Fluorescence-Guided Cancer Diagnosis and Surgery by a Zero Cross

Subscriber access provided by University of Winnipeg Library

Article

Fluorescence-Guided Cancer Diagnosis and Surgery by a Zero CrossTalk Ratiometric Near-Infrared #-Glutamytranspeptidase Fluorescent Probe Juan Ou-Yang, Yongfei Li, Wen-Li Jiang, Shuang-Yan He, Hong-Wen Liu, and Chun-Yan Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04416 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Fluorescence-Guided Cancer Diagnosis and Surgery by a Zero CrossTalk Ratiometric Near-Infrared γ-Glutamytranspeptidase Fluorescent Probe Juan Ou-Yang,† Yongfei Li,†,‡ Wen-Li Jiang,† Shuang-Yan He,§ Hong-Wen Liu,*,† and Chun-Yan Li*,† †Key

Laboratory for Green Organic Synthesis and Application of Hunan Province, Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, PR China. ‡College of Chemical Engineering, Xiangtan University, Xiangtan, 411105, PR China. §Hunan SJA Laboratory Animal Co., Ltd, Changsha, 400125, PR China. *Corresponding Author. E-mail: [email protected] (Chun-Yan Li), [email protected] (Hong-Wen Liu). ABSTRACT: The ability to early detect cancer in an accurate and rapid fashion is of critical importance for cancer diagnosis and accurate resection in surgery. γ-Glutamyltranspeptidase (GGT) is overexpressed in several human cancers while keeps low expression in normal microenvironment, and thus recognized as an important cancer biomarker. To date, rational design of a zero cross-talk ratiometric near-infrared (NIR) GGT fluorescent probe for efficiently cancer diagnosis in various biological samples is still a big challenge. In this work, a zero cross-talk ratiometric NIR GGT fluorescent probe named Cy-GSH is developed. Cy-GSH shows highly sensitive to GGT, which is desired for cancer early diagnosis. Upon additional GGT, a large emission shift from 805 nm to 640 nm is observed, which is suitable for visualizing deeply located cancer in vivo. In addition, successful monitoring GGT activity in bloods, cells, tissues and in vivo, makes Cy-GSH possess great potentials for the clinical cancer early diagnosis. Furthermore, accurate visualizing tumors and metastases in mouse models illuminates that the probe may be a convenient tool for fluorescence-guided cancer surgery. To our knowledge, this is the first report to describe the strategy of a zero cross-talk ratiometric NIR GGT fluorescent probe for cancer early diagnosis and fluorescence-guided surgery.

INTRODUCTION According to the WHO statistics for 2017, there were 16.9 million new cancer cases and 6.0 million cancer deaths globally.1-3 Of those deaths associated with cancer, approximately 30% of people could have been saved if their cancer could be detected earlier. The early detection and treatment of cancer are highly significant to increase the chance of being cured and improve the cancer survival rates. The challenges in early diagnosis and accurate localization of malignant cancers highly call for detecting and imaging of cancer biomarkers sensitively and selectively. Among various potential biomarkers, enzymes have attracted much attention because of their upregulated activities in cancers, which makes diagnosis events be identified rapidly, selectively and sensitively. γ-Glutamyltranspeptidase (GGT) is a cell-surfacebound enzyme that can catalyze the cleavage of γ-glutamate bond in extracellular glutathione (GSH).4-6 As malignant cells are highly dependent on cysteine, GGT mediated metabolism of extracellular GSH to produce cysteine, which will confer growth and survival advantages for tumour cells. It has been well documented that the overexpressed levels of GGT are relative with tumorigenesis in a number of human cancers such as hepatic, colorectal, ovarian cancers, etc.7-10 Thus, GGT is recognized as an important cancer biomarker. However, analyzing GGT activity in biological samples still involves two main challenges. One is that the enzyme activity is

susceptible to disturbance by environmental factors. So it is necessary to develop an efficient detection method suitable for biological samples. The other is that the precise determination of GGT activity with high sensitivity is of importance in both clinical diagnosis and biomedical researches due to low GGT level in the primary of cancer. The universal methods for monitoring GGT activity in cancer including colorimetric assays,11,12 high-performance liquid chromatography (HPLC)13 and fluorescence techniques.14-33 Each technique is irreplaceable and has its own characteristics. However, colorimetric assays and HPLC techniques are not suitable for real-time biological imaging applications. In contrast, fluorescent probe-based imaging techniques with remarkable characteristics including high sensitivity, noninvasiveness and facile visualization, can offer an accurate and specific detection of GGT activity in clinic blood samples, pathological tissues, which will help to be used for cancer early diagnosis.34-39 And the fluorescence imagingguided GGT-activable probe is helpful for surgeons by visualizing accurate borders between cancer and normal tissue during surgery. Up to now, many fluorescent probes have been developed for accurate measurement and efficient imaging of GGT activity both in living cancer cells and xenograft tumor.14-33 However, on the one hand, most of them show a “turn-on” green to red fluorescence emission.14-29 Among them, a

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fluorescent probe, developed by Urano et al., exhibited a dramatically “turn-on” green fluorescent signal for in vitro or in vivo imaging of GGT in cancers.15 Impressively, this probe was successfully applied for imaging-guided cancer surgery. However, this probe possessed short excitation and emission wavelengths (< 550 nm), leading to excessive autofluorescence and shallow penetration depth. By contrast, fluorescent probes with the emission in the near-infrared (NIR) region, which are much less absorbed by biomolecules and penetrates deeper into tissues, so as to minimize the interference from background fluorescence and increase the collection efficiency.40-43 So, NIR fluorescent probes are more desired for the detection and imaging of GGT in biological samples. On the other hand, ratiometric fluorescent probes simultaneously regulating two emission signals can provide built-in correction for quantitative analysis, which may be used for the accurate detection of GGT activity in cancers.44-46 However, most ratiometric fluorescent probes based on intramolecular charge transfer (ICT) or förster resonance energy transfer (FRET) mechanism, show a major deficiency small emission shift (usually < 90 nm), which is easily subject to cross-talk of the two emission signals.30-32 Therefore, development of GGT-activable ratiometric NIR fluorescent probes with large emission shift for imaging-guided cancer diagnosis and surgery in an accurate and rapid fashion is still challenging. Aim to the above considerations, we designed and synthesized a zero cross-talk ratiometric NIR GGT fluorescent probe, termed Cy-GSH, for precisely cancer diagnosis and imaging-guided surgery (Scheme 1). In this probe, GSH unit is selected as the substrate to target GGT and is conjugated to a NIR fluorophore (cyanine). The probe itself exhibits strong fluorescence emission at 805 nm. Upon the addition of GGT, enzymatic cleavage of γ-glutamyl linkage occurs and the chemical structure of the probe transforms into Cy-N with the fluorescence emission shift from 805 nm to 640 nm. To our delight, the ratiometric NIR emission with zero cross-talk (165 nm emission shift) is beneficial for highly sensitive detecting GGT activity in biological samples. Owing to the good performance of this ratiometric NIR fluorescent probe, CyGSH was successful applied for monitoring GGT activity in blood samples, cells, tissues and in vivo. This provides basic information that would be helpful in the early cancer diagnosis. Benefit from the zero cross-talk ratiometric NIR emission, this probe clearly indicated accurate borders between cancer and normal tissue, when sprayed onto tissue surfaces that are suspected of harboring tumors. Moerover, Cy-GSH could visualize metastases as small as 1 mm in diameter inside the peritoneal cavity of human colon cancer model. The results obviously demonstrate preclinical potential value of probe CyGSH for fluorescence-guided diagnosis and surgery of cancers. Scheme 1. Design of probe Cy-GSH to GGT em = 805 nm

