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Sensors

Tailor-Made Micro-Object Optical Sensor Based on Mesoporous Pellets for Visual Monitoring and Removal of Toxic Metal Ions from Aqueous Media

Sherif A. El-Safty , * M. A. Shenashen , and A. Shahat

88

Methods for the continuous monitoring and removal of ultra-trace levels of toxic inorganic species (e.g., mercury, copper, and cadmium ions) from aqueous media such as drinking water and biological fl uids are essential. In this paper, the design and engineering of a simple, pH-dependent, micro-object optical sensor is described based on mesoporous aluminosilica pellets with an adsorbed dressing receptor (a porphyrinic chelating ligand). This tailor-made optical sensor permits ultra-fast (≤ 60 s), specifi c, pH-dependent visualization and removal of Cu 2 + , Cd 2 + , and Hg 2 + at sub-picomolar concentrations ( ∼ 10 − 11 mol dm − 3 ) from aqueous media, including drinking water and a suspension of red blood cells. The acidic active acid sites of the pellets consist of heteroatoms arranged around uniformly shaped pores in 3D nanoscale gyroidal mesostructures densely coated with the chelating ligand. The sensor can be used in batch mode, as well as in a fl ow-through system in which sampling, target ion recognition and removal, and analysis are integrated in a highly automated and effi cient manner. Because the pellets exhibit long-term stability, reproducibility, and versatility over a number of analysis/regeneration cycles, they can be expected to be useful for the fabrication of inexpensive sensor devices for naked-eye detection of toxic pollutants.

1. Introduction

The determination and removal of ultra-trace levels of metal

ions (such as mercury, copper, and cadmium ions) have

attracted great interest because of their high toxicity to bio-

logical systems as well as their bioaccumulative, mutagenic,

and suspected carcinogenic properties. [ 1 ]

© 2013 Wiley-VCH Vewileyonlinelibrary.com

DOI: 10.1002/smll.201202407

Prof. S. A. El-Safty, Dr. M. A. Shenashen, Dr. A. ShahatNational Institute for Materials Science (NIMS)1-2-1 Sengen, Tsukuba-shi Ibaraki-ken, 05-0047, JapanTel: + 81-298592135; Fax: + 81-298592025 E-mail: sherif.elsafty@nims.go.jp; sherif@aoni.waseda.jp

Prof. Sherif A. El-SaftyGraduate School for Advanced Science and EngineeringWaseda University3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-8555, Japan

According to the US Environmental Protection Agency

(US EPA) and World Health Organization (WHO), the per-

missible levels of inorganic Hg 2 + and Cd 2 + ions in drinking

water are around 2 and 3 parts per billion (ppb), respectively.

Despite copper being essential components in the environ-

ment, the Cu 2 + ions are considered toxic with permissible level

in drinking water of 1.3–2 mg/L (parts per million, ppm). [ 2 ]

When mercury is dispersed in the environment, it is geo-

chemically transformed into toxic species, and the effects of

these species on humans have been extensively studied. [ 3 ] The

toxicity of Hg 2 + arises mainly from its high binding affi nity

for thiol (–SH) groups of proteins. [ 3d ] For example, interac-

tion of Hg 2 + with–SH groups in red blood cell (RBC) mem-

branes induces hemolysis. [ 4 ] Low-level exposure to Hg 2 + has

been linked to subtle neurodevelopmental disabilities, likely

resulting from its ability to induce reactive oxygen species.

Mercury also induces apoptosis, reportedly by causing the

exposure of phosphatidylserine receptors on the external

membranes of nucleated and nonnucleated cells such RBCs. [ 3a ]

For freshwater organisms, the acute toxicity thresholds for

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Optical Sensor Based on Mesoporous Pellets

inorganic mercury (typically, HgCl 2 ) vary considerably, from

5–230 μ g L − 1 for crustaceans to 60–800 μ g L − 1 for fi sh, which

accumulate methyl mercury in muscle and other tissues. [ 5 ]

Copper is essential for the maintenance of normal growth

during pregnancy and lactation in humans, and disorders of

Cu 2 + metabolism are strongly associated with life-threatening

neurological diseases, such as Menkes syndrome and Wilson’s

disease. [ 6 ] Moreover, defects in Cu 2 + metabolism that result in

a higher-than-normal Cu 2 + levels are associated with physi-

ological disturbances in humans. [ 7 ] Aquatic species are partic-

ularly sensitive to environmental Cu 2 + exposure. [ 8 ] Cadmium

is a rare element. Anthropogenic sources of cadmium in the

environment include mining and smelting of metal ores, fossil

fuel combustion, and metal industries. [ 9 ] Chronic cadmium

exposure causes renal dysfunction, a bone-related syndrome

called itai-itai disease, metabolic disorders, and increased inci-

dence of certain forms of cancer, possibly owing to the direct

inhibition of DNA mismatch remediation by cadmium. [ 10 ]

Because these bioaccumulative metal ions are hazardous

to the environment and to humans, sensitive, reliable, and

cost-effective methods for their determination in and removal

from aqueous media are highly desirable. Although various

methods (such as electrothermal atomic absorption spectrom-

etry, inductively coupled plasma–optical emission spectrom-

etry, and inductively coupled plasma–mass spectrometry) can

be used to monitor toxic metal ions even at very low concen-

trations (parts per billion), [ 11 ] these methods require expen-

sive equipment and trained personnel, [ 12 ] which prevents their

widespread use, especially in developing countries. Therefore,

the development of optical nanosensors [ 13 ] has attracted great

interest for the detection and removal of toxic metal ions.

However, slow reaction kinetics and limited detection sensi-

tivity have restricted the practical use of such nanosensors. [ 14 ]

The development of optical sensor devices would overcome

some of these limitations and permit the monitoring of ultra-

Scheme 1 . (A) 3D Geometrical model of cubic Ia3d aluminosilica mesostructures. (B) The atomic charge distribution of 3D cubic Ia3d network cluster units of aluminosilica sensor pellets. The Si/Al ratio used in this cluster is equal to 9. (C) Aluminasilica sensor pellet synthesis with stepwise immobilization of TMPyP followed by sensing/removal of metal ions, such as mercury. The design indicates the feasibility of the capturing/stripping process inside the mesopores of pellets.

trace levels of metal ions by means of one-

step cycling of environmental samples.

