Cholesterol modified OPE functionalized film: Fabrication, fluorescence behavior and sensing performance

Article abstract of DOI:10.1039/c2jm16637b

A novel oligo(p-phenyleneethynylene) with cholesterol on its side chains (CHOL-OPE) was designed and synthesized. Film 1 and Film 2 were fabricated by immobilizing, separately, the oligomer as prepared and its control, OPE, which contains no side chains,

Full text of DOI:10.1039/c2jm16637b

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PAPER
Cholesterol modified OPE functionalized film: fabrication, fluorescence
behavior and sensing performance†
*
Hongyue Wang, Gang He, Xiangli Chen, Taihong Liu, Liping Ding and Yu Fang
Received 16th December 2011, Accepted 13th February 2012
DOI: 10.1039/c2jm16637b
A novel oligo(p-phenyleneethynylene) with cholesterol on its side chains (CHOL-OPE) was designed
and synthesized. Film 1 and Film 2 were fabricated by immobilizing, separately, the oligomer as
prepared and its control, OPE, which contains no side chains, on glass plate surfaces via utilization of
a self-assembled monolayer (SAMs) technique. Fluorescence studies revealed that the profile of the
fluorescence emission spectrum of Film 1 is dependent upon the composition of a mixture of THF and
water. In contrast, Film 2 shows no such effect. Sensing performance studies demonstrated that the
fluorescence of Film 1 is super-sensitive to the presence of 2,4,6-trinitrophenol (picric acid, PA), and
sensitive to 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT) and nitrobenzene (NB) in the
aqueous phase, whereas the nitroaromatics (NACs) as studied show little effect on the fluorescence
emission of Film 2. The results from fluorescence quenching studies and the fact that copper salts
(copper nitrate and copper acetate) show no detectible effect on the fluorescence emission of Film 1
show that the oligomer and the short linker, connecting the oligomer and the substrate, of the CHOL-
OPE functionalized film may adopt a compact structure. Interestingly, the sensing process is fully
reversible and free of interference from commonly found compounds, including methanol, THF,
toluene, dichloromethane, ammonia, HCl, NaOH, NaCl, copper salts and seawater, etc. Fluorescence
lifetime measurements revealed the static nature of the quenching process. The superior sensing
performance of Film 1 for the analytes in the aqueous phase guarantees that the film may have the
potential to be developed into a sensor device for the detection of PA and other NACs in groundwater.
cumbersome pretreatment of samples, interference from other
Introduction
compounds, or the need for sophisticated instrumentation.5
Fluorescence methods are recommended because of their sensi-
tivity, selectivity and low cost in instrumentation.9
Nitroaromatic compounds (NACs) are commonly found in
military, homeland security and industry due to their wide
ranging applications from explosives to intermediates of dyes.1–3
For security and environmental protection reasons, the fast and
sensitive detection of NACs in the liquid phase, in particular
2,4,6-trinitrophenol (PA), 2,4,6-trinitrotoluene (TNT) and 2,4-
dinitrotoluene (DNT), has attracted much attention over the
past ten years. In fact, various methods, including amperometry,4
high-pressure liquid chromatography,5 surface enhanced Raman
spectrometry,6 and electrochemical methods7,8 have been devel-
oped for the detection of NACs in the liquid phase. However,
these methods have suffered from drawbacks such as
Conjugated polymers (CPs) or oligomers have been widely
adopted as sensing fluorophores in the development of both
chemical and biological sensors. This is because they are not only
fluorescence active but also possess the so-called super-quench-
ing effect, since their backbone can act as a molecular wire,
enabling the rapid propagation of an exciton throughout the
individual polymer chain.1,10–12 However, most of the sensing
films reported were prepared by spin coating, dip coating, casting
or other physical methods. These films suffer from slow response
due to diffusion problems encountered by the analytes, and
sample contamination due to fluorophore leaking when they are
used in the liquid phase.13–17 To solve these problems, a self-
assembled monolayer (SAMs) chemistry-based method has been
introduced to fabricate fluorescent films with CPs as their sensing
fluorophores.18
Key Laboratory of Applied Surface and Colloid Chemistry (Ministry of
Education), School of Chemistry and Chemical Engineering, Shaanxi
Normal University, Xi’an 710062, P. R. China. E-mail: yfang@snnu.edu.
