Surface functionalization for subsequent receptor couplingon inorganic nanoparticles

Abbas Abdulameer Salman • Thorsten Heidelberg

Received: 18 February 2014 / Accepted: 11 April 2014 / Published online: 26 April 2014

� Springer Science+Business Media New York 2014

Abstract A two-stages process for an effective coupling of

inorganic nanoparticles with biological receptor molecules

is reported. Initial particle surface functionalization applies

an ethylene glycol-based phosphonic acid or a corresponding

ester analog with an azide functional group for subsequent

receptor coupling under mild click chemistry conditions. A

simple carbohydrate was applied as model receptor, while a

luminescent LaPO4:Ce,Tb with dimensions of 5–7 nm was

chosen for the nanoparticle. Analysis of the particle surface

applied IR and TGA, while effects of the surface modifica-

tion on the particle core were investigated by XRD, TEM,

SAXS, and fluorescence spectroscopy. The receptor content

was determined using a photometric assay, leading to a

surface loading of*40 receptors per particle. This translates

to a surface area of *6.5 nm2 per receptor based on the

inorganic particle core.

Abbreviations

EDX Energy-dispersive X-ray spectroscopy

FESEM Field emission scanning electron microscope

SAXS Small angle X-ray scattering

TEHP Tris-(2-ethyl-hexyl) phosphate

TEM Transition electron microscope

TGA Thermo-gravimetric analysis

TMS Trimethylsilyl

XRD X-ray scattering diffraction

Introduction

Nanoparticles provide unique features as they enable the

combination of bulk material properties with a high surface

area and the ability to form practically homogenous dis-

persions owing to their size. Particularly precious metals

have been proposed for potential medical applications [1,

2]. Their small size enables them to circulate in mamma-

lian blood systems, thus reaching practically the entire

organism [3]. Selective surface modification can enforce

attraction of suitable functionalized particles to selective

cell surfaces, as shown in Fig. 1. This interaction enables

the use of physical properties of the nanomaterial core for

diagnostic purposes, e.g., as contrast reagent for radio

imaging [4, 5]. As the latter increases with the atomic size,

heavy atoms, mostly referring to metals, are particularly

interesting components. Clustering of these in an inorganic

lattice of low dissociative solubility avoids toxic effects of

heavy metals [6, 7] and could further enhance the contrast.

Effective functionalization of nanoparticle surfaces [8]

requires strong interactions between the surface ligands

and the base particle. Covalent binding is considered most

effective, as merely physically adsorbed receptors may

easily be replaced by body fluid components and hence lost

[9, 10]. Due to limited chemical reactivity on the surface of

inorganic nanoparticles, the chemical functionalization

usually requires relative harsh conditions [11] and excess

of modification reagent. Application of this strategy for the

binding of receptor ligands on the particle surface is dis-

favored based on costs for the functionalized ligands. More

promising is a two-stage-approach, based on an initial

functionalization of the particle surface for subsequent

receptor coupling under mild conditions. Easy operations,

high efficiency, and compatibility with a wide range of

functional groups, as well as reaction media favors a

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10853-014-8247-7) contains supplementarymaterial, which is available to authorized users.

A. A. Salman � T. Heidelberg (&)

Chemistry Department, Faculty of Science, University of

Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia

e-mail: heidelberg@um.edu.my

123

J Mater Sci (2014) 49:5388–5397

DOI 10.1007/s10853-014-8247-7

Huisgen-based dipolar cycloaddition (click chemistry) for

the coupling of functionalized nanoparticle and comple-

mentary modified receptor ligands [12–16]. Functionali-

zation of nanoparticles for subsequent click coupling has

been reported previously. For the initial binding of the

click-precursor, either an alkyne or an azide, functionalized

silanes [17–19], multiple carboxylic acids [20], as well as

phosphonates [21, 22] has been applied. Although multi-

step processes have been described involving the conver-

sion of a primary functional group, e.g., an organic halide,

on the nanoparticle into the click function [17, 19, 22], the

application of a surface modification reagent with pre-

formed click functionality is favorable.

Materials and methods

Materials

Reagents and solvents were obtained from various com-

mercial sources and used without further purification.

Previously reported base nanoparticles of composition

LaPO4:Ce,Tb [23–25] were used in the investigation, based

on their small and homogeneous size, as well as their

luminescent behavior, which adds an additional ‘‘bulk

feature’’ to the material thus widening the potential appli-

cation scope.

