A simple fluorescent probe for biothiols detection and imaging of living cells in vivo based on a hemicyanine derivative

A simple fluorescent probe for biothiols detection and imaging of living cells in vivo based on a hemicyanine derivative | 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 simple fluorescent probe for biothiols detection and imaging of living cells in vivo based on a hemicyanine derivative Xia Gao, Xuehan Liu, Xinping Zheng, Yaxin Liu, Jinming Lin, Pengbo Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6233684/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 11 You are reading this latest preprint version Abstract Biothiols play essential roles in various biological processes and are closely associated with diseases such as cancer, neurodegenerative disorders, cardiovascular diseases and so on. To monitor biothiols in organisms and living cells, we present the synthesis of a novel intramolecular charge transfer (ICT)-based probe, HCD, by coupling a hemicyanine dye with 2,4-dinitrobenzenesulfonyl chloride. The HCD exhibits remarkable sensitivity, with a low detection limit of 0.32µM, 0.72µM and 0.27 µM for Cys, Hcy, and GSH, respectively, and demonstrates high selectivity for biothiols in the presence of various interfering species. The detection mechanism was thoroughly validated using high-resolution mass spectra (HRMS), UV and fluorescence spectra. Moreover, the applicability of HCD was successfully demonstrated in human urine samples and in vivo settings using RAW264.7 cells, confirming its potential as a powerful tool for monitoring endogenous biothiols in biological systems. Biothiols Fluorescent probe Fluorescence imaging Visual detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction In modern biological research and medical diagnostics, detecting and imaging biomolecules in living systems are critical. Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are vital for maintaining redox balance, mitigating oxidative stress, and regulating enzymatic activities [ 1 , 2 ]. Abnormal biothiol levels are linked to a variety of diseases. For example, altered levels of biothiols have been found in cancer, neurodegenerative disorders, and cardiovascular diseases [ 3 – 7 ]. Therefore, developing sensitive and selective methods for biothiols detection is essential for understanding the pathophysiology of these diseases and for developing effective diagnostic and therapeutic strategies. Traditional methods employed for biothiols detection frequently encounter limitations. They often fall short in sensitivity, fail to provide high specificity, and lack compatibility with live-cell imaging [ 8 – 10 ]. Fluorogenic techniques have emerged as powerful tools for biothiols detection, presenting notable benefits in comparison with traditional approaches [ 11 ]. These benefits include adjustable optical characteristics, swift response durations, exceptional sensitivity (even when the concentrations are at the micromolar level), cost-effectiveness and straightforward operational procedures [ 11 – 13 ]. Fluorogenic techniques exploit the unique reactivity of biothiols with specific fluorogenic probes. This interaction triggers a cascade of events that ultimately amplifies the fluorescence signals, thereby enabling highly sensitive and selective detection of biothiols [ 14 ]. Recent advancements in fluorogenic probe designed and synthesised have further expanded the utility of these techniques for biothiols analysis in complex biological samples [ 15 – 17 ]. For example, Wang et al. reported a mitochondria-targeted fluorescent probe for discrimination of biothiols by dual-channel imaging in living cells and zebrafish [ 18 ]. Wang et al. reported an ortho-activation strategy to develop NIR fluorescent probe for rapid imaging of biothiols in vivo [ 19 ]. However, many existing probes suffer from limitations such as cumbersome synthesis steps, poor water solubility, difficulty in imaging living cells in vivo and so on. Herein, we present a simple fluorescent probe (HCD) for biothiols detection and live-cell imaging based on the condensation of hemicyanine dye with 2,4-dinitrobenzenesulfonamide. Hemicyanine dyes exhibit strong visible-to-NIR absorption/emission, enabling deep tissue penetration and reduced background interference [ 20 , 21 ]. HCD selectively reacts with biothiols via 2,4-dinitrobenzenesulfonamide cleavage, inducing spectral shifts, fluorescence enhancement and live-cell imaging. The probe is expected to offer high sensitivity and selectivity for biothiols detection, as well as good stability and biocompatibility. To evaluate the performance of HCD, a series of experiments are conducted. Its selectivity and sensitivity for biothiols detection are assessed by testing its response to various biomolecules and analytes. The fluorescence response to different concentrations of biothiols is measured to determine its detection limit and dynamic range. In addition, HCD is used to detect Hcy levels in human urine samples and image biothiols in living cells. The development of this simple fluorescent probe for biothiols detection and imaging of living cells in vivo holds great promise for advancing our understanding of biological processes and improving the diagnosis and treatment of diseases. It provides a valuable tool for researchers and clinicians to study the roles of biothiols in health and disease and to monitor the response to therapeutic interventions. 2 Experimental section 2.1 Reagents and apparatus 2,4-Dinitrobenzenesulfonyl chloride, p-aminobenzaldehyde, L-homocysteine (Hcy), L-Cysteine(Cys) and 1,2,3,3-tetramethyl − 3H -indolium iodide were obtained from Sigma-Aldrich Company. Disodium hydrogen phosphate (Na 2 HPO 4 ), sodium dihydrogen phosphate(NaH 2 PO 4 ), glutathione (GSH), L-tyrosine(Tyr), leucine(Leu), L-threonine(Thr), L-phenylalanine(Phe), valine(Val), DL-aspartic acid(Asp), L-tryptophan(Try), DL-proline(Pro), L-glutamic acid(Glu), L-lysine(Lys), L-histidine(His), L-arginine(Arg), glycine(Gly), DL-serine(Ser) and β-alanine(β-Ala) were obtained from Shanghai Bide Pharmaceutical Technology Co., Ltd. Dimethylsulfoxide (cell culture level), penicillin-streptomycin, Trypsin-EDTA Digest (0.25%, no phenol red) and phosphate buffered saline buffer (PBS buffer) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Fetal cattle serum (FCS) was received from Biological Industries (Israel); Dulbecco's Modified Eagle Medium ( DMEM, high Glucose) was purchased from Hyclone Biochemical Products Co., Ltd.(America); Cell Counting Kit-8(CCK-8)was purchased from MedChem Express Biotechnology Co., Ltd. All reagents were of analytical grade and used without further purification. Twice-deionized water was further distilled in the presence of KMnO 4 . 1 H nuclear magnetic resonance (NMR) spectra were obtained via using the Bruker AV III 400M, 13 C NMR spectra were measured on Bruker AV III 500M spectrometer and tetramethyl silane (TMS) as internal standard. MS analyses were obtained on a Bruker Daltonics Bio-TOF-Q mass spectrometer by the ESI method. Column chromatography was performed on silica gel (200–300 mesh). A T9 UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., Beijing, China) and a 1 cm quartz cell were used for recording absorption spectra and measuring absorbance; Fluorescence was measured using a Agilent Cary Eclipse Fluorescence Spectrophotometer (Agilent, America). Cell culture was carried out in a CO 2 cell incubator (Thermo Fisher Scientific, America). The pH was measured with a Model EL20 meter (METTLER TOLEDO, China). 2.2 Synthesis of the new hemicyanine dye The synthetic procedure for the new hemicyanine dye (HCD) was illustrated in Scheme 1 . 2.2.1 Synthesis of compound 1. Compound 1 was synthesized as follows: 2.1375 g (8 mmol) of 2,4-dinitrobenzenesulfonyl chloride and 0.4840 g (4 mmol) of p-aminobenzaldehyde were added, then, dichloromethane (DCM, 40 mL) dissolved with 0.6328 g (8 mmol) of pyridine was added slowly, the reaction mixture was refluxed for 2h under argon atmosphere, and allowed to cool to room temperature. After being extracted with ethyl acetate for three times, the organic phase was rotary evaporated and purified by flash chromatography with petroleum ether /ethyl acetate (2/1, V/V) as eluent to afford 1(0.8736 g, yield: 31.11%). The newly synthesized compound was characterized using 1 H NMR and 13 C NMR. The results obtained were as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ11.76 (s, 1H), 9.86 (s, 1H), 8.92 (d, J = 2.3 Hz, 1H), 8.60 (dd, J = 8.7, 2.3 Hz, 1H), 8.31 (d, J = 8.7 Hz, 1H), 7.96–7.73 (m, 2H), 7.42–7.18 (m, 2H) (Fig. S 1 ). 