A new strategy for fluorometric detection of ascorbic acid based on hydrolysis and redox reaction

Article abstract of DOI:10.1039/c4ra04822a

In this study, a near-infrared, indole-cyanine probe, i.e. Cy5-HD, was designed for the detection of ascorbic acid for the first time. The probe has a hydrazone moiety that can be hydrolyzed by Cu²⁺ to induce the fluorescence quenching of Cy5-H

Full text of DOI:10.1039/c4ra04822a

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A new strategy for fluorometric detection of
ascorbic acid based on hydrolysis and redox
Cite this: RSC Adv., 2014, 4, 35112
reaction†
Received 22nd May 2014
Accepted 18th July 2014
*
Yirong Zhao, Yinhui Li, Yijun Wang, Jing Zheng and Ronghua Yang
DOI: 10.1039/c4ra04822a
www.rsc.org/advances
In this study, a near-infrared, indole-cyanine probe, i.e. Cy5-HD, was further application. Thus, it is of great importance to develop
designed for the detection of ascorbic acid for the first time. The probe new uorescent probe to detect AA.
has a hydrazone moiety that can be hydrolyzed by Cu2+ to induce the
Herein, we designed and synthesized a near-infrared, indole-
fluorescence quenching of Cy5-HD. However, the fluorescence cyanine probe, i.e., Cy5-HD, and proposed a new strategy to
quenching ability of Cu2+ will be weakened or even disabled in the detect AA by the degree of repression on uorescence quench-
presence of ascorbic acid, thus the fluorescence recovers. The prac- ing. The sensing mechanism is based on AA inhibiting the
tical applicability of Cy5-HD was tested by the determination of quenching behavior of Cu2+ toward the uorescence of Cy5-HD
ascorbic acid in human urine samples, and satisfactory results were (Scheme 1). The uorescence intensity was efficiently quenched
obtained.
via the Cu2+-promoted hydrolysis of hydrazones21,22 in the
absence of AA, as the new product Cy5–CHO has a low quantum
yield resulting from an electron-withdrawing aldehyde group.
However, in the presence of AA, the efficient inhibition of
uorescence quenching could be ascribed to the reduction of
Cu2+ to Cu+ by AA. As a result, the detection of AA could be
achieved by measuring the saving uorescence. The present
probe functions in a turn-on mode. To the best of our knowl-
edge, this is the rst functional uorescent probe based on the
combination of an incidental hydrolysis reaction and a redox
reaction.
The synthesis of Cy5-HD is outlined in Scheme S1.† Ben-
zohydrazide (compound 2) was synthesized through the
transesterication of ethyl benzoate (compound 1) with
hydrazine hydrate in EtOH solution in 85% yield. Triformyl-
methane (compound 3) and 3-formyl-5-(1,3,3-trimethylindolin-
2-ylidene)penta-1,3-dienyl-1,3,3-trimethyl-3H-indolium iodide
(compound 4) were prepared according to a published proce-
dure.23 Then, the synthesis of probe Cy5-HD was preformed via
the Knoevenagel condensation of compound 2 and 4 with a
yield of 65%.
Ascorbic acid (AA), an important anti-oxidant, a coenzyme
factor as well as a component of enzymes related to neuro-
transmitters in the human body, plays a key role in health care
by promoting the development of healthy cells1,2 and down-
regulating the synthesis of cholesterol and triglyceride.3 The
deciency of ascorbic acid will cause scurvy or cardiovascular
diseases such as stroke. However, excessive intake of AA can
also result in diarrhoea, hyperacidity and kidney calculi.4
Therefore, it is important to develop a rapid and sensitive
method for monitoring AA in biological samples.
In the past few decades, several techniques have been
employed to detect AA, including chromatography,5 spectro-
photometry,6,7 optical sensor,8,9 chemiluminescence,10,11 electro-
chemical method12–16 and colorometric methods,17,18 the
majority of which are based on the electrochemical method
because of its low cost and high sensitivity. However, the
problems of weak reproducibility, high background and
complex preparation of electrodes prompted us to develop new
methods. Fluorescent detection methods possess advantages
such as simple operation, high sensitivity and rapid response.
Recently, Yang19 and Long20 took advantage of the uorescence
of QDs to detect AA; however, the toxicity of QDs prevented its
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, Hunan University, Changsha, China. E-mail:
yinhuili16@hnu.edu.cn; Fax: +86-731-88822523; Tel: +86-731-88822523
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra04822a
Scheme 1 Schematic illustration of fluorescence detection of AA by
using a hydrolysis reaction and a redox reaction.