Surface modification reagents

With respect to aqueous application targets, oligo-ethylene

glycols were selected as surface modification base mate-

rials [26]. Low-molecular weight materials were chosen to

avoid coiling based inaccessibility of coupling linkers for

the receptor binding. A terminal phosphonate aimed for an

effective interaction of the modification reagent with the

nanoparticle, either by a covalent pyrophosphate linkage or

ionic binding to surface lanthanides [27, 28]. The second

end of the ethylene glycol was converted to an azide, which

enables mild coupling to a propargylated receptor in click

chemistry fashion [12, 29–31]. The selected approach bears

advantages over the reverse coupling scheme, as the azide

is a sensitive IR-probe that can easily be detected on a

particle. The core structure of the surface modification

reagents is shown in Fig. 2.

The synthesis of the surface modification reagent

applied an Arbusov reaction [32] on commercially avail-

able dichloride analogs of oligo-ethylene glycol, as dis-

played in Fig. 3. Single substitution was favored based on

Fig. 1 Binding of receptor functionalized nanoparticles to target cell

membranes

Fig. 2 Structure of primary nanoparticle surface modification reagent

Fig. 3 Synthesis of nanoparticle surface modification reagent

J Mater Sci (2014) 49:5388–5397 5389

123

excess of chloride 1, which can easily be separated from

the mono-phosphonate 2 by distillation. Subsequent

replacement of the remaining chloride with sodium azide

[33] provided the azido-phosphono-ester 3. Owing to

Staudinger-type side reactions of organic azides with tri-

valent phosphorous compounds, the order of substitutions

cannot be reversed. Final saponification of the phosphonate

in a two-step procedure using TMS-bromide and methanol

[34] furnished the surface modification reagent 4. Details

are provided in supplementary information.

Particle surface modification

The modification of the nanoparticle surface applied a two-

stage process, involving initial particle surface functional-

ization followed by click coupling [35] of resulting nano-

particle 10 with a complementary functionalized model

receptor model 9, as shown in Fig. 4. In order to avoid too

close proximity of the receptor and the click reaction site,

an ethylene glycol-based spacer was applied. Ethylene

glycol oligomers 6 were converted into the mono-propar-

gyl ethers [36] 7 by applying large excess of the alcohol,

which can be removed by an extraction process. A simple

carbohydrate, i.e., glucose, resembled the model receptor.

The receptor anchor 7 was attached to glucose by glyco-

sylation providing the activated receptor model 9. Details

of the model-receptor synthesis are provided in the sup-

plementary material.

Nanoparticle functionalization

Base LaPO4:Ce,Tb nanoparticles 5 [23–25] (0.25 g) were

heated with surface modification reagent 4b (3.3 g) and

tetraglyme (1 mL to reduce the viscosity) to 120 �C until

the initial suspension cleared into a solution. Heating was

continued for about 6 h at 135 �C. After cooling the par-

ticles were precipitated with methanol and isolated by

centrifugation. Repeated washing with methanol removed

absorbed surface modification reagent. The process fur-

nished 0.20 g nanoparticles 10 after drying at high vacuum.

Fig. 4 Process for model-

receptor binding on nanoparticle

surface

5390 J Mater Sci (2014) 49:5388–5397

123

Alternative particle modification applied the phosphonic

ester 3b (5 g/0.6 g 5) instead of the corresponding phos-

phonic acid 4b to provide 0.5 g nanoparticles 10. In this

case, the addition of tetraglyme can be omitted.

Model-receptor coupling

Functionalized nanoparticle 10 (150 mg based on treat-

ment with 3b) and model receptor 9 (100 mg) were dis-

persed in aqueous DMSO (10 mL, 80 %) using

ultrasonication to break particle agglomerates [35].

CuSO4 9 5 aq (14 mg) and sodium ascorbate (33 mg)

were added, and the reaction was kept stirring at 60 �C for

24 h. Particles were precipitated with MeOH and isolated

by centrifugation at 12,5009g for 10 min. After washing

with MeOH, the nanoparticles 11 were dried at high

vacuum.

Particle analysis

Particle sizes were estimated based on SAXS measure-

ments measured on a PANalytical Empyrean diffractome-

ter and analyzed by the EasySAXS software. The same

instrument was used to record the XRD patterns of the

nanoparticles. The investigation was complemented by

HRTEM images taken on a Jeol JEM-2100F transmission

electron microscope on amorphous carbon-coated copper

grids. Preparation of samples for the latter involved thermal

treatment with dodecylamine, followed by dispersion in

chloroform [37].

