Where, and How, Does a NanowireBreak?Dongxu Wang, Jianwei Zhao,* Shi Hu, Xing Yin, Shuai Liang, Yunhong Liu, andShengyuan Deng

School of Chemistry and Chemical Engineering, Nanjing UniVersity,Nanjing, China 210008

Received December 14, 2006; Revised Manuscript Received February 26, 2007

ABSTRACT

Using molecular dynamics (MD) simulation, we studied the structural transformation and breaking mechanism of a single crystalline coppernanowire under continuous strain. At a certain strain rate, an ensemble of relaxed initial states of the nanowire can preferentially go throughone or more paths of deformation. In each deformation path, disordered atoms can be generated at the specific positions of the nanowire,where necking and breaking take place afterward. Such a breaking position is not predetermined; multiple initial states lead to a strain-rate-dependent, statistical distribution of breaking positions.

Nanoscale, defect-free materials like nanowires are of greatinterest due to their potential applications in micromechanicaland electronic systems (MEMS), molecular electronic de-vices, and other fields of nanotechnology. During the pastdecade, a great deal of effort has been made to elucidate thestructural and mechanical properties of nanowires. A numberof studies focused on the problem of nanowire breaking, buta comprehensive understanding has not yet been achieved.Ikeda et al.1 discovered that, under sufficiently high strainrate (∼5% ps-1) at 300 K, Ni nanowire is elastic up to 7.5%strain, after which the continuous strain induces a transitionfrom the crystalline phase to the amorphous phase. Koh etal.2 found that, for Pt nanowires at 300 K, the critical strainrate to induce a complete amorphous deformation is 4.0%ps-1, while the onset of amorphization occurred at a lowerstrain rate, 0.4% ps-1. Amorphous nanowires recrystallizewhen the strain rate was slowed down.1,3 For lower strainrate, dislocation and relaxation steps repeat.2,4 During thisrepeated process, global crystalline order changed to localclose-packed atomic arrangements,5 and the nanowire’ssurface reconstructed.6 Various disorders, faults, and vacan-cies were then created in the nanowires, and they played animportant role in nanowire elongation and breaking.5-9 Radialdistribution functions (RDF) and stress-strain relationshipscharacterize these different deformation mechanisms. Thedeformation mechanism also depends on nanowire’s size,shape, surface orientation, etc, as well as strain rate.9,10

Experimentally, a nanowire can form at nanocontacts, andnecking phenomenon can be observed.11,12

Although much progress has been made, the study ofnanoscale objects can still lead to new findings, which may

be governed by the microscopic statistics. A typical problemis: where does a finite-length nanowire break under tension?This has not been answered yet. One might intuitively thinkthat a nanowire would break at its middle along the straindirection if both the wire and the tension are symmetrical.Yet according to a number of atomic configuration plots,2,3,7

there is no evidence that breaking always takes place at themiddle. These plots suggest that there is a probability for ananowire to break at a certain position. As nanowires candeform by different mechanisms,13 such tendencies may becorrelated with the mechanism that come into play withdifferent strain rates.

It is not easy to find such a probability distribution. Thecommon “strain-and-relax” method used in simulation of ananowire under tension makes the study of breaking positiondifficult. In this model,1-3,8 strain is exerted at a certain time,and then the nanowire relaxes to a new equilibrium statebefore strain is exerted again. This quasi-equilibrium methodignores the difference in the initial states, which would great-ly influence the final atomic configuration due to the charac-teristics of many-body problems. In a dynamic, continuouspulling method,7,14 the nanowire is strained from a certainequilibrium state and evolves to a final atomic configuration.Strain from another equilibrium state can lead to a differentfinal atomic configuration. If strain is exerted on an ensembleof equilibrium states, the evolution of the whole ensemblecan be revealed. This will yield the distribution of breakingpositions for a nanowire at certain conditions.

To address the question about breaking, we performedmolecular dynamics simulations of nanowires strained bycontinuous pulling. Single crystalline copper nanowires(Figure 1), positioned with{100} surfaces facing out, are* Corresponding author. E-mail: zhaojw@nju.edu.cn.

NANOLETTERS

2007Vol. 7, No. 51208-1212

10.1021/nl0629512 CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 03/28/2007

Coordination Chemistry Reviews 251 (2007) 1951–1972

Review

Design of artificial metallonucleases with oxidative mechanism

Qin Jiang a, Nan Xiao a, Pengfei Shi b, Yangguang Zhu a, Zijian Guo a,∗a State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China

b Chemistry Department, Huaihai Institute of Technology, Lianyungang 222005, PR China

Received 26 September 2006; accepted 19 February 2007Available online 23 February 2007

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19522. Mechanism of oxidative cleavage of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1953

2.1. Reactive oxygen or metal-oxo species (ROS/RMOS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19532.2. Nucleobase modification or sugar-hydrogen abstraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1955

3. Methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19573.1. Investigation of enzymatic action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1957

3.1.1. Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19573.1.2. Atomic force microscopy (AFM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1957

3.2. Identification of reaction species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19573.2.1. Mass spectrometry (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19573.2.2. Electron paramagnetic resonance (EPR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19583.2.3. Nuclear magnetic resonance (NMR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19593.2.4. Other techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

3.3. Theoretical calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19594. Factors influencing oxidative cleavage efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

4.1. Metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19604.1.1. Metal centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19604.1.2. Redox potential of metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1960

4.2. Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19604.2.1. Structural requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19604.2.2. Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19604.2.3. Planarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19614.2.4. Geometric configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19624.2.5. Steric factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1962

4.3. Co-factors and reaction conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19634.3.1. Co-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19634.3.2. pH values and ionic strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1965

Abbreviations: AFM, atomic force microscopy; Arg, arginine; Asc, ascorbic acid; BDEs, bond dissociation energies; BLM, bleomycin; bpy, bipyridine;BQQ, benzoquinoquinoxaline; DL, dinucleating ligand; dppt, 3-(1,10-phenanthrolin-2-yl)-5,6-diphenylas-triazine; dppz, dipyrido[3,2-a:2′,3′-c]phenazine; dpq,dipyrido[3,2-d:2′,3′-f]quinoxaline; ds, double strand; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; GGH,glycine-glycine-histidine; Gly, glycine; Gs, guanosine; GSH, glutathione; HAT, hydrogen abstraction transfer; Hapt, 5-amino-3-pyridin-2-yl-1,2,4-triazole; His,histidine; HPLC–MS, high performance liquid chromatography–mass spectrometry; HTH, helix-turn-helix; KGHK, lysine-glycine-histidine-lysine; lc, linear con-former; MALDI, matrix-assisted laser desorption ionization; MCD, magnetic circular dichroism; MPA, 3-mercaptopropionic acid; MS, mass spectrometry; NMR,nuclear magnetic resonance; NS oligo, non-specific oligonucleotide; oc, open-circular conformer; ODNs, oligodeoxyribonucleotides; ONT, oligonucleotide; OP,1,10-phen-anthroline; ORNs, oligoribonucleotides; PAGE, polyacrylamide gel electrophoresis; PCET, proton-coupled electron transfer; phen, 1,10-phenanthroline;pta, 3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]acenaphthylene; RMOS, reactive metal-oxo species; ROS, reactive oxygen species; salen, N,N′-ethylenebis (sali-cylidene aminato); sc, supercoiled conformer; SECM, scanning electrochemical microscopy; SET, single electron transfer; TFO, triple-helix-forming oligonucleotide;TL, trinucleating ligand; Xaa, amino acid; XAS, X-ray absorption spectroscopy

∗ Corresponding author. Tel.: +86 25 83594549; fax: +86 25 83314502.E-mail address: zguo@nju.edu.cn (Z. Guo).

0010-8545/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ccr.2007.02.013

Ionic-Liquid-Doped Polyaniline Inverse Opals:Preparation, Characterization, and Application for theElectrochemical Impedance Immunoassay of HepatitisB Surface Antigen

By Xing-Hua Li, Lin Dai, Yan Liu, Xiao-Jun Chen, Wei Yan, Li-Ping Jiang,*

and Jun-Jie Zhu*

1. Introduction

Currently, with the development of nanoscience and nanotechnol-ogy,more andmore interest has been focused on using assemblednanoparticles to fabricate biosensors.[1–3] Among these assemblednanostructures, 3D orderedmacroporous (3DOM)materials withinterconnectedmacropores (the so-called ‘‘inverse opals’’) showedpotential applications in the field of bioreactors due to their well-designed structures, huge surface area, and unique properties.[4,5]

Self-assembled colloidal crystals, which are close-packed self-assemblies of monodisperse spheres, stand out as ideal templatesfor 3DOM fabrication by the infiltration of an opal with othermaterials and subsequent removal of the spheres. Among the

templates, silica with a spherical shape is anideal candidate material since it can beobtained conveniently in the desired size.Furthermore, commercial silica spheres areavailable and can be simply removed bydissolution with an aqueous HF solution. Sofar, various types of inverse opals have beenavailable by utilizing these colloidal crystaltemplates, for instance, metals,[4,6–9] inor-ganic oxides,[10–12] semiconductors,[13,14] andpolymers.[15–21] Among these materials, con-ducting polymers with easily adjustableproperties, especially polyaniline (PANI),are much more desirable as inverse opalsdue to simplicity in the synthesis from aninexpensive monomer, stability in ambientconditions, and unique electrochemicalproperties such as tunable redox proper-ties.[22,23]

Recently, some efforts have been made toapply macroporous PANI in the bio-electro-chemical field to exploit the advantages

provided by the highly ordered porous structure and the hugesurface area. Because the PANI obtained by electropolymerizationcan present a great amount of amine groups with positive charges,it can offer electrostatic anchoring points for nanoparticles orbiomolecules with negatively charged surfaces.[24–27] Meanwhile,because of its favorable storage stability, PANI can act as animmobilization platform for biocomponents. It has been reportedthat themacrosporousPANI is able to electrocatalyze the oxidationof reduced b-nicotinamide adenine dinucleotide (NADH)[28,29]

and catalyze the electrochemical reduction of nitrite,[30] and it canalso be used to fabricate a enzyme electrode for the detection ofglucose.[31]However, PANIdoesnot keep its conductive propertiesin nonacid media[32,33] and a neutral condition is required forimmunoreactions. The loss of redox activity of PANI in neutralsolutions also brings the limitation of using pure PANI inverseopals for immunoassays. As a result, electropolymerization ofPANI is preferentially carried out in the presence of dopants. It hasbeendemonstrated that the redox activity ofPANI canbe sustainedin neutral solutions by doping.[34–36] The inclusion of the dopantmaintains electrical neutrality in the oxidized form of the polymerand also leads to an increase in its structural stability andconductivity over a broader range of pH values.

FULLPAPER

www.afm-journal.de

[*] Dr. L.-P. Jiang, Prof. J.-J. Zhu, Dr. X.-H. Li, L. Dai, Y. Liu, Dr. X.-J. Chen,Dr. W. YanKey Lab of Analytical Chemistry for Life Science (MOE)School of Chemistry and Chemical EngineeringNanjing UniversityNanjing, 210093 (P. R.China)E-mail: Jianglp@nju.edu.cn; jjzhu@nju.edu.cn

DOI: 10.1002/adfm.200901003

A 3D ordered macroporous (3DOM) ionic-liquid-doped polyaniline (IL-PANI)

inverse opaline film is fabricated with an electropolymerization method and

gold nanoparticles (AuNPs) are assembled on the film by electrostatic

adsorption, which offers a promising basis for biomolecular immobilization

due to its satisfactory chemical stability, good electronic conductivity, and

excellent biocompatibility. The AuNP/IL-PANI inverse opaline film could be

used to fabricate an electrochemical impedance spectroscopy (EIS)

immunosensor for the determination of Hepatitis B surface antigen (HBsAg).

The concentration of HBsAg is measured using the EIS technique by

monitoring the corresponding specific binding between HBsAg and HBsAb

(surface antibody). The increased electron transfer resistance (Ret) values are

proportional to the logarithmic value of the concentration of HBsAg. This

novel immunoassay displays a linear response range between 0.032 pg mL�1

and 31.6 pg mL�1 with a detection limit of 0.001 pg mL�1. The detection of

HBsAg levels in several sera showed satisfactory agreement with those using

a commercial turbidimetric method.

3120 � 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2009, 19, 3120–3128

FULL

DOI: 10.1002/adfm.200701340 P

APER

Gold Nanoparticle–Colloidal Carbon Nanosphere Hybrid Material:Preparation, Characterization, and Application for an AmplifiedElectrochemical Immunoassay**

By Rongjing Cui, Chang Liu, Jianming Shen, Di Gao, Jun-Jie Zhu,* and Hong-Yuan Chen

A rapid microwave-hydrothermal method has been developed to prepare monodisperse colloidal carbon nanospheres from

glucose solution, and gold nanoparticles (AuNPs) are successfully assembled on the surface of the colloidal carbon nanospheres

by a self-assembly approach. The resultingAuNP/colloidal carbon nanosphere hybridmaterial (AuNP/C) has been characterized

and is expected to offer a promising template for biomolecule immobilization and biosensor fabrication because of its satisfactory

chemical stability and the good biocompatibility of AuNPs. Herein, as an example, it is demonstrated that the as-prepared

AuNP/C hybrid material can be conjugated with horseradish peroxidase-labeled antibody (HRP-Ab2) to fabricate

HRP-Ab2-AuNP/C bioconjugates, which can then be used as a label for the sensitive detection of protein. The amperometric

immunosensor fabricated on a carbon nanotube-modified glass carbon electrode was very effective for antibody immobilization.

The approach provided a linear response range between 0.01 and 250 ng mL�1 with a detection limit of 5.6 pg mL�1. The

developed assay method was versatile, offered enhanced performances, and could be easily extended to other protein detection

as well as DNA analysis.

1. Introduction

The development of nanoscience and nanotechnology has

brought a great momentum to electrochemical bioassay.

Analysts in this field are always enthusiastic in finding new

materials with good biocompatibility for improving the

behavior of bioassays. It has been reported that nanoparticle

(NP)-based amplification platforms and amplification pro-

cesses dramatically enhance the intensity of the electroche-

mical signal and lead to ultrasensitive bioassays.[1–3] Metal

NPs, for example, were once directly used as electroactive

labels to amplify the electrochemical detection of DNA and

proteins.[4,5] Meanwhile, some nanomaterials, such as carbon

nanotubes[6,7] and silicon nanoparticles[8–11] have been used as

efficient carriers to load a large amount of electroactive species

(enzyme, guanine, etc.) as reporters/markers for amplifying the

detection of biomolecules. However, as-prepared nanomater-

ials usually have relatively inert surfaces, which makes surface

modification almost unavoidable before use as supports.[12–15]

[*] Prof. J.-J. Zhu, R. J. Cui, C. Liu, J. M. Shen, D. Gao, Prof. H. Y. ChenMOE Key Laboratory of Analytical Chemistry for Life ScienceSchool of Chemistry and Chemical Engineering, Nanjing UniversityNanjing 210093 (P. R. China)E-mail: jjzhu@nju.edu.cn

[**] This project was supported by the National Natural Science Founda-tion of China (20635020, 20575026, and 20521503) and the NationalBasic Research Program of China (2006CB933201). We are grateful tothe financial support of the European Community Sixth FrameworkProgram through a STREP grant to the SELECTNANO Consortium,Contract No. 516922.

