RESEARCH PAPER

Green and facile synthesis of water-soluble ZnS quantumdots nanohybrids using chitosan derivative ligands

Fabio P. Ramanery • Alexandra A. P. Mansur •

Fernanda G. L. M. Borsagli • Herman S. Mansur

Received: 5 April 2014 / Accepted: 5 June 2014 / Published online: 24 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Semiconductor quantum dots (QDs) are

fluorescent nanocrystals with great potential for use in

biomedical and environmental applications. However,

eliminating the potential cytotoxicity of the QDs

comprised of a core containing heavy metals and using

a green chemical process are still challenges faced by

the research community. Thus, the aim of this work

was to develop a novel green and facile route for

synthesizing biocompatible water-soluble QDs using

chemically modified chitosan as a capping ligand in

aqueous media, with their chemical and optical

properties tuned by the nanoparticle size. The synthe-

sis of ZnS QDs capped by carboxymethylchitosan

(CMC) was performed using a single-step aqueous

colloidal process at room temperature. The nanohy-

brids were extensively characterized by several imag-

ing and spectroscopic techniques, and the results

demonstrated that ultra-small ZnS nanocrystals were

produced with average nanoparticles ranging from 3.2

to 4.2 nm. In addition, the luminescent properties of

ZnS QDs were influenced by the pH during the

synthesis due to the size distribution of the

nanoparticles produced. Hence, new ‘‘heavy metal

free’’ nanohybrids were successfully developed based

on ZnS QDs directly surface-functionalized by bio-

polymer exhibiting fluorescent activity that may be

potentially used in a large number of eco-friendly and

biomedical applications.

Keywords Nanoparticle � Quantum dot �Nanomaterials � Colloids � Chitosan � Nanohybrid

Introduction

In the last two to three decades, innovation in nanotech-

nology and nanoscience for biology, medicine, and

environmental applications has been a major driving

force in the creation of new nanocomposites, nanohy-

brids, and nanoconjugates. This new class of materials

may bring together the intrinsic functionalities of

inorganic nanoparticles combined with the biocompat-

ibility and eco-friendly interfaces offered by biomole-

cules and biopolymers, such as carbohydrates,

glycoconjugates, proteins, and enzymes (Emerich

2005; Tan et al. 2007; Etheridge et al. 2013). Among

the alternatives of nanomaterials, ultra-small semicon-

ductor nanocrystals, which are known as quantum dots

(QDs), have lately emerged as an advanced class of

materials with unique physico-chemical, electronic, and

optical properties. Despite exhibiting interesting prop-

erties, the use of QDs in environmental applications has

only recently attracted attention from scientists (Zhang

F. P. Ramanery � A. A. P. Mansur �F. G. L. M. Borsagli � H. S. Mansur (&)

Center of Nanoscience, Nanotechnology and Innovation -

CeNano2I, Department of Metallurgical and Materials

Engineering, Federal University of Minas Gerais, Av.

Antonio Carlos, 6627 – Escola de Engenharia,

Bloco 2 – Sala 2233, Belo Horizonte,

MG 31270-901, Brazil

e-mail: hmansur@demet.ufmg.br

123

J Nanopart Res (2014) 16:2504

DOI 10.1007/s11051-014-2504-1

et al. 1998; Rajabi et al. 2013). As a major drawback,

most of the experimentally studied and commercial QDs

are synthesized via traditional organometallic methods,

using highly toxic elements, such as cadmium, lead and

mercury, and use organic solvents and ligands at high

temperatures, which are undesirable in eco-friendly

processes. Compared with organic-phase synthesis,

aqueous-phase process is much simpler, greener, and

cheaper. Currently, the most commonly used QDs are

made with Cd2? (CdS, CdSe, and CdTe), which is a

heavy metal severely harmful to the environment

because of its toxicity to plants, animals, and micro-

organisms. As a result, cadmium-free QDs, such as zinc-

based nanoparticles, are very promising alternatives due

to the environmental abundance and natural presence of

Zn2? in the human body (Lim et al. 2013; Rajabi et al.

2013). For that reason, zinc sulfide (ZnS) has been the

most frequent QD semiconductor for the application in

biomedical and chemical catalysis, taking advantage of

both the low toxicity and the huge surface area-to-

volume ratio of QDs compared with their bulk counter-

parts (Rajabi et al. 2013). However, QDs exhibit a strong

thermodynamic tendency to agglomerate and grow

during operation that is highly unfavorable for the

overall stability of the system. Therefore, the develop-

ment of molecular ligands for the synthesis of QDs is

increasingly important because they will play a crucial

role in determining the overall physico-chemical stabil-

ity and environmentally friendly nanoconjugates. In

view of the large number of available alternatives to

produce core–shell (inorganic–organic) hybrids, carbo-

hydrates, such as cellulose, chitosan, and starch, have

been often a common choice of ligand, due to their

biocompatibility, physico-chemical and mechanical

properties, and relative chemical solubility and stability

in aqueous physiological environment (Tan et al. 2007;

Dash et al. 2011). Chitosan (poly-b(1?4)-2-amino-2-

deoxy-D-glucose) is one of the most abundant polysac-

charides (semi-processed) from natural sources, second

only to cellulose (Tan et al. 2007; Dash et al. 2011).

Chitosan and its derivatives are promising options as

capping ligands for stabilizing nanosize particles for a

wide range of applications. They contain a large number

of chemical groups, such as hydroxyls and amino

groups, and other functionalities may also be added to

the polymer chain, resulting in strong interactions and

adsorption properties with different molecules and ions.

