Enhanced Sensitivity for Hydrogen Peroxide Detection: Polydiacetylene Vesicles with Phenylboronic Acid Head Group

Article abstract of DOI:10.1007/s10895-015-1691-1

It was recently reported that, besides UV irradiated polymerization, polymerization of diacetylene compounds could also been initiated by radicals generated from enzyme catalyzed hydrogen peroxide (H₂O₂) decomposition. A new optical

Full text of DOI:10.1007/s10895-015-1691-1

J Fluoresc
DOI 10.1007/s10895-015-1691-1
ORIGINAL ARTICLE
Enhanced Sensitivity for Hydrogen Peroxide Detection:
Polydiacetylene Vesicles with Phenylboronic Acid Head
Group
Chen Jia1 & Jie Tang2 & Shengguo Lu3 & Yuwang Han2 & He Huang3
Received: 20 July 2015 /Accepted: 7 October 2015
#
Springer Science+Business Media New York 2015
Abstract It was recently reported that, besides UV irradiated
polymerization, polymerization of diacetylene compounds
could also been initiated by radicals generated from enzyme
catalyzed hydrogen peroxide (H2O2) decomposition. A
new optical sensing method for H2O2 was proposed
based on this phenomenon. However, the sensitivity of
this method is relatively lower than existed ones. In the
present work, phenylboronic acid (PBA) functionalized
10, 12-pentacosadiynoic acid (PDA-PBA) was synthesized
and its vesicles were formed successfully as colorimetric sen-
sor for H2O2 detection. It was found that color change during
the polymerization of vesicles composed of the PBA modified
monomer is much stronger than that of the non-modified one.
The response of PDA-PBA vesicles to H2O2 is 16 times more
sensitive than that of the PDA. The absorption of PDA-PBA at
650 nm is linearly related to the concentration of H2O2 and a
detection limit of ~5 μM could be achieved.
Introduction
Hydrogen peroxide (H2O2) has been widely used in
bleaching, cleaning and disinfection [1]. It is also a
by-product of oxidative metabolism [2]. The detection
of H2O2 is of practical importance in food, clinical, modern
medicine, environmental monitoring and various other indus-
tries [3, 4]. Many methods such as chemiluminescence [5, 6],
electrochemical methods [7–9] and chromatography [10] have
been developed for the detection of H2O2. However, these
detection methods need either expensive instruments or re-
agents. Therefore, optical biosensors are of the significant
interest for the detection of H2O2 due to their low cost and
simple instruments [11].
Recently, conjugated polymer colorimetric biosensor has
become popular due to its high sensitivity, selectivity and
practicality [12–16]. We previously reported a free radical
triggered polymerization of 10, 12-pentacosadiynoic acid
(PCDA) vesicles as a colorimetric reagent for H2O2 detection
[17]. The polymerization of well-packed diacetylene mono-
mers can be induced by the hydroxyl radical produced from
the horseradish peroxidase (HRP) catalyzed H2O2 decompo-
sition reaction [18–20]. The color of the reaction mixture
changes from colorless to blue during the polymerization. Un-
der the optimal conditions, the colorimetric detection of H2O2
can be completed with 20 μg/mL HRP (pH = 7.0) at 30 °C in
30 min. However, the previously reported PCDA-based meth-
od showed a relatively higher detection limit of ca. 80 μM
which has no advantage compared to other methods [17].
The topotactic polymerization of diacetylene is generally
known to be acutely sensitive to the molecular order of mo-
lecular assemblies. So, the molecular assemblie of the
diacetylene determines not only the ability to form stable ves-
icles but also its chromic behavior. Besides the total chain
length and position of the diacetylenic unit within the chain,
.
.
.
Keywords Polydiacetylene Hydrogen peroxide Vesicles
.
.
Modification Phenylboronic acid UV light
* He Huang
biotech@njtech.edu.cn
1
State Key Laboratory of Materials-Oriented Chemical Engineering,
College of Chemistry and Chemical Engineering, Nanjing Tech
University, Nanjing 211816, People’s Republic of China
2
3
College of Science, Nanjing Tech University, Nanjing 211816,
People’s Republic of China
State Key Laboratory of Materials-Oriented Chemical Engineering,
College of Pharmaceutical Sciences, Nanjing Tech University,
Nanjing 211816, People’s Republic of China