Adv. Funct. Mater. 2008, 18, 2197–2204 � 2008 WILEY-VCH Verlag

Recently, Li and coworkers[16] developed a controllable

hydrothermal synthetic route tomonodisperse colloidal carbon

spheres in aqueous glucose solutions. The approach is an

absolute ‘green’ method, and the synthetic procedure involves

none of the organic solvents, initiators, or surfactants that are

commonly used for the preparation of polymer micro- or

nanospheres. The colloidal carbon spheres represent excellent

candidates for application in bioassays by virtue of their high

chemical stability, and the convenient and absolutely ‘green’

preparation method.[16] In particular, the as-formed colloidal

spheres inherit large numbers of functional groups from

the starting materials and have reactive surfaces, which

facilitate loading with noble-metal nanoparticles and make it

possible for further bioassay application.

In this work, we present a new method for the rapid syn-

thesis of colloidal carbon spheres in aqueous glucose solutions

by a microwave-hydrothermal technique. Citrate-stabilized

gold nanoparticles (AuNPs) are assembled on the surface of

colloidal carbon spheres to fabricate a core–shell hybrid

material by the self-assembly approach. This material retains

the colloids’ stability and inherits the advantages from its

parent materials, such as good solubility and dispersibility

in water. Furthermore, since carbon is far more stable than

polymers with respect to high-temperature stability and

resistance to acids, bases, and solvents, these hybrid struc-

tures should have superior advantages over noble-metal

nanoparticle-decorated polymer spheres in fields such as

catalysis or protein and DNA detection.

The advantages of the core–shell AuNP-colloidal carbon

sphere (AuNP/C) hybrids make them suitable for biomolecule

loading, e.g., with horseradish peroxidase-labeled antibody

GmbH & Co. KGaA, Weinheim 2197

DOI: 10.1002/adfm.200800492

Cadmium(II) (8-Hydroxyquinoline) Chloride Nanowires:Synthesis, Characterization and Glucose-Sensing Application**

By Hongcheng Pan, Hongyu Lin, Qingming Shen, and Jun-Jie Zhu*

1. Introduction

Metal 8-hydroxyquinoline (Mqn) chelates have been the

focus of many studies because of their wide range of

applications in photoluminescence, electroluminescence, and

field-emission. Anzenbacher and co-workers recently reported

the synthesis of a series of emission-color-tunable Alq3complexes with arylethynyl substituents.[1] The substituents

were found to affect the emission color and fluorescence

quantum yield of the resulting AlIII complex. A new d-phase of

Alq3 was identified and its blue luminescence studied.[2,3] Brett

and co-workers fabricated chiral thin films, consisting of sub-

micrometer scaled helical Alq3 structures that generate

circularly polarized photoluminescence.[4] As electrolumines-

cent materials, Mqn and its derivatives have been widely used

in organic light-emitting devices (OLEDs).[5–10] Alq3 has been

used frequently because of its good electronic conductivity and

strong electroluminescence emission.[11,12] Many other Mqnchelates have also been demonstrated to be useful emitter

materials. For instance, the Gaq3 devices have a power

efficiency that is approximately 50% higher than Alq3structures, suggesting that Gaq3 might be a superior emitter

material for display applications. The operating voltage of

(Znq2)4-based OLEDs is lower than that of identical devices

made with Alq3.[6] Due to the unique optoelectronic properties

of nanostructured organic materials, more attention is, there-

fore, turning to nanostructuredMqn chelates. For example, the

electroluminescent device fabricated by Alq3 nanowires

showed an obvious size-dependent performance.[5] In addition,

Alq3 nanostructures, such as nanowires, nanorods, and

nanometer-scaled crystalline films, exhibited field emission

with a relatively low turn-on voltage.[13–15] Currently, most

research work has focused on the nanostructure of Alq3 and

Znq2. Only a few investigations of other metal 8-hydroxyqui-

noline nanostructures have been carried out, partly because it

is not easy to synthesize a well-defined nanostructure.

Recently, luminescent Cdq2 has receivedmuch attention. As

most-frequently reported in the literature, Cdq2 can be

synthesized by the reaction of the cadmium ion with excess

8-hydroxyquinoline.[16,17] For instance, Cdq2 nanorods and

nanoflowers were synthesized in an oleic acid/sodium oleate/

ethanol/hexane/H2O system at a Cd2þ/q ratio of 1:2.[18] The

reaction time, temperature, and reagent concentrations are the

key factors in controlling the morphology of the Cdq2nanostructure at a q-excess condition. However, it is interest-

ing to investigate the situation where q reacts with excess

cadmium salt. A new structure might be obtained because of

the effect of the cadmium-salt anion on the formation of Cdq2.

Herein, we report a sonochemical route to synthesize well-

defined CdqCl nanowires in the presence of a highly excessive

amount of cadmium salt (CdCl2), while Cdq2 flakes were

obtained at a q-excess condition. By increasing the ratio of

CdCl2/q, the morphology and composition of the products

could be transformed from Cdq2 micrometer-scaled flakes to

CdqCl nanowires. Furthermore, the prepared CdqCl nano-

wires can be developed into a fluorescence sensor for H2O2 and

FULLPAPER

[*] Prof. J. J. Zhu, Dr. H. C. Pan, H. Y. Lin, Q. M. ShenKey Lab of Analytical Chemistry for Life Science (MOE)School of Chemistry and Chemical EngineeringNanjing University, Nanjing 210093 (P. R. China)E-mail: jjzhu@nju.edu.cn

[**] This work is supported by the National Natural Science Foundation ofChina (NSFC; No. 20325516, 20635020, 90606016), NSFC for CreativeResearch Group (No. 20521503), and the China Scholarship Council(No. 20073020). We also thank the support of the National BasicResearch Program of China (2006CB933201).

Luminescent cadmium(II) (8-hydroxyquinoline) chloride (CdqCl) complex nanowires are synthesized via a sonochemical

solution route. The results of X-ray photoelectron spectroscopy, energy dispersive X-ray analysis, infrared spectroscopy,

elemental analysis (EA), and atomic absorption spectroscopy demonstrate that the chemical composition of the product is

Cd(C9H6NO)Cl. Transmission electron microscopy and scanning electron microscopy images show that the CdqCl product is

wire-like in structure, with a diameter of approximately 50 nm and an approximate length of 2–4mm. The morphology and

composition of the product can be transformed fromCdq2micrometer-scaled flakes to CdqCl nanowires by increasing the ratio of

CdCl2/q. A new fluorescent sensing strategy for detecting H2O2 and glucose is developed and is based on the combination of the

luminescent nanowires and the biocatalytic growth of Au nanoparticles. The quenching effects of Au nanoparticles and AuCl�4 on

the fluorescence of CdqCl nanowires are investigated. The dominant factor for the fluorescence quenching of CdqCl nanowires is

that the Stern–Volmer quenching constant of Au nanoparticles is larger than that of AuCl�4 .

3692 � 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2008, 18, 3692–3698

DOI: 10.1002/adfm.200600491

Conductive Mesocellular Silica–Carbon Nanocomposite Foamsfor Immobilization, Direct Electrochemistry, and Biosensingof Proteins**

By Shuo Wu, Huangxian Ju,* and Ying Liu

1. Introduction

Over the last two decades considerable attention has been paidto the development of new biocompatible materials with goodconductivity,[1,2] suitable hydrophilicity,[1] high porosity,[3] andlarge surface area[4] for protein immobilization. The electrochem-ical biosensing applications of such materials have been exploreddue to the inherent advantages of electrochemical methods, suchas low cost, portability, high sensitivity, and good selectivity.[5] In-organic materials are attractive for protein immobilization due totheir chemical and thermal stability, good mechanical strength,biocompatibility, and their potential for improving the stability ofthe enzymes and/or proteins under extreme conditions.[6,7] Or-dered mesoporous materials are a typical example of inorganicmaterials that have been used for this purpose. These materialshave high surface areas, large pore volumes, and narrow pore size

distributions. Up till now, several different kinds of mesoporousmolecular sieves composed of silicates,[8,9] metal oxides,[10] andcarbon[11–13] have been used for protein immobilization; thesematerials generally exhibit excellent performance. The chemicalcomposition, surface characteristics,[9,14] morphology,[15] as well asthe mesopore distribution,[16] can be designed to improve thebiocompatibility and hydrophilicity of the materials for the im-mobilization of different proteins. Among these mesoporousmaterials, mesoporous silicates are the most extensively used bio-compatible materials in the area of electrochemical biosensing.Many heme proteins such as hemoglobin, myoglobin, horserad-ish peroxide, and cytochrome c show direct electron transfer in aseries of mesoporous silica materials.[3,4,17]

Mesoporous carbon is another important versatile materialdue to its low cost and conductive properties. In recent years,many researchers have tried to immobilize proteins in orderedmesoporous carbon materials.[11–13,18] However, very few re-ports have focused on the electrochemical biosensing applica-tions of these materials due to their limited pore diameters ofless than 5 nm and their high hydrophobicity.[12,19] The hydro-phobicity makes it difficult for analytes[1] in aqueous solutionto approach the active enzyme sites on the electrode surface.In this work, we have designed a new mesoporous silica–car-bon nanocomposite foam (MSCF) by combining the advan-tages of mesoporous carbon with the good biocompatibilityand hydrophilicity of silica materials.

Porous silica–carbon nanocomposites have been developedas solar energy absorbers by two general strategies which leadto different carbon cluster sizes. Silica is used as a transparentbinder and carbon nanoparticles are employed as the light-ab-

Adv. Funct. Mater. 2007, 17, 585–592 © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 585

–[*] Prof. H. X. Ju, Dr. S. Wu, Y. Liu

Key Laboratory of Analytical Chemistry for Life ScienceMinistry of Education of ChinaSchool of Chemistry and Chemical EngineeringNanjing UniversityNanjing 210093 (P.R. China)E-mail: hxju@nju.edu.cn

[**] We gratefully acknowledge the National Science Funds for Distin-guished Young Scholars (20 325 518) and Creative Research Groups(20 521 503), the Key Program (20 535 010) from the National NaturalScience Foundation of China, and the Creative Program for Postgrad-uates in Jiangsu Universities for financial support of this research.Supporting Information is available online from Wiley InterScience orfrom the author.

A mesocellular silica–carbon nanocomposite foam (MSCF) is designed for the immobilization and biosensing of proteins. Theas-prepared MSCF has a highly ordered mesostructure, good biocompatibility, favorable conductivity and hydrophilicity, largesurface area, and a narrow pore-size distribution, as verified by transmission electron microscopy (TEM), IR spectroscopy,electrochemical impedance spectroscopy (EIS), nitrogen adsorption–desorption isotherms, pore size distribution plots, andwater contact angle measurements. Using glucose oxidase (GOD) as a model, the MSCF is tested for immobilization of redoxproteins and the design of electrochemical biosensors. GOD molecules immobilized in the mesopores of the MSCF show directelectrochemistry with a fast electron transfer rate (14.0 ± 1.7 s–1) and good electrochemical performance. Based on a decreaseof the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linearresponse to glucose concentrations ranging from 50 lM to 5.0 mM with a detection limit of 34 lM at an applied potential of–0.4 V. The biosensor shows good stability and selectivity and is able to exclude interference from ascorbic acid (AA) and uricacid (UA) species that always coexist with glucose in real samples. The nanocomposite foam provides a good matrix for proteinimmobilization and biosensor preparation.

FULL

PAPER

Two-Dimensional Pentacene:3,4,9,10-PerylenetetracarboxylicDianhydride Supramolecular Chiral Networks on Ag(111)

Wei Chen,*,† Hui Li,‡ Han Huang,† Yuanxi Fu,‡ Hong Liang Zhang,† Jing Ma,*,‡ andAndrew Thye Shen Wee*,†

Department of Physics, National UniVersity of Singapore, 2 Science DriVe 3, 117542 Singapore,and Institute of Theoretical and Computational Chemistry, Department of Chemistry, Nanjing

UniVersity, Hankou Road 22, Nanjing, 210093 P.R. China

Received March 3, 2008; E-mail: phycw@nus.edu.sg (W.C.); majing@netra.nju.edu.cn (J.M.); phyweets@nus.edu.sg (A.T.S.W.)

Abstract: Self-assembly of the binary molecular system of pentacene and 3,4,9,10-perylenetetracarboxylicdianhydride (PTCDA) on Ag(111) has been investigated by low-temperature scanning tunneling microscopy,molecular dynamics (MD), and density functional theory (DFT) calculations. Well-ordered two-dimensional(2D) pentacene:PTCDA supramolecular chiral networks are observed to form on Ag(111). The 2D chiralnetwork formation is controlled by the strong interfacial interaction between adsorbed molecules and theunderlying Ag(111), as revealed by MD and DFT calculations. The registry effect locks the adsorbedpentacene and PTCDA molecules into specific adsorption sites due to the corrugation of the potentialenergy surface. The 2D supramolecular networks are further constrained through the directional CdO · · ·H-Cmultiple intermolecular hydrogen bonding between the anhydride groups of PTCDA and the peripheralaromatic hydrogen atoms of the neighboring pentacene molecules.

Introduction

Self-assembled molecular superstructures with well-definedorientation, conformation, and two-dimensional (2D) organiza-tion on surfaces have potential applications in organic1 andmolecular electronics2 and biosensors.3 In particular, thedevelopment of molecular nanodevices relies on the controlledpositioning and assembly of functional molecules on surfaces,which crucially depends on the interplay between variouschemical and physical bonds formed and lateral interactions ofdifferent strength and length scales.4 Directional intermolecularinteractions, such as hydrogen-bonding (H-bonding) andmetal-ligand interactions, have been widely used in supramo-

lecular chemistry to fabricate engineered molecular crystals and,more recently, surface-supported one-dimensional (1D) or 2Dhighly periodic supramolecular assemblies, including molecularsupergrating,5 honeycomb networks,6 and various rationallydesigned molecular nanostructures.7-9 In some cases, molecularself-assembly on surfaces can lead to the formation of chiralsupramolecular structures.12 Such chiral effects can arise fromthe adsorption of chiral molecules on substrates, where thechirality of the adsorbates is transferred to the surface as-semblies,13 or the adsorption on the high-index chiral metal

† National University of Singapore.‡ Nanjing University.

(1) (a) Dimitrakopoulos, C. D.; Malenfant, P. R. L. AdV. Mater. 2002,14, 99. (b) Coropceanu, V.; J.; Cornil, J.; da Silva, D. A.; Olivier, Y.;Silbey, R.; Bredas, J. L. Chem. ReV. 2007, 107, 926.

(2) (a) Joachim, C.; Ratner, M. A. Proc. Natl. Acad. Sci. U.S.A. 2005,102, 8801. (b) Heath, J. H.; Ratner, M. A. Phys. Today 2003, 56, 43.(c) Joachim, C.; Gimzewski, J. K.; Aviram, A. Nature 2000, 408, 541.

