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Photosynthesis
Conjugated Polymer Nanoparticles to Augment Photosynthesis of
Chloroplasts
Yunxia Wang, Shengliang Li, Libing Liu,* Fengting Lv, and Shu Wang*
Abstract: By coating chloroplasts with conjugated polymer
nanoparticles (CPNs), a new bio-optical hybrid photosynthesis
system (chloroplast/CPNs) is developed. Since CPNs possess
unique light harvesting ability, including the ultraviolet part
that chloroplasts absorb less, chloroplast/CPN complexes can
capture broader range of light to accelerate the electron
transport rates in photosystem II (PS II), the critical protein
complex in chloroplasts, and augment photosynthesis beyond
natural chloroplasts. The degree of spectral overlay between
emission of CPNs and absorption of chloroplasts is critical for
the enhanced photosynthesis. This work exhibits good potential
to explore new and facile nanoengineering strategy for
reforming chloroplast with light-harvesting nanomaterials to
enhance solar energy conversion.
Compared with other energy sources, solar energy is the
most abundant, eco-friendly, and sustainable source.[1, 2]
Enhancing solar energy conversion is increasingly crucial.
Green plants can convert solar energy into chemical fuels by
photosynthesis. In natural photosynthesis systems, chloro-
plasts containing photoreactive elements are the critical
organelles. However, the absorbed light of chloroplasts is
limited to visible range of the spectrum in photosynthesis
process.[3] Thus, researchers have focused on constructing
artificial chloroplasts or chloroplast hybrid systems for
extending the range of solar light utilization.[4] Importantly,
transforming ultraviolet light to visible light required by
chloroplasts might be a good approach to improve the solar
energy conversion efficiency of chloroplasts.[5] Notably, light-
harvesting materials can effectively capture full range of
sunlight and transform into other energy, such as electrons
and photons. From this perspective, developing novel and
efficient light-harvesting materials for enhancing photosyn-
thesis is imperative.
Conjugated polymers (CPs), as outstanding light-harvest-
ing materials, possess a delocalized electronic structure and
large extinction coefficient, and thus they have been exten-
sively applied in optoelectronic devices, including field effect
transistors, light-emitting diodes, and photovoltaic devices.[6]
To prepare thin films with nanoscale domains in optoelec-
[*] Y. Wang, Dr. S. Li, Prof. L. Liu, Prof. F. Lv, Prof. S. Wang
Beijing National Laboratory for Molecular Sciences, Key Laboratory
of Organic Solids, Institute of Chemistry, Chinese Academy of
Sciences
Beijing 100190 (P.R. China)
E-mail: liulibing@iccas.ac.cn
wangshu@iccas.ac.cn
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201702376.
5392 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemie
Deutsche Ausgabe: DOI: 10.1002/ange.201702376
Internationale Ausgabe: DOI: 10.1002/anie.201702376
tronic devices, conjugated polymer nanoparticles (CPNs) are
further developed.[7] In view of their superior properties, such
as high brightness and quantum yield, excellent photostability
and low cytotoxicity,[8] CPNs have also been widely applied in
biosensors, optical imaging, and biomedicine.[9] These biolog-
ical applications generally combine the light-harvesting and
light-amplifying properties with biocompatibility of CPNs.
Among these CPNs, those made of polyfluorene have strong
absorption to ultraviolet light and emit intense blue light. This
characteristic exactly meets the demands for improving the
photosynthesis of chloroplasts. Inspired by the aforemen-
tioned findings, exploring light-harvesting CPNs for augment-
ing biological photosynthesis is promising. Herein we de-
scribe a new strategy for improving photosynthesis by
reforming chloroplast with CPNs. In this bio-optical hybrid
system, CPNs with large absorption cross section can absorb
and convert the ultraviolet light to visible light that could be
absorbed by chloroplasts, thus enhancing their photosynthetic
activity by accelerating the electron transport rates in photo-
system II.
