Hg2+ wettability and fluorescence dual-signal responsive switch based on a cysteine complex of piperidine-calix[4]arene

Article abstract of DOI:10.1039/c3ob41794h

The recognition of the mercury(ii) ion (Hg²⁺) is essential because of its extreme toxicity in the environment and food. Hence we reported a novel cysteine (Cys) complex of piperidine-calix[4]arene (L) as a convenient and effective dual-signal responsive switch for Hg²⁺. This switch system exhibited excellent selectivity toward Hg²⁺ by fluorescence (FL), ¹H NMR spectroscopy and the atomic force microscopy (AFM). More importantly, the Hg²⁺-responsive switch had an important and potential application by water contact angle (CA) on a functional micro-nano silicon surface, including intelligent microfluidic and laboratory-on-chip devices, controllable drug delivery, and self-cleaning surfaces.

Full text of DOI:10.1039/c3ob41794h

Organic &
Biomolecular Chemistry
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Hg2+ wettability and fluorescence dual-signal
responsive switch based on a cysteine complex of
Cite this: Org. Biomol. Chem., 2013, 11,
8262
piperidine-calix[4]arene†
Xiaoyan Zhang, Haiyang Zhao, Xianliang Cao, Ningmei Feng, Demei Tian and
Haibing Li*
The recognition of the mercury(II) ion (Hg2+) is essential because of its extreme toxicity in the environ-
ment and food. Hence we reported a novel cysteine (Cys) complex of piperidine-calix[4]arene (L) as a con-
venient and effective dual-signal responsive switch for Hg2+. This switch system exhibited excellent
selectivity toward Hg2+ by fluorescence (FL), 1H NMR spectroscopy and the atomic force microscopy
(AFM). More importantly, the Hg2+-responsive switch had an important and potential application by
water contact angle (CA) on a functional micro–nano silicon surface, including intelligent microfluidic
and laboratory-on-chip devices, controllable drug delivery, and self-cleaning surfaces.
Received 3rd September 2013,
Accepted 3rd October 2013
DOI: 10.1039/c3ob41794h
www.rsc.org/obc
and a hydrophilic character have aroused great attention due
to a wide range of potential applications, including intelligent
Introduction
Mercury (Hg) is a non-essential element of living, which is microfluidic and laboratory-on-chip devices, controllable drug
associated with chemical substances: metallic mercury, in- delivery, and self-cleaning surfaces.5 Many external stimuli,
organic and organic mercury. Among them, inorganic mercury such as pH, light, temperature, electric potential and solvents,
such as mercuric sulfide, mercuric chloride and mercuric exhibit excellent and reversible wettability on the functional
nitrate can easily pass through the skin, respiratory, and surfaces.6 Among them, the design and synthesis of response
gastrointestinal tissues, which leads to DNA damage, mitosis molecules play a key role in constructing wettable functional
impairment, and permanent damage to the central nervous surfaces. As is known to all, the calixarene with a “cup-like”
system.1 So the mercury(II) ion (Hg2+) is one of the most shape as the third generation of supramolecules has an impor-
dangerous and ubiquitous pollutants, which raises serious tant application, because its cavity is adjustable by varying the
environmental and health concerns.2 At present, the “on–off” shape and size or by modifying the substituents of the upper
fluorescent probe is an extraordinarily common method to and lower rims.7 They are widely applied as complexing
recognize Hg2+. For example, Mandal et al. described a novel reagents for many metals, organic molecules, and drugs.8 Just
imine-based receptor as a turn-on fluorescence response for recently, we reported a switchable wettability sensor for ion
the detection of Hg2+ in aqueous media.3 Wu et al. have pairs based on calix[4]azacrown by the click reaction on a
reported a novel “off–on” fluorescent probe based on rhod- silicon surface.9 Compared with responsive wettability surfaces
amine for the detection of Hg2+ in aqueous solution.4 However, for recognizing small molecules, surfaces for recognizing
all of the above fluorescent switches were just a single output cations have been barely reported. This may be because the
signal in the solution phase. Accordingly, the development of a cations themselves are without hydrophilic or hydrophobic
Hg2+ dual-signal responsive switch has attracted great interest groups. Considering that, we can detect cations through the
because of potential applications as chips or materials which synergy between functional surfaces based on calixarenes and
could detect environmental pollution.
some substances with hydrophilic and hydrophobic groups.