J Fluoresc
chemical nature of the headgroup is a key structural factor for
diacetylene compounds. Interactions between neighboring
diacetylene molecules via their headgroups are especially im-
portant for signal amplification in sensors and this could be
tailored by synthesis [21]. Niwa et al. prepared LB film of 10,
12-pentacosadiynamide phenylboronic acid (DA-PBA) and
got a blue polymer film upon UV irradiation [22]. It is found
that dehydration of boronic acid groups constrained in posi-
tion on the surface of a two dimensional monolayer assembly
would form cross-linked boronic anhydrides. Lee et al. also
successfully prepared phenylboronic acids functionialized
diacetylene network films on solid substrates [23]. In the pres-
ent work, a polydiacetylene vesicle bearing phenylboronic
acid group was prepared for the first time and used as a more
sensitive colorimetric biosensor for H2O2 detection.
24 h under 350 rpm mechanical stirring in the dark. The reac-
tion solution was then evaporated to dryness with the rotary
evaporator. The dried residue was re-dissolved in 30 mL
chloroform and extracted with 20 mL saturated sodium
chloride solution 6 times. The organic phase was collected
and dried over anhydrous magnesium sulfate for 12 h.
The product was dissolved in 50–70 mL chloroform. Ten
milliliters ethylenediamine (4 mmol in dichloromethane)
was added to the product solution dropwise. The pH of
the solution was adjusted to 9.0 with triethylamine. The
reaction was allowed to precede under 300 rpm mechani-
cal at 30 °C in the dark. The reaction solution was then
evaporated to dryness and the product, APCDA, was pu-
rified on a silica gel column with an eluant of chloroform:
methyl alcohol (5:1). The yield of APCDA is 83–94 %.
1
Figure 2 is the HNMR of APCDA and data are as follows:
1HNMR (300 MHz, CDCl3): δ(ppm) 0.78–0.81(t, J = 9, 3 H),
1.11–1.85 (m, 36 H), 2.14–2.16 (t, J = 6, 3 H), 2.24–2.26
(t, J = 6, 2 H), 3.31–3.34(dd, J = 9, 2 H), 7.19(brs, 1 H).
Synthetic Routes
Purification of PCDA
Synthesis of APCDA-PBA
The commercial PCDA was purified for further use. Briefly,
PCDA solid powder was dissolved in methylene chloride and
filtered through 0.45 μm membrane to remove the poly-
PCDA. The methylene chloride was evaporated with a rotary
evaporator and the resultant PCDA monomer was stored at
−20 °C in the dark.
The synthesis route of APCDA-PBA is present in Fig.3. Ten
grams of 4-carboxyphenylboronic acid (PBA, 60.3 mmol)
were added into a two-necked flask. Thionyl chloride
(150 ml) was then added to the flask in a N2 atmosphere.
The mixture was magnetically stirred (350 rpm) for 24 h at
88 °C. The reaction mixture was then evaporated to dryness
and the dry residue was re-dissolved in 60 ml anhydrous tet-
rahydrofuran. Chloroform (20 ml) was added dropwise to
3 mmol of APCDA in a N2 atmosphere with an ice-bath. A
drop of dimethyl formamide (DMF) was added after 30 min
and kept in the ice-bath for another 2 h in the dark. After that,
the obtained mixture was added dropwise to the previous pre-
pared PBA solution and allowed to react for 20 h in the dark.
The reaction mixture was evaporated to dryness with the ro-
tary evaporator and the product was purified on a silica gel
column with an eluant of chloroform: methyl alcohol (7:1).
The yield of APCDA-PBA is between 91 and 94 %. Figure 4
Synthesis of APCDA
Figure 1 shows the synthesis route for the mediate product 10,
12-pentacosadiynoic acid 2-aminoacetamide (APCDA).
Briefly, 2.45 mmol purified PCDA monomer in 20 mL tetra-
hydrofuran was mixed with 2.4 mmol N-hydroxysuccinimide
and 2.45 mmol 1-ethyl-3(3-dimethyl ammonium propyl)-
carbodiimide. The mixed solution was allowed to react for
1
is the HNMR of APCDA-PBA and data are as follows:
1HNMR (300 MHz, CDCl3): δ(ppm) 0.78–0.81(t, J = 9,
3 H), 1.18–1.58(m, 36 H), 2.14–2.17(t, J = 9, 4 H), 2.35–
2.37(t, J = 6, 2 H), 2.87(s, 1 H), 2.94(s, 1 H), 7.05–7.06
(d, J = 3, 2 H), 7.19(brs, 2 H), 7.84–7.86 (d, J = 6, 2 H)..
Preparation of APCDA-PBAVesicle
The produced APCDA-PBA (14.98 mg) was dissolved in
4 ml DMSO and 36 ml distilled water, heated to 70 °C and
sonicated for 15 min. The solution was filtered and stored at
4 °C in the dark overnight.
Fig. 1 The synthesis route of APCDA

