicating that Nap-Mem-GSH has the sensitivity to detect GSH. 5. Conclusions In this work, a novel naphthalimide-based cell membrane-targeted fluorescent chemosensor was developed that carried a 2-hydroxyquinoline fragment on the naphthalimide dye as a recognition site for selective detection of GSH. The probe Nap-Mem-GSH not only selectively responds to GSH. Moreover, it has good cell membrane targeting properties. Simultaneous GSH detection and cell membrane targeting allowed verification of both intra- and extra-membrane recycling of GSH. This low toxicity probe had been successfully used to detect glutathione on cell membranes in living cells, as well as zebrafish imaging. Declarations 6.Acknowledgement This work was financially supported by by the Natural Science Foundation of Henan Province (No. 252300423095), National Natural Science Foundation of China (21801145), Major Scientific Research Project for the Construction of State Key Lab (No. 2025ZDGZ02). Author Contribution Lin Li and Jian-Yong Wang: Conceptualization, Resources, Writing - Review & Editing, Supervision, Project Administration.Shao-Bin Sun: Formal Analysis, Resources, Project Administration, Writing original draft preparation. Dan Qiao and Jing Zhou: Formal Analysis, Data Curation, Software. Lin-Qing Wang: Resources, Software. References Scirè, A., Cianfruglia, L., Minnell, C., et al. (2019). Glutathione compartmentalization and its role in glutathionylation and other regulatory processes of cellular pathways. 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Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 23 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers invited by journal 02 Apr, 2026 Editor assigned by journal 02 Apr, 2026 Submission checks completed at journal 25 Mar, 2026 First submitted to journal 24 Mar, 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. We do this by developing innovative software and high quality services for the global research community. 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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-9217551","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619076458,"identity":"9e229bf2-b28b-4dc3-9672-53744c992304","order_by":0,"name":"Lin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACPmbmhgMMFTYgNuODhIoawlrYmBmBWs6kgdjMBg/OHCNCCwNjAwNj22EwW/JhCzMRWtgZGw8XsB3OM7h2+FlFYgMbA397dwJBhx2ewZNebHA7zexG4g4ZBokzZzcQ1sIjYZ244XYO243EM2wMBhK5xGgxYAZrKUhsYyZWS4IzWAsDCVoOpBVL3k4zlkg4c4yHoF/4+Q8f/sz7zyaP73byw48/Kmrk+Nt78WuBgQQYg4co5ShaRsEoGAWjYBRgAABqg0f0yVMZuAAAAABJRU5ErkJggg==","orcid":"","institution":"Zhengzhou Normal University","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Li","suffix":""},{"id":619076459,"identity":"8e63f4ba-cc7f-46ac-8925-8a34deb539b6","order_by":1,"name":"Shao-Bin Sun","email":"","orcid":"","institution":"Qi Lu University of Technology (Shandong Academy of Sciences)","correspondingAuthor":false,"prefix":"","firstName":"Shao-Bin","middleName":"","lastName":"Sun","suffix":""},{"id":619076460,"identity":"9c5d5d56-1450-435e-aba0-1b3372af136c","order_by":2,"name":"Dan Qiao","email":"","orcid":"","institution":"Zhengzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Qiao","suffix":""},{"id":619076461,"identity":"b88c3a77-11e2-4164-be1f-7a89a182fd87","order_by":3,"name":"Jing Zhou","email":"","orcid":"","institution":"Qi Lu University of Technology (Shandong Academy of Sciences)","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhou","suffix":""},{"id":619076462,"identity":"54d67515-9c64-4318-a159-e850c824c51b","order_by":4,"name":"Lin-qing Wang","email":"","orcid":"","institution":"Zhengzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lin-qing","middleName":"","lastName":"Wang","suffix":""},{"id":619076463,"identity":"c366c07d-7007-4ca5-a448-b6102fa940be","order_by":5,"name":"Jian-Yong Wang","email":"","orcid":"","institution":"Qi Lu University of Technology (Shandong Academy of Sciences)","correspondingAuthor":false,"prefix":"","firstName":"Jian-Yong","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-03-25 03:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9217551/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9217551/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106533249,"identity":"633a35cb-082b-4b8e-9ba1-7dc6341ff23d","added_by":"auto","created_at":"2026-04-09 14:56:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51620,"visible":true,"origin":"","legend":"\u003cp\u003eA. Fluorescence intensity (λ\u003csub\u003eex\u003c/sub\u003e= 390 nm) of \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM, λ\u003csub\u003eem \u003c/sub\u003e= 545 nm) after 10 min incubation with GSH, Cys, Hcy and NaSH, respectively. B. Absorption spectra of\u003cstrong\u003e Nap-Mem-GSH\u003c/strong\u003e (10 μM, black curve) before and after the addition of GSH (100 μM, red curve), incubated for 120 min at 25 ℃ in DMSO/PBS buffer (1:1, v/v, 10 mM, pH = 7.4). C. \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM, λ\u003csub\u003eem \u003c/sub\u003e= 545 nm) in increasing GSH concentrations (final concentrations: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 μM) in DMSO/PBS buffer (1:1, v/v, 10 mM, pH = 7.4) at 25 ℃. D. After 10 min of incubation, the fluorescence intensity of \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (λ\u003csub\u003eem \u003c/sub\u003e= 545 nm) showed a linear calibration curve with GSH concentration in the range of 0 ~ 130 μM.