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Protein conformation driven biomimetic synthesis of semiconductornanoparticles

Debasmita Ghosh, Somrita Mondal, Srabanti Ghosh and Abhijit Saha*

Received 3rd August 2011, Accepted 3rd October 2011

DOI: 10.1039/c1jm13730a

The present investigation demonstrates the role of protein conformation in synthesizing nanoparticles

(NPs) through biomimetic route. Highly water-soluble and biocompatible CdS and CdSe nanoparticles

in bovine serum albumin (BSA) matrix have been synthesized using a simple and controllable method

at room temperature. Fourier transform infrared (FTIR) data are used to envisage the binding of the

semiconducting particles with amide and OH groups of the protein molecule. Optical absorption and

emission spectra confirm that particles formed lie within the size quantization regime. Circular

dichroism spectroscopy reveals that BSA adopts different conformations at different pH which in turn

controls the particle size. Further, addition of sodium borohydride (NaBH4) in BSA solution results in

breakage of disulfide bonds generating increased number of thiolate groups which provide better

stabilization and increased passivation of electronic defects on particle surface. In the process, better

quality semiconductor NPs with higher quantum yield are produced. Thus, by modulating the protein

conformation, the size and quality of the nanoparticles can be controlled.

Introduction

Semiconductor nanocrystals have generated great research

interest in the past two decades because of their unique size-

dependent photophysical and photochemical properties.1–4

Recently, biosynthesis and biological surface modification of

semiconductor5–8 and metal nanoparticles9,10 with biomolecules

such as proteins and nucleic acids have added a new dimension to

nanoparticle research. Binding of certain biomolecules on the

nanoparticle surface could impart biocompatibility to these

particles for their subsequent use in various biological applica-

tions.11–14 The availability of these novel biomodified nano-

structures can greatly facilitate development of in situ probes and

biosensors. DHLA-coated core–shell CdSe–ZnS semiconductor

quantum dots have been used for stabilization of vaterites,

resulting in intrinsic fluorescent scaffolds.15 Using these DHLA-

quantum dot-stabilized vaterites, FRET-based biosensing of

biological targets including fluorescent proteins and streptavidin

conjugated quantum dots has been done. In the framework of

colloidal chemistry, the two general existing strategies of nano-

crystal preparations are high temperature organometallic

synthesis16,17 and synthesis in aqueous media using different

stabilizing agents18–22 which cap the nanoparticle surface thereby

preventing agglomeration and passivating surface electronic

defects. Different bioactive end groups such as thiol, –NH2,

–COOH etc.23–25 have been used to synthesize biocompatible

UGC-DAE Consortium for Scientific Research, Kolkata Centre, III-LB/8Bidhannagar, Kolkata, 700 098, India. E-mail: abhijit@alpha.iuc.res.in;Fax: +913323357008; Tel: +913323351866

This journal is ª The Royal Society of Chemistry 2012

nanoparticles. The conjugation of nanoparticles with biological

molecules represents combination of nanotechnology and

biotechnology where novel hybrid materials can be synthesized

by incorporating the unique optical and electronic properties of

nanoparticles and highly selective binding of proteins and

oligonucleotides.

In recent years, biomimetic synthesis has become a hot topic.

There are many studies, which show that biological macromol-

ecules, such as amino acids, proteins, DNA and RNA, are

capable of controlling nucleation and growth of nanomaterials

to different degrees.26 The biomolecule-conjugated nano-

materials can provide bioactive functionalities on the nano-

crystal surface for further biological interactions or couplings

and these materials can be used in life sciences for luminescence

tagging, drug delivery, and many other aspects.

