Quantum Dot Containingpolymerparticleswith

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Quantum dot-containing polymer particles with

thermosensitive fluorescence

Alla N. Generalova a, Vladimir A. Oleinikov a,b, Alyona Sukhanova b,c, Mikhail V. Artemyev b,Vitaly P. Zubov a, Igor Nabiev b,c,n

a ShemyakinOvchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russian Federationb Laboratory of Nano-Bioengineering, Moscow Engineering Physics Institute, 31 Kashirskoe sh., 115409 Moscow, Russian Federationc European Technological Platform Semiconductor Nanocrystals, Institute of Molecular Medicine, Trinity College Dublin, Jamess Street, Dublin 8, Ireland

a r t i c l e i n f o

Article history:

Received 6 June 2012

Received in revised form

17 July 2012

Accepted 18 July 2012Available online 25 July 2012

Keywords:

Colloidal nanocrystals

Thermosensitive polymer

Biosensing

Quantum dots

Optical encoding

Local temperature

a b s t r a c t

Composite polymer particles consisting of a solid poly(acrolein-co-styrene) core and a poly

(N-vinylcaprolactam) (PVCL) polymer shell doped with CdSe/ZnS semiconductor quantum dots (QDs)

were fabricated. The temperature response of the composite particles was observed as a decrease in

their hydrodynamic diameter upon heating above the lower critical solution temperature of the

thermosensitive PVCL polymer. Embedding QDs in the PVCL shell yields particles whose fluorescence is

sensitive to temperature changes. This sensitivity was determined by the dependence of the QD

fluorescence intensity on the distances between them in the PVCL shell, which reversibly change as a

result of the temperature-driven conformational changes in the polymer. The QD-containing thermo-

sensitive particles were assembled with protein molecules in such a way that they retained their

thermosensitive properties, including the completely reversible temperature dependence of their

fluorescence response. The composite particles developed can be used as local temperature sensors,

as carriers for biomolecules, as well as in biosensing and various bioassays employing optical detection

schemes.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Dispersions of polymer particles are characterized by a large

specific surface area and are easy to produce and functionalize;

therefore, they are widely used in analytical chemistry, biosen-

sing, and clinical diagnosis (Bangs, 1996). In the past decades,

increasing attention has been paid to the preparation of smart

functionalized polymer particles reversibly responding to slight

environmental changes, such as variations of temperature, pH,

and ionic strength (Takata et al., 2003). In particular, unique

properties of thermosensitive polymer particles (TPPs) make

them suitable for many applications, especially in biology, suchas the measurement of the local temperature in a single cell or in

volumes smaller than 1018 l (Duracher et al., 2000; Snowden

et al., 1994; Vihola et al., 2007).

At present, optical detection schemes become a technological

frontier in biosensing (Bachmann et al., 2008). In this connection,

the development of thermosensitive polymer particles containing

an optical label with temperature-dependent properties attracts

much attention. For example, photoluminescent (PL) nano-

crystals, such as CdSe/ZnS quantum dots (QDs), are considered

promising as labels for optical detection based on changes in the

fluorescence intensity and/or peak position (Sukhanova et al.,

2002, 2004). QDs are characterized by a high quantum yield and

exceptional resistance to both chemical degradation and photo-

degradation. Another advantage is that QDs of different sizes can

be excited at the same wavelength while emitting PL with a

narrow symmetrical spectrum at distinctly different wavelengths

in the visible or near-IR region. This allows multicolor detection

using nanoprobes (Oleinikov et al., 2007; Nabiev et al., 2008) and

multiple optical encoding of microparticles with nanocrystals(Sukhanova et al., 2007; Sukhanova and Nabiev, 2008). QDs may

also be included in complex superstructures (Sukhanova et al.,

2006) and used in fluorescent resonance energy transfer (FRET)

schemes (Wargnier et al., 2004).

The QD fluorescence intensity exhibits a linear temperature

response (Liu et al., 2006) that is sensitive to QD local environ-

ment (Kalyuzhny and Murray, 2005). Interestingly, QD-capping

agents are known to make QD fluorescence temperature-insensi-

tive. For example, QD fluorescence intensity becomes indepen-

dent of temperature both when denatured ovalbumin is used as a

capping agent (Wang et al., 2008) and when QDs are embedded in

polymer particles (Stsiapura et al., 2004; Joumaa et al., 2006).

Contents lists available at SciVerse ScienceDirect

journal homepage: www.elsevier.com/locate/bios

Biosensors and Bioelectronics

0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.bios.2012.07.030

n Corresponding author at: the Institute of Molecular Medicine, Trinity College

Dublin,Jamess Street, Dublin 8, Ireland. Tel.: 33 631 259 180; fax: 33 326 918 127.