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The UV/visible absorption and emission spectra of Cy5-HD
were measured in the optimized condition of DMSO–PBS
mixture (9 : 1, v/v, 20 mM, pH 7.4) in which Cy5-HD exhibited
the absorption (lmax) and emission (lmax) maxima at 628 nm
and 660 nm, respectively (Fig. S1–4†). Prior to applying the
probe in the uorescence sensing of AA, the effects of different
Cu2+ concentrations on the uorescence of Cy5-HD were rst
studied. Fig. 1 displays the typical uorescence spectra of Cy5-
HD as a function of the concentration of Cu2+ from 0 to 2.0 ꢀ
10ꢁ5 M. It is easily seen that the uorescence intensity of Cy5- Fig. 2 (A) Fluorescence emission spectra (lex ¼ 628 nm) of Cy5-HD
(1.0 mM) in DMSO solution. Pink: Cy5-HD, blue: Cy5-HD + 10 eq. Cu2+
,
HD was quenched gradually upon the addition of increasing
concentrations of Cu2+. With the concentration of Cu2+
increased up to 10.0 equiv. of Cy5-HD, an 8.2-fold uorescence
quenching was observed and a further increase in Cu2+
concentration caused no further change. However, it did not
generate quenching effect similar to Cy5-HD in the presence of
other metal ions, such as Ni2+, Zn2+, Fe2+, Fe3+, Al3+, Cd2+, Ca2+,
Mg2+ and K+, indicating that Cy5-HD is highly and selectively
red: Cy5-HD + 10 eq. Cu2+ + H2O (100 ml), black: Cy5-HD + Cu2+
+
H2O + EDTA. (B) Fluorescence spectra of Cy5-HD (1.0 mM) in DMSO–
PBS (9 : 1 v/v, PBS, 20 mM, pH 7.4). Red: Cy5-HD, blue: Cy5-HD + 20
eq. AA, green: Cy5-HD + 20 eq. AA + 10 eq. Cu2+, black: Cy5-HD + 10
eq. Cu2+
.
quenched by Cu2+. In particular, there was nearly no difference at 412.2422, which corresponds well with the expected molec-
in the uorescence spectra upon the addition of Cu+, which ular weight of the hydrolyzed product Cy5–CHO. It was further
provided a feasible basis for sensing AA as per our proposed conrmed by 1H NMR as shown in Fig. S8,† a characteristic
strategy.
peak of –CHO group at d 10.23 ppm appeared to show the
The strong quenching ability of Cu2+ on Cy5-HD in DMSO– existence of an aldehyde group.
H2O (9 : 1, v/v) may be explained by the hydrolysis of Cu2+. From
Following the detection mechanism, AA can reduce Cu2+ to
Fig. 2A, in pure DMSO solvent, the uorescence spectrum of Cu+, leading to the inhibition of the hydrolysis reaction;
Cy5-HD did not undergo any changes when Cu2+ was added. consequently, weaken the quenching ability of Cu2+. Next, the
However, when 100 ml H2O was then added into the solution, feasibility of Cy5-HD for AA was veried as shown in Fig. 2B. As
the uorescence of Cy5-HD was obviously quenched, which was expected, Cy5-HD with 10.0 equiv. Cu2+ in DMSO–H2O solution
also in good accordance with the time scan experiment showed very weak uorescence. However, when 20.0 equiv. AA
(Fig. S5†), proving that the quenching behavior of Cu2+ toward was added to the solution containing Cy5-HD in advance, hardly
Cy5-HD needs the assistance of H2O. However, the addition of a did the uorescence change on further addition of 10.0 equiv.
Cu2+ complexing agent EDTA could not cause a recovery in the Cu2+ because Cu2+ had been reduced to Cu+ in the presence of
uorescence, which indicates the quenching behavior might excess AA. This demonstrated that Cu2+ has the priority to react
have resulted from the hydrolysis reaction but not the para- with AA rather than Cy5-HD and thus inhibited the uorescence
magnetism character of Cu2+. Moreover, the possibility of Cy5- quenching. It was also conrmed by the absorption spectra
HD self-hydrolyzing could be excluded due to its high stability (Fig. S9†), in which absorption of Cy5-HD at 628 nm increased
in H2O (Fig. S6†). This quenching mechanism was studied by with increasing amount of AA. These together proved that it was
mass spectrometry (Fig. S7†). In a system without H2O, the feasible to construct a uorescence sensor for AA detection
molecular ion peak of Cy5-HD was highly stable against Cu2+
However, when H2O was added, the molecular ion peak of Cy5-
.
based on our proposed mechanism.
