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QD-‘‘Onion’’-Multicode silica nanospheres with remarkable stability as pHsensors†

Iv�an Castell�o Serrano,a Qiang Maab and Emilio Palomares*ac

Received 6th July 2011, Accepted 8th September 2011

DOI: 10.1039/c1jm13125g

We present a flexible reverse microemulsion method using hydrophobic QDs for multiplexed encoded

nanobeads encapsulating layers of different coloured quantum dots, called QD-‘‘Onion’’-Multicode

bead (QOM). This novel system has monodisperse, photostable, and excellent luminescence properties

that are stable at different pH values. The protection that the silica layers confer to the QDs allows them

to be used as a ratiometric pH sensor by measuring the ratio of PL intensity of QDs from the different

layers.

Introduction

Micro- and nanometre-sized particles with embedded spectro-

scopic signatures are of considerable interest in analytical

chemistry and bioengineering due to their potential applications

in multiplexed bioassays, biotechnology and bioimaging.1–4

Advances by several groups in the last decade have led to a burst

of activities regarding so-called optical ‘‘bar coding’’ based on the

use of nanostructures, semiconductor quantum dots (QDs)

among them.5–9 QDs represent a particularly interesting class of

probes well-suited for advanced fluorescence imaging applica-

tions, such as multiplexed quantitative analysis of cellular

phenotypes, real-time monitoring of intracellular processes, and

in vivo molecular imaging.10–17 Compared to conventional fluo-

rophores, QDs exhibit many interesting characteristics, including

size-tunable and spectrally narrow light emission along with

different light absorption throughout a wide spectrum, improved

brightness with outstanding resistance to photobleaching and

degradation, and extremely large Stokes shift.13 Therefore, QDs

greatly expand the capabilities of fluorescence imaging. The

multiplexing capability of QDs is completed by efficient light

absorption over a broad spectral range, as essentially any photon

in the UV-visible range with energy exceeding the band gap can

be absorbed without damaging the nanoparticle. Unlike organic

fluorophores, the molar extinction coefficient of QDs gradually

increases toward shorter wavelength, allowing multicolor QDs to

be simultaneously excited by a single high-energy light source,

eliminating the need for multiple excitation sources. This coupled

aInstitute of Chemical Research of Catalonia (ICIQ), Avda. Pa€ısosCatalans, 16, Tarragona, E-43007, Spain. Fax: +34 977920223; Tel:+34977929241bChemistry College, Jinlin University, 130012, ChinacICREA, Avda. Llu�ıs Companys, 23, Barcelona, E-08010, Spain. E-mail:epalomares@iciq.es

† Electronic supplementary information (ESI) available. See DOI:10.1039/c1jm13125g

This journal is ª The Royal Society of Chemistry 2011

with the narrow emission makes them extremely attractive. In

principle, multiple colors and intensities can be used to encode

thousands of biomolecules and small-molecule compounds.5

Recent work has used silica beads, in which are embedded the

QDs, to improve the QD fluorescence for the coding signal. Silica

is one of the most popular inert materials for surface coating,

which has several advantages compared to other materials.

Firstly, silica is non-toxic and can be easily modified with func-

tionalized groups that can form covalent bonds with biomole-

cules. Secondly, degradation can be avoided due to the resistance

of silica to both aqueous (except extremely high pH solutions)

and non-aqueous solvents.18 Finally, silica nanoparticles are

easily separated by centrifugation during preparation, function-

alization, and other treatment processes due to their high

density.19 Many attempts have been reported in the scientific

literature to achieve multiplexed color encoded silica nano-

spheres.20–22 Nevertheless, because of the use of hydrophilic QDs

in these methods, lower emission quantum yield and poorer

stability are usually observed. Moreover, these methods do not

generally satisfy the requirements of multi-color encoded spheres

and only single color spheres are obtained. Several authors have

incorporated the QDs onto the surface of micro- or nanospheres,

however the quantity of QDs that can be incorporated is

limited.23,24

Herein we employ a reverse microemulsion method for the

encapsulation of hydrophobic QDs bringing together the best

properties reported for multiplexed encoded materials, using

silica nanospheres, that we call Quantum Dot-‘‘Onion’’-Multi-

code (QOM), following our previous work published in Chem.