Herein, we selected CdS nanocrystals and BSA as model

systems for investigating different aspects of the biomimetic

synthesis of semiconductor nanocrystals. Bovine serum albumin

(BSA), one of the most widely studied proteins,27 has been

frequently adopted to synthesize various nanocrystals. BSA is an

excellent foaming agent. It is known that aqueous foam is an

excellent template for the growth of nanoparticles over a range of

chemical compositions. There are many reports on synthesis of

various metal nanoparticles,28 minerals29 and oxide nano-

particles29 in a foam template. For example, there are reports on

biomimetic synthesis of protein capped Ag,30,31 Au,30 Pt,31 Ag–

Au,30 Ag–Pt31 nanomaterials in aqueous BSA foam, BSA-conju-

gated Ag2S nanorods,32,33 HgS,34 PbS,35 CuS nanoparticles,36

CuSe nanosnakes37 etc.These synthesized 1D nanomaterials have

unique electrical, optoelectronic, biological, and mechanical

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properties with fundamental significance and great potential in

applications such as electrochemical storage cells, solar cells,

solid-state electrochemical sensors, semiconducting optical

devices, catalyst, superionic materials, and biomedical engi-

neering.38–41Hence, fast developingmethodologies for fabrication

of these types of materials have spurred interest in the field of

materials, micro-electronics, and nanotechnology in recent years.

Despite the synthesis of a number of materials through biomi-

metic route, little attention is paid to unravelling the intricacy of

the protein conformation in determining the characteristics of the

synthesized nanocrystals. Hence, manoeuvring the different

conformations of proteins as assistant media to fabricate nano-

particles with controllable size, shape and unique properties is still

a great challenge. It is well known that BSA has different

conformations at different pH values.42,43 In the present study, we

have endeavoured to look into the role of conformation of BSA in

controlling the size and quality of nanoparticles. We have

synthesized CdS and CdSe nanocrystals in the BSA matrix by

varying pH in the media. In addition, borohydride has been

incorporated in the media, which can open up secondary/tertiary

structure of the protein molecule thereby enabling a greater

exposure of thiol stabilization of the growing nanocrystals. The

properties of these as-prepared products have been investigated

by UV–vis spectroscopy, photoluminescence spectroscopy (PL),

high-resolution transmission electron microscopy (TEM),

selected-area electron diffraction (SAED), dynamic light scat-

tering (DLS) and circular dichroism (CD) spectroscopy. We have

demonstrated that size and size distribution of the particles and

luminescence quantum efficiency can be manipulated by

controlling the protein structure in the biomimetic method.

Materials and methods

Materials

CdCl2, Na2S and sodium-borohydride were purchased from

Seisco Research Laboratory, India. BSA was purchased from

Sigma-Aldrich, USA. All chemicals used were of analytical grade

or of high purity available. Milli-Q water (Millipore) was used as

a solvent.

Synthesis of CdS and CdSe nanoparticles

The synthesis of CdS NPs was carried out following two different

pathways, one with adding NaBH4 as disulfide cleavage agent

and other one without adding borohydride. In the first proce-

dure, an aqueous solution containing Cd2+ ions and BSA of

appropriate amount was prepared and foam was generated while

nitrogen was bubbled through the solution to remove dissolved

oxygen. An aqueous solution of Na2S was added into the mixture

and a yellow colored solution of CdS NPs was obtained. In the

second procedure, NaBH4 was added to the BSA solution in

nitrogen purged conditions prior to the addition of an aqueous

solution of Cd2+ ions and kept for 1 h. Thus, in order to study the

influence of BSA on CdS crystallization, the concentration of

BSAwas varied. In a typical synthesis, 4 ml BSA solution (3.0 mg

ml�1) was mixed with 1 ml of 2� 10�3 M CdCl2 solution in water;

the solution was left at 25 �C for some time to facilitate

a chelating balance. To this reaction mixture, an aqueous solu-

tion of fresh Na2S (1 � 10�3 M) was added. The reaction system

700 | J. Mater. Chem., 2012, 22, 699–706

was left at 25 �C overnight. The obtained CdS nanocrystals were

separated from the yellowish colloid by centrifugation, then

washed with water and ethanol three times, respectively,

and dried in vacuum. In all preparations, the final molar ratio of

Cd2+ : S2� was kept as 1 : 0.5. In order to see pH effect, synthesis

was carried out at different pH values, such as pH 2, pH 4 and

pH 6.

BSA capped CdSe NPs were also synthesized by the same

procedure using NaHSe solution as the selinide source. In the

preparation of NaHSe solution, selenium powder was suspended

in minimum amount of water (�0.2 ml) and the solution was

heated with borohydride under continuous flow of nitrogen gas

until it becomes colourless. Then the colourless solution was kept

in an ice bath for 2–3 h, after which a white precipitate of sodium

tetraborate came out. Now the clear supernatant containing

NaHSe free from contamination of borates was used for the

synthesis.