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/f406ecb142686e11b7431f3b.jpg"},{"id":106533272,"identity":"4bbe3541-a4dc-4184-a16b-17d3b987ad44","added_by":"auto","created_at":"2026-04-09 14:56:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32161,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of pH on the fluorescence intensity (λem = 390 nm) of \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM, λex = 545 nm) in DMSO/PBS buffer upon addition of 100 μM GSH after incubation at 25 ℃ for 120 min.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/cd31e1b8b966dc3b44c5f8a4.jpg"},{"id":106533118,"identity":"d5b417ab-0d62-4f7e-a14c-d5799608987b","added_by":"auto","created_at":"2026-04-09 14:56:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85600,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence intensity at 545 nm (λex=390 nm) of \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM) in the presence of various analytes in DMSO/PBS buffer (1:1, v/v, 10 mM, pH=7.4) at 25 ℃ prior to (blue bars) and after (red bars) addition of 100 μM GSH to the individual \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e /analyte solution. (1) blank, (2) NaSH, (3) Cys, (4) Hcy, (5) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; (6) Cl\u003csup\u003e-\u003c/sup\u003e, (7) Cu\u003csup\u003e2+\u003c/sup\u003e, (8) HClO, (9) Glu, (10) NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, (11) TBHP, (12) Fe\u003csup\u003e2+\u003c/sup\u003e, (13) Ag\u003csup\u003e+\u003c/sup\u003e.\u003csup\u003e \u003c/sup\u003e(200 μM). Data were recorded 10 min after addition of analytes/GSH.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/38e8691b236edc2a81d6b222.jpg"},{"id":106533076,"identity":"b8ca3179-a00a-4c6b-aeff-45369c0a84e8","added_by":"auto","created_at":"2026-04-09 14:56:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40544,"visible":true,"origin":"","legend":"\u003cp\u003e(A)Fluorescence imaging of live HeLa cells: control cells; (B)cells incubated with \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM)+GSH; (C)cells incubated with \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM)+NEM; (D)cells incubated with \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e (10 μM)+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/238dcf75b2ee297c558374cd.jpg"},{"id":106533057,"identity":"21970053-c272-4dc6-a769-0e9d34ad9e98","added_by":"auto","created_at":"2026-04-09 14:56:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76155,"visible":true,"origin":"","legend":"\u003cp\u003eImages of HeLa cells under confocal laser scanning microscopy with \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e(10 μM, 20 min) and DiD perchlorate(0.5 μM, 20 min)\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/de79a400d94181bbfc62eb18.jpg"},{"id":106533007,"identity":"277eb3ce-0895-4d69-991d-0c22dea162dc","added_by":"auto","created_at":"2026-04-09 14:56:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":34611,"visible":true,"origin":"","legend":"\u003cp\u003eImaging of zebrafish with the probe\u003cstrong\u003e Nap-Mem-GSH\u003c/strong\u003e, and changes in fluorescence intensity at the same site in the same zebrafish.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/2943850cd5ea15539db2718c.jpg"},{"id":106533420,"identity":"a0c8e34e-1f02-4857-bcd0-d4172261faa1","added_by":"auto","created_at":"2026-04-09 14:57:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1231851,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/936aa2e3-911d-41e6-99cb-39de36defc90.pdf"},{"id":106533159,"identity":"81cd1547-8819-4291-83c8-70499edfa5ba","added_by":"auto","created_at":"2026-04-09 14:56:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":524594,"visible":true,"origin":"","legend":"","description":"","filename":"NapMemGSHSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/c4ecacb6714275aef104af3e.docx"},{"id":106533121,"identity":"26b987b3-c096-4437-883b-92dff7d2a7e2","added_by":"auto","created_at":"2026-04-09 14:56:25","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e. Synthetic route of \u003cstrong\u003eNap-Mem-GSH\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9217551/v1/19e953e417542113547403df.