Commun.25 This novel procedure has several main features.

Firstly, it is facile and straightforward, avoiding complex

chemical reactions that may quench the CdSe/ZnS QDs lumi-

nescence. Secondly, QD loading and composition can be

controlled simply through the number of silica layers and QDs

selection respectively, thereby providing a means to tune the

nanosphere’s optical properties. Finally, the use of inert silica for

J. Mater. Chem., 2011, 21, 17673–17679 | 17673

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coating QDs avoids the leakage of heavy metal ions into the

environment and enhances the chemical stability of CdSe QDs.

Furthermore, the lack of toxicity is also an ideal platform for

bioapplications. Scheme 1 shows the general procedure for the

preparation of these QOM silica nanospheres, explained in the

Experimental section. Previously, a number of groups have built

QD-based pH sensors because QDs are sensitive to chemicals in

the surrounding environment such as acids, bases, ions and

proteins.26 However, under dynamic conditions such as QD

endocytosis and exocytosis, it would be very difficult to correlate

pH values with absolute QD fluorescence. A classier and more

robust approach is to use ratiometric measurements such as

using QD-dye FRET pairs, because fluorescence intensity ratios

are independent of changes in probe quantity, excitation inten-

sity and detector sensitivity.27,28 We perform this QOM in order

to protect the QDs from the environmental conditions.

Experimental

Materials

Selenium powder (�100 mesh, 99.99%), technical-grade tri-

octylphosphine (TOP; 90%), anhydrous toluene (99.8%), tech-

nical-grade trioctylphosphine oxide (TOPO, 90%), cadmium

acetate dihydrate (98%), sulfur precipitated puriss, metal basis

cadmium oxide (CdO, >99.99%,), technical grade 1-octadecene

(90%), tetraethyl orthosilicate (TEOS, 99.999 metal basis), citric

acid, boric acid, phosphoric acid, and HEPES-sodium salt (99%)

were all purchased from Sigma-Aldrich. Paraffin oil and

ammonia were purchased from Fluka. Zinc acetate dihydrate

(98.0–101.0%) was purchased from Alfa Aesar. All the rest of the

reagents were purchased with analytical grade. For acidic pH

Scheme 1 Illustration of the procedure used to prepar

17674 | J. Mater. Chem., 2011, 21, 17673–17679

values, including pH 7, several BR (borate) buffers were used.

50 mL of BR buffer 0.04 M was prepared by mixing 0.385 of

citric acid, 0.124 g of boric acid and 0.230 g of phosphoric acid in

water and adjusting the pH with NaOH 0.2 M or 0.5 M. For

basic pH values, several HEPES buffers were used. 50 mL of

HEPES buffer was prepared by dissolving 130 mg of HEPES in

water and adjusting the pH with HCl 4 M.

Instruments

The UV-visible and fluorescence spectra were recorded, using a 1

cm path length quartz cell in a Shimadzu UV spectrophotometer

1700 and an Aminco-Bowman Series 2 luminescence spectrom-

eter. Transmission electron microscopy (TEM) measurements

were performed using a JEOL 1011 microscope in order to

calculate the average size of the nanospheres.

Synthesis of CdSe QDs

We followed Peng’s method39 with some modifications. 0.4 mL

of selenium powder, 10 mL of TOP and 0.2 mL of toluene were

mixed in a flask and stirred under argon. A mixture of 20 g of

TOPO and 0.25 g of cadmium acetate was prepared in another

flask, stirred and heated to 150 �C under argon and then the

temperature increased to 320 �C, added 10 mL of selenium

solution and then cooled to 270 �C. The reaction was run for

a specific time (depending on the size of the QD that we expected

to achieve: 1, 10 and 20 minutes for green, yellow and orange

respectively). After stopping the reaction, samples were cooled

and washed with methanol and acetone 3 times and stored in

chloroform.

e multiplexed QOM encapsulating QD multilayers.