Luminescence investigations were performed in chlo-

roform solution after pretreatment of the respective nano-

particles with dodecylamine. Clear dispersions were

obtained upon filtration through 0.2 lm membranes and

adjusted to an optical density of 0.30 at 274 nm. Fluores-

cence spectra were recorded in 1 cm cells on a Varian Cary

Eclipse at a resolution of 1 nm.

Particle surface analysis

Monitoring of chemical changes at the particle surface was

based on IR-spectroscopy. Repeated intensive washing of

the particles with methanol, in which all used reagents are

well soluble, was applied to remove loosely adsorbed

reagents. IR spectra were recorded in KBr at a resolution of

4 cm-1. Complementary elementary composition analyses

were performed by EDX, taken under FESEM on a Jeol

JSM-7600F scanning electron microscope and recorded as

average out of triplicate measurements with a relative

standard deviation below 10 %. TGA measurements, per-

formed on a PerkinElmer TGA4000, complemented the

surface investigations. The latter applied heating rates of

10 �C min-1 under nitrogen atmosphere. An additional

NMR investigation applied highly diluted particle disper-

sions in CDCl3 on a Bruker Avance 400 spectrometer.

Particles were pretreated with dodecylamine to ensure a

homogeneous dispersion. For quantification of the model-

receptor loading, a photometric phenolic sugar assay was

applied [38]. For this a sample of the biofunctionalized

particles 11 was digested in concentrated hydrochloric

acid, and the solution was subsequently assayed on its

glucose content. Details are provided in the supplementary

information.

Results and discussion

Thermal treatment of the base nanoparticles (LaPO4:

Ce,Tb) with surface modification reagents 3b and 4b,

respectively, did not alter the particle dispersion behavior

significantly. This refers both, to the precipitation of par-

ticles from methanol and their unchanged ability to form a

clear dispersion in chloroform after treatment with dode-

cylamine [37] and reflects previously observed behavior for

both post-synthetic [39] and in situ surface modification

[40] of lanthanide-based nanoparticles. Particularly the first

is of practical value, as it enables an easy isolation of the

particles compared to alternative size filtration techniques.

IR investigations confirmed the attachment of the surface

modification reagent, as shown in Fig. 5. The characteristic

azide vibration of 3b is clearly noticeable. However, the

vibration is not intense, thus suggesting a relatively low

Fig. 5 IR spectra of nanoparticles before and after initial surface

modification; a functionalization with phosphonic acid 4b, b func-

tionalization with phosphonic ester 3b

J Mater Sci (2014) 49:5388–5397 5391

123

loading on the particle surface. This can also explain the

missing effect of the surface modification on the dispersion

properties. In view of the targeted application for biologi-

cal recognition, it avoids dense clustering of receptors, thus

ensuring their accessibility for effective interaction with

biological samples, while reducing costs on required

receptor amounts at the same time.

Compared to the corresponding ester 3b, the phosphonic

acid 4b is a slightly less efficient surface modification

reagent. This is reflected in the reduced intensity of the

azide vibration, as shown in Fig. 5. An explanation may be

found in a reduced chemical reactivity of the phosphonic

acid for the formation of pyrophosphate linkages [41]. It

suggests that the surface modification reagent rather binds

to a phosphate-dominating particle surface than interacts

with surface lanthanide ions. The interpretation is in line

with the particle preparation, which applied excess of

phosphoric acid.

The presence of the surface modification reagent does not

prove a covalent binding on the nanoparticle surface. This

would require information on the chemical environment of

the phosphonate group of the modification reagent, which

could not be obtained from the IR. However, the presence of

the azide vibration despite extensive washing processes

indicates strong association with the surface. For practical

purposes a covalent binding, though most effective, is not

essential, as long as the association is sufficiently strong to

avoid loss of the anchor in the biological target system.

Attempts to monitor the particle functionalization by

NMR spectroscopy failed to provide substantial informa-

tion. Only very dilute dispersions could be investigated.

Samples involving more than *20 mg/mL of particles

could not be shimmed, probably due to the paramagnetic

particle core. While the 1H-NMR (supplementary material)

supports the presence of ethylene glycol substructures, the31P-NMR spectrum did not show any peaks. Solid-state

conditions in combination with the presence of paramag-

netic metal cations and varying environment of phosphate

ions due to the small particle size are the most likely rea-

sons for the missing signals.