Such sensors might also be applicable as

monitoring devices in households, where

instrument-dependent analytical methods

are prohibitively expensive. [ 15 ]

However, problems with the fabrica-

tion of optical nanosensor devices, namely,

time-consuming and complicated syntheses,

as well as high capital and operating costs,

need to be solved. Most nanosensor mate-

rials are obtained as precipitates that must

be reshaped for their fi nal use in sensing

devices. [ 13–17 ] Therefore, the production

of nanosensors into different morpho-

logical shapes and fi lm-like membranes is

needed for simple, easy-to-use, and multi-

functional cleanup optical sensors. Nano-

engineered mesoporous aluminosilica

materials show a diverse and expanding

range of applications, particularly for

adsorption, separation, and catalysis. [ 18 , 19 ]

The incorporation of aluminum into the

mesoporous framework generates cationic

© 2013 Wiley-VCH Verlag Gmbsmall 2013, 9, No. 13, 2288–2296

Brønsted or Lewis acid sites corresponding to tetrahedrally

coordinated aluminum species (AlO 4 − ) in the aluminosilica

framework. [ 20 ] We suspected that mesoporous aluminosilica

pellets with uniformly shaped pores arranged in three-dimen-

sional nanoscale gyroidal structures could be used as scaf-

folds for the construction of practical and inexpensive optical

sensors for determination and removal of toxic metals in

aqueous media.

In this paper, we describe a simple, pH-dependent,

micro-object optical sensor system based on a scaffold of 3D

gyroidal mesoporous aluminosilica pellets with an adsorbed

dressing receptor (a porphyrinic ligand) without prior modi-

fi cation of the scaffold surface with thiol or silane coupling

agents. This tailor-made system permitted ultra-fast and

selective visualization and removal of multiple toxic metals

from aqueous media (including a suspension of RBCs). An

important advantage of this design is its ability to sensitively

detect multiple analytes. To the best of our knowledge, there

have been no previous reports of micro-object optical sensors

based on aluminosilica pellets with a single dressing receptor

for specifi c visualization and removal of multiple toxic metals

from aqueous media. The sensor showed a remarkable ability

to selectively remove Hg 2 + , Cd 2 + , and Cu 2 + ions from drinking

water. Furthermore, the sensor suppressed the toxicity of

Hg 2 + ions (by ∼ 50%) in a suspension of RBCs, without

exerting any toxicological effects on the RBCs.

2. Results and Discussion

For use in the tailor-made micro-object sensor, we prepared

robust disk-like pellets with mechanically stabilized shapes,

micrometer-sized particles and tunable periodic scaffolds

consisting of cubic Ia3d mesoporous aluminosilica with low

alumina content (Si/Al = 9; Schemes 1 and S1 and Figure S1).

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The pellets had large surface area-to-volume ratios and uni-

formly shaped pores in three-dimensional nanoscale gyroidal

structures.

The synthesis protocol and characteristics of cubic Ia3d

aluminosilica mesocylinders as carriers are of particular inter-

ests due to the following:

a) Hand-operating and easy-to-make synthesis of mesopo-

rous pellets with micrometric particle sizes was achieved

using our simple method that requires shorter time and

simple composition.

b) Our strategy enabled fabrication of an optical gel-like

material in a graduate ingot, in which acquired the shape

and size of the cylindrical casting vessel, leading to control

the pellet-like materials without use of well- equipment

control (Scheme S1).

c) The development of ordered mesoporous pellets that fea-

ture 3D structures, 3D cylinder-shaped and uniform pore

sizes, monodispersed porosities into large particle sizes

with ultra- or micrometer-scale morphologies signifi cantly

led to densely coated with the chelating ligand (Scheme 1 ).

The inner and outer pore walls of pellets could be

directly modifi ed with a charged porphyrinic chelating

ligand, α , β , γ , δ -tetrakis(1-methylpyridinium-4-yl)porphyrin

p -toluenesulfonate (TMPyP), without the need for surface-

mounted coupling agents. The surface acidity (see Supporting

Information) of the pellets facilitated the uniform modifi -

cation of the pore walls by the TMPyP molecules without

prior decoration of the surface supports with silane or thiol

coupling agents. Strong ion-pair interactions between the

0

Figure 1 . (A,B) HRTEM micrographs and FTD (insets) patterns of cubic Ia3d aluminosilica pellets with a Si/Al ratio of 9. HRTEM and FTD patterns were recorded along the [311] (A), and the [111] (B) zone axes, respectively. (C) 3D TEM micrograph of pellets recorded along the [311] direction with 45 ° tilt. In general, there are smooth and well-ordered network surfaces over wide-range domains of cubic Ia3d structures. (D) Bright-fi eld STEM of sensor pellets.

pyridinium ion–containing TMPyP mole-

cules and the negatively charged AlO 4 −

units of the aluminosilica host material

prevented leaching of TMPyP from the

pore surfaces during the sensing assays.

Scheme 1 A shows the electron density

distribution on a structural model of the

aluminosilica pellets and the effect of the

atom arrangement in the 3D lattice on the

pore orientation is shown in Scheme 1 B

(see Supporting Information). The oxygen

atoms in the Si–O–Al linkages were more

negatively charged than the oxygen atoms

in the other linkages, which indicate that

the surface sites in the former linkages

were more acidic than other sites in the

framework. The surface acidity was con-

fi rmed by 27 Al NMR magic-angle spinning

spectroscopy and NH 3 –temperature pro-

grammed desorption studies (Figures S2

and S3, SI). Because of the acid-site sur-

faces, the amount of TMPyP adsorbed

on the pellet matrices (40 mg g − 1 ) was

suffi cient for the generation of a color

change that was visible to the naked eye

in response to the formation of complexes

between TMPyP and toxic metal ions. The

high loading capacity and the immobili-

zation of TMPyP ligand into functional

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pellets were evident from scanning transmission electron

microscopy (STEM)–energy dispersive spectroscopy map-

ping and thermogravimetry/differential thermal analyzer

(TG/DTA), (Figures S4, S5). The key to designing pellets

for optical sensors is to ensure that the accommodation of

the chelating agent (a porphyrin in this case) is densely and

uniformly immobilized on the 3D gyroidal mesopore pellet

matrices, which leads to rapid binding of the target metal

ions during the sensing assay.

The most important feature of the pellets was the uniform

arrangement and continuous ordering (that is, without distor-

tion) of gyroidally cubic Ia3d mesostructures in all directions.

