Fabrication of a nanohybrid of conjugated polymer nanoparticles and graphene oxide for biosensing of trypsin

Article abstract of DOI:10.1002/pola.27215

Conjugated polymers containing triphenylamine group are synthesized via Suzuki coupling polymerization. Fluorescent-conjugated polymer nanoparticles (CPN) are prepared by reprecipitation method using the newly synthesized conjugated polymer. CPN can be encapsulated with polyarginine by electrostatic interaction. The CPN modified with polyarginine exhibit excellent interaction with graphene oxide (GO) which is chemically modified with hydrophilic groups that possesses negative charge, which, in turn, induces the quenching of the fluorescence of CPN upon formation of CPN-GO nanohybrid. Upon exposure to trypsin, the quenched fluorescence is recovered by release of CPN from the nanohybrid, because trypsin cleaves the polyarginine linkage, resulting in weakening of interaction between CPN and GO. Copyright

Full text of DOI:10.1002/pola.27215

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Fabrication of a Nanohybrid of Conjugated Polymer Nanoparticles
and Graphene Oxide for Biosensing of Trypsin
Jaeguk Noh,1 Byung-Jae Chae,2 Bon-Cheol Ku,3 Taek Seung Lee1
1Organic and Optoelectronic Materials Laboratory, Department of Advanced Organic Materials and Textile System Engineering,
Chungnam National University, Daejeon 305-764, Korea
2Department of Chemistry, Chonbuk National University, Jeonju 561-756, Korea
3Institute of Advanced Composites Materials, Korea Institute of Science and Technology, Jeonbuk 565-905, Korea
Correspondence to: T. S. Lee (E-mail: tslee@cnu.ac.kr)
Received 20 February 2014; accepted 9 April 2014; published online 29 April 2014
DOI: 10.1002/pola.27215
ABSTRACT: Conjugated polymers containing triphenylamine
group are synthesized via Suzuki coupling polymerization.
Fluorescent-conjugated polymer nanoparticles (CPN) are pre-
pared by reprecipitation method using the newly synthesized
conjugated polymer. CPN can be encapsulated with polyargi-
nine by electrostatic interaction. The CPN modified with polyar-
ginine exhibit excellent interaction with graphene oxide (GO)
which is chemically modified with hydrophilic groups that pos-
sesses negative charge, which, in turn, induces the quenching
of the fluorescence of CPN upon formation of CPN–GO nano-
hybrid. Upon exposure to trypsin, the quenched fluorescence
is recovered by release of CPN from the nanohybrid, because
trypsin cleaves the polyarginine linkage, resulting in weakening
C
V
of interaction between CPN and GO. 2014 Wiley Periodicals,
Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1898–1904
KEYWORDS: sensors; fluorescence; conjugated polymer nano-
particles; conjugated polymers; trypsin; graphene oxide
and DNAs can facilely adsorb on to the GO surface via noncova-
lent interactions, which can be cleaved by external target spe-
cies with more favorable interaction with GO.