Characterization

Absorbance spectra of CdS NPs were taken using a Shimadzu

(UV1601PC) spectrophotometer. Photoluminescence (PL)

measurements were performed at room temperature using

a Perkin Elmer (LS 55) Luminescence spectrometer. Photo-

luminescence quantum efficiency (PLQE) of CdS nanocrystals

was estimated following the procedure described in the litera-

ture,44,45 by taking quinine sulphate in 0.1 M H2SO4 as the

standard and comparing the integrated intensity for the cor-

rected PL spectra of the two fluorophores.

FTIR spectroscopic measurements of crystalline BSA and

BSA-capped CdS in solid precipitated form were recorded using

a FTIR spectrometer (Perkin Elmer, Spectra GX). Samples for

FTIR measurements were prepared in the form of pellets by

mixing 200 mg of IR spectroscopic grade potassium bromide

with 2 mg of dried sample (i.e., BSA and BSA-capped CdS). The

spectra were recorded in transmission mode over 20 scans with

a resolution of 4 cm�1.

TEM was carried out on a FEI, Technai S-twin with an

acceleration voltage of 200 kV. A drop of aqueous solution of

CdS NPs was placed on a carbon-coated copper grid of 400 mesh

and dried before putting it into the sample chamber of the TEM.

Next, size distribution measurements of the nanoparticles were

made by dynamic light scattering (Model DLS-nanoZS, Zeta-

sizer, Nanoseries, Malvern Instruments). Samples were filtered

several times through a 0.22 mm Millipore membrane filter prior

to measurements.

The conformation changes in the secondary structures of BSA

in the reaction system were determined by circular dichroism

(CD) spectroscopy using a Jasco-810 spectropolarimeter, fitted

with a xenon lamp and calibrated with (+)-D-10-camphor

sulfonic acid. The light path length of the cell used was 5 mm in

the near-UV region.

Determination of average particle size

Semiconductor nanoparticles exhibit a blue shift in their

absorption spectra as the size is reduced below the characteristic

Bohr exciton diameter of the bulk material.46,47 The quantitative

relationship between absorption spectra and particle size is now

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well understood.48 The band gap (Eg) was calculated from

absorption onset (lonset) in the UV–Vis absorption spectrum of

each nanoparticle solution using the relationship, Eg ¼ hc/lonset,

where h is Planck’s constant and c the speed of light. The average

size of nanoparticles (d) was obtained using the correlation of

band gap shift (DEg ¼ Eg(nanocrystal) � Eg(bulk)), and the

particle size was deduced by using the tight-binding approxi-

mation48 (eqn (1)).

DEg ¼ a1e�d/b1 + a2e

�d/b2 (1)

The values of the parameters for CdS nanoparticles are a1 ¼2.83, b1 ¼ 8.22, a2 ¼ 1.96, b2 ¼ 18.07 and those for CdSe

nanoparticles are a1 ¼ 7.62, b1 ¼ 6.63, a2 ¼ 2.07, b2 ¼ 28.88. The

particle size determined by this optical method was found to be in

good agreement with the size obtained from TEM

measurements.

Fig. 2 (a) UV–vis absorption spectra and (b) PL spectra of CdS–BSA

for different concentrations of BSA.

Results

UV–visible and photoluminescence spectroscopy

Fig. 1 illustrates the UV–vis absorption spectra of pure BSA,

BSA–Cd2+, and BSA–CdS in the wavelength range of 200–

600 nm. The spectrum of pure BSA showed an intrinsic

absorption at 280 nm primarily due to tryptophan and tyrosine

groups. The spectrum of BSA in the presence of Cd2+ showed no

significant shift in absorption peak at 280 nm, but absorbance at

this wavelength increased considerably.

In order to investigate the dependence of different parameters

for the size control of nanoparticles in the biomimetic route, the

evolution of particle growth as a function of protein concentra-

tion was followed. Fig. 2(a) shows the variation of particle size of

CdS nanoparticles synthesized with different concentrations of

BSA at pH 6. The average particle size of CdS NPs at different

concentrations of BSA was determined from absorption onset

using the correlation of the bandgap as described in the char-

acterization section. Fig. 2(b) represents the PL spectra of CdS–

BSA NPs synthesized with different concentrations of BSA

solution.