The integrity of the cubically ordered frameworks in the

micrometer-sized particle and the gyroidal mesostructures

of the pellets lead to attain the porphyrin ligand interior the

3D pore-like pocket (Scheme 1 ), as shown by high-resolution

transmission electron microscopy (HRTEM), STEM, three-

dimensional TEM (3DTEM; Figure 1 ), small-angle X-ray

scattering, and N 2 isotherm profi les ( Figure 2 ). The HRTEM,

3DTEM, and STEM micrographs along the zone axes of the

pellets revealed regular arrays running along a large area of

the [311] and [111] directions, which allowed the analyte ions

to diffuse homogeneously and interact with the binding sites,

even after the pellets had been stored for a long period (one

year to date). The small-angle X-ray scattering images of the

pellets exhibited well-resolved Bragg peaks with d-spacing

ratios of 9.4, 8.1, 6.1, and 5.7, indicating highly ordered cubic

Ia3d nanophase domains (Figure 2 A). The N 2 isotherms

featured an H1-type hysteresis loop with a sharp infl ection,

which is characteristic of architectures with uniform and

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Optical Sensor Based on Mesoporous Pellets

Figure 3 . pH-dependent curves of the sensor pellets in the measurement of refl ectance spectra ‘signal’ response of the M-to-TMPyP complex during the detection/removal of Cu 2 + , Cd 2 + and Hg 2 + ions at 552 (a), 620 (b), and 636 (c) nm, respectively.

Figure 2 . SAXS patterns (A) and N 2 adsorption/desorption isotherms at 77 K (B) of the highly ordered cubic Ia3d aluminosilica scaffolds with Si/Al ratio of 9 (a), TMPyP-modifi ed pellet sensors (b), and after reuse cycles (c). Insert (A) shows the cubic Ia3d lattices ( a = d 211 √6) and (B) the textural parameters, such as surface area (S BET ), pore size (D), and pore volume (Vp).

open cylindrical pores. [ 21 ] The N 2 isotherms indicate that the

important textural parameters (e.g., surface area and pore

size and volume; Figure 2 B, inset) of the mesostructures

were retained upon noncovalent binding of a substantial of

amount of TMPyP on the inner pore surfaces, as evidenced

by the shift in the adsorption branch toward a lower relative

pressure (P/Po) and the slight reduction of the surface area

and pore volume upon functionalization with TMPyP.

We constructed a micro-object optical sensor system by

installing the singular pellet in a fl ow-through module in

which sampling, target ion recognition and removal, and anal-

ysis occurred (Scheme S2, SI). The pellets were placed in the

central reactor cavity of a sandwich-like glass cell. Laminar

fl ow predominated in this setup, thereby permitting precise

control of the sensing reaction conditions. In addition, the

minimal reagent use, controlled metal-to-ligand binding, and

containment of the pellets in the module ensured that haz-

ardous substances could be analyzed safely. The color change

that occurred upon binding of the target metal ions to the

TMPyP-containing pellets was monitored visually as well as

by UV–vis refl ectance spectroscopy. In addition, the solution

emerging from the outlet of the fl ow-through module was

analyzed by inductively coupled plasma–mass spectrometry

(ICP-MS) to determine the extent to which the target ions

were removed.

The specifi city of the pellets for the target metal ions

was controlled by means of pH adjustments. The porphyrinic

ligand immobilized on the pellets has four charged pyrid-

inium nitrogens and two pyrrole nitrogens, which can be

neutral or protonated depending on the pH of the medium

(see Supporting Information S6–S8). Therefore, by varying

the pH, we were able to control the visual detection and

selective removal of Cu 2 + , Cd 2 + , and Hg 2 + ions. In both batch

experiments (see Supporting Information) and fl ow-through

experiments conducted with solutions of Cu 2 + , Cd 2 + , and Hg 2 +

ions, we observed a rapid signaling response ( ≥ 60 s) based

on the change in the specifi c color map and the refl ectance

intensities at λ max values of 552, 620, and 636 nm for the

M–TMPyP complexes that formed in the pellets at pH values

of 5.5, 9.5, and 11.5, respectively ( Figure 3 ). These metal com-

plexes formed as a result of a defi ciency in the d-orbitals of

the metal ion centers, which caused the metals to bind fi rmly

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheismall 2013, 9, No. 13, 2288–2296

to the TMPyP donor molecules to form

symmetrical fi ve-membered rings in octa-

hedral complexes with high stability con-

stants (Scheme 1 ). [ 22 ]

A major advantage of the pellet sensor

in both micro-object and batch sensing

systems is its selectivity to the target Cu 2 + ,

Cd 2 + , and Hg 2 + ions, thus preventing hin-

drance from actively interfering compo-

nents, particularly the competitive ions

and species. These interfering components

may make the sensing/recovery of metal

target ions from a mixture containing sev-

eral competitive components diffi cult. The

selectivity of the pellet sensor for Cu 2 + ,

Cd 2 + , and Hg 2 + ions was checked in both

micro-object and batch sensing systems.

The interfering cations and anions were initially added at

high concentration range to the pellet sensor ( Figures 4 , S6

and S7) at ion-sensing conditions of pH 5.5, 9.5, and 11.5 for

Cu 2 + , Cd 2 + , and Hg 2 + , respectively. First, we studied the spe-

cifi c behavior of the pellets that permitted the selective deter-

mination of Cu 2 + , Cd 2 + , and Hg 2 + , by separately adding each

of a series of interfering transition metal ions and group I and

II metal ions at pH values of 5.5, 9.5, and 11.5, respectively

(Figures 4 and S6). We found that alkali and alkaline earth

metals (including Na + , Li + , Mg 2 + , and Ca 2 + ions) at a concen-

tration of 10 ppm did not exhibit any appreciable interfer-

ence. In contrast, the addition of an equivalent amount of

transition metal ions (Cu 2 + , Ni 2 + , Co 2 + , Pb 2 + , and Zn 2 + ) to the

Hg(II) ion-sensing system interfered slightly ( ± 2%) with the

quantitative determination and removal of the target ions. At

high doses (up to 10 times of target concentration), competi-

tive cations (particularly Ni 2 + , Fe 3 + , Pd 2 + , and other Cu 2 + and

Cd 2 + target ions) produced positive errors in the refl ectance

signal of the Hg(II) ion-sensor pellets. The addition of 0.05 M

oxalate, citrate, thiosulphate, or tartrate to the sensing system

increased the tolerable concentration of the active targets

at each ion-sensing by up to 20 times the concentration of

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Figure 4 . Effect of the additive cations and anions as interfered components on the refl ectance spectra at 636 nm and color response, respectively, of Hg-to-TMPyP complexes, recorded using a 20 mg/L sensor pellet at pH 11.5, and with the addition of 1 ppm of Hg 2 + ions. Note the interfered (10 ppm) alkali and alkaline-earth metals (that is, Na + , K + , Li + , and Mg 2 + ions). The other cations’ concentrations were at 1 ppm. In addition, the interfered [50 ppm] anions from the four groups (G-b to G-e) are as follows: G-b (CTAB, SDS, Triton X, NO 3 − , and SO 3 2 − ), G-c (C 2 O 4 2 − , C 8 H 4 O 4 − , and C 4 H 4 O 6 2 − ), G-d (C 6 H 5 O 7 3 − , CO 3 2 − , and NO 2 − ) , and G-e (CH 3 COO − , SO 4 2 − , and PO 4 3 − ), respectively.