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(6) (a) Theobald, J. A.; Oxtoby, N. S.; Phillips, M. A.; Champness, N. R.;Beton, P. H. Nature 2003, 424, 1029. (b) Theobald, J. A.; Oxtoby,N. S.; Champness, N. R.; Beton, P. H.; Dennis, T. J. S. Langmuir2005, 21, 2038. (c) Perdigao, L. M. A.; Perkins, E. W.; Ma, J.; Staniec,P. A.; Rogers, B. L.; Champness, N. R.; Beton, P. H. J. Phys. Chem.B 2006, 110, 12539. (d) Staniec, P. A.; Perdigao, L. M. A.; Saywell,A.; Champness, N. R.; Beton, P. H. ChemPhysChem 2007, 8, 2177.

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(8) (a) Stepanow, S.; Lingenfelder, M.; Dmitriev, A.; Spillmann, H.;Delvigne, E.; Deng, X.; Cai, C.; Barth, J. V.; Kern, K. Nat. Mater.2004, 3, 229. (b) Stepanow, S.; Lin, N.; Barth, J. V.; Kern, K. Chem.Commun. 2006, 2153.

Published on Web 08/23/2008

10.1021/ja801577z CCC: $40.75 2008 American Chemical Society J. AM. CHEM. SOC. 2008, 130, 12285–12289 9 12285

Technical Notes

Analysis of Nonadherent Apoptotic Cells by aQuantum Dots Probe in a Microfluidic Device forDrug Screening

Liang Zhao, Peng Cheng, Jianxin Li, Yue Zhang, Miaomiao Gu, Jun Liu, Jianrong Zhang,* andJun-Jie Zhu*

MOE Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering,Nanjing University, Nanjing 210093, P. R. China

This technical note describes a facile technique to screensome anticancer drugs and evaluate their effects onnonadhesive leukemic cells in an easily fabricated mi-crofluidic device by utilizing the Annexin V conjugatedquantum dots as apoptosis detection probes. The cellimmobilizing structures and gradient-generating channelswere integrated within the device which was fabricated inone-single step. The nonadhesive leukemic HL-60 cellscan be felicitously immobilized and cultured on the damstructures at a proper lateral pressure. We then deliveredAnnexin V functionalized quantum dots which can readilybind to the outer membrane of apoptotic cells anddistinguish the apoptosis from unaffected cells with singlecell level resolution. The diffusion time of quantum dotsreduced to 5 min before imaging. The capabilities ofevaluating drug effect on HL-60 cell line have been shownin both population way and individual cell level. Thetechnique presented herein can bridge the gap betweenthe quantum dots based in vitro cell imaging and theanalysis of individual apoptotic cell in a microfluidicsystem, allows an easy operating protocol to screen someclinically available anticancer drugs.

Apoptosis refers to a specific form of programmed or suicidalcell death, which takes place in tissues to guarantee the welfareof the whole organism though the elimination of unwanted cells.1,2

Dysfunction in apoptosis can lead to disease states includingneurodegenerative diseases (e.g., Alzheimer, Parkinson), autoim-mune diseases (e.g., lupus erythematosus, rheumatoid arthritis),and cancer.3-5 The techniques for detection apoptotic cell aretherefore highly interesting, and much effort has been taken tomeasure apoptosis in a specific and a sensitive way. Rapid andsensitive detection of apoptotic cell is of considerable interest to

biological, pharmarceutical, and chemical researchers.6-8 Nowa-days, a number of methods are available to determine pro-grammed cell death.9-12 However, in most cases, expertise andsophisticated equipments were involved in these methods, whichare very labor intensive and sample-consuming. Moreover, con-ventional techniques are not suitable for single cell level analysisor incapable of real-time monitoring for understanding theheterogeneity during apoptosis.

Microfluidic device have shown great potential toward realizingthese goals in recent development.13-24 Albert van den Berg andco-workers successfully developed a silicon-glass microfluidicdevice which realized real time apoptotic dynamics measuring ina single cell level with the advantage of easily transporting cellsin the channel and discriminating different dynamic stages during

* To whom correspondence should be addressed. E-mail: jjzhu@nju.edu.cn(J.-J.Z.); jrzhang@nju.edu.cn. (J.Z.). Fax: +86-25-83594976.

(1) Kerr, J. F. R.; Wyllie, A. H.; Currie, A. R. Br. J. Cancer 1972, 26, 239–257.(2) Ravichandran, K. S. Cell 2003, 113, 817–820.(3) Haunstetter, A.; Izumo, S. Circ. Res. 1998, 82, 1111–1129.(4) Carson, D. A.; Ribeiro, J. M. Lancet 1993, 341, 1251–1254.(5) Savill, J.; Gregory, C.; Haslett, C. Science 2003, 302, 1516–1517.

(6) Vermes, I.; Haanen, C.; Reutelingsperger, C. J. Immunol. Methods 2000,243, 167–190.

(7) van Tilborg, G. A. F.; Mulder, W. J. M.; Chin, P. T. K.; Storm, G.;Reutelingsperger, C. P.; Nicolay, K.; Strijkers, G. J. Bioconjugate Chem.2006, 17, 865–868.

(8) Wang, G.; Song, E.; Xie, H.; Zhang, Z.; Tian, Z.; Zuo, C.; Pang, D.; Wu, D.;Shi, Y. Chem. Commun. 2005, 4276–4278.

(9) Lapotko, D. Cytometry 2004, 58A, 111–119.(10) Cotter, T. G.; Martin, S. J. Techniques in Apoptosis: A User’s Guide; Portland

Press Ltd.: London, 1996.(11) Vermes, I.; Hannen, C.; Steffens-Nakken, H.; Reutelingsperger, C. J. Im-

munol. Methods 1995, 184, 39–51.(12) Zhang, J.; Xu, M. Trends Cell Biol. 2002, 12, 84–89.(13) Dittrich, P. S.; Manz, A. Nat. Rev. Drug Discovery 2006, 5, 210–218.(14) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403–411.(15) Qin, J.; Ye, N.; Liu, X.; Lin, B. Electrophoresis 2005, 26, 3780–3788.(16) Tamaki, E.; Sato, K.; Tokeshi, M.; Sato, K.; Aihara, M.; Kitamori, T. Anal.

Chem. 2002, 74, 1560–1564.(17) Wu, H. K.; Wheeler, A.; Zare, R. N. Proc. Natl. Acad. Sci. U.S.A. 2004,

101, 12809–12813.(18) Munoz-Pinedo, C.; Green, D. R.; van den Berg, A. Lab Chip 2005, 5, 628–

633.(19) Di Carlo, D.; Aghdam, N.; Lee, L. P. Anal. Chem. 2006, 78, 4925–4930.(20) Nevill, J. T.; Cooper, R.; Dueck, M.; Breslauer, D. N.; Lee, L. P. Lab Chip

2007, 7, 1689–1695.(21) Wang, Z.; Kim, M. C.; Marquez, M.; Thorsen, T. Lab Chip 2007, 7, 740–

745.(22) Jian, G.; Yin, X.; Fang, Z. Lab Chip 2004, 4, 47–52.(23) Li, X.; Li, P. C. H. Anal. Chem. 2005, 77, 4315–4322.(24) Thompson, D. M.; King, K. R.; Wieder, K. J.; Toner, M.; Yarmush, M. L.;

Jayaraman, A. Anal. Chem. 2004, 76, 4098–4103.

Anal. Chem. 2009, 81, 7075–7080

10.1021/ac901121f CCC: $40.75 2009 American Chemical Society 7075Analytical Chemistry, Vol. 81, No. 16, August 15, 2009Published on Web 07/27/2009

Synthesis of Gelatin-Stabilized Gold Nanoparticlesand Assembly of Carboxylic Single-Walled CarbonNanotubes/Au Composites for Cytosensing andDrug Uptake

Jing-Jing Zhang, Miao-Miao Gu, Ting-Ting Zheng, and Jun-Jie Zhu*

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), School of Chemistry andChemical Engineering, Nanjing University, Nanjing 210093, P. R. China

Gelatin-stabilized gold nanoparticles (AuNPs-gelatin) withhydrophilic and biocompatible were prepared with asimple and “green” route by reducing in situ tetrachlo-roauric acid in gelatin. The nanoparticles showed theexcellent colloidal stability. UV-vis spectra, transmissionelectron microscopy (TEM), and atomic force microscopyrevealed the formation of well-dispersed AuNPs withdifferent sizes. By combination of the biocompatibility ofAuNPs and excellent conductivity of carboxylic single-walled carbon nanotubes (c-SWNTs), a novel nanocom-posite was designed for the immobilization and cytosens-ing of HL-60 cells at electrodes. The immobilized cellsshowed sensitive voltammetric response, good activity,and increased electron-transfer resistance. It can be usedas a highly sensitive impedance sensor for HL-60 cellsranging from 1 × 104 to 1 × 107 cell mL-1 with a limitof detection of 5 × 103 cell mL-1. Moreover, thenanocomposite could effectively facilitate the interac-tion of adriamycin (ADR) with HL-60 cells and remark-ably enhance the permeation and drug uptake ofanticancer agents in the cancer cells, which couldreadily lead to the induction of the cell death ofleukemia cells.

For the last 2 decades, there has been a considerable interestin the design of biosensors for the detection of cells and theiractivity monitoring. Some methods such as scanning electro-chemical microscopy (SECM),1 electrochemical impedance spec-troscopy (EIS),2 electric cell-substrate impedance sensing (ECIS),3

and oxygen electrode4 have been developed for monitoring cellviability and proliferation. Among these methods, electrochemicalcell-based biosensors have attracted increasing attention due totheir remarkable advantages such as low cost, convenient opera-tion, rapid detection, and good sensitivity.5-7 Anchoring cells onthe electrode for producing electrochemical signals is a key step

in the development of electrochemical biosensors. However, manylimitations were observed during the immobilization of cells onelectrode surface by conventional methods, such as instability,additional diffusional barrier, and decrease of cell viability.8-10

Recent research has focused on the design of biomaterials forcell adhesion.11-13

With the development of nanoscience and nanotechnology,gold nanoparticles (AuNPs) have been extensively used as amatrix and cytochemical label for the respective cancer diagnosticsand tumor target treatment.14-16 To date, a variety of methodshave been developed to generate monodisperse gold nano-particles.17-20 One of the most widely used methods is thereduction of tetrachloraurate ions by NaBH4 or other reductivereagents. Recently, AuNPs synthesized with polymers as boththe reducing and stabilizing agent were reported to improvetheir stability and biocompatibility and enhance the capabilityfor the immobilization due to the superior properties of theformed nanocomposites.21 Gelatin is the thermally and hydro-lytically denatured product of collagen, which has beenextensively applied as the immobilization matrix for thepreparation of biosensors.22,23 It has a triple-helical structure and

* To whom correspondence should be addressed.(1) Kaya, T.; Torisawa, Y. S.; Oyamatsu, D. Biosens. Bioelectron. 2003, 18,

1379–1383.(2) K’Owino, I. O.; Sadik, O. A. Electroanalysis 2005, 17, 2101–2113.(3) Heiskanen, A. R.; Spegel, C. F.; Kostesha, N. Langmur 2008, 24, 9066–

9073.(4) Lahdesmaki, I.; Park, Y. K.; Carroll, A. D. Analyst 2007, 132, 811–817.(5) Du, D.; Ju, H. X.; Zhang, X. J.; Chen, J.; Cai, J.; Chen, H. Y. Biochemistry

2005, 44, 11539–11545.

(6) Hua, H. L.; Jiang, H.; Wang, X. M.; Chen, B. A. Electrochem. Commun.2008, 10, 1121–1124.

(7) Shen, Q.; You, S. K.; Park, S. G.; Jiang, H.; Guo, D. D.; Chen, B. A.; Wang,X. M. Electroanalysis 2008, 20, 2526–2530.

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11–14.(10) Zheng, J.; Northrup, S. R.; Hornsby, P. J. In Vitro Cell. Dev. Biol.: Anim.

1998, 34, 679–684.(11) Ionescu, R. E.; Abu-Rabeah, K.; Cosnier, S. Electroanalysis 2006, 18, 1041–

1046.(12) Zhang, Z.; Chen, S. F.; Jiang, S. Y. Biomacromolecules 2006, 7, 3311–3315.(13) Gu, H. Y.; Chen, Z.; Sa, R. X. Biomaterials 2004, 25, 3445–3451.(14) Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.;

Mirkin, C. A. Nano Lett. 2007, 7, 3818–3821.(15) Souza, G. R.; Christianson, D. R.; Staquicini, F. I.; Ozawa, M. G.; Snyder,

E. Y.; Sidman, R. L.; Miller, J. H.; Arap, W.; Pasqualini, R. Proc. Natl. Acad.Sci. U.S.A. 2006, 103, 1215–1220.

(16) Murphy, C. J. Nano Lett. 2007, 7, 116–119.(17) Guo, S. J.; Wang, E. K. Inorg. Chem. 2007, 46, 6740–6743.(18) Zheng, N.; Fan, J.; Stucky, G. D. J. Am. Chem. Soc. 2006, 128, 6550–6551.(19) Lu, X. M.; Tnan, H. Y.; Korgel, B. A. Chem.sEur. J. 2008, 14, 1584–1591.(20) Li, Z. G.; Friedrich, A.; Taubert, A. J. Mater. Chem. 2008, 18, 1008–1014.(21) Sardar, R.; Park, J. W.; Shumaker-Parry, J. S. Langmuir 2007, 23, 11883–

11889.(22) Li, N.; Xu, J. Z.; Yao, H.; Zhu, J. J.; Chen, H. Y. J. Phys. Chem. B 2006,

110, 11561–11565.

Anal. Chem. 2009, 81, 6641–6648

10.1021/ac900628y CCC: $40.75 2009 American Chemical Society 6641Analytical Chemistry, Vol. 81, No. 16, August 15, 2009Published on Web 07/14/2009

Channel and Substrate Zone Two-DimensionalResolution for Chemiluminescent MultiplexImmunoassay

Zhifeng Fu,† Zhanjun Yang,† Jinhai Tang,‡ Hong Liu,† Feng Yan,*,‡ and Huangxian Ju*,†

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry, NanjingUniversity, Nanjing 210093, and Jiangsu Institute of Cancer Research, Nanjing 210009, P.R. China

A two-dimensional resolution system of channels andsubstrate zones was proposed for multiplex immunoassayperformed with a designed multichannel chemilumines-cent (CL) detection device coupled with a single photo-multiplier. Using carcinoma antigen 125 (CA 125),carcinoma antigen 153 (CA 153), carcinoma antigen 199(CA 199), and carcinoembryonic antigen (CEA) as twocouples of model analytes, two couples of capture anti-bodies were immobilized in two channels, respectively.With a sandwich format, the CL substrates for alkalinephosphatase and horseradish peroxidase were deliveredinto the channels sequentially to perform a multipleximmunoassay after the sample and tracer antibodies wereintroduced into the channels for on-line incubation. CA125, CA 153, CA 199, and CEA could be assayed in theranges of 0.50-80, 2.0-100, and 5.0-150 U/mL and1.0-70 ng/mL with limits of detection of 0.15, 0.80, and2.0 U/mL and 0.65 ng/mL at 3σ, respectively. The wholeassay process including regeneration of the device couldbe completed in 37 min. The proposed system showedacceptable detection and fabrication reproducibility, andthe results obtained were in acceptable agreement withthose from parallel single-analyte test of practical clinicalsera. This technique provides a new strategy for a simple,automated, and near-simultaneous multianalyte immu-noassay.