INTRODUCTION Recently, considerable demands on nano-
structured materials such as fibers, particles, rods, tubes,
and disks have been emerged for a variety of applications,
including optoelectronics, multimodal imaging, and nanome-
dicine.1 In particular, nanoparticles fabricated from conju-
gated polymers with unique properties,2 such as high
fluorescence, excellent photostability, good biocompatibility,
and low cytotoxicity have provided new fluorescent probes
for sensing and cellular imaging with proper surface modifi-
cations. The surface modification of CPN is known to be
important in biology-related studies because the colloidal
stability of CPN in aqueous solution is a primary concern.3
As one of the proteases, trypsin is known to be an important
digestive enzyme, and is produced in the pancreas as the
proenzyme trypsinogen. The concentration of trypsin in the
human body increases with some pancreatic diseases.6 Thus,
it is known that trypsin is overexpressed in pathological con-
ditions of cancer and inflammation.7 Moreover, as a kind of
protease, trypsin can selectively cleave the carboxyl chain of
amino acids such as lysine or arginine.8
In this work, we demonstrate a new, versatile CPN material
for the analysis of a specific protein, trypsin, while GO is
used as an effective quencher. The fluorescence quenching
by GO follows a surface energy transfer process, rather than
GO is a two-dimensional carbonaceous nanomaterial that can
be readily prepared by an exfoliation process that forms mono-
layer sheets. Usually, GO has oxygen-based functional groups,
including carboxylic acids, alcohols, and epoxides, leading to
uniform dispersions in aqueous-based media, which have many
applications.4 Because GO nanosheets in aqueous dispersion
possess negative charges on a hydrophobic backbone, positively
charged molecules can be promptly adsorbed on the GO surface
through electrostatic and p2p interactions. Using this surface
property of GO, many attempts to detect biologically related
species have been carried out via turn-on or turn-off modes.5 In
these systems, charged species, such as polypeptides, proteins,
€
a Forster resonance energy transfer (FRET) mechanism,
which does not require the spectral overlap between the
energy donor and the acceptor, enabling wide selection win-
dow of the energy donor.9 Fabrication of a nanohybrid, com-
prised CPN and GO, was successfully carried out through
electrostatic assembly, via preparation of surface-modified
CPN with polyarginine (pArg), which made the CPN posi-
tively charged and thus acted as a linker for both CPN and
Additional Supporting Information may be found in the online version of this article.
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GO. By constructing a hybrid of GO and pArg-encapsulated
CPN, quenching could be observed because of the presence
of the energy-accepting GO. Because trypsin selectively
degrades the linker pArg, recovery of the initial fluorescence
of CPN can be expected, thereby a selective trypsin detection
method can be envisaged. The fluorescence-quenching prop-
erty of GO depends on the ability of fluorescent species to
immobilize on the GO surface, thus opening the possibility of
constructing “turn-on” assay.5,10(a–c) To our knowledge, stud-
ies on the pArg-encapsulated CPN have seldom been repor-
final product was yellowish powder (yield 0.35 g, 47%). 1H
NMR (300 MHz, CDCl3) d 5 9.9 (s), 7.8–6.9 (m), 4.1–3.7 (m),
2.8–2.0 (m), 1.6–1.0 (m) ppm. 13C NMR (CDCl3): d 5 191.2,
146.6, 137.6, 133.1, 129.4, 112.7, 51.2, 31.1, 29.2, 27.9, 22.6.
FT-IR (cm21): 3030 (CAH), 1697 (CH@O), 1591 (C@C),
1462(C@C), 1284 (CAN). Anal. calcd for
C50.92H72.25
N0.27O0.27: C 88.0, H 10.0, N 0.52; found: C 87.5, H 9.92,
N 0.50.
CP2: 3
ted.10(d,e) Besides, it is rare to introduce CPN into
a
(0.215 g, 0.50 mmol), 9,9-dioctyl-2,7-dibromofluorene(0.274
g, 0.50 mmol), and 9,9-dioctylfluorene-2,7-diboronic acid
bis(1,3-propanediol)ester (0.67 g, 1.20 mmol) were dissolved
in a mixture of THF (10 mL) and 2 M aqueous potassium
carbonate solution (4 mL). After addition of tetrakis(triphe-
nylphosphine) palladium(0) (3.50 mg, 0.003 mmol), the reac-
tion mixture was stirred under argon at 100 ꢀC for 48 h.
The reaction and work-up procedures are the same as the
case for CP1. The final product was yellowish powder (yield
quenching- and recovery-based fluorescence-sensing system
with the aid of GO for the detection of protease.
EXPERIMENTAL
Materials
9,9-Dioctyl-2,7-dibromofluorene,
9,9-dioctylfluorene-2,7-
diboronic acid bis(1,3-propanediol)ester, and tetrakis(tri-
phenyl phosphine)-palladium(0) were purchased from
Sigma-Aldrich and used without further purification. Polyar-
ginine (mol. wt. 15,000–70,000) and trypsin (mol. wt.