Our strategy for enhancing photosynthetic activity of
chloroplasts with CPNs is illustrated in Scheme 1. As a proof
of concept, two typical light-harvesting conjugated polymers
with backbone structure containing fluorene units, poly[2,7-
(9,9-dihexylfluorene)-co-alt-p-phenylene] (PFP) and
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1’,3}-
thiadiazole)] (PFBT), are employed to prepare PFP-NPs and
PFBT-NPs, respectively. Under light irradiation, photosyste-
m II (PS II), the critical protein complex in chloroplasts, can
capture light to split water into protons, oxygen, and
electrons.[3, 4] After reforming chloroplast with CPNs, the
coated CPNs on the surface of chloroplasts can strongly
absorb incident light, especially ultraviolet light, and then
emit visible light that chloroplasts could absorb. Owing to the
superior light-harvesting property of CPNs, PS II can capture
more light transformed by CPNs to oxidize water, which
generates more electrons in electron-transport chain as
compared to that of natural chloroplasts, resulting in obvi-
ously improved photosynthetic activity.
Conjugated polymer PFP was synthesized as described in
a previous report[10] (Supporting Information, Figure S1), and
PFBT is commercial available. As shown in Figure 1 a, the
carboxyl-functionalized CPNs are prepared by the nano-
precipitation method[11] through rapidly co-precipitating con-
jugated polymers with poly(styrene-co-maleic anhydride)
(PSMA). Transmission electron microscopy (TEM) images
reveal the spherical morphology of the CPNs (Figure 1 b,c),
and dynamic light scattering (DLS) measurements show the
average diameters of PFP-NPs and PFBT-NPs are about
40 nm and 70 nm, respectively (Figure 1 d,e). From the optical
Angew. Chem. 2017, 129, 5392 –5395
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electron microscopy (SEM). As shown in Figure 2 b, upon

compositing with CPNs, the surface of chloroplast becomes
rough as compared with that of unmodified chloroplast, which
demonstrates that CPNs successfully on chloroplast mem-
brane possibly. The fluorescence lifetimes of chlorophyll and
CPNs were also investigated (Supporting Information, Fig-
ure S3). Since the fluorescence lifetimes of two components
were almost unchanged, it indicates that CPNs cannot enter
chloroplasts to interact with inside thylakoid. The result is
consistent with that of aforementioned experiments. To
further investigate driving force for interactions between
CPNs and chloroplast, isothermal titration calorimetry (ITC)
experiment was carried out (Figure 2 c). The binding affinity
constant between chloroplast and CPNs was 3.7 X 105 m @1,
which was calculated by fitting the ITC curve with one set of
site binding model. Considering that DH is of large positive
value while TDS is of comparable positive value, the
complexation of chloroplast with CPNs is an entropy-driven
process. Therefore the thermodynamic information shows
that CPNs interact with chloroplast membrane mainly
through hydrophobic interactions.
The light absorbed by chlorophyll molecules have three
Scheme 1. Illustration of the strategy for augmenting light reaction of
isolated chloroplasts utilizing conjugated polymer nanoparticles fates: driving photosynthesis, heat, and chlorophyll fluores-
(CPNs). The chemical structures of conjugated polymers for CPNs cence. These three pathways occur in competition. Consider-
construction are also shown. PS = photosystem, PFP = poly[2,7-(9,9- ing that the emission spectra of CPNs overlays the absorption
dihexylfluorene)-co-alt-p-phenylene], PFBT = poly[(9,9-dioctylfluorenyl- of chloroplast, fluorescence spectra of chloroplast/CPN com-
2,7- diyl)-co-(1,4-benzo-{2,1’,3}-thiadiazole)], DCPIP = 2,6-dichlorophe- plexes were measured at a maximum excitation wavelength of
nolindophenol dye, PQ = plastoquinone.
CPNs. As shown in Figure 3 a,b, the emissions of PFP-NPs
and PFBT-NPs are significantly quenched, while the emer-
gence of chloroplast emission indicates that chloroplast can
spectra shown in Figure 1 f,g, it can be
seen that PFP-NPs possess good
absorption of ultraviolet light (300–
400 nm), while antenna pigments of
chloroplast do not. PFBT-NPs possess
good absorption towards to ultraviolet
(300–400 nm) and visible light (400–
550 nm). Furthermore, the emission
spectra of both CPNs overlay well
with the absorption of chloroplast,
thus light transformed by CPNs is
beneficial for plant photosynthesis.[12]
Upon compositing with CPNs, the
absorption spectrum of chloroplasts
become broader, which provides the
possibility to promote photosynthetic
activity of chloroplast (Supporting
Information, Figure S2).