13 C NMR (101 MHz, DMSO- d6 ) δ191.75, 150.32, 147.83, 141.87, 135.83, 132.36, 131.74, 131.26, 127.50, 120.64, 119.24(Fig. S 2 ). 2.2.2 Synthesis of fluorescent probe HCD Compound HCD was synthesized as follows: 0.1766 g (0.5 mmol) of compound 1 and 0.2275 g (0.75 mmol) of 1,2,3,3-tetramethyl-3H-indole iodide were dissolved in 3 mL of ice acetic acid, the reaction mixture was refluxed for 4h under N 2 atmosphere, and allowed to cool to room temperature. Then the reaction mixture was filtered and the solid was washed three times with 2 mL of acetone and then fully dried to afford a yellow-orange solid HCD (0.1777 g, yield: 56.05%). The purity of HCD was confirmed by MS, 1 H NMR and 13 C NMR spectra as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ11.76 (s, 1H), 8.94 (d, J = 2.3 Hz, 1H), 8.61 (dd, J = 8.8, 2.3 Hz, 1H), 8.42–8.24 (m, 2H), 8.15 (d, J = 8.6 Hz, 2H), 7.95–7.79 (m, 2H), 7.65–7.50 (m, 3H), 7.28 (d, J = 8.5 Hz, 2H), 4.10 (s, 3H), 1.75 (s, 6H) (Fig. S 3 ). 13 C NMR (101 MHz, DMSO- d6 ) δ181.58, 151.80, 150.27, 147.88, 143.54, 141.82, 140.79, 135.84, 132.13, 131.76, 130.69, 129.36, 128.97, 127.46, 122.89, 120.50, 119.54, 115.20, 112.41, 52.12, 34.60, 25.29 (Fig. S 4 ). ESI-MS m/z calculated value for C 19 H 21 N 2 S + [M-I] + , 277.1699, found 277.1700 (Fig. S 5 ). 2.3 Standard procedures for sensing biothiols To a 10.0 mL volumetric ask 100 µL stock solution of HCD (20 µM) in DMSO, 1.00 mL of phosphate buffer solution (PB, 20 mM, pH 7.40), an appropriate working solution of biothiols were transferred in turn, and the mixture was diluted to 5.0 mL with distilled water and mixed thoroughly. The mixture was left undisturbed for 40 min (for Cys) or 60 min (for Hcy and GSH) to allow HCD and biothiols to interact fully and improve the reproducibility and stability of the test results. Then the absorbance intensities and fluorescence intensities of the sample and the blank (prepared in a similar manner without biothiols) were determined by measuring the absorbance at 465 nm and the fluorescence at 558nm. 2.4 Cells culture RAW264.7 macrophages were purchased from China Center for Type Culture Collection (CCTCC) and cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37 ℃ in a 5% CO 2 /95% air incubator. One day before imaging, the cells were detached and replanted on glass-bottomed dishes. 3 Results and Discussion 3.1 Spectral characteristics HCD is a hemicyanine dye with good stability, which only recognizes biothiols with high specificity. As shown in Fig. 1 (A), free HCD in PB buffer (pH 7.40) solution exhibited the maximum absorption wavelength at 465 nm. However, the maximum absorption wavelength shifted to 480 nm with the decline of the absorption peak at 465 nm after addition of Cys, Hcy and GSH. Along with the changes of absorption spectra, the fluorescence intensity of HCD at 558 nm increased significantly upon excitation at 465 nm (Fig. 1 (B)) with the addition of Cys, Hcy and GSH. This phenomenon was ascribed to the strong interaction between HCD and biothiols. 3.2 Optimization of the buffer system, pH, temperature and incubation time To obtain the best experimental conditions, some buffer systems such as tris–HCl buffer, HEPES buffer and phosphate buffer were examined in terms of the analytical sensitivity and linear range of the calibration curve, and it was found that phosphate buffer (PB) was better than the others (Fig. 2 (A)). Then the influence of pH on the sensitivity were investigated. The results showed the best absorbance changes (A 0 -A, where A 0 and A are the absorbance at 465 nm in the absence and presence of biothiols, respectively) were obtained at pH 7.40 (Fig. 2 (B)). Therefore, pH 7.40 of PB buffer was selected to control the acidity of the system in this study. Effects of temperature on the response system is shown in Fig. 3 . It can be clearly seen from the figure that the response of HCD to Cys is most sensitive at 37°C, while that is most sensitive at 42°C for GSH and Hcy. Considering the subsequent cell experiments, 37°C was selected as the temperature for the subsequent experiments. The effect of incubation time on the absorbance changes at 465 nm was shown in Fig. 4 , the system basically reached equilibrium within 40 min following Cys addition and 60 min following Hcy and GSH addition. Moreover, the response system remained constant for at least 80 min. Thus, it was suggested that the measurement should start 40 min after addition of Cys, 60 min after addition of Hcy or GSH, and finish within 80 min. 3.3 Selectivity and interference of foreign substances The specific recognition ability is a key indicator for evaluating the performance of fluorescent probes. We investigated the specific recognition ability of probe HCD for Cys, GSH, and Hcy. The results are shown in Fig. 5 (A). As can be clearly seen from the figure, among many amino acids, the absorbance intensity of probe HCD is significantly changed only when Cys, GSH or Hcy is added. This indicates that the probe HCD has a high selectivity for Cys, GSH, and Hcy, reflecting its specific recognition ability for biothiols. To further validate the method, competitive experimentation of HCD for Cys, GSH, and Hcy was carried out by adding nearly the same concentration of Cys, GSH, Hcy and other amino acids (Fig. 5 (B)). It could be seen that other amino acids had no obvious effects on the spectral response of HCD for Cys, GSH, and Hcy. Detection results of selective and competitive experiments showed that the probe had excellent selectivity towards Cys, GSH, and Hcy compared with amino acids. 3.4 Calibration graphs The calibration graphs of three kinds of biothiols were constructed under the optimal conditions. There were good linear relationships between the increments of fluorescence intensity and the concentration of biothiols, and the results are given in Fig. 6 and Table 1 . Table 1 Analytical parameters for the determination of biothiols using this method Biothiols Normal level in the body Linear range Linear equation Detection limit Cys 30–200 µM 6.0×10 − 7 -6.0×10 − 4 M ΔI = 716.03C Cys -4.2953 3.02×10 − 7 M GSH 1–10 mM 6.0×10 − 7 -2.0×10 − 4 M ΔI = 1279.10C GSH + 2.6253 2.65×10 − 7 M Hcy 5–12 µM 8.0×10 − 7 -1.2×10 − 3 M ΔI = 467.09C Hcy + 0.2600 7.62×10 − 7 M To examine the potential applicability in simulated physiological media, we take Cys as an example and investigate the fluorescence titrations in the presence of biologically interfering ions such as Na + , K + , Mg 2+ , Ca 2+ , Cl − , HCO 3 − , H 2 PO 4 − , and HPO 4 2− . Under the testing conditions, the interferences from these species are negligible in the titration by Cys compared with those obtained in the presence of Cys alone (Fig.S 6 ). 3.5 Endogenous biothiols imaging in live cells with HCD The above studies indicated that HCD was highly suitable for detecting biothiols under simulated physiological conditions (Fig.S 6 ). To examine the feasibility of probe HCD as an imaging reagent, its potential toxicity to cells was evaluated by a standard CCK-8 (Cell Counting Kit-8) assay. The results demonstrated that even when treated with 10 µM HCD at 37°C for 48 h, the cell viability did not change significantly (Fig. S 7 ), implying the low cytotoxicity and good biocompatibility of probe HCD. Subsequently, the applicability of HCD for imaging RAW 264.7 macrophages was explored. The results of fluorescence imaging detection of biothiols in cell medium by HCD are shown in Fig. 6 . As we can see, when RAW264.7 cells are treated with a sulfhydryl scavenger (NEM) and then added a 10 µM probe HCD solution, no obvious fluorescence signal is observed [Fig. 7 (A1)]; while when the probe HCD solution is directly added, a relatively obvious fluorescence signal is observed [Fig. 7 (B1)], and the fluorescence intensity is 3.0 times stronger than that of the group pretreated with NEM. These experimental results fully indicate that HCD can be used for the visual detection of endogenous biothiols in cells. 