Figure 5 . Refl ectance spectra for aluminasilica sensor pellets with different concentrations of Hg 2 + ions at the optimum pH = 11.5. (A, insert) Calibration plots of the refl ectance spectra for our fabricated sensor with various concentrations of Hg 2 + ions at λ 636 nm (contact time of 1 minute, at 25 ° C), based on the relationship between the R–R o and metal ion concentrations. Calibration was done by measuring the relative refl ectance of Hg-TMPyP complexes (R) formed with respect to a blank solid of sensor (R 0 ) at the specifi c λ of 636 nm. The inserts show the amplifi cation for the linear colorimetric response, and the calculated limit of detection for mercury ions was 1.4 × 10 − 9 M. The error bars denote a relative standard deviation of approximately 0.03% for the analytical data of ten replicate analyses.

the Cu 2 + , Hg 2 + , and Cd 2 + target ions. Second, we investigated

the selectivity of the TMPyP-modifi ed pellet sensors in the

presence of interfering surfactants [cetyl trimethylammo-

nium bromide (CTAB), Triton X, and sodium dodecyl sulfate

(SDS)] and organic [citrate: C 6 H 5 O 7 3 − , oxlate: C 2 O 4

2 − , tar-

trate: C 4 H 4 O 6 2 − , phthalate: C 8 H 4 O 4 − ,and acetate: CH 3 COO − ]

and inorganic [NO 2 − , NO 3

− , SO 3 2 − , SO 4

2 − , CO 3 2 − , and PO 4

3 − ]

anions, respectively, that may coexist with our target ions in

water and the environment. In our study, the experiment was

carried out by separately studying the effect of each inter-

fering surfactants and anions (Figure S7), and then studying

their effect as a group (Figure 4 C). Interestingly, we found

that the addition of inorganic anions (i.e., NO 2 − , NO 3

− , SO 3 2 − ,

SO 4 2 − , CO 3

2 − , and PO 4 3 − ), which we thought might impede the

detection and removal processes, had no appreciable effect on

the TMPyP-modifi ed pellets; these anions were tolerated at

www.small-journal.com © 2013 Wiley-VCH V

concentrations of up to 50-times the concentrations of Cu 2 + ,

Cd 2 + , and Hg 2 + ions (Figure 4 C and S7). In addition, there is

no signifi cant interference or effect from the used surfactants

and organic anions on the signal responses or color profi les

of TMPyP-modifi ed pellet sensors (Figure S7), indicating the

ion-selective sensor with high tolerable limits of interfering

matrix concentrations. The selectivity of the pellets can be

ascribed to their high binding affi nity for the target metal

ions, the stability of the formed [M-TMPyP] n + complexes (see

Figure S6 and S7), and TMPyP-to-M complexation affi nities

among all interfered metals at optimized pH conditions.

We measured UV–vis refl ectance spectra to monitor the

change in the pellets upon formation of the M–TMPyP com-

plexes in the micro-object sensor system ( Figures 5 and S6,

SI). We measured the refl ectance spectra of the complexes

at λ max values of 552, 620, and 636 nm for Cu 2 + , Cd 2 + , and

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Optical Sensor Based on Mesoporous Pellets

Figure 6 . Feasible application of sensor pellets on the batch sensing/capturing process of Cd 2 + , Cu 2 + , and Hg 2 + ions from real water with sequential color changes upon interaction with each metal at the optimized pH values.

Hg 2 + ions, respectively, to monitor the

uptake of M n + target ions by the TMPyP

ligand (Figure S6, SI). In addition, metal

ion concentrations in the sample solu-

tions before and after exposure to the pel-

lets were measured by means of ICP-MS

(Scheme S2). The wavelength of the

charge-transfer refl ection band of the com-

plexes depended on the ligand-binding

affi nity of the central metal ion and on

the nature of the complex formed under

the sensing conditions. Moreover, the vis-

ible color change provided a simple way to

sensitively and selectively detect M n + ions

without the need for sophisticated instru-

ments. The rapid detection and extraction

and the M n + -to-TMPyP binding events

that formed on the pellet surfaces resulted

in the separation and preconcentration of

divalent metal ions even at trace concen-

trations. We prepared calibration curves

by analyzing standard solutions with a

wide range of Cu 2 + , Cd 2 + , and Hg 2 + con-

centrations (0.005 μ g dm − 3 to 5 mg dm − 3 ;

10 replicates for each concentration)

at the optimum pH for each metal ion

(Figure S6, SI). The standard deviation for the quantitative

analysis data for Cu 2 + , Cd 2 + , and Hg 2 + ions was 0.02–0.06%, as

indicated by the curve fi tted to the calibration data (Figures 5

and S6, SI). The limits of detection (LOD) of the pellets were

estimated from the linear regions of the calibration curves

(Figures 5 and S6, insets). The LOD values indicated that the

sensing system could detect Cu 2 + , Cd 2 + , and Hg 2 + ions at con-

centrations down to ∼ 10 − 11 mol dm − 3 , even in the presence

of the matrices (as shown by calibration curves (Figure 5 B)

dotted lines).