ex = 730 nm HO

O

H N O

N

HO

GGT cleavege

H2N

O

O

OH

N H

em = 640 nm

ex = 540 nm O

HN O

N

SH N

GGT

S

N

N

I

Cy-GSH

Cy-N

Large emission shift: 165 nm

EXPERIMENTAL SECTION

Reagents and Instruments. IR-780 iodide, glutathione (GSH), human plasma (HP), fetal bovine serum (FBS), trypsin (Try), pepsase (Pep), collagen hydrolase (Hyd), chymotrypsin (Chy), amyloglucosidase (Amy), aprotinin (Apr), alkaline phosphatase (ALP), γ-glutamytranspeptidase (GGT), 6-diazo5-oxo-L-norleucine (DON) and acivicin were purchased from Sigma-Aldrich and used as received. Nuclear magnetic resonance (NMR) spectra and Mass spectra (MS) were recorded on Bruker Avance II NMR spectrometer (Germany) and Bruker Autoflex MALDI-TOF mass spectrometer (Germany), respectively. The fluorescence spectra and absorption spectra were obtained on Perkin Elmer LS-55 fluorescence spectrometer (USA) and Perkin Elmer Lambda 25 UV/Vis spectrophotometer (USA), respectively. HPLC was performed on an Agilent 1260 LC system with a C18 column (USA). Fluorescence imaging of cells, tissues and mice were conducted by Olympus FV1000 confocal fluorescence microscope (Japan) and IVIS Lumina XR small animal optical in vivo imaging system (USA), respectively. Fluorescence measurement for GGT Activity. The fluorescence measurement for GGT activity was operated as following. Different activities of GGT from 1 to 90.0 U were added into the solution (1 mL) containing 10 μM Cy-GSH and 10 mM PBS buffer solution (pH 7.4) at 37 °C. After adding GGT, the fluorescence spectra for the solutions were recorded with the excitation at 540.0 nm and 730.0 nm, and the emission at 600.0-740.0 nm and 740.0-850.0 nm. Detection of GGT Activity in blood samples. The blood samples were obtained from normal mice and colorectal cancer mice. And then, the blood samples were diluted 10-fold with PBS buffer solution (10 mM, pH 7.4) and treated with Cy-GSH (10 μM). The fluorescence spectra for the samples were measured at the excitation at 540.0 nm and 730.0 nm, and the emission at 600.0-740.0 nm and 740.0-850.0 nm. Fluorescence Imaging in Living Cells. The living LO2 (human normal liver cells), FHC (human colorectal mucosal cells), HCT116 (human colorectal cancer cells) and HepG2 (human liver cancer cells) were acquired from the Biomedical Engineering Center of Hunan University (Changsha, China), which were cultured in DMEM supplemented with 10% FBS at 37 º C and 5% CO2. The four kinds of cells were treated with the Cy-GSH (10 μM) at 37 °C for 30 min, and then washed by PBS buffer solution three times for imaging. For inhibitor investigation, the cancer cells (HepG2 and HCT116) were incubated with acivicin (GGT inhibitor, 1 mM) at 37 °C for 30 min, followed by treating with Cy-GSH (10 μM) for another 30 min, and then washed by PBS buffer solution three times for imaging. Fluorescence imaging was carried out on confocal fluorescence microscope. Fluorescence Imaging in Mice. Six week-old male BALB/C nude mice were chosen and used in all the experimental process. All animal operation was performed according to the regulations issued by the Ethical Committee of Xiangtan University. The mice were separated into two groups and handled separately. The mice in the first group were subcutaneously injected with 1×106 HCT116 cells to establish subcutaneous tumor model. The mice in the second group were injected with 1×106 HCT116 cells into colon site by surgery to establish in suit colon tumor model. Tumors were allowed to grow to 5-6 mm in diameter and then used for experimental usage.


Page 2 of 9

confirmed by MS and HPLC (Figure S4 and S5). Thus, the emission peak at 805 nm of the probe decreases with the increase of GGT concentration, and the emission peak at 640 nm (λex = 540 nm) of the probe increases gradually. The remarkable spectral shift (165 nm) confirms that the disruption of the pull-push π-conjugation system in the cyanine dye. Utilizing the property, the NIR ratiometric detection of GGT can be established with Cy-GSH by the fluorescence intensity ratio (F640 / F805). As shown in Figure 1D, the ratio (F640 / F805) increases from 0.2 to 5.4 and the probe exhibits a good linearity of emission ratio (F640 / F805) in the range of 0.1 to 90.0 U/L. The detection limit is thus determined to be 0.03 U/L (3σ/k), which is low enough for determining GGT levels in early stage of cancer. All these results strongly indicate that Cy-GSH has high sensitivity for quantification of GGT activity. a b

.6

.2 0.0 500

1000 (C)

600

700

(B) 600

200

760

800

Wavelength (nm)

800

F640/F805

GGT

400 200

780

800

820

840

Wavelength (nm)

4 3 2 1 0

0

0

620 640 660 680 700 720 740

(E)

Wavelength (nm)

6 (F)

90 U/L

20

40

60

80

100

GGT (U/L)

5

4

0 U/L

3

(D)

5

600

6

GGT

400

F640/F805

2

4 3 2 1

1

0

0

0

5

10

Time (min)