However, chitosan is reasonably soluble only in acidic

water solutions with poor solubility above pH 6.5

(pKa = 6.5). The solubility of chitosan can be improved

by introducing chemical groups, as it has reactive amino,

primary hydroxyl, and secondary hydroxyl functional

groups, which can be used for chemical modifications

under relatively mild reaction conditions. Consequently,

chitosan derivatives like carboxymethylated chitosans

(O-carboxymethylchitosan and N-carboxymethyl chito-

san), which are water-soluble in acidic, alkaline, and

physiologic circumstances, may be good candidates as

ligands for preparing colloidal semiconductor nanopar-

ticles (Chen and Park 2003; Mansur et al. 2013). These

combined features of chitosan and its derivatives offer

limitless possibilities for developing water-dispersible

QD conjugates for biological, pharmaceutical (Mansur

et al. 2012, 2013; Santos et al. 2013), and environmental

purposes (Jiang et al. 2012). Unexpectedly, reports on

the surface bio-functionalization of colloidal QDs with

chitosan and its derivatives are scarcely found in the

literature (Tan et al. 2007; Chang et al. 2011), and only

recently the direct synthesis of QDs using chitosan in

aqueous colloidal media was published by our group

(Mansur et al. 2012). Mansur and collaborators pio-

neered the direct synthesis of CdS quantum dots using

chitosan and derivatives as capping ligands for potential

biomedical applications (Mansur et al. 2012; Santos

et al. 2013). Nevertheless, no study was found in the

accessed literature where water-soluble ZnS QDs were

directly produced with carboxyl-functionalized chitosan

using strictly green colloidal chemistry.

Thus, this research focused on the synthesis of ZnS

quantum dots directly capped by carboxymethylchitosan

(CMC) using a green, facile, reproducible, and single-step

aqueous process at room temperature. In addition, the

nanohybrid systems were extensively characterized, and

the influence of pH on the formation of the semiconductor

nanocrystals and their fluorescent response was investi-

gated. The novel colloidal bio-functionalized water-

soluble nanoconjugates made of ZnS QDs/CMC are

potentiallynontoxicand,combined with their luminescent

properties, may offer potential use in various biomedical

and environmentally friendly applications.

Experimental

Materials

All reagents and precursors, such as zinc chloride

(Sigma-Aldrich, USA, C98 %, ZnCl2), sodium sulfide

2504 Page 2 of 14 J Nanopart Res (2014) 16:2504

123

(Synth, Brazil,[98 %, Na2S�9H2O), sodium hydrox-

ide (Merck, USA, C99 %, NaOH), acetic acid (Synth,

Brazil, C99.7 %, CH3COOH), hydrochloric acid

(Sigma-Aldrich, USA, 36.5–38.0 %, HCl), monochlo-

roacetic acid (Sigma-Aldrich, USA, 99 %, ClCH2-

COOH), ethanol (Synth, Brazil, 99.8 %, CH3CH2OH),

and isopropanol (Sigma-Aldrich, USA, 99.5 %,

(CH3)2CHOH), were used as-received. Chitosan

powder (Aldrich, USA, MM = 310,000 to

[375,000 g/mol, DD C 75.0 %, and viscosity

800–2,000 cPoise, at 1 % in 1 % acetic acid) was

used as the reference ligand. Deionized water (Milli-

pore SimplicityTM) with resistivity of 18 MX�cm was

used in the preparation of all solutions. All preparations

and synthesis were performed at room temperature

(23 ± 2 �C) unless specified.

Synthesis of carboxymethylchitosan (CMC)

and ZnS quantum dots (ZnS-CMC)

A similar method to that reported by Liu et al. (2001)

was used to prepare carboxymethylchitosan (CMC) at

room temperature. Approximately 3.0 g of chitosan

powder was suspended in 70.8 mL of isopropanol.

After 30 min of magnetic stirring, 8.6 g of NaOH

dissolved in 10.0 g of deionized water (45 % in

water), and 10 mL of isopropanol were added to the

suspension and kept under stirring for 1 h. Next,

14.4 g of monochloroacetic acid/isopropanol solution

(1:1 in mass) was added to the suspension. The

reaction proceeded for 4 h under magnetic stirring at

room temperature and stopped by adding 100 mL of

ethanol. Subsequently, the suspension was filtered,

and the solid filtrate (Na-CMC) was washed with

ethanol/water mixtures of increasing ethanol content

(from 70 to 90 %). The neutralization was conducted

by suspending 1.0 g of Na-CMC salt in 80 % ethanol/

aqueous solution (100 mL), adding hydrochloric acid

(1 mL, 37 %) and stirring for 30 min. In the sequence,

the suspension was filtered, and the solid filtrate was

rinsed in 70 to 90 % ethanol to neutral, and then

vacuum dried, leading to the formation of CMC

derivative. Finally, the CMC solution (1.0 % w/v) was

prepared by dissolving 0.5 g of CMC flakes in 50 mL

of deionized water under moderate magnetic stirring

until complete solubilization occurred.

ZnS nanoparticles were synthesized via an aqueous

route at room temperature as follows: 2 mL of the

CMC solution (1 % w/v) and 45 mL of deionized

water were added to a flask. The pH value of this

solution was adjusted to 4.0 ± 0.2 or 10.0 ± 0.2 with

NaOH (1.0 mol L-1). Under magnetic stirring,

4.0 mL of the zinc precursor solution (ZnCl2,

8 9 10-3 mol L-1) and 2.5 mL of the sulfur source

solution (Na2S�9H2O, 1.0 9 10-2 mol L-1) were

added to the flask (S:Zn = 1:2) and stirred for

60 min. ZnS QDs colloidal suspensions were referred

to as ZnS-CMC_4 and ZnS-CMC_10, as a function of

the pH of quantum dots synthesis. Sampling aliquots

of 3.0 mL were collected at different time intervals for

UV–Vis spectroscopy measurements to evaluate the

colloidal stability.