Fig. 1 UV–vis absorption spectra of (a) BSA, (b) BSA–Cd2+ and (c)

BSA–CdS.

This journal is ª The Royal Society of Chemistry 2012

Since pH can control the BSA conformation, it can also

contribute to the particle qualities. Thus, pH dependent UV–Vis

spectra of BSA–CdS NPs were recorded (Fig. 3). It is observed

Fig. 3 UV–vis absorption spectra and inset: PL spectra of BSA–CdS

NPs at different pH of BSA.

J. Mater. Chem., 2012, 22, 699–706 | 701

Fig. 5 (a) and 5a: inset are UV–vis and PL spectra, respectively, of BSA–

CdS. (b) and 5b: inset are UV–vis and PL spectra, respectively, of BSA–

CdSe.

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that the UV profile is more structured when CdS NPs were

synthesized in BSA solution of pH 6. Further, the observed

average particle size was found to vary with the pH of the BSA

solution (4 nm at pH 6, 5.2 nm at pH 4 and 6.4 nm at pH 2).

BSA capped CdS nanoparticles also show pH dependent

luminescence. CdS NPs show maximum fluorescence intensity in

the case of BSA solution of pH 2, as shown in Fig. 3 (inset).

Besides CdS NPs, the effect of pH was also investigated in the

formation of CdSe NPs in BSA matrix (Fig. 4 and inset). The

trends in the pH dependence of the particle size and the lumi-

nescence intensity were found to be similar to those observed for

the formation of CdS–BSA.

Further, to look into the role of NaBH4, a well-known

reducing agent, in the protein-mediated synthesis of nano-

particles, we carried out the synthesis processes for both CdS and

CdSe nanoparticles in the presence of borohydride. Fig. 5(a) and

5(b) show the UV–vis and PL spectra of BSA capped CdS and

CdSe nanoparticles in the presence of NaBH4, respectively. If we

compare the UV–vis spectra in the presence and in the absence of

NaBH4 at a particular pH, then, it is seen that in the presence

of NaBH4, we get a better UV–vis profile indicating narrowing of

the size distribution. A simple, but accurate, approach has been

used to estimate the size distribution of the nanocrystals from the

UV–vis absorption spectrum.49 This approach has been found to

work well for a variety of semiconductor nanocrystals in the

quantum confinement regime.50 Because of NaBH4, the relative

percentage distribution, as calculated, decreased from 10% to

3.5% in the case of BSA–CdS and from 12% to 4% in the case of

BSA–CdSe at pH 6. Comparing PL spectra of BSA–CdS or

BSA–CdSe in the presence (Fig. 5(a): inset and 5(b): inset) and

absence of NaBH4 (Fig. 3: inset and Fig. 4: inset), it was observed

that the presence of NaBH4 caused a significant increase in the

PL intensity of BSA capped nanoparticles at all pH values

studied. Photoluminiscence quantum efficiency (PLQE) also

increased in presence of NaBH4. In the case of BSA–CdS, PLQE

goes up from 10% to 16%, in the presence of NaBH4 and in the

case of CdSe, PLQE goes up from 20% to 36%. However, when

the pH dependence effect on the size and luminescence of NPs

was followed in the presence of NaBH4, the trend was similar to

that observed in the absence of NaBH4.

Fig. 4 UV–vis absorption spectra and inset: PL spectra of BSA–CdSe

NPs at different pH of BSA.

702 | J. Mater. Chem., 2012, 22, 699–706

TEM measurements

A typical TEM image for CdS NPs is shown in Fig. 6. The CdS

NPs are approximately spherical in shape. The average particle

size determined is 4 nm. The related SAED pattern (Fig. 6(a):

inset) shows principal rings corresponding to the 111 and 200

planets of cubic CdS. The HRTEM image in Fig. 6(b) provides

further insight into the structure of the products. The image

exhibits lattice fringes with d spacing of 2.05 �A, which corre-

sponds to 220 planes in cubic CdS crystallites.