15

20

Bla n GG k T AL P Ap Am r y Ch y Hy d Pe p Try HP FB S

Spectral Response of Cy-GSH to GGT. Cy-GSH was synthesized following the synthetic route displayed in Scheme S1. The detailed synthetic steps and characterization were given in the Supporting Information (Figure S1-S3). With the probe in hand, the absorption spectra of Cy-GSH before and after the addition of GGT were tested in PBS buffer solution. As shown in Figure 1A, Cy-GSH itself exhibited a maximal absorbance at 786 nm. Upon the addition of GGT, the absorption spectra demonstrated a sharp decrease at 786 nm and an obvious increase at 544 nm. Thus, a large blue shift (242 nm) is observed and concomitantly the color of the solution changes from blue to pink. Next, the titration experiments of the enzymatic reaction were monitored to explore the ability of Cy-GSH for the detection of GGT activity quantitatively. As shown in Figure 1B and 1C, Cy-GSH itself shows the fluorescence spectra with the peak at 805 nm (λex = 730 nm). The emission is at NIR region (805 nm) since there is large π-conjugation system in the probe. After the addition of GGT, GGT-triggered cleavage of the γ-glutamyl linkage in Cy-GSH induces the release of amino group in cysteinyl-glycine residue (compound 1). The released negatively charged amino group in compound 1 would undergo a nucledeophilic substitution reaction to yield an amino-substituted product (compound 2), followed by a possible charge transfer from indocyanine to the nucleophilic nitrogen to obtain Cy-N.47,48 The process was detailedly displayed in Scheme S2 and the formation of Cy-N was well

b

a

.4

5

RESULTS AND DISCUSSION

Fluorescence Intensity

(A)

.8

Absorbance

Subcutaneous tumor model: a normal and a HCT116xenograft mouse given an intratumoral injection of probe CyGSH (10 μM), respectively, then imaged by small animal optical in vivo imaging system. Ex vivo: The normal liver was sacrificed from a normal mouse. The normal muscle next to the HCT116 tumor, the pathological liver and the HCT116 tumor were sacrificed from a HCT116-xenograft mouse. Then, the four types of organs were treated with Cy-GSH (10 μM), respectively, and imaged by small animal optical in vivo imaging system. Tissue slices: the normal liver tissue slices were prepared from a normal mouse, the normal muscle tissue slices, the pathological liver tissue slices and the HCT116 tumor were prepared from a HCT116-xenograft mouse. Then the four types of tissue slices were treated with the Cy-GSH (10 μM) respectively, and then imaged by confocal fluorescence microscope. Orthotopic tumor model: The tumor mice were separated into five groups and handled separately. The mice in the first group were given an intraperitoneal injection of probe CyGSH (10 μM) for 30 min. The mice in the second group were anesthetized with chloral hydrate (10 % in saline) and the abdominal cavity was exposed, then spraying of Cy-GSH (10 μM) onto peritoneum for 30 min. The mice in the third group were anesthetized with chloral hydrate (10 % in saline) and the abdominal cavity was exposed, then co-spraying of the inhibitor acivicin (1 mM) and Cy-GSH (10 μM) for 30 min. The mice in the fourth group were sacrificed with CO2 and the mesentery was exposed, then spraying of Cy-GSH (10 μM) onto peritoneum for 30 min. The mice in the fifth group were sacrificed with CO2 and the mesentery was exposed, then cospraying of the inhibitor acivicin (1 mM) and Cy-GSH (10 μM) for 30 min. The images were obtained by small animal optical in vivo imaging system.

Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


F640/F805

Page 3 of 9

Figure 1. (A) Absorption spectra of (a) Cy-GSH (10 μM), (b) Cy-GSH (10 μM) reacting with GGT (90 U/L). (B) Fluorescence spectra (λex = 730 nm) and (C) Fluorescence spectra (λex = 540 nm) of Cy-GSH (10 μM) after adding GGT (0, 0.1, 2.5, 5.0, 10, 15, 25, 30, 40, 55, 70, 90 U/L). (D) The linear relationship between the fluorescence intensity ratio (F640 / F805) and GGT activity. (E) Time-dependent fluorescence intensity ratio (F640 / F805) of Cy-GSH (10 µM) upon the addition of GGT (0, 10, 30, 50, 70, 90 U/L). (F) The fluorescence intensity ratio (F640 / F805) of Cy-GSH (10 µM) toward different enzymes and biological fluids recorded at 4, 8, 10, 16 min. Furthermore, time-dependent fluorescence intensity ratio (F640 / F805) of Cy-GSH with various GGT activities was studied. As shown in Figure 1E, the fluorescence ratio signal of Cy-GSH increases over time and arrived at a plateau in 16 min. Thus, 16 min is adopted as suitable incubation time for GGT assay. The effect of pH on the fluorescence ratiometric response of Cy-GSH was also evaluated (Figure S6). The results reveal that a maximum fluorescence intensity ratio of Cy-GSH for GGT is attained at pH 7.4, which is applied in following experiments and beneficial for its utilization in