E-mail address: [email protected] (I. Nabiev).

Biosensors and Bioelectronics 39 (2013) 187193
http://www.elsevier.com/locate/bioshttp://www.elsevier.com/locate/bioshttp://localhost/var/www/apps/conversion/tmp/scratch_15/dx.doi.org/10.1016/j.bios.2012.07.030mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_15/dx.doi.org/10.1016/j.bios.2012.07.030http://localhost/var/www/apps/conversion/tmp/scratch_15/dx.doi.org/10.1016/j.bios.2012.07.030mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_15/dx.doi.org/10.1016/j.bios.2012.07.030http://localhost/var/www/apps/conversion/tmp/scratch_15/dx.doi.org/10.1016/j.bios.2012.07.030http://localhost/var/www/apps/conversion/tmp/scratch_15/dx.doi.org/10.1016/j.bios.2012.07.030http://www.elsevier.com/locate/bioshttp://www.elsevier.com/locate/bios


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The excellent temperature-detection properties of QDs were

explored in superstructures engineered from ensembles of QDs

connected with molecular springs (Lee and Kotov, 2007). Ensem-

bles of QDs can be obtained by modifying their surface and linking

them via surface functional groups, with polymer molecules

serving as linkers. An example of such a superstructure whose PL

intensity undergoes reversible changes in response to temperature

variation has been described by Lee et al. (2005). These authors

have developed a reversible nanothermometer based on a dynamicsuperstructure of two types of nanoparticlesa central gold core

and corona-like superstructures of CdTe QDs. These are linked

via a polyethylene glycol (PEG) spacer acting as a temperature-

dependent molecular spring in the aqueous medium. However, not

only is the gold core expansible, but its diameter is practically

invariable.

We have attempted to design a superstructure based on an

advanced architecture consisting of a colloidal polymer core and a

shell containing QDs. The size of the synthetic polymer core can

be controlled by varying the conditions of its synthesis. In order to

make the distance between QDs, which determines the fluores-

cence intensity, changeable, we covered the core with a shell of a

temperature-sensitive smart polymer. Such polymers exhibit

critical phenomena, such as phase transitions, in response to

external stimuli, including changes in temperature (Cole et al.,

2009). They undergo reversible conformational or phase-

separation changes at temperatures above the so-called lower

critical solution temperature (LCST) (Kirsh, 1998). We chose poly-

(acrolein-co-styrene) as the material for this core, because its

outer layer bears double bonds, which make it easy to form a shell

over it. The shell consisted of the temperature-sensitive polymer

poly(N-vinylcaprolactam) (PVCL).

Fluorescent TPP can be obtained by embedding QDs in the

PVCL shell. Temperature variation around LCST causes conforma-

tional changes in PVCL and, hence, changes in the distances

between QDs, which, in turn, results in fluorescence changes

(Fig. 1). These fluorescence changes may serve as a basis for

optical detection methods in bioassays.

Thus, the purpose of this study was to design polymer particles

with thermosensitive fluorescence and study their properties, in

particular, as carriers of biologically active compounds (exemplified

in this study by bovine serum albumin, BSA) and as nanotherm-

ometers for measuring changes in the local temperature (exemplified

by monitoring the temperature in the course of chemical reactions).

2. Materials and methods

2.1. Materials

Acrolein (H2CQCHCHO) was purchased from Fluka, Germany.

It was distilled three times at the atmospheric pressure, and the

fraction with a boiling point of 56 1C, r4200.806 g/cm3, and nD

20

1.40 was used. Styrene was also purchased from Fluka, Germany,

purified with a 5% sodium hydroxide aqueous solution to remove

the stabilizer, rinsed with water until pH became neutral, dried

over calcium chloride, and distilled twice in vacuum. The fraction

with a boiling point of 51 1C (2.1 kPa), r4200.906 g/sm3, and

nD201.54 was used.

The following materials were purchased from SigmaAldrich

and used without further purification: N-vinylcaprolactam (VCL),

potassium persulfate (PP), a,a0-azo-isobutyronitrile (AIBN), sodium

chloride, sodium borate buffer, bovine serum albumin (BSA), andsodium azide. Ethanol, methanol, propanol-2, and chloroform

(Aldrich) were of analytical grade.

Semiconductor CdSe/ZnS coreshell nanocrystals were synthe-

sized as described earlier (Wargnier et al., 2004). In the present

study, hydrophobic nanocrystals with diameters of 3.5 nm (with a

PL emission peak at 554 nm), and 6 nm (with a PL emission peak

at 610 nm) were used. Their PL was excited at lex480 nm in

all cases.

