Highly sensitive detection of cysteine over glutathione and homo-cysteine: New insight into the Michael addition of mercapto group to maleimide

• Published in: Biosensors & bioelectronics 2017-05, Vol.91, p.553-559
• Main Authors: Chen, Zhaoyang, Sun, Qian, Yao, Yuhua, Fan, Xiaoxiang, Zhang, Weibing, Qian, Junhong
• Format: Article
• Language: English
• Subjects: Animals; Bio-imaging application; Biosensing Techniques - methods; Cattle; Cell Line; Coumarins - chemistry; Cysteine - analysis; Cysteine detection; Fluorescent Dyes - chemistry; Fluorescent probe; Glutathione - analysis; Homocysteine - analysis; Limit of Detection; Maleimide; Maleimides - chemistry; Mice; Models, Molecular; Optical Imaging - methods; Sensing mechanism; Serum Albumin, Bovine - chemistry; Spectrometry, Fluorescence - methods; Sulfhydryl Compounds - analysis
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2017.01.013

A fluorescence “off–on” probe CMP for thiols was designed with coumarin as the fluorophore and maleimide as the receptor. The fluorescence of the coumarin was quenched through photoinduced electron transfer (PET) from the fluorophore to maleimide group. The Michael addition of the mercapto group toward maleimide formed a thioether with relatively weak fluorescence. The intramolecular nucleophilic substitution of amino group in cysteine (Cys) to alkylthio produced a much stronger fluorescent amino adduct, which was supported by UPLC-MS and NMR titration. The above sensing mechanism ensured CMP a highly sensitive probe toward Cys over GSH and Hcy. The fluorescence intensity at 495nm was linear with Cys concentration over the range of 0–10μM with a detection limit of 14nM and a rapid response time of 20min. High selectivity and good competition of the probe toward thiols over other biologically relevant species enabled us to monitor mercapto-containing proteins as well as fluorescence imaging Cys in living cells. A fluorescence “off–on” probe CMP for thiols was designed with coumarin as the fluorophore and maleimide as the receptor. The sensing mechanism of Michael addition followed by intramolecular nucleophilic substitute ensures highly sensitive detection of Cys over GSH and Hcy as well as bioimaging of Cys in living cells. [Display omitted] •An “off-on” probe CMP for selective and sensitive detection of biothiols was designed.•The probe exhibited highly selective response to cysteine over glutathione and homo-cysteine.•CMP could be applied in the detection of mercapto-containing proteins as well as bioimaging of thiols in living cells.•A sensing mechanism of Michael addition followed by intramolecular nucleophilic substitution was proposed.