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ZnS coating of CdSe QDs

The CdSe QDs were coated with ZnS following a similar method

previously described40 with some minor modifications. A mixture

of 5 mL of QDs in chloroform, 0.0273 g of sulfur, 0.1866 g of zinc

acetate dihydrate and 50 mL of paraffin oil was prepared in

a round-bottomed flask and heated to 80 �C for 20 minutes.

When the mixture appeared homogeneous it was heated to

145 �C and kept at this temperature for 50 minutes. The solution

was then removed and cooled to room temperature. The CdSe/

ZnS (core/shell) was precipitated with copious amounts of

methanol and collected by centrifugation and decanting. The

CdSe/Zn was then resuspended in chloroform. The coating

procedure was repeated several times and in the final step of

centrifugation and decanting the CdSe/ZnS was resuspended and

stored in chloroform solution.

Synthesis of ZnxCd1 � xS

For a typical preparation of ZnxCd1 � xS,41 a mixture of 0.0032 g

of CdO, 0.0041 g of ZnO, 2.5 mL of oleic acid, and 20 mL of

octadecene was heated to 80 �C and degassed under argon for 20

min. The reaction vessel was then filled with argon, and its

temperature was increased to 310 �C. After the CdO and ZnO

precursors were dissolved completely to form a clear colorless

solution, the temperature was lowered to 300 �C. A solution of

0.016 g of sulfur in 5 mL of octadecene was quickly injected into

this hot solution, and the reaction mixture was kept at 300 �C for

the subsequent growth and annealing of the resulting nano-

crystals. Aliquots of the sample were taken at different time

intervals, and UV-vis and PL spectra were recorded for each

aliquot. These sampling aliquots were quenched in cold chloro-

form (25 �C) to terminate the growth of the particles

immediately.

QOM

The encapsulation of QDs into silica beads was performed

following our previously reported method.25 Amixture of 2.6 mL

of Tergitol NP7 and 15 mL of cyclohexane was prepared in

a flask and left to stir for 15 minutes. To this solution 800 mL of

QDs in chloroform solution and 640 mL of TEOS were added to

the previous mixture and left to stir for another 30 minutes. From

this moment on the reaction was conducted in the dark. 200 mL

of aqueous ammonia solution (30%) were added to start the

hydrolysis and left to stir for 24 hours. 800 mL of QD in chlo-

roform solution was then added to the previous mixture and left

to stir for 30 minutes. Then 100 mL of TEOS were added to the

reaction and left to stir for 24 hours. Another 800 mL of QDs in

chloroform solution was added to the reaction and left to stir for

30 minutes and 100 mL of TEOS were added to the mixtures and

left to stir for 24 hours. Acetone was added to stop the reaction

and the mixture was centrifuged for 20 minutes with ethanol 3

times at 3300g. The samples were stored in ethanol.

pH sensitivity

1 mL of silica-coated QDs (core) was added to 1 mL of several

buffers with different pH values from 2 to 11. They were incu-

bated for 1 h with continuous shaking. The pH stability test was

This journal is ª The Royal Society of Chemistry 2011

performed following the procedure described above, adding 1

mL of 1-layer QOM to 1 mL of several buffers with different pH

values from 4 to 9. The QOM was formed with a core with

590 nm CdSe/ZnS embedded QDs and an additional layer of

silica embedded with 520 nm green CdSe/ZnS QDs. They were

incubated for 1 h with continuous shaking.