J Fluoresc
Fig. 2 1HNMR of APCDA
Results and Discussion
the ethylene diamine monomer was successfully coupled
with the PCDA. APCDA-PBA showed three new absorp-
tion peaks at 1810 cm−1, 860 cm−1 and 1551 cm−1, which
were assigned to the stretching vibrations of C = C and
stretching vibrations of C-H and B-O, respectively. These
indicate that phenylboronic acid was coupled with the
APCDA successfully.
The microstructures of poly-PCDA and poly-APCDA-
PBA vesicles were imaged with a SEM (Fig.6). The poly-
PCDA vesicles are composed of large spherical particles,
while poly-APCDA-PBA vesicles composite of smaller rod-
shaped and square particles. This can be explained that am-
phiphilic monomers with achiral headgroups usually form
spherical liposomes and chiral amphiphiles form non-
spherical structures such as helices and tubules [24]. Similar
small particles can also be found in polydiacetylene-amino/1,
2-dimyristoyl-SN-glycero-3 -phosphocholine (PDA/DMPC)
mixed vesicles, which can improve the UV-Vis absorption of
the vesicles and enhance the color change [25]. The PDA/
Characterization of APCDA-PBA
The reaction substrate (PCDA), mediate product (APCDA)
and product (APCDA-PBA) were confirmed with FTIR
(Fig.5). The asymmetric stretching vibrations of CH2 groups
of the diacetylene side chains can be found at 2928 cm−1 and
2852 cm−1. The peak at 2349 cm−1 can be ascribed to the
vibrations of the unsaturated double bond CH = CH in the
diacetylene monomer. These feature FTIR peaks are shared
by all three different diacetylene derivatives. APCDA showed
an absorption peak at 3324 cm−1, which was assigned to the
stretching vibrations of N-H bond in amide. The absorption
peaks at 1697 cm−1 and 1632 cm−1 can be assigned to the
stretching vibrations of hydrogen-bonded carbonyl in carboxy
bond and the stretching vibrations of hydrogen-bonded car-
bonyl in amide, respectively. The new peak at 1070 cm−1 is
ascribed to the C-N bond in amide. These results indicate that
Fig. 3 The synthesis route of
APCDA-PBA

J Fluoresc
Fig. 4 1HNMR of APCDA-PBA
DMPC mixed vesicle coupled with antigen has a flat
shape, which can be attributed to its inherent variability
and instability in preparation process [26]. Morphology
change of vesicles can usually affect their detection sensitivi-
ties and stabilities [27–29]. The smaller vesicles provide more
surface binding sites and thus improve the detection sensitivity
of the colorimetric method [30].
peak appeared at 550 nm. A650 did not show significantly
change. Thus, 1 min UV irradiation time is enough for the
polymerization of APCDA-PBA. These also indicate that
the polymerization of APCDA-PBA is easier to be trigged
UV Light Trigged Polymerization
The polymerization of diacetylene vesicles is initiated by UV
light. The effects of irradiation time on the polymerization
process of PCDA and APCDA-PBA were investigated. The
absorption of the poly-PCDA at 650 nm (A650) increased from
0.06 to 0.21 when the irradiation time was increased from
1 min to 2 min (Fig.7a). Meanwhile, A650 of poly-APCDA-
PBA reached as high as 0.65 with 1 min irradiation. When
irradiation time was increased to 2 min, the blue phase of poly-
APCDA-PBA shifted to the purple phase and an absorption
Fig. 6 SEM images of poly-PCDA vesicles (a) and poly-APCDA-PBA
vesicles (b) The polymerization was initiated with 254 nm UV light at a
distance of 30 cm
Fig. 5 FTIR spectra of PCDA (red line), APCDA (blue line) and
APCDA-PBA (green line)