DLS measurements

The average sizes of the synthesized particles were determined

by DLS measurements (Fig. 7(a)) as 5, 6 and 8 nm at pH 6, 4

and 2, respectively, which are slightly larger than the sizes

determined by UV–vis absorption spectroscopy. This is

consistent with the established notion that it is the hydrody-

namic diameter of the colloidal particles that gets measured by

the DLS technique. Again the size of BSA at different pH was

also determined (Fig.7(b)).

FT-IR measurements

To study the formation mechanism of CdS NPs in BSA matrix,

the FTIR spectra of pure BSA and BSA–CdS powders were

This journal is ª The Royal Society of Chemistry 2012

Fig. 6 (a) TEM image of CdS NPS, inset: SAED pattern and (b)

HRTEM.

Fig. 7 DLS measurements of (a) BSA–CdS and (b) BSA at different pH

values.

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determined. The FTIR spectra and the data of the main peaks

are demonstrated in Fig. 8. The IR peaks of pure BSA at 3430,

1653, and 1531 cm�1 are assigned to the stretching vibration of

–OH, amide I (mainly C]O stretching vibrations), and amide II

(the coupling of bending vibrations of N–H and stretching

vibrations of C–N) bands, respectively.36,37

Fig. 8 FTIR spectra of BSA and BSA–CdS.

Circular dichroism spectroscopy

To further study the change in conformation of BSA, the

secondary structures of BSA in the reaction system were deter-

mined by CD spectroscopy (Fig. 9), which is a valuable spec-

troscopic technique for studying protein and its complex.

Circular dichroism is observed when molecules absorb left and

right circularly polarized light to different extents. The amide

chromophore of the peptide bond in protein dominates their CD

spectra below 250 nm. In the a helix structure of protein,

a negative band near 220 nm is observed due to the strong

hydrogen-bonding environment of this conformation, a second

transition at 190 nm is split into a negative band near 208 nm and

This journal is ª The Royal Society of Chemistry 2012

a positive peak near 192 nm. The CD spectra of b sheet display

a negative band near 216 nm, a positive band between 195 and

200 nm, and a negative band near 175nm. The CD spectra of

pure BSA and BSA–CdS solutions at different pH values are

J. Mater. Chem., 2012, 22, 699–706 | 703

Fig. 9 CD spectra at different pH for (a) BSA, (b) BSA in the presence of NaBH4, (c) BSA–CdS and (d) BSA–CdS in the presence of NaBH4.

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given in Fig. 9. From the figure, it can be seen that the CD curve

of BSA is different from that of BSA–CdS.

Fig. 10 The three conformers of BSA, which are N (Normal), F (Fast)

and E (Expanded) at pH 6, 4 and 2, respectively.

Discussion

We have described here the role of BSA conformation in

controlling the size and quality of nanoparticles. BSA has

different functional groups. In the process of synthesising CdS

particles, Cd2+ ions can possibly attach initially with –NH,

and/or –OH groups of tryptophan and/or tyrosine, respec-

tively, of the BSA molecule. This may result in increased

absorbance [Fig. 1]. However, when Na2S was added in the

solution mixture of BSA and Cd2+, the absorption peak shifted

to a higher wavelength at about 340 nm, which is due to the

formation of CdS nanocrystals in the BSA matrix.51 The

difference between the IR spectrum of pure BSA and that of

BSA–CdS is obvious from the shift of the characteristic peak

of the –NH groups, suggesting a coordination interaction

between CdS and –NH groups of BSA and this may play an

important role in the formation of CdS nanoparticles.

Comparing the IR spectra of BSA–CdS with those of pure

BSA, the characteristic peak of the –OH groups shifts to

a higher wavenumber and it has broadened in the case of

BSA–CdS. The results indicate that there might be conjugate

bonds between the CdS nanoparticles and –OH groups and

–NH groups of BSA.

It can be seen from light scattering measurements [Fig.7] that,

the as-synthesized particles are of almost the same sizes as those

for pure BSA at different pH. So, it can be reasonably assumed

that the particle formation occurs in the core of BSA, which

controls the growth of the nanoparticles.