To reveal the full scope of AA response on Cy5-HD, the
HD disappeared, accompanied by the appearance of a new peak quantitative detection of AA was carried out in DMSO–PBS
mixture (9 : 1, v/v, 20 mM, pH 7.4). Fig. 3 reveals that Cy5-HD
can be developed as a uorescent probe for AA. The free Cy5-HD
exhibited a strong uorescence emission peak at 660 nm.
However, upon addition of different concentrations of AA into
the Cy5-HD solution in the presence of 10.0 equiv. Cu2+, the
uorescence intensity at 660 nm showed various degrees of
quenching. With the increasing AA concentration, the
quenching was better inhibited, giving stronger uorescence
intensity. This conrmed that the addition of AA could inhibit
the hydrolysis reaction between Cy5-HD and Cu2+. Moreover,
the uorescence ratio F/F0 at 660 nm showed a linear relation in
the range of 0.1–4.0 mM of AA (Fig. 3A). The regression equation
Fig. 1 (A) Fluorescence emission spectra (lex ¼ 628 nm) of Cy5-HD
was F/F0 ¼ 1.283 + 8.526C (mM) with a linear coefficient efficient
of 0.9972. F is the uorescence intensity of Cy5-HD at 660 nm
upon addition of AA and Cu2+ one aer another, and F0 is
uorescence intensity of Cy5-HD at 660 nm in the presence of
(1.0 mM) in the presence of increasing amounts of Cu2+ (0, 2.0, 3.0, 4.0,
5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0 mM) in DMSO–PBS (9 : 1 v/v, 20
mM, pH 7.4). (B) The fluorescence intensity of Cy5-HD observed at
660 nm upon addition of different metal ions.
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Conclusions
In summary, a new method with high selectivity and sensitivity
has been developed based on the incidental hydrolysis reaction
and redox reaction. This method can be easily adopted to other
cyanine-based uorescent dyes to develop different uorescent
probes for other analytes. The present probe possesses an
extremely low detection limit, as low as 26 nM, and displays fast
response by the degree of uorescence recovery. Furthermore,
this probe is also proved useful for the determination of AA in
human urine samples with satisfactory results. We anticipate
that this novel method will be helpful to expand the uores-
cence detection technique for food, pharmaceutical, and blood
analysis, where analysis of redox species like ascorbic acid, as
well as it could be essential for the examination of contents in
food and drug along with the monitoring of human health.
Fig. 3 (A) Fluorescence emission spectra of Cy5-HD (1.0 mM) upon
addition of various concentrations of AA (0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,
8.0, 9.0, 10.0 mM) and Cu2+ (10.0 mM) one after another in DMSO–PBS
(9 : 1 v/v, 20 mM, pH 7.4). Arrow shows fluorescence enhancement
with increasing concentration of AA. Inset shows fluorescence ratio of
F/F0 as a function of AA concentration. (B) The selectivity of this system
upon addition of 10.0 mM Glu, urea, L-Cys, UA, DA, AA, mix,
respectively.
Cu2+. The detection limit that was taken to be 3 times the
standard derivation of a blank solution, and it was estimated to
be 26 nM. The results suggested that the proposed approach
was potentially appropriate for the quantication of AA in real
samples.
Acknowledgements
We are grateful for the nancial support from the National
Natural Science Foundation of China (21305036, 21135001 and
J1103312), the Foundation for Innovative Research Groups of
NSFC (21221003), and the Fundamental Research Funds for the
Central Universities.
Selectivity is an important property for probe application in
real samples. The selectivity of Cy5-HD was studied by exam-
ining other analogues such as D-glucose (Glu), uric acid (UA),
urea, dopamine (DA) and L-cysteine (Cys). As shown in Fig. 3B,
almost no enhancement was seen in the presence of Glu, UA,
urea, and Cys, even at a high concentration. Though dopamine
Notes and references
1 M. W. Davey, M. V. Montagu, D. Inze, M. Sanmartin,
A. Kanellis, N. Smirnoff, I. Benzie, J. J. Strain, D. Favell and
J. J. Fletcher, J. Sci. Food Agric., 2000, 80, 825–860.
2 M. Levine and D. Downing, J. Nutr. Environ. Med., 1992, 3,
361–362.
can partly deteriorate the hydrolysis of Cy5-HD caused by Cu2+
,
its efficiency was pale compared to that of AA. The results
revealed that this response of Cy5-HD was highly selective
toward AA compared with other interfering agents.