cn; Fax: +86-29-85310097; Tel: +86-29-85300081
† Electronic supplementary information (ESI) available: Solvent
composition dependence of the fluorescence emission of Film 3, TNT
quenching of the fluorescence emission of Film 1, ATR-IR spectra of
Film 1 and Film 2 in virgin state and PA treated state, and NaCl or
NaOH effects on the fluorescence quenching of PA on Film 1. See
DOI: 10.1039/c2jm16637b
SAMs with functional termini represent a promising method
to fabricate fluorescent films because formation of SAMs on
a substrate surface and attachment of fluorophores to the func-
tional ends of SAMs are chemical linkages, which provide films
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as prepared with high stability and ensure the films’ usefulness in
harsh environments.19 At the same time, sensing molecules are
immobilized, in theory, in a monolayer fashion on a substrate
surface, and thereby resistance to the diffusion of analytes within
the films, as encountered by films fabricated by physical
methods, could be minimized. Our group has been seeking to
develop a new family of fluorescent film sensors using SAMs of
polycyclic aromatics on substrate surfaces over the past decade.
On the basis of these studies, a series of fluorescent film sensors
for various analytes, including nitromethane, dicarboxylic acids,
organic copper salts, NACs, etc., have been successfully devel-
oped.20–22 A few years ago, we started to explore the combination
of CPs and SAMs in order to prepare highly sensitive and stable
fluorescent film sensors. As expected, combination of the
advantages of CPs and those of SAMs resulted in several fluo-
rescent films with superior sensing performances to NACs and
formaldehyde in the vapor phase.18
preparation of the samples were performed using standard
vacuum line and Schlenk techniques under a purified argon
atmosphere. THF and toluene were distilled from sodium
benzophenone ketyl under argon prior to use. NACs including
PA, TNT, DNT, NB were of analytical grade and used directly
without further purification. Caution: TNT and other NACs used
in the present study are highly explosive and should be handled only
in small quantities.
Measurements
Fluorescence measurements were performed at room tempera-
ture on a time-correlated single photon counting Edinburgh FLS
920 fluorescence spectrometer with a front-face method. The
fabricated film was inserted into a quartz cell with its surface
facing the excitation light source. Then to the cell 2.5 mL of water
was added and then, if necessary, a given volume of the standard
solution of a specific quencher in ethanol or water was added
with stirring. Finally, the fluorescence emission spectrum of the
system was recorded. It is to be noted that the position of the film
was kept constant during each set of measurements. Pressed KBr
discs for the powder samples were used for the transmission
infrared (FTIR) spectroscopy measurements, and their FTIR
spectra were obtained with a Bio-Rad FTIR spectrometer.
Attenuated total reflection infrared (ATR-IR) spectroscopy: the
spectra were measured with a Bruker VERTEX 70v spectrom-
eter. Contact angles of the films were measured on a Dataphysics
OCA20 contact-angle system at ambient temperature. X-Ray
photoelectron spectroscopy (XPS) measurements were carried
out on an ESCAPHI5400 photoelectron spectrometer. 1H NMR
spectra were measured on Bruker AV 300 NMR spectrometers.
The MALDI-TOF mass spectra were recorded in a Microflex
mass analyzer (Bruker, USA) by using dithranol as the MALDI
matrixes.