Characterization of CMC and ZnS-CMC colloidal

quantum dots

The presence of characteristic functional groups of

chitosan (CHI) and the introduction of carbo-

xymethylchitosan (CMC) were evaluated by Fourier

transform infrared spectroscopy (FTIR) and thermal

analysis. FTIR was performed over the range of

650–4,000 cm-1 (Thermo Fischer, Nicolet 6700)

using the attenuated total reflectance spectroscopy

method (ATR-FTIR). Chitosan and functionalized

chitosan derivative (CMC) flakes were placed on the

ATR crystal prism (ZnSe), and 16 scans were acquired

at a 2 cm-1 resolution with a background subtraction.

Thermogravimetric (TG) and differential scanning

calorimetry (DSC) analyses were performed using

SDT Q-600 simultaneous TGA/DSC instrument (TA

Instruments). Samples of approximately 9.0 ± 1.0 mg

were used for the experiments at a heating rate of

10 �C min-1 to temperatures of up to 700 �C. The

samples were loaded into an open platinum crucible.

The TG, DTG (derivative thermogravimetric ana-

lysis), and DSC curves were recorded simultaneously

with 0.1 lg sensitivity. The analysis was performed

under the continuous flow of dry nitrogen gas

(100 mL min-1).

The water solubility of CMC as a function of the pH

was evaluated by the turbidity method using UV–Vis

(ultraviolet–visible) spectroscopy based on the

reported literature (Abreu and Campana-Fillho 2009;

Chen and Park 2003). Briefly, the CMC samples were

dissolved at a concentration of 1.5 g L-1 in deionized

water, and the solutions were prepared at different pH

values by adjusting with HCl or NaOH (0.5 %

aqueous solution). The transmittance of the solution

J Nanopart Res (2014) 16:2504 Page 3 of 14 2504

123

was recorded on a Lambda EZ-2100 spectrophotom-

eter (Perkin-Elmer) using a quartz cell with an optical

path length of 10 mm. The spectra were acquired in

the range 190 nm B k B 800 nm, and the sample was

considered insoluble when the transmittance was

lower than 85 % at k = 450 nm.

The nucleation, growth, and stabilization of colloi-

dal ZnS quantum dots in CMC were monitored by

UV–Vis spectroscopy, and their physico-chemical and

optical properties were assessed using photolumines-

cent spectroscopy (PL), fluorescent microscopy,

FTIR, transmission electron microscopy (TEM), and

Zeta potential (ZP).

UV–Vis spectroscopy measurements were con-

ducted in transmission mode with samples in a quartz

cuvette (optical path length of 10 mm) over a wave-

length range from 190 to 600 nm (Lambda EZ-210,

Perkin-Elmer). All of the experiments were conducted

in triplicates (n = 3) unless otherwise noted; the data

are presented as the mean ± standard deviation.

ZnS-CMC conjugates fluorescence emissions and

relative intensities were acquired by using Nanodrop

3300 fluorospectrometer (Thermo Scientific, UV LED

with kexcitation = 365 ± 10 nm). All the PL spectra

were collected at room temperature, and the measure-

ments of fluorescence intensities were reported in

relative fluorescent units (RFU). The relative activity

was calculated by subtracting the background of

samples without the presence of the colloidal QDs. In

order to avoid errors and deviation in the data gener-

ated, all experiments were performed by sampling 2 lL

of the homogeneous QD medium using precision

pipette with precision tips, and the absence of bubbles

was guaranteed (minimum of four replicates, n C 4). In

addition, QD colloidal media were dropped in a glass

slide, and the fluorescence response of each system was

characterized by inverted fluorescence microscopy

(fluorescence microscope Ti-U, Nikon, illuminating

system 130 W/Hg lamp, objective 409, eyepiece 109)

coupled to a refrigerated CCD camera (DS Qi1-U3,

Nikon) with 1,280 9 1,024 resolution (1.5 Mpixels).

The filter cube (C-FL UV-2E/C DAPI, blue excitation)

had an excitation filter: 340–380 nm; a dichroic mirror:

400 nm; and an emission filter: 435–485 nm.

Nanostructural characterizations of the QD nano-

conjugates were based on the images and selected area

electron diffraction patterns (SAED) obtained using a

Tecnai G2-20-FEI transmission electron microscope

at an accelerating voltage of 200 kV. Energy-

dispersive X-ray spectra (EDX) were collected using

the TEM for element chemical analysis. In all of the

TEM analyses, the samples were prepared by dropping

the colloidal dispersion onto a porous carbon grid. The

QD size and size-distribution data were obtained

based on the TEM images by measuring at least 100

randomly selected nanoparticles using an image

processing program (ImageJ, version 1.44, public

domain, National Institutes of Health).

ZnS-CMC_4 and ZnS-CMC_10 quantum dots were

analyzed by diffuse reflectance infrared Fourier

transform spectroscopy (DRIFTS) method (Thermo

Fischer, Nicolet 6700) over the range of

400–4,000 cm-1 using 64 scans and a 2 cm-1 reso-

lution. These samples were prepared by placing a

droplet of the chitosan solution or ZnS-CMC disper-

sions onto KBr powder and drying at the temperature

of (60 ± 2 �C) for 24 h. CMC solutions at the same

pH values (4.0 and 10.0) were used as references.