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Observation of glutathione intra- and extracellular membrane with probe modified by long alkyl chains","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eReduced glutathione (GSH), a biomarker of oxidative stress, is the most abundant intracellular non-protein thiol species\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. GSH is a tripeptide composed of glutamate (Glu), cysteine (Cys) and glycine (Gly) \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Glutathione plays not only a role in maintaining the balance of cellular redox homeostasis through antioxidant protection and thiol-disulfide exchange of peptides and proteins, but also in the regulation of cell signaling and gene expression. Additionally, it is involved in detoxification of toxic compounds and the synthesis of eicosanoids\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Abnormal glutathione levels are associated with a variety of diseases such as AIDS, cancer, liver injury and neurodegenerative diseases\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Furthermore, high levels of GSH can lead to increased organismal resistance\u003csup\u003e[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, the real-time detection of GSH is of great interest.\u003c/p\u003e \u003cp\u003eGlutathione production and metabolism involves an intracellular and extracellular cycle, which is a cycle of six enzymatic reactions involving γ-glutamate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The first stage is the formation of peptide bonds between cysteine and glutamate, catalyzed by γ-glutamate ligase (γ-GCL), to form a polypeptide substance of cysteine and glutamate, which plays a rate-limiting role in glutathione synthesis. The second stage is a reaction catalyzed by glutathione synthase (GS), which leads to the production of glutathione due to the binding of glycine to γ-glutamyltransferase\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. GSH is transported from intracellular to extracellular via a transporter protein on the cytoplasmic membrane. Importantly, γ-glutamyltransferase (γ-GT) is an important enzymatic substance in the glutathione cycle that hydrolyzes some specific bonds between glutamate and cysteine residues in the glutathione molecule, which is mainly distributed in the outer cytoplasmic membrane of specific cell types, and its role is to facilitate the transfer of γ-glutamine residues to neutral amino acids for their transport into the cell. The GSH outside the cell membrane is catalyzed by γ-GT and re-entered into the cell to achieve the GSH cycle. Using the cellular GSH cycle, a number of modern intelligent drug delivery systems can be developed based on changes in intracellular and extracellular GSH -concentrations\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo date, there have been numerous analytical techniques for the determination of glutathione, including colorimetric methods\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, high-performance liquid\u003csup\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e chromatography\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, electrochemistry, and spectroscopy\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, these methods possess limitations. For instance, they are unable to accurately measure GSH levels in organisms and often come with a high cost. Compared with conventional detection methods, small-molecule fluorescent probes have attracted extensive research interest due to their low cost, fast response time, remarkable sensitivity, and simple operation \u003csup\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe cell membrane is the boundary of the cell, separating the various substances and organelles within the cell from the external environment\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Cell membranes are responsible for signal transduction, transport of substances and maintaining the stability of the intracellular environment. Cell membranes are also associated with oxidative stress, which can cause peroxidative breakdown and property changes of membrane lipids. Therefore, probes for targeting cell membranes and detecting substances inside and outside cell membranes have been developed and widely used in biochemistry and medicine\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Hence, it is critical for monitoring substances inside and outside cell membranes.