The measurement of disease markers has been showing itssignificance in early screening of disease, evaluating the extentof disease, and monitoring the response of disease to therapy.However, a single marker is often not sufficient for diagnosispurpose due to its limited specificity and sensitivity. The assay ofmarkers panel can improve their diagnostic value.1 Thus, multipleximmunoassay has attracted considerable interest to meet thegrowing demand for diagnostic application. Furthermore, multi-plex immunoassay can offer higher sample throughput, lesssample consumption, shorter assay time and lower cost than thetraditional parallel single-analyte immunoassay.2

So far, two dominant modes have been adopted to realize thegoal of a multiplex immunoassay. The first mode is the spatialresolution of different immunoreaction areas using a universallabel for fluorescent,3,4 chemiluminescent (CL),5,6 spectrophoto-metric,7,8 electrochemical,1,9,10 and piezoelectric11 detection ofantigen or antibody array. These methods are commonly per-formed with expensive array detectors, such as a charge-coupleddevice (CCD) camera or multichannel electrochemical worksta-tion, and an electrochemical sensor array often suffers from cross-talk potentially occurring due to the diffusion of electroactiveproduct generated at one electrode to a neighboring electrode.1,10

A CL multichannel immunosensor has been designed for simul-taneous immunoassay of three analytes by using three parallelphotodetectors.12 Some nonarray detector-based immunoassaysystems have also been proposed for spatial-resolved multiplexdetection, including an immunosensor with three channels for thespectrophotometric detection of biological warfare agents,13 anaffinity microcolumn composed of discrete segments of beadsbearing distinct receptors for the simultaneous detection of twoanalytes,14 and two multiple-band disposable optical capillaryimmunosensors for pesticides15 and hormones.16 The second mode

* Corresponding authors. Tel./Fax: +86-25-83593593. E-mail: yanfeng2007@sohu.com. E-mail: hxju@nju.edu.cn.

† Nanjing University.‡ Jiangsu Institute of Cancer Research.

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(3) Sapsford, K. E.; Rasooly, A.; Taitt, C. R.; Ligler, F. S. Anal. Chem. 2004,76, 433-440.

(4) Rissin, D. M.; Walt, D. R. Anal. Chim. Acta 2006, 564, 34-39.(5) Fall, B. I.; Eberlein-Konig, B.; Behrendt, H.; Niessner, R.; Ring, J.; Weller,

M. G. Anal. Chem. 2003, 75, 556-562.(6) Knecht, B. G.; Strasser, A.; Dietrich, R.; Martlbauer, E.; Niessner, R.; Weller,

M. G. Anal. Chem. 2004, 76, 646-654.(7) Christodoulides, N.; Tran, M.; Floriano, P. N.; Rodriguez, M.; Goodey, A.;

Ali, M.; Neikirt, D.; McDevitt, J. T. Anal. Chem. 2002, 74, 3030-3036.(8) Fernandez-Sanchez, C.; McNeil, C. J.; Rawson, K.; Nilsson, O.; Leung, H.

Y.; Gnanapragasam, V. J. Immunol. Methods 2005, 307, 1-12.(9) Shi, M. H.; Peng, Y. Y.; Zhou, J.; Liu, B. H.; Huang, Y. P.; Kong, J. L. Biosens.

Bioelectron. 2006, 21, 2210-2216.(10) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikebukuro, K.; Karube, I.

Anal. Chem. 2003, 75, 1116-1122.(11) Luo, Y.; Chen, M.; Wen, Q. J.; Zhao, M.; Zhang, B.; Li, X. Y.; Wang, F.;

Huang, Q.; Yao, C. Y.; Jiang, T. L.; Cai, G. R.; Fu, W. L. Clin. Chem. 2006,52, 2273-2280.

(12) Yacoub-George, E.; Meixner, L.; Scheithauer, W.; Koppi, A.; Drost, S.; Wolf,H.; Danapel, C.; Feller, K. A. Anal. Chim. Acta 2002, 457, 3-12.

(13) Koch, S.; Wolf, H.; Danapel, C.; Feller, K. A. Biosens. Bioelectron. 2000,14, 779-784.

(14) Piyasena, M. E.; Buranda, T.; Wu, Y.; Huang, J. M.; Sklar, L. A.; Lopez, G.P. Anal. Chem. 2004, 76, 6266-6273.

(15) Mastichiadis, C.; Kakabakos, S. E.; Christofidis, I.; Koupparis, M. A.; Willetts,C.; Misiakos, K. Anal. Chem. 2002, 74, 6064-6072.

(16) Petrou, P. S.; Kakabakos, S. E.; Christofidis, I.; Argitis, P.; Misiakos, K.Biosens. Bioelectron. 2002, 17, 261-268.

Anal. Chem. 2007, 79, 7376-7382

7376 Analytical Chemistry, Vol. 79, No. 19, October 1, 2007 10.1021/ac0711900 CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 08/23/2007

Anodic Electrochemiluminescence of CdTeQuantum Dots and Its Energy Transfer forDetection of Catechol Derivatives

Xuan Liu, Hui Jiang, Jianping Lei, and Huangxian Ju*

MOE Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University,Nanjing 210093, P.R. China

This work reported for the first time the anodic electro-chemiluminescence (ECL) of CdTe quantum dots (QDs)in aqueous system and its analytical application based onthe ECL energy transfer to analytes. The CdTe QDs weremodified with mercaptopropionic acid to obtain water-soluble QDs and stable and intensive anodic ECL emis-sion with a peak value at +1.17 V (vs Ag/AgCl) in pH 9.3PBS at an indium tin oxide (ITO) electrode. The ECLemission was demonstrated to involve the participationof superoxide ion produced at the ITO surface, whichcould inject an electron into the 1Se quantum-confinedorbital of CdTe to form QDs anions. The collision betweenthese anions and the oxidation products of QDs led to theformation of the excited state of QDs and ECL emission.The ECL energy transfer from the excited CdTe QDs toquencher produced a novel methodology for detection ofcatechol derivatives. Using dopamine and L-adrenalin asmodel analytes, this ECL method showed wide linearranges from 50 nM to 5 µM and 80 nM to 30 µM for thesespecies. Both ascorbic acid and uric acid, which arecommon interferences, did not interfere with the detectionof catechol derivatives in practical biological samples.

Semiconductor nanocrystals, or quantum dots (QDs), arebright and photostable materials with broad excitation spectra butnarrow Gaussion emission superior to conventional organicfluorescence dyes. Furthermore, their emission positions aretunable in a wide emission range from UV to NIR due to thequantum size effect. Thus, they have been widely used asmulticolored photoluminescent probes and biological luminescentlabels in bioassays1-4 and bioimagings,5,6 especially in enzymaticprocesses3 and immunoassays4 since two breakthrough works

were reported in 1998.7,8 Multifarious water-soluble QDs modifiedwith different compounds have been synthesized to show differentoptical properties, biocompatibilities, and cellular toxicities,9-13

which lead to their different applications. One important applica-tion is based on the light emission of the excited state of QDs,which can be produced either by absorption of a quantum of lightgiven rise to photoluminescence (PL), by electrical injection ofan electron-hole pair (electroluminescence), by electron impactresulting in cathodoluminescence,14 or by chemical reaction ofQDs as chemiluminescent emitters.15 The fluorescence resonanceenergy transfer from the excited state of the CdSe/ZnS core/shell16 or CdTe17 QDs to acceptors has rapidly been used formonitoring purposes. In recent years, the fluorescence resonanceenergy transfer from donors to CdSe/ZnS core/shell QDs4 andthe chemiluminescence resonance energy transfer from theexcited oxidation product of luminol to CdTe QDs18 to produceexcited QDs have also been suggested. Some methods based onthe chemical or physical interaction between analytes and CdSQDs to change their surface charges or components, which canaffect the efficiency of the core electron-hole recombination andthus the luminescent emission, have been developed for sensitivedetection of metal cation, such as copper cation19 and cyanideanion.20 All these transfers and interactions afford new methodolo-

* Corresponding author. Tel./Fax: +86-25-83593593. E-mail: hxju@nju.edu.cn.(1) Gill, R.; Freeman, R.; Xu, J. P.; Willner, I.; Winograd, S.; Shweky, I.; Banin,

U. J. Am. Chem. Soc. 2006, 128, 15376-15377.(2) Zhelev, Z.; Bakalova, R.; Ohba, H.; Jose, R.; Imai, Y.; Baba, Y. Anal. Chem.

2006, 78, 321-330.(3) Jin, T.; Fujii, F.; Sakata, H.; Tamura, M.; Kinjo, M. Chem. Commun. 2005,

4300-4302.(4) Charbonniere, L. J.; Hildebrandt, N.; Ziessel, R. F.; Lohmannsroben, H. G.

J. Am .Chem. Soc. 2006, 128, 12800-12809.(5) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005,

4, 435-446.(6) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.;

Wise, F. W.; Webb, W. W. Science 2003, 300, 1434-1436.

(7) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018.(8) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science

1998, 281, 2013-2016.(9) Wang, Q.; Kuo, Y. C.; Wang, Y. W.; Shin, G.; Ruengruglikit, C.; Huang, Q.

R. J. Phy. Chem. B 2006, 110, 16860-16866.(10) Jin, T.; Fujii, F.; Yamada, E.; Nodasaka, Y.; Kinjo, M. J. Am. Chem. Soc.

2006, 128, 9288-9289.(11) Hoshino, A.; Fujioka, K.; Oku, T.; Suga, M.; Sasaki, Y. F.; Ohta, T.; Yasuhara,

M.; Suzuki, K.; Yamamoto, K. Nano Lett. 2004, 4, 2163-2169.(12) Duan, H. W.; Nie, S. M. J. Am. Chem. Soc. 2007, 129, 3333-3338.(13) Choi, J. H.; Chen, K. H.; Strano, M. S. J. Am. Chem. Soc. 2006, 128, 15584-

15585.(14) Rodriguez-Viejo, J.; Jensen, K. F.; Mattoussi, H.; Michel, J.; Dabbousi, B.

O.; Bawendi, M. G. Appl. Phys. Lett. 1997, 70, 2132-2134.(15) Poznyak, S. K.; Talapin, D. V.; Shevchenko, E. V.; Weller, H. Nano Lett.

2004, 4, 693-698.(16) Medintz, I. L.; Trammell, S. A.; Mattoussi, H.; Mauro, J. M. J. Am. Chem.

Soc. 2004, 126, 30-31.(17) Xu, L.; Xu, J.; Ma, Z. Y.; Li, W.; Huang, X. F.; Chen, K. J. Appl. Phys. Lett.

2006, 89, No.033121.(18) Huang, X. Y.; Li, L.; Qian, H. F.; Dong, C. Q.; Ren, J. C. Angew. Chem., Int.

Ed. 2006, 45, 5140-5143.(19) Chen, Y. F.; Rosenzweig, Z. Anal. Chem. 2002, 74, 5132-5138.(20) Jin, W. J.; Fernandez-Arguelles, M. T.; Costa-Fernandez, J. M.; Pereiro, R.;

Sanz-Medel, A. Chem. Commun. 2005, 883-885.

Anal. Chem. 2007, 79, 8055-8060

10.1021/ac070927i CCC: $37.00 © 2007 American Chemical Society Analytical Chemistry, Vol. 79, No. 21, November 1, 2007 8055Published on Web 10/02/2007

CdS Nanocrystal-BasedElectrochemiluminescence Biosensor for theDetection of Low-Density Lipoprotein byIncreasing Sensitivity with Gold NanoparticleAmplification

Guifen Jie, Bo Liu, Hongcheng Pan, Jun-Jie Zhu,* and Hong-Yuan Chen

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), School of Chemistry and ChemicalEngineering, Nanjing University, Nanjing 210093, P. R. China

Mercaptoacetic acid (RSH)-capped CdS nanocrystals (NCs)was demonstrated to be electrochemically reduced duringpotential scan and react with the coreactant S2O8

2- togenerate strong electrochemiluminescence (ECL) in aque-ous solution. Based on the ECL of CdS NCs, a novel label-free ECL biosensor for the detection of low-density lipo-protein (LDL) has been developed by using self-assemblyand gold nanoparticle amplification techniques. The bio-sensor was prepared as follows: The gold nanoparticleswere first assembled onto a cysteamine monolayer on thegold electrode surface. This gold nanoparticle-coveredelectrode was next treated with cysteine and then reactedwith CdS NCs to afford a CdS NC-electrode. Finally, apoB-100 (ligand of LDL receptor) was covalently conjugatedto the CdS NC-electrode. The modification procedure wascharacterized by cyclic voltammetry, electrochemical im-pedance spectroscopy, and atomic force microscopy,respectively. The resulting modified electrode was testedas ECL biosensor for LDL detection. The LDL concentra-tion was measured through the decrease in ECL intensityresulting from the specific binding of LDL to apoB-100.The ECL peak intensity of the biosensor decreased linearlywith LDL concentration in the range of 0.025-16 ngmL-1 with a detection limit of 0.006 ng mL-1. The CdSNCs not only showed high ECL intensity and good bio-compatibility but also could provide more binding sitesfor apoB-100 loading. In addition, the gold nanoparticleamplification for protein ECL analysis was applied to theimprovement of the detection sensitivity. Thus, the bio-sensor exhibited high sensitivity, good reproducibility,rapid response, and long-term stability.

In recent years, semiconductor nanocrystals (NCs) withexcellent luminescent properties have attracted increased attentionbecause of its application in many areas of fundamental andtechnical importance.1-3 Recent works have indicated the semi-

conductor NCs are electrically excitable in nonaqueous media4-7

and aqueous systems8 containing supporting electrolyte. Efficientand stable electrogenerated chemiluminescence (ECL) from CdSeNCs in aqueous solution can be obtained by applying a cathodicpotential to the CdSe nanocrystal films.8 The electrochemicallyreduced and oxidized Si NCs4 or CdSe NCs5 can react with thecoreactants to generate ECL. Moreover, semiconductor quantumdots (QDs) have been widely used in bioconjugates1,9-10 andoptical biosensing.11 Chan and Nie used water-soluble QDs as thebiological labels.2 Therefore, semiconductor NCs or QDs havegreat potential for development of novel ECL biosensors.

In addition, a rapid, highly specific method of detecting andquantifying biological substances is increasingly needed. Thesematerials can be determined by a binding method with a highdegree of specificity, such as antigen-antibody, nucleic acidhybridization, and protein-ligand. Electrochemical,12,13 optical,14

ECL,15-22 and photoelectrochemical methods23 have been usedas special detection techniques. Among them, the ECL sensor

* Corresponding author. Tel: +86-25-83594976. Fax: +86-25-83317761. E-mail: jjzhu@netra.nju.edu.cn.(1) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998,

281, 2013-2016.

(2) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018.(3) Chen, S. W.; Truax, L. A.; Sommers, J. M. Chem. Mater. 2000, 12, 3864-

3870.(4) Ding, Z.; Quinn, B. M.; Haram, S. K.; Pell, L. E.; Korgel, B. A.; Bard, A. J.

