激活型近红外荧光探针用于肝癌中二肽基肽酶活性检测

doi:10.6023/cjoc202404028

有机化学 ›› 2024, Vol. 44 ›› Issue (11): 3417-3426.DOI: 10.6023/cjoc202404028 上一篇下一篇

研究论文

激活型近红外荧光探针用于肝癌中二肽基肽酶活性检测

张飞, 梅煜, 丁雪峰, 李再峰^*(), 张鹏^*(), 丁彩凤^*()

青岛科技大学化学与分子工程学院光电传感与生命分析教育部重点实验室山东省生化分析重点实验室山东省生命分析高校重点实验室山东青岛 266042

收稿日期:2024-04-19 修回日期:2024-05-20 发布日期:2024-06-13
基金资助:
国家自然科学基金(21904077); 国家自然科学基金(22074074); 山东省高校优秀青年创新团队项目(2022KJC004); 山东省泰山学者计划(tstp20231227); 青岛市自然科学基金(23-2-1-175-zyyd-jch)

A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers

Fei Zhang, Yu Mei, Xuefeng Ding, Zaifeng Li^*(), Peng Zhang^*(), Caifeng Ding^*()

Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science (Ministry of Education), College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, Shandong 266042

Received:2024-04-19 Revised:2024-05-20 Published:2024-06-13
Contact: *E-mail:lizfengphd@126.com;pengzhang@qust.edu.cn;dingcaifeng@qust.edu.cn
Supported by:
National Natural Science Foundation of China(21904077); National Natural Science Foundation of China(22074074); Youth Innovation Team Development Program of Shandong Higher Education Institutions(2022KJC004); Taishan Scholar Program of Shandong Province(tstp20231227); Natural Science Foundation of Qingdao City(23-2-1-175-zyyd-jch)

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摘要/Abstract

二肽基肽酶(DPP-IV)作为一种癌细胞中过量表达的跨膜糖蛋白, 常被用于肿瘤早期检测的重要生物标志物. 开发了一种能够灵敏检测肝癌细胞中DPP-IV的开启型近红外荧光探针MB-DPP. 该探针以亚甲基蓝为荧光团, 以对氨基苯甲醇为连接臂, 通过脱水缩合反应键合甘氨酰-L-脯氨酸作为DPP-IV的特异性酶切位点, 从而实现DPP-IV触发的荧光“开启”反应, 探针MB-DPP与DPP-IV作用后在691 nm处发出强烈的近红外荧光. 光谱实验表明, 探针MB-DPP对DPP-VI表现出较高的选择性和检测灵敏度(检测限为1.12 ng/mL). 生物成像研究显示, MB-DPP不仅能实现了肝癌细胞内DPP-IV的活性荧光成像分析, 也可用于肝癌活体肿瘤内DPP-IV的活性成像. 因此探针MB-DPP的开发将有助于肝癌的早期诊断.

关键词: 二肽基肽酶, 近红外荧光探针, 亚甲基蓝, 肝癌, 活细胞成像, 活体成像

Dipeptidyl peptidase IV (DPP-IV), a transmembrane glycoprotein overexpressed in cancer cells, is often used as an important biomarker for early detection of tumors. A near-infrared turn ON fluorescence probe (MB-DPP) was developed for the sensitively detection of DPP-IV in liver cancer cells. MB-DPP employs methylene blue as fluorophore and p-aminobenzyl alcohol as the connecting arm to bond glycyl-L-proline as the specific cleavage site of DPP-IV through dehydration condensation reaction, thereby achieving the fluorescence “turn on” reaction triggered by DPP-IV. Probe MB-DPP emits strong near-infrared fluorescence at 691 nm after the incubation with DPP-IV. Spectral experiments showed that MB-DPP exhibited high selectivity and detection sensitivity (with a detection limit of 1.12 ng/mL) for DPP-VI. Bio-imaging studies showed that MB-DPP could not only achieve fluorescence imaging analysis of DPP-IV activity in liver cancer cells, but also be used for imaging of DPP-IV in liver cancer tumors. Therefore, the development of probe MB-DPP will contribute to the early diagnosis of liver cancer.

Key words: dipeptidyl peptidase, near infrared fluorescent probe, methylene blue, liver cancer, living cell imaging, in vivo imaging

引用此文

张飞, 梅煜, 丁雪峰, 李再峰, 张鹏, 丁彩凤. 激活型近红外荧光探针用于肝癌中二肽基肽酶活性检测[J]. 有机化学, 2024, 44(11): 3417-3426.

Fei Zhang, Yu Mei, Xuefeng Ding, Zaifeng Li, Peng Zhang, Caifeng Ding. A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers[J]. Chinese Journal of Organic Chemistry, 2024, 44(11): 3417-3426.

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图/表 11

图式1. 探针MB-DPP对DPP-IV响应的传感机制

Scheme 1. Sensing mechanism of MB-DPP to DPP-IV

图式2. 探针MB-DPP的合成路线

Scheme 2. Synthetic route of probe MB-DPP

图1. 探针MB-DPP在不含(黑线)或含有(红线)80 ng/mL DPP-IV的PBS [pH=7.4, 含有体积分数为0.5%的二甲基亚砜(DMSO)]中于37 ℃孵育90 min后的(a)紫外-可见吸收光谱和(b)荧光光谱([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 1. (a) Absorption and (b) fluorescence spectra of MB-DPP before (black line) and after (red line) the incubation with 80 ng/mL DPP-IV in PBS (pH=7.4, containing 0.5% DMSO) at 37 ℃for 90 min ([MB-DPP]=10 μmol/L, λex=600 nm)