Wettable responsive switches on the functional surface that Cys is a heavy metal detoxification agent due to its strong
can be reversibly changed between a hydrophobic character binding affinity toward Hg2+.10 The affinity is closely related
with the interaction of Hg2+ and sulfhydryl (–SH) groups.
Therefore, we designed a wettable responsive switch based on
Key Laboratory of Pesticide and Chemical Biology (CCNU), Ministry of Education,
a calixarene receptor and Cys complex for the recognition of
College of Chemistry, Central China Normal University, Wuhan, 430079,
Hg2+. As a consequence of its outstanding properties, the
P. R. China. E-mail: lhbing@mail.ccnu.edu.cn; Fax: (+86) 27 67866423
Hg2+-responsive switch based on the calixarene and Cys
†Electronic supplementary information (ESI) available: Characterized (EIS, IR,
complex has formed attractive applications in many fields,
CA); copies of NMR of new compounds. See DOI: 10.1039/c3ob41794h
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especially on a silicon surface. To the best of our knowledge, a recognition for Hg2+ was carried out using an excitation wave-
Hg2+ wettability and fluorescence dual-signal responsive length of 350 nm. The Cys receptor property of L was explored
switch has been largely unexplored.
by fluorescence experiments and absorption titrations of the
In order to achieve this goal, we synthesized a novel deep [L + Cys] complex in CH3CN–H2O solution with 7 different
cavity of calix[4]arene with piperidine groups as the upper rim metal ions, including heavy metal ions, viz., Hg2+, Pb2+, Cd2+,
(L). On the basis of the interaction between the piperidine Cu2+, Ni2+, Zn2+ and Ba2+ with nitrates as counter anion. Since
groups and Cys, we obtained an [L + Cys] complex with a con- both the receptor L and Cys had no fluorescence, the reaction
jugation ratio of 1 : 1, by FL, 1H NMR spectroscopy and density of dansyl chloride and Cys produced sulfa-drug derivatives
functional calculations (DFT) studies. Further, the in situ [L + (Cys-F) which had strong and stable fluorescence signals, as
Cys] complex was added to 7 important heavy metal ions, viz., reported in the literature.14 The fluorescence of Cys-F
Hg2+, Pb2+, Cd2+, Cu2+, Ni2+, Zn2+ and Ba2+. From this it was weakened with the addition of the receptor L in CH3CN–H2O
then found that this complex acted as a recognition ensemble (v/v, 1/1) because of their interaction. This indicated that the
1
toward Hg2+, by switch-off fluorescence, H NMR spectroscopy [L + Cys] complex was successfully obtained. Subsequently, the
and AFM. Even further, the Hg2+-responsive switch based on selective recognition of 7 different heavy metal ions was moni-
the [L + Cys] complex had been successfully applied on a func- tored by FL. Among all of these ions, only Hg2+ showed a
tional micro–nano silicon surface by the simple click reac- fluorescence enhancement with the addition of increasing
tion.11 Moreover, the [L + Cys] complex could act as a concentrations of this ion, and the enhancement approached
convenient and effective wettability switch for Hg2+ by its (56.6 0.5)-fold at saturation compared with the other metal
contact angle (CA) measurement on the silicon surface.
ions when the titration was carried out in CH3CN–H2O (v/v,
1/1) (Fig. 1a and 1b). A plot of the relative fluorescence inten-
sity (I/I0) versus the molar ratio (n[Hg2+]/n[L-Cys-F]) suggested a
stoichiometric ratio of 1 : 1 between the [L + Cys] complex and
Hg2+. Furthermore, the binding stoichiometry of the complex
formed between L and Cys was also 1 : 1 from Job’s plot by
Results and discussion
The synthetic strategy for the complex L was depicted in
Scheme 1. Calix[4]arene (C4DT) and sodium methylate were
stirred and refluxed in acetonitrile for 0.5 h, and then propar-
gyl bromide was added.12 The mixture was stirred at room
temperature for 8 h, and the crude product was purified by
column chromatography to afford propinyl-calix[4]arene
(C4AM) (Fig. S1†) in 85% yield. Subsequently, C4AM, piper-
idine, formaldehyde and acetic acid were stirred in THF at
room temperature for 48 h.13 The evaporated crude product
was then purified by column chromatography to give a white
powder (L) in 86% yield. Complex L was totally characterized
by NMR (Fig. S2 and S3†) and ESI-MS (Fig. S4†). As shown in
Fig. S2†, the 1H NMR spectrum of L exhibited a pair of
doublets for the bridging methylene groups at 4.06 and
4.48 ppm, a single peak for the alkynyl group at 2.61 ppm,
and then two singlets for the piperidine groups at 1.40 and
1.53 ppm. All of these indicated the cone conformation of L.