704 | J. Mater. Chem., 2012, 22, 699–706

From Fig. 2(a), it was observed that for a given concentration

of Cd2+ ions (2.0 mM), the size of CdS particles varied from 7 nm

to 3.8 nm with increasing concentration of BSA from 2 mg ml�1

to 4 mg ml�1. Further increase in BSA did not affect the particle

size. It appears from these results that size of CdSNPs in the BSA

matrix gradually decreases with the increase of BSA concentra-

tion and reaches a limiting value. So, we can easily control size-

tunable optical properties of nanoparticles through altering the

concentration of BSA.

Fig. 2(b) indicates that the PL intensity increases with the

increase of BSA concentration. Higher concentrations of BSA

offer a greater number of functional groups such as thiol, –NH2,

–OH, –COOH which can help in passivating the electronic

defects on the surface of the nanoparticles.

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BSA has reversible conformational isomerization in different

pH conditions. When the pH value is lower than 4.7, BSA

undergoes another expansion with a loss of the intra-domain

helices (10) of domain I, which is, connected to helix (1) of

domain II and that of helix (10) of domain II connected to helix

(1) of domain III.27,28 Thus, BSA has three conformers, which are

N (Normal), F (Fast) and E (Expanded) at pH 6, 4 and 2,

respectively (Fig. 10).

These conformational variations result in the formation of

different sizes of semiconductor nanoparticles. Nanoparticles

bound with the N form have the smallest size and particles bound

with the E form have the largest size due to the expanded

conformation of BSA.

Again, NaBH4 acts as a disulfide cleavage agent. Thus, it

breaks the disulfide bonds in BSA resulting in increased avail-

ability of thiol groups which are very efficient stabilizing agents.

So, due to better stabilization in the presence of NaBH4, particles

of smaller size and improved luminescence quantum efficiency

are produced in comparison to those obtained in the absence of

NaBH4.

Since BSA conformation is more open at low pH, NaBH4

can access more disulfide bonds, and thus more sulfide groups

are produced at lower pH. From CD spectroscopic results, it is

observed that the conformation of BSA is changed due to

variation of pH of the solution or the presence of NaBH4 or

formation of BSA–CdS nanocomposites [Fig.9]. With the

decrease in pH of the solution, more and more a helix were

stretched and transformed into b sheets, which could contribute

to the impairment or break of hydrogen bonds.34 the b sheet

secondary structure forms a suitable conformation for the

oriented growth of CdS NPs which result in CdS NPs

uniformly coated with BSA. Again, NaBH4 breaks disulfide

bonds of BSA. So, in the presence of NaBH4, the –S� groups of

BSA take part in formation of BSA-capped CdS NPs. The

interaction of thiolate with the surface of the CdS NPs

passivates the surface traps arising from electronic defects. As

more such functional groups are present, more surface traps

gets passivated. It prevents non-radiative emission of the

absorbed energy thereby increasing the luminescence. With the

decrease of pH, more a helix are stretched making more

functional groups, like –OH, –NH, –COOH, accessible to

interact with the surface of NPs, and more disulfide bonds

become accessible to NaBH4 which reduces the disulfide bonds

and produces BSA with more –S� groups. Therefore, lumi-

nescence intensity of the as-prepared materials is high at low

pH owing to improved surface passivation of the particles. The

luminescence has been further improved in the presence of

NaBH4 as a result of greater exposure of thiol groups due to

borohydride-induced disulphide cleavage in BSA.

Conclusions

CdS and CdSe nanoparticles were synthesized at room temper-

ature using BSA as matrix. The different conformations of BSA

at different pH values control the size and quality of the

synthesized nanoparticles. Addition of NaBH4 to BSA causes

breakage of disulfide bonds, and produces thiolate ions, which

gives better stabilization. This has led to the formation of better

quality semiconductor NPs with higher PLQE. Thus, by

This journal is ª The Royal Society of Chemistry 2012

manoeuvring the conformation of protein, the size and quality of

nanoparticles can be controlled.

Acknowledgements

The authors are grateful to the Saha Institute of Nuclear Physics

and the Central Glass & Ceramic Research Institute for

providing the Electron Microscope Facility. One of the authors

(S. M.) is thankful to the University Grants Commission for

a NET fellowship.

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