With excellent selectivity and sensitivity in an optimized
condition, the proposed method was employed for AA detection
in urine samples. The samples were voluntarily provided by
healthy people and stored at 4 ꢂC. Before uorescence detec-
tion, samples were centrifuged and diluted 10-fold with a DMSO
solution, and then Cy5-HD and 10.0 eq. Cu2+ were added. The
results are shown in Table 1, which revealed a high consistency
in the determination of AA in urine samples by the proposed
method and the standard colorimetric method using 2,4-dini-
trobenzene hydrazine as substrate,24 demonstrating the excel-
lent performance of this probe in practical applications.
3 A. Bossi, S. A. Piletsky, E. V. Piletska, P. G. Righetti and
A. P. F. Turner, Anal. Chem., 2000, 72, 4296–4300.
4 L. K. Massey, M. Liebman and S. A. Kynast-Gales, J. Nutr.,
2005, 135, 1673–1677.
´
´
´
´
5 L. Novakova, D. Solichova, S. Pavlovicova and P. Solich, J.
Sep. Sci., 2008, 31, 1634–1644.
˘
¨
¨
¨
6 R. Apak, K. Guclu, M. Ozyurek and B. Bekta¸soglu, Anal.
Chim. Acta, 2007, 588, 88–95.
7 Z. Aydogmusx and S. M. Cetin, Turk. J. Chem., 2002, 26, 697–
704.
8 N. Li, Y. Li, Y. Han, W. Pan, T. Zhang and B. Tang, Anal.
Chem., 2014, 86, 3924–3930.
9 H. W. Park, S. M. Alam, S. H. Lee, M. M. Karim,
S. M. Wabaidur, M. Kang and J. H. Choi, Luminescence,
2009, 24, 367–371.
Table 1 Detection of AA in urine samples
Proposed
10 S. Zhao, Y. Huang and Y. M. Liu, J. Chromatogr. A, 2009, 1216,
6746–6751.
Sample
AA added/mM
SM/mM
method/mM
Recovery/%
1
2
3
4
0
1
2
3
—
—
—
11 N. Inoue, K. Akasaka, H. Arimoto and H. Ohrui, Biosci.,
Biotechnol., Biochem., 2006, 70, 1517–1520.
12 S. Y. Qiu, S. Gao, L. D. Xie, H. Q. Chen, Q. Liu, Z. Y. Lin,
B. Qiu and G. N. Chen, Analyst, 2011, 136, 3962–3966.
13 M. N. Zhang, K. Liu, L. Xiang, Y. Q. Lin, L. Su and L. Q. Mao,
Anal. Chem., 2007, 79, 6559–6565.
0.99 ꢃ 0.5a
1.89 ꢃ 0.16
2.95 ꢃ 0.14
0.95 ꢃ 0.15
2.1 ꢃ 0.25
2.8 ꢃ 0.12
95.0
105.0
93.3
a
Mean ꢃ standard deviation of three determinations. SM indicates the
2,4-dinitrobenzene hydrazine method.
35114 | RSC Adv., 2014, 4, 35112–35115
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14 N. Nasirizadeh, Z. Shekari, H. R. Zare, M. R. Shishehbore, 20 C. Kong, D. W. Li, Y. Li, R. Partovi-Nia, T. D. James,
A. R. Fakhari and H. Ahmar, Biosens. Bioelectron., 2013, 41,
608–614.
15 M. H. Pournaghi-Azar, H. Dastangoo and R. Fadakar,
Electroanalysis, 2010, 22, 229–235.
16 J. B. Raoof, R. Ojani, H. Beitollahi and R. Hossienzadeh,
Electroanalysis, 2006, 18, 1193–1201.
17 A. Wahba, D. A. Yassa and R. S. Labir, Analyst, 1974, 99, 397–
402.
Y. T. Long and H. Tian, Analyst, 2012, 137, 1094–1096.
´
¨
21 J. Kovacs, T. Rodler and A. Mokhir, Angew. Chem., Int. Ed.,
2006, 45, 7815–7817.
22 M. Kumar, N. Kumar, V. Bhalla, P. R. Sharma and T. Kaur,
Org. Lett., 2012, 14, 406–409.
23 X. Peng, Z. Yang, J. Wang, J. Fan, Y. He, F. Song, B. Wang,
S. Sun, J. Qu, J. Qi and M. Yan, J. Am. Chem. Soc., 2011,
133, 6626–6635.
18 R. Q. Thompson, Anal. Chem., 1987, 59, 1119–1121.
19 S. S. Yang, C. L. Ren, Z. Y. Zhang, J. J. Hao, Q. Hu and
X. J. Chen, J. Fluoresc., 2011, 21, 1123–1129.
24 J. H. Roe and C. A. Kuether, J. Biol. Chem., 1943, 147, 399–
407.
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