Results and discussion

Incorporation of CdSe/ZnS QDs into silica beads forming QOM

There are two main types of routes to prepare silica spheres, the

St€ober method23,29–31 and the reverse microemulsion process. The

former is hard to apply if the nanoparticles are insoluble in

alcohol–water solution. For this reason, ligand exchange is

usually required prior to commencing the St€ober process for

QDs synthesized by organometallic methods. This exchange is

commonly associated with a decrease of fluorescent efficiency of

QDs and it therefore requires more sensitive fluorescence

measurement systems. In the case of aqueous QDs, the St€ober

method can be directly used to synthesize QDs@SiO2 to obtain

spheres with a controllable thickness of the silica shell over the

range from a few nanometres to several micrometres, but

multistep procedures32 are required and the size distribution of

QDs/SiO2 is not very narrow. An alternative method is the

reverse microemulsion method that uses water-in-oil micro-

emulsions where the silica spheres are synthesized by the

hydrolysis of tetraethoxysilane (TEOS), followed by their

condensation on water nanodroplets.33 Nanocomposites32,34 and

dye molecules35 can be encapsulated by the silica spheres as long

as they are soluble in water. Recently,36,37 a variant reverse

micelle-based approach was performed to synthesize silica-

coated hydrophobic QDs and other hydrophobic nanoparticles.

NP-7 is a surfactant that causes the microemulsion because of its

amphiphilic nature and it can also exchange the TOPO from

QDs to make them water-soluble to allow the QDs to be inside

the mini-pool or bubbles that will be surrounded by TEOS.

Ammonia reacts with TEOS to produce silica via hydrolysis, as

shown in Scheme 1. The effects of water and ammonia are

complex. Water catalyzes the hydrolysis and increases the

nucleation rate of silica particles. Lower water content reduces

supersaturation and favors the growth of the QD seed particles

over the nucleation of fresh silica. This is favoured by solvents

with higher dielectric constants (such as water), which favour

ionization of silanol groups and enhance electrostatic repulsion

between particles. Ammonia is also a catalyst for TEOS hydro-

lysis. Notwithstanding this the rate of spontaneous nucleation

was found to increase as the ammonia concentration was

lowered.

The UV-Vis absorption and emission spectra of synthesized

CdSe/ZnS and CdSe/ZnS@SiO2 QDs are presented in Fig. 1 and

show well-defined absorption peaks and small full width at half

maximum (FWHM), indicating a rather uniform and narrow size

distribution. Without the silica coating (see Fig. S1† in the ESI),

the colloidal solutions lose their homogeneity in less than a week

due to aggregation and precipitation of the QDs. We can observe

a blue shift due to corrosion of CdSe/ZnS during the embedding

process in which hydrolysis occurs.

J. Mater. Chem., 2011, 21, 17673–17679 | 17675

Fig. 1 Comparison of the UV-Vis absorption and emission spectra of

colloidal CDSe/ZnS QDs (continuous line) and the same QDs incorpo-

rated into silica bead (dotted line). Notice that the fluorescence is

normalized.

Fig. 3 TEM images of QOM after incubation at (A) pH 2, the scale bar

corresponds to 200 nm, (B) pH 7, the scale bar corresponds to 100 nm,

and (C) pH 11, the scale bar corresponds to 100 nm.

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The beads were directly visualized by transmission electron

