English

• As one of the important organs for maintaining life activities, the liver has a variety of functions, such as metabolic synthesis function, detoxification function and immune function [1-3]. Liver damage has become a usual illness in modern medicine, which can progress to hepatocirrhosis or hepatoma, thus causing serious impact on human health and life. Drug-induced liver injury (DILI) is the most common liver injury, which has received extensive attention in modern medicine [4-6]. At present, the existing diagnostic methods of DILI mainly include liver biopsy, histopathological examination, and serum biochemistry [7,8]. However, most of these technologies have disadvantages such as high risk, false positives, and complicated operation steps. Therefore, there is an urgent need to develop a new method for non-invasive, rapid and accurate diagnosis of liver injury.

  Cysteine (Cys) is one of the sulfur-containing α-amino acids commonly found in organisms [9]. Within the body, Cys is mainly synthesized from homocysteine through the transmethylation of methionine in the liver, and it participates in the adjustment of redox processes and phospholipid metabolic process [10-12]. In addition, the study found that the increase of Cys concentration in serum was also closely related to the severity of liver injury [13-16]. Therefore, non-invasive in situ detecting Cys concentration is completely significant for the diagnosis and treatment of liver injury.

  Near-infrared fluorescence (NIR, 650~900 nm) imaging technology has been widely used to detect the level of Cys in vivo due to its advantages of non-invasive and high-sensitivity [17-22]. However, the limited penetration depth of NIR fluorescence probes makes it difficult to image Cys in deep tissues in vivo [23-26]. Photoacoustic imaging (PAI) is an emerging non-intrusive biomedical imaging technology with promising outlook, which integrates the superiorities of conventional optics with acoustics imaging, overcome the limitations of conventional optical imaging and achieve high spatial definition imaging of deep tissues in living animals [27-30]. Therefore, near-infrared fluorescence (NIRF) and photoacoustic (PA) dual-mode imaging are expected to achieve the non-invasive and accurate detection of Cys level in deep liver tissues and facilitate the early diagnosis and treatment of DILI. However, there is still a lack of a PA/NIRF bimodal activated probe with deep-penetrating and rapid-response to reveal the role of Cys in drug-induced liver injury. Therefore, based on the advantages of NIRF and PA dual-modality imaging, it is very important to design a fast and robust probe to detect the change of Cys concentration in deep tissues in vivo. We hope that this strategy would provide a powerful tool for penetrating deep organization, high spatial resolution and high response rate imaging in liver damage.

  Hence, we rationally designed and synthesized a new NIRF/PA dual-modality small molecule probe CDR for noninvasive detection of Cys and diagnosis of liver injury in vivo. The dual-modality CDR is composed of an acrylate-caged NIR quinolinium-xanthene dye. CDR initially has no NIRF and PA signals because it is in a passivation state, weakening the ability of the oxygen atoms to provide electrons. In the presence of Cys, CDR can be specifically activated by Cys to turn on its NIRF/PA signals. Due to the complementary advantages of NIRF/PA imaging, the probe CDR enable highly sensitive and deep imaging of Cys level at the site of liver injury. Therefore, this NIRF/PA dual-mode probe is expected to provide a new approach for the in-depth study, diagnosis and treatment of liver injury.

  The probe CDR consists of NIR quinoline-xanthene dye (CDR-OH) and an acrylate (Cys response sites). CDR itself would be no NIRF₇₆₅/PA₇₂₅ signals due to the intramolecular charge transfer (ICT) effect is suppressed by acrylate groups. When CDR reacts with Cys, the acrylate moiety of CDR will be hydrolyzed and the hydroxyl group will be released, leading to the recovery of the ICT process and the release of NIRF₇₆₅/PA₇₂₅ signals (Scheme 1). The synthetic route of CDR is described in Scheme S1 (Supporting information). Detailed synthesis steps and characterization are given in the Supporting information.

  Scheme1

  

  Scheme1.  Rational designs of the NIRF/PA probe CDR reporting Cys.