To evaluate the practical applicability of the pellets, we

performed batch detection and removal of Cu 2 + , Cd 2 + , and

Hg 2 + ions from samples of environmental fl uids at 25 ° C and

pH values of 5.5, 9.5, and 11.5, respectively. ICP-MS was used

to determine the concentrations of the various constituents

before and after each experiment. The samples contained

alkali and alkaline earth metal ions (12.8–31.1 mg dm − 3 ) and

traces of Zn 2 + , Mn 2 + , Sn 2 + , Fe 2 + , and Fe 3 + ions (0.02–0.083 mg

dm − 3 ). The samples were spiked with Cu 2 + , Hg 2 + , and Cd 2 +

ions (0.001–0.5 mg dm − 3 ). Langmuir isotherms were used to

verify the effi ciency of metal ion absorption by the TMPyP-

bearing pellets in terms of the loading capacities and binding

effi ciencies of Cu 2 + , Hg 2 + , and Cd 2 + ions at ultra-trace concen-

trations. [ 23 ] Our results indicate that the use of the pellets for

colorimetric assays in the fi eld and in the laboratory is a viable

alternative to the currently used laboratory methodology in

terms of time and cost savings ( Figure 6 ). The linear adsorp-

tion curves (Figure S7, SI) revealed two features of the sensor

system: (1) metal ions at a wide range of concentrations could

be removed without the need for preconcentration of the

samples, and (2) the metal ions formed a monolayer on the

interior pore surfaces of the pellets in this sensor system. The

practical adsorption capacity ( q m ) and the Langmuir coverage

© 2013 Wiley-VCH Verlag Gmbsmall 2013, 9, No. 13, 2288–2296

constant ( K L ) were obtained from the slope and intercept of

the linear regions of the Langmuir plots. The metal ions could

be removed from the aqueous medium with high adsorption

effi ciencies (94–96%). Thus, removal of 0.114–0.14 g of Cu 2 + ,

Hg 2 + , or Cd 2 + ions from an aqueous solution would require

1 g of TMPyP-containing pellets. The pellets displayed sub-

stantial adsorption effi ciency, comparable to that of commer-

cial adsorbents such as nanoporous silica, activated carbon,

sphagnum moss peat, TiO 2 , coconut shell charcoal, organi-

cally modifi ed clays, polypyrrole/reduced graphene oxide,

and ion-exchange resins. [ 23 ] In addition, these adsorbents are

complicated to design and complicated to use for detection

and removal of metal ions. [ 23 ]

If these pellets are to be used for waste management and

decontamination of polluted media, they must exhibit repro-

ducible high performance, recyclability, and durability. [ 24 ] To

demonstrate that the pellets could be recycled by decompl-

exation of the bound metal ions, we treated the metal-con-

taining pellets with a solution of 0.02 M ClO 4 − or 0.05 M

ethylenediaminetetraacetic acid “stripping agents” to remove

the Cu 2 + , Hg 2 + , and Cd 2 + ions. The solution of stripping agents

was injected into the pellets containing 0.6 ppm of metal

target ions (with a syringe) for several hours (Scheme 1 ). The

gradual change in the pellet color as the regeneration time

was increased was visible to the naked eye and was quanti-

tatively confi rmed by UV–vis refl ectance spectroscopy and

ICP-MS. Approximately 98% ( ± 2.3%) of the initial amount

of metal ions was recovered. The pellets could generate and

transduce an optical color signal and exhibit fast TMPyP–M

binding (on the order of minutes), even after multiple regen-

eration/reuse cycles ( Figure 7 ), with only a slight decrease in

effi ciency as the number of cycles was increased, indicating

that the mesostructured functionality and features were

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Figure 7 . Evaluation of the effi ciency changes for the recognition and capturing properties of 1 ppm of Hg 2 + , Cu 2 + , and Cd 2 + ions using 20 mg/L of TMPyP-modifi ed sensor pellet after six regeneration/reuse cycles at 25 ° C.

retained. The retention of functionality was confi rmed by

TEM and fi eld emission SEM ( Figure 8 ), which indicated that

the three-dimensional architecture ordering and the pellet

morphology (size and shape) were retained to a high degree.

These features could lead to long-term stability, reproduc-

ibility, and versatility of the pellets over a number of reuse/

cycles (Figure 8 ).

We also evaluated the use of the pellets to remove metal

ions from a physiological fl uid. Mercury ions interact rapidly

with–SH groups in RBC membranes, leading to peroxida-

tion of membrane lipids and, eventually, to cellular lysis. [ 4 , 25 ]

Therefore, we investigated the removal of Hg 2 + ions from a

suspension of RBCs to determine whether removal would

inhibit Hg 2 + -induced RBC hemolysis (Figure S8, SI). Mer-

cury ions (3 μ M) were added to a suspension of RBCs, which

was incubated for 12 h with ground pellets (0.02%, 0.05%

and 0.07% w/v). The absorbance of released hemoglobin

at 540 nm ( n = 3) was used as a marker of RBC hemolysis.

Upon the addition of the ground sensor pellets (0.05% w/v)

at physiological pH (7.4),–ve effect of the sensor on RBCs

was observed. Furthermore, the Hg 2 + -induced hemolysis

decreased by approximately 40% (Figure S8), and we attrib-

uted the decrease to the removal of some of the Hg 2 + ions

by the pellets. This fi nding revealed that the Hg 2 + ions might

have been removed if the process had been carried out at

www.small-journal.com © 2013 Wiley-VCH V

Figure 8 . (A) TEM, (B) FESEM micrographs of sensor pellets after releasingCd 2 + ions from the sensor surfaces.

the optimal pH for Hg 2 + (11.5, as described above) instead

of at physiological pH 7.4. In general, such inhibition of the

mercury-induced hemolysis is the key to exploring the appli-

cability of micro-object-device sensor in controlling the tox-

icity of mercury in blood through the removal and capture of

mercury ions.

3. Conclusion

In this study, we monitored and removed ultra-trace concen-

trations Cu 2 + , Hg 2 + , and Cd 2 + ions from aqueous solutions and

a suspension of RBCs by means of a tailor-made micro-object

optical sensor that integrated sampling, chemical reaction, sep-

aration, detection, and data processing in a highly automated

and effi cient system. The system enabled ultra-fast and specifi c

detection and removal of multiple toxic metals by means of

mesoporous aluminosilica pellets bearing an adsorbed single

dressing receptor (a porphyrinic ligand). The pellets had large

surface area-to-volume ratios and uniformly shaped pores

in three-dimensional nanoscale gyroidal structures and per-

mitted rapid detection ( ≥ 60 s) of ultra-trace concentrations

( ∼ 10 − 11 mol dm − 3 ) of the target metals by means of a colori-

metric signal visible to the naked eye, as well as by means of

UV–vis refl ectance spectroscopy. In addition, the metal ions

captured by the pellets could be removed from the pore sur-

faces via chemical stripping, and the pellets could be reused.

This capture-and-release process concentrates on the col-

lected elements up to 98% effi ciency in controlled waste man-

agement. Pellet-based micro-object sensing devices like the

one described here can be expected to be important for use in

portable and remote sensing systems, especially for household

use, where instrumental methods are prohibitively expensive.

4. Experimental Section

Details on the fabrication and characterization of the sensor pel-lets, as well as their use to remove metal ions from a suspension of RBCs, are presented in the Supporting Information.