However, the creation of new CPs-based fluorescent films is
not as easy as that by using low-molecular mass fluorophores,
such as polycyclic aromatics, as sensing fluorophores. This is
because for routine fluorophores new films can be created in
a relatively simple way via variation of the structures of the
linkers, which connect the sensing fluorophores to the SAMs
termini groups. This strategy is not applicable to the creation of
new films when CPs is used as a sensing fluorophore because of
the limitations of the rigorous reaction conditions. To solve this
problem or avoid this obstacle, we proposed to develop new films
by introducing side chains to the backbones of CPs.23 It is
believed that in this way new fluorescent films can be created by
variation of the structures of side chains of a given CP. Following
this strategy, a long flexible alkyl, hexadecyl, was introduced as
side chains in poly(p-phenyleneethynylene) (PPEs) for the
fabrication of a PPE-based fluorescent film recently.23 As
expected, variation in the conformation of the side chains
induced aggregation or disaggregation of the backbones of CPs,
resulting in changes in the fluorescence behavior of the film.
Clearly, this discovery lays the foundation for establishing a new
principle in the design of CPs-based fluorescent film sensors.
Cholesterol is hydrophobic, possesses a rigid skeleton, several
stereogenic centers, and has a strong tendency to form aggregates
via van der Waals interactions as revealed by scientists working
in the molecular gel field.24–27 It is expected that the rich supra-
molecular properties possessed by cholesterol may bring the CPs,
upon which it is chemically attached, and the CPs-based films
some unusual properties. Driven by this, cholesterol was
purposely introduced into oligo(p-phenyleneethynylene)s
(CHOL-OPEs) as side chains, and the oligomer was chemically
immobilized on a glass plate surface via a SAMs route. This
article reports the details of the fabrication, fluorescence
behavior and sensing performance of the film.
Synthetic procedure
Synthesis of compound 1. A mixture of cholesterol (6 g,
15.5 mmol), 1,8-dibromooctane (14.29 mL, 77.5 mmol), KOH
(3.48 g, 62 mmol), and dry THF(50 mL) was stirred under argon
at 80 ꢀC for 48 h. The solution was concentrated, the residue was
diluted with CH2Cl2 (50 mL), washed with brine (50 mL), and
then dried over MgSO4. The MgSO4 was removed by vacuum
filtration, and the filtrate was evaporated to dryness. The
resulting yellow oil was purified by column chromatography on
silica gel column with petroleum ether : CH2Cl2 (v : v, 1 : 1) as
eluent to afford compound 1 as a white solid (5.8 g, 64%).
Melting point: 69–71 ꢀC. IR (KBr, cmꢁ1): 2931, 2852, 1461, 1381,
1103. 1H NMR (d (ppm), 300 MHz, CDCl3): 5.33 (s, 1H), 3.46–
3.37 (m, 4H), 3.10–3.12 (m, 1H), 2.34–0.67 (m, 55H).
Synthesis of compound 2. A mixture of compound 1 (2 g,
3.5 mmol), 2,5-diiodobenzene-1,4-diol (0.5 g, 1.4 mmol),28 KOH
(0.63 g, 11.2 mmol), dry THF (10 mL) and dry DMSO (30 mL)
was stirred under an argon atmosphere at room temperature for
48 h. Then, the reaction mixture was poured onto water
(300 mL), resulting in a white solid precipitate. The residue was
collected, washed with water, dried, and purified by column
chromatography on silica gel column with petroleum
Experimental section
Materials
4-Bromobenzaldehyde (Alfa, 99%), phenylacetylene (Alfa, 99%),
1,4-dimethoxybenzene (Sigma, 99%), (3-aminopropyl)tri-
methoxysilane (APTMS, Alfa, 97%), Pd(PPh3)4 (Alfa, 99%) and
CuI (Alfa, 98%) were used as received. All manipulations for the
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ether : CH2Cl2 (v : v, 2 : 3) to afford compound 2 as a white solid
(1.1 g, 55%). Melting point: 149–151 C. IR (KBr, cmꢁ1): 2931,
amounts of toluene and CH2Cl2, and then dried under a gentle
stream of nitrogen.