2.2. Methods

Optical and fluorescent characteristics were measured using

an UV/VIS Beckman DU700 spectrophotometer, a Shimadzu

RF551 spectrofluorimeter, and a BioDoc-IT System UV-Transil-luminator. The FT-IR spectra were recorded using a Varian 3100

FT-IR spectrophotometer.

2.2.1. Synthesis of poly(acrolein-co-styrene) core particles

Emulsifier-free radical copolymerization was carried out in

distilled water at a comonomer-to-water ratio of 1:9 and an

acrolein-to-styrene molar ratio of 10:1. A homogeneous styrene

water mixture (at a monomer-to-water ratio of 1:10 v/v) was

prepared, acrolein was added into the reactor. The reaction

mixture was deoxygenated by purging with N2 for 30 min, and

PP (0.5 wt% relative to the monomer mixture) was added as an

initiator. The temperature of the polymerizing mixture was

adjusted at 65 1C. Polymerization was carried out under nitrogen

for 12 h while stirring.

2.2.2. Radical polymerization of N-vinylcaprolactam in the presence

of core particles

The dispersion of poly(acrolein-co-styrene) core particles to be

used as seeds was purified by centrifugation in the presence of

excess water. VCL (0.5 ml) at a seed particle to VCL ratio of 1:0.1,

1:0.2, 1:0.5, 1:1, or 1:1.5 (w/w) in a 20:1 waterpropanol-2

mixture and 0.15 ml of PP or AIBN (0.2 wt% relative to VCL) in

the same solvent was added to 1 ml of a 1-wt% dispersion of seed

particles. The mixture was purged with nitrogen and stirred for

0.5 h. Then, the flask was placed to a water bath, and the mixture

was stirred under nitrogen at 70 1C for 1, 2, or 3 h.

2.2.3. Radical seed polymerization of N-vinylcaprolactamVCL (0.5 ml) at a seed particle to VCL ratio of 1:0.1, 1:0.2, 1:0.5,

1:1, or 1:1.5 (w/w) in a 20:1 waterpropanol-2 mixture was

added to 1 ml of a 1-wt% dispersion of seed particles and left for

swelling at 4 1C for 12 h. Then, 0.15 ml of PP or AIBN (0.2 wt%

relative to VCL) in a 20:1 waterpropanol-2 mixture was added,

and the temperature of the polymerizing mixture was adjusted

to 70 1C. Polymerization was carried out under nitrogen for 1, 2,

or 3 h while stirring.

2.2.4. Measurement of the hydrodynamic radius of polymer particles

The hydrodynamic radius (R) of the polymer particles was

measured using the dynamic light scattering technique. The

dispersion was diluted with water to obtain the concentration

required for the light scattering experiments according to the

Fig. 1. Engineering of thermosensitive polymer particles. Poly(acrolein-co-styr-

ene) particles were used as a solid core (the red sphere). A thermosensitive shell

(green) around the solid core was obtained via radical polymerization of

vinylcaprolactam (VCL). Embedding QDs (pink) in this thermosensitive shell

resulted in fluorescent particles whose fluorescence changed due to variations of

the distance between QDs as a result of changes in the PVCL conformation at the

lower critical solution temperature. (For interpretation of the references to color in

this figure caption, the reader is referred to the web version of this article.)

A.N. Generalova et al. / Biosensors and Bioelectronics 39 (2013) 187 193188


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manufacturers recommendations and then poured into a cuvette

(Lines, 1985). The cuvette holder was kept at the desired tem-

perature between 20 and 45 1C. The particle size was measured

using a Coulter N4-MD sub-micron particle analyzer.

2.2.5. Measurement of the acrolein oligomer concentration

The acrolein oligomer concentration in the supernatant obtained

after centrifugation of the polymer suspension was measured

against water at lmax273 nm using a Beckman DU-70 spectro-photometer (Margel and Rembaum, 1980). The results obtained

(in absorbance units) were represented as the oligomer mass using

a calibration graph of the optical absorption of known quantities of

oligomer dissolved in water.

2.2.6. Incorporation of quantum dots into thermosensitive polymer

particles

Solvents for QD incorporation into TPPs were selected among

water, methanol, ethanol, propanol, propanol-2, butanol, hexane,

chloroform, and their mixtures at ratios of 1:1, 5:1, and 10:1.

It was required that the solvent do not affect the size of TPPs, their

aggregation, or colloid formation upon incubation.