Science 2002, 296, 1293-1297.(5) Myung, N.; Ding, Z.; Bard, A. J. Nano Lett. 2002, 2, 1315-1319.(6) Myung, N.; Bae, Y.; Bard, A. J. Nano Lett. 2003, 3, 1053-1055.(7) Bae, Y.; Myung, N.; Bard, A. J. Nano Lett. 2004, 4, 1153-1161.(8) Poznyak, S. K.; Talapin, D. V.; Shevchenko, E. V.; Weller, H. Nano Lett.

2004, 4, 693-698.(9) Wang, D.; Rogach, A. L.; Caruso, F. Nano Lett. 2002, 2, 857-861.

(10) Ding, S. Y.; Jones, M.; Tucker, M. P.; Nedeljkovic, J. M.; Wall, J.; Simon,M. N.; Rumbles, G.; Himmel, M. E. Nano Lett. 2003, 3, 1581-1585.

(11) Sapsford, K. E.; Medintz, I. L.; Golden, J. P.; Deschamps, J. R.; Uyeda, H.T.; Mattoussi, H. Langmuir 2004, 20, 7720-7728.

(12) Mikkelsen, S. R. Electroanalysis 1996, 8, 15-19.(13) Wang, J. Anal. Chim. Acta 2002, 469, 63-71.(14) Christodoulides, N.; Tran, M.; Floriano, P. N.; Rodriguez, M.; Goodey, A.;

Ali, M.; Neikirk, D.; McDevitt, J. T. Anal. Chem. 2002, 74, 3030-3036.(15) Lee, W. Y. Mikrochim. Acta 1997, 127, 19-39.(16) Fahnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-559.(17) Miao, W. J.; Bard, A. J. Anal. Chem. 2003, 75, 5877-5885.(18) Namba, Y.; Usami, M.; Suzuki, O. Anal. Sci. 1999, 15, 1087-1093.(19) Knight, A. W.; Greenway, G. M. Analyst 1995, 120, 2543-2747.(20) Xu, X. H.; Jeffers, R. B.; Gao, J.; Logan, B. Analyst 2001, 126, 1285-1292.(21) Zhou, M.; Roovers, J.; Robertson, G. P.; Grover, C. P. Anal. Chem. 2003,

75, 6708-6717.(22) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036.

Anal. Chem. 2007, 79, 5574-5581

5574 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 10.1021/ac062357c CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 07/06/2007

Technical Notes

Indirect Determination of Sulfide at UltratraceLevels in Natural Waters by Flow Injection On-LineSorption in a Knotted Reactor Coupled withHydride Generation Atomic FluorescenceSpectrometry

Yan Jin, Hong Wu, Ye Tian, Luhong Chen, Jiongjia Cheng, and Shuping Bi*

School of Chemistry and Chemical Engineering, State Key Laboratory of Coordination Chemistry and Key Laboratory ofMOE for Life Science, Nanjing University, Nanjing 210093, China

A simple and sensitive nonchromatographic approach forindirect determination of sulfide at ultratrace levels innatural waters based on its selective precipitation withHg2+ on the inner wall of a knotted reactor (KR) wasdeveloped for flow injection on-line sorption coupled withhydride generation atomic fluorescence spectrometry(HG-AFS). With the Hg2+ pH kept at 2.0, the HgSprecipitation was formed in the KR after a reaction timeof 120 s. A 10% (v/v) HCl was introduced to elute theremnant inorganic mercury and to merge with the KBH4

solution (0.05% m/v) for HG-AFS detection. Under theoptimal experimental conditions, the sample throughputswere 20 h-1. The detection limit was found to be 0.05 µgL-1, and the relative standard deviation (RSD, n ) 11)for determination of 2.0 µg L-1 sulfide was 3.3%. Thedeveloped method was successfully applied to the deter-mination of sulfide in a variety of natural water samplesand wastewater samples with the gas-phase separationand sorption apparatus.

Because its toxicity and causticity increase awfully whenprotonated, sulfide has gained sufficient attention from thebiological and industrial points of view.1-3 Thus, it is important todevelop a rapid and sensitive method for immediate sulfidemonitoring in natural waters.

Analytical methods for sulfide determination include spectros-copy, chromatography, and electrochemical detection. The me-thylene blue (MB) method plays a great role in spectroscopicallydetermining sulfide, and the performance is improved whencombined with flow injection4 such as flow-injection solid-phase

spectrophotometry,4e when a detection limit was achieved as lowas 1.7 µg L-1. Some other methods are based on the measurementof generated hydrogen sulfide to quantify sulfide and gain highsensitivity when measured by sulfur-specific flame photometricdetection5 with a detection limit of 64 µg L-1. Direct spectroscopicdetection such as ultraviolet spectrophotometery6 also obtainedoutstanding analytical performance for sulfide determination.Chromatography was employed for sulfide speciation. Someresearches reported sulfide speciation in natural waters taken byion chromatography (IC)7,8 as well as estuarine water9 andseawaters10 measured by high-performance liquid chromatography(HPLC), which could detect sulfide as low as 0.067 µg L-1.9

Electrochemical detection also offers more choices for sulfidemeasurement,11 and the detection limit can even reach 0.016 µgL-1.11h

* Corresponding author. Phone: 86-25-86205840. Fax: 86-25-83317761.E-mail: bisp@nju.edu.cn.(1) Howard, E. C.; Henriksen, J. R.; Buchan, A.; Reisch, C. R.; Burgmann, H.;

Welsh, R.; Ye, W. Y.; Gonzalez, J. M.; Mace, K.; Joye, S. B.; Kiene, R. P.;Whitman, W. B.; Moran, M. A. Science 2006, 314, 649-652.

(2) Meinrat, O. A. Science 2007, 315, 50-51.(3) Pierce, D. T.; Applebee, M. S.; Lacher, C.; Bessie, J. Environ. Sci. Technol.

1998, 32, 1734-1737.

(4) (a) Ferrer, L.; Estela, J. M.; Cerda, V. Anal. Chim. Acta 2006, 573, 391-398. (b) Ferrer, L.; de Armas, G.; Miro, M.; Estela, J. M.; Cerda, V. Analyst2005, 130, 644-651. (c) Ferrer, L.; de Armas, G.; Miro, M.; Estela, J. M.;Cerda, V. Talanta 2004, 64, 1119-1126. (d) De Armas, G.; Ferrer, L.;Miro, M.; Estela, J. M.; Cerda, V. Anal. Chim. Acta 2004, 524, 89-96. (e)Cassella, R. J.; Teixeira, L. S. G.; Garrigues, S.; Costa, A. C. S.; Santelli, R.E.; de la Guardia, M. Analyst 2000, 125, 1835-1838. (f) Singh, V.; Gosain,S.; Mishra, S.; Jain, A.; Verma, K. K. Analyst 2000, 125, 1185-1188.

(5) Howard, A. G.; Yeh, C. Y. Anal. Chem. 1998, 70, 4868-4872.(6) Guenther, E. A.; Johnson, K. S.; Coale, K. H. Anal. Chem. 2001, 73, 3481-

3487.(7) Miura, Y.; Fukasawa, K.; Koh, T. J. Chromatogr., A 1998, 804, 143-150.(8) Montegrossi, G.; Tassi, F.; Vaselli, O.; Bidini, E.; Minissale, A. Appl.

Geochem. 2006, 21, 849-857.(9) Tang, D. G.; Santschi, P. H. J. Chromatogr., A 2000, 883, 305-309.

(10) Gru, C.; Sarradin, P. M.; Legoff, H.; Narcon, S.; Caprais, J. C.; Lallier, F. H.Analyst 1998, 123, 1289-1293.

(11) (a) Salimi, A.; Roushani, M.; Hallaj, R. Electrochim. Acta 2006, 51, 1952-1959. (b) Zen, J. M.; Chang, J. L.; Chen, P. Y.; Ohara, R.; Pan, K. C.Electroanal. 2005, 17, 739-743. (c) Hassan, S. S. M.; Marzouk, S. A. M.;Sayour, H. E. M. Anal. Chim. Acta 2002, 466, 47-55. (d) Prodromidis,M. I.; Veltsistas, P. G.; Karayannis, M. I. Anal. Chem. 2000, 72, 3995-4002. (e) Muller, B.; Stierli, R. Anal. Chim. Acta 1999, 401, 257-264. (f)Giuriati, C.; Cavalli, S.; Gorni, A.; Badocco, D.; Pastore, P. J. Chromatogr.,A 2004, 1023, 105-112. (g) Giovanelli, D.; Lawrence, N. S.; Jiang, L.; Jones,T. G. J.; Compton, R. G. Analyst 2003, 128, 173-177. (h) Alfarawati, R.;Vandenberg, C. M. G. Mar. Chem. 1997, 57, 277-286.

Anal. Chem. 2007, 79, 7176-7181

7176 Analytical Chemistry, Vol. 79, No. 18, September 15, 2007 10.1021/ac070699s CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 08/18/2007

Electrogenerated Chemiluminescence Detection ofAmino Acids Based on Precolumn DerivatizationCoupled with Capillary Electrophoresis Separation

Jianguo Li, Qinyi Yan, Yunlong Gao, and Huangxian Ju*

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry,Nanjing University, Nanjing 210093, P. R. China

A novel method for highly sensitive detection of primaryand secondary amino acids with selective derivatizationusing acetaldehyde as a new derivatization reagent wasproposed by capillary electrophoresis (CE) coupled withelectrogenerated chemiluminescence (ECL) of tris(2,2′-bipyridine)ruthenium(II). The precolumn derivatizationof these amino acids with acetaldehyde was performed inaqueous solution at room temperature for 1 h. Uponoptimized derivatization, the ECL intensities and detectionsensitivities of the amino acids were significantly en-hanced by 20-70 times. Using four amino acids, argin-ine, proline, valine, and leucine, as model compounds,their derivatives could be completely separated by CE andsensitively detected by ECL within 22 min. The linearranges were 0.5-100 µM for arginine and proline and5-1000 µM for valine and leucine with the detectionlimits of 1 × 10-7 (0.5 fmol, arginine), 8 × 10-8 (0.4fmol, proline), 1 × 10-6 (5 fmol, valine), and 1.6 × 10-6

M (8 fmol, leucine) at a signal-to-noise ratio of 3. Thederivatization reactions and ECL process of amino acidswere also proposed based on in situ Fourier transforminfrared and ultraviolet spectrometric analyses.

Since capillary electrophoresis (CE) was presented in the1980s,1 it has been developed as an efficient separation techniquedue to its high efficiency, powerful resolution potential, relativelyshort analysis time, and small sample volume. Different kinds ofdetection methods compatible with CE, including UV-visibleabsorbance, refractive index, thermooptical absorbance, fluores-cence, chemiluminescence, mass spectrometry, electrochemistry,and electrochemiluminescence or electrogenerated chemilumi-nescence (ECL), have been developed.2 ECL detection, knownas a useful analytical technique, has attracted considerable interestfor development of highly sensitive and selective detectionmethods.3 The most important ECL system is the reaction ofruthenium complexes, particularly tris(2,2′-bipyridine)ruthenium-

(II) (Ru(bpy)32+) and its derivatives, which has become a powerful

tool for the determination of different compounds containingtertiary amine or diketone groups,4 amino acids,5 oxalate,6 variousdrugs,7 and NADH.8 Recently, the development of miniaturizedECL detection cells has extended greatly the application rangeof both ECL and CE.3b,5,9 This work developed a novel, sensitivemethod for simultaneous detection of different amino acids byCE coupled with ECL of Ru(bpy)3

2+.Reactions between Ru(bpy)3

3+ and tertiary amines have led tothe development of ECL-based detection devices for a variety ofbiologically important molecules.3b,10 According to the mechanismof Ru(bpy)3

2+ ECL, Ru(bpy)32+ is oxidized to form Ru(bpy)3

3+,which further reacts with analyte, accompanied by light emission.Based on this mechanism, several subtle CE-ECL cells have beenreported to detect amino acids,5,11,12 peptides,13 aliphatic amines,3b,14

* Corresponding author. Phone: +86-25-83593593. Fax: +86-25-83593593.E-mail: hxju@nju.edu.cn.(1) (a) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-216.

(b) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272.(2) St. Claire, R. L., III. Anal. Chem. 1996, 68, 569R-586R.(3) (a) Fahnrich, K. A.; Pravda, M.; Guilbaut, G. G. Talanta 2001, 54, 531-

559. (b) Chiang, M. T.; Lu, M. C.; Whang, C. W. Electrophoresis 2003, 24,3033-3039.

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(5) (a) Wang, X.; Bobbit, D. R. Anal. Chim. Acta 1999, 383, 213-220. (b)Huang, X. J.; Wang, S. Li.; Fang, Z. L. Anal. Chim. Acta 2002, 456, 167-175.

(6) (a) Skotty, D. R.; Lee, W.-Y.; Nieman, T. A. Anal. Chem. 1996, 68, 1530-1535. (b) Skotty, D. R.; Nieman, T. A. J. Chromatogr., B 1995, 665, 27-36.

(7) (a) Forbes, G. A.; Nieman, T. A.; Sweedler, J. V. Anal. Chim. Acta 1997,347, 289-293. (b) Cao, W. D.; Liu, J. F.; Qiu, H. B.; Yang X. R.; Wang, E.K. Electroanalysis 2002, 14, 1571-1576. (c) Liu, J. F.; Cao, W. D.; Qiu, H.B.; Sun, X. H.; Yang X. R.; Wang, E. K. Clin. Chem. 2002, 48, 1049-1058.(d) Cao, W. D.; Yang, X. R.; Wang, E. K. Electroanalysis 2004, 16, 169-174. (e) Sun, X. H.; Liu, J. F.; Cao, W. D.; Yang, X. R.; Wang E. K.; Fung Y.S. Anal. Chim. Acta 2002, 470, 137-145.

(8) (a) Liang, P.; Sanchez, R. I.; Martin, M. T. Anal. Chem. 1996, 68, 2426-2431. (b) Martin, A. F.; Nieman, T. A. Anal. Chim. Acta 1993, 281, 475-481.

(9) (a) Liu, J. F.; Yan, J. L.; Yang, X. R.; Wang. E. K. Anal. Chem. 2003, 75,3637-3642. (b) Cao, W. D.; Liu, J. F.; Yang, X. R.; Wang, E. K. Electrophoresis2002, 23, 3683-3691. (c) Yin, X. B.; Qiu, H. B.; Sun, X. H.; Yan, J. L.; Liu,J. F.; Wang, E. K. Anal. Chem. 2004, 76, 3846-3850. (d) Tsukagoshi, K.;Obata, Y.; Nakajima, R. J. Chromatogr., A 2002, 971, 255-260.

(10) (a) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999,378, 1-41. (b) Noffsinger, J. B.; Danielson, N. D. Anal. Chem. 1987, 59,865-868. (c) Greeway, G. M.; Knight, A. W.; Knight, D. J. Analyst 1995,120, 2549-2552. (d) Downey, T. M.; Nieman, T. A. Anal. Chem. 1992, 64,261-268.