23,300) were purchased from Sigma-Aldrich and used as
received. 4-(Bis(4-bromophenyl)amino) benzaldehyde (3)
was synthesized using previously published methods.11 GO
was prepared from graphite powder based on Hummer’s
method.12
1
0.42 g, 51%). H NMR (300 MHz, CDCl3) d 5 9.8 (s), 7.7–6.8
(m), 4.2–3.7 (m), 3.7–3.2 (m), 2.6–3.0 (m), 2.5–1.8 (m), 1.6–
1.0 (m) ppm. 13C NMR (CDCl3): d 5 191.2, 146.7, 137.6,
133.1, 129.3, 128.4, 112.8, 51.2, 27.9 ppm. FT-IR (cm21):
3028 (CAH), 1698 (CH@O), 1592 (C@C), 1461 (C@C), 1275
(CAN). Anal. calcd for C50.4H69.0N0.4O0.4: C 88.2, H 10.1, N
0.85; found: C 87.9, H 9.92, N 0.81.
CP3: 3
(0.30 g, 0.70 mmol), 9,9-dioctyl-2,7-dibromofluorene(0.17 g,
0.30 mmol), and 9,9-dioctylfluorene-2,7-diboronic acid
bis(1,3-propanediol)ester (0.67 g, 1.20 mmol) were dissolved
in a mixture of THF (10 mL) and 2 M aqueous potassium
carbonate solution (4 mL). After addition of tetrakis(triphe-
nylphosphine) palladium(0) (3.50 mg, 0.003 mmol), the reac-
tion mixture was stirred under argon at 100 ꢀC for 48 h.
The reaction and work-up procedures are the same as the
case for CP1. The final product was yellowish powder (yield
Characterization
The 1H NMR and 13C NMR spectra were obtained on a
Bruker DRX-300 spectrometer (Korea Basic Science Insti-
tute). Elemental analysis was performed with a CE Instru-
ments EA-1110 elemental analyzer. The FT-IR spectra were
recorded on a Bruker Tensor 27 spectrometer. The scanning
electron microscope (SEM) images were obtained from Hita-
chi S-4800. The transmission electron microscope (TEM)
images were obtained from JEOL JEM-3010. The image of
atomic force microscope (AFM) was recorded on VEECO
Nanoman. The UV–vis absorption spectra were recorded on
a Perkin Elmer Lambda 35 spectrometer. The photolumines-
cence spectra were taken using a Varian Cary Eclipse spec-
1
0.31 g, 43%). H NMR (300 MHz, CDCl3) d 5 9.9 (s), 8.0–6.8
(m), 4.1–3.5 (m), 2.8–2.0 (m), 1.7–0.8 (m) ppm. 13C NMR
(CDCl3): d 5 191.1, 146.6, 137.3, 133.5, 129.7, 112.9, 51.1,
29.3, 27.9, 22.6. FT-IR (cm21): 3028 (CAH), 1697 (CH@O),
1593 (C@C), 1462 (C@C), 1323 (CAN). Anal. calcd for
trophotometer equipped with
source.
a xenon lamp excitation
C52.41H66.25N0.51O0.51: C 89.0, H 9.31, N 1.10; found: C 88.59,
H 9.23, N 1.01.
Synthesis of Polymers
CP1: 3
Preparation of CPN
Green-emitting CPN were prepared by reprecipitation
method.13 All experiments were performed in aqueous
medium at room temperature. Typically, CP2 was first dis-
solved in THF to obtain 1 mg/mL stock solution. The CP2
solution (1 mL) was quickly added to Milli-Q water (12 mL)
under vigorous sonication. Then, THF was removed by nitro-
gen stripping. The solution was filtered with 0.45-mm syringe
filter. The aqueous medium containing dispersed CPN was
transparent and stable for 2 weeks without any aggregation.
(0.13 g, 0.30 mmol), 9,9-dioctyl-2,7-dibromofluorene (0.38 g,
0.70 mmol), 9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-
propanediol)ester (0.67 g, 1.20 mmol) were dissolved in a
mixture of THF (10 mL) and 2 M aqueous potassium carbon-
ate solution (4 mL). After addition of tetrakis(triphenylphos-
phine) palladium(0) (3.50 mg, 0.003 mmol), the reaction
mixture was stirred under argon at 100 ꢀC for 48 h. After
the reaction, the reaction mixture was cooled and poured
into methanol (500 mL), and resulting precipitates were iso-
lated by filtration. The precipitates were washed with metha-
nol and extracted with acetone for 48 h in a Soxhlet
apparatus to remove oligomers and catalyst residues. The
Preparation of pArg-Encapsulated CPN (pArg@CPN)
pArg was dissolved in water to prepare a stock solution (1
mg/mL). Then, 1 mL quantity of the pArg solution was
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SCHEME 1 Synthetic routes for monomers and polymers.
added to aqueous CPN solution (10 mL) under a vigorous
sonication. The mixture was further sonicated for 2 h. The
aqueous solution of pArg@CPN was stored in a refrigerator
to be used for the next step.