图2. (a) 探针MB-DPP与80 ng/mL DPP-IV在不同pH (4.0~9.0)的PBS中于37 ℃孵育60 min后的荧光光谱及(b)不同pH (4.0~9.0)的PBS溶液中MB-DPP与DPP-IV孵育前(蓝线)后(红线)于691 nm处的荧光强度([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 2. (a) Fluorescence spectra of MB-DPP upon incubated with 80 ng/mL DPP-IV at 37 ℃ for 60 min in PBS of different pH from 4.0~9.0 and (b) fluorescence intensity of MB-DPP at 691 nm before (blue line) and after (red line) the incubation with DPP-IV in PBS of different pH values (4.0~9.0) ([MB-DPP]=10 μmol/L, λex=600 nm)

图3. (a) MB-DPP与80 ng/mL DPP-IV在pH=7.4的PBS中于不同温度(5~60 ℃)下孵育60 min后的荧光图及(b)在5~60 ℃的PBS中MB-DPP与DPP-IV孵育前(蓝线)后(红线)在691 nm处的荧光强度([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 3. (a) Fluorescence spectra of MB-DPP incubated with 80 ng/mL DPP-IV in PBS of pH 7.4 at different temperatures for 60 min and (b) fluorescence intensity of MB-DPP at 691 nm before (blue line) and after (red line) the incubation with DPP-IV in PBS at 5~60 ℃ ([MB-DPP]=10 μmol/L, λex=600 nm)

图4. (a) MB-DPP与80 ng/mL DPP-IV在pH 7.4的PBS中于37 ℃下孵育0~120 min后的荧光图及(b)在pH 7.4的PBS中MB-DPP与DPP-IV于37 ℃孵育前(蓝线)后(红线)在691 nm处的荧光强度([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 4. (a) Fluorescence spectra of MB-DPP incubated with 80 ng/mL DPP-IV at 37 ℃ in PBS of pH 7.4 for 0~120 min and (b) fluorescence intensity of MB-DPP at 691 nm before (blue line) and after (red line) the incubation with DPP-IV in PBS of pH 7.4 at 37 ℃ ([MB-DPP]=10 μmol/L, λex=600 nm)

图5. (a) MB-DPP与不同浓度的DPP-IV (0~80 ng/mL)在pH=7.4的10 mmol/L PBS中于37 ℃孵育90 min后的荧光光谱及(b) MB-DPP在691 nm处荧光强度与DPP-IV浓度在0~80 ng/mL范围内的线性关系([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 5. (a) Fluorescence spectra of MB-DPP after the incubation with different concentrations of DPP-IV (0~80 ng/mL) in 10 mmol/L PBS of pH 7.4 at 37 ℃ for 90 min and (b) linear relationship between the fluorescence intensity of MB-DPP at 691 nm and the concentrations of DPP-IV from 0 to 80 ng/mL ([MB-DPP]=10 μmol/L, λex=600 nm)

图6. (a)不同浓度的西他列汀(0~0.01 μmol/L)对80 ng/mL DPP-IV活性的抑制效率及(b)抑制效率与西他列汀浓度(0~0.01 μmol/L)的对数之间的线性关系([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 6. (a) Inhibition efficiency of different concentrations of sitagliptin (0 to 0.01 μmol/L) for 80 ng/mL DPP-IV activity and (b) linear relationship between the logarithm of concentrations of sitagliptin (0 to 0.01 μmol/L) and the inhibition efficiency ([MB-DPP]=10 μmol/L, λex=600 nm)

图7. (a~d)不同浓度的MB-DPP (0~15 μmol/L)与Hep G2细胞孵育30 min的实时荧光成像及(e)图a~d的相对荧光强度(λex=628 nm, λem: 660~720 nm, 标尺: 100 μm)

Figure 7. (a~d) Real-time fluorescence imaging of Hep G2 cells after incubated with MB-DPP (0~15 μmol/L) for 30 min and (e) relative fluorescence intensities of figures a~d (λex=628 nm, λem: 660~720 nm, ruler bar=100 μm)

图8. (a~d) Hep G2细胞与10 μmol/L MB-DPP孵育0~90 min的实时荧光成像、(e)用0.1 μmol/L西他列汀预先处理Hep G2细胞30 min后再将探针与其孵育30 min的实时荧光成像、(f) HEK 293细胞与10 μmol/L MB-DPP孵育30 min的实时荧光成像及(g)图a~f的相对荧光强度(λex=628 nm, λem: 660~720 nm, 标尺: 100 μm)

Figure 8. (a~d) Real-time fluorescence imaging of Hep G2 cells incubated with 10 μmol/L MB-DPP for 0~90 min, (e) fluorescence imaging of Hep G2 cells pre-treated with 0.1 μmol/L sitagliptin for 30 min and then incubated with MB-DPP for 30 min, (f) fluorescence imaging of HEK 293 cells incubated with 10 μmol/L MB-DPP for 30 min, and (g) relative fluorescence intensities of figures a~e (λex=628 nm, λem: 660~720 nm, ruler bar=100 μm)

图9. (a~h)荷瘤小鼠皮下DPP-IV活性的荧光图像(伪彩图)及(i)图a~h相对荧光强度

Figure 9. (a~h) Fluorescence images of subcutaneous DPP-IV activity in tumor-bearing mice (pseudo-color image) and (i) relative fluorescence intensities of figures a~h The mice were incubated with MB-DPP (2.5 mmol/L, 100 μL) for (a) 0, (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0, (f) 2.5, (g) 3.0 and (h) 24 h. For the inhibition experiments, the mice were pretreated with sitagliptin (0.1 mmol/L, 100 μL) for 30 min, and then treated with MB-DPP. λex=600 nm, λem: 680~720 nm

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激活型近红外荧光探针用于肝癌中二肽基肽酶活性检测

A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers

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