(11) (a) Dickson, J. A.; Ferris, M. M.; Milofsky, R. E. J. High Resolut. Chromatogr.1997, 20, 643-646. (b) Cao, W. D.; Jia, J. B.; Yang, X. R.; Dong, S. J.;Wang, E. K. Electrophoresis 2002, 23, 3692-3698. (c) Smith, J. T.Electrophoresis 1999, 20, 3078-3083.

(12) (a) Qiu, H. B.; Yan, J. L.; Sun, X. H.; Liu, J. F.; Cao, W. D.; Yang, X. R.;Wang, E. K. Anal. Chem. 2003, 75, 5435-5440. (b) Chiang, M. T.; Whang,C. W. J. Chromatogr., A 2001, 934, 59-66.

(13) Hendrickson, H. P.; Anderson, P.; Wang, X.; Pittman, Z.; Bobbitt, D. R.Microchem. J. 2000, 65, 189-195.

Anal. Chem. 2006, 78, 2694-2699

2694 Analytical Chemistry, Vol. 78, No. 8, April 15, 2006 10.1021/ac052092m CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 03/23/2006

Flow-Through Multianalyte ChemiluminescentImmunosensing System with Designed SubstrateZone-Resolved Technique for Sequential Detectionof Tumor Markers

Zhifeng Fu, Hong Liu, and Huangxian Ju*

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry,Nanjing University, Nanjing 210093, PR China

A novel flow-through immunosensing system for perform-ing a multianalyte chemiluminescent determination in asingle run was designed. A new analytical strategy ofsubstrate zone-resolved technique was proposed. Usingcarcinoma antigen 125 (CA 125) and carcinoembryonicantigen (CEA) as model analytes, the capture antibodiesfor CA 125 and CEA were immobilized on an UltraBindaldehyde-actived membrane to act as an immunoreactor,to which the mixture of CA 125, CEA, and their corre-sponding tracers, horseradish peroxidase (HRP)-labeledanti-CA 125 and alkaline phosphatase (ALP)-labeled anti-CEA, was introduced for on-line incubation. The sub-strates for HRP and ALP were then delivered into thedetection cell sequentially to perform substrate zone-resolved immunoassay by a sandwich format. Underoptimal conditions, CA 125 and CEA could be assayedin the ranges of 5.0-100 units/mL and 1.0-120 ng/mL,respectively. The whole assay process including incuba-tion, wash, detection, and regeneration could be com-pleted in 35 min. The serum samples from the clinic wereassayed with the proposed method, and the results werein acceptable agreement with the reference values. Thismethod and the strategy of substrate zone-resolved tech-nique could be further developed for high-throughputmultianalyte immunoassay.

Immunoassay, as a promising approach for selective andsensitive analysis, has recently gained increasing attention indifferent fields including environmental monitoring, food safety,and clinical diagnosis. Multianalyte immunoassay in a single runis often necessary to monitor or quantitate several componentsin a complex system for diagnostic purposes. Compared withparallel single-analyte immunoassay methods, a multianalyteimmunoassay offers some remarkable advantages, such as highsample throughput, improved assay efficiency, low sample con-sumption, and reduced overall cost per assay.1 Thus, considerableeffort has been paid to the development of a multianalyte assay.The developed multianalyte immunoassay can be classified into

two main modes. The first mode is based on spatially separatedimmunoreaction areas, i.e., array immunoassay, coupled withelectrochemical,2,3 fluorescent,4,5 and chemiluminescent6 deter-mination. This mode includes antibody arrays for achievingquantitative protein profiling in a multiplex format.7 Similarly, twoarrays of flow-through multichannel immunosensors have alsobeen proposed for multianalyte detection.8,9 Although this modecan screen large numbers of analytes, accurate quantitative datain these arrays are usually limited or difficult to obtain.10 The arrayimmunoassay system often needs an expensive array detectorsuch as a charge-coupled device (CCD) camera for opticaldetection4-7 and multichannel potentiostat for electrochemicalmeasurement.3,10

The second mode is performed using antibodies or antigenswith different labels (one per analyte), including radioisotopes,11,12

enzymes,13,14 fluorescence compouds,15-17 and metal compounds.18-20

Usually, the detectors used in this mode include radiometric,

* To whom correspondence should be addressed. E-mail: hxju@nju.edu.cn.Tel. and Fax: +86-25-83593593.(1) Kricka, L. J. Clin. Chem. 1992, 38, 327-328.

(2) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikebukuro, K.; Karube, I.Anal. Chem. 2003, 75, 1116-1122.

(3) Wilson, M. S.; Nie, W. Y. Anal. Chem. 2006, 78, 2507-2513.(4) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681-5687.(5) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.;

MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 1999, 71, 3846-3852.

(6) Knecht, B. G.; Strasser, A.; Dietrich, R.; Martlbauer, E.; Niessner, R.; Weller,M. G. Anal. Chem. 2004, 76, 646-654.

(7) Barry, R.; Soloviev, M. Proteomics 2004, 4, 3717-3726.(8) Koch, S.; Wolf, H.; Danapel, C.; Feller, K. A. Biosens. Bioelectron. 2000,

14, 779-784.(9) Yacoub-George, E.; Meixner, L.; Scheithauer, W.; Koppi, A.; Drost, S.; Wolf,

H.; Danapel, C.; Feller, K. A. Anal. Chim. Acta 2002, 457, 3-12.(10) Wilson, M. S. Anal. Chem. 2005, 77, 1496-1502.(11) Wlans, F. H.; Dev, J., Jr.; Powell, M. M.; Heald, J. I. Clin. Chem. 1986, 32,

887-890.(12) Gow, S. M.; Caldwell, G.; Toft, A. D.; Beckett, G. J. Clin. Chem. 1986, 32,

2191-2194.(13) Blake, C.; Al-Bassam, M. N.; Gould, B. J.; Marks, V.; Bridges, J. W.; Riley,

C. Clin. Chem. 1982, 28, 1469-1473.(14) Bates, D. L.; Bailey, W. R. Dual enayme assay. International Patent WO89/

06802, 1989.(15) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.;

Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684-688.(16) Swartzman, E. E.; Miraglia, S. J.; Mellentin-Michelotti, J.; Evangelista, L.;

Yuan, P. M. Anal. Biochem. 1999, 271, 143-151.(17) Wu, F. B.; Han, S. Q.; Xu, T.; He, Y. F. Anal. Biochem. 2003, 314, 87-96.(18) Liu, G. D.; Wang, J.; Kim, J.; Jan, M. R.; Collins, G. E. Anal. Chem. 2004,

76, 7126-7130.(19) Bordes, A. L.; Limoges, B.; Brossier, P.; Degrand, C. Anal. Chim. Acta 1997,

356, 195-203.

Anal. Chem. 2006, 78, 6999-7005

10.1021/ac0610560 CCC: $33.50 © 2006 American Chemical Society Analytical Chemistry, Vol. 78, No. 19, October 1, 2006 6999Published on Web 08/25/2006

Current Medicinal Chemistry, 2006, 13, 525-537 525

Copper in Medicine: Homeostasis, Chelation Therapy and Antitumor DrugDesign

Tuo Wang and Zijian Guo*

State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, P.R. China

Abstract: As one of the most important essential transition metals, copper is involved in a variety ofbiological processes such as embryo development, connective tissue formation, temperature control and nervecell function. It is also related to severe diseases such as Wilson’s and Menkes diseases and some neurologicaldisorders. Novel components of copper homeostasis include copper-transporting P-type ATPases, Menkes andWilson proteins, and copper chaperones in humans have been identified and characterized at the molecularlevel. These findings have paved the way towards better understanding of the role of copper deficiency orcopper toxicity in physiological and pathological conditions. Therefore, organic compounds that can interferewith copper homeostasis may find therapeutic application in copper-dependent diseases. The antitumoractivity of copper complexes was reported several decades ago, and many new complexes have demonstratedgreat antitumor potential. Copper complexes may have relatively lower side effects than platinum-based drugs,and are suggested to be able to overcome inherited or acquired resistance of cisplatin. In this overview, themost recent advances in copper homeostasis, copper-related chelation therapy and design of copper-basedantitumor complexes will be summarized.

1. INTRODUCTION 2. COPPER HOMEOSTASIS AND COPPER-RELATED DISEASES

Copper, as an essential element, forms an integralcomponent of many metalloproteins and enzymes and playsa vital role in electron transfer reactions of many cellularprocesses [1-2]. However, excessive copper can be very toxicand results in severe diseases. Recently, some importantprogresses have been made in the field of copperhomeostasis and copper in medicine [3-4]. Research onWilson’s and Menkes diseases, the well-known geneticdisorders resulting in copper deficiency and excess, hasprovided new insights into the understanding of intracellulartrafficking and distribution of copper at molecular level [5-7]. These have been crucial for the therapeutic developmentof copper-related diseases.

Copper homeostasis is maintained by a conserved groupof proteins that often contain copper binding domains rich incysteine, methionine or histidine residues [13]. It is mainlyrelated to three continuous processes, the uptake, subcellulardistribution and efflux. The key pathways relevant to copperhomeostasis are depicted in Scheme (1).

2.1. Uptake

Copper(II) is normally absorbed by the intestinal tractand then enters the blood circulation from the intestinal wallthrough mainly the action of ATP7A protein (or designatedas Menkes protein). Then it binds to human serum albumin(HSA) and L-histidine that form an exchangeable pool ofcopper [14]. It is suggested that the ternary complex Cu(II)-HSA-His may be the major species for copper(II) transport[15-16].

Since the clinical success of cisplatin (cis-[PtCl2(NH3)2])for the treatment of several human tumors, thousands ofplatinum and other metal-based compounds have been testedfor their antitumor activity for the search of more effectiveand less toxic drugs than cisplatin [8-10]. Copper complexesof thiosemicarbazone (TSC) and derivatives were amongstthe early non-platinum compounds tested for antitumorpotential and the first related study could be traced back toearly 1960s [11]. Some recent reports showed that themodification of TSC could lead to some interestingcompounds with potent cytotoxicity and low toxicity tonormal cells [12].

During the uptake process, copper(II) is reduced tocopper(I) through a number of mechanisms [17-18] andenters the cell through the transmembrane transporters. Oneof such transporters is human copper transporter 1 (hCtr1)[19], which belongs to a family of high affinity coppertransporters including two yeast proteins Ctr1 (yCtr1) andCtr3 (yCtr3), murine Ctr1 (mCtr1), etc [20-22]. The Ctr1gene was first discovered to be essential for iron traffickingin Saccharomyces cerevisiae [20,23]. However, it was foundlater that the yCtr1 protein is specific for copper and its“involvement” in iron transport is actually due to therequirement of copper for Fet3, a ferroxidase that facilitatesiron uptake by catalyzing the oxidation of Fe2+ to Fe3+ byO2 [24]. This group of copper transporters has also beenfound essential for the survival of mammalian embryo [25].

In this review, we focus on the current understanding ofhow copper was controlled by the intracellular proteins, thedevelopment of copper related chelation therapy and thedesign of novel copper-based anticancer compounds.

*Address correspondence to this author at the State Key Laboratory ofCoordination Chemistry, Nanjing University, Nanjing 210093, P.R. China;Tel: 86-25-83594569; Fax: 86-25-83314502; E-mail: zguo@nju.edu.cn

The hCtr1 can be categorized as a copper(I) transportermainly because of the following two reasons: (1) hCtr1 can

0929-8673/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

A new MOF-505 analog exhibiting high acetylene storagew

Yunxia Hu,aShengchang Xiang,

bWenwei Zhang,*

aZhuxiu Zhang,

aLei Wang,

a

Junfeng Bai*aand Banglin Chen*

b

Received (in Cambridge, UK) 18th August 2009, Accepted 22nd October 2009

First published as an Advance Article on the web 9th November 2009

DOI: 10.1039/b917046d

A new microporous metal–organic framework Cu2(EBTC)(H2O)2�xG (EBTC = 1,10-ethynebenzene-3,3 0,5,50-tetracarboxylate;

G = guest molecule) was rationally designed with a NbO net,

exhibiting significantly high acetylene storage of 252 and

160 cm3 g�1 at 273 and 295 K under 1 bar, respectively.

The MOF-505 series of porous MOFs have been emerging as

very promising materials for gas storage.1–9 The prototype

MOF-505 of NbO topology is self-assembled from square

planar copper paddle-wheel Cu2(COO)4 secondary building

units (SBUs) with distorted square planar 3,30,5,50-biphenyl-

tetracarboxylate (BPTC) organic linkers.1 Because of the high

surface area, suitable pore size and open copper sites for gas

adsorption within MOF-505, extensive efforts have been made

to increase the surface area, optimize the pore size and

immobilize specific binding sites within the MOF-505 series

to maximize their gas storage capacities. In fact, by the

incorporation of naphthalene and anthracene into the original

BPTC organic backbone, the new MOF-505 analogs

NOTT-103 and PCN-14 take up hydrogen and methane at

extraordinarily high levels of 7.22 wt% at 77 K and 60 bar,

and 220 v/v at 298 K and 35 bar, respectively.7,8

The very explosive nature of acetylene has limited the safe

storage of acetylene under a pressure of 0.2 MPa at room

temperature,9 thus specific sites need to be immobilized within

porous MOFs to enhance their interactions with acetylene

molecules and thus maximize the storage capacities.10–21

Recently we have realized the very important role of copper

open metal sites to direct their bonding with acetylene

molecules, as exemplified in HKUST-1 and MOF-505, high-

lighting the promise of the MOF-505 series as potential

practical acetylene storage media because of the open copper

sites, high surface area and tunable pore structure within such

NbO type frameworks.22 To incorporate a triple CRC bond

into the original BPTC organic backbone,23 herein we report

the synthesis and X-ray single crystal structure of a new highly

porous MOF-505 analog, MOF 1, Cu2(EBTC)(H2O)2�xG(EBTC = 1,10-ethynebenzene-3,30,5,50-tetracarboxylate; G =

guest molecule). The activated MOF 1a has Langmuir

and BET surface areas of 2844 and 1852 m2 g�1, takes up

286 cm3 g�1 (2.58 wt%) H2 at 77 K and 1 bar, 178 cm3 g�1

CO2 and 31 cm3 g�1 CH4 at 273 K and 1 bar. Most significant

is the highest ever reported acetylene uptake of 252 cm3 g�1

at 273 K under 1 bar and an adsorption enthalpy of

34.5 kJ mol�1 (at a coverage of 1 mmol g�1).

MOF 1 was synthesized by the solvothermal reaction of

H4EBTC and Cu(NO3)2�3H2O in a mixture of N,N-dimethyl-

formamide (DMF), dimethyl sulfoxide (DMSO) and HNO3 at

65 1C for 24 h to give cyan block-shaped crystals.zy It was

formulated as [Cu2(EBTC)(H2O)2]�8H2O�DMF�DMSO by

elemental analysis and single-crystal X-ray diffraction studies,

and the phase purity of the bulk material was independently

confirmed by powder X-ray diffraction (PXRD) and thermal

gravimetric analysis (TGA).