  DownLoad: Full-Size Img PowerPoint

  First, the absorption and fluorescence spectra of CDR before and after adding Cys were studied. As shown in the Figs. S1A and B (Supporting information), CDR had the maximum absorption peak at 560 nm. After the addition of Cys, a new absorption peak appeared at 725 nm, and the color changed from purple to blue. Moreover, CDR itself has almost no fluorescence because the acrylate inhibits the ICT process. After the CDR reacted with Cys, the NIR fluorescence were observed at 765 nm. These spectral data imply the release of the dye CDR-OH. High resolution mass spectrometry (HRMS) data further confirmed the reaction between CDR and Cys. The results are shown below. As show in Fig. S2 (Supporting information), a new mass peak appeared at m/z 416.1419 after CDR reacted with Cys, which is identical to CDR-OH. Based on the results of the above data we can speculate that CDR could react with Cys to release the dye CDR-OH with strong NIR fluorescence signals.

  In order to test the recognition effect of Cys by CDR, different concentrations of Cys (0~55 µmol/L) were added to CDR for reaction, and the absorption and fluorescence emission spectra were recorded. We can get an intuitive data graph. As shown in Fig. 1A, The absorption intensity at 725 nm (Abs₇₂₅) enhanced with the concentration of Cys increase and reached a plateau at about 45 µmol/L Cys concentration. The Abs₇₂₅ intensity has a good linear correlation with Cys concentration in the range of 0~45 µmol/L (y = 0.00772x + 0.01146, R² = 0.99344) and the detection limit was calculated to be 1.25 µmol/L by the equation 3σ/k (Fig. S3A in Supporting information). In addition, when CDR reacted with different concentrations of Cys (0~45 µmol/L), the NIR fluorescence intensity at 765 nm (NIRF₇₆₅) gradually increased (Fig. 1B). The NIRF₇₆₅ intensity has a good linear correlation with Cys concentration in the range of 0~45 µmol/L (y = 9.20227x + 58.93716, R² = 0.9988), and the detection limit was calculated to be 0.7584 µmol/L by the equation 3σ/k (Fig. S3B in Supporting information). These results indicate that CDR has an excellent response to Cys in vitro. The photoacoustic signal of CDR was measured under different excitation wavelengths. As shown in (Fig. 1C), the pure probe showed no obvious signal changes, while the probe showed the maximum photoacoustic signal intensity at 725 nm under the action of cysteine. Next, we further investigated the PA responses of CDR to different concentrations of Cys. As the concentration of Cys increased, the strength of PA₇₂₅ gradually increased, and the color of the reaction liquid changed from purple to blue (Fig. 1D). The PA₇₂₅ intensity has a good linear correlation with concentration of Cys (0~45 µmol/L) (y = 20.54714x + 21.6999, R² = 0.9983), the detection limit was calculated as 2.07 µmol/L by the 3σ/k equation (Fig. S3C in Supporting information). We next investigated the response time of CDR to Cys in vitro. As show in Fig. 1E and Fig. S4 (Supporting information), the Abs₇₂₅ and NIRF₇₆₅ signal enhanced with the increase of the reaction time between the probe CDR and Cys, and reached a plateau within 5 min, indicating CDR can detect Cys rapidly. To evaluate the selectivity of CDR, we investigated the probe recognition of other biologically relevant analytes, such as biothiols, reactive oxygen species (ROS) (Fig. 1F), no absorption response was observed, indicating that CDR has high selectivity for Cys. These results demonstrate that CDR had a good photoacoustic/fluorescent response to Cys, which is expected to provide a method for detecting of Cys in deep tissues in vivo.