3.6 The molecular mechanism of HCD to recognize biothiols As shown in Scheme 1 , HCD is a typical “Donor-π-Accepter” (“D-π-A”) molecule with excellent optical properties due to intramolecular charge transfer (ICT). When HCD is treated with 2,4-dinitrobenzenesulfonyl chloride, the charge density of the “Donor” is decreased, weakening its ICT process and thus resulting in shortwave absorption (with an absorbance maximum at 465 nm) and low fluorescence intensity at 558 nm. When HCD reacts with biothiols (Cys), the absorbance maximum will be redshifted to 480 nm and the fluorescence intensity will be enhanced. The specific interaction process between HCD and Cys is shown in Scheme 2 . As depicted in Scheme 2 , when Cys is added to the detection system containing HCD, a nucleophilic substitution reaction occurs between HCD and Cys, leading to the cleavage of the sulfonamide bond and the generation of the fluorophore ATI (2-(4-aminostyrenyl)-1,3,3-trimethyl-3H-indole iodonium, ATI). The amino group in ATI increases the charge density of the “Donor” and causes a redshift of the absorption spectra and an enhancement of fluorescence intensity. This assumption is further supported by the HRMS of ATI (Fig. S 8 ), which shows a strong peak at m/z 277.1700, assigned to C 19 H 21 N 2 + ([M-I] + : 277.1699). This clearly indicates that the reaction of HCD with Cys generates ATI, thereby causing changes in the absorption spectra and fluorescence spectra. 3.7 Practical application of the method in human urine samples Relevant studies indicate that urinary Hcy levels can directly reflect renal function. To explore the potential applicability of this method in complex biofluids, we utilized it for Hcy detection in human urine. Specifically, 30 mL urine samples were collected from five early renal injury patients (from the Third Affiliated Hospital of Xinxiang Medical University) and five healthy volunteers. After centrifugation at 12000 rpm for 10 min, 15 mL of supernatant from each sample was collected and diluted to 30 mL. The proposed method and the Ellman method were then employed to measure Hcy content in each urine sample. As shown in Table 2 , the results obtained by the developed method closely matched those from the Ellman method. Additionally, both methods yielded results consistent with literature findings [ 22 ]. These outcomes, along with the data presented in Fig. S 6 and Fig. 7 , confirm that the proposed method is reliable and practical for determining biothiols in complex biofluids. Table 2 Detection of Hcy in human urine Samples Proposed method found a (µM) Hcy added(µM) Hcy found(µM) Recovery (%) Ellman method found(µM) 1 14.26 ± 0.1 2.00 16.33 ± 0.1 104.0 14.29 ± 0.1 2 11.22 ± 0.1 2.00 13.32 ± 0.1 105.0 11.28 ± 0.1 3 13.65 ± 0.1 2.00 15.52 ± 0.1 93.5 13.68 ± 0.1 4 11.85 ± 0.1 2.00 13.69 ± 0.1 92.0 11.89 ± 0.1 5 13.26 ± 0.1 2.00 15.28 ± 0.1 101 13.34 ± 0.1 6 18.35 ± 0.1 2.00 20.30 ± 0.1 97.5 18.28 ± 0.1 7 16.68 ± 0.1 2.00 18.66 ± 0.1 99.0 16.71 ± 0.1 8 16.85 ± 0.1 2.00 18.69 ± 0.1 92.0 16.88 ± 0.1 9 17.38 ± 0.1 2.00 19.39 ± 0.1 100.0 17.33 ± 0.1 10 18.01 ± 0.1 2.00 20.10 ± 0.1 105.0 18.06 ± 0.1 a 1-5: Representing healthy volunteers; 6–10: Representing patients with early renal injury. 4 Conclusion In summary, a novel fluorescent probe, HCD, was successfully synthesized by condensing a hemicyanine dye with 2,4-dinitrobenzenesulfonyl chloride. This probe exhibits high sensitivity and specificity for detecting biothiols in aqueous solutions and allows for the visualization of biothiols within cellular environments. Its practicality and accuracy were further validated through real sample measurements.The proposed method is characterized by its simplicity, rapidity, and exceptional sensitivity and selectivity for biothiol detection. Its potential application for in situ monitoring of biothiol levels in biological systems could provide critical data to support and advance human health protection efforts. Declarations Author Contributions X.G.established the characterization procedures using spectroscopy techniques;X.H.L.synthesized the fluorescent probe following the designed route and characterized the probe by performing fluorescence and other spectroscopic measurements; X.P.Z.carried out tests on the probe in biological cells ;Y.X.L.analyzed all the experimental data to evaluate the probe's performance;J.M.L.secured the research funding and review the manuscript;P.B.Z.designed the synthetic method for the probe. Acknowledgements The authors are grateful for the fund support by the Key Scientific Research Projects of Universities of Henan Province (23A330002), the Scientific and Technological Project of Henan Province (252102310090), the National Natural Science Foundation of China (grant no.22201242) and the Research Foundation for Distinguished Professor’s Team of Xinxiang Medical University (no.505527). Data availability The data supporting the findings of this study are available upon request from the corresponding author. Ethics Approval and Consent to Participate Urine samples were collected from five patients with early renal injury (from the Third Affiliated Hospital of Xinxiang Medical University) and five healthy volunteers. The participants were all informed explicitly about the plan, protocol, and procedure for the study, and written consent was obtained. This consent procedure was reviewed and approved by the Medical Ethics Committee of Xinxiang Medical University. All experiments in this work were performed in compliance with the relevant laws and institutional guidelines and approved by the Medical Ethics Committee of Xinxiang Medical University. Consent for Publication Not Applicable. Competing Interests The authors declare no competing interests References Huang N, Yang D, Chen HH, Xiao Y, Wen JH, Long YJ, Zheng HZ (2023) Colorimetric detection of biothiols and Hg 2+ based on the peroxidase-like activity of GTP. Spectrochim Acta Mol Biomol Spectrosc 290:122263 Gong YJ, Wang PP, Zhai HC, Xiao Y, Wang Q, Ma NN, Zhang GS, Zhang H (2024) Equivalent Response Strategy for Sensing Total Biothiols in Human Serums and Living Cells Using a Hemicyanine-Based Self-Immolative Probe. Anal Chem 96:1009–1018 Niu HY, Liu JW, O’Connor HM, Gunnlaugsson T, James TD, Zhang H (2023) Photoinduced electron transfer (PeT) based fluorescent probes for cellular imaging and disease therapy. Chem Soc Rev 52:2322–2357 Zhang CJ, Qin Y, Deng CF, Zhu N, Shi YN, Wang W, Qin L (2023) GSH-specific fluorescent probe for sensing, bioimaging, rapid screening of natural inhibitor Celastrol and ccRCC theranostics. Anal Chim Acta 1248:340933 Liu CY, Zhang Y, Sun WM, Zhu HC, Su MJ, Wang X, Rong XD, Wang K, Yu MH, Sheng WL, Zhu BC (2023) A novel GSH-activable theranostic probe containing kinase inhibitor for synergistic treatment and selective imaging of tumor cells. Talanta 260:124567 Wang W, Peng ZH, Ji M, Chen JQ, Wang P (2022) Highly selective fluorescent probe based on AIE for identifying cysteine/homocysteine. Bioorg Chem 126:105902 Lu WY, Li HJ, Wu YC (2024) Assessment of environmental and biological stress using mitochondria-targeted red-emitting and near-infrared fluorescent probes for biothiol analysis: a review. Environ Chem Lett 22:3135–3169 Millan S, Jeffery DW, Dall'Acqua S, Masi A (2021) A novel HPLC-MS/MS approach for the identification of biological thiols in vegetables. Food Chem 339:12780 Sun Y, Yao T, Guo XC, Peng Y, Zheng J (2016) Simultaneous assessment of endogenous thiol compounds by LC-MS/MS. J Chromatogr B 1029–1030:213–221 Kostal V, Katzenmeyer J, Arriaga Edgar A (2008) Capillary electrophoresis in bioanalysis. Anal Chem 80:4533–4550 Pak YL, Swamy KMK, Yoon J (2015) Recent Progress in Fluorescent Imaging Probes. Sensors 15:24374–24396 Jung HS, Chen X, Kim JS, Yoon J (2013) Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem Soc Rev 42:6019–6031 Lippert AR, New EJ, Chang CJ (2011) Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells. J Am Chem Soc 133:10078–10080 Asghar R, Li Y, Huo F, Yin C (2024) Sensing mechanism of cysteine specific fluorescence probes and their application of cysteine recognition. Chem Biomed Imaging 4:250–269 Dai J, Ma C, Zhang P, Fu Y, Shen B (2020) Recent progress in the development of fluorescent probes for detection of biothiols. Dyes Pigm 177:108321 Ding S, Liu M, Hong Y (2018) Biothiol-specific fluorescent probes with aggregation-induced emission characteristics. Sci China Chem 61:882–891 Kim Y, Kim J, An JM, Park CK, Kim D (2023) All-nontoxic