\u003c/p\u003e \u003cp\u003eIn recent years, many fluorescent probes had been developed for imaging GSH\u003csup\u003e[\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. However, conventional probes can only target certain organelles or subpopulations within cells and detect intracellular GSH levels. Although a number of fluorescent probes have been available to detect biothiols, there are fewer specific assays for GSH, Cys and Hcy\u003csup\u003e[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. There are no probes capable of detecting extracellular GSH. In this paper, a novel naphthylamine based fluorescent probe (\u003cb\u003eNap-Mem-GSH\u003c/b\u003e) was firstly developed for the detection of GSH inside and outside the cell membrane. The probe \u003cb\u003eNap-Mem-GSH\u003c/b\u003e could respond selectively with glutathione and insignificantly with other amino acids, common metal ions and other analytes. The long alkyl chain conferred \u003cb\u003eNap-Mem-GSH\u003c/b\u003e the ability of cell membrane targeting, and GSH detection and cell membrane targeting could be performed simultaneously, allowing verification of the intra- and extra-membrane recycling of GSH. This low toxicity probe had been successfully used to detect glutathione on cell membranes in living cells, as well as for imaging zebrafish.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cp\u003eDetailed methods are provided in the ESI.\u003c/p\u003e"},{"header":"3. Synthesis","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1\u003c/b\u003e Synthesis of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003e4-Bromo-1,8-naphthalic anhydride (2.0 g, 8.6 mmol) and 1-dodecylamine (3.2 g, 17.2 mmol) were dissolved in Ethanol (30.0 mL). After cooling to room temperature, the crude product was purified by filtration and column chromatography to give \u003cb\u003e1\u003c/b\u003e ( 2.356 g, 62% yield). Then, \u003cb\u003e1\u003c/b\u003e (443.0 mg, 1.0 mmol) and 2-Hydroxyquinoline (291.0 mg, 2.0 mmol) were dissolved in DMF (5.0 mL). Sodium \u003cem\u003etert\u003c/em\u003e-butoxide (48.0 mg, 0.5 mmol) was added, reacted under nitrogen atmosphere for 12 h and cooled to room temperature. The residue was obtained by filtration and purified by silica gel column to give a yellow solid ( 228.6 mg, 45% yield). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) δ 8.57\u0026ndash;8.43 (m, 2H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 4.4 Hz, 1H), 7.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 1H), 7.77 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 1H), 7.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 1H), 7.50 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 1H), 7.31 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 Hz, 2H), 6.50 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.4 Hz, 1H), 4.00 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), 1.60 (s, 2H), 1.30 (s, 4H), 1.21 (s, 14H), 0.84 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO): δ\u0026thinsp;=\u0026thinsp;164.30, 159.43, 133.70, 131.91, 129.82, 129.04, 127.72, 125.58, 123.62, 122.65, 122.31, 114.30, 110.05, 40.50, 31.92, 29.63, 28.22, 27.22, 22.70, 14.21. HRMS (ESI\u003csup\u003e+\u003c/sup\u003e): m/z calcd for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H\u003csup\u003e+\u003c/sup\u003e]\u003csup\u003e+\u003c/sup\u003e:509.2804, found: 509.2808\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Design strategy of probe \u003cb\u003eNap-Mem-GSH\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e2-Hydroxyquinoline and its derivatives are an important class of alkaloids, and many compounds containing the 2-hydroxyquinoline group have a variety of effects, and have a wide range of applications in anticancer\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, antibiotic\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, antiviral\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, antibacterial\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, and blood pressure lowering\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, and so on, so 2-hydroxyquinoline derivatives have a broad application prospect\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMost cytoplasmic membrane probes such as DiI(1,1'-Di-n-octadecyl-3,3,3',3'-tetraMethylindocarbocyanine perchlorate) and DiO(3,3\u0026rsquo;-dioctadecyloxacarbocyanine) have long Long alkyl chains. Long alkyl chains are highly hydrophobic, and the introduction of long aliphatic chains allows the probe to interact hydrophobically with the phospholipids of the cytoplasmic membrane of the cell and immobilize in the cytoplasmic membrane of the cell\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, \u003cb\u003eNap-Mem-GSH\u003c/b\u003e can be used to detect the sensitivity of cell membranes. 4-Bromo-1,8-naphthalene anhydride was reacted with 1-dodecylamine to form compound \u003cb\u003e1\u003c/b\u003e, and then with 2-Hydroxyquinoline to obtain the probe \u003cb\u003eNap-Mem-GSH.