  Figure 1

  

  Figure 1.  Spectroscopic experiments of CDR. (A) Absorption spectra of CDR (10 µmol/L) with varied concentrations of Cys (0~55 µmol/L). All spectra were tested after 20 min at 25 ℃ in PBS buffer (pH 7.4, 25% DMSO, v/v). (B) Fluorescent spectrum of CDR (10 µmol/L) with varied concentrations of Cys (0~55 µmol/L). (C) PA intensity of CDR with and without Cys (45 µmol/L) for different wavelength excitations. (D) The color image after the reacting of each solution. (E) Time-dependent Abs₇₂₅ changes with the addition of Cys (45 µmol/L). (F) Absorbance of CDR (10 µmol/L) at 725 nm for various analytes. 1, Cys; 2, Pro; 3, Ser; 4, Phe; 5, Ala; 6, Met; 7, GSH; 8, Arg; 9, Tyr; 10, Asp; 11, His; 12, KCl; 13, ZnCl₂; 14, H₂O₂; 15, NaOH; 16, NaClO; 17, Na₂SO₄; 18, FeSO₄. Concentration: Cys, GSH, Hcy, H₂O₂, 150 µmol/L; other analytes, 300 µmol/L.

  DownLoad: Full-Size Img PowerPoint

  In addition, the response of the probes to Cys was also evaluated under different pH conditions. The Abs₇₂₅ and NIRF₇₆₅ signals of CDR remained almost unchanged at pH 3.0~6.0. After adding Cys, CDR exhibited a good signal enhancement of Abs₇₂₅ and NIRF₇₇₅ in the pH range of 6.0~7.5, indicating that CDR could detect Cys under a wide range of pH physiological conditions (Figs. S5A and B in Supporting information). These results indicate that CDR has a fast NIRF/PA dual signal response to Cys in vitro, it has the potential of detecting cysteine sensitively in living cells and deep tissues in vitro.

  With the results of optical experiment, we further studied CDR imaging of Cys in living cells by NIRF/PA imaging technique. The biocompatibility of the probe was fist studied before cell imaging [31]. Human normal hepatocytes cells (HL-7702) and human hepatoma cells (HepG2) were cultured with different concentrations of CDR (0–50 µmol/L) for 24 h, and then the cell activity was detected by methyl thiazolyl tetrazolium (MTT) assay. When the 50 µmol/L CDR was incubated with cells, the HL-7702 and HepG2 cells viability was still higher than 80%, this indicated that CDR has low toxicity to cells and can be used to image Cys in vivo (Fig. S6 in Supporting information). It is well known that Acetaminophen (APAP) is a commonly used medicine for reducing fever, pain and colds. However, long-term or excessive use of APAP could lead to liver damage and even death [32,33]. To validate the potential of probe for NIRF/PA imaging in vivo, NIRF/PA imaging of cells was first performed. After incubation with CDR for 30 min, HL-7702 cells showed no obvious NIRF and PA signals. However, when cells were pretreated with APAP (50 µL, 1 mmol/L) for 1 h and cultured with the CDR for 30 min, high NIRF and PA signals were observed. When cells were stimulated with APAP and then treated with 3 mmol/L NAC for 2 h, (NAC is N-acetyl-l-cysteine, a therapeutic agent for liver injury) [34-36], the PA and NIRF signals were significantly reduced (Figs. S7A-C in Supporting information). These results confirmed that the turn-on of the NIRF/PA response should be attributed to the presence of endogenous Cys in HL-7702 cells, and NAC can be used to treat cell injury resulted in significantly enhanced of NIRF/PA. These results indicate that CDR has good biocompatibility and could be used for NIRF/PA dual-modality imaging of Cys in living cells.