A previously disclosed patent describes the modification

of the particle surface of LaPO4:Ce,Tb during the particle

synthesis, i.e., in situ [40]. However, attempts to apply the

concept on reagent 4b failed to provide a noticeable azide

vibration on the generated nanoparticles. The result is

surprising, giving the fact that other phosphonates are

incorporated under the reaction conditions. It is possible

that the ethylene glycol-based (polar) reagent 3b interacts

less effectively on the alkyl-phosphate dominated particle

surface than reagents exhibiting a more hydrophobic

structure. On the other hand, a chemical reaction of the

azide during the particle synthesis offers an alternative

explanation for the missing receptor anchor.

Subsequent coupling of the functionalized nanoparticle

10 with the complementary activated model receptor 9

applied the Cu(I) mediated Huisgen coupling (click

chemistry). In order to enable a wide application of the

approach, aqueous DMSO was chosen as reaction medium.

The solvent enables the dispersion of the functionalized

particles 10 in an aqueous medium, despite their resistance

to disperse in water. IR investigations confirm the receptor

coupling, as shown in Fig. 6; the practical vanishing of the

azide vibration, indicating the surface modification reagent,

is accompanied by the appearance of a carbonyl vibration

from the model receptor. The intensity of the latter, how-

ever, is so low that a spectral expansion is required to see

it. The most likely reason is for this is a partial deacety-

lation of the carbohydrate receptor model. Indications for

this side reaction can be found in the IR of 11 based on a

significant increase of absorption for hydroxyl vibrations

(broad peak around 3400 cm-1). Carbohydrates are easily

deacetylated in aqueous medium, particularly at higher pH

[42]. Basic reaction conditions may arise either from

coordination of the catalytic Cu(I) species or from the

formation of the triazole in the click coupling. Owing to the

elevated reaction temperature, a very minor increase of the

pH appears to be sufficient to promote significant hydro-

lysis of the esters [43].

Owing to the low carbonyl vibration of the receptor-

coupled nanoparticles 11, we looked for an additional

evidence for the coupling. Unlike the azide, the triazole

linkage provides no characteristic IR vibrations, which can

be utilized as structural proof. In lieu of this, the elemental

composition of the receptor-coupled particles was deter-

mined by EDX. The data, shown in Table 1, indicate an

unchanged amount of nitrogen for the functionalized and

the receptor-coupled nanoparticles 10 and 11, respectively,

while no nitrogen was detected in the base nanoparticles.

These results indirectly confirm the coupling. The com-

position with respect to the lanthanides reflects the molar

ratio of the reagents (40:45:15). However, the phosphorous

content of the base nanoparticles exceeds with nearly 1.5

Fig. 6 IR spectra of nanoparticles before and after coupling with the

model receptor

5392 J Mater Sci (2014) 49:5388–5397

123

equivalents the phosphoric acid equivalents applied during

the particle preparation. This corresponds with a significant

carbon content, originating from the reaction medium, i.e.,

tris-(2-ethylhexyl) phosphate (TEHP). Upon surface func-

tionalization, the phosphor content is reduced to about 1.1

equivalents of the lanthanides. This indicates a significant

loss of associated TEHP-based organic content during the

surface modification. Besides a partial replacement of

organic phosphates on the particle surface by the phos-

phonate reagent, the loss is likely reflecting physically

adsorbed TEHP or reaction product thereof, which could

not be removed by the initial methanol washings. On the

other hand, the phosphorous content remains practically

constant upon coupling of the receptor model, thus con-

firming a strong binding of the remaining phosphates and

phosphonates to the nanoparticle. The significantly

increased oxygen content upon model-receptor coupling

reflects the presence of the carbohydrate, which also may

give rise to associated water on the particle surface. While

the EDX investigation provides valuable qualitative

insights to surface processes on the nanoparticle, the

model-receptor loading cannot be quantified due to adsor-

bed moisture and solvents, esp. TEHP from the particle

synthesis.

Systematic TGA investigations on the nanoparticles 5,

10 and 11, as shown in Fig. 7 and summarized in Table 2,

indicate increasing organic content upon both surface

modification and receptor coupling, thus further strength-

ening the coupling assumption. A comparison of the TGAs

for nanoparticles before and after functionalization reveals

an increased degradation temperature for the organic con-

tent. This reflects a higher chemical stability of the surface

modification reagent 4b compared to the solvent (TEHP)-

based organic matrix from the particle synthesis. The slight

temperature decrease for the degradation start upon

receptor coupling is likely associated with the elimination

reactions on the carbohydrate component.