J Fluoresc
Fig. 7 a UV-Vis absorption
spectra of poly-PCDA and poly-
APCDA-PBA vesicles radiated
with UV light for 1 min and
2 min. b The poly-APCDA-PBA
vesicles radiated with UV light
for 1 min and poly-PCDA vesi-
cles radiated with UV light for
2 min. Both polymerizations were
inidiated with 254 nm UV light at
a distance of 30 cm
than that of PCDA alone. The much higher absorption of the
poly-APCDA-PBA in blue region (Fig.7b) indicates the high
sensitive of colorimetric method with APCDA-PBA vesicle.
is consistent with the results of its UV-induced polymerization
reaction (Fig.7b). Figure 8c shows the images of APCDA-
PBA vesicles being polymerized with the different concentra-
tion of H2O2 from 5 μM to 100 mM. It is clear that the inten-
sity of the blue color is significantly increased with the in-
crease of the concentration of H2O2 and the color change of
APCDA-PBA vesicles is more dramatic than that of PCDA
vesicles [17] under same conditions.
The possibility of the quantification of H2O2 with APCDA-
PBA vesicles was explored. The relationship between the ab-
sorption of poly-APCDA-PBA at 650 nm and the concentra-
tion of H2O2 is fitted in a linear regression logA = −0.719+
0.187logc (R2 = 0.991, n = 7) (Fig.8b), where A is the absor-
bance of poly-APCDA-PBA at 650 nm and c is the concen-
tration of H2O2 in μM. The lowest detectable concentration of
H2O2 is ~5 μM.
Detection of H2O2 with APCDA-PBAVesicle
The potential application of APCDA-PBA vesicles in the
quantification of H2O2 was explored. Figure 8a shows the
UV-Vis absorption spectra of APCDA-PBAvesicles polymer-
ized with various concentration of H2O2 ranging from 0 to
100 mM. It is clear the peak absorption at 650 nm increased
with the increase of H2O2 concentration. APCDA-PBA vesi-
cles can respond to as low as 5 μM H2O2, which is 16 times
more sensitive than PCDAvesicles. In addition, the absorption
of APCDA-PBA vesicles at 650 nm is significantly higher
than that of PCDAvesicles [17] under same conditions, which
Fig. 8 a UV-Vis absorption
spectra of the APCDA-PBA ves-
icles polymerized with various
concentrations of H2O2. b The
linear calibration curve of the ab-
sorption of APCDA-PBA at
650 nm to the concentration of
H2O2. Error bars represent the
standard deviation for three
indenpent measurements. c Typi-
cal images of the APCDA-PBA
vesicles polymerized with differ-
ent concentrations of H2O2 in the
presence of 20 μg/mL HRP. The
polymerization was conduct at
pH = 7.0 and 30 °C for 30 min

J Fluoresc
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In summary, the response of polydiacetylene vesicles to
H2O2 can be effectively enhanced by the introduction of
phenylboronic acid group. The absorption of PDA-PBA
vesicles at 650 nm and the concentration of H2O2 are
linearly related. The detection limit of the PDA-PBA vesicle
based colorimetric method is ~5 μM H2O2, which is 16 times
lower than that of the PDA vesicles based colorimetric
method. The PDA-PBA vesicle is a promising tool for
the detection of H2O2 in modern medicine, environmental
and biotechnology monitoring.
Acknowledgments This work was supported by the National Science
Foundation for Distinguished Young Scholars of China (No.21225626)
and the Key Program of the National Natural Science Foundation of
China (No.20936002).
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