  Before performing in vivo PA imaging, the probe in vivo toxicity was first investigated. All animal experiments were performed in compliance with the relevant laws and approved by the Animal Care and Experiment Committee of Guangxi University (protocol number: Gxu-2021–115). Compared with the control group, mice treated with CDR showed no casualties and a slight increase in body weight, indicating that CDR has good biocompatibility (Fig. S8 in Supporting information). CDR and Cys were injected into the high region of interest (ROI) L of mouse legs to test the ability of CDR to recognize Cys in vivo. When injected with CDR (100 µL, 30 µmol/L) for 30 min, ROI L showed obvious PA₇₂₅ signals (Fig. 2A). However, when the ROI R region was pretreated with 1 mmol/L N-ethylmaleimide (NEM, a cysteine inhibitor) for 3 h, followed by subcutaneous injection of probes for 30 min, the PA signal was significantly attenuated compared with the only CDR injected group. These results indicate that the probe could achieve PA imaging of Cys in live mice. However, after injection of exogenous Cys (50 µL, 1 mmol/L), the ROI R region exhibited stronger PA signal than the pure CDR group (Figs. 2A and B). The results suggest that CDR is suitable for PA imaging of endogenous and exogenous Cys in vivo.

  Figure 2

  

  Figure 2.  Endogenous and exogenous fluorescence imaging and PA imaging in living mice. (A) Exogenous PA imaging of mouse leg. (ROI) L was injected with CDR (100 µL, 30 µmol/L) for 30 min, (ROI) R was injected with NEM (100 µL, 1 mmol/L) for 3 h and then injected with CDR (100 µL, 30 µmol/L) for 30 min after imaged inject of exogenous Cys (50 µL, 1 mmol/L). (B) Image of experimental data of exogenous PA in mice. (C) Fluorescence imaging of mice with different degrees of liver injury. Three rats were injected CDR (100 µL, 30 µmol/L) through the tail vein and then injected separately with APAP (0, 150 and 300 mg/kg) through the tail vein to establish liver injury, and collect real-time fluorescence signal images under the excitation of 720 nm laser. (D) NIRF₇₆₅ signal intensity values from (C).

  DownLoad: Full-Size Img PowerPoint

  Next, the ability of CDR for real-time monitoring of endogenous Cys and non-invasive in-situ visualization of early DILI diagnosis was evaluated. First, three groups of healthy mice were injected with different doses of APAP (0, 150, 300 mg/kg) through the tail vein. As shown in (Fig. 2C), there were slight change in fluorescence signal in the liver of healthy mice after injected of CDR (control group). This indicates that CDR could be enriched in the liver and identify of endogenous Cys in vivo. Initially, no fluorescence signal was given to the liver region of mice with a small dose of APAP (150 mg/kg), but as the drug was administered over time, the fluorescent signal in the liver region gradually enhanced, and reached a maximum intensity of 4 h. We found that when mice were injected with high dose of APAP (300 mg/kg) for 4 h, the PA signal intensity of liver became observably higher than the pure CDR group, suggesting that the concentration of Cys in the liver increased with the aggravation of DILI. These experiments demonstrate that CDR can image Cys level during the development of APAP-induced acute liver injury (Figs. 2C and D). It is worth noting that the fluorescence signal gradually decreased after 4 h, which we speculated was because CDR was excreted from the body by metabolism. These experiments demonstrate that CDR could enable real-time in situ identification of Cys level during the development of APAP-induced early liver injury.

  To further demonstrate that the probe can image Cys level in the liver, six important organs of the control group and the APAP-induced liver injury model group (2 h, 4 h and 7 h) were dissected for in vitro fluorescence imaging. The spleen, kidney, lung, liver, stomach, and heart were subjected to fluorescence imaging at 720 nm excitation as shown in Fig. S9 (Supporting information). The experimental results showed that only the liver had fluorescent signal, and the fluorescent signal of APAP group were stronger than those of control group, which further indicated that APAP-induced early liver injury would lead to the increase of Cys level. Therefore, this probe CDR can be used to diagnose APAP-induced early liver injury process.

  The ability of CDR to monitor endogenous Cys and non-invasively diagnose DILI was assessed. The experimental results are shown in Fig. 3A, there were no obvious PA signal in liver when injected of CDR (100 µL, 30 µmol/L) for 3 h. After pretreatment with 300 mg/kg APAP for 4 h and injected with CDR (100 µL, 30 µmol/L) for 3 h, the liver PA signal intensity was greatly increased compared with the control group. Notably, when the hepatoprotective agent NAC (100 µL, 3 mmol/L) was injected into DILI mice for 4 h, a significant attenuation of the PA signal at the liver site was observed, which was consistent with the NIRF/PA imaging results of NAC-treated cells (Fig. 3B). This indicates that the probe could be used for pathological diagnosis and therapeutic evaluation of early liver injury mice by non-invasive PA imaging technology.