biological sample, as most of such samples exist at physiological environment. Then, the effect of temperature on fluorescence ratio of Cy-GSH was also investigated (Figure S7). The result shows that the change of fluorescence intensity ratio reaches maximum at 37 °C. Therefore, 37 °C is applied for the detection of GGT activity, which is consistent with the environment in human body. In addition, Cy-GSH with various concentrations was treated in a fixed GGT concentration in order to optimize the concentration of CyGSH used in the study. The enzyme kinetics of Cy-GSH for GGT was studied by Michaelis-Menten equation and the profile was presented in Figure S8A. The kinetic data are calculated by converting the curve to a linear plot using Lineweaver-Burke analysis (Figure S8B). Vmax and Km are determined to be 1.26 µM/min and 9.87 µM, which are in keeping with those reported for GGT calculated by other methods.23 Thus, 10 µM is chosen as the ideal Cy-GSH concentration for GGT assay. The selectivity of probe Cy-GSH were evaluated by a range of specific enzymes including trypsin (Try), pepsase (Pep), collagen hydrolase (Hyd), chymotrypsin (Chy), amyloglucosidase (Amy), aprotinin (Apr), alkaline phosphatase (ALP). The results show that no measurable fluorescence alteration can be caused by the above enzymes (Figure 1F). Moreover, the selectivity of Cy-GSH was studied by detecting various possible interfering materials including amino acids (Glu, Gly, Leu, Tyr, Phe, Arg, Lys and Cys) and ions (Na+, K+, Mg2+, Ca2+, Al3+, NO3-, CO32-and PO43-) (Figure S9). It could be seen that the amino acids and ions have limited interference on the detection of GGT. All the results indicate that Cy-GSH is particularly selective to GGT. The stability of Cy-GSH in 10% human plasma (HP) or 10% fetal bovine serum (FBS) was also examined (Figure 1F). Negligible hydrolyzation under these conditions was observed, indicating that the specific recognition and cleavage are induced by GGT. Moreover, almost no detectable signal change is observed over 24 h, indicating that Cy-GSH is stable (Figure S10). GGT Inhibitor Investigation. To develop a simple and reliable method to evaluate GGT inhibitor is obviously necessary because GGT inhibitor may be of great potential as anticancer drugs. DON and acivicin (two common inhibitors for GGT) was then investigated to verify this possibility of Cy-GSH for the screening of GGT inhibitors (Figure S11). As depicted in Figure S11A-C, 1 mM DON could decease 78 % of the fluorescence intensity ratio while 1 mM acivicin could completely inhibit the activity of GGT. Furthermore, as shown in Figure S11D-E, the IC50 value of DON and acivicin are estimated to be 0.44 mM and 0.16 mM, respectively. All these results clearly indicate that the inhibition ability of acivicin is stronger than that of DON, suggesting that Cy-GSH possesses great potential for screening of GGT inhibitors. Detection of GGT Activity in Mice Blood. To some extent, the level of blood GGT is a significant diagnostic index for various diseases such as cancers.17,29 The common assay in clinic is usually accomplished by absorbance measurement, which unfortunately presented very low sensitivity and required a large number of blood samples. What’s more, fluorescent probe-based assays exhibited strong background fluorescence interference owing to the blood samples which are rich in those biomolecules with high background fluorescence such as haemoglobin. Hence, a ratiometric NIR

Page 4 of 9

fluorescent probe Cy-GSH is anticipated to be useful for assaying blood GGT with high sensitivity. The assay by three blood samples from normal mice and six blood samples from colorectal cancer mice was carried out (Table 1). The activity of GGT measured by the probe is close to that tested by ELISA kit (the commercial assay kit for GGT), which demonstrates the reliability and accuracy of the probe. Moreover, the GGT level in different mice was precisely calculated by using two signal channels output and thus with high sensitivity. As depicted in Table 1, the GGT level in the healthy mice blood samples are below 60 U/L, while the GGT level in the colorectal cancer mice blood samples are over 90 U/L. The results illuminate that the probe with high sensitivity not only can differentiate normal blood samples and cancer blood samples, but also holds great application potential for detecting GGT activity in clinical use. Table 1. Measurement of GGT Level in Mice Blood Samples Sample

This probe

ELISA kit

1 47.96 ± 4.27 48.85 ± 5.15 2 53. 31 ± 4.82 54. 66 ± 5.91 3 43.39 ± 4.19 44.33 ± 4.23 4 131.08 ± 6.13 132.33 ± 6.73 5 123.31 ± 6.55 124.44 ± 7.38 6 101.53 ± 6.32 102.96 ± 7.21 7 135.53 ± 5.97 135.91 ± 6.15 8 133.13 ± 6.34 133.85 ± 6.93 9 126.55 ± 7.12 127.17 ± 7.44 Note: The blood samples 1-3 were acquired from normal mice. The blood samples 4-9 were acquired from colorectal cancer mice. Fluorescence Imaging in Living Cells. Before fluorescence imaging, the cytotoxicity of Cy-GSH in living cells was firstly evaluated. The cytotoxicity measurements were conducted in normal cells (FHC and LO2) and cancer cells (HCT116 and HepG2) by conventional MTT assay (Figure S12). High cell viability was observed after the cells were treated with various concentrations of Cy-GSH from 0 to 30.0 μM, demonstrating the minimal cytotoxicity of Cy-GSH. Next, to investigate whether the probe could efficiently response of GGT in living cells, Cy-GSH was used for detecting GGT activity in both cancer and normal cells by using two signal channels (Figure 2). As depicted in Figure 2a-f, the normal cells (FHC and LO2) treated with Cy-GSH showed strong fluorescence in Channel 1 and no fluorescence in Channel 2. The ratio of fluorescence intensity (Channel 2 / Channel 1) was 1.0 for FHC cells and 1.1 for LO2 cells, respectively, which clearly suggests that GGT activity remains very low in normal cells (Figure 2C).16,18 In contrast, when cancer cells (HepG2 and HCT116) were treated with Cy-GSH, by contrast, almost no fluorescence in Channel 1 and strong emission in Channel 2 were observed. The ratios increased to 15.4 for HepG2 cells and 12.0 for HCT116 cells, respectively, which obviously demonstrates that GGT is overexpressed in these cancer cells (Figure 2g-l). These results proved that this zero cross-talk ratiometric NIR fluorescent probe has the capability for discriminating normal and cancer cells. In order to further verify the fact that the fluorescence change indeed come from GGT cleavage, inhibitor investigation was studied. Just as expected, the cancer cells (HepG2 and HCT116 cells)


treated with Cy-GSH, and then incubated with acivicin, exhibit a marked increase of fluorescence signal in Channel 1 and a noticeable reduction of fluorescence signal in Channel 2. The ratio is severely decreased, displaying that GGT activity can be efficiently inhibited by acivicin (Figure 2m-r). In addition, the time-dependent fluorescence responses to endogenous GGT in HepG2 cells (as a representative) were surveyed at different time points (0, 4, 8, 12, 16 and 20 min). As shown in Figure S13, the ratio of fluorescence intensity (Channel 2 / Channel 1) in HepG2 cells gradually increased over time and reaches a maximum at 16 min, which is consistent with the result from the in vitro study. All in all, these imaging experiments illuminate that Cy-GSH with zero cross-talk ratiometric fluorescence not only can work well to monitor endogenous GGT activity in living cells, but also remarkably discriminate between normal cells and cancer cells. Fluorescence Imaging of Subcutaneous Tumor in Vivo. Besides the living cell study, the ability of probe Cy-GSH to ratiometric response of GGT activity in subcutaneous tumor mouse models was also examined (Figure 3). HCT116 cells were grafted into the BALB/c mouse to produce murine xenograft subcutaneous tumor models. As shown in Figure 3A, the normal mouse was given an intratumoral injection of CyGSH, showing strong fluorescence in Channel 1 and rather weak fluorescence in Channel 2. On the other hand, the HCT116-xenograft mouse given an intratumoral injection of Cy-GSH, there was almost no fluorescence in Channel 1 and intense signals in Channel 2. There is a 9.3-fold enhancement of the fluorescence intensity ratio (Channel 2 / Channel 1) in tumor mouse compared with normal mouse (Figure 3B). These results demonstrate that probe Cy-GSH can be used to zero cross-talk ratiometric imaging GGT activity in subcutaneous tumor mouse with high contrast.