QDs (0.2 mg) were purified from TOP/TOPO by dispersing in

chloroform and precipitating with methanol (at a chloroform-to-methanol ratio of 1:3). The purified QDs were dispersed in 1 ml of

propanol-2 and added to 0.5 ml of a 1 wt% TPP dispersion in a

20:1 waterpropanol-2 mixture. The mixture was stirred vigor-

ously, sonicated for 2 min, incubated for 20 min while stirring

(this procedure was repeated three times), shaken for 1 h at room

temperature, and centrifuged at 7000 rpm for 10 min with addi-

tion of water (this procedure was repeated five times to remove

free QDs). The pellet was then dispersed in 0.5 ml of water. To

remove propanol-2, the obtained TPPs embedded with QDs were

dialyzed against the waterpropanol-2 mixture.

2.2.7. Bovine serum albumin immobilization on polymer particles

An aliquot (0.125 ml) of a 1-wt% dispersion of TPPs containing

QDs was incubated with BSA (1.615 mg/g polymer solids) in a0.1 M sodium borate buffer solution pH 8.2 at 20 1C for 2 h and in

a water bath at 40 1C for 0.5, 1, or 2 h. To block the groups that

had not reacted, 0.5 ml of a glycine solution in 0.1 M sodium

borate (10 mg/ml buffer) was added. Then, the reaction mixture

was washed by centrifugationdispersion three times to remove

the excess protein, and the pellet was dispersed in 1 ml of a

glycine solution buffered with 0.1 M sodium borate (10 mg/ml).

The concentration of unbound BSA was determined by Bradfords

method at l595 nm, with allowance for dilution during the

adsorption procedure.

3. Results and discussions

3.1. Preparation of thermosensitive polymer particles

Thermosensitive composite particles were obtained using a

two-stage reaction: first, core particles were synthesized via

emulsifier-free radical copolymerization; then, the particles were

modified with the thermosensitive polymer (Fig. 1).

The first step was the synthesis of core particles based on the

copolymer of styrene and acrolein. This type of cores possessed

the properties of polystyrene particles (Yen et al., 1976); in

addition, the polyacrolein component provided hydrophilicity of

the surface and contained double bonds due to the specific

characteristics of acrolein polymerization (Slomkowski, 1998).

The particle size can be easily varied by changing the ratio of

the polymerized monomers (Generalova et al., 2007). We used

emulsifier-free radical copolymerization of acrolein and styrene

in water with an acrolein-to-styrene monomer ratio of 10:1 in the

presence of K2S2O8 (PP) for preparing polymer particles with a

hydrodynamic diameter of 185715 nm. This diameter was suffi-

ciently small to preclude spontaneous sedimentation during

measurements.

The second step was the formation of the PVCL thermosensi-

tive shell. We used two approaches:

(1) Radical polymerization of VCL in the presence of core parti-cles, which served as seed particles. This seed polymerization

procedure made it possible to graft PVCL polymer chains on

the surface of seed particles owing to the initiator inducing

VCL polymerization in the dispersion. The resultant PVCL was

water-insoluble, because it was formed at 70 1C and adsorbed

on the surface of the seed particles, after which it formed

bonds with them, with the activated double bonds of poly-

acrolein involved in the process (Eliseeva, 1988).

(2) Radical seed polymerization of VCL. This polymerization was

carried out after swelling of seed particles with VCL in the

presence of the initiator. Under these conditions, the grafting

of the formed PVCL molecules was mainly due to the double

bonds of polyacrolein. This type of polymerization allowed

the formation of composite particles, with few, if any, new

particles been generated (Gardon, 1973).

The effects of different factors, such as the ratio between the

seed-particle and VCL concentrations (1:0.1, 1:0.2, 1:0.5, 1:1, or

1:1.5, w/w), duration of polymerization (1, 2, or 3 h), dispersion

medium (water, watermethanol, or waterpropanol-2), type of

the initiator (the water-soluble PP or oil-soluble AIBN), were

estimated in terms of optimizing the conditions of TPP preparation.

The thermosensitive properties of the obtained particles were

evaluated by measuring the dynamic light scattering. It is note-

worthy that the hydrodynamic particle sizes of the TPPs decreased

with increasing temperature above the LCST due to conformational

changes of PVCL from a hydrated coil to a collapsed hydrophobic

globule (Yi and Xu, 2005). TPPs collapsed remarkably at 32 1C,

which was the LCST of PVCL. It was found that the preferable

medium for the second step was a 20:1 waterpropanol-2 mixture.

The results (Supplementary Table S1) show that the TPPs with

optimal properties were prepared using seed polymerization by

method (1) in the presence of the water-soluble initiator PP (TPP I)

and seed polymerization by method (2) in the presence of the

oil-soluble initiator AIBN (TPP II). Both approaches to obtaining

TPPs could be used under identical conditions, namely, a 20:1

waterpropanol-2 mixture as a dispersion medium, a seed particle

to VCL ratio of 1:0.5, and a polymerization duration of 3 h.