fluorescent probe for biothiols and its clinical applications for real-time glioblastoma visualization. ACS Sens 8:1723–1732 Wu ZJ, Xu NG, Zhang D, Liu H, Li LL, Wang FY, Ren J, Wang EF (2024) A mitochondria-targeted fluorescent probe for discrimination of biothiols by dual-channel imaging in living cells and zebrafish. Spectrochim Acta Mol Biomol Spectrosc 322:124846 Yu L, Xie ML, Chen M, Yang HR, Chen L, Xing PF, Tian ZY, Wang CJ (2024) An ortho-activation strategy to develop NIR fluorescent probe for rapid imaging of biothiols in vivo. Talanta 266:125110 Bi KY, Tan R, Hao RT, Miao LX, He YQ, Wu XH, Zhang JF, Xu R (2019) A carbazole-hemicyanine dye based ratiometric fluorescent probe for selective detection of bisulfite(HSO 3 – ) in cells and C.elegans. Chin Chem Lett 30:545–548 Xiang K, Pan JB, Yu JJ, Xiao LH, Sun SK, Cheng R (2024) A hemicyanine-based near-infrared fluorescent probe with large Stokes shift for non-invasive bioimaging of brown adipose tissue. Anal Methods 16:5272–5279 Ren CN, Chao DU, Qi YU, Zhang Y (2019) Diagnostic value of urine homocysteine and retinol binding protein detection in the early renal injury. China Mod Med 26:131–133 Schemes Schemes 1 and 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files biothiolsSupplementaryInformationxuehan.docx GraphicalabstractandSchemes.docx Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 01 Apr, 2025 Reviews received at journal 27 Mar, 2025 Reviews received at journal 27 Mar, 2025 Reviewers agreed at journal 24 Mar, 2025 Reviews received at journal 24 Mar, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers agreed at journal 21 Mar, 2025 Reviewers invited by journal 21 Mar, 2025 Editor assigned by journal 19 Mar, 2025 Submission checks completed at journal 19 Mar, 2025 First submitted to journal 15 Mar, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6233684","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434912357,"identity":"e50bf1cc-7361-4c98-8cfa-e2521a764dbb","order_by":0,"name":"Xia Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACNvmHDQcSKmxgXCK08DMkNz74cCaNBC2SDenNhjPbDpOgxeDAwTZp3rbziRuunTFg+FB2mIF/dgMBLQcb26R5zt02lpydY8A449xhBok7BwhoOcwI1FJ2W45fOseAmRfoQgOJBAJajoG0sJ3jYQNp+UuMFskexmbDGW0HILYwEqOFX4IRFMjJQL+kFRzsOZfOI3GDgBY2CfYHwKi0S9xwO3njgx9l1nL8MwhoQQEHgJiHBPWjYBSMglEwCnABAN6KQ72yt2bMAAAAAElFTkSuQmCC","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xia","middleName":"","lastName":"Gao","suffix":""},{"id":434912358,"identity":"0aeb0bdc-21f9-44d2-bf99-9f9770ab9597","order_by":1,"name":"Xuehan Liu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuehan","middleName":"","lastName":"Liu","suffix":""},{"id":434912359,"identity":"1a1b2f2b-39d4-48d4-8437-bdfd606be468","order_by":2,"name":"Xinping Zheng","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinping","middleName":"","lastName":"Zheng","suffix":""},{"id":434912360,"identity":"fbb3a755-480c-4c22-868d-88482c1dc5ef","order_by":3,"name":"Yaxin Liu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yaxin","middleName":"","lastName":"Liu","suffix":""},{"id":434912361,"identity":"e8995197-4f76-4e3a-a624-1ad3af41290c","order_by":4,"name":"Jinming Lin","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinming","middleName":"","lastName":"Lin","suffix":""},{"id":434912362,"identity":"1ed622e7-0c5a-4595-b945-fef189f8050e","order_by":5,"name":"Pengbo Zhang","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Pengbo","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-15 15:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6233684/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6233684/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-025-04350-7","type":"published","date":"2025-04-30T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79658360,"identity":"d9688083-a76d-48b7-b9f3-6630290ea9a6","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88168,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Absorbance spectra of HCD as recorded in PB buffer (pH 7.40) in the presence or absence of Cys, Hcy and GSH. (b) Fluorescence spectra of HCD in the absence and presence of Cys, Hcy and GSH. Concentration of HCD 2.00×10\u003csup\u003e-5\u003c/sup\u003e M, Cys, Hcy and GSH 2.00×10\u003csup\u003e-5\u003c/sup\u003e M , Other conditions are the same as those described in the procedure\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/5223ba33db1a450d506b27fd.png"},{"id":79658750,"identity":"e29c34d0-1124-4be9-a146-6a377d70a586","added_by":"auto","created_at":"2025-04-01 09:20:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72810,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of buffer system(A) and pH(B)([HCD]= 2.00×10\u003csup\u003e-5\u003c/sup\u003e M， [Cys, Hcy or GSH] = 1.00×10\u003csup\u003e-4\u003c/sup\u003e M，response at 37°C for 40 min)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/beec67ba5b25b0d63fbbc163.png"},{"id":79658362,"identity":"e697c3a5-85f6-4cfc-969c-b8e2a60edd57","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42492,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of temperature ([HCD]= 2.00×10\u003csup\u003e-5\u003c/sup\u003e M， [Cys, Hcy or GSH ] = 1.00×10\u003csup\u003e-4\u003c/sup\u003e M，response at 37°C for 40 min)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/fe7dd9f3e55df9f9056db615.png"},{"id":79658363,"identity":"b265a314-154b-4943-8c07-587c3555b485","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38574,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of incubation time on the HCD-biothiols ensembles\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/f13d839d146ef4ff7a4b5041.png"},{"id":79658372,"identity":"7d0e2c9a-5ce9-4427-bc58-1c9ccf07a46f","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154236,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Absorption response of HCD to Cys, Hcy and GSH and other amino acids ; (b) Absorbance response of HCD to Cys, Hcy and GSH in the presence of competitive substance.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/8eb34f60752949e96a12d3a8.png"},{"id":79658367,"identity":"e0ffec87-aafd-4aae-8154-422e3073a6b7","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46888,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence titration curves for spectrophotometric determination of biothiols.([HCD]= 2.00×10\u003csup\u003e-5\u003c/sup\u003e M , for Cys : PB buffer solution of pH 7.40, response at 37°C for 40 min; for GSH and Hcy:PB buffer solution of pH 8.00, response at 42°C for 60 min )\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/6d494040ecf62aef83c09fc7.png"},{"id":79658375,"identity":"eb854d1b-b5bf-4530-9723-8e244e727ab4","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":162452,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/0951f08df174f7e99592810e.png"},{"id":81987443,"identity":"02c8e37b-39e5-4c1a-9239-3c3b84ffd6e7","added_by":"auto","created_at":"2025-05-05 16:02:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1459755,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/a74f958d-8f8d-4381-a2a9-936afb955b2d.pdf"},{"id":79658366,"identity":"59a5fe10-a766-450e-9497-53f2de0a32e1","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":360715,"visible":true,"origin":"","legend":"","description":"","filename":"biothiolsSupplementaryInformationxuehan.docx","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/81d7145163caa09327e107e7.docx"},{"id":79658368,"identity":"94271624-7765-49d9-b4f3-7bce19ec6af5","added_by":"auto","created_at":"2025-04-01 09:12:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":559573,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractandSchemes.docx","url":"https://assets-eu.researchsquare.com/files/rs-6233684/v1/ae7579a8c396362b026b7188.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A simple fluorescent probe for biothiols detection and imaging of living cells in vivo based on a hemicyanine derivative","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eIn modern biological research and medical diagnostics, detecting and imaging biomolecules in living systems are critical. Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are vital for maintaining redox balance, mitigating oxidative stress, and regulating enzymatic activities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Abnormal biothiol levels are linked to a variety of diseases. For example, altered levels of biothiols have been found in cancer, neurodegenerative disorders, and cardiovascular diseases [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, developing sensitive and selective methods for biothiols detection is essential for understanding the pathophysiology of these diseases and for developing effective diagnostic and therapeutic strategies.\u003c/p\u003e \u003cp\u003eTraditional methods employed for biothiols detection frequently encounter limitations. They often fall short in sensitivity, fail to provide high specificity, and lack compatibility with live-cell imaging [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Fluorogenic techniques have emerged as powerful tools for biothiols detection, presenting notable benefits in comparison with traditional approaches [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These benefits include adjustable optical characteristics, swift response durations, exceptional sensitivity (even when the concentrations are at the micromolar level), cost-effectiveness and straightforward operational procedures [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Fluorogenic techniques exploit the unique reactivity of biothiols with specific fluorogenic probes. This interaction triggers a cascade of events that ultimately amplifies the fluorescence signals, thereby enabling highly sensitive and selective detection of biothiols [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recent advancements in fluorogenic probe designed and synthesised have further expanded the utility of these techniques for biothiols analysis in complex biological samples [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For example, Wang \u003cem\u003eet al.\u003c/em\u003e reported a mitochondria-targeted fluorescent probe for discrimination of biothiols by dual-channel imaging in living cells and zebrafish [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Wang \u003cem\u003eet al.\u003c/em\u003e reported an ortho-activation strategy to develop NIR fluorescent probe for rapid imaging of biothiols in vivo [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, many existing probes suffer from limitations such as cumbersome synthesis steps, poor water solubility, difficulty in imaging living cells in vivo and so on.\u003c/p\u003e \u003cp\u003eHerein, we present a simple fluorescent probe (HCD) for biothiols detection and live-cell imaging based on the condensation of hemicyanine dye with 2,4-dinitrobenzenesulfonamide. Hemicyanine dyes exhibit strong visible-to-NIR absorption/emission, enabling deep tissue penetration and reduced background interference [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. HCD selectively reacts with biothiols via 2,4-dinitrobenzenesulfonamide cleavage, inducing spectral shifts, fluorescence enhancement and live-cell imaging. The probe is expected to offer high sensitivity and selectivity for biothiols detection, as well as good stability and biocompatibility.\u003c/p\u003e \u003cp\u003eTo evaluate the performance of HCD, a series of experiments are conducted. Its selectivity and sensitivity for biothiols detection are assessed by testing its response to various biomolecules and analytes. The fluorescence response to different concentrations of biothiols is measured to determine its detection limit and dynamic range. In addition, HCD is used to detect Hcy levels in human urine samples and image biothiols in living cells. The development of this simple fluorescent probe for biothiols detection and imaging of living cells in vivo holds great promise for advancing our understanding of biological processes and improving the diagnosis and treatment of diseases. It provides a valuable tool for researchers and clinicians to study the roles of biothiols in health and disease and to monitor the response to therapeutic interventions.\u003c/p\u003e"},{"header":"2 Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents and apparatus\u003c/h2\u003e \u003cp\u003e2,4-Dinitrobenzenesulfonyl chloride, p-aminobenzaldehyde, L-homocysteine (Hcy), L-Cysteine(Cys) and 1,2,3,3-tetramethyl \u0026minus;\u0026thinsp;3H -indolium iodide were obtained from Sigma-Aldrich Company. Disodium hydrogen phosphate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), sodium dihydrogen phosphate(NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), glutathione (GSH), L-tyrosine(Tyr), leucine(Leu), L-threonine(Thr), L-phenylalanine(Phe), valine(Val), DL-aspartic acid(Asp), L-tryptophan(Try), DL-proline(Pro), L-glutamic acid(Glu), L-lysine(Lys), L-histidine(His), L-arginine(Arg), glycine(Gly), DL-serine(Ser) and β-alanine(β-Ala) were obtained from Shanghai Bide Pharmaceutical Technology Co., Ltd. Dimethylsulfoxide (cell culture level), penicillin-streptomycin, Trypsin-EDTA Digest (0.25%, no phenol red) and phosphate buffered saline buffer (PBS buffer) were purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. Fetal cattle serum (FCS) was received from Biological Industries (Israel); Dulbecco's Modified Eagle Medium ( DMEM, high Glucose) was purchased from Hyclone Biochemical Products Co., Ltd.(America); Cell Counting Kit-8(CCK-8)was purchased from MedChem Express Biotechnology Co., Ltd. All reagents were of analytical grade and used without further purification. Twice-deionized water was further distilled in the presence of KMnO\u003csub\u003e4\u003c/sub\u003e. \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance (NMR) spectra were obtained via using the Bruker AV III 400M,\u003csup\u003e13\u003c/sup\u003eC NMR spectra were measured on Bruker AV III 500M spectrometer and tetramethyl silane (TMS) as internal standard. MS analyses were obtained on a Bruker Daltonics Bio-TOF-Q mass spectrometer by the ESI method. Column chromatography was performed on silica gel (200\u0026ndash;300 mesh). A T9 UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., Beijing, China) and a 1 cm quartz cell were used for recording absorption spectra and measuring absorbance; Fluorescence was measured using a Agilent Cary Eclipse Fluorescence Spectrophotometer (Agilent, America). Cell culture was carried out in a CO\u003csub\u003e2\u003c/sub\u003e cell incubator (Thermo Fisher Scientific, America). The pH was measured with a Model EL20 meter (METTLER TOLEDO, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis of the new hemicyanine dye\u003c/h2\u003e \u003cp\u003eThe synthetic procedure for the new hemicyanine dye (HCD) was illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Synthesis of compound 1.\u003c/h2\u003e \u003cp\u003eCompound 1 was synthesized as follows: 2.1375 g (8 mmol) of 2,4-dinitrobenzenesulfonyl chloride and 0.4840 g (4 mmol) of p-aminobenzaldehyde were added, then, dichloromethane (DCM, 40 mL) dissolved with 0.6328 g (8 mmol) of pyridine was added slowly, the reaction mixture was refluxed for 2h under argon atmosphere, and allowed to cool to room temperature. After being extracted with ethyl acetate for three times, the organic phase was rotary evaporated and purified by flash chromatography with petroleum ether /ethyl acetate (2/1, V/V) as eluent to afford 1(0.8736 g, yield: 31.11%). The newly synthesized compound was characterized using \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR. The results obtained were as follows:\u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ11.76 (s, 1H), 9.86 (s, 1H), 8.92 (d, J\u0026thinsp;=\u0026thinsp;2.3 Hz, 1H), 8.60 (dd, J\u0026thinsp;=\u0026thinsp;8.7, 2.3 Hz, 1H), 8.31 (d, J\u0026thinsp;=\u0026thinsp;8.7 Hz, 1H), 7.96\u0026ndash;7.73 (m, 2H), 7.42\u0026ndash;7.18 (m, 2H) (Fig. S\u003csub\u003e1\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ191.75, 150.32, 147.83, 141.87, 135.83, 132.36, 131.74, 131.26, 127.50, 120.64, 119.24(Fig. S\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Synthesis of fluorescent probe HCD\u003c/h2\u003e \u003cp\u003eCompound HCD was synthesized as follows: 0.1766 g (0.5 mmol) of compound 1 and 0.2275 g (0.75 mmol) of 1,2,3,3-tetramethyl-3H-indole iodide were dissolved in 3 mL of ice acetic acid, the reaction mixture was refluxed for 4h under N\u003csub\u003e2\u003c/sub\u003e atmosphere, and allowed to cool to room temperature. Then the reaction mixture was filtered and the solid was washed three times with 2 mL of acetone and then fully dried to afford a yellow-orange solid HCD (0.1777 g, yield: 56.05%). The