The absorption and emission spectra of the conjugated poly-
mers in THF solutions are exhibited in Figure 1. The main
absorption bands at 382 nm for CP1, 381 nm for CP2, and
383 nm for CP3 are attributed to the absorption of p2p*
transitions of the polymer backbones, which are independent
of the chemical composition of the backbone. In the fluores-
cence spectra, a major emission band at 490–501 nm
assigned to aldehyde-containing triphenylamine units was
observed for all the conjugated polymers. The intensity
around 420 nm, assigned to the polyfluorene moiety,
increases with the increase in the fluorene content in the
polymers; its effect is dominant in CP1. In CP2 and CP3,
stronger bluish green emissions from the aldehyde-
containing triphenylamine groups are observed because of
the energy transfer from fluorene units to aldehyde-
containing triphenylamine groups.14 Thus, the presence of
electron-withdrawing aldehyde group is essential for exhibit-
ing various emission colors by simple variation in feed ratios
Trypsin Sensing
The sensing studies were carried out in a quartz cuvette
with a path length of 10 mm. GO (20 mL) aqueous solution
(100.1 mg/mL) was added to a dispersion of pArg@CPN (2.5
mL) in 20 mM HEPES buffer (pH 5 7.4) and then the mix-
ture was incubated for 5 min at room temperature. Then,
trypsin was added into the solution. Subsequently, the solu-
tion was incubated at 37 ꢀC for 40 min. Photoluminescence
spectra of GO-pArg@CPN in the presence of the trypsin with
various concentration were investigated.
RESULTS AND DISCUSSION
Scheme 1 shows the synthesis of the polymers, which are
composed of a triphenylamine unit with a pendant aldehyde
group and a fluorene moiety with long alkyl chains, exhibit-
ing a reaction of 3, 9,9-dioctylfluorene-2,7-diboronic acid
bis(1,3-propanediol)ester, and 9,9-dioctyl-2,7-dibromofluor-
ene via Suzuki coupling polymerization in the presence of a
palladium catalyst. The structure of the polymer was con-
firmed by NMR and elemental analysis. The molar composi-
tion of each polymer was determined by elemental analysis
and is tabulated in Table 1. All the polymers were soluble in
common organic solvents such as THF, chloroform, and DMF.
The molecular weights of the polymers were determined by
gel permeation chromatography.
TABLE 1 Properties of Conjugated Polymers
Feed
ratio (m:n) Compositiona Mn
Mw
Polydispersity
b
b
CP1 0.3:0.7
CP2 0.5:0.5
CP3 0.7:0.3
0.27:0.73
0.40:0.60
0.52:0.48
6300
10,570 49,470 4.68
6680 19,220 2.88
20,320 3.22
a
Determined by elemental analysis.
Determined by GPC.
b
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Subsequently, CPN of CP2 was prepared, using the conven-
tional reprecipitation method, via the rapid addition of THF
solution of CP2 to water. The spherical nanoparticles were
successfully fabricated by the self-assembly of the hydropho-
bic polymer in the aqueous solution as shown in Figure 2(a).
The particle size distribution of the CPN of CP2, determined
by dynamic light scattering (DLS), is illustrated in Supporting
Information Figure S1(a) and Supporting Information Table
S1. A unimodal distribution can be observed for the solution
of the CPN with a mean diameter of 37.02 nm. The photo-
physical properties of CP2 in THF solution and the CPN of
CP2 in aqueous solution are compared in Supporting Infor-
mation Figure S2. Both CP2 in THF solution and CPN in
water showed very similar UV absorption (380 nm) and fea-
tureless, broad emission spectra centered at 491 nm, which
emitted a bluish green light because of the broad emission
in the long wavelength region. The quantum yields of CP2 in
THF and CPN in water were measured as 43 and 23%,
respectively, relative to rhodamine B in water.