The 13C NMR spectrum data further corroborated the cone
conformation of L in the presence of a peak for the methylene
resonances. At the same time, the m/z peak of L was at 753 in
the ESI mass spectrum.
ultraviolet-visible (UV) spectrum, which had
a peak at
286.5 nm with a molar fraction of 0.5 (Fig. S7†). However,
when this was titrated with the other metal ions, no fluore-
scence change was observed (Fig. 1c and Fig. S6†). These
results clearly suggested that the [L + Cys] complex could selec-
tively recognize Hg2+. The sensitivity of the [L + Cys] complex
for Hg2+ had been evaluated by measuring the lowest detect-
able concentration. The fluorescence titration was carried out
between the [L + Cys] and Hg2+ by maintaining a 1 : 1 ratio.
This showed that 2.00 μM of Hg2+ is the lowest detectable con-
centration in CH3CN–H2O (Fig. S5†).
To further study the interaction of L with Cys carried out by
Hg2+, a fluorescence cycling experiment was done using an
excitation wavelength of 350 nm (Fig. 2a). Under the above
To provide support for the binding between L and Cys,
and then the removal of Cys from the [L + Cys] complex by
Hg2+, some verifying experiments were done in the solution
phase. First of all, a fluorescence experiment of the selective
Fig. 1 (a) FL spectra for the [L + Cys] complex with 7 different metal ions,
which showed the highly selective recognition for Hg2+ based on the [L + Cys]
complex in CH3CN–H2O (v/v, 1/1). Note: all metal salts were nitrates. (b) FL vari-
ation [ΔI = I0/(I0 − I)] (where I0 is the FL intensity of L-Cys-F) histogram for the
[L + Cys] complex with 7 different metal ions at 350 nm. More clearly, this also
showed the highly selective recognition for Hg2+ based on the [L + Cys]
complex. (c) Relative fluorescence titration intensity (I/I0) versus molar ratio
(n[cation]/n[L-Cys-F]), λex = 350 nm. It suggested a stoichiometric ratio of 1 : 1
between the [L + Cys] complex and Hg2+, and no fluorescence change was
noted when this was titrated with the other metal ions. Note: others stood for
Scheme 1 Synthesis of L.
Pb2+, Cd2+, Cu2+, Ni2+, Zn2+ and Ba2+
.
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spectrum (Fig. S9†) revealed intermolecular nuclear Overhau-
ser effects (NOEs) between the piperidine groups of L and
the –CH2 groups of CS, which showed that the –NH2 moiety of
CS had an interaction with the piperidine groups of L instead
of the sulfhydryl (–SH) moiety of CS.
To understand how the microscopic structural features of L
differed from its in situ complex and the complex with Hg2+,
atomic force microscopy (AFM) techniques were used. AFM
images of L, the [L + Cys] complex and the complex with Hg2+
demonstrated that the particles were well distributed over the
mica sheet. The morphological features of L alone and in the
presence of Cys were quite different in terms of size. Complex
L alone formed spherical particles of 270–370 nm in size.
However, the [L + Cys] complex was 130–230 nm in size. Fur-
thermore, the [L + Cys] complex with Hg2+ produced uniform
spherical particles of 230–330 nm. The spherical particle sizes
of the [L + Cys] complex with Hg2+ were similar to those of
L only, which also showed that Cys was removed from the
[L + Cys] complex by Hg2+ (Fig. 4).