\u003c/b\u003e The probe\u003cb\u003eNap-Mem-GSH\u003c/b\u003e was structurally characterized comprehensively via standard \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003e C NMR and HRMS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.2\u003c/b\u003e Spectra response towards biothiols\u003c/h2\u003e \u003cp\u003eResponse tests were conducted using various common biothiols, such as Cys, Hcy, GSH and H\u003csub\u003e2\u003c/sub\u003eS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). During the screening of the corresponding substances, It was found that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e responded well to GSH, while other biothiols had less effect on \u003cb\u003eNap-Mem-GSH\u003c/b\u003e. The response of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e to GSH led us to the next detailed study.\u003c/p\u003e \u003cp\u003eDMSO/PBS buffer was configured at room temperature, then GSH was added and \u003cb\u003eNap-Mem-GSH\u003c/b\u003e (10 \u0026micro;M) was tested in UV, and it could find that the maximum absorption wavelength of the solution without the addition of GSH to \u003cb\u003eNap-Mem-GSH\u003c/b\u003e solution was 475 nm, After the addition of GSH, the absorption intensity at 390 nm increased and the UV absorption intensity at 475 nm decreased. Also, testing the fluorescence emission of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e in the presence or absence of GSH, using 390 nm as the excitation wavelength, it could be found that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e showed weak emission in the absence of GSH, while the fluorescence intensity of the solution increased significantly to 40 times the original one with the addition of 100 \u0026micro;M GSH and incubation.\u003c/p\u003e \u003cp\u003eIn DMSO/PBS buffer, the GSH concentration was increased from 1 \u0026micro;M to 140 \u0026micro;M and reacted with \u003cb\u003eNap-Mem-GSH\u003c/b\u003e (10 \u0026micro;M). The fluorescence spectra were recorded and the fluorescence intensity at 545 nm was plotted against the concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.). The fluorescence intensity was linearly proportional to the GSH concentration in the range of 1 \u0026micro;M\u0026thinsp;~\u0026thinsp;130 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD.).\u003c/p\u003e \u003cp\u003eThe strong emission signal corresponding to GSH compared to other biological thiols indicated that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e is capable of detecting GSH, which provides the possibility of cellular imaging of intracellular concentrations of GSH at 1\u0026ndash;10 mM. This GSH cycle was simultaneously present outside the cell membrane. To investigate the temporal stability changes of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e and also to investigate whether \u003cb\u003eNap-Mem-GSH\u003c/b\u003e can respond rapidly to GSH detection. The probe \u003cb\u003eNap-Mem-GSH\u003c/b\u003e was tested for temporal stability, and over time the fluorescence intensity was relatively stable before the addition of GSH, while it rose to a maximum within 2 minutes after the addition of GSH, allowing rapid adaptation to intracellular conditions. As shown in Fig. S2, the response of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e to intracellular GSH concentration resulted in a rapid increase in fluorescence and good fluorescence stability over 100 min. A further possibility for validating cell imaging was provided.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.3\u003c/b\u003e The effect of pH\u003c/h2\u003e \u003cp\u003eResponsiveness of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e to GSH was tested as a function of pH, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. It was found that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e itself has the strongest and most stable fluorescence intensity in the pH range of 7.0\u0026ndash;8.0. Because the reactivity of GSH increases with increasing pH, Meanwhile, \u003cb\u003eNap-Mem-GSH\u003c/b\u003e was affected under acidic conditions, leading to a decrease in the activity of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e in reaction with glutathione, which resulted in a weak fluorescence intensity. \u003cb\u003eNap-Mem-GSH\u003c/b\u003e reacted best with GSH in the pH range of 7.0\u0026ndash;8.0. T More assuredly, \u003cb\u003eNap-Mem-GSH\u003c/b\u003e was able to adapt to the cellular environment and enable cellular imaging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.4\u003c/b\u003e Selective response of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e to GSH\u003c/h2\u003e \u003cp\u003e \u003cb\u003eNap-Mem-GSH\u003c/b\u003e was then tested for its selectivity for other analytes, including other amino acids and common biometallic ions. It was found that only the addition of GSH to the \u003cb\u003eNap-Mem-GSH\u003c/b\u003e solution significantly enhanced the fluorescence intensity. Other substances did not trigger fluorescence enhancement. Thus \u003cb\u003eNap-Mem-GSH\u003c/b\u003e had high selectivity for GSH.