Zeta potential measurements were performed on

quantum dot colloidal media using a Zetaplus instru-

ment by applying the laser light diffusion method

(Brookhaven Instruments). This instrument uses the

laser Doppler electrophoresis technique (35 mW red

diode laser at k = 660 nm). All tests were performed

using a minimum of three replicates (n C 3), and the

values were averaged (Table 1).

Results and discussion

The FTIR spectra of chitosan (CHI) and carbo-

xymethylchitosan (CMC) are showed in Fig. 1. The

main bands associated with chitosan and CMC

observed in the FTIR spectra are summarized and

listed in Table 2. Changes in the macromolecule of

chitosan after the carboxymethylation reaction were

detected by infrared spectroscopy. Unlike the chitosan

spectrum (Fig. 1(a)), a characteristic band is observed

at approximately 1,731 cm-1, which is assigned to

carboxyl (–COOH) groups. Protonation of the amine

group in CMC causes the appearance of two new

bands. The first band at 1,640–1,620 cm-1, associated

with the antisymmetric –NH3? stretching overlaps the

amide I band, and the second band at

1,550–1,500 cm-1 is related to the –NH3? symmetric

deformation. These bands indicated the H-form CMC

(H-CMC). In addition, the absorbance bands at 1,588

and 1,412 cm-1 were attributed to the symmetric and

2504 Page 4 of 14 J Nanopart Res (2014) 16:2504

123

asymmetric axial deformations of COO-, respec-

tively, which are the characteristics of sodium carbo-

xymethylchitosan (Na-CMC). Based on the results,

the acid form and the Na-salt of CMC were produced.

Because CMC was prepared in alkaline solution, the

carboxyl groups are deprotonized. After the neutral-

ization procedure, both forms of CMC (Na-CMC and

H-CMC) were detected, indicating that the complete

conversion of the salt form to the free acid form was

not achieved even when washing to acid condition (pH

3.0).

From Fig. 2 (TG/DTG), which shows the thermal

analyses of chitosan and CMC, it was possible to

determine the temperatures for the occurrence of the

main thermal events associated with losses of mass.

Two steps can be observed in the TG/DTG curve of

chitosan (Fig. 2A). The first region of temperature

ranges from 25 to 110 �C with a peak centered at

52 �C and mass loss of approximately 10.7 %, which

was due to the evaporation of water physically

adsorbed in the polymeric network. The second region

starts at the temperature of approximately 250 �C and

ends at 400 �C with a corresponding mass loss of

approximately 52 %, which is attributed to the thermal

degradation of polymeric chain (Jiang et al. 2010).

Both stages are associated with endothermic peaks in

the DSC curve (not showed). Figure 2B indicates that

CMC derivative also exhibited a first stage of mass

loss in the range of 25–125 �C, primarily associated

with water removal. The total mass loss in this step

was 12.9 %, which is higher than that calculated for

the parent chitosan, and the temperature correspond-

ing to the maximum loss is 49 �C. These data could be

attributed to the higher hydrophilicity of CMC due to

the character of the negatively charged carboxymethyl

groups incorporated in the chitosan backbone, con-

sidering that the gain of water is mostly related to the

concentration of hydrophilic groups in the polymer

(Ujang et al. 2011). For CMC, the region of the TG

Table 1 Optical and morphological parameters of ZnS quantum dots capped by CMC as a function of the pH during the synthesis

Sample pH kexc (nm) EQD (eV) Blue-shift (eV) Bulka = 3.61 Size—2r (nm)

ZnS-CMC_4 4.0 ± 0.2 281 ± 2 3.83 ± 0.02 0.22 ± 0.02 4.2 ± 0.1

ZnS-CMC_10 10.0 ± 0.2 268 ± 2 4.10 ± 0.02 0.49 ± 0.02 3.2 ± 0.1

a Reference of the bulk value ZnS (cubic crystalline structure, Cooper et al. 2011)

1900 1800 1700 1600 1500 1400 1300

-CH 1380

Abs

orba

nce

(a.u

.)

Wavenumber (cm-1)

(a)

(b)

-NH+

3

-COOH1731

1640-1620

1588-COO-

1550-1500

-NH+

3

1412-COO-

-C=OAmide I

1650 -NH -NH

2 and Amide II

1575

-CNAmide III

1320-CH 1422

Fig. 1 The FTIR spectra of chitosan (a) and CMC (b)

Table 2 FTIR vibrational bands associated with CHI and

CMC (Chen and Park 2003; Lawrie et al. 2007; Abreu and

Campana-Filho 2009; Yan et al. 2011; Mansur et al. 2013)

Wavenumber

(cm-1)

Band assignment Material

1,740–1,730 –COOH stretch H-CMC

1,650 –C=O bend (Amide I) CHI and

CMC

1,640–1,620 –NH3? antisymmetric stretch H-CMC

1,600–1,580 –COO- symmetric stretch Na-CMC

1,590–1,550 –NH bend (NH2 and Amide II) CHI and

CMC

1,550–1,500 –NH3? symmetric stretch H-CMC

1,422 –CH3 antisymmetric

deformation

CHI and

CMC

1,412 –COO- asymmetric stretch Na-CMC

1,380 –CH3 symmetric deformation CHI and

CMC

1,320 –CN stretch (Amide III) CHI and

CMC

1,120–1,090 –C–O stretch (C3-OH

secondary alcohol)

CHI and

CMC

1,060–1,020 –C–O stretch (C6-OH primary

alcohol)

CHI and

CMC

J Nanopart Res (2014) 16:2504 Page 5 of 14 2504

123

curve related to degradation of polymer ranges from

200 to 450 �C (mass loss equal to 47 %) and consists

of three endothermic peaks at 275, 302, and 370 �C

corresponding to the decomposition of carboxylic

groups, glucosamine units, and sodium salt carboxy-

methyl species, respectively (Ujang et al. 2011). These

data reveal the introduction of carboxymethyl groups

in the chitosan structure and the reduction of the

thermal stability of the polymer derivative as the

temperature corresponding to the onset of the degra-

dation step decreased (Ujang et al. 2011).