Further, the binding of L and Cys was also examined by
computational calculations at the B3LYP/6-31G(d) level using
Gaussian03.16 Partial hydrogen atoms of the host and guest
were omitted for clarity. The host L was yellow, the guest Cys
was purple, oxygen atoms were red, nitrogen atoms were blue,
sulfur atoms were dark yellow, and hydrogen atoms were
white. The results from the molecular mechanics calculations
were generally consistent with FL, 1H NMR and AFM experi-
mental results. Fig. 5 shows the top and side views of the
optimized structure of the host–guest complex. According to
the calculation formula ΔE(binding energy) = E(host + guest) −
[E(host) + E(guest)], the binding energy values for the two pro-
cesses in which the –SH groups of Cys were placed in the
bottom and upper of L were −0.0183468 a.u. and −40.3052274
a.u. (Fig. S10†), respectively. Comparing above binding ener-
gies, the stable structure was that of the carboxyl inserted into
the lower rim of the cavity and the sulfhydryl exposed outside
the cavity of L (Fig. 5).
Fig. 2 (a) The corresponding cycling experiment of the fluorescence. (b) The
linear graph of the fluorescence cycle. They showed the fluorescence recovery
for 6 switching cycles. Moreover, Cys was carried out by Hg2+ from the [L + Cys]
complex in the solution phase.
conditions, the same equiv. of Hg2+ was added for its inter-
action with Cys-F, which showed the fluorescence recovery over
6 switching cycles (Fig. 2b). As there were more errors since
the volume of the mixture was gradually increasing, a better
fluorescence recovery could not be achieved later.
To obtain further insight into the interaction, 1H NMR
experiments were completed. The binding of Cys and L
1
was studied by H NMR by keeping a fixed concentration of L
and Cys to reach up to 2 equiv., respectively. During the study,
significant changes which mainly included the chemical shifts
of the hydrogen atoms from the piperidine groups were
observed in the 1H NMR spectra of L upon addition of Cys.
The result also indicated that the [L + Cys] complex was suc-
cessfully formed. 1H NMR was carried out to support the
removal of Cys from the [L + Cys] complex to generate the free
L. For this purpose, cysteamine (CS) was used instead of Cys
because of the poor solubility of the latter when taken in
DMSO-d6.15 During the experiment, the signals corresponding
to the complex started to disappear whereas those corres-
ponding to the free L started to appear as the concentration of
CS was increased (Fig. 3), and thus it clearly supported the
removal of Cys from the complex. Further, a 2D NOESY
Fig. 4 AFM images and corresponding particle size distribution plots of (a) L,
(b) [L + Cys] complex, and (c) {[L + Cys] + Hg2+}. Complex L alone formed spheri-
cal particles of 270–370 nm in size; however, the [L + Cys] complex particles
were 130–230 nm in size. Furthermore, the [L + Cys] complex with Hg2+ pro-
duced uniform spherical particles of 230–330 nm. This size range was just
similar to L alone, which also showed that Cys was removed by Hg2+ from the
[L + Cys] complex.
Fig. 3 1H NMR spectroscopy of (a) L, (b) [L + CS] complex, (c) [L + CS] complex
with Hg2+ (cycling experiment of 1H NMR spectroscopy), which showed the
interaction L with Cys carried out by Hg2+ (DMSO, 600 MHz, 298 K). Note:
cysteamine (CS) was used instead of Cys because of the poor solubility of the
latter when taken in DMSO-d6.
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Fig. 5 (a) The top view on the optimized structure of the [L + Cys] complex. (b)
The side view on the optimized structure of the [L + Cys] complex. The stable
structure was that the carboxyl inserted into the lower rim of the cavity and the
sulfhydryl exposed outside the cavity of L.
Fig. 7 (a) Clicking L onto the silicon surface, which indicated that piperidine-
calix[4]arene (L) was successfully modified on the silicon surface by the click
reaction. (b) Constructing the [L + Cys] complex, which indicated a significant
change in the CA between hydrophobicity and hydrophilicity.
the rough silicon surface before and after modification with L,
the results indicated that L was successfully modified on the
silicon surface. Moreover, the water-drop profiles showed
different CAs on bare silicon wafer and the L-SAMs surface
(Fig. S12†). This also proved the above conclusion.
Fig. 6 The formation process of the functional L-SAM, which indicated that L
was successfully modified on a silicon surface by the click reaction.