\u003c/p\u003e \u003cp\u003eTo further demonstrate the selective recognition of GSH by \u003cb\u003eNap-Mem-GSH\u003c/b\u003e. Competition experiments between GSH and other substances were further tested by adding 20 \u0026micro;M aliquots of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e to a 200 \u0026micro;M solution of other analytes for testing, followed by 20 \u0026micro;M of GSH for testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, it was found that the other analytes did not affect the detection of GSH by \u003cb\u003eNap-Mem-GSH\u003c/b\u003e. GSH can also be easily detected in the presence of other analytes at the same time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.5\u003c/b\u003e The cell imaging of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo demonstrate the feasibility of the designed probe for glutathione imaging in intracellular medium using cell cultures. We performed MTT experiments to test the cytotoxicity of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e (Fig. S3). The results showed that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e had good cell viability, even when the probe concentration reached more than 20 \u0026micro;M. And \u003cb\u003eNap-Mem-GSH\u003c/b\u003e had no effect on cell growth, indicating that the probe should be used in biological cell experiments.\u003c/p\u003e \u003cp\u003eAccording to the practical application requirements, HeLa cells were selected as a cell model to analyze the performance of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e for the detection of intracellular GSH and GSH on cell membrane. Then, confocal laser scanning microfluorescence imaging studies were performed. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, HeLa cells could see brighter yellow fluorescence after the addition of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e (10 \u0026micro;M) and incubation for 20 minutes. When HeLa cells were pretreated with 5 mM of \u003cem\u003en\u003c/em\u003e-ethylmaleimide (NEM, a well-known biological thiol scavenger) \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e and incubated with \u003cb\u003eNap-Mem-GSH\u003c/b\u003e, only a small amount of yellow fluorescence was seen. To further investigate the cell membrane targeting of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e, In co-localization studies, imaging was performed using a widely used commercial cell membrane targeting dye, perchlorate DiD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). the signal generated by \u003cb\u003eNap-Mem-GSH\u003c/b\u003e overlapped very well with the fluorescence of DiD perchlorate. the Pearson's co-localization coefficient (describing the correlation of the intensity distribution between the two channels) was calculated to be 0.95, confirming that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e was specifically located in the cell membrane of live HeLa cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.6\u003c/b\u003e The zebrafish imaging of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFurther, \u003cb\u003eNap-Mem-GSH\u003c/b\u003e was investigated in zebrafish. The same part of the same zebrafish was imaged by fluorescence confocal microscopy test \u003cb\u003eNap-Mem-GSH\u003c/b\u003e. Yellow fluorescence was seen to appear in zebrafish, and then, by adding exogenous GSH, it was found enhanced yellow fluorescence in zebrafish, demonstrating the ability of \u003cb\u003eNap-Mem-GSH\u003c/b\u003e to detect exogenous GSH in zebrafish. Then added NEM exogenous to the solution while detecting the change in fluorescence intensity, and it was found that the fluorescence intensity in zebrafish diminished in vivo, indicating that \u003cb\u003eNap-Mem-GSH\u003c/b\u003e has the sensitivity to detect GSH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this work, a novel naphthalimide-based cell membrane-targeted fluorescent chemosensor was developed that carried a 2-hydroxyquinoline fragment on the naphthalimide dye as a recognition site for selective detection of GSH. The probe \u003cb\u003eNap-Mem-GSH\u003c/b\u003e not only selectively responds to GSH. Moreover, it has good cell membrane targeting properties. Simultaneous GSH detection and cell membrane targeting allowed verification of both intra- and extra-membrane recycling of GSH. This low toxicity probe had been successfully used to detect glutathione on cell membranes in living cells, as well as zebrafish imaging.