Although both EDX and TGA support the coupling of

the model receptor, the data do not enable a quantization of

the receptor loading of the particles. In order to achieve the

latter, a photometric assay on glucose [38] was applied.

The result indicated a lanthanide-glucose ratio of *29:1.

Based on previously reported findings on the dimensions of

the LaPO4:Ce,Tb base particles [23, 24] this translates to a

receptor loading of *40 sugars per particle and a core

particle surface area of *6.5 nm2 per receptor, which is

significantly larger than 1 nm2, which had been reported

for a similar surface modification reagent on ferrous oxide

nanoparticles [21]. The particle surface area per receptor

does not consider the phosphate-organic shell around the

inorganic particle core and, hence, is underestimated.

Nonetheless, the data enable a reasonable estimation on a

potential receptor surface concentration. The occupied

surface area is at least one magnitude smaller than the

carbohydrate’s effective surface area in the range of

30–40 A2 [44]. This suggests good accessibility of the

receptors, thus a potentially effective interaction with tar-

get cells. The number of potential binding sites, on the

other hand, is sufficiently large to ensure strong binding

due to the clustering of interaction sites [45].

Investigations of the particle core revealed no detectable

effect of either surface functionalization or receptor model

coupling. Practically identical XRDs were obtained for all

nanoparticles, see Fig. 8, matching previously reported

results for the base nanoparticle 5. SAXS measurements, as

shown in Fig. 9, confirmed the unchanged particle core

Table 1 Particle composition (m/m); due to adsorbed solvent and

moisture the EDX-based data only provide qualitative information on

the surface modification

Element 5 (%) 10 (%) 11 (%)

C 14 12 10

N n.d. 1 1

O 34 18 29

P 12 13 12

La 15 22 18

Ce 19 25 22

Tb 6 9 8

Elemental composition determined by EDX for base (5), surface-

functionalized (10), and model-receptor-coupled nanoparticles (11)

n.d. not detected

Fig. 7 TGA spectra for base (5), functionalized (10), and receptor-

coupled nanoparticles (11)

Table 2 Organic (surface) content

5 10 11

Degradation (%) 5 10 15

Tstart (�C) *200 *260 *230

Total mass loss (%) 10 19 25

TGA analysis of base (5), surface-functionalized (10), and model-

receptor-coupled nanoparticle (11)

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123

throughout the surface modification process. Based on the

shape of the scattering curve, the data were analyzed

according to a scattering type 3 to represent particles of an

average diameter of 4–5 nm with narrow size distribution.

These results are in good agreement with TEM images for

5 and 10, see Fig. 10, which confirm the previously

reported narrow size distribution of ellipsoid particles with

diameters of 4–5 and 6–7 nm, respectively [23, 24].

In order to monitor the effect of the surface modification

and subsequent model-receptor coupling on the lumines-

cent behavior of the nanomaterial, fluorescence spectra

were recorded. High quantum yields have been reported for

the base nanoparticles 5 [23]. However, the reported

sample preparation involves treatment of the particles with

a strong alkaline medium, which effectively removes most

of the organic layer around the LaPO4:Ce,Tb core. Since

this procedure is expected to remove the surface modifi-

cation as well, a more moderate sample treatment was

chosen [37]. Figure 11 displays the fluorescence spectra for

5, 10, and 11 in clear chloroform dispersion. Owing to the

significantly reduced fluorescence of 11, the corresponding

spectrum is doubled in intensity for better comparison.

The fluorescence in chloroform is drastically reduced

compared with those reported by Riwotzki et al. for alkali-

treated alcoholic dispersions [23]. This particularly affects

the Tb-based emission, which remains below 10 % of its

reported value. On the other hand, Fig. 11 indicates a

significant amount of organic-based fluorescence (around

350 nm), overlapping with the Ce–emission from

LaPO4:Ce,Tb. Only minor differences were observed in the

fluorescence of nanoparticles 5 and 10, as shown in

Table 3. The slight increase in the fluorescence of 10, and

in particular, the moderate increase of the Tb-based emis-

sion, probably reflects removal of some surface-attached

impurities due to the additional purification (precipitation)