  Figure 3

  

  Figure 3.  (A) In vivo PA imaging of mouse liver injury model. (a) There was no obvious PA signal in liver when injected CDR (100 µL, 30 µmol/L) into caudal vein for 3 h. (b) A liver injury model was established by APAP (300 mg/kg) for 4 h and then CDR (100 µL, 30 µmol/L) was injected into caudal vein for 3 h. PA imaging showed strong fluorescence. (c) After a caudal vein injection of APAP for 4 h to establish a liver injury model, the PA imaging signal was significantly weakened after caudal vein injection of NAC (100 µL, 3 mmol/L) for 4 h and tail vein injection of CDR for 3 h. (B) The Photoacoustic signal intensity in mice.

  DownLoad: Full-Size Img PowerPoint

  In conclusion, we constructed a novel probe CDR for diagnosing drug-induced early liver injury in vivo by NIRF and PA dual-mode imaging, for the first time. CDR exhibited obvious NIRF/PA dual-signal responses to Cys with high sensitivity with fast response speed and high sensitivity. In addition, the probe CDR has been successfully performed NIRF/PA imaging of intracellular Cys level. More importantly, in the APAP-induced early-stage liver injury model in mice, CDR could not only diagnosed liver injury by in situ monitoring of hepatic Cys level, but also evaluated the repair effect of NAC drugs on liver injury. Based on the excellent performance of the NIRF/PA dual-modality CDR, we hope this work would provide new ideas for the early diagnosis and pathogenesis of DILI.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This work was funded by the National Natural Science Foundation of China (Nos. 21672083, 21877048, 22077048), Natural Science Foundation of Guangxi (Nos. 2021GXNSFDA075003, AD21220061), and the Startup Fund of Guangxi University (No. A3040051003).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.107845.

  
  
• 1. [1]

     K. Ray, Nat. Rev. Gastro. Hepat. 15 (2018) 130–131. doi: 10.1038/nrgastro.2018.13

  2. [2]

     T. Akaike, T. Ida, F.Y. Wei, et al., Nat. Commun. 8 (2017) 1177. doi: 10.1038/s41467-017-01311-y

  3. [3]

     M. Fellner, E. Siakkou, A.-.S. Faponle, et al., J. Biol. Inorg. Chem. 21 (2016) 501–510. doi: 10.1007/s00775-016-1360-0

  4. [4]

     Y. Zhou, J. Wang, D. Zhang, et al., Chin. Med. UK 16 (2021) 135. doi: 10.1186/s13020-021-00543-x

  5. [5]

     R.J. Andrade, N. Chalasani, E.S. Björnsson, et al., Nat. Rev. Dis. Primers. 5 (2019) 58. doi: 10.1038/s41572-019-0105-0

  6. [6]

     J.S. Au, V.J. Navarro, S. Rossi, Aliment. Pharm. Ther. 34 (2011) 11–20. doi: 10.1111/j.1365-2036.2011.04674.x

  7. [7]

     R.A. Nathwani, S. Pais, T.B. Reynolds, N. Kaplowitz, Hepatology 41 (2005) 380–382. doi: 10.1002/hep.20548

  8. [8]

     J. Ozer, M. Ratner, M. Shaw, W. Bailey, S. Schomaker, Toxicology 245 (2008) 194–205. doi: 10.1016/j.tox.2007.11.021

  9. [9]

     X. Chen, Y. Zhou, X. Peng, J. Yoon, Chem. Soc. Rev. 39 (2010) 2120–2135. doi: 10.1039/b925092a

  10. [10]

      G.B. Steventon, S. Khan, S.C. Mitchell, J. Pharm. Pharmacol. 70 (2018) 1069–1077. doi: 10.1111/jphp.12944