In view of the extraordinary structure of L, we designed a
Cys complex of piperidine-calix[4]arene on the functional
silicon surface, and then the above metal ions were selected as
guests because of the strong interaction between some metal
ions and Cys. This process from hydrophilicity to hydrophobi-
city was proved by CA. Cys aqueous solution was added drop-
wise onto the modified silicon surface; 15 min later, the CA
was 47.3 2.0°, then the [L + Cys] complex was constructed
(Fig. 7b). The complex acted as a recognition ensemble toward
Hg2+ against Pb2+, Cd2+, Cu2+, Ni2+, Zn2+ and Ba2+. The
complex showed a wettability response and selectivity for Hg2+
by CA measurements (Fig. 8a). At the same time, the corres-
ponding CA variation [ΔCA = (CA0 − CA)/CA0] (CA0 stood for
contact angle of the [L + Cys] complex) histogram for the
[L + Cys] complex with 7 different metal ions revealed a highly
selective switch for Hg2+ (Fig. 8b). In addition, the L-SAMs
surface which was immersed in Cys aqueous solution and
sequentially reverted to Hg2+ aqueous solution could switch
More importantly, the Hg2+-responsive switch had an
important and potential application by measurement of the
contact anlge (CA) on a functional micro–nano silicon surface.
Because micro/nano structured and functional silicon surfaces
can amplify the signal output with respect to the alteration of
wettability,17 it could reversibly switch Cys and Hg2+ between a
hydrophobic character and a hydrophilic character. At first,
the L-SAMs (self-assembled monolayers) were constructed by
the click reaction between the Si–N3 SAMs and L in ethanol
(Fig. 6 and Fig. S11†).
The properties of the L-immobilized silicon surfaces were
studied by CA. The bare rough silicon wafer was superhydro-
philic (5.0 2.0°) (Fig. S12†) and the CA for the Si–N3-immobi-
lized surface was 74.6
2.0°. After the click reaction, the
L-SAMs surface became hydrophobic (122.6 2.0°) (Fig. 7a)
because of the hydrophobic properties of the piperidine moi-
eties within L. Therefore, we concluded that the functional
L-SAMs were constructed perfectly.
reversibly between hydrophobicity (122.6
2.0°) and hydro-
philicity (47.3 2.0°). According to these results, a cycling
The L-modified silicon surfaces were characterized by X-ray
photoelectron spectroscopy (XPS) and CA measurements
(Fig. S13†). The concentration of carbon had a significant
increase and the concentration of oxygen had an obvious
decrease in the XPS-derived atomic concentration analysis for
the SAMs after the click reaction. This indicated that the click
reaction had occurred.
Through scanning electron microscopy (SEM), the rough
substrate exhibited a regular array of square silicon micro-
convexes (bright squares), and then the L-modified substrate
showed a thin film on the surface. Comparing SEM images of
experiment (Fig. S14†) between Cys and Hg2+ on the L-modi-
fied silicon surface was carried out, which switched cycles
6 times (Fig. 8c) and indicated a good reversible change
between hydrophobicity and hydrophilicity. These results also
indicated that Cys was successfully removed by Hg2+ from the
[L + Cys] complex. Furthermore, the CAs of the [L + Cys]
complex with various concentrations of Hg2+ (between 1.0 ×
10−2 and 1.0 × 10−6 mol L−1) from hydrophobicity to hydrophi-
licity clearly showed that the detection limit for Hg2+ was 1.0 ×
10−6 mol L−1 (Fig. S15†).
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suggested that the [L + Cys] complex was selective only for
Hg2+ owing to its unique Hg2+⋯SH interaction. The highly
selective wettability switch of the [L + Cys] complex toward
Hg2+ had been demonstrated by the above methods. The
addition of Cys to L showed significant a switch off CA, and
the CA was regained when Hg2+ was added to result in the
switch on mode. The result clearly demonstrated the reversible
property of the [L + Cys] complex toward Hg2+.