ꢀ
1
2852, 1461, 1381, 1213, 1103. H NMR (d (ppm), 300 MHz,
CDCl3): 7.16 (s, 2H), 5.33 (s, 2H), 4.01–3.95 (m, 4H), 3.44–3.39
(m, 4H), 3.10–3.12 (m, 2H), 2.34–0.67 (m, 110H). Anal. Calcd.
for C76H124O4I2: C, 67.34; H, 9.22; found: C, 66.99; H, 9.50%.
Chemical coupling of OPE on a glass plate surface
The APTMS-modified glass plate was further macerated into
a CHCl3 solution of compound 4 or 5 (1 mM) under reflux for
12 h to generate the corresponding Schiff base. Then the glass
plate was washed with CHCl3 and directly reduced with NaBH4
in anhydrous methanol for 1 h. To remove unreacted compound
4 or 5, the glass plate was rinsed with plenty of CHCl3, acetone
and water, respectively.
Synthesis of compound 3. To a solution of diisopropylamine
(3 mL) in toluene (7 mL) were added compound 2 (500 mg,
0.37 mmol), 4-ethynylbenzaldehyde (16 mg, 0.12 mmol),
Pd(PPh3)4 (7.1 mg, 0.006 mmol), and CuI (1.2 mg, 0.006 mmol).
ꢀ
The reaction mixture was stirred under argon at 70 C for 48 h.
The reaction mixture was diluted with CH2Cl2 (50 mL), washed
with brine (50 mL), and then dried over MgSO4. The MgSO4 was
removed by vacuum filtration, and the filtrate was evaporated to
dryness. The resulting yellow solid was purified by column
chromatography on silica gel column with CH2Cl2 to afford
compound 3 as a yellow solid (0.06 g, 35%). Melting point: 98.2–
99.5 ꢀC. IR (KBr, cmꢁ1): 2931, 2852, 2730, 2208, 1461, 1381,
1213, 1103. 1H NMR (d (ppm), 300 MHz, CDCl3): 10.0 (s, 1H),
7.87–7.84 (d, 2H), 7.66–7.64 (d, 2H), 7.32 (s, 1H), 6.90 (s, 1H),
5.33 (s, 2H), 4.01–3.95 (m, 4H), 3.44–3.39 (m, 4H), 3.10–3.12 (m,
2H), 2.34–0.67 (m, 110H). MS (m/z, [M+NaꢁI]+): calculated:
1253.89, found: 1254.01.
Results and discussion
Characterization of the films
Contact angles with water at ambient temperature of the surfaces
at different stages of the functionalization process have been
measured, and the results are shown in Fig. 1. Reference to the
figure reveals that the advancing contact angle decreased signif-
icantly from 18.7 ꢃ 0.7ꢀ to 10.9 ꢃ 0.5ꢀ after treatment with the
‘‘piranha solution’’. Further treatment with APTMS resulted in
a sharp increase to 68.3 ꢃ 0.3ꢀ, indicating that the wettability of
the surface had reversed. The angle increased further after
treatment with CHOL-OPE and reached a value of 80.7 ꢃ 1.0ꢀ,
a typical hydrophobic surface. These results are consistent with
the expectation from the chemical composition of the surface, as
shown in Scheme 1.
Synthesis of compound 4 (CHOL-OPE). To a solution of di-
isopropylamine (3 mL) in toluene (7 mL) were added compound
3 (100 mg, 0.07 mmol), phenylacetylene (0.04 mL, 0.37 mmol),
Pd(PPh3)4 (4.3 mg, 0.004 mmol), and CuI (1 mg, 0.004 mmol).
Chemical coupling of CHOL-OPE on the glass plate surface
was further confirmed by XPS measurements. Fig. 2a depicts the
XPS spectra of the glass plate with different surface compositions
and structures. It can be seen that the signal of C1s and N1s of
the amino group covered substrate is much stronger than that of
the one covered with hydroxyl groups, indicating that the
silanizing reagent is somewhat exposed to the outer surface.
Compared with the APTMS-modified glass plate, the XPS
spectrum of the CHOL-OPE treated glass plate surface is char-
acterized by two N1s signals, appearing at 399.8 and 401.6 eV,
respectively (Fig. 2b), indicating the presence of nitrogen-con-
taining molecules on the substrate surface, direct evidence for the
successful coupling of the CHOL-OPE on the substrate surface.