microscopy (TEM). In Fig. 2 the comparison of the emission

spectra of different QOMs is shown. In the case of Fig. 2A, we can

see the emission of a 2-layer QOM with a core and an additional

layer of silica embedded with green CdSe/ZnS QDs and another

silica layer embedded with orange CdSe QDs. In Fig. 2B and C,

the emission spectrum belonging to a 2-layer QOM with the

inversed code is observed, where the core and an additional layer

of silica are embeddedwith orangeCdSe and another silica layer is

Fig. 2 Comparison of the emission spectra and TEM image of multilayer Q

(core/shell/shell). (B) The QOM code is 6 : 10 for the QDs525nm/QDs580nm/QD

QDs580nm/QDs580nm (core/shell/shell). (D) The code is 10 : 8 (orange : blue) f

QDs600nm/QDs525nm/QDs465nm (core/shell/shell). (F) TEM image of 2-layer Q

17676 | J. Mater. Chem., 2011, 21, 17673–17679

embedded with green QDs. Fig. 2D shows the emission of a 1-

layer QOM with orange CdSe/ZnS embedded in the core and an

additional layer of silica with blue ZnxCd1 � xS QDs. Fig. 2E

shows the emission spectrum of a 2-layer QOM, where the core is

embedded with orange CdSe/ZnS QDs, the first silica layer is

embedded with green CdSe/ZnS QDs and the second silica layer

with blue ZnxCd1 � xSQDs. The PL intensity is directly related to

the ratio of green-orange QDs; playing with different ratios and

number of layers we can get many intensities as a bar code.

Silica protection

For biological applications, QDs should be at least stable

between pH 4 and 8. This is because most bioconjugation reac-

tions are performed at this pH range and pH values found in the

human body also fall into this range. TEM images in Fig. 3A and

B show samples at different pH values indicating that silica

protects the QDs from the different pH environments. None-

theless, at pH 11, silica becomes unstable (Fig. 3C) and we could

not measure the PL intensity directly because the embedded QDs

with TOPO are not water-soluble.

OMs. (A) The QOM code is 10 : 6 for the QDs580nm/QDs525nm/QDs525nms580nm (core/shell/shell). (C) The QOM code is 2 : 10 for the QDs525nm/

or the QDs600nm/QDs465nm (core/shell). (E) The code is 10 : 4 : 3 for the

OM. The scale bar corresponds to 200 nm.

This journal is ª The Royal Society of Chemistry 2011

Fig. 6 Normalized PL intensity of silica beads containing green emissive

QDs as a function of pH monitored after periods of 1 h, 3 h, and 5 h, and

1–7 days.

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The amount of initially added TEOS was found to be impor-

tant, irrespective of the composition of the mixture: if the TEOS

concentration was high, larger silica particles with multiple QDs

were synthesized. The optimal amount of TEOS for our experi-

ments corresponded to a silica shell with a thickness of about 10–

15 nm. The thickness of the silica shells could be increased by

subsequent addition of more TEOS (as discussed below). The

higher the amount of TEOS, the higher the protection of the QDs

inside the beads. The thickness of the beads was measured by

TEM (Fig. 4) to be: 60, 130, and 180 nm, on average, for the 1�,

2�, and 3� TEOS amounts, respectively. So we can conclude

that the thicker the silica layer, the more stable the PL intensity

of the QDs. We can vary this thickness either by increasing the

amount of TEOS during the synthesis or by adding more layers

of silica (as shown in Fig. S3†, see ESI). Fig. 5 compares the

normalized photoluminescence intensity of the silica beads with

different amounts of TEOS used during synthesis. The normal-

ization was done with the PL intensity at pH 7 for all samples.

The behaviour of the PL intensities of the silica beads at

different pH values was measured after different time periods

and is shown in Fig. 6. The PL intensity is stable in the pH range

4–10, agreeing with the obtained results from Gao and Hu.38 All

the samples have the same response to the pH, with increasing PL

intensity at basic pH stable from 4 to 10. However, silica becomes

Fig. 4 TEM images of QOM with different amounts of TEOS used

during synthesis. (A) 640 mL of TEOS, (B) 1280 mL of TEOS, and (C)

1920 mL of TEOS. The scale bar corresponds to 50 nm.

Fig. 5 Comparison of normalized PL intensity of different silica beads

synthesized with different amounts of TEOS (x¼ 640 mL) at different pH

values.

This journal is ª The Royal Society of Chemistry 2011

unstable at pH 11 and the QDs are released with an irreversible

loss of PL. Normalized PL intensity of different silica beads

measured as a function of time at different pH values (Fig. 7 and

Table S1†, see ESI) shows that PL intensity is more or less stable

for a full week as silica beads keep at least 60% of the initial PL

intensity.