The micrometric sensor pellets were synthesized as follows. Cubic Ia3d mesocylinder aluminosilica pellets (Scheme S1, SI) were used as solid scaffolds for functionalization with a porphy-rinic ligand, α , β , γ , δ -tetrakis(1-methylpyridinium-4-yl)porphyrin

erlag GmbH & Co. KGaA,

the Hg 2 + , Cu 2 + , and

p -toluenesulfonate (TMPyP). TMPyP in ethanol solution was immobilized on the pellets by means of a dispersion process at 45–50 ° C. The immobilization process was repeated several times until the equilibrium adsorption capacity of the pellets for TMPyP molecules was saturated, as indicated by spectrophotom-etry. The solid sensor pellets were thoroughly washed with deionized water until no elution of the TMPyP was observed. We determined the equilibrium adsorption capacity of the pel-lets for TMPyP by measuring its absorbance at various time intervals. The amount of TMPyP adsorbed onto the scaffolds was estimated to be 40 mg g − 1 .

Weinheim small 2013, 9, No. 13, 2288–2296

Optical Sensor Based on Mesoporous Pellets

Next, we tested the utility of the sensor pellets for detection and removal of ultra-trace concentrations of Cu 2 + , Cd 2 + , and Hg 2 + ions in aqueous solutions at pH 5.5, 9.5, and 11.5, respectively. The concentrations of the metal ions were monitored in a fl ow-through module, which was connected to pellets housed in a sandwich-like glass cell with a central reactor cavity that was 3 mm high and ∼ 15 mm in diameter (Scheme S2, SI). Laminar fl ow pre-dominated in this setup, which permitted precise control of reac-tion parameters, such as concentration gradients, temperature, and pH. The sample solutions were pumped into the reactor cavity containing the pellets at a fl ow rate of 1.5 μ L min − 1 by means of an autosampler pump through silicon tubes (inner diameter 1 mm). The glass cell device was connected to a deuterium-halogen light source (AvaLight-DH-S, Avantes), which has a deep UV bulb (190–400 nm), a deuterium lamp (215–400 nm), and a halogen lamp (360–1,500 nm). A fi ber optic spectrometer system (CIMPS-Abs-UV, Zahner, Germany; AvaSpec-2048 × 14, Avantes) was used to measure the signal from the molecular recognition event.

The optical color intensity of the solid TMPyP sensor pellet was estimated with a UV–vis spectrometer (CIMPS-Abs-UV, Zahner, Ger-many) installed with AvaSoft 7.6 software (Avantes; Scheme S2, SI). ICP-MS was used to determine the effi ciency of Cu 2 + , Cd 2 + , and Hg 2 + ion removal by the pellets by measuring the metal ion concentrations in the aqueous samples after exposure to the pel-lets and comparison of the measured concentrations to the initial concentrations.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

M. Shenashen and A. Shahat thank the Egyptian Petroleum Research Institute (EPRI), and Suez University, Egypt, respectively, for granting a leave of absence.

[ 1 ] a) R. S. Pohren , J. A. V. Rocha , K. A. Leal , V. M. F. Vargas , Environ. Int. 2012 , 44 , 40 – 52 ; b) J.-M. Moulis , F. Thévenod , Biometals 2010 , 23 , 763 – 768 ; c) H. H. Harris , I. Pickering , G. N. George , Science 2003 , 301 , 1203 ; d) J. Liu , W. Qu , M. B. Kadiiska , Toxicol. Appl. Pharm. 2009 , 238 , 209 – 214 ; e) N. Cerullia , L. Campanellab , R. Grossib , L. Politic , R. Scandurrac , S. Giovanni , F. Galloe , S. Damianie , A. Alimontif , F. Petruccif , S. J. Carolif , Trace Elements Med. Bio. 2006 , 20 , 171 – 179 ; f) X. Chen , G. Zhu , S. Gu , T. Jin , C. Shao , Environ. Toxicol. Phar. 2009 , 28 , 232 – 236 ; g) V. C. Ezeh , C. Todd , T. C. Harrop , Inorg. Chem. 2012 , 51 , 1213 – 1215 .

[ 2 ] 2012 Edition of the Drinking Water Standards and Health Advi-sories, EPA 822-S-12-001, US Environmental Protection Agency , Washington, DC , April, 2012 ; Cadmium in Drinking-water, Background document for development of WHO Guidelines for Drinking-water Quality, WHO/SDE/WSH/03.04/80/Rev/1, WHO , 2011 ; Mercury in Drinking-water, Background document for development of WHO Guidelines for Drinking-water Quality WHO/SDE/WSH/05.08/10, WHO , 2005 .

© 2013 Wiley-VCH Verlag GmbHsmall 2013, 9, No. 13, 2288–2296

[ 3 ] a) K. Eisele , P. A. Lang , D. S. Kempe , B. A. Klarl , O. Niemöller , T. Wieder , S. M. Huber , C. Duranton , F. Lang , Toxicol. Applied Pharma. 2006 , 210 , 116 – 122 ; b) S. H. Kim , R. P. Sharma , Tox-icol. Appl. Pharmacol. 2004 , 196 , 47 – 57 ; c) A. Stacchiotti , F. Morandinib , F. Bettonib , I. Schenab , A. Lavazzac , P. Giovanni , G. P. Apostolie , R. Rezzania , M. F. Aleob , Toxicology 2009 , 264 , 215 – 224 ; d) S. Ceccatelli , M. Aschner , Methylmercury and Neuro-toxicity , Springer , New York 2012 .

[ 4 ] a) G. Liu , Y. Cai , N. O’Driscoll , Environmental Chemistry and Toxi-cology of Mercury , John Wiley & Sons, Inc ., Hoboken, New Jersey , 2012 ; b) K.-M. Lim , S. Kim , J.-Y. Noh , K. Kim , W.-H. Jang , O.-N. Bae , S.-M. Chung , J.-H. Chung , Environ. Health Perspect. 2010 , 118 , 928 – 935 .

[ 5 ] E. Guallar , M. I. Sanz-Gallardo , P. van’t Veer , P. Bode , A. Aro , J. Gómez-Aracena , J. D. Kark , R. A. Riemersma , J. M. Martín-Moreno , F. J. Kok , N. Engl. J.Med. 2002 , 347 , 1747 – 1754 .

[ 6 ] a) E. Madsen , J. D. Gitlin , Annu. Rev. Neurosci. 2007 , 30 , 317 – 337 ; b) J. Y. Uriu-Adams , R. E. Scherr , L. Lanoue , C. L. Keen , Bio Factors 2010 , 36 , 136 – 152 .