4 2

j

b

e

h

k

n

q

c

f

i

l

0

r

Ratio

Channel 1

HepG2 + Acivicin p

g

Ratio

(C) 16 14 12 10 8 6 4 2 0

c

f

i

l

o

r

Figure 2. Fluorescence imaging in cells. (A) Normal cells (FHC, LO2): (a-c) FHC cells incubated with Cy-GSH (10 μM), (d-f) LO2 cells incubated with Cy-GSH (10 μM). (B) Cancer cells (HCT116, HepG2): (g-i) HCT116 cells incubated with Cy-GSH (10 μM). (j-l) HepG2 cells incubated with Cy-GSH (10 μM). (m-o) HCT116 cells pretreated with Cy-GSH (10 μM), and then incubated with acivicin (1 mM). (p-r) HepG2 cells pretreated with Cy-GSH (10 μM), and then incubated with acivicin (1 mM). (C) The ratio of fluorescence intensity (Channel 2 / Channel 1) and the ratio of image c is set as 1.0. Channel 1: λex = 640 nm, λem = 750-850 nm; Channel 2: λex = 560 nm, λem = 580-680 nm. Scale bar: 10 μm.

(1)

Cancer

(2)

(4)

(3)

(D) 16

14 12 10 8 6 4 2 0

a

b

c

d

a

b

c

d

(Tissue slices) Normal (6)

(7)

Cancer

(8)

Channel 2 Channel 1

6

Cancer cells HCT116 + Acivicin m

HepG2

d

Ratio

Channel 2 Channel 1

Channel 1 Channel 2 8

HCT116

LO2

a

Normal

(5)

10

(B)

Normal cells FHC

(Ex vivo)

(C)

(E)

(B)

(A)

(F) 14 Ratio

(In vivo) Normal Cancer

(A)

Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Channel 2

Page 5 of 9

12 10 8 6 4 2 0

0

Normal

Cancer

Figure 3. Fluorescence imaging of subcutaneous tumor mouse models. (A) In vivo: a normal and HCT116-xenograft mouse given an intratumoral injection of probe Cy-GSH (10 μM). (B) The ratio of fluorescence intensity (Channel 2 / Channel 1) is obtained from the image A and the ratio of normal mouse is defined as 1.0. (C) Ex vivo: the separated organs sacrificed from normal and HCT116-xenograft mice. (1) A normal liver, (2) a normal muscle, (3) a pathological liver and (4) a HCT116 tumor treated with CyGSH (10 μM). (D) The ratio of fluorescence intensity (Channel 2 / Channel 1) is obtained from the image B and the ratio of image (a) is defined as 1.0. (E) Tissue slices. (5) A normal liver tissue slice, (6) a normal muscle tissue slice, (7) a pathological liver tissue slice and (8) a HCT116 tumor tissue slice treated with Cy-GSH (10 μM). (F) The ratio of fluorescence intensity (Channel 2 / Channel 1) is obtained from the image E and the ratio of image e is set as 1.0. Channel 1: λex = 735 nm, λem = 750-850 nm; Channel 2: λex = 560 nm, λem = 580-680 nm.



Moreover, the separated normal tissues (a normal liver and a normal muscle) treated with Cy-GSH produce great fluorescence in Channel 1 and scarcely any fluorescence in Channel 2 (Figure 3C). The ratio of fluorescence intensity (Channel 2 / Channel 1) is 1.0 for the normal liver and 0.78 for the normal muscle, which suggests that GGT activity is low in normal tissues (Figure 3D). While, a pathological liver (from the tumor-bearing mouse) and a HCT116 tumor treated with Cy-GSH display almost no fluorescence signal in Channel 1 and strong fluorescence signal in Channel 2 (Figure 3C). The ratio is 12.9 for the pathological liver and 11.6 for the HCT116 tumor, respectively, which indicates that GGT activity is high in these tissues (Figure 3D). Thus, obviously different phenomena of ex vivo imaging further clearly demonstrates that probe Cy-GSH has the ability of visualizing GGT activity in tissues and discriminating normal tissues and cancer tissues. (A)

White light

Fluorescence Channel 1 Channel 2

Cy-GSH

Cy-GSH+ acivicin

(B)

Fluorescence White light

Channel 1

Channel 2

Cy-GSH

Cy-GSH

Cy-GSH+ acivicin

Figure 4. Fluorescence imaging of orthotopic colon tumor mouse models. (A) In vivo: representative white light and fluorescence images of the mouse peritoneum after spraying probe Cy-GSH (10 μM). (B) Ex vivo: representative white light and fluorescence images of the mesentery of in suit colon tumor mouse after spraying probe Cy-GSH (10 μM). Channel 1: λex = 735 nm, λem = 750-850 nm; Channel 2: λex = 560 nm, λem = 580-680 nm. Note: circle represents tumors, arrow represents disseminated cancer nodules. Furthermore, to study the depth-distribution of this ratiometric NIR fluorescence probe in tissues, the fluorescence images at different depths in the liver tissue slice of HCT116-tumor bearing mice incubated with CyGSH were collected in the Z-scan mode (Figure S14). It is meaningful that Cy-GSH is able to image GGT activity in tissues with the imaging depth at 40~120 μm. The results