purity of HCD was confirmed by MS, \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra as follows:\u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ11.76 (s, 1H), 8.94 (d, J\u0026thinsp;=\u0026thinsp;2.3 Hz, 1H), 8.61 (dd, J\u0026thinsp;=\u0026thinsp;8.8, 2.3 Hz, 1H), 8.42\u0026ndash;8.24 (m, 2H), 8.15 (d, J\u0026thinsp;=\u0026thinsp;8.6 Hz, 2H), 7.95\u0026ndash;7.79 (m, 2H), 7.65\u0026ndash;7.50 (m, 3H), 7.28 (d, J\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), 4.10 (s, 3H), 1.75 (s, 6H) (Fig. S\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ181.58, 151.80, 150.27, 147.88, 143.54, 141.82, 140.79, 135.84, 132.13, 131.76, 130.69, 129.36, 128.97, 127.46, 122.89, 120.50, 119.54, 115.20, 112.41, 52.12, 34.60, 25.29 (Fig. S\u003csub\u003e4\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eESI-MS m/z calculated value for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e[M-I] \u003csup\u003e+\u003c/sup\u003e, 277.1699, found 277.1700 (Fig. S\u003csub\u003e5\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Standard procedures for sensing biothiols\u003c/h2\u003e \u003cp\u003eTo a 10.0 mL volumetric ask 100 \u0026micro;L stock solution of HCD (20 \u0026micro;M) in DMSO, 1.00 mL of phosphate buffer solution (PB, 20 mM, pH 7.40), an appropriate working solution of biothiols were transferred in turn, and the mixture was diluted to 5.0 mL with distilled water and mixed thoroughly. The mixture was left undisturbed for 40 min (for Cys) or 60 min (for Hcy and GSH) to allow HCD and biothiols to interact fully and improve the reproducibility and stability of the test results. Then the absorbance intensities and fluorescence intensities of the sample and the blank (prepared in a similar manner without biothiols) were determined by measuring the absorbance at 465 nm and the fluorescence at 558nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cells culture\u003c/h2\u003e \u003cp\u003eRAW264.7 macrophages were purchased from China Center for Type Culture Collection (CCTCC) and cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e/95% air incubator. One day before imaging, the cells were detached and replanted on glass-bottomed dishes.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Spectral characteristics\u003c/h2\u003e \u003cp\u003eHCD is a hemicyanine dye with good stability, which only recognizes biothiols with high specificity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(A), free HCD in PB buffer (pH 7.40) solution exhibited the maximum absorption wavelength at 465 nm. However, the maximum absorption wavelength shifted to 480 nm with the decline of the absorption peak at 465 nm after addition of Cys, Hcy and GSH. Along with the changes of absorption spectra, the fluorescence intensity of HCD at 558 nm increased significantly upon excitation at 465 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(B)) with the addition of Cys, Hcy and GSH. This phenomenon was ascribed to the strong interaction between HCD and biothiols.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optimization of the buffer system, pH, temperature and incubation time\u003c/h2\u003e \u003cp\u003eTo obtain the best experimental conditions, some buffer systems such as tris\u0026ndash;HCl buffer, HEPES buffer and phosphate buffer were examined in terms of the analytical sensitivity and linear range of the calibration curve, and it was found that phosphate buffer (PB) was better than the others (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A)). Then the influence of pH on the sensitivity were investigated. The results showed the best absorbance changes (A\u003csub\u003e0\u003c/sub\u003e-A, where A\u003csub\u003e0\u003c/sub\u003e and A are the absorbance at 465 nm in the absence and presence of biothiols, respectively) were obtained at pH 7.40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(B)). Therefore, pH 7.40 of PB buffer was selected to control the acidity of the system in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEffects of temperature on the response system is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It can be clearly seen from the figure that the response of HCD to Cys is most sensitive at 37\u0026deg;C, while that is most sensitive at 42\u0026deg;C for GSH and Hcy. Considering the subsequent cell experiments, 37\u0026deg;C was selected as the temperature for the subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of incubation time on the absorbance changes at 465 nm was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the system basically reached equilibrium within 40 min following Cys addition and 60 min following Hcy and GSH addition. Moreover, the response system remained constant for at least 80 min. Thus, it was suggested that the measurement should start 40 min after addition of Cys, 60 min after addition of Hcy or GSH, and finish within 80 min.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Selectivity and interference of foreign substances\u003c/h2\u003e \u003cp\u003eThe specific recognition ability is a key indicator for evaluating the performance of fluorescent probes. We investigated the specific recognition ability of probe HCD for Cys, GSH, and Hcy. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A). As can be clearly seen from the figure, among many amino acids, the absorbance intensity of probe HCD is significantly changed only when Cys, GSH or Hcy is added. This indicates that the probe HCD has a high selectivity for Cys, GSH, and Hcy, reflecting its specific recognition ability for biothiols.\u003c/p\u003e \u003cp\u003eTo further validate the method, competitive experimentation of HCD for Cys, GSH, and Hcy was carried out by adding nearly the same concentration of Cys, GSH, Hcy and other amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B)). It could be seen that other amino acids had no obvious effects on the spectral response of HCD for Cys, GSH, and Hcy. Detection results of selective and competitive experiments showed that the probe had excellent selectivity towards Cys, GSH, and Hcy compared with amino acids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Calibration graphs\u003c/h2\u003e \u003cp\u003eThe calibration graphs of three kinds of biothiols were constructed under the optimal conditions. There were good linear relationships between the increments of fluorescence intensity and the concentration of biothiols, and the results are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalytical parameters for the determination of biothiols using this method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiothiols\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal level in the body\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinear range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinear equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDetection limit\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u0026ndash;200 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e-6.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔI\u0026thinsp;=\u0026thinsp;716.03C\u003csub\u003eCys\u003c/sub\u003e-4.2953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.02\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;10 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e-2.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔI\u0026thinsp;=\u0026thinsp;1279.10C\u003csub\u003eGSH\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2.6253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.65\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHcy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026ndash;12 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e-1.2\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔI\u0026thinsp;=\u0026thinsp;467.09C\u003csub\u003eHcy\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.2600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.62\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo examine the potential applicability in simulated physiological media, we take Cys as an example and investigate the fluorescence titrations in the presence of biologically interfering ions such as Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, and HPO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e. Under the testing conditions, the interferences from these species are negligible in the titration by Cys compared with those obtained in the presence of Cys alone (Fig.S\u003csub\u003e6\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Endogenous biothiols imaging in live cells with HCD\u003c/h2\u003e \u003cp\u003eThe above studies indicated that HCD was highly suitable for detecting biothiols under simulated physiological conditions (Fig.S\u003csub\u003e6\u003c/sub\u003e). To examine the feasibility of probe HCD as an imaging reagent, its potential toxicity to cells was evaluated by a standard CCK-8 (Cell Counting Kit-8) assay. The results demonstrated that even when treated with 10 \u0026micro;M HCD at 37\u0026deg;C for 48 h, the cell viability did not change significantly (Fig. S\u003csub\u003e7\u003c/sub\u003e), implying the low cytotoxicity and good biocompatibility of probe HCD. Subsequently, the applicability of HCD for imaging RAW 264.7 macrophages was explored.