3.2. Characterization of thermosensitive polymer particles

The FT-IR technique was used to control the desired surface

modification of TPPs I and II (Supplementary Fig. S1). In thesespectra, one can see an adsorption peak at 1650 cm1, which is

characteristic of CQO amide groups (Silverstein and Webster,

1998). This peak was more intense for particles modified by

method (2). These results provide evidence for the grafting of

PVCL onto poly(acrolein-co-styrene) cores.

The obtained particles remained stable for a long time and

were unaffected by electrolyte (0.15 M NaCl, physiological saline).

This stability was also preserved at high temperatures, when the

PVCL particles were shrunken. It may be concluded that methods

(1) and (2) of seed polymerization are methods of choice for

obtaining TPPs that do not coagulate or precipitate in solutions

with a high ionic strength.

The modification of copolymer particles with PVCL made it

possible to decrease the amount of low-molecular-weight products



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in dispersion media. These products are formed because of partial

degradation of polyacrolein during storage, with oligomers

released into the dispersion medium (Rembaum et al., 1984). The

amounts of low-molecular-weight products in the cases of TPPs I

(0.21 mg/ml) and II (0.11 mg/ml) were found to be, respectively,

half as much and quarter as much compared to unmodified

copolymer particles (0.46 mg/ml).

Fig. 2A shows the temperature dependence of the hydro-

dynamic radius of TTPs of types I and II. With increasing

temperature, the radii of both types of TPPs gradually decreased

when heated to 29 1C and then decreased abruptly as the

temperature further increased to 32 1C. This sharp decrease was

due to hydrophobic aggregation of PVCL chains. The results

indicated that the particles drastically collapsed at 32 1C, which

was the LCST for the PVCL polymer. Thus, colloidally and chemi-

cally stable TPPs can be obtained by methods 1 (TPP I) and 2

(TPP II). The following part of the study was aimed at obtaining

fluorescent TPPs and studying their properties.

3.3. Thermosensitive polymer particles embedded with QDs

Semiconductor QDs emitting light at about 555 nm were incorpo-

rated into the PVCL shells of TPPs from a 1:20 chloroformpropanol-

2 mixture after removal of TOPO as described earlier (Generalova

et al., 2009). The resultant TPPs doped with QDs displayed intense

green fluorescence. Efficient incorporation of QDs into TPPs was

proved by the absence of free QDs in the dispersion medium after

centrifugation of the TPP suspension: the fluorescence intensity

of the supernatant fraction after centrifugation was found to be

negligible.

Microfluorescence and transmission electron microscopic photo-

graphs of the resultant thermosensitive polymer particles doped

with semiconductor QDs are shown in Supplementary Fig. S2.

Note that QDs had almost no effect on the thermosensitiveproperties of TPPs. Fig. 2B shows that both types of TPPs contain-

ing QDs responded to heating in about the same way as TPPs

without QDs. However, the embedding of QDs in both TPP types

resulted in a slight LCTS shift towards lower temperatures.

This phenomenon can be explained in terms of the effect of QDs

as a hydrophobic component of the composite particle on the

conformation of the aqueous associates of PVCL, which results in

breakage of cross-linking hydrogen bonds (Kirsh, 1998).

In Fig. 3A, the fluorescence spectra of the obtained TPPs at

various temperatures are compared with the fluorescence spectra

of seed poly(acrolein-co-styrene) particles doped with the same

amount of QDs. The swelling procedure used for doping was

described earlier (Generalova et al., 2007). Note that heating

of TPPs I and II reduced their fluorescence intensity, but the

fluorescence intensity of seed copolymer particles was almost

unchanged at higher temperatures (Fig. 3A). Moreover, the

intensity of TPP fluorescence recorded at 20 1C was almost four-

fold higher compared to that of seed particles doped with QDs by

swelling. Thus, the inclusion of QDs into TPPs seems to be

preferable over their inclusion into seed copolymer particles. As

noted by Nida et al. (2008), the ZnS shell may be damaged by a

solvent (e.g., chloroform), which results in coordinative unsatura-

tion (the surface emitting state) of QDs and, consequently,

fluorescence quenching. PVCL around each QD is likely to occupy

the vacant coordinate sites on the QD surface and efficiently

passivate the surface emitting state (as compared to the copoly-

mer chains of seed particles), which results in an increase in the

TPP fluorescence.

The temperature effect on the fluorescence intensity of TPPs is

confirmed by the fact that only the peak corresponding to TPPs

was decreased (Fig. 3B) upon heating the mixture containing

QD-embedded TPPs (lem550 nm) and QD-embedded unmodified

seed copolymer particles (lem610 nm).