Conclusions
In summary, a novel piperidine-calix[4]arene complex (L) had
been successfully synthesized for the first time. It exhibited
strong binding toward Cys. Subsequently, the [L + Cys]
complex was constructed. This had been demonstrated by FL,
1H NMR spectroscopy and DFT computational studies. The
[L + Cys] complex was selective toward Hg2+, over six other
important heavy metal ions, viz., Pb2+, Cd2+, Cu2+, Ni2+, Zn2+
and Ba2+. This was verified by FL, CA, 1H NMR spectroscopy
and AFM experiments. Thus, the present study demonstrated
the utility of the [L + Cys] complex as a highly selective switch
for Hg2+ by exhibiting dual-signal reversible cycles. Most
importantly, the [L + Cys] complex could act as a convenient
and effective wettability switch for Hg2+ on the functional
micro–nano silicon surface. The recognition process and
method can be used in many fields, such as biomimetics,
environmental monitoring, clinical medicine and so on.18
Fig. 8 (a) CAs for the [L + Cys] complex (control) with 7 different metal ions.
The error range of CAs was 2.0°. Note: the following error range of CAs was
the same. (b) CA variation [ΔCA = (CA0 − CA)/CA0] (CA0 stood for contact angle
of the [L + Cys] complex) histogram for the [L + Cys] complex with 7 different
metal ions, which showed a highly selective switch for Hg2+ based on the Cys
complex. (c) A cycling experiment of the wettability switch between Cys and
Hg2+ on the L-modified silicon surface, which exhibited a selective switch for
Hg2+ through multiple reversible cycles on the functional silicon surface.
To substantiate the role of the piperidine moiety within
L in the recognition process, the control experiments were
undertaken. The reference compound C4AM was adopted in
the same method with Cys. CA and 1H NMR measurements for
C4AM in the presence of Cys were studied. As can be seen
from Fig. S17 and S8,† they showed only small change upon
addition of Cys. Thus, the results of the control experiments
indicated that the piperidine moiety played an important role
in the efficient interaction with Cys. To obtain further insight
into the interaction, some other control experiments were per-
formed. Moreover, some control experiments of CA were
carried out with other biologically relevant molecules contain-
ing –SH, viz., cysteamine (CS), mercaptoacetic acid (MPA) and
N-acetyl-L-cysteine (NAC) (Fig. 9 and Fig. S16†). These results
Experimental section
1. Materials and instruments
1H NMR and 13C NMR spectra were recorded using a Varian
Mercury VX400 instrument at ambient temperature with TMS
as the internal standard. ESI-MS was carried out using a Finni-
gan LCQ-Advantage instrument. The static water contact
angles were measured at 25 °C by means of an OCA 20 contact
angle system (Dataphysics, Germany). FL spectra were recorded
using a Type Cary Eclipse instrument. XPS was recorded on a
KRATOS XSAM800 Electron spectrometer (FRR mode).
All chemicals were of A.R. grade and were purified by stan-
dard procedures. Milli-Q water was used to prepare all solu-
tions in this study.
2. The synthesis of organic compounds
The procedure for the synthesis of calix[4]arene (C4AM).
Calix[4]arene (C4DT) (2.4 mmol) was completely dissolved in
acetonitrile and then sodium methylate (2.8 mmol) was added
before stirring and refluxing for 0.5 h. Subsequently, propargyl
bromide (0.5 mL) was added, then the mixture was stirred for
8 h. The crude product was purified by column chromato-
graphy (SiO2, petroleum ether–chloroform = 3/1) to afford pro-
pinyl-calix[4]arene (C4AM) in 85% yield. 1H NMR (400 MHz,
CDCl3): δ 9.67 (s, 1H, ArOH), 9.08 (s, 2H, ArOH), 7.09–6.98 (m,
9H, ArH), 6.69 (s, 3H, ArH), 4.95 (s, 2H, ArOCH2), 4.49–4.46 (d,
Fig. 9 (a) The structure of different thiols. (b) The histogram showed CA
changes of Hg2+ with different thiols. These results suggested that the [L + Cys]
complex was selective for only Hg2+ owing to its unique Hg2+⋯SH interaction.