It can also be seen that the signal of C1s (284.6 eV) became much
stronger on the CHOL-OPE covered surface. Simple calculation
using the ratio data from the different N1s signals, one corre-
sponding to the amino nitrogen, and the other to imino nitrogen,
shown in the XPS spectrum for Film 1, reveals that about 43.7 ꢃ
2.9% of the amino groups (Fig. 2b) reacted with CHOL-OPE,
a suitable loading density for sensing as validated by our trial
and error test. For Film 2, the measurement reveals that about
12.7 ꢃ 1.5% of the amino groups (Fig. 2c) reacted with OPE
ꢀ
The reaction mixture was stirred under argon at 70 C for 48 h.
The reaction mixture was diluted with CH2Cl2 (50 mL), washed
with brine (50 mL), and then dried over MgSO4. The MgSO4 was
removed by vacuum filtration, and the filtrate was evaporated to
dryness. The resulting yellow solid was purified by column
chromatography on a silica gel column with CH2Cl2 to afford
compound 4 (CHOL-OPE) as a yellow solid (0.07 g, 75%).
Melting point: 104–106 ꢀC. IR (KBr, cmꢁ1): 2931, 2852, 2730,
1
2208, 1461, 1381, 1213, 1103. H NMR (d (ppm), 300 MHz,
CDCl3): 10.0 (s, 1H), 7.88–7.85 (d, 2H), 7.68–7.65 (d, 2H), 7.54–
7.53 (d, 2H), 7.35–7.34 (d, 3H), 7.02–7.01 (d, 2H), 5.33 (s, 2H),
4.05–4.01 (m, 4H), 3.44–3.39 (m, 4H), 3.10–3.12 (m, 2H), 2.34–
0.67 (m, 110H). MS (m/z, [M+Na]+): calculated: 1355.06, found:
1355.02.
Synthesis of compound 5 (OPE). Compound 5 was synthesized
according to literature methods.29,30
Activation and silanization of the glass plate surface
A glass plate (0.9 cm ꢂ 2.5 cm) was treated in a ‘‘piranha solu-
tion’’ (3/7, v/v, 30% H2O2/98% H2SO4) (Warning: piranha solu-
tion should be handled with extreme caution because it can react
violently with organic matter) at 98 ꢀC for 1 h, rꢀinsed thoroughly
with plenty of water, and finally dried at 100 C in a dust-free
oven for 1 h. The activated glass plate was placed in a flask
charged with a warm (50 ꢀC) toluene solution (30 mL) of
APTMS (0.15 mL) containing a trace amount of water (10 mL)
for 3 h. After being cooled to room temperature, the plate was
removed from the flask, successively washed with copious
Fig. 1 Static contact angles (q) with water for various glass plate
surfaces, where the meanings of (1), (2), (3) and (4) are the same as those
explained in Scheme 1.
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Scheme 1 Preparation of OPE with cholesterol in its side-chains and immobilization of the OPE and its derivatives onto an APTMS modified glass
plate surface.
showed no detectable fluorescence emission, suggesting that
leaking is negligible for both films, indirect but strong evidence to
support chemical coupling of CHOL-OPE or OPE on the
substrate surface.
Steady-state excitation and emission spectra of the films
The fluorescence excitation and emission spectra of the two films
immersed in water or THF were recorded separately and the
normalized spectra are shown in Fig. 3. It can be seen that for
Film 1, the maximum excitation and emission in water appears at
370 nm and 455 nm, respectively. Surprisingly, the maximum
emission blue-shifts to 445 nm, while the excitation shows no
Fig. 2 (a) XPS traces of Film 1 and Film 2 (5), where the meanings of
(2), (3) and (4) are the same as those explained in Scheme 1; (b) the N1s
signal of Film 1, and (c) the N1s signal of Film 2.