Page 6 of 9

indicated that Cy-GSH with near-infrared fluorescence emission has outstanding tissue penetrating and staining ability. And then, the normal tissue slices (liver tissue slice and muscle tissue slice) were treated with Cy-GSH, respectively, strong fluorescence in Channel 1 and negligible fluorescence in Channel 2 are observed (Figure 3E). The ratio of fluorescence intensity (Channel 2 / Channel 1) is 1.0 for the normal liver tissue slice and 0.79 for the normal muscle tissue slice (Figure 3F). By contrast, when a pathological liver tissue slice from the tumor-bearing mouse and a HCT116 tumor tissue slice were incubated with CyGSH, respectively, there were no fluorescence in Channel 1 and bright fluorescence in Channel 2 (Figure 3E). The ratio is 9.9 for the pathological liver tissue slice and 13.7 for the HCT116 tumor tissue slice (Figure 3F). These results indicate that the liver of tumor-bearing mouse have pathological changes. Efficient ratiometric monitoring GGT activity in subcutaneous tumor mouse models makes probe Cy-GSH be suitable for the early cancer diagnoses. Fluorescence Imaging of Orthotopic Colon Tumor in Vivo. As we had confirmed that the ratiometric fluorescence of probe Cy-GSH could be activated by tumor-associated GGT in vivo, the suitability for cancer diagnose of this probe was then examined in orthotopic HCT116 colon tumor mouse model. As shown in Figure S15, after intraperitoneal injection of Cy-GSH, there was almost no fluorescence in Channel 1 and rather strong fluorescence in Channel 2. The result proves that the probe can be applied for in vivo imaging of tumor, which may benefit for pre-operative whole-body imaging. We next investigated whether tumorspecific ﬂuorescence imaging could be achieved simply by spraying Cy-GSH onto peritoneum of orthotopic colon tumor mice. As shown in Figure 4A, using white light imaging, it is difficult to recognize the minute pleural dissemination and lymph node metastases, which are less than 2-3 mm in diameter. However, these lesions are clearly visualized by ratiometric fluorescence imaging after spraying of Cy-GSH. It is obviously found that the normal regions exhibit significant fluorescence in Channel 1 and no fluorescence in Channel 2. Conversely, the tumors and disseminated cancer nodule regions display no fluorescence in Channel 1 and rather strong fluorescence in Channel 2. Further, upon co-spraying of the inhibitor acivicin and CyGSH, strong fluorescence could be observed in Channel 1 for the normal regions and the tumors and disseminated cancer nodules regions, while almost no fluorescence in Channel 2 for either the normal regions or the tumors and disseminated cancer nodules regions are noticed. These results suggest that tumors and even metastases as small as 1 mm in diameter inside the peritoneal cavity can be clearly and specifically visualized by Cy-GSH. To evaluate the possibility of probe Cy-GSH has the ability of visualizing the tumors and disseminated cancer nodules during surgery, the ex vivo imaging of the mesentery of orthotopic colon tumor mouse models were also investigated. As shown in Figure 4B, for the normal regions, there is significant fluorescence in Channel 1 and no fluorescence in Channel 2. By contrast, for the tumors and disseminated cancer nodules regions, there are almost no fluorescence in Channel 1 and much enhanced fluorescence in Channel 2. By using this ratiometric fluorescence probe, the tumors and disseminated cancer nodules could be readily distinguished, which benefit for aiding surgeons in detecting tiny cancerous nodules


Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry during accurate tumor resection. To ensure the authenticity of these data, repetitive experiment was carried out and expected result was obtained (the second row in Figure 4B). In addition, the inhibitor experiment was further proved these results (the third row in Figure 4B). Based on the fact that the orthotopic model is much closer to the usual clinical situation, and thus, the results in the orthotopic tumorbearing mice directly suggest that probe Cy-GSH with no cross-talk ratiometric fluorescence have clear potential for NIR fluorescence guidance of tumor diagnosis and surgical

resection.

CONCLUSIONS In conclusion, we have reported a novel ratiometric NIR GGT fluorescent probe for efficient cancer diagnosis with no cross-talk in various biological samples. The large emission shift (165 nm) of Cy-GSH with GGT is implemented by modulation of the conjugated π-electron system of cyanine dye. The probe shows high sensitivity with high ratio (F640 / F805) enhancement from 0.2 to 5.4 and low detection limit (0.03 U/L). Cy-GSH is also successfully applied in evaluating GGT inhibitors. In particular, the probe can be used to monitoring GGT activity in various biological samples, including bloods, cells, tissues and in vivo, with the results indicating that the probe can be applied for cancer early diagnosis. What’s more, successfully visualizing tumor and metastases in mouse models illuminates that the probe are practically translatable as convenient tools for fluorescence imaging guiding surgery of human cancer.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental section, supplemental spectroscopic data, and additional fluorescence images in biological samples.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Chun-Yan Li) *E-mail: [email protected] (Hong-Wen Liu)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21775133), Hunan Provincial Natural Science Foundation (2018JJ2385), State Key Laboratory of Chemo/Biosensing and Chemometrics Foundation (2017001), Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization.

REFERENCES (1) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; LortetTieulent, J.; Jemal, A. Global Cancer Statistics, 2012. CA cancer J. clin. 2015, 65, 87-108. (2) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2017. CA cancer J. clin. 2017, 67, 7-30. (3) Miller, K. D.; Siegel, R. L.; Lin, C. C.; Mariotto, A. B.; Kramer, J. L.; Rowland, J. H.; Jemal, A. Cancer Treatment and Survivorship Statistics, 2016. CA cancer J. clin. 2016, 66, 271-289.