The solubility tests based on UV–Vis transmittance

revealed that CMC solutions were soluble under acidic

and alkaline conditions, and only insoluble in the

range of 6.0 \ pH \ 7.5. This is one of the major

advantages of CMC derivative over chitosan because

it is soluble in both acid and alkaline aqueous media,

and chitosan is insoluble at pH higher than 6.5. The

solubility range of chitosan and chitosan derivatives

may be explained based on the overall balance of

negative and positive charges on the polymer chain.

Under acidic conditions (pH \ pKa & 6.5) (Sonia

and Sharma 2011), the amine group of chitosan is

protonated to various degrees (Eq. 1). Protonated

amine groups repeal each other, favoring the chitosan-

water interactions, which overcomes the associative

hydrogen bonds and hydrophobic interactions

between chains. In a moderately acidic to neutral

medium, the number of –NH3? species and the net

result of the interchain repulsive electrostatic forces

are reduced and solubility reduces.

CHI� NH2 ðsÞ þ HþðaqÞ ! CHI� NHþ3 ðaqÞ: ð1Þ

The CMC synthesized in this study presents both

acid (CHI–CH2COOH) and sodium salt (CHI–CH2-

COONa) forms of CMC. In this sense, the sodium salt

is readily available for dissociation in aqueous solu-

tion (Eq. 2), because it is practically independent of

the pH. On the other hand, the dissociation of the acid

form of CMC is pH-dependent (Eq. 3). In acidic

solutions, two species of protonated groups are present

in CMC, carboxyls (–COOH), and amines (–NH3?).

As the pH is raised (i.e., by adding OH-), the

carboxylic groups (–COOH) are firstly deprotonated

forming carboxylates (–COO-, Eq. 3) and then

followed by the deprotonation of amines from

–NH3? to –NH2 (Wang et al. 2008) (Eq. 4). The

schematic representation of the expected chemical

steps of deprotonation of CMC by increasing the pH is

showed in Eq. 5.

CHI� CH2COONaðsÞ þ H2OðlÞ! CHI� CH2COO�ðaqÞ þ NaþðaqÞ ð2Þ

CHI� CH2COOHðsÞ þ OH�ðaqÞ! CHI� CH2COO�ðaqÞ þ H2OðlÞ ð3Þ

CHI� NHþ3 ðaqÞ þ OH�ðaqÞ ! CHI� NH2 ðaqÞ

þ H2OðlÞ ð4Þ

CHICH2COOH

NHþ3

���!þOH�CHICH2COO�

NHþ3

���!þOH�CHICH2COO�

NH2

pH1 \ pH2 \ pH3

:

ð5ÞBecause the main factor determining the solubility

of the polymer at a given pH is the net result of positive

and negative charges, the insolubility of the polymer is

predominantly due to an insufficient excess of charge

(Abreu and Campana-Filho 2009). As a result, the

effect of carboxymethyl groups incorporation in

chitosan chain (CMC) was a slight shift to lower pH

20

40

60

80

100

Temperature (oC)

Mas

s lo

ss (

%)

(a)

(b)

300oC

52oC

10.7%

-2

0

2

4

6

8

10

12

14

Derivative m

ass (%/m

in)

0 100 200 300 400 500 600 700

0 100 200 300 400 500 600 70020

40

60

80

100

Temperature (oC)

Mas

s lo

ss (

%)

(a)

(b)

49oC

275oC

302oC

371oC

0

1

2

3

4

5

6

Derivative m

ass (%/m

in)

12.3%

(A)

(B)

Fig. 2 TG (a) and DTG (b) curves of chitosan (A) and CMC (B)

2504 Page 6 of 14 J Nanopart Res (2014) 16:2504

123

(from 6.5 in CHI to 6.0 in CMC) of the low solubility

range in water, probably caused by the presence of

dissociated –COO- side chains balancing the ammo-

nium groups (Chen and Park 2003; Abreu and

Campana-Filho 2009). In the pH range from 6.0 to

7.5, it is expected that the polymer was uncharged or

slightly positive or negative, resulting in lower

solubility. Below pH 6.0 the protonated amine positive

charges predominate and promote CMC solubility.

Additionally, above 7.5, the carboxymethyl groups

250 300 350 400 450 500 550

ZnS-CMC_4 ZnS-CMC_10

Wavelength (nm)

Ab

sorb

ance

(a.

u.)