As expected, the framework is composed of paddle wheel

dinuclear Cu2 units which are bridged by EBTC to form a 3D

NbO type structure in which there exists one-dimensional

pores along the c axis (Fig. S1w). Careful examination of the

structure reveals that there are two types of cages which are

alternately stacked with each other along this pore axis. The

small cage of about 8.5 A in diameter is encapsulated by six

paddle wheel Cu2(COO)4 clusters (Fig. 1a), while the large

irregular elongated cage of about 8.5 � 21.5 A is surrounded

by twelve Cu2(COO)4 clusters (Fig. 1b). The accessible pore

volume from the single crystal structure is 73% as calculated

using the Platon program.

About 100 mg of methanol-exchanged MOF 1 (6–8 times)

was activated at 120 1C overnight until the outgas rate was less

than 4 mmHg min�1 to form anhydrous MOF 1a. As shown in

Fig. 2a, 1a exhibits a type I reversible sorption isotherm and

takes up a significant amount of N2 at 77 K (652 cm3 g�1 at

1 bar), featuring Langmuir and BET surface areas of 2844 and

1852 m2 g�1, respectively. The gas storage capacities of MOF

1a are 286 cm3 g�1 (2.58 wt%) H2 at 77 K and 1 bar,

561 cm3 g�1 (5.01 wt%) H2 at 77 K and 20 bar, 178 cm3 g�1

CO2 and 31 cm3 g�1 CH4 at 273 K and 1 bar, respectively. The

nitrogen uptake of 652 cm3 g�1 in MOF 1a at 77 K and 1 bar is

much higher than that of 448 cm3 g�1 in MOF-505 because of

the incorporation of the CRC bond into the original BPTC

organic backbone. Accordingly, the hydrogen storage capacity

of MOF 1a (5.01 wt%) is also higher than that of MOF-505

(4.02 wt%) at 77 K and 20 bar.1,2 The most significant feature

is its extraordinarily high acetylene uptakes of 252 and

160 cm3 g�1 under 1 bar at 273 K and 295 K, respectively

(Fig. 2b), which are systematically higher than those of

177 and 148 cm3 g�1 in MOF-505.22 The uptake of 160 cm3 g�1

highlights the second highest acetylene storage material at

295 K and 1 bar, while that of 252 cm3 g�1 is the highest one

ever reported at 273 K and 1 bar.22 The storage capacities of

a State Key Laboratory of Coordination Chemistry,School of Chemistry and Chemical Engineering, Nanjing University,Nanjing 210093, ChinaE-mail: wwzhang@nju.edu.cn, bjunfeng@nju.edu.cn

bDepartment of Chemistry, University of Texas at San Antonio,San Antonio, TX 78249-0698, USA. E-mail: banglin.chen@utsa.edu

w Electronic supplementary information (ESI) available: Experimentaldetails, crystal, adsorption, desorption and TGA data and hydrogenisotherm of 1a. CCDC 744108. For ESI and crystallographic data inCIF or other electronic format see DOI: 10.1039/b917046d

This journal is �c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 7551–7553 | 7551

COMMUNICATION www.rsc.org/chemcomm | ChemComm

Facile synthesis of nickel oxide nanotubes and their antibacterial,

electrochemical and magnetic propertiesw

Huan Pang,a Qingyi Lu,*a Yecheng Lia and Feng Gao*b

Received (in Cambridge, UK) 22nd July 2009, Accepted 14th October 2009

First published as an Advance Article on the web 6th November 2009

DOI: 10.1039/b914898a

Nickel oxide nanotubes with great antibacterial activities,

electrochemical capacitance, and magnetic properties have been

synthesized through a precursor method with dimethylglyoxime

as precipitant for the precursor, and the method has been

developed for the synthesis of Ni/C nanorods.

Due to low mass density, high porosity, and increased surface

area, hollow inorganic nanomaterials constitute an important

family of nanostructures with interesting properties and

potential applications, such as catalysts, host materials,

sensors, drug-delivery carriers, acoustic insulation, biomedical

diagnosis agents, and chemical reactors.1–4 Up to now, some

synthetic strategies have been developed to fabricate hollow

inorganic nanostructures, such as the Kirkendall effect,5

self-curling mechanism,6,7 template-directed synthesis,8,9 etc.

However, these methods need complicated multi-steps such as

removal of the template or selective etching in an appropriate

solvent, which would make the product impure and limit the

extensive applications of the nanotubes.

Nickel oxide (NiO) has been under extensive investigations

as an important functional inorganic material and has diverse

technological applications in fuel cell electrodes, hetero-

geneous catalysis, antiferromagnetic layers, etc.10–13 The

valuable functions greatly depend on the composition and

structure. Of the wide range of nanostructures, nanotubes

represent a perfect one-dimensional topology that hold the

foreground as functionalized materials as a result of both

shape-specific and quantum size effects. However, to the best

of our knowledge there are few reports about the production

of NiO nanotubes.14,15 In the study presented here, we

developed a simple and convenient approach for the synthesis

of NiO nanotubes through one-dimensional metal complex

bis(dimethylglyoximato) nickel(II), Ni(dmg)2. The obtained

NiO nanotubes show good antibacterial properties compared

to commercial NiO and synthetic NiO nanoflowers. Also, the

NiO nanotubes exhibit effective electrochemical capacitance,

which can be used as a supply of energy in the exothermic

system. The magnetic measurement demonstrates that

unlike antimagnetic bulk NiO,16 the NiO nanotubes are

magnetic and may be used in fields of MR imaging and

magnetic drug delivery.

Dimethylglyoxime CH3(CQNOH)2CH3 is a common

organic precipitant for Ni2+ in industry.17 Herein, the

precursor was easily synthesized by mixing dimethylglyoxime

and Ni(OAc)2 in aqueous solution with vigorous stirring

(see ESIw for experimental details). X-ray diffraction (XRD)

analysis and scanning electron microscopy (SEM) observation

confirm that the obtained product is Ni(dmg)2 and has a

one-dimensional morphology, with an average diameter of

600 nm (XRD pattern and SEM images are shown in Fig. 1a

and ESIw Fig. SI1, respectively). Dimethylglyoxime would

chelate with Ni2+ ion to form a square planar configuration

with Ni2+ in the center. The central metal Ni2+ ions interact

strongly with the adjacent molecules and the square planes

stack face-to-face, resulting in a one-dimensional columnar

structure as shown in Fig. SI2.w18 This inherent columnar

structure would lead to the formation of Ni(dmg)2 nanorods.

After the precursor was calcined at 450 1C in air, NiO

nanotubes can be obtained. The XRD analysis (Fig. 1b)

confirms that the Ni(dmg)2 nanorod precursor has been

completely transformed to NiO (JCPDS-780643). The XRD

peaks are very broad, indicating that the NiO nanocrystallites

are in the range of the nanometre scale. The rod-like morphology

is maintained on a large scale, as evidenced by SEM image in

Fig. 2a. But unlike the precursor, these 1D crystals are tubes,

which can be clearly seen from Fig. 2b as there are holes of the

broken ends. Fig. 2c displays an SEM image with a higher

magnification, confirming that the product is one-dimensional

nanotubes, and the surface is very rough, suggesting that the

nanotubes might be composed of NiO nanoparticles. The

Fig. 1 XRD patterns of (a) Ni(dmg)2 nanorods; (b) NiO nanotubes;

(c) commercial NiO; (d) NiO nanoflowers.

a State Key Laboratory of Coordination Chemistry,Coordination Chemistry Institute, Nanjing National Laboratory ofMicrostructures, Nanjing University, Nanjing, 210093, P. R. China.E-mail: qylu@nju.edu.cn

bDepartment of Materials Science and Engineering, NanjingUniversity, Nanjing, 210093, P. R. China. E-mail: fgao@nju.edu.cn

w Electronic supplementary information (ESI) available: Experimentaldetails; SEM images, molecular structure, and TG curve of Ni(dmg)2nanorods; SEM images of NiO nanoflowers and commercial NiO;variation of specific capacitance with cycle number; XRD pattern, SEMand TEM images of Ni/C nanocomposite. See DOI: 10.1039/b914898a

7542 | Chem. Commun., 2009, 7542–7544 This journal is �c The Royal Society of Chemistry 2009

COMMUNICATION www.rsc.org/chemcomm | ChemComm

Spiropyran-linked dipeptide forms supramolecular hydrogel with dual

responses to light and to ligand–receptor interactionw

Zhenjun Qiu, Haitao Yu, Jinbo Li, Yu Wang and Yan Zhang*

Received (in Cambridge, UK) 18th December 2008, Accepted 6th March 2009

First published as an Advance Article on the web 30th March 2009

DOI: 10.1039/b822840j

Integration of photo-sensitive spiropyran with dipeptide

D-Ala–D-Ala in one small molecule resulted in a hydrogelator

which can form supramolecular hydrogel with responses not only

to light but to ligand–receptor interaction.

Supramolecular hydrogels formed by self-assembly of low

molecular weight molecules are useful soft materials with

diverse applications.1 Stimuli-responsive hydrogels, especially

photo-responsive hydrogels, are of particular interest in

material science and engineering because their property and

function are subject to spatio-temporal manipulation.2

Compared with photo-responsive polymer hydrogels which

have been extensively studied, only a few examples of photo-

responsive small molecule hydrogels have been established up

to now.3 Different from other ‘‘smart’’ supramolecular hydrogels

with response to pH,4 chemicals,5 enzymes6 or biologically-

relevant stimuli,7 photo-reponsive supramolecular hydrogels

often undergo photo-induced reaction or isomerization which

induces change in molecular packing and triggers photo-

response. Organic molecules whose structure or conformation

can be regulated by light have been widely used as ‘‘light

switches’’ to develop photo-responsive functional materials or

biologically-active molecules.8 Peptides have been proved to

be ideal building blocks for stimuli-responsive supramolecular

hydrogels with diverse applications.9 Incorporation of bio-

active short peptides with photo-sensitive functional groups

should be an efficient method to develop novel photo-responsive

small molecule hydrogelators with bio-functions.

Spiropyran analogues are one of the most widely used

photochromic species which have found interesting applica-

tions in light-regulated molecular devices,10 photoswitchable

nanoparticles,11 photo-switched binding with DNA or RNA12

and photo-responsive polymers.13 The reversible isomeriza-

tion between the non-planar spiropyran (SP) form and the

planar merocyanine (MC) form can be regulated by UV (SP to

MC) or visible light (MC to SP) irradiation. It has been found

that the planar MC isomer has a strong tendency to form an

aggregate-like structure due to intermolecular p–p stacking,

while the non-planar SP molecule does not have such inter-

action.14 Therefore it is rational to include a spiropyran

moiety in small molecule hydrogelators to realize photo-

response by regulating the intermolecular p–p stacking. To

develop novel photo-responsive supramolecular hydrogels

as candidates for multi-functional biomaterials, we tried to

integrate biomimetic small molecules with photo-sensitive

spiropyran components. Dipeptide residue D-Ala–D-Ala was

linked to the spiropyran moiety because of its biomimetic

interaction with the antibiotic vancomycin.15 Based on the

molecule integrated with these two components, here we report

the first example of small molecule hydrogel with responses not

only to light but also to ligand–receptor interaction.

The chemical structure of the spiropyran substituted

dipeptide 1 is shown in Scheme 1. The N-terminal of

D-Ala–D-Ala was linked to the amino group on spiropyran

via succinic acid. The synthetic route to 1-SP has been

optimized and finally the compound was prepared from

commercially available protected D-alanine and 1,3,3-tri-

methyl-2-methyleneindoline. The chemical structure of the

compound has been confirmed by NMR and MS (ESIw).Similar to other spiropyran derivatives, isomerization between

1-SP and 1-MC has been observed upon light irradiation.

The hydrogelation ability of the compound was then tested.

As we expected, 1-SP could not form a stable hydrogel due to

the non-planar conformation of the molecule. However, when

the non-planar 1-SP was converted to its isomer 1-MC, whose

merocyanine moiety has a strong tendency to form p–pstacking, the molecule became a hydrogelator which could

gel water at a concentration of around 11 mM and pH

around 3 (Fig. 1). Similar to other C-terminal short peptide

hydrogelators,5 compound 1 showed good solubility in neutral

or basic aqueous solution. The dark red solution with 1-MC as

the major component could be turned into hydrogel by care-

fully adding diluted hydrochloric acid to adjust the pH to

around 3. Although the hydrogelator turned out to be

Scheme 1 Chemical structure of the spiropyran-linked dipeptide in

SP form (1-SP) and MC form (1-MC).

School of Chemistry and Chemical Engineering, Key Lab ofAnalytical Chemistry for Life Science, Ministry of Education ofChina, Nanjing University, Nanjing, 210093, P. R. China.E-mail: njuzy@nju.edu.cnw Electronic supplementary information (ESI) available: Syntheticroute, NMR and MS data of 1-SP, CD spectra of the gel form by1-MC and the control compound. See DOI: 10.1039/b822840j

3342 | Chem. Commun., 2009, 3342–3344 This journal is �c The Royal Society of Chemistry 2009

COMMUNICATION www.rsc.org/chemcomm | ChemComm

Tetraalkylammonium amino acids as functionalized ionic liquids of lowviscosity{

Ying-Ying Jiang, Guan-Nan Wang, Zheng Zhou, You-Ting Wu,* Jiao Geng and Zhi-Bing Zhang

Received (in Cambridge, UK) 5th September 2007, Accepted 22nd October 2007

First published as an Advance Article on the web 9th November 2007

DOI: 10.1039/b713648j

Four of nine tetraalkylammonium-based amino-acid ionic

liquids prepared in this work show lower viscosities than all

amino acid-based ionic liquids found in the literature and their

reversible CO2 absorption approaches 0.5 mol per mol ionic

liquid.

Since Wilkes and Zaworotko1 synthesized the air- and water-stable

ionic liquid (IL) [emim]BF4 (1-ethyl-3-methylimidazolium tetra-

fluoroborate), ILs have attracted considerable attention from the

industrial and academic communities as ‘green solvents’. The study

of air- and moisture-stable ionic liquids has become the subject of

an increasing number of scientific investigations documented in the

literature.2–5 Particularly, after Fukumoto et al.6 succeeded in

synthesizing ionic liquids from 20 natural amino acids in 2005,

many amino acid-based ionic liquids have been prepared including

amino acids as anions,7,8 amino acids as cations9 or amino acid

derivatives.10 These ILs are high-quality functionalized liquids that

have chiral centers, biodegradable characteristics, and high

biocompatibility. Thus, in the fields of industrial chemistry and

pharmaceutical chemistry, amino acid-based ILs (AAILs) should

provide a variety of applications, such as intermediates for peptide

syntheses, chiral solvents, absorbents for acidic gases and other

functional materials.

Although most of the AAILs found and prepared in the

literature are liquids at room temperature, their uses as liquid

materials or solvents are still limited due to their viscous nature.