(4) Grimm, C.; Hofstetter, G.; Aust, S.; Mutz-Dehbalaie, I.; Bruch, M.; Heinze, G.; Polterauer, S. Association of GammaGlutamyltransferase with Severity of Disease at Diagnosis and Prognosis of Ovarian Cancer. Brit. J. Cancer 2013, 109, 610-614. (5) Terzyan, S. S.; Burgett, A. W.; Heroux, A.; Smith, C. A.; Mooers, B. H.; Hanigan, M. H. Human Gamma-Glutamyl Transpeptidase 1: Structures of the Free Enzyme, Inhibitor-Bound Tetrahedral Transition States and Glutamate-Bound Enzyme Reveal Novel Movement within the Active Site during Catalysis. J. Biol. Chem. 2015, 290, 17576-17586. (6) Kushwaha, N.; Srivastava, S. Gamma-Glutamyl Transpeptidase from Two Plant Growth Promoting Rhizosphere Fluorescent Pseudomonads. Anton. Leeuw. Int. J. G. 2014, 105, 4556. (7) Strasak, A. M.; Rapp, K.; Brant, L. J.; Hilbe, W.; Gregory, M.; Oberaigner, W.; Ulmer, H. Association of γGlutamyltransferase and Risk of Cancer Incidence in Men: a Prospective Study. Cancer Res. 2008, 68, 3970-3977. (8) Ueo, H.; Shinden, Y.; Tobo, T.; Gamachi, A.; Udo, M.; Komatsu, H.; Sakimura, S. Rapid Intraoperative Visualization of Breast Lesions with γ-Glutamyl Hydroxymethyl Rhodamine Green. Sci. Rep. 2015, 5, 12080-12085. (9) Shimane, T.; Aizawa, H.; Koike, T.; Kamiya, M.; Urano, Y.; Kurita, H. Oral Cancer Intraoperative Detection by Topically Spraying a γ-Gglutamyl Transpeptidase Activated Fluorescent Probe. Oral oncol. 2016, 54, 16-18. (10) Kawakami, K.; Fujita, Y.; Matsuda, Y.; Arai, T.; Horie, K.; Kameyama, K.; Mizutani, K. Gamma-Glutamyltransferase Activity in Exosomes as a Potential Marker for Prostate Cancer. BMC cancer, 2017, 17, 316-327. (11) Del Corso, A.; Cappiello, M.; Buono, F.; Moschini, R.; Paolicchi, A.; Mura, U. J. Colorimetric Coupled Enzyme Assay for γ-Glutamyltransferase Activity Using Glutathione as Substrate. Biochem. Biophys. Methods 2006, 67, 123-130. (12) Tateishi, N.; Higashi, T.; Nomura, T.; Naruse, A.; Nakashima, K.; Shiozaki, H.; Sakamoto, Y. Higher Transpeptidation Activity and Broad Acceptor Specificity of γGlutamyltransferases of Tumors. Gan. 1976, 67, 215-222. (13) Kiuchi, K.; Kiuchi, K.; Nagatsu, T.; Togari, A.; Kumagai, H. Highly Sensitive Assay for γ-Glutamyltranspeptidase Activity by High-Performance Liquid Chromatography with Electrochemical Detection. J. Chromatogr. A 1986, 357, 191-198. (14) Luo, Z.; An, R.; Ye, D. Recent Advances in the Development of Optical Imaging Probes for γGlutamyltranspeptidase. Chem. Bio. Chem. 2018, doi:10.1002/cbic.201800370. (15) Urano, Y.; Sakabe, M.; Kosaka, N.; Ogawa, M.; Mitsunaga, M.; Asanuma, D.; Kobayashi, H. Rapid Cancer Detection by Topically Spraying a γ-Glutamyltranspeptidase-Activated Fluorescent Probe. Sci. Transl. Med. 2011, 3, 110ra119-110ra119. (16) Zhang, P.; Jiang, X. F.; Nie, X.; Huang, Y.; Zeng, F.; Xia, X.; Wu, S. A Two-Photon Fluorescent Sensor Revealing DrugInduced Liver Injury via Tracking γ-Glutamyltranspeptidase (GGT) Level in Vivo. Biomaterials 2016, 80, 46-56. (17) Li, L.; Shi, W.; Wu, X.; Gong, Q.; Li, X.; Ma, H. Monitoring γ-Glutamyl Transpeptidase Activity and Evaluating Its Inhibitors by a Water-Soluble Near-Infrared Fluorescent Probe. Biosens. Bioelectron. 2016, 81, 395-400. (18) Hou, X.; Zeng, F.; Wu, S. A Fluorescent Assay for γGlutamyltranspeptidase via Aggregation Induced Emission and Its Applications in Real Samples. Biosens. Bioelectron. 2016, 85, 317323. (19) Tong, H.; Zheng, Y.; Zhou, L.; Li, X.; Qian, R.; Wang, R.; Wang, W. Enzymatic Cleavage and Subsequent Facile Intramolecular Transcyclization for in Situ Fluorescence Detection of γ-Glutamyltranspetidase Activities. Anal. Chem. 2016, 88, 10816-10820. (20) Luo, Z.; Feng, L.; An, R.; Duan, G.; Yan, R.; Shi, H.; Ye, D. Activatable Near-Infrared Probe for Fluorescence Imaging of γGlutamyl Transpeptidase in Tumor Cells and In Vivo. Chem. Eur. J. 2017, 23, 1-9.



(21) Park, S.; Lim, S. Y.; Bae, S. M.; Kim, S. Y.; Myung, S. J.; Kim, H. J. Indocyanine-Based Activatable Fluorescence Turn-On Probe for γ-Glutamyltranspeptidase and Its Application to the Mouse Model of Colon Cancer. ACS Sensors 2016, 1, 579-583. (22) Wang, P.; Zhang, J.; Liu, H. W.; Hu, X. X.; Feng, L. L.; Yin, X.; Zhang, X. B. An Efficient Two-Photon Fluorescent Probe for Measuring γ-Glutamyltranspeptidase Activity During the Oxidative Stress Process in Tumor Cells and Tissues. Analyst 2017, 142, 1813-1820. (23) Li, L.; Shi, W.; Wang, Z.; Gong, Q.; Ma, H. Sensitive Fluorescence Probe with Long Analytical Wavelengths for γGlutamyl Transpeptidase Detection in Human Serum and Living Cells. Anal. Chem. 2015, 87, 8353-8359. (24) Luo, Z.; Huang, Z.; Li, K.; Sun, Y.; Lin, J.; Ye, D.; Chen, H. Y. Targeted Delivery of a γ-Glutamyl Transpeptidase Activatable Near-Infrared-Fluorescent Probe for Selective Cancer Imaging. Anal. Chem. 2018, 90, 2875-2883. (25) Iwatate, R. J.; Kamiya, M.; Umezawa, K.; Kashima, H.; Nakadate, M.; Kojima, R.; Urano, Y. Silicon Rhodamine-Based Near-Infrared Fluorescent Probe for γ-Glutamyltransferase. Bioconjugate Chem. 2018, 29, 241-244. (26) Iwatate, R. J.; Kamiya, M.; Urano, Y. Asymmetric Rhodamine-Based Fluorescent Probe for Multicolour in Vivo Imaging. Chem. Eur. J. 2016, 22, 1696-1703. (27) Park, S.; Bae, D. J.; Ryu, Y. M.; Kim, S. Y.; Myung, S. J.; Kim, H. J. Mitochondria-Targeting Ratiometric Fluorescent Probe for γ-Glutamyltranspeptidase and Its Application to Colon Cancer. Chem. Commun. 2016, 52, 10400-10402. (28) Wang, F.; Zhu, Y.; Zhou, L.; Pan, L.; Cui, Z.; Fei, Q.; Zhao, C. Fluorescent in Situ Targeting Probes for Rapid Imaging of Ovarian-Cancer-Specific γ-Glutamyltransferase. Angew. Chem. Int. Ed. 2015, 54, 7349-7353. (29) Hou, X.; Yu, Q.; Zeng, F.; Yu, C.; Wu, S. Ratiometric Fluorescence Assay for γ-Glutamyltranspeptidase Detection Based on a Single Fluorophore via Analyte-Induced Variation of Substitution. Chem. Commun. 2014, 50, 3417-3420. (30) Zhang, H.; Wang, K.; Xuan, X.; Lv, Q.; Nie, Y.; Guo, H. Cancer Cell-Targeted Two-Photon Fluorescence Probe for the RealTime Ratiometric Imaging of DNA Damage. Chem. Commun. 2016, 52, 6308-6311. (31) Li, H.; Yao, Q.; Xu, F.; Xu, N.; Duan, R.; Long, S.; Peng, X. Imaging γ-Glutamyltranspeptidase for Tumor Identification and Resection Guidance via Enzyme-Triggered Fluorescent Probe. Biomaterials 2018, 179, 1-14. (32) Ou-Yang, J.; Li, Y.; Wu, P.; Jiang, W. L.; Liu, H. W.; Li, C. Y. Detecting and Imaging of γ-Glutamytranspeptidase Activity in Serum, Live Cells and Pathological Tissues with a High SignalStability Probe by Releasing a Precipitating Fluorochrome. ACS Sens. 2018, 3, 1354-1361. (33) Liu, H. W.; Chen, L.; Xu, C.; Li, Z.; Zhang, H.; Zhang, X. B.; Tan, W. Recent Progresses in Small-Molecule Enzymatic Fluorescent Probes for Cancer Imaging. Chem. Soc. Rev. 2018, 47, 7140-7180. (34) Fu, Q.; Chen, G.; Liu, Y.; Cao, Z.; Zhao, X.; Li, G.; You, J. In Situ Quantification and Evaluation of ClO- / H2S Homeostasis in Inflammatory Gastric Tissue by Applying a Rationally Designed Dual-Response Fluorescence Probe Featuring a Novel H+-Activated Mechanism. Analyst 2017, 142, 1619-1627. (35) Zhang, Q.; Yu, D.; Ding, S.; Feng, G. Q. A Low Dose, Highly Selective and Sensitive Colorimetric and Fluorescent Probe