ZnS bulk 343 nm

λexc

= 281 nm

λexc

= 268 nm

416 nm435 nm

470 nm(a)

(b)

(a)

(b)

0

50

100

150

200

250 Relative F

luo

rescence U

nits (R

FU

)

2.0 2.5 3.0 3.5 4.0 4.5 5.00

5000

10000

15000

20000

25000

30000

EQD

= 3.83 eVBlue Shift = 0.22 eV2R = 4.2 nm

ZnS-CMC_4 ZnS-CMC_10

(αhν

)2 ( e

V.c

m-1)2

hν (eV)

EQD

= 4.10 eVBlue Shift = 0.49 eV2R = 3.2 nm

(a) (b)

ZnS bulk3.61 eV

(A)

(B)

Inset

ZnS

Zn+2

Zn+2

Zn+2

Zn+2

Zn+2

Fig. 3 UV-Vis and PL spectra (A) and the optical band gap using ‘‘Tauc’’ relation (B) for ZnS conjugates synthesized at different pH:

(a) pH 4.0 and (b) pH 10.0. Inset: blue luminescence under UV excitation. (Color figure online)

J Nanopart Res (2014) 16:2504 Page 7 of 14 2504

123

impart an anionic polyelectrolyte character to the

polymer expanding solubility range to alkaline media.

UV–Visible spectroscopy is a powerful tool to

evaluate the effectiveness of the CMC as a capping

agent to stabilize the nucleation and growth of ZnS

quantum dot systems in aqueous media. The UV–Vis

absorption spectra of the colloidal ZnS nanoparticles

produced using CMC as the stabilizing ligand (ZnS-

CMC) are showed in Fig. 3A. The curves exhibit a

broad absorption band from 250 to 300 nm associated

with the first excitonic transition indicating nucle-

ation/growth of ZnS nanoparticles within the ‘‘quan-

tum confinement regime’’. It was observed the so-

called ‘‘blue-shift’’ for the curves when compared with

the ‘‘bulk’’ value (Fig. 3A, arrow at k = 343 nm)

(Brus 1984). Therefore, the optical band gap energy of

a QD (EQD) is larger than that of the original bulk

material (Eg) (Brus 1984).

The optical band gap (absorbance onset) and the

‘‘blue-shift’’ values were determined from the absorp-

tion coefficient data as a function of wavelength using

the ‘‘Tauc relation’’ (Tauc and Menth 1972) extracted

from the UV–Vis spectra. The results are summarized

in Fig. 3B, which support the formation of ZnS

quantum dots in the chitosan media because the band

gap energy of the semiconductor (EQD equal to

3.83 ± 0.02 eV and 4.10 ± 0.02 eV for ZnS-

CMC_4 and ZnS-CMC_10, respectively) was signif-

icantly greater than the ZnS bulk (i.e., 3.61 eV)

(Cooper et al. 2011).

The average ZnS nanoparticle size (r) was esti-

mated from an empirical model published in the

literature (Jaiswal et al. 2011) using the optical band

gap value (EQD) according to Eq. 6. Therefore, ZnS

QDs were produced and stabilized with an estimated

diameter (2r) of 4.2 ± 0.1 nm and 3.2 ± 0.1 nm at

pH 4.0 and 10.0, respectively.

r EQDð Þ ¼ 0:32� 2:9� EQD � 3:49ð Þ1=2h i

=

2� 3:50� EQDð Þ:ð6Þ

Note that, based on a literature review, despite

several studies reporting the synthesis of ZnS quantum

dots, there has been no previous studies that developed

potentially nontoxic, water-soluble, and bio-function-

alized ZnS/CMC nanohybrids via a green ‘‘one-step’’

aqueous colloidal chemistry at room temperature for

potential use in eco-friendly and biomedical

applications.

Figure 3A (right y-axis) also shows the photolumi-

nescence spectra of the ZnS-CMC colloidal nanopar-

ticles collected at room temperature (RT) with their

relative intensities. Based on the absorbance curves

and the band gap energies evaluated under excitation,

ZnS-Chitosan nanoconjugates were expected to emit

light in the UV range (Eg C 3.6 eV). However, band

edge recombination was not detected, and other bands

in the violet-blue range were observed (inset in

Fig. 3A). These results are in agreement with the

reported emission spectra of ZnS nanoparticles, which

typically exhibit radiative recombination in the

400–550 nm wavelength range arising from trap states

associated with point and surface defects (Wageh et al.

2003; Chang et al. 2011). The emission band identified

at approximately 416 nm is due to transitions involv-

ing Zn2? interstitial states (IZn), while the emission

band at 435 nm is assigned to sulfur vacancy states

(VS), compatible with the procedure used in this work

using a stoichiometric molar ratio of Zn2?:S2- = 2:1.

The band at approximately 470 nm, observed for all

systems (independent to the pH of the synthesis), may

be assigned to surface defects (Denzler et al. 1998;

Wageh et al. 2003). In addition, photoluminescence

properties were also affected by the pH. The PL

relative efficiency value (RFU) of the CMC-ZnS

bioconjugates was higher under acidic conditions (pH

4.0) of synthesis (220 ± 12 RFU at 416 nm) than in

alkaline medium (pH 10.0, 94 ± 5 RFU at 416 nm). It

can be explained taking into account that ZnS

nanoparticles were smaller at pH 10.0 than under

low pH, and the amine groups were fully deprotonated

(pH [ pKa). That means, as the ZnS nanoparticle size

decreases, surface disorder and dangling bonds will

possibly dominate the luminescence properties, creat-

ing non-radiative pathways that dissipate quantum

dots emission, which resulted in the reduction of the

PL intensity (Landes et al. 2011). Considering spher-

ical QDs, as the average nanoparticle diameter (2r)

reduces from 4.2 nm (pH 4.0) to 3.2 nm (pH 10.0), the

relative surface area (surface/volume = 3/r) is signif-

icantly increased (*31 %) leading to more surface

defects. Additionally, amine groups (i.e., electron pair

donor) can act as hole scavengers, which may quench

the photoluminescence (Baker and Kamat 2010).