The maximum variation of the fluorescence intensity corre-

sponds to the largest changes in the TPPs radius at temperatures

between 26 and 32 1C for TPPI and between 27 and 31 1C for

TPPII (Supplementary Table S1). The sensitivity of temperature

measurement within this range has been found to be about 0.1 1C.

Note that the region of the maximum sensitivity of temperature

measurement may be varied by changing the type of the thermo-

sensitive polymer used. For example, the use of the copolymer of

PVCL and poly-N-vinylpyrrolidone allows shifting the range of the

maximum sensitivity toward higher temperatures, whereas the

use of the copolymer of PVCL and vinyl alcohols results in a

downshift of the region of the maximum sensitivity.

The fluorescence intensities of TPPs I and II were also mea-

sured in heatingcooling cycles with the temperature varying

between 25 and 40 1C (Fig. 4). Note that the process was totallyreversible for both TPP I and TPP II, showing negligible photo-

degradation in every temperature cycle (20 min).

Fig. 4B shows the dependence of the fluorescence intensity in a

heatingcooling cycle on the time as a dynamic characteristic of

TPPs. The maximum decrease in the fluorescence intensity upon

heating a 1-ml sample containing 0.1 mg of TPPs from 25 to 40 1C

was observed for both TPP types within 5 min of heating. This

level of fluorescence remained unchanged at 40 1C for at least

30 min. Upon cooling, TPPs I and II behaved somewhat differ-

ently: TPPs I recovered their fluorescence intensity to the initial

value within 5 min, whereas this took almost 10 min in the case

of TPPs II.

The fluorescence of TPP supernatants after centrifugation was

at a vanishingly low level, which confirmed that the QDs were not

80

90

100

110

120

130

140

150

20 25 30 35 40 45 50

temperature, C

radius,nm

TPP I

TPP II

shell (PVCL)

80

90

100

110

120

130

140

150

20 25 30 35 40 45 50

temperature, C

radius,nm

I

II

core (seed particle)

TPP I

TPP II

core (seed particle)

shell (PVCL)

Fig. 2. The effect of temperature on the hydrodynamic radii of (A) thermosensitive polymer particles and (B) thermosensitive polymer particles doped with QDs.



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lost from the TPPs during heatingcooling cycles. The sensors

developed were very stable, with no more than 10% of the

fluorescence lost during 10 heatingcooling cycles, 10 min per

cycle. The shelf life of the TPPs was found to be more than 2 years

without any effect on their fluorescence properties.

The phenomenon of reversible temperature-dependent fluor-

escence of TPPs can be explained as follows. The rise of tempera-

ture altered the conformation of PVCL on the surface of seed

copolymer particles, resulting in the formation of hydrophobic

globules (Songa et al., 2011). As mentioned above, this may be

observed as shrinkage of the PVCL layer and, hence, a decrease in

the particle size. The shrinkage of the PVCL layer seems to

decrease the distances between embedded QDs, which is the

crucial factor in quenching the QD fluorescence (Zaharchenko

et al., 2005). In addition, the peaks were slightly red-shifted at

temperatures above the LCST, which also indicated that QDs were

located close to one another.

Our calculations of the distance between QDs before and after

heating confirmed this suggestion. Since our previous data

showed that QDs could not be incorporated into seed copolymer

particles without preliminarily swelling for at least 1 h, the

distance was calculated on the assumption on predominant QD

penetration into the PVCL shell (Generalova et al., 2007). UVvis

measurements show that about 104 QDs could be incorporated

into each TPP, which gives a mean distance between QD centers

of about 9.5 nm at 20 1C. According to Chistyakov et al. (2008),

this distance is typical of films with a relatively low QD density.

Heating to 40 1C induced shrinkage of the PVCL layer, and the

distance between QDs was decreased to 4.2 nm. This agrees with

the data obtained by Chistyakov et al. (2008) for films of densely

packed QDs (4.04.1 nm). Thus, the PVCL shells on copolymer

particles analyzed at different temperatures may be regarded as

films with different QD densities. The films containing QDs at a

low density were characterized by narrow fluorescence spectral

bands and a relatively intense fluorescence that can be quenched

by increasing the density of QDs accompanied by a red shift of the

emission peak (Murray et al., 2000; Sukhanova et al., 2006). This

quenching resulted from nonradiative excitation transfer between

QDs (Murray et al., 2000) and interaction of the dipole moments

related to the QD asymmetry (Colvin et al., 1994). To summarize,

we may conclude that the above analogy between PVCL shells

containing QDs and QD films supports our strategy of the

formation of TPPs with temperature-dependent fluorescence.This strategy is based on variations of the distances between

QDs caused by temperature-dependent conformational changes

of PVCL.