The surface morphology of CPN in the presence of pArg was
further investigated with TEM [Fig. 2(b)]. The CPN were suc-
cessfully encapsulated by pArg (denoted as pArg@CPN); in
this material, the pArg surrounds each CPN. It can be
deduced that the negatively charged CPN were enveloped by
positively charged pArg through electrostatic interaction. As
expected, the average diameters of CPN determined by DLS
changed from 37.02 to 53.26 nm after modification with
pArg (Supporting Information Fig. S1 and Supporting Infor-
mation Table S1). For better insight into the interaction
between CPN and pArg, the zeta potentials for CPN before
and after treatment of pArg were investigated. As depicted
in Supporting Information Table S1, the net negative charge
of the CPN (234.6 mV) changed to 147.8 mV upon encapsu-
lation with pArg. The strongly negatively and positively
charged surfaces of the CPN and the resulting pArg@CPN,
respectively, imply good stability in solution over a long
period without precipitation.15 The positive charges on the
surface of pArg@CPN enabled further electrostatic interac-
tion with negative GO.
GO was prepared using the modified Hummers method.12
The prepared material nearly had a single layer with a topo-
graphic height of less than 2 nm according to atomic force
microscopy and SEM characterization (Supporting Informa-
tion Fig. S3). The chemically prepared GO was readily dis-
persible in water, mainly because of the presence of
hydrophilic groups such as hydroxyl (3420 cm21) and car-
boxylic groups (1740 and 1250 cm21) at the GO surface;
these were confirmed by FT-IR [Supporting Information Fig.
S3(c)].
FIGURE 1 UV absorption (filled) and normalized fluorescence
spectra (empty) of (a) CP1, (b) CP2, and (c) CP3 in THF solution
(1 3 1025 M for absorption of solutions). Excitation wavelength
kex 5 380 nm.
The working principle of our strategy is represented in
Scheme 2. pArg is introduced on the surface of CPN via elec-
trostatic interaction to obtain pArg@CPN. The nanohybrids
were fabricated by assembling pArg@CPN on GO nanosheets
via electrostatic interaction. The pArg on the surface of the
CPN serves as a linker to improve the immobilization of CPN
of monomers. Furthermore, different from other electron-
withdrawing groups, including carboxylic acid and sulfonic
acid, aldehyde groups do not have electrolyte property,
resulting in prevention of unfavorable, nonspecific interac-
tion with biomolecules.
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can be released from the hybrid because the surrounding
pArg is selectively hydrolyzed by the enzymatic action of
trypsin, resulting in a weakening of the electrostatic interac-
tion between GO and pArg@CPN. Finally, the nonemissive
nanohybrid disrupts and the released pArg@CPN becomes
re-emissive because of its trypsin activity.
Once the positive pArg@CPN is immobilized to the surface
of the negative GO, the nanohybrid can be easily formed via
electrostatic interaction. As a result, the fluorescence of the
resultant GO-pArg@CPN nanohybrid is quenched because of
charge–transfer interactions between the pArg@CPN and
GO.10(a),16 Figure 3 shows the emission spectral change of
GO-pArg@CPN according to the concentration of GO. About
80% quenching is observed in the presence of GO, caused by
photo-induced electron or energy transfer. Further complete
quenching is possible using GO. In such a case, the facile
recovery of fluorescence in the presence of trypsin will be
problematic because of the large amount of GO.