(details of the calculation of the standard deviation are in
the ESI†).
Leaching is almost always a substantial problem to limit
solution sensing applications of fluorescent films, particularly
when they are fabricated in physical ways. For our films, either
water or THF from 24 h immersion of either Film 1 or Film 2
Fig. 3 The excitation and emission spectra of Film 1 and Film 2
recorded in water and THF, respectively.
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significant change when water is replaced by THF. On the
contrary, the profiles of the excitation and emission spectra of
Film 2 recorded in either water or THF are very similar. The
observed different spectral variation between Film 1 and Film 2
may be correlated to the structural difference of the side-chains in
the two oligomers and their different responses to the
microenvironments.
the other hand, the ratio values go up when the THF composi-
tion increases, indicating that THF helps to disaggregate the
surface-confined OPE molecules. This phenomenon could be
correlated with the solubility of the OPE molecules. Water is
a poor solvent for both the OPE backbone and the side chains,
and condensed side chains are expected to form in poor solvent
and lead to a more aggregated state of the OPE molecules. On the
other hand, THF is a good solvent for both the OPE backbone
and the side chains, and the side chains may extend to the outer
of the OPE layer and disaggregate the surface-confined OPE
molecules (Scheme 2).
To have a detailed understanding of the microenvironment
dependence of the profile of the fluorescence emission of Film 1,
its emission spectrum was recorded in THF–water mixtures with
different compositions. For explaining the trends clearly, the
profile of an emission spectrum as recorded can be represented by
a ratio of the intensities of the spectrum at 445 to 500 nm. The
measured effect of solvent composition on I445/I500 of Film 1 is
depicted in Fig. 4. For comparison, similar measurements for
Film 2 (as a control) and Film 3 (a previously reported film)23
were also conducted and the results were presented in Fig. 4 and
Fig. S1, respectively.†
This assumption was supported by the similar results found for
Film 3. From Fig. S1,† small intensity ratio values for more
aggregated states were observed when the ethanol (the poor
solvent) composition was large, and large ratio values for less
aggregated states were found when the THF (the good solvent)
composition was large. This suggests that the conformation of
the side chains adjusted by the solvent composition has a signif-
icant influence on the aggregation states of the surface-confined
OPE molecules. Therefore, it is not difficult to understand that
no obvious emission profile variation was observed for Film 2
since there are no side chains in the OPE molecules used for this
film. The variation of the solvent composition leads to negligible
alteration of the space of the OPE backbones.
With reference to the results presented in Fig. 4 and Fig. S1,† it
is revealed that: (1) only the profiles of the fluorescence emission
of the films possessing side chains (Film 1 and Film 3) show
significant response to changes of the solvent composition; (2) for
Film 3, the ratio of the intensities of the emission at the two
wavelengths is almost linearly dependent upon the composition
of the mixture solvent within a range from 20 to 80% of ethanol
in the ethanol–THF mixture solvent, but for Film 1, the depen-
dence is complicated, the ratio of the intensities of the emission at
the two selected wavelengths exhibits a sharp variation with the
composition instead of a linear dependence on the solvent
composition.
The different dependence on the binary solvent composition
between Film 1 and Film 3, where the former is partially
reversible and the latter is fully reversible, could be attributed to
the different side chains they employed. For Film 3, as the
extension and contraction of the conformation of the long alkyl
side chains is a continuous process,23 thus the profile change of
the fluorescence emission of Film 3 induced by addition of THF
to ethanol is also a gradual change process. For Film 1, however,
there may be only two states (Scheme 2). One, in which the
cholesterol units extend to the outer space of the OPE layer,
corresponds to more space for the OPE backbones, and another
corresponds to less space due to occupation of the cholesterol
units within the layer. Clearly, introduction of THF favors
transition of the second state to the first state, but the content of
THF in the mixture solvent must exceed a critical value. Simi-
larly, addition of water favors the reverse process. But the
transition occurs only when water content in the mixture solvent
exceeds another critical value. It is the two state structure of the
CHOL modified OPE functionalized film that makes the profile
of the fluorescence emission of the film change in a way very
different from that modified with simple long alkyl chains.