of 10 after the surface modification. More pronounced is

the effect of the receptor coupling on the luminescent

behavior; the Tb-fluorescence in 11 is reduced to about half

of its value in 10, while the ratio of Tb-emission and total

fluorescence remains practically unchanged. The latter

suggests that the fluorescence depression might be caused

by absorption of excitation light by the triazole, this way

reducing the excitation of the particles. An investigation of

the absorption and fluorescence behavior of the non-parti-

cle bound surface ligand in 11, prepared from 3b and 9 as

described in the supplementary material, confirms this

assumption. Figure 12 does not only indicate a significant

absorption of the ligand triazole at the excitation wave-

length but also confirms fluorescence of the ligand in the

range of the Ce-emission for LaPO4:Ce,Tb particles. The

excitation for the nanoparticle core appears to match a

maximum of the ligands own excitation.

Conclusions

A new class of surface modification reagents for nano-

particles has been prepared. The application of ethylene

glycol-based azido-phosphonates and -phosphonic acids,

respectively, enables an efficient coupling of biomolecules

on nanoparticles under mild conditions. The phosphonic

acid derivative ensures binding of the azide-anchor on the

particle surface, which subsequently can be coupled with

an alkyne-modified bio-molecule in copper-assisted click

coupling. Unlike a previously reported approach [21], the

initial nanoparticle modification was performed in a single

step, avoiding the stripping of the particle from organic

ligands prior to the anchor binding. Besides the operational

advantage, this leads to a lower, yet still sufficiently

effective, anchor density, which is economically favored if

expensive receptors are to be applied in life science

applications. Recently a click coupling for the modification

of a silicon surface using thiols has been reported [46, 47].

While this approach has high potential for the coupling of

biological material and nanoparticles due to the natural

Fig. 8 XRD of surface-functionalized nanoparticles 10 and reference

reflections for CePO4; practically identical spectra (see supplementary

material) were obtained for the precursor 5 and the model-receptor-

coupled particle 11

Fig. 9 SAXS scattering curve for surface-functionalized nanoparti-

cles 10; practically identical curves (see supplementary material)

were obtained for the precursor 5

5394 J Mater Sci (2014) 49:5388–5397

123

availability of thiol groups on proteins, the functionaliza-

tion of particles is difficult to monitor and even more dif-

ficult to quantify, owing to the low spectroscopic

sensitivity of the alkyne compared to an azide, as applied in

this investigation.

Triazole-linked receptor coupling can affect the fluo-

rescence of luminescent nanoparticles due to a ligand-

based absorption of excitation light. While the effect can be

overcome by increased excitation density, the triazole-

based reduction of particle fluorescence illustrates a limi-

tation of the LaPO4:Ce,Tb system as luminescent probe for

bio-related systems; the excitation is easily affected by

organic material, e.g., biological contents. Therefore, a

more effective luminescent probe requires a red-shift of the

excitation wavelength.

Supporting information

Experimental details on the synthesis of surface modifica-

tion reagents and the receptor model, including images of

Fig. 10 TEM image of surface-

functionalized nanoparticles 10;

similar images were obtained

for the precursor 5 in agreement

with previous reports [23, 24]

Fig. 11 Fluorescence spectra of base, surface, and model-receptor-

coupled nanoparticles 5, 10, and 11; the intensity for 11 is doubled for

better comparison of the spectra shapes

Table 3 Fluorescence

comparison of LaPO4:Ce,Tb

nanoparticles upon surface

modification and subsequent

‘‘click’’ coupling

a Relative standard

5

(%)

10

(%)

11

(%)

Total

emission

100a 106 52

Tb-em.

(abs.)

1.8 2.7 1.2

Tb-em.

(ratio)

1.8 2.5 2.4

Fig. 12 Absorption and fluorescence spectra of organic surface

structure in 11, based on ‘‘click’’ coupling of modification reagent 3band model receptor 9

J Mater Sci (2014) 49:5388–5397 5395

123

spectra to support the purity of the reagents, are provided

as supplementary material, besides a 1H NMR investiga-

tion on model-receptor-coupled nanoparticles 11. It also

contains details for the photometric determination of the

glucose model-receptor and a subsequent calculation-based

estimation on model-receptor density on the particle sur-

face, as well as additional SAXS measurements and EDX

spectra.

Acknowledgements Financial support for this work by the Uni-

versity of Malaya under research grants RG026-09AFR, RP024-

2012B and PS380-2010B is gratefully acknowledged.

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