The cooling procedure resulted in expansion of PVCL chains,

and the distance between QDs probably returned to the initial

value, which resulted in fluorescence recovery. This behavior

ensured reversibility of the fluorescence intensity changes. The

difference between TPPs I and II in the duration of fluorescence

recovery may be accounted for by differences in the morphology

of the PVCL layer (chain length, flexibility, conformation, etc.) and

the characteristics of its grafting onto the particle surface owing

to the specificity of seed polymerization used in methods (1) and (2).

Thus, incorporation of fluorescence labels, including QDs, into

TPPs facilitates the production of optically sensitive polymer

0

20

40

60

80

500 520 540 560 580 600 620 640

wavelength, nm

fl.

intensity,a.u.

t ~ 20 C

t > 40 C

t < 40 C

0

20

40

60

80

500 520 540 560 580 600 620

wavelength, nm

fl.intensity,a.u.

1

2

3

4

Fig. 3. The fluorescence spectra of poly(acrolein-co-styrene) particles and mixtures of thermosensitive polymer particles embedded with QDs and seed copolymer particles

prepared by the swelling procedure. (A) The fluorescence spectra of poly(acrolein-co-styrene) particles at (1) 20 1C and (2) 40 1C and thermosensitive polymer particles at

(3) 20 1C and (4) 40 1C. (B) Temperature dependence of the fluorescence spectra of a mixture of thermosensitive polymer particles embedded with QDs (lem550 nm) and

unmodified seed copolymer particles embedded with QDs (lem610 nm) using the swelling procedure described by Generalova et al. (2007).

Fig. 4. Cyclic heating (40 1C)cooling (25 1C) temperature variation in solutions of

thermosensitive polymer particles (TPPs) of types I and II (A) and corresponding

changes in the fluorescence of these solutions (B). In Panel C, the dependence of

the fluorescence intensities on the time of heating (40 1C) and cooling is

represented as the ratio of the TPP fluorescence intensity at the given moment(It) to the initial level at 20 1C (I20). dl is the region of the maximum temperature

sensitivity of TPPs of both types.



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particles with temperature-dependent fluorescence. In addition,

the fluorescence intensity of these particles is reversible during a

heatingcooling cycle, which is promising in terms of the devel-

opment of optical detection methods for bioassays.

3.4. Bovine serum albumin immobilization on thermosensitive

polymer particles

For all bioanalytical applications, particles should be conju-

gated with a specific bioligand, preferably protein or peptide. It is

known that PVCL is capable of complexing with various com-

pounds. In the case of its interaction with proteins, PVCL amide

groups form hydrogen bonds with carboxyl or amino groups of

proteins. The conditions and efficiency of this complexing were

estimated using the model of BSA immobilized at concentrations

from 1.6 to 15 mg/g polymer (Kirsh, 1998).

It can be seen in Fig. 5A that the amount of the adsorbed

protein increased with increasing protein concentration in the

solution until signal saturation is reached at a concentration of

10 mg/g polymer. Apparently, the plateau corresponded to the

situation where the surfaces of the polymer particles were

completely covered with the attached protein macromolecules,

and there was no free space left for more protein. Note that the

increase in the amount of immobilized BSA to 10 mg/g caused a

decrease in the TPP fluorescence intensity (Fig. 5A), which

remained practically unchanged as the BSA concentration further

increased. The adsorption processes for TPPs I and II were almost

identical. BSA at the saturating surface concentration seemed to

form a complex with PVCL, which gave rise to conformational

changes and decreased the PVCL capacity for passivating the

surface emitting states of QDs. After saturation of the surface

with BSA (at a concentration of 10 mg/g), the PVCL shell probably

underwent no further conformational changes, and the fluores-

cence remained practically unvaried. In addition, the BSA adsorp-tion had almost no effect on the thermosensitive properties of

TPPs embedded with QDs: in the vicinity of 32 1C, the hydro-

dynamic radii of TPPs I and II decreased by about 45 and 48 nm,

respectively. Therefore, we estimated the amount of BSA added

that corresponded to saturation.

We studied different conditions of BSA immobilization on

TPPs, including incubation at 20 1C for 1 h and incubation at

40 1C for 0.5, 1, and 2 h. The maximum amount of adsorbed BSA

($80% of the amount added) on both types of TPPs was found in

the case of incubation at 40 1C for 1 h. With increasing tempera-

ture, the hydrophobic interactions between PVCL and BSA became

stronger. It is known that deformation of protein molecules due to

their interaction with PVCL, which is facilitated by the rise of

temperature, promotes mechanical entrapment of BSA during the

shell shrinkage (Songa et al., 2011). This effect increased the

amount of adsorbed BSA at 40 1C as compared to that at 20 1C

(Fig. 5B).