The differences in the dependences of the profiles of the fluo-
rescence emissions of the three films can be attributed to the
differences in their structures (cf. Scheme 1). Film 1 possesses
side chains containing cholesterol structure, however, Film 3 is
characterized by long alkyl side chains, and Film 2, as the
control, has no side chains. There is no doubt that the profile of
the fluorescence emission spectrum of an OPE modified film
should be a reflection of the balance between the disaggregated
state and the aggregated state of the surface-immobilized OPE
molecules. More aggregates correspond to lower values of the
ratio, and vice versa. It can be seen from Fig. 4 that small values
of the ratio are observed when the water composition is high,
suggesting water facilitates the formation of more aggregates. On
Fluorescence quenching studies
Given the structure of the fluorophore layer of Film 1, it is
expected that the film should show an enrichment effect to
hydrophobic compounds in the aqueous phase, and the
compounds could be probed if they are quenchers of the fluo-
rescence emission of the film. Accordingly, the effects of a series
of representative fluorescence quenchers, including PA, TNT,
DNT, NB, Cu(NO3)2 and Cu(Ac)2, on the fluorescence emission
of the film were examined in the aqueous phase. As expected, all
the NACs quenched, with different efficiencies, the emission of
the CHOL-OPE functionalized film, but the two copper salts
showed a slight effect on the emission of the film.
Fig. 4 Plots of the ratios of Ix/Iy of a given fluorescent film (Film 1 and
Film 2) against the compositions of the mixture solvents in which the
fluorescence measurements were conducted (for Film 1, x ¼ 445 nm, y ¼
500 nm; for Film 2, x ¼ 374 nm, y ¼ 420 nm).
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Scheme 2 The two states adopted by the molecules of CHOL-OPE in water and THF, respectively.
Fig. 5 depicts the fluorescence emission spectra of Film 1 in the
presence of different concentrations of PA. Clearly, addition of
5 mM of PA results in nearly 75% of reduction of the initial
emission of the film. The quenching data were subjected to the
Stern–Volmer equation, I0/I ¼ 1 + KSV[PA], where I0 and I are
the fluorescence intensities of the film in the absence and presence
of PA, respectively, and KSV is the Stern–Volmer quenching
constant. The results are shown as the inset of Fig. 5. It can be
seen that the Stern–Volmer plot is down-curved, suggesting that
some of the surface-tethered fluorophores are unaccessible or
difficult to access by the quenchers. This phenomenon could be
attributed to the heterogeneous structure of the fluorophore
layer, as discussed by Lakowicz and co-workers.31
TNT reduces nearly 78% of the initial emission of the film
(Fig. S2†). DNT and NB also quench the emission of the film in
the aqueous phase, but the quenching efficiencies are much
lower. For comparison, similar experiments were conducted for
Film 2, and it was found that almost all of the NACs tested show
no effect on the fluorescence emission of the control film (Fig. 6).
When THF was used as a medium for the test, it was found that
all the chemicals except PA (in the case of Film 1) show little
effect on the fluorescence emission of the two films. With refer-
ence to the figure, it reveals that PA quenches the fluorescence
emission of Film 1 in THF, and the quenching efficiency is only
one third of that in water. For copper salts, no obvious
quenching effect was observed in THF as in the case of water.
The differences in the sensing performances of the two films
may be understood by considering the so-called ‘‘two-dimen-
sional solution’’ model or ‘‘spacer-layer screening effect’’, which
was proposed by us in the studies of the sensing performances of
single-layer chemistry-based low-molecular mass fluorophore
functionalized fluorescence films.22,32 For Film 1, the backbones
of OPE, the linker connecting OPE and the substrate surface,
and the side chains including the CHOL residues may be
considered as an organic layer, of which the properties are
different from either the substrate, a typical inorganic material,
or the bulk phase, a typical liquid. Clearly, this layer should be
very thin and possess hydrophobic properties, and thereby it
endows the film specific affinity for hydrophobic analytes, such
as NACs, when the measurement is conducted in the aqueous
phase (cf. Fig. S3† and discussion presented there). As for the
weak response of the control film to the presence of the analytes,
this could be attributed to the absence of the so-called ‘‘two-
dimensional solution’’22,32 due to the lack of side chains and the
limited density of OPE (Fig. 2c), which results in no specific
affinity for the analytes in either of the two solvents (Fig. S3†).