  11. [11]

      T. Rehman, M.A. Shabbir, M. Inam-Ur-Raheem, et al., Food. Sci. Nutr. 8 (2020) 4696–4707. doi: 10.1002/fsn3.1818

  12. [12]

      C.M. Hunt, J.I. Papay, V. Stanulovic, A. Regev, Hepatology 66 (2017) 646–654. doi: 10.1002/hep.29152

  13. [13]

      S.A. Seifert, D. Kovnat, V.E. Anderson, et al., Toxicol. Lett. 54 (2016) 282–285. doi: 10.3109/15563650.2015.1134798

  14. [14]

      A. Pastore, A. Alisi, G. Di Giovamberardino, et al., Int. J. Mol. Sci. 15 (2014) 21202–21214. doi: 10.3390/ijms151121202

  15. [15]

      L. Yue, H. Huang, W. Song, W. Lin, Chem. Eng. J. 441 (2022) 135981. doi: 10.1016/j.cej.2022.135981

  16. [16]

      W. Li, R. Li, R. Chen, et al., Anal. Chem. 93 (2021) 8978–8985. doi: 10.1021/acs.analchem.1c01568

  17. [17]

      J. Xu, J. Pan, Y. Zhang, et al., Sens. Actuators B 238 (2017) 58–65. doi: 10.1016/j.snb.2016.07.038

  18. [18]

      R. Zhang, J. Yong, J. Yuan, Z. Xu, Coordin. Chem. Rev. 408 (2020) 213182. doi: 10.1016/j.ccr.2020.213182

  19. [19]

      L. Yuan, W. Lin, K. Zheng, L. He, W. Huang, Chem. Soc. Rev. 42 (2013) 622–661. doi: 10.1039/C2CS35313J

  20. [20]

      S. Cai, C. Liu, X. Jiao, L. Zhao, X. Zeng, J. Mater. Chem. B 8 (2020) 2269–2274. doi: 10.1039/c9tb02609f

  21. [21]

      H. Ren, F. Huo, Y. Zhang, S. Zhao, C. Yin, Sens. Actuators B 319 (2020) 128248. doi: 10.1016/j.snb.2020.128248

  22. [22]

      Y. Yang, T. Zhou, M. Jin, et al., J. Am. Chem. Soc. 142 (2020) 1614–1620. doi: 10.1021/jacs.9b12629

  23. [23]

      A. Tarai, Y. Li, B. Liu, et al., Coordin. Chem. Rev. 445 (2021) 214070. doi: 10.1016/j.ccr.2021.214070

  24. [24]

      X. Wu, W. Shi, X. Li, H. Ma, Acc. Chem. Res. 52 (2019) 1892–1904. doi: 10.1021/acs.accounts.9b00214

  25. [25]

      X. Li, X. Liang, J. Yin, W. Lin, Chem. Soc. Rev. 50 (2021) 102–119. doi: 10.1039/D0CS00896F

  26. [26]

      T. Li, F. Huo, J. Chao, C. Yin, Chem. Commun. 56 (2020) 11453–11456. doi: 10.1039/d0cc04839a

  27. [27]

      R. Li, W. Li, R. Chen, W. Lin, Sens. Actuators B 347 (2021) 130616. doi: 10.1016/j.snb.2021.130616

  28. [28]

      X.P. Fan, W. Yang, T.B. Ren, et al., Anal. Chem. 94 (2022) 1474–1481. doi: 10.1021/acs.analchem.1c05026

  29. [29]

      C. Zhang, Z. Qiu, L. Zhang, et al., Chem. Sci. 12 (2021) 4883–4888. doi: 10.1039/d0sc06573k

  30. [30]

      W. Li, R. Li, R. Chen, et al., Anal. Chem. 94 (2022) 7996–8004. doi: 10.1021/acs.analchem.2c01048

  31. [31]