We first employed confocal laser
scanning microscopy (CLSM) to inves-
tigate the coating of CPNs on chloro-
plast membrane. As shown in Fig-
ure 2 a, blue fluorescent PFP-NPs and
green fluorescent PFBT-NPs are
around the perimeter of red fluorescent
chloroplasts, which indicates that both
Figure 1. Preparation and characterization of CPNs: a) Illustration for the preparation process of
CPNs are coated on the membrane of CPNs; b),c) TEM images of PFP-NPs (b) and PFBT-NPs (c); d),e) hydrodynamic diameters of PFP-
chloroplasts. The distribution pattern NPs (d) and PFBT-NPs (e) measured by DLS; f) absorption and g) emission spectra of PFP-NPs,
was further confirmed by scanning PFBT-NPs, and chloroplasts.

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Figure 2. a) Confocal images of chloroplast/CPN complexes; blue fluorescence is from PFP-NPs
(lex = 405 nm, lem = 415–465 nm), red fluorescence is from chloroplasts (lex = 559 nm, lem = 650–
750 nm), and green fluorescence is from PFBT-NPs (lex = 488 nm, lem = 500–545 nm). b) SEM
images of chloroplast, chloroplast/PFP-NPs, and chloroplast/PFBT-NP complexes. c) ITC fitting
data for the titration of chloroplasts to PFP-NPs.
absorb light transformed from CPNs to possibly conduct light
reaction. To confirm this hypothesis, photosynthetic activity
of chloroplast/CPN complexes was investigated by measuring
reduction rate of the dye 2,6-dichlorophenolindophenol
(DCPIP). DCPIP is an artificial electron acceptor that can
capture electrons transported from PS II to PS I in the
photoreaction of chloroplasts. Therefore the DCPIP reduc-
tion assay is a usual method to estimate the activity of PS II.[13]
Considering blue light is involved in a broad range of plant
processes and associated with the expression of sun-type
characteristics such as a high photosynthetic capacity at the
chloroplast level,[14] blue-emitting PFP-NPs were firstly
investigated. The experiments were conducted with the
optimal CPNs concentration (10 mg mL@1) and light intensity
(4 mW cm@2), which is 30 % of the light saturation for
photosynthesis (Supporting Information, Figure S2).[15] As
shown in Figure 3 c, under light irradiation, the absorbance at
600 nm gradually decreases with prolonged irradiation time,
which implies that the photosynthesis of chloroplasts them-
selves leads to DCPIP reduction. For the chloroplast/CPN
complex, the reduction rate of DCPIP is obviously acceler-
ated in comparison to chloroplast itself. CPNs could not
reduce DCPIP without chloroplasts, demonstrating the accel-
erated reduction rate of DCPIP is attributed to the combi-
nation of chloroplast and PFP-NPs. By binding to the
membrane of chloroplasts, PFP-NPs with good light-harvest-
ing ability strongly absorb ultraviolet light and transform to
blue light that could be absorbed by chloroplasts, thereby
enhancing the sunlight utilization of chloroplasts. As a con-
sequence, CPNs endow the chloroplast/PFP-NP system with
better solar energy conversion than natural chloroplast. As
shown in Figure 3 d, chloroplast/PFBT-NPs could also accel-
erate the reduction rate of DCPIP compared with chloroplast
5394 www.angewandte.de T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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itself. These results demonstrate that
the light transformed from CPNs to
chloroplast is critical for enhancing
photosynthetic activity of chloroplasts.
As illustrated in Figure 3 e, PS II can
split water into oxygen, protons, and
electrons, thus generating a proton
gradient for ATP synthase to produce
ATP under light irradiation, which is
termed photophosphorylation. To
investigate ATP production of chlor-
oplast and chloroplast/CPN complex,
the luciferin/luciferase assay was
employed, and result is shown in Fig-
ure 3 f. Apparently, the chloroplast/
CPN complex can produce more
ATP than chloroplast under light illu-
mination. It is consistent with the
result of DCPIP reduction assay
experiment. All these points indicate
that chloroplast/CPN complex show
enhanced photosynthetic activity than
chloroplast.
