Dyes and Pigments
Discriminative detection of cysteine/homocysteine and glutathione in HeLa
cells by dual-channel fluorescent probe
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Yu Chen, Yumin Wang, Xiang Hua Wu , Bo Liu , Jun Feng Zhang
College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming, 650500, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play important roles in various
physiological and pathological processes. However, it is still a great challenge to selectively detect Cys/Hcy and
GSH in biological samples due to the similarity of structures and re-activities of these biothiols. In this paper, a
new dual-channel near infrared-7-nitro-1,2,3-benzoxadiazole (NIR-NBD) fluorescent probe has been developed
for the discriminative detection of Cys/Hcy and GSH. The probe is composed of two functional parts: a near-
infrared dye of dicyanoisophorone, as fluorophore precursor, and NBD as both fluorophore precursor and
sensing group. It was observed that NIR-NBD probe displayed two different fluorescent signals in response to
biothiols, green-red for Cys/Hcy and red for GSH. It also showed a near infrared emission (756 nm), low
detection limit, fast response, high stability, selectivity and sensitivity. Furthermore, NIR-NBD was effectively
applied for the discriminative detection of Cys/Hcy and GSH in HeLa cells.
Fluorescent probe
Green-red signals
Biothiols
Cellular imaging
1
. Introduction
Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), the three
[15]. Fluorescent probes have attracted great attention due to its
fast-response, simple operation, non-invasion, good selectivity and high
sensitivity characteristics [16–22]. In the past decade, many fluorescent
probes have been developed for the detection of biothiols in living cells
[23,24]. However, it is still a great challenge to achieve selective
detection of Cys/Hcy and GSH in biological samples due to the similarity
of structures and reactivities of these biothiols [25]. Recently, Tu et al.
developed a fluorescent probe for the discriminative detection of
Cys/Hcy and GSH based on blue-green emission channels from
combining NBD (7-nitro-1,2,3-benzoxadiazole) with coumarin dye [26].
However, a disadvantage of the coumarin dye is its relatively short
emission wavelength (about 460 nm), which can be exposed to multi-
factorial interference by other compounds in biological materials or
samples. In order to achieve selective or discriminative detection of
Cys/Hcy and GSH, it was of interest to find a near infrared dye with
emission wavelength greater than 700 nm.
common biothiols in biosystems, play important roles in various phys-
iological and pathological processes [1,2]. Cys, a proteinogenic amino
acid, is a precursor of GSH, and participates in many enzymatic reactions
due to its highly reactive sulfhydryl group [3]. The abnormal concen-
tration of Cys can cause many diseases such as retarded growth, skin
lesions, liver damage, edema and Parkinson’s diseases [4–6]. Hcy, a
non-protein amino acid, is a precursor of Cys, and is biosynthesized by
transsulfuration or methionine-conserving pathways. Therefore, over-
concentration of Hcy has been associated with many diseases, such as
cardiovascular disease, neuropsychiatric illness, Alzheimer’s disease
and cancer [7–9]. GSH, the most abundant intracellular thiols, is a vital
endogenous antioxidant, which plays an important role in anti-oxidative
stress, signal conduction and regulation of apoptosis [10,11]. Abnormal
concentration of GSH has been associated with many diseases, such as
leucocyte loss, liver damage, AIDS, Alzheimer’s disease and cancer
In this study, we designed and synthesized a new dual-channel,
fluorescent NIR-NBD probe for the discriminative detection of Cys/
Hcy and GSH in HeLa cells. The probe is composed of two functional
parts, including near infrared dye of dicyanoisophorone as fluorophore
precursor and NBD as both fluorophore precursor and sensing group
[
12–14].
The discriminative detection of different biothiols is of great interest
in terms of early diagnostics and pathological analysis of some diseases
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Corresponding author.
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* Corresponding author.
Received 11 August 2020; Received in revised form 12 November 2020; Accepted 17 November 2020
Available online 25 November 2020
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143-7208/© 2020 Elsevier Ltd. All rights reserved.