[ 7 ] a) L. Finney , S. Vogt , T. Fukai , D. Glesne , Clin. Exp. Phar-macol. Physiol. 2009 , 36 , 88 – 94 ; b) T. Wu , C. T. Sempos , J. L. Freudenheim , P. Muti , E. Smit , Ann. Epidemiol. 2004 , 14 , 195 – 201 ; c) Y. Zhao , K. Lin , W. Zhang , L. Liu , J. Environ. Sci. 2010 , 22 , 1987 – 1992 .

[ 8 ] a) R. D. Handy , Comp. Biochem. Phys. A—Mol. Integrative Phys. 2003 , 135 , 25 – 38 ; b) H. Jiang , H. Yang , X. Kong , S. Wang , D. Liu , S. Shi , Life Sci. J. 2012 , 9 , 233 – 245 .

[ 9 ] a) M. H. Lee , K. K. Cho , A. P. Shah , P. Biswas , Environ. Sci. Technol. 2005 , 39 , 8481 – 8489 ; b) C. T. McMurray , J. A. Tainer , Nat. Genet. 2003 , 34 , 239 – 241 ; c) J. Goel , K. Kadirvelu , C. Rajagopal , V. K. Garg , Ind. Eng. Chem. Res. 2006 , 45 , 6531 – 6537 .

[ 10 ] J. A. Hansen , J. Lipton , P. G. Welsh , J. Morris , D. Cacela , M. J. Suedkamp , Aquatic Toxicol. 2002 , 58 , 175 – 188 .

[ 11 ] a) M. R. Gomez , S. Cerutti , L. L. Sombra , M. F. Silva , L. D. Martìnez , Food Chem. Toxicology 2007 , 45 , 1060 – 1064 ; b) S. P. Dolan , D. A. Nortrup , P. M. Bolger , S. G. Capar , J. Agric. Food Chem. 2003 , 51 , 1307 – 1312 ; c) S. Steely , D. Amarasiriwardena , J. Jones , J. Yanez , Microchem. J. 2007 , 86 , 235 – 240 ; d) J. Buffl e , M.-L. Tercier-Waeber , Trends Anal. Chem. 2005 , 24 , 172 – 191 .

[ 12 ] B. B. Kebbekus , Sample Preparations Techniques in Analytical Chemistry (Ed: S. Mitra ), John Wiley and Sons, Inc. , New Jersey 2003 .

[ 13 ] a) M. E. Moragues , R. Martínez-Máñez , F. Sancenón , Chem. Soc. Rev. 2011 , 40 , 2593- 2643 ; b) T. Balaji , S. A. El-Safty , H. Matsunaga , T. Hanaoka , F. Muzukami , Angew. Chem. 2006 , 118 , 7360 – 7366 ; c) S. A. El-Safty , A. A. Ismail , H. Matsunaga , T. Hanaoka , F. Mizukami , Adv. Funct. Mater. 2008 , 18 , 1485 – 1500 ; d) J. V. Ros-Lis , R. Casasús , M. Comes , C. Coll , M. D. Marcos , R. Martínez-Máñez , F. Sancenón , J. Soto , P. Amorós , J. El Haskouri , N. Garró , K. Rurack , Chem. Eur. J. 2008 , 14 , 8267 – 8278 ; e) S. J. Lee , D. R. Bae , W. S. Han , S. S. Lee , J. H. Jung , Eur. J. Inorg. Chem. 2008 , 1559 – 1564 ; f) H. Y. Lee , D. R. Bae , J. C. Park , H. Song , W. S. Han , J. H. Jung , Angew. Chem. Int. Ed. 2009 , 48 , 1239 – 1243 ; g) E. Kim , S. Seo , M. L. Seo , J. H. Jung , Analyst 2010 , 135 , 149 – 156 .

[ 14 ] a) S. Tatay , P. Gavina , E. Coronado , E. Palomares , Org. Lett. 2006 , 8 , 3857 – 3860 ; b) Z. Guo , W. Zhu , L. Shen , H. Tian , Angew. Chem. Int. Ed. 2007 , 46 , 5549 – 5553 ; c) J. S. Kim , M. G. Choi , K. C. Song , K. T. No , S. A. Ahn , S.-K. Chang , Org. Lett. 2007 , 9 , 1129 – 1132 ; d) R. Martínez-Máñez , F. Sancenón , M. Hecht , M. Biyikal , K. Rurack , Anal. Bioanal. Chem. 2011 , 399 , 55 – 74 ; e) S. Liu , H. Nie , J. Jiang , G. Shen , R. Yu , Anal. Chem. 2009 , 81 , 5724 ; f) J. Gong , T. Zhou , D. Song , L. Zhang , X. Hu , Anal. Chem. 2010 , 82 , 567 .

[ 15 ] a) G. Aragay , J. Pons , A. Merkoçi , Chem. Rev. 2011 , 111 , 3433 – 3458 ; b) L. H. Keith , L. U. Gron , J. L. Young , Chem. Rev. 2007 , 107 , 2695 – 2708 ; c) O. S. Wolfbeis , J. Mater. Chem. 2005 , 15 ,

2295www.small-journal.com & Co. KGaA, Weinheim

S. A. EI-Safty et al.

22

full papers

2657 ; d) S. Nagl , O. S. Wolfbeis , Analyst 2007 , 132 , 507 – 511 ; e) R. Niessner , Trends Anal. Chem. 1991 , 10 , 310 ; f) H. N. Kim , W. X. Ren , J. S. Kim , J. Yoon , Chem. Soc. Rev. 2012 , 41 , 3210 – 3244 ; g) P. Buhlmann , E. Pretsch , E. Bakker , Chem. Rev. 1998 , 98 , 1593 – 1687 .

[ 16 ] a) M. Comes , G. R. Lopez , M. D. Marcos , R. M. Manez , F. Sancenon , J. Soto , L. A.Villaescusa , P. Amoros , D. Beltran , Angew. Chem. Int. Ed. 2005 , 44 , 2918 ; b) M. Comes , M. D. Marcos , F. Sancenon , J. Soto , L. A.Villaescusa , P. Amoros , D. Beltran , Adv. Mater. 2004 , 16 , 1783 ; c) A. B. Desacalzo , K. Rurack , H. Weisshoff , R. M. Manez , M. D. Marcos , P. Amoros , K. Hoffmann , J. Soto , J. Am. Chem. Soc. 2005 , 127 , 184 ; d) N. A. Carrington , Z.-L.Xue , Acc. Chem. Res. 2007 , 40 , 343 – 350 ; e) S. A. El-Safty , D. Prabhakaran , A. A. Ismail , H. Matsunaga , F. Muzukami , Adv. Funct. Mater. 2007 , 17 , 3731 .