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J = 13.2 Hz, 2H, ArCH2Ar), 4.29–4.25 (d, J = 18.9 Hz, 2H, 20 μm high, 9 μm long and with a spacing of 12 μm between
ArCH2Ar), 3.49–3.46 (d, J = 13.8 Hz, 4H, ArCH2Ar), 2.75 (s, 1H, the silicon pillars.19
CuCH). Anal. Calc. for C31H26O4: C, 80.50; H, 5.67. Found: C,
Preparation of the Si–N3-modified silicon substrates. Silicon
80.42; H, 5.52%.
substrates cut into 1 cm × 1 cm square pieces were soaked in
The procedure for the synthesis of piperidine-calix[4]arene chromosulfuric acid solution for 30–60 min and then rinsed
(L). C4AM (1.0 mmol) was completely dissolved in THF and with double distilled water and dried under a stream of N2 gas.
then acetic acid (5.0 mmol) was added. Subsequently, piper- The cleaned wafers were immersed in aqueous NaOH
idine (5.0 mmol) was added dropwise into the system. Later, (0.1 mol L−1
) for 6 min and subsequently in HNO3
formaldehyde (5.0 mmol) was added and the mixture was (0.1 mol L−1) for 12 min to generate surface hydroxyl groups.
stirred for 8 h. Finally, complex L was produced. The product After the silicon substrates had been washed with an excess of
was purified by column chromatography (SiO2, ethyl acetate– double distilled water and dried under a stream of N2 flow,
petroleum ether = 2/10) giving a white powder L with a yield of they were immersed in a refluxing solution of 5 wt% Si–N3 in
86%. 1H NMR (600 MHz, DMSO): δ 6.99 (s, 2H, ArH), 6.87–6.79 dry toluene (10 mL) at 110 °C for 6 h. Then they were washed
(d, J = 16.0 Hz, 4H, ArH), 6.71–6.69 (d, J = 7.3 Hz, 2H, ArH), with toluene and ethanol to remove the excess Si–N3 and dried
6.48 (s, 1H, ArH), 4.55 (s, 2H, ArOCH2), 4.47 (d, J = 11.8 Hz, under a stream of N2 gas.
2H, ArCH2Ar), 4.05 (d, J = 12.3 Hz, 2H, ArCH2Ar), 3.44 (s, 6H,
The click reaction between Si–N3 and L on the silicon sub-
ArCH2N), 3.13–3.08 (m, 4H, ArCH2Ar), 2.61 (s, 1H, CuCH), strates. The Si–N3-modified silicon surfaces were immersed in
1.53 (m, 20H, (CH2)2N), 1.40–1.01 (m, 10H, (CH2)2N). 13C NMR L solution in ethanol at 10−2 M, then the mixture of copper
(150 MHz, DMSO): δ 156.70, 156.34, 155.59, 135.89, 132.59, sulfate (10−6 M) and sodium ascorbate (10−7 M) were added
132.42, 132.15, 132.07, 131.59, 130.76, 129.51, 124.44, 80.56, into this solution, which was then heated at 75 °C for 8 h.
76.90, 64.04, 63.32, 54.51, 54.34, 49.48, 49.34, 49.13, 49.05, Then the silicon wafer was washed with a little ethanol and
48.91, 35.05, 32.97, 25.55, 25.51, 25.35, 24.53, 24.15. EI (+) MS dried under a stream of N2 gas.
m/z = 753.4 ([M] + 80%) Anal. Calc. for C49H59N3O4: C, 78.05;
H, 7.89; N, 5.57. Found: C, 77.94; H, 7.79; N, 5.43%.
Conflict of interest
3. Fluorescence experiments
The authors declare no competing financial interest.
All experiments of fluorescence cycles were carried out at an
excitation wavelength of 350 nm using a fluorescence spectro-
meter and a 1 cm quartz cell. A bulk solution (1.0 × 10−3 M,
2 mL) of Cys-F was freshly made before each set of experi- Acknowledgements
ments. L solution (1.0 × 10−2 M) was made by dissolving L in
This work was financially supported by the National Natural
CH3CN (1.2 mL). The Hg2+ solutions were made at 1.0 ×
Science Foundation of China (21072072, 21102051), PCSIRT
10−2 M in water (1.2 mL). The fluorescence titrations were
(no. IRTO953), Program for New Century Excellent Talent in
carried out by exciting the solution at 350 nm after adding the
University (NCET-10-0428).
appropriate volume (20 μL) of L solution to measure the fluore-
scence. Then the Hg2+ solution (20 μL) was added to the above
mixed solution to measure the fluorescence, which showed
fluorescence recovery over 6 switching cycles.
Notes and references
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