Although the BSA adsorption at higher temperatures decreased

the fluorescence intensity of TPPs, this decrease was smaller than

in the case of BSA absorption at 20 1C (Fig. 5B). In addition, the

smallest change in the TPP fluorescence after BSA adsorption was

observed in the case of BSA immobilization on TPP II at 40 1C for

1 h. Fig. 5B also shows the reversible fluorescence dependence on

temperature during a coolingheating cycle for both TPPs I and II.

Thus, QD-containing TPPs I and II could be efficiently assembled

with protein molecules (as exemplified by BSA) in such a manner

that TPPs retained their thermosensitive properties, including

the reversible dependence on temperature, with a relatively

small loss of fluorescence intensity under the optimal protein

adsorption conditions.

4. Conclusions

We have developed an approach to obtaining QD-based

reversibly temperature-sensitive superstructures with a synthetic

colloidal polymer core of desirable functionality, whose diameter

is easily controllable by the synthesis procedure. The super-

structure is based on an advanced architecture consisting of the

polymer core and a shell containing QDs.

In order to make the distance between QDs, which determines

the fluorescence intensity, changeable, we made the shell

from a temperature-sensitive smart polymer undergoing rever-

sible conformational changes at temperatures above the LCST.

The solid polymer core is composed of poly(acrolein-co-styrene)

particles, which have double bonds in the outer layer due to the

characteristics of acrolein polymerization. These double bonds

allow the fabrication of a shell of the thermosensitive polymerPVCL, whose polymer chain undergoes conformational changes

from a hydrated coil to a collapsed globule at LCST (Songa et al.,

2011). The coil-to-globule transition of PVCL, which entails inter-

and intra-chain bonding resulting in a loss of solubility and

hydrophobic aggregation, has been detected at a temperature of

about 32 1C. This temperature is assumed to be the LCST of this

polymer in water (Lau and Wu, 1999). It is close to the physio-

logically normal temperature in higher mammals. In addition,

PVCL can adsorb, and form complexes with, protein molecules

(Kirsh, 1998). Thus, PVCL forms a functionalized layer binding

protein molecules on the surface of polymer particles, which

makes them promising for the use in bioassays.

The use of particles consisting of a thin thermosensitive shell

over a solid core instead of bulk thermosensitive particles makes

Fig. 5. Dependences of the amount of bovine serum albumin (BSA) adsorbed on thermosensitive polymer particles (TTPs) and their fluorescence intensity on the amount of

BSA added (A) and changes in the intensities of fluorescence of TPPs of types I and II during a heatingcooling cycle after BSA immobilization (B). BSA was immobilized at

20 1C or 40 1C for 1 h. IBSA is the fluorescence intensity of TPPs after BSA immobilization. I0 is the initial fluorescence intensity of TPPs.



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it possible to accelerate the response to temperature changes:

heating above the LCST induces a globule conformation of PVCL,

shrinkage of the TPP shell, and decrease in the distances between

QDs resulting in fluorescence quenching.

The potential for TPP application as carriers of biopolymers has

been studied using BSA as a model. The effects of the temperature,

time of BSA immobilization, and BSA concentration on the TPP

fluorescence have been evaluated. The optimal adsorption condi-

tions allowed us to obtain BSA-tagged particles with a reversiblytemperature-dependent fluorescence.

Finally, bioanalytical applications of developed TPPs were illu-

strated by two examples of their use for real-time remote mon-

itoring the local temperature of a reaction mixture in the course of

exothermic chemical reactions: enzymatic hydrolysis of BSA and

cross-linking of chitosan (see Supplementary Information). These

data show that the TPPs developed may be used for measurement

of the local temperature, as carriers for biomolecules, and in

bioassays employing optical detection schemes.

Acknowledgments

This study was partly supported by the European Commission

through the FP7 Cooperation Program (grant no. NMP-2009-4.0-3-246479 NAMDIATREAM) and the Ministry of Higher Education

and Science of the Russian Federation (grant no. 11.G34.31.0050

to I.N). V.A.O. and V.P.Z. acknowledge the support of the Russian

Foundation for Basic Research (RFBR, grant nos. 10-04-00393 and

12-04-00779) and the Ministry of Higher Education and Science

of the Russian Federation (grant 11.519.11.2005).

Appendix A. Supporting information

Supplementary data associated with this article can be found in

the online version at http://dx.doi.org/10.1016/j.bios.2012.07.030.

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