In summary, a new bio-optical
hybrid photosynthesis system is devel-
oped by coating chloroplasts with
conjugated polymer nanoparticles. The light-harvesting
CPNs were applied as optical amplifier to augment photo-
synthesis beyond natural chloroplasts. CPNs possess broad
light absorption, including the ultraviolet part; the chloro-
plast/CPN complex can capture broader range of light to
accelerate the electron transport rates in photosystem II in
chloroplasts and improve the photosynthetic activity. The
degree of spectral overlay between emission of CPNs and
absorption of chloroplasts is critical for the enhanced photo-
synthesis. This work exhibits good potential to explore new
and facile nanoengineering strategy for reforming chloroplast
with light-harvesting nanomaterials to enhance solar energy
conversion.
Experimental Section
Preparation of the chloroplast/CPN complex: A suspension of
chloroplast (5 mg mL@1) and CPNs (10 mg mL@1) was mixed suffi-
ciently for 1 minute. The free CPNs were removed by centrifugation
of the suspension (3000 rpm, 3 min). The precipitate was resuspended
with sucrose buffer (pH 7.3) to concentrate to 50 mg mL@1. A
suspension (10 mL) of chloroplast/CPN complex was added into
clean glass slides, followed by slightly covering coverslips for
immobilization. Confocal laser scanning microscopy images were
then taken. For SEM measurement, a suspension (4 mL) of chlor-
oplast/CPN complex was dropped on clean silicon slices and allowed
to evaporate at room temperature. After the specimens dried, 0.5 %
glutaraldehyde was added for fixation overnight. Next, 0.5 %
glutaraldehyde was removed and the specimens were washed with
sterile water for 2 times. Ethanol was added in a graded series (20, 40,
50, 70, 90, and 100 % for 6 min, respectively) followed by natural
drying in the air. After coated with platinum, the specimens were
measured by SEM.
Photosynthesis measurement of the chloroplast/CPN complex:
For experimental groups, a suspension of chloroplast (5 mg mL@1),
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Figure 3. a) Fluorescence spectra of PFP-NPs, chloroplast, and chlor-
oplast/PFP-NP complex with an excitation of 380 nm. b) Fluorescence
spectra of PFBT-NPs, chloroplast, and chloroplast/PFBT-NP complex
with an excitation of 475 nm. c),d) The absorption changes of DCPIP
at 600 nm for chloroplast (black line), CPNs (red line), and chloro-
plast/CPNs (blue line) upon irradiation with a light intensity of
4 mWcm@2 for 5 min. e) Illustration of photophosphorylation of chlor-
oplast under light irradiation. f) ATP production of PFP-NPs, chloro-
plast, and chloroplast/PFP-NP complex under light irradiation.
DCPIP (60 mm), and CPNs (0, 1, 5, 10 mg mL@1) was mixed sufficiently
for 1 minute. For the control group, there was no chloroplast in the
solution. All of the suspensions were divided into six groups. Upon
light irradiation for certain time (0, 1, 2, 3, 4, and 5 min, respectively),
the absorbance of the solution at 600 nm was measured. The light
intensity for irradiation is 1, 4, and 8 mW cm@2, respectively.
Acknowledgements
The authors are grateful to the National Natural Science
Foundation of China (Nos. 91527306, 21533012, 21473220),
and the Strategic Priority Research Program of the Chinese
Academy of Sciences (XDA09030306).
Conflict of interest
The authors declare no conflict of interest.
Angew. Chem. 2017, 129, 5392 –5395 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Keywords: chloroplasts · conjugated polymers ·
light harvesting · photosynthesis · polymer nanoparticles
How to cite: Angew. Chem. Int. Ed. 2017, 56, 5308 – 5311
Angew. Chem. 2017, 129, 5392 – 5395
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Manuscript received: March 6, 2017
Final Article published: April 5, 2017
www.angewandte.de 5395
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