After preparation of the nanohybrid of GO-pArg@CPN, trypsin
was added to the mixture solution and the changes in fluores-
cence intensity were observed as the concentration of trypsin
was increased. In the concentration range of 0–25 mg/mL in
HEPES buffer solutions, the fluorescence intensity of GO-
pArg@CPN at 496 nm increased to 80% of the original inten-
sity of pArg@CPN [Fig. 4(a)]. The plot of the trypsin concen-
tration is linearly proportional to the intensity change with a
correlation coefficient (R) of 0.996 [Fig. 4(b)]. The limit of
detection (3r/slope) was found to be 0.827 mg/mL, as esti-
mated according to a method reported previously.17 The spec-
tral response was clearly attributed to the enzymatic activity
FIGURE 2 TEM images of (a) CPN from CP2 and (b)
pArg@CPN (scale bar 100 nm).
on GO. Simultaneously, the original green emission of
pArg@CPN is almost quenched because of energy transfer to
the electron-accepting GO. Upon exposure to trypsin, CPN
SCHEME 2 Schematic illustration for the detection of trypsin using nanohybrid of pArg@CPN and GO: (a) Green-emitting CPN; (b)
pArg@CPN; (c) fluorescence-quenched pArg@CPN via interaction between GO and pArg@CPN; (d) revival of green fluorescence of
pArg@CPN via hydrolysis of linker pArg by enzymatic action of trypsin, leading to releasing pArg@CPN. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
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GO-pArg@CPN. The relative selectivity of this system toward
trypsin detection is illustrated in Figure 5, which shows that
more than twofold emission enhancement can be attained
compared with other proteins. To the best of our knowledge,
the nanohybrid of GO and pArg-encapsulated CPN using the
specific activity of trypsin has not been exploited previously
for the turn-on detection of trypsin.
CONCLUSIONS
This work demonstrates a new assay based on the control of
interaction between GO and pArg-encapsulated CPN for tryp-
sin detection, in which CPN were prepared from a newly
synthezised conjugated polymer. By means of nanohybrid
formation of GO and pArg@CPN, the fluorescence of the
FIGURE 3 (a) Changes in emission intensity and (b) linear rela-
tionship of pArg@CPN in the presence of GO with various con-
centrations in 20 mM HEPES buffer at pH 5 7.4 (top to bottom
[GO] 5 0; 33.3; 50.1; 66.7; 83.4; 100.1; 116.8 mg/mL). Excitation
wavelength kex 5 380 nm. Io and I correspond to emission
intensity at 491 nm in the absence and presence of GO, respec-
tively. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
of trypsin. In this mechanism, the trypsin cleaves a peptide
chain at the carboxyl side of the pArg@CPN and, thus, weak-
ens the electrostatic interaction between the pArg@CPN and
GO, resulting in the release of CPN from GO.
To evaluate the effect of trypsin activity further, the increase
in fluorescence was investigated according to incubation time.
About 40 min of incubation time is sufficient for trypsin activ-
ity for this emission-enhancement system [Supporting Infor-
mation Fig. S4(a)]. To investigate whether this detection
method can enhance the selectivity, control experiments were
carried out using lysozyme, glucose oxidase (GOx), bovine
serum albumin (BSA), and pepsin instead of trypsin in HEPES
buffer solutions, as shown in Supporting Information Figure
S4. Other proteins and proteases did not show obvious fluo-
rescence enhancement, because other proteins cannot hydro-
lyze the pArg and, thus, cannot release the CPN from the
FIGURE 4 (a) Changes in emission intensity and (b) linear rela-
tionship of GO-pArg@CPN in the presence of trypsin with vari-
ous concentrations in 20 mM HEPES buffer at pH 5 7.4.
[GO] 5 100.1 mg/mL; [trypsin] 5 0; 5; 10; 15; 20; 25 mg/mL (from
bottom to top). Excitation wavelength kex 5 380 nm. Io and I
correspond to emission intensity at 491 nm in the absence and
presence of GO, respectively. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 5 Selectivity for trypsin compared to other proteins
and enzymes in 20 mM HEPES buffer at pH 5 7.4. [GO] 5 100.1
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pArg@CPN was effectively suppressed by GO. The presence
of trypsin selectively degraded pArg and, in turn, destroyed
the fluorescence-quenched nanohybrid assay, resulting in the
release of CPN. Therefore, fluorescence could be recovered
according to the concentration of trypsin. Because pArg was
employed as the linker of CPN and GO as well as selective
degrading sites by trypsin, unique selectivity was observed
over other proteins, which indicates this system can be an
efficient tool for potein sensing.
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Financial support from National Research Foundation of Korea
(NRF) funded by Korean government via Basic Science
Research Program (2012R1A2A2A01004979) is gratefully
acknowledged.
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