Figure 4 shows representative images of ZnS QDs

produced with the CMC at pH 4.0 ± 0.2 (a) and pH

10.0 ± 0.2 (b) with spherical shape. The chemical

analysis of the nanocrystals based on the EDX spectra

2504 Page 8 of 14 J Nanopart Res (2014) 16:2504

123

revealed that Zn and S are the major elements

(Fig. 4C), excluding the copper related to the TEM

grid, the oxygen and carbon peaks associated with the

polymer stabilizer, and Si from the detector. The

lattice fringes with an interplanar distance of approx-

imately 0.30 ± 0.02 nm can be assigned to the (111)

0

5

10

15

20

25

30

Fre

quen

cy (

%)

Diameter (nm)

Energy (keV)

C

O

CuZn

Si S

Cu

Zn Cu Zn

2.2 2.4 2.6 2.8 3.0 3.2

0.0 0.5 1.0 1.5 2.0 7.5 8.0 8.5 9.0 9.5 10.0

3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.20

5

10

15

20

25

30

Fre

quen

cy (

%)

Diameter (nm)

2R = 4.3±0.3 nm 2R = 2.7±0.2 nm

d = 0.30±0.2 nm

(A) (B)

(C)

Inset

(D) (E)

Fig. 4 TEM images of ZnS-CMC_4 (A) and ZnS-CMC_10 (B). Inset nanocrystal plane spacing. Typical EDX spectrum of the QDs

(C). Histograms of the nanoparticle size distribution of QDs synthesized at pH 4.0 (D) and pH 10.0 (E)

J Nanopart Res (2014) 16:2504 Page 9 of 14 2504

123

plane of ZnS with a cubic crystalline structure (JCPDS

05-0566) as showed in the inset of Fig. 4B. The

histogram of the size distribution of ZnS-CMC_4

nanoconjugates (Fig. 4D) indicates a monodisperse

distribution with an average size of 4.3 ± 0.3 nm.

Similarly, the ZnS-CMC_10 samples exhibited rea-

sonably monodisperse nanoparticles, with an average

size centered at approximately 2.7 ± 0.2 nm

(Fig. 4E). Thus, the TEM results demonstrated that

ZnS quantum dots were properly stabilized by CMC,

in reasonable agreement with the values obtained from

the UV–Vis optical absorbance in the previous section

for ZnS-CMC_4 (2r = 4.2 ± 0.1 nm) and ZnS-

CMC_10 (2r = 3.2 ± 0.1 nm).

Figure 5A, B (curves (b)) show the FTIR spectra of

the ZnS-CMC_4 and ZnS-CMC_10 conjugates,

respectively, compared with their ligand solutions

(CMC at pH 4 and pH 10, curves (a)). The FTIR

spectra of CMC were compatible with the amphiprotic

behavior of CMC. At pH 4, the vibration mode

associated with protonated amines (–NH3?) is over-

lapped with the characteristic band of amide I. The

C=O stretch band of the carboxyl group is absent from

the spectrum due to deprotonation of carboxylic group

from H-CMC in aqueous solution at this pH. Instead,

the band that is assigned to carboxylates (COO-)

stretching mode associated with both deprotonated

forms and dissociated Na-CMC is observed. For CMC

Abs

orba

nce

(a.u

.)

(b)

(a)

Amide I

COO-

-NH+

3

C6-OH

(a)

(b)

C3-OH

Abs

orba

nce

(a.u

.)

Amide I

COO-

(a)

(b)

1800 1750 1700 1650 1600 1550 1500 1150 1100 1050 1000 950

C3-OH

C6-OH

(a)

(b)

1800 1750 1700 1650 1600 1550 1500 1150 1100 1050 1000 950

(A)

(B)

Wavenumber (cm-1)

Wavenumber (cm-1)

Fig. 5 FTIR spectra results for (a) CMC and (b) ZnS-CMC conjugates at pH 4.0 (A) and pH 10.0 (B). Vibrational spectral regions:

1,800–1,500 cm-1 (left) and 1,150–950 cm-1 (right)

2504 Page 10 of 14 J Nanopart Res (2014) 16:2504

123

solution at pH 10, as amine groups from chitosan are

fully deprotonated, the band at 1,640–1,620 cm-1 is

not present, emphasizing the carboxylate band at

1,600–1,580 cm-1. In addition to amines, amides, and

carboxylate groups, chitosan and CMC also have

hydroxyls as possible reactive sites (Riva et al. 2011).

The absorptions at 1,020–1,060 cm-1 and

1,090–1,120 cm-1 indicate the C–O stretching vibra-

tion in CMC, which are associated with the C6-OH

primary alcohol and the C3-OH secondary alcohol,

respectively (Table 2).