Page 8 of 9

for Biothiols and Its Application in Bioimaging. Chem. Commun., 2014, 50, 14002-14005. (36) Zeng, R.; Gao, Q.; Cheng, F.; Yang, Y.; Zhang, P.; Chen, S.; Long, Y. A Near-Infrared Fluorescent Sensor with Large Stokes Shift for Rapid and Highly Selective Detection of Thiophenols in Water Samples and Living Cells. Anal. Bioanal. Chem. 2018, 410, 2001-2009. (37) Feng, W.; Li, M.; Sun, Y.; Feng, G. Near-Infrared Fluorescent Turn-On Probe with a Remarkable Large Stokes Shift for Imaging Selenocysteine in Living Cells and Animals. Anal. Chem. 2017, 89, 6106-6112. (38) Li, S. J.; Li, C. Y.; Li, Y. F.; Fei, J.; Wu, P.; Yang, B.; OuYang, J.; Nie, S. X. Facile and Sensitive Near-Infrared Fluorescence Probe for the Detection of Endogenous Alkaline Phosphatase Activity in Vivo. Anal. Chem. 2017, 89, 6854-6860. (39) Fan, C.; Lv, X.; Liu, F.; Feng, L.; Liu, M.; Cai, Y.; Wang, H. Silver Nanoclusters Encapsulated into Metal-Organic Frameworks with Enhanced Fluorescence and Specific Ion Accumulation toward the Microdot Array-Based Fluorimetric Analysis of Copper in Blood. ACS sensors 2018, 3, 441-450. (40) Wu, Y.; Wang, J.; Zeng, F.; Huang, S.; Huang, J.; Xie, H.; Wu, S. Pyrene Derivative Emitting Red or Near-Infrared Light with Monomer/Excimer Conversion and Its Application to Ratiometric Detection of Hypochlorite. ACS Appl. Mater. Interfaces 2016, 8, 1511−1519. (41) Feng, L. L.; Wu, Y. X.; Zhang, D. L.; Hu, X. X.; Zhang, J.; Wang, P.; Tan, W. Near Infrared Graphene Quantum Dots-Based Two-Photon Nanoprobe for Direct Bioimaging of Endogenous Ascorbic Acid in Living Cells. Anal. Chem. 2017, 89, 4077-4084. (42) Wei, Y.; Cheng, D.; Ren, T.; Li, Y.; Zeng, Z.; Yuan, L. Design of NIR Chromenylium-Cyanine Fluorophore Library for “Switch-On” and Ratiometric Detection of Bio-Active Species in Vivo. Anal. Chem. 2016, 88, 1842-1849. (43) Huang, Y.; Yu, F.; Wang, J.; Chen, L. Near-Infrared Fluorescence Probe for in Situ Detection of Superoxide Anion and Hydrogen Polysulfides in Mitochondrial Oxidative Stress. Anal. Chem. 2016, 88, 4122-4129. (44) Zhou, L.; Wang, Q.; Zhang, X. B.; Tan, W. Through-Bond Energy Transfer-Based Ratiometric Two-Photon Probe for Fluorescent Imaging of Pd2+ Ions in Living Cells and Tissues. Anal. Chem. 2015, 87, 4503-4507. (45) Xu, J.; Pan, J.; Jiang, X.; Qin, C.; Zeng, L.; Zhang, H.; Zhang, J. F. A Mitochondria-Targeted Ratiometric Fluorescent Probe for Rapid, Sensitive and Specific Detection of Biological SO2 Derivatives in Living Cells. Biosens. Bioelectron. 2016, 77, 725732. (46) Liu, Y.; Yu, D.; Ding, S.; Xiao, Q.; Guo, J.; Feng, G. Rapid and Ratiometric Fluorescent Detection of Cysteine with High Selectivity and Sensitivity by a Simple and Readily Available Probe. ACS Appl. Mater. Interfaces 2014, 6, 17543-17550. (47) Guo, Z.; Kim, G. H.; Shin, I.; Yoon, J. A Cyanine-Based Fluorescent Sensor for Detecting Endogenous Zinc Ions in Live Cells and Organisms. Biomaterials 2012, 33, 7818-7827. (48) Lim, S. Y.; Hong, K. H.; Kim, D. I.; Kwon, H.; Kim, H. J. Tunable Heptamethine-Azo Dye Conjugate as an NIR Fluorescent Probe for the Selective Detection of Mitochondrial Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2014, 136, 70187025.


Page 9 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Only for TOC


Fluorescence-Guided Cancer Diagnosis and Surgery by a Zero Cross

Recommend Documents