The affinity towards hydrophobic analytes is expected to
diminish when the measurements are conducted in the organic
Meanwhile, the response of Film 1 to the presence of TNT was
also tested in the aqueous phase, and it was shown that 50 mM of
Fig. 5 Fluorescence emission spectra of Film 1 in the presence of
different concentrations of PA (from top to bottom, 0, 0.2, 0.4, 0.6, 0.8, 1,
2, 3, 4 and 5 mM) in an aqueous medium (lex ¼ 370 nm).
7534 | J. Mater. Chem., 2012, 22, 7529–7536
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Fig. 6 Quenching efficiencies of various NACs on the fluorescence emission of Film 1 and Film 2 in water and THF, respectively, and the interferences
of commonly found interferents in the sensing of Film 1 (concentration of NACs and interferents are 50 mM).
phase because the hydrophobic analytes tend to stay in the bulk
phase. This should explain why the film shows a more sensitive
response to the presence of the analytes in the aqueous phase. In
addition, proton transfer from PA to the amine group in the
linker may also play an important role for PA sensing. This is
because the quenching efficiency is significantly decreased when
NaCl is introduced or the PA is neutralized by NaOH as shown
in Fig. S4.† A similar observation was obtained in the studies of
PA sensing of a pyrene functionalized fluorescent film.33,34 It is to
be noted that it is the synergistic effect of the enrichment and the
specific binding of the fluorophore layer that makes Film 1 more
sensitive to PA than to other analytes. As for copper salts, they
are too hydrophilic to be combined by the films, and thereby
show no obvious quenching effect of the fluorescence emission of
the two films.
from time-resolved measurements. Examining the plots shown in
Fig. 7, the lifetime ratio, s0/s, is almost independent of the
concentration of PA and nearly equals 1, indicating that the
quenching is static in nature. In other words, formation of a non-
fluorescent OPE-PA complex is the origin of the quenching.
Reversibility of the quenching process
The procedures adopted for examination of the reversibility of
the sensing process are as follows: firstly, the film was inserted
into a cell with 2.5 mL of distilled water, and the fluorescence
emission of the film was recorded. Secondly, 10 mL PA solution
(2.5 mM) was added, and after 10 min equilibration the
Interference of other chemicals
To test the selectivity of the response of Film 1 to NACs, the
effects of commonly found interferents, including methanol,
THF, toluene, dichloromethane, ammonia, HCl, NaOH, NaCl
solution, copper salts and seawater on the fluorescence emission
of Film 1 were examined in the same conditions as those used for
the examination of the sensing performance of the film for
NACs. It was found that no significant change in the fluorescence
emission of the film was observed upon exposing the film to
aqueous solutions of the interferents as mentioned above (Fig. 6).
Quenching mechanism of the sensing process
The quenching mechanism was also studied by comparing the
quenching results from steady-state measurements and those
Fig. 7 Plots of I0/I and s0/s as functions of PA concentration.
J. Mater. Chem., 2012, 22, 7529–7536 | 7535
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Project of Shaanxi Province (2010-ZDKG-89) and Program for
Changjiang Scholars and Innovative Research Team in Univer-
sity (IRT1070) for financial support.
Notes and references
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Acknowledgements
The authors thank the Natural Science Foundation of China
(Nos. 20927001, 20803046, 91027017 and 21173142), the 13115
7536 | J. Mater. Chem., 2012, 22, 7529–7536
This journal is ª The Royal Society of Chemistry 2012