This is very attractive for bioapplications where stability is

required for up to several hours. In agreement with Gao,

a unique silica shell is not enough to protect QDs from acid- or

chemical-induced quenching, but several shells make them

exhibit remarkable stability for the biological pH range.

Although their system shows stability over a wide range, our

system is stable enough to be used in bioassays, not only against

biological pH conditions but also along with time.

pH sensitivity test

Fig. 8A shows a comparison of the emission spectra of different 1-

layer QOMs. This system is formed from a core of 590 nm CdSe/

ZnS embedded QDs and an additional layer of silica embedded

Fig. 7 Comparison of normalized PL intensity of different silica beads

containing green emissive QDs measured as a function of time at different

pH values.

J. Mater. Chem., 2011, 21, 17673–17679 | 17677

Fig. 8 (A) Comparison of normalized PL intensity of QOM with two

different coloured embedded CdSe/ZnS (520 nm/590 nm) (core/shell) at

different pH values. (B) Ratio between the PL intensity at 590 nm and PL

intensity at 520 nm.

Fig. 9 Photographs of a series of multiplexed optical encoded silica

beads based on bicolor QOM under daylight (above) and under UV light

(below), respectively.

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with 520nmgreenCdSe/ZnSQDs at different pHvalues (pH4–9).

As we can see, the more basic the pH, the more quenched the PL

intensity of the orange peak (590 nm). This result is in agreement

with our previous results in which we could see that PL intensity

increases at higher pH values, at least until pH 10.

As sigmoidal behaviour is observed in the ratio between the PL

intensity of both peaks (Fig. 8B), it can be concluded that both

acidic and basic conditions affect the PL intensity more or less to

the same degree. Therefore, the QOM appears to be functioning

as a ratiometric pH sensor and, indeed, a rather stable one.

Fig. 9 shows the comparison of a series of 2-layer QOMs with

two different colour QDs (QDs520nm/QDs520nm/QDs590nm) at

different pH values under daylight and under UV light. The

emitted colour changes depend on the pH value because the ratio

between the PL intensity at 520 and 590 nm changes. The

QDs520nm in the core are protected by a double silica layer, which

can protect from the pH and maintain a stable signal. On the

other hand, the QDs590nm, which are protected by a thin silica

layer, can be easily affected by pH. This makes our system useful

17678 | J. Mater. Chem., 2011, 21, 17673–17679

as a ratiometric pH sensor. Furthermore, the higher the 590 nm

PL intensity the more the red-shifted emission and the emitted

colour changes from green to yellow.

Conclusions

We have developed a new method for the preparation of stable

QDs by an encapsulation method based on the formation of

several silica layers. Hydrophobic QDs were first encapsulated

with silica shell based on a well-established reverse micro-

emulsion method. We went a further step and added more layers

of silica with embedded QDs following the same procedure.

Based on this new series of stable embedded QDs, we show that

they can be used for pH sensor applications. The silica matrix

used plays an important role in making them water-soluble and

protecting them from PL quenching, at least in the pH range

useful for biological applications (between pH 4 and pH 8). The

greater the silica layer thickness or higher the number of silica

layers the greater the protection offered to the QDs inside. The

ratio of PL intensity from two populations of QDs has been

shown to correspond to the pH value in the media, making our

system a potential ratiometric pH sensor. This method has

a good potential to develop a wide range of sensors by varying

the functional QDs and other components either embedded or on

the silica surface.

Acknowledgements

We would like to thank the financial support from ICIQ, ICREA

and the Spanish MICINN project CTQ2010-18859. EP also

thanks the EU for the ERCstg Polydot. QM is also grateful to

Prof. Xingguang Su and the National Natural Science Founda-

tion of China (no. 21005029).

Notes and references

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