[ 17 ] a) C. Wu , M. K. K. Oo , X. Fan , ACS Nano 2010 , 4 , 5897 ; b) X. Guo , C. Wang , Y. Fang , L. Chen , S. Chen , J. Mater. Chem. 2011 , 21 , 1124 ; c) T. Linssen , F. Mees , K. Cassiers , P. Cool , A. Whittaker , E. F. Vansant , J. Phys. Chem. B 2003 , 107 , 8599 – 8606 ; d) J. Liu , Y. Lu , J. Am. Chem. Soc. 2005 , 127 , 12677 – 12683 ; e) J.-S. Lee , M. S. Han , C. A. Mirkin , Angew. Chem., Int. Ed. 2007 , 46 , 4093 – 4096 .

[ 18 ] a) X. Fang , Z. Liu , M.-F. Hsieh , M. Chen , P. Liu , C. Chen , N. Zheng , ACS Nano 2012 , 6 , 4434 – 4444 ; b) S. A. El-Safty , M. A. Shenashen , M. Ismael , M. Khairy , Adv. Funct. Mater. 2012 22 , 3013 – 3021 ; c) H. Yang , Q. Liu , Z. Liu , Z. Xie , Micr. Meso. Mater. 2010 , 127 , 213 ; d) K. Niesz , P. Yang , G. A. Somorjai , Chem. Commun. 2005 , 15 , 1986 – 1987 ; e) S. M. Morris , P. E. Fulvio , M. Jaroniec , J. Am. Chem. Soc. 2008 , 130 , 15210 – 15216 ; f) Q. Yuan , A.-X. Yin , C. Luo , L.-D. Sun , Y.-W. Zhang , W.-T. Duan , H.-C. Liu , C.-H. Yan , J. Am. Chem. Soc. 2008 , 130 , 3465 – 3472 ; g) C. Marquez-Alvarez , N. Zilkova , J. Perez-Pariente , J. Cejka , Catal. Rev. 2008 , 50 , 222 – 286 .

[ 19 ] a) S. A. El-Safty , Trends Anal. Chem. 2011 , 30 , 447 ; b) S. A. El-Safty , A. Shahat , W. Warkocki , M. Ohnuma , Small 2011 , 7 , 62 ; c) N. D. Hoa , S. A. El-Safty , Chem. Eur. J. 2011 , 17 , 12896 – 1290 ; d) S. A. El-Safty , M. Mekawy , A. Yamaguchi , A. Shahat , K. Ogawa , N. Teramae , Chem. Commun. 2010 , 46 , 3917 – 3919 .

96 www.small-journal.com © 2013 Wiley-VCH V

[ 20 ] a) A. Corma , S. Iborra , S. Miquel , J. Primo , J. Catal. 1996 , 161 , 713 – 719 ; b) S. A. El-Safty , A. Shahat , K. Ogawa , T. Hanaoka , Micro. Meso. Mater. 2011 , 138 , 51 – 62 .

[ 21 ] a) S. A. El-Safty , T. Hanaoka , Adv. Mater. 2003 , 15 , 1893 ; b) S. A. El-Safty , T. Hanaoka , Chem. Mater. 2004 , 16 , 384 .

[ 22 ] a) G. D. Christian , Analytical Chemistry , 6 th Ed , John Wiley & Sons, Inc. , NY , 2003 ; b) E. B. Sandell , Colorimetric Determina-tion of Traces of Metals , 3 th Ed , Interscience Publisher, Inc. , NY , 1959 , p.326; c) G. Wirnsberger , B. J. Scott , G. D. Stucky , Chem. Commun. 2001 , 119 – 120 ; d) Y.-F. Lee , F.-H. Nan , M.-J. Chen , H.-Y. Wu , C.-W. Ho , Y.-Y. Chen , C.-C. Huang , Anal. Methods 2012 , 4 , 1709 – 1717 .

[ 23 ] [ 23 ] D. Pérez-Quintanilla , I. del Hierro , M. Fajardo , I. Sierra , Mater. Res. Bull. 2007 , 42 1518 – 1530 ; b) L. Khezami , R. Capart , J. Hazard. Mater. 2005 , 123 , 223 ; c) Z.-Q. Zhao , X. Chen , Q. Yang , J.-H. Liu , X.-J. Huang , Chem , Commun. 2012 , 48 , 2180 – 2182 ; d) N. L. D. Filho , D. R. do Carmo , Talanta 2006 , 68 , 919 – 927 ; e) A. M. Khan , S. Khan , S. A. Ganai , Biol. Med. 2009 , 1 , 127 – 133 .

[ 24 ] a) F. Long , C. Gao , H. C. Shi , M. He , A. N. Zhu , A. M. Klibanov , A. Z. Gu , Biosen. Bioelectro. 2011 , 26 , 4018 – 4023 ; b) S. A. El-Safty , M. Khairy , M. Ismael , Sens. Actut. B—Chem. 2012 , 166 , 253 – 263 ; c) S. A. El-Safty , M. Shenashen , Trends Anal. Chem. 2012 , 48 , 98 – 115 .

[ 25 ] a) Y. Xudong , R. L. Chapman , W. Zhiqian , Phytochem. Anal. 2011 , 22 , 189 – 198 ; b) Y. Issa , D. C. Watts , A. J. Duxbury , P. A. Brunton , M. B Watson , C. M. Biomaterials 2003 , 24 , 981 – 987 ; c) S. H. Kim , R. P. Sharma , Toxicol. Appl. Pharmacol. 2004 , 196 , 47 – 57 ; d) K. Eisele , P. A. Lang , D. S. Kempe , B. A. Klarl , O. Niemöller , T. Wieder , S. M. Huber , C. Duranton , F. Lang , Toxicol. Appl. Pharm. 2006 , 210 , 116 – 122 ; e) K.-M. Lim , S. Kim , J.-Y. Noh , K. Kim , W.-H. Jang , O.-N. Bae , S.-M. Chung , J.-H. Chung , Environ. Health Perspect. 2010 , 118 , 928 .

Received: September 30, 2012 Revised: November 6, 2012Published online: January 29, 2013

erlag GmbH & Co. KGaA, Weinheim small 2013, 9, No. 13, 2288–2296