\u003c/p\u003e \u003cp\u003eThe results of fluorescence imaging detection of biothiols in cell medium by HCD are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As we can see, when RAW264.7 cells are treated with a sulfhydryl scavenger (NEM) and then added a 10 \u0026micro;M probe HCD solution, no obvious fluorescence signal is observed [Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(A1)]; while when the probe HCD solution is directly added, a relatively obvious fluorescence signal is observed [Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(B1)], and the fluorescence intensity is 3.0 times stronger than that of the group pretreated with NEM. These experimental results fully indicate that HCD can be used for the visual detection of endogenous biothiols in cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 The molecular mechanism of HCD to recognize biothiols\u003c/h2\u003e \u003cp\u003eAs shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, HCD is a typical \u0026ldquo;Donor-π-Accepter\u0026rdquo; (\u0026ldquo;D-π-A\u0026rdquo;) molecule with excellent optical properties due to intramolecular charge transfer (ICT). When HCD is treated with 2,4-dinitrobenzenesulfonyl chloride, the charge density of the \u0026ldquo;Donor\u0026rdquo; is decreased, weakening its ICT process and thus resulting in shortwave absorption (with an absorbance maximum at 465 nm) and low fluorescence intensity at 558 nm. When HCD reacts with biothiols (Cys), the absorbance maximum will be redshifted to 480 nm and the fluorescence intensity will be enhanced. The specific interaction process between HCD and Cys is shown in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As depicted in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, when Cys is added to the detection system containing HCD, a nucleophilic substitution reaction occurs between HCD and Cys, leading to the cleavage of the sulfonamide bond and the generation of the fluorophore ATI (2-(4-aminostyrenyl)-1,3,3-trimethyl-3H-indole iodonium, ATI). The amino group in ATI increases the charge density of the \u0026ldquo;Donor\u0026rdquo; and causes a redshift of the absorption spectra and an enhancement of fluorescence intensity. This assumption is further supported by the HRMS of ATI (Fig. S\u003csub\u003e8\u003c/sub\u003e), which shows a strong peak at m/z 277.1700, assigned to C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ([M-I] \u003csup\u003e+\u003c/sup\u003e: 277.1699). This clearly indicates that the reaction of HCD with Cys generates ATI, thereby causing changes in the absorption spectra and fluorescence spectra.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Practical application of the method in human urine samples\u003c/h2\u003e \u003cp\u003eRelevant studies indicate that urinary Hcy levels can directly reflect renal function. To explore the potential applicability of this method in complex biofluids, we utilized it for Hcy detection in human urine. Specifically, 30 mL urine samples were collected from five early renal injury patients (from the Third Affiliated Hospital of Xinxiang Medical University) and five healthy volunteers. After centrifugation at 12000 rpm for 10 min, 15 mL of supernatant from each sample was collected and diluted to 30 mL. The proposed method and the Ellman method were then employed to measure Hcy content in each urine sample. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the results obtained by the developed method closely matched those from the Ellman method. Additionally, both methods yielded results consistent with literature findings [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These outcomes, along with the data presented in Fig. S\u003csub\u003e6\u003c/sub\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, confirm that the proposed method is reliable and practical for determining biothiols in complex biofluids.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetection of Hcy in human urine\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProposed method found\u003csup\u003ea\u003c/sup\u003e(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHcy added(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHcy found(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRecovery\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEllman method\u003c/p\u003e \u003cp\u003efound(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e104.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e14.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e105.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e13.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e13.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e18.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e17.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e105.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e18.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e1-5: Representing healthy volunteers; 6\u0026ndash;10: Representing patients with early renal injury.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn summary, a novel fluorescent probe, HCD, was successfully synthesized by condensing a hemicyanine dye with 2,4-dinitrobenzenesulfonyl chloride. This probe exhibits high sensitivity and specificity for detecting biothiols in aqueous solutions and allows for the visualization of biothiols within cellular environments. Its practicality and accuracy were further validated through real sample measurements.The proposed method is characterized by its simplicity, rapidity, and exceptional sensitivity and selectivity for biothiol detection. Its potential application for in situ monitoring of biothiol levels in biological systems could provide critical data to support and advance human health protection efforts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eX.G.established the characterization procedures using spectroscopy techniques;X.H.L.synthesized the fluorescent probe following the designed route and characterized the probe by performing fluorescence and other spectroscopic measurements; X.P.Z.carried out tests on the probe in biological cells ;Y.X.L.analyzed all the experimental data to evaluate the probe\u0026apos;s performance;J.M.L.secured the research funding and review the manuscript;P.B.Z.designed the synthetic method for the probe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful for the fund support by the Key Scientific Research Projects of Universities of Henan Province (23A330002), the Scientific and Technological Project of Henan Province (252102310090), the National Natural Science Foundation of China (grant no.22201242) and the Research Foundation for Distinguished Professor\u0026rsquo;s Team of Xinxiang Medical University (no.505527).