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003e6.Acknowledgement\u003c/h2\u003e \u003cp\u003eThis work was financially supported by by the Natural Science Foundation of Henan Province (No. 252300423095), National Natural Science Foundation of China (21801145), Major Scientific Research Project for the Construction of State Key Lab (No. 2025ZDGZ02).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLin Li and Jian-Yong Wang: Conceptualization, Resources, Writing - Review \u0026amp; Editing, Supervision, Project Administration.Shao-Bin Sun: Formal Analysis, Resources, Project Administration, Writing original draft preparation. Dan Qiao and Jing Zhou: Formal Analysis, Data Curation, Software. Lin-Qing Wang: Resources, Software.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eScir\u0026egrave;, A., Cianfruglia, L., Minnell, C., et al. (2019). Glutathione compartmentalization and its role in glutathionylation and other regulatory processes of cellular pathways. \u003cem\u003eBiofactors (Oxford, England)\u003c/em\u003e, \u003cem\u003e45\u003c/em\u003e, 152\u0026ndash;168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong, J. S., Steinauer, K. K., Hornung, B., et al. (2002). Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. \u003cem\u003eCell Death \u0026amp; Differentiation\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 252\u0026ndash;263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTownsend, D. M., Tew, K. D., \u0026amp; Tapiero, H. (2003). 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A., et al. (2010). Second generation analogues of the cancer drug clinical candidate tipifarnib for anti-Chagas disease drug discovery. \u003cem\u003eJournal of medicinal chemistry\u003c/em\u003e, \u003cem\u003e53\u003c/em\u003e(10), 3887\u0026ndash;3898.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBecker, P. S., Cohen, C. M., \u0026amp; Lux, S. E. (1986). The effect of mild diamide oxidation on the structure and function of human erythrocyte spectrin. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e, \u003cem\u003e261\u003c/em\u003e(10), 4620\u0026ndash;4628.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"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":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Glutathione (GSH), large Stokes shift, Cell membrane targeting, Cell imaging, Zebrafish imaging","lastPublishedDoi":"10.21203/rs.3.rs-9217551/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9217551/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA naphthylimide dye-based fluorescent probe (\u003cb\u003eNap-Mem-GSH\u003c/b\u003e) was synthesized for the detection of Glutathione inside and outside cell membranes. This probe carried a 2-Hydroxyquinoline fragment as a recognition site on top of a naphthalimide dye, which exhibits better selective and fluorimetric response toward Glutathione in natural media. The long alkyl chain confers a cell membrane targeting role to \u003cb\u003eNap-Mem-GSH\u003c/b\u003e. Therefore, the detection of intra- and extracellular glutathione inside and outside the cell membrane can be realized by \u003cb\u003eNap-Mem-GSH\u003c/b\u003e. Meanwhile, it not only has a large Stokes shift (160 nm), but also has the advantages of low cytotoxicity and good membrane permeability to living cells, and has been successfully applied to effectively detect and image intracellular glutathione by confocal fluorescence imaging.\u003c/p\u003e","manuscriptTitle":"Observation of glutathione intra- and extracellular membrane with probe modified by long alkyl chains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 14:54:09","doi":"10.21203/rs.3.rs-9217551/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T07:05:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T04:02:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T09:39:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115750858040457449790137670270771260937","date":"2026-04-07T15:27:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62381815972958866153861209599151835397","date":"2026-04-02T10:36:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-02T10:00:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T09:26:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-25T17:41:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photochemical \u0026 Photobiological Sciences","date":"2026-03-25T03:07:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e768a5ae-ade9-4665-8407-c70c2928ce30","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T07:11:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 14:54:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9217551","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9217551","identity":"rs-9217551","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}