      H. Yan, F. Huo, Y. Yue, J. Chao, C. Yin, J. Am. Chem. Soc. 143 (2021) 318–325. doi: 10.1021/jacs.0c10840

  32. [32]

      D. Chen, H. -. M. Ni, L. Wang, et al., Hepatology 69 (2019) 2164–2179. doi: 10.1002/hep.30422

  33. [33]

      X. Shi, H. Bai, M. Zhao, et al., Transl. Res. 196 (2018) 31–41. doi: 10.1016/j.trsl.2018.02.003

  34. [34]

      Y. Ntamo, K. Ziqubu, N. Chellan, et al., Oxid. Med. Cell. Longev. 2021 (2021) 3320325.

  35. [35]

      G.P. Sreekanth, J. Panaampon, A. Suttitheptumrong, et al., Antiviral. Res. 166 (2019) 42–55.

  36. [36]

      H. Jaeschke, J.Y. Akakpo, D.S. Umbaugh, A. Ramachandran, Toxicol. Sci. 174 (2020) 159–167. doi: 10.1093/toxsci/kfaa002

• 

  Scheme1  Rational designs of the NIRF/PA probe CDR reporting Cys.

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Spectroscopic experiments of CDR. (A) Absorption spectra of CDR (10 µmol/L) with varied concentrations of Cys (0~55 µmol/L). All spectra were tested after 20 min at 25 ℃ in PBS buffer (pH 7.4, 25% DMSO, v/v). (B) Fluorescent spectrum of CDR (10 µmol/L) with varied concentrations of Cys (0~55 µmol/L). (C) PA intensity of CDR with and without Cys (45 µmol/L) for different wavelength excitations. (D) The color image after the reacting of each solution. (E) Time-dependent Abs₇₂₅ changes with the addition of Cys (45 µmol/L). (F) Absorbance of CDR (10 µmol/L) at 725 nm for various analytes. 1, Cys; 2, Pro; 3, Ser; 4, Phe; 5, Ala; 6, Met; 7, GSH; 8, Arg; 9, Tyr; 10, Asp; 11, His; 12, KCl; 13, ZnCl₂; 14, H₂O₂; 15, NaOH; 16, NaClO; 17, Na₂SO₄; 18, FeSO₄. Concentration: Cys, GSH, Hcy, H₂O₂, 150 µmol/L; other analytes, 300 µmol/L.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Endogenous and exogenous fluorescence imaging and PA imaging in living mice. (A) Exogenous PA imaging of mouse leg. (ROI) L was injected with CDR (100 µL, 30 µmol/L) for 30 min, (ROI) R was injected with NEM (100 µL, 1 mmol/L) for 3 h and then injected with CDR (100 µL, 30 µmol/L) for 30 min after imaged inject of exogenous Cys (50 µL, 1 mmol/L). (B) Image of experimental data of exogenous PA in mice. (C) Fluorescence imaging of mice with different degrees of liver injury. Three rats were injected CDR (100 µL, 30 µmol/L) through the tail vein and then injected separately with APAP (0, 150 and 300 mg/kg) through the tail vein to establish liver injury, and collect real-time fluorescence signal images under the excitation of 720 nm laser. (D) NIRF₇₆₅ signal intensity values from (C).

  下载: 全尺寸图片 幻灯片
  

  Figure 3  (A) In vivo PA imaging of mouse liver injury model. (a) There was no obvious PA signal in liver when injected CDR (100 µL, 30 µmol/L) into caudal vein for 3 h. (b) A liver injury model was established by APAP (300 mg/kg) for 4 h and then CDR (100 µL, 30 µmol/L) was injected into caudal vein for 3 h. PA imaging showed strong fluorescence. (c) After a caudal vein injection of APAP for 4 h to establish a liver injury model, the PA imaging signal was significantly weakened after caudal vein injection of NAC (100 µL, 3 mmol/L) for 4 h and tail vein injection of CDR for 3 h. (B) The Photoacoustic signal intensity in mice.

  下载: 全尺寸图片 幻灯片
•