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUrine samples were collected from five patients with early\u0026nbsp;renal\u0026nbsp;injury (from the Third Affiliated Hospital of Xinxiang Medical University) and five healthy volunteers. The participants were all informed explicitly about the plan, protocol, and procedure for the study, and written consent was obtained. This consent procedure was reviewed and approved by the Medical Ethics Committee of Xinxiang Medical University. All experiments in this work were performed in compliance with the relevant laws and institutional guidelines and approved by the Medical Ethics Committee of Xinxiang Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuang N, Yang D, Chen HH, Xiao Y, Wen JH, Long YJ, Zheng HZ (2023) Colorimetric detection of biothiols and Hg\u003csup\u003e2+\u003c/sup\u003e based on the peroxidase-like activity of GTP. Spectrochim Acta Mol Biomol Spectrosc 290:122263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong YJ, Wang PP, Zhai HC, Xiao Y, Wang Q, Ma NN, Zhang GS, Zhang H (2024) Equivalent Response Strategy for Sensing Total Biothiols in Human Serums and Living Cells Using a Hemicyanine-Based Self-Immolative Probe. Anal Chem 96:1009\u0026ndash;1018\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu HY, Liu JW, O\u0026rsquo;Connor HM, Gunnlaugsson T, James TD, Zhang H (2023) Photoinduced electron transfer (PeT) based fluorescent probes for cellular imaging and disease therapy. Chem Soc Rev 52:2322\u0026ndash;2357\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang CJ, Qin Y, Deng CF, Zhu N, Shi YN, Wang W, Qin L (2023) GSH-specific fluorescent probe for sensing, bioimaging, rapid screening of natural inhibitor Celastrol and ccRCC theranostics. Anal Chim Acta 1248:340933\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu CY, Zhang Y, Sun WM, Zhu HC, Su MJ, Wang X, Rong XD, Wang K, Yu MH, Sheng WL, Zhu BC (2023) A novel GSH-activable theranostic probe containing kinase inhibitor for synergistic treatment and selective imaging of tumor cells. Talanta 260:124567\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Peng ZH, Ji M, Chen JQ, Wang P (2022) Highly selective fluorescent probe based on AIE for identifying cysteine/homocysteine. Bioorg Chem 126:105902\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu WY, Li HJ, Wu YC (2024) Assessment of environmental and biological stress using mitochondria-targeted red-emitting and near-infrared fluorescent probes for biothiol analysis: a review. Environ Chem Lett 22:3135\u0026ndash;3169\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMillan S, Jeffery DW, Dall'Acqua S, Masi A (2021) A novel HPLC-MS/MS approach for the identification of biological thiols in vegetables. Food Chem 339:12780\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Yao T, Guo XC, Peng Y, Zheng J (2016) Simultaneous assessment of endogenous thiol compounds by LC-MS/MS. J Chromatogr B 1029\u0026ndash;1030:213\u0026ndash;221\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKostal V, Katzenmeyer J, Arriaga Edgar A (2008) Capillary electrophoresis in bioanalysis. Anal Chem 80:4533\u0026ndash;4550\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePak YL, Swamy KMK, Yoon J (2015) Recent Progress in Fluorescent Imaging Probes. Sensors 15:24374\u0026ndash;24396\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung HS, Chen X, Kim JS, Yoon J (2013) Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem Soc Rev 42:6019\u0026ndash;6031\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLippert AR, New EJ, Chang CJ (2011) Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells. J Am Chem Soc 133:10078\u0026ndash;10080\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsghar R, Li Y, Huo F, Yin C (2024) Sensing mechanism of cysteine specific fluorescence probes and their application of cysteine recognition. Chem Biomed Imaging 4:250\u0026ndash;269\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai J, Ma C, Zhang P, Fu Y, Shen B (2020) Recent progress in the development of fluorescent probes for detection of biothiols. Dyes Pigm 177:108321\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing S, Liu M, Hong Y (2018) Biothiol-specific fluorescent probes with aggregation-induced emission characteristics. Sci China Chem 61:882\u0026ndash;891\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim Y, Kim J, An JM, Park CK, Kim D (2023) All-nontoxic fluorescent probe for biothiols and its clinical applications for real-time glioblastoma visualization. ACS Sens 8:1723\u0026ndash;1732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu ZJ, Xu NG, Zhang D, Liu H, Li LL, Wang FY, Ren J, Wang EF (2024) A mitochondria-targeted fluorescent probe for discrimination of biothiols by dual-channel imaging in living cells and zebrafish. Spectrochim Acta Mol Biomol Spectrosc 322:124846\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu L, Xie ML, Chen M, Yang HR, Chen L, Xing PF, Tian ZY, Wang CJ (2024) An ortho-activation strategy to develop NIR fluorescent probe for rapid imaging of biothiols in vivo. Talanta 266:125110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi KY, Tan R, Hao RT, Miao LX, He YQ, Wu XH, Zhang JF, Xu R (2019) A carbazole-hemicyanine dye based ratiometric fluorescent probe for selective detection of bisulfite(HSO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e) in cells and C.elegans. Chin Chem Lett 30:545\u0026ndash;548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang K, Pan JB, Yu JJ, Xiao LH, Sun SK, Cheng R (2024) A hemicyanine-based near-infrared fluorescent probe with large Stokes shift for non-invasive bioimaging of brown adipose tissue. Anal Methods 16:5272\u0026ndash;5279\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen CN, Chao DU, Qi YU, Zhang Y (2019) Diagnostic value of urine homocysteine and retinol binding protein detection in the early renal injury. China Mod Med 26:131\u0026ndash;133\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biothiols, Fluorescent probe, Fluorescence imaging, Visual detection","lastPublishedDoi":"10.21203/rs.3.rs-6233684/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6233684/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiothiols play essential roles in various biological processes and are closely associated with diseases such as cancer, neurodegenerative disorders, cardiovascular diseases and so on. To monitor biothiols in organisms and living cells, we present the synthesis of a novel intramolecular charge transfer (ICT)-based probe, HCD, by coupling a hemicyanine dye with 2,4-dinitrobenzenesulfonyl chloride. The HCD exhibits remarkable sensitivity, with a low detection limit of 0.32µM, 0.72µM and 0.27 µM for Cys, Hcy, and GSH, respectively, and demonstrates high selectivity for biothiols in the presence of various interfering species. The detection mechanism was thoroughly validated using high-resolution mass spectra (HRMS), UV and fluorescence spectra. Moreover, the applicability of HCD was successfully demonstrated in human urine samples and in vivo settings using RAW264.7 cells, confirming its potential as a powerful tool for monitoring endogenous biothiols in biological systems.\u003c/p\u003e","manuscriptTitle":"A simple fluorescent probe for biothiols detection and imaging of living cells in vivo based on a hemicyanine derivative","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 09:12:34","doi":"10.21203/rs.3.rs-6233684/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-01T17:10:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-27T14:53:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-27T04:19:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334670016126602179197174089259274550669","date":"2025-03-24T13:06:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-24T08:52:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321738417032102525315025038704799764968","date":"2025-03-24T03:15:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31367738208787370236693134391302398973","date":"2025-03-21T22:14:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T18:11:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-19T17:40:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-19T17:38:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-03-15T15:13:51+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":"8374d76e-8be2-4ca1-95fb-958cd9c8c934","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T15:58:31+00:00","versionOfRecord":{"articleIdentity":"rs-6233684","link":"https://doi.org/10.1007/s10895-025-04350-7","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-04-30 15:57:02","publishedOnDateReadable":"April 30th, 2025"},"versionCreatedAt":"2025-04-01 09:12:34","video":"","vorDoi":"10.1007/s10895-025-04350-7","vorDoiUrl":"https://doi.org/10.1007/s10895-025-04350-7","workflowStages":[]},"version":"v1","identity":"rs-6233684","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6233684","identity":"rs-6233684","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}