Some changes in the relative intensities and/or

energies of the bands assigned to these reactive species

in the FTIR spectra may be expected after conjugation

with the nanoparticles (Cooper et al. 2011). Such a

trend may be associated with the formation of metal

ion complexes with CMC caused by ion exchange,

chelating, and electrostatic interactions in the coordi-

nation compounds (metal ions–ligands) (Deacon and

Phillips 1980; Cooper et al. 2011). The relative

‘‘excess’’ of zinc ions compared with sulfides used in

the synthesis ([S2-]:[Zn2?] = 1:2) was designed for

stabilizing the ZnS nanoparticles with the electroneg-

ative groups of the capping ligands (i.e., amines and

hydroxyls in CHI and carboxylates in CMC). Based on

the results (Fig. 5), in the FTIR spectra of both

0-200 -150 -100 -50 50 100 150 2000.0

0.2

0.4

0.6

0.8

1.0

Pow

er

Zeta Potential (mV)

-33.0+/-2.1 +32.9+/2.0

(a)(b)

-

- -

-

- -

--

-

-

-

-

-

-

-

- +

+ +

+ +

+ +

+

+

+ +

++

+ ZnS “core”

-NH3+

-NH3+

-NH3+

CMC “shell”

Zn2+

-NHCH2COO- ---

-NHCH2COO----

(c) Zn2+

-NHCH2COO-

-NHCH2COO-

-NHCH2COO-

+

+

Fig. 6 Zeta potential curve of CMC solutions at pH 4.0 (a) and pH 10.0 (b). Schematic representation of the zeta potential and the

interactions at the ZnS-CMC nanointerfaces (not to scale) (c)

J Nanopart Res (2014) 16:2504 Page 11 of 14 2504

123

conjugated QDs, no considerable changes were

detected in the relative intensity and wavenumber of

the amines, amides, and hydroxyls. The major change

observed after CMC conjugation with ZnS was in the

relative intensity of the carboxylate band vibration that

weakened or even disappeared. Hence, it may be

assumed that the carboxylate groups of CMC are

involved in the formation and stabilization of ZnS

quantum dots. The differences between the FTIR

spectra of CMC before and after conjugation with ZnS

nanocrystals can be assigned to the formation of

coordination complexes Zn2?–(COO-) on the surface

of the QDs.

As a consequence of the stabilization of ZnS nuclei

mostly by carboxylate species, a significant influence

of pH on the formation/growth/stabilization of the ZnS

QDs in CMC colloidal solution was observed. Based

on the UV–Vis spectroscopy results, when the pH was

raised from 4 to 10, the average nanocrystal size was

decreased by approximately 30 % (from 4.2 to

3.2 nm). The decrease in size could be attributed to

the sum of several contributions toward the formation

of the nanoconjugates made by the ZnS ‘‘core’’ and the

CMC ‘‘shell’’. At relatively lower pH (pH 4), most of

the amine groups of the chitosan derivative are

protonated; as a result, the positively charged transi-

tion metal ions [Zn2?] must compete with the

electrostatic attraction between NH3?���COO- reduc-

ing the sites for metal–ligand interactions. However,

as the pH increases up to 10, amine and carboxylic

groups are fully deprotonated, and all COO- become

available in the CMC chain for complexation with

Zn2?, favoring the stabilization of the ZnS nanocrys-

tals at smaller dimensions due to the increase of the

number of nucleation sites.

The zeta potential (ZP) calculated for ZnS-CMC_4

and ZnS-CMC_10 was ?32.9 ± 2.0 mV (Fig. 6a)

and -33.0 ± 2.1 mV (Fig. 6b), respectively, indicat-

ing that stabilization of these nanoparticles rely both

on electrostatic repulsion and steric hindrance of the

polymer chains adsorbed physically and chemically to

prevent close contact between the fluorescent QDs

(Jiang et al. 2009).

These results indicated that the CMC conjugates

possess positive surface charges at pH 4 and negative

surface charges at pH 10. At pH 4, the carboxylic

groups are mostly involved in the formation of

coordination complexes at ZnS nanointerfaces, leaving

the protonated amines positively charged facing out-

wards in the polymeric ‘‘shell’’, which are available for

anions adsorption/chelation. In contrast, at alkaline

medium, all amines are deprotonated and carboxylates

control both the nucleation and growth of the ZnS

quantum dots and the stabilization of the ‘‘core–shell’’

nanohybrids. In addition, the negatively charged shell

is available for electrostatic cations uptake. Figure 6c

summarizes the results of the zeta potential analysis

with a schematic representation of the interactions at

interface based on the FTIR results. Based on the

results, it may be suggested that the nanohybrids made

of ZnS QD-carboxymethylchitosan may be designed

and tailored to improve the adsorption of cations and

anions in a broad range of pH in water media.

Furthermore, considering the known biocompatibility

of chitosan and its derivatives, the nanoconjugates

produced in this study could be possibly utilized as

cationic/anionic sorbents for environmental and sus-

tainable applications, with prospective recovery due to

their fluorescent property at the same time.

Conclusions

In the present work, ZnS QDs directly bio-function-

alized by carboxymethylchitosan were synthesized

using a green single-step colloidal process in aqueous

medium at room temperature. The results indicated

that varying the pH within a wide range, from acid (pH

4.0) to alkaline (pH 10.0) CMC solutions, significantly

affects the average size and zeta potential of ZnS

nanocrystals produced. By increasing pH from 4.0 to

10.0, the diameter decreased from 4.2 to 3.2 nm and

the ZP changed from ?33 to -33 mV, leading to

changes in the luminescent properties of the ZnS

nanohybrids. In addition, the results indicated that the

stabilization mechanism of the ZnS nanoconjugates

was predominantly determined by the interactions of

the carboxylate functional groups of CMC with Zn2?

at the surface of the QDs.

Acknowledgments The authors acknowledge the financial

support from CAPES, FAPEMIG, and CNPq. The authors

express their gratitude to the staff of the Microscopy Center/

UFMG for TEM analysis.

Conflict of interests The authors declare that they have no

competing interests.

2504 Page 12 of 14 J Nanopart Res (2014) 16:2504

123

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