For example, AAILs containing 1-ethyl-3-methylimidazolium as a

cation ([emim][amino acid] ILs) were obtained as liquids at room

temperature, but the hydrogen bonding at the 2-position of the

imidazolium cation ring with its amino acid anions caused these

imidazolium-based AAILs to have a minimum viscosity as high as

486 mPa s at 25 uC.6 Tetrabutylphosphonium-based AAILs

([TBP][amino acid]) were also found to be more viscous than

344 mPa s, even though their thermal stability was generally better

than that of [emim][amino acid] ILs.7 The AAILs that were

prepared using amino acids as cations8 or using amino acid

derivatives were either solids or liquids of extremely high viscosity,

up to 4180 mPa s at room temperature.10 Until now, no AAILs

that have viscosity lower than 200 mPa s at 25 uC have ever been

reported in the literature. Since viscosity is one of the most

important characteristics that determine the efficiencies of heat and

mass transfer processes, AAILs of low viscosity are eagerly sought

for use as process solvents. In this work, as part of the search for

AAILs of low viscosity, novel AAILs using symmetric tetraalkyl-

ammonium ([TAA]) as cation are prepared. All these newly-

prepared tetraalkylammonium-based AAILs are found to be

liquid below 50 uC and two of them are liquids with viscosities

lower than 140 mPa s. In particular, among them tetraethyl-

ammonium a-alanine ([N2222][L-Ala]) is found to be the AAIL that

has the lowest known viscosity of 81 mPa s. This paper is the first

to report the existence of AAILs of low viscosities, especially when

symmetric tetraalkylammoniums are used as the cations of AAILs.

The details of the preparation and characterization experiments

can be seen in the ESI.{Table 1 summarizes the properties of nine [TAA][amino acid]

ILs obtained in this work. It is clearly shown that seven of the nine

products are room temperature ILs and all products are ILs below

50 uC. [N1111][Ala] and [N1111][Val] showed only a melting point

(Tm) near 40 uC, five AAILs showed only a glass transition

temperature (Tg), and two species showed both a melting point

and a glass transition temperature. As is known from the

literature, ILs that consist of symmetric tetraalkylammonium

cations (alkyl chain length not larger than C4) and a conventional

anion such as tetrafluoroborate (BF42), hexafluorophosphate

(PF62) and bis(trifluoromethylsulfonyl)imide (Tf2N

2) are solids

and have high melting points. The [TAA][amino acid] ILs in this

paper behave completely in contrast to the traditional [TAA] ILs

that contain a common anion such as BF42, PF6

2 or Tf2N2. This

difference may be due to the symmetric property of the anion

structure, that is, the more symmetric the anion structure the

higher the melting point. The Tm values of [N1111][BF4],

[N1111][Tf2N] and tetramethylammonium 2,2,2-trifluoro-N-(tri-

fluoromethylsulfonyl)acetamide ([N1111][TSAC]) appear to be

related to the relative anion symmetric property. The trend of

the highest to lowest Tm values is [N1111][BF4] (.300 uC)11 .

[N1111][Tf2N] (133 uC)12 . [N1111][TSAC] (64 uC),13 which is

School of Chemistry and Chemical Engineering, Nanjing University,Nanjing, 210093, China. E-mail: ytwu@nju.edu.cn;Fax: +86-25-83593772; Tel: +86-25-81638601{ Electronic supplementary information (ESI) available: Experimentaldetails. See DOI: 10.1039/b713648j

Table 1 Properties of tetraalkylammonium type amino acid ionicliquids

IL Tg/uC Tm/uC Tdec/uC r/g cm23 g/mPa s

[N1111][L-Ala] ND 42 219 ND NA[N2222][L-Ala] 280 ND 185 0.9984 81[N1111][b-Ala] 282 ND 193 1.0877 668[N2222][b-Ala] 285 ND 184 1.0133 132[N4444][b-Ala] 273 ND 180 0.9545 465[N1111][Gly] 268 ND 181 1.0887 304[N4444][Gly] 271 16 179 0.9406 214[N1111][Val] ND 40 223 ND NA[N4444][Val] 269 25 185 0.9470 660a ND: not detected. NA: Solid or glass at 25 uC.

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This journal is � The Royal Society of Chemistry 2008 Chem. Commun., 2008, 505–507 | 505

Supermolecule density functional calculations on the water exchange of

aquated Al(III) species in aqueous solutionw

Zhaosheng Qian, Hui Feng, Wenjing Yang, Qiang Miao, Lina He and Shuping Bi*

Received (in Cambridge, UK) 15th April 2008, Accepted 21st May 2008

First published as an Advance Article on the web 3rd July 2008

DOI: 10.1039/b806433d

Supermolecule density functional calculations suggest the dis-

sociative (D) mechanism for the water exchange of aquated

Al(III) species in aqueous solution and the calculated results

agree well with experimental data.

The water exchange of aquated metal ions between the first

hydration sphere and the bulk water is significant to understand

the reactivity of metal ions in solution. Although the experi-

mental methods can determine the water exchange rate, they are

not accessible for the detailed microscopic nature of the under-

lying reaction mechanism.1,2 Recent theoretical works3–5 simu-

lated the water exchange of metal ions using the cluster model

in which the solvent was neglected or treated with self-consis-

tent reaction fields, but this model did not reproduce properties

accurately for the metal ions with strong second shell solvation

such as Al(III).6 In this communication, we simulated the water

exchange on aquated Al(III) species (eqn (1)) successfully.

[M(H2O)n]z+ + nH2O* 2 [M(H2O*)n]

z+ + nH2O (1)

For understanding complicated reactions and those at mineral

surfaces,7,8 the rate constants and activation parameters for water

exchange on Al(H2O)63+ and Al(H2O)5(OH)2+ were determined

from kinetic 17ONMR experiments.9–11 For the detailed informa-

tion the water exchange mechanisms on Al(H2O)63+ were simu-

lated based on the gas-phase cluster model.12 The models only

involved the gas-phase clusters which neglected the solvent effect.

Recently, Hanauer et al.13 using the gas-phase cluster model with

four additional water molecules investigated the water exchange

of Al(H2O)63+ and Al(H2O)5(OH)2+. Since their model did not

consider the bulk solvent effects, their computed activation

energies with the consideration of the second hydration sphere

deviated from the corresponding experimental values.10,11

In the current study, we employed a supermolecule model on

the water exchange of Al(H2O)63+ and Al(H2O)5(OH)2+ by

density functional theory calculations. In the proposed super-

molecule model, six explicit solvent water molecules are added to

core complexes (Al(H2O)63+ and Al(H2O)5(OH)2+) for consid-

eration of the explicit solvent effect induced by the hydrogen

bonding interaction between the first and second coordinated

water molecules, and the remaining solvent water is modeled as a

polarizable dielectric continuum medium surrounding the super-

molecular reaction system. For comparison, the gas-phase model

(core complexes with one additional water molecule) calculations

were also performed at the same level. All the structures of the

gas-phase species and supermolecular species were fully optimized

in the gas phase at B3LYP/6-311+G** level13–16 using density

functional theory, and then single-point PCM17,18 calculations

were performed in aqueous solution. E(aq) and Es(aq) indicate

bulk solvent effect on the optimized gas-phase species and super-

molecular species, respectively. Vibrational frequency calculations

were carried out to confirm the stable structures and the transition

state. To obtain accurate energies, single-point energies were

further calculated using MP219 at 6-311+G** level. The Gaus-

sian 03 suite of programs was used throughout.20 The reactants,

transition states and products in the gas-phase reaction system for

water exchange on Al(H2O)63+ and Al(H2O)5(OH)2+ are de-

noted as R1, TS1, P1, R2, TS2, and P2, respectively, and those in

the supermolecular reaction system are denoted as Rs1, TSs1, Ps1,

Rs2, TSs2, and Ps2, respectively. The parameters used for struc-

tural changes comprise average bond length between aluminium

and the first coordinated water molecules r(Al–O), the bond

distance between aluminium and the leaving water molecule

r(Al–OL), the bond distance between the leaving water and

hydrogen of the neighboring water in the first coordination sphere

r(H–OL), and the sum of the distance between aluminium and the

water molecules in the first and second spheresP

r(Al–O).

Concerning the number of the nonbulk water molecules

included in the supermolecular model to describe the explicit

solvent effect, six explicit solvent water molecules were considered

in the present study. Until now, the solvation sphere around

hydrated metal ions has usually only been considered explicitly

for the first and to some extent for the second coordination

spheres, usually for only one or at most four additional water

molecules.13 For confirmation of the sufficiency of the adopted

explicit structure models, we also modeled the supermolcules with

the consideration of four and five explicit water molecules for the

water exchange on Al(H2O)63+. As shown in Table 1, the

calculated energy barriers with the consideration of five and six

explicit water molecules are very close and in good agreement

with the experimental data, and thereby the convergence with

respect to the number of water molecules is satisfactory for n4 5.

In addition, a large number of explicit water molecules might

produce a very large number of orientations associated with local

minima, which may lead to biased conclusions. Nevertheless, the

supermolcular model in this study should be less effected by the

orientation problem due to two reasons. Firstly, the orientations

of the explicit water molecules are simply determined by the

solute–solvent hydrogen-bonding. Secondly, the orientations of

School of Chemistry and Chemical Engineering, State Key Laboratoryof Coordination Chemistry of China & Key Laboratory of MOE forLife Science, Nanjing University, Nanjing 210093, People’s Republicof China. E-mail: bisp@nju.edu.cn; Fax: +86-25-83317761;Tel: +86-25-86205840w Electronic supplementary information (ESI) available: Optimizedstructures and structural parameters for the gas-phase reaction system,energy profiles for water exchange on the aquated Al(III) species andcartesian coordinates of the supermolecules. For ESI see DOI:10.1039/b806433d

3930 | Chem. Commun., 2008, 3930–3932 This journal is �c The Royal Society of Chemistry 2008

COMMUNICATION www.rsc.org/chemcomm | ChemComm

An iron(III) phosphonate cluster containing a nonanuclear ring{

Hong-Chang Yao,a Jun-Jie Wang,a Yun-Sheng Ma,a Oliver Waldmann,*b Wen-Xin Du,c You Song,a

Yi-Zhi Li,a Li-Min Zheng,*a Silvio Decurtins*b and Xin-Quan Xina

Received (in Cambridge, UK) 18th January 2006, Accepted 27th February 2006

First published as an Advance Article on the web 13th March 2006

DOI: 10.1039/b600763e

This communication reports the first example of cyclic ferric

clusters with an odd number of iron atoms capped by

phosphonate ligands, namely, [Fe9(m-OH)7(m-O)2(O3PC6H9)8-

(py)12]. The magnetic studies support a S = 1/2 ground state

with an exchange coupling constant of about J # 230 K.

Cyclic iron cages are attractive not only because of their capability

to accommodate alkali-metal cations1,2 but also because of their

unprecedented magnetic behavior.3 Having been regarded initially

as ideal models for studying 1D anti-ferromagnetic materials it

subsequently became clear that their magnetism is best described

by a quantized rotation of the Neel vector and is more similar to

that of 2D and 3D materials.4 Moreover, quantum phenomena

such as Neel vector tunneling were also demonstrated.5 However,

all cyclic iron cages recorded so far are even-membered wheels

with 6,1,2 8,2,6 10,3,7 128 and 169 metal ions in which carboxylate

ligands are usually involved in linking the metal ions. Synthesizing

odd-membered iron rings is an interesting goal, not only from a

chemical perspective but also because the odd number of magnetic

electrons induce quantum spin-frustration effects.10 Recently, a

nonanuclear wheel consisting of one Ni(II) and eight Cr(III) ions

has been synthesized.11 Because of the even number of electrons,

the frustration situation in such a wheel is fundamentally different

from that in systems with an odd number of electrons.12 In recent

years iron clusters involving phosphonate ligands have attracted

much attention. Several complexes of iron phosphonates with Fe4,

Fe6, Fe7 and Fe14 cages have been obtained.13,14 The oxo-centered

[Fe3(m3-O)]7+ triangle units are found in these cages and the

carboxylates appear as coligands. Herein, we report a novel

nonanuclear iron(III) cage complex, [Fe9(m-OH)7(m-O)2-

(O3PC6H9)8(py)12]?6H2O (1?6H2O), which contains a nine iron

ring of {Fe9(m-OH)7(m-O)2} capped by phosphonate ligands.

Treatment of [Fe3O(O2CMe)6(H2O)3]NO3 (0.262 g, 0.4 mmol)

with two equivalents of C6H9PO3H2 (0.130 g, 0.8 mmol) in

pyridine (10 mL) gave a light brown solution from which green–

yellow block crystals were formed in good yield (46%) by diffusing

Et2O into the filtrate.{ The purity was judged by comparing

powder XRD with the crystallographic result. The 57Fe

Mossbauer spectrum of 1 was recorded in zero field at room

temperature. The observed broad doublet can be best fitted to two

doublets with an area ratio of 2 : 1 with isomer shifts (d) and

quadrupole splitting (DEQ) of 0.359, 0.579 mm s21 and 0.447,

0.578 mm s21, respectively, corresponding to two types of

high-spin ferric centers.15

Single-crystal structural analysis reveals that complex 1?6H2O§

crystallizes in the monoclinic space group C2. The asymmetric unit

contains half of [Fe9(m-OH)7(m-O)2(O3PC6H9)8(py)12]. The lattice

water molecules reside on seven crystallographic sites with partial

occupancies which are summed to ‘‘three’’. There are five

crystallographically different Fe atoms in the structure. The

Fe(1) and O(5) atoms lie on a twofold axis; the other four Fe

atoms are on general positions. Two types of Fe atoms, A and B,

can be distinguished, see Fig. 1(top). In type A [Fe(2), Fe(3),

Fe(5)], the distorted octahedral sphere of each Fe atom is

completed by three phosphonate oxygens, two m-OH (or m-O)

oxygens and one pyridine nitrogen atom, forming an FeO5N

coordination environment. In type B [Fe(1), Fe(4)], the octahedral

sphere of each Fe is completed by two phosphonate oxygens, two

m-OH (or m-O) oxygens and two pyridine nitrogen atoms, forming

a coordination mode of FeO4N2. The molar ratio of type A and B

Fe atoms is 2 : 1, in agreement with the Mossbauer result. The

aState Key Laboratory of Coordination Chemistry, CoordinationChemistry Institute, Nanjing University, Nanjing, 210093, P. R. China.E-mail: lmzheng@nju.edu.cnbDepartment of Chemistry and Biochemistry, University of Bern,Freiestrasse 3, CH-3012, Bern, Switzerland.E-mail: oliver.waldmann@iac.unibe.ch; silvio.decurtins@iac.unibe.chcState Key Laboratory of Structure Chemistry, Fujian Institute ofResearch on the Structure of Matter, The Chinese Academy of Sciences,Fuzhou, 350002, P. R. China{ Electronic supplementary information (ESI) available: XRD, TG, IRand Mossbauer spectrum of complex 1?6H2O. See DOI: 10.1039/b600763e

Fig. 1 Top: asymmetric unit of 1 (ellipsoids at 30% probability). Bottom:

inorganic core of 1. Hydrogen atoms are omitted.

COMMUNICATION www.rsc.org/chemcomm | ChemComm

This journal is � The Royal Society of Chemistry 2006 Chem. Commun., 2006, 1745–1747 | 1745

Biosensors and Bioelectronics 24 (2009) 3693–3697

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Short communication

Near infrared sensing based on fluorescence resonance energy transfer betweenMn:CdTe quantum dots and Au nanorods

Guo-Xi Liang, Hong-Cheng Pan, Ye Li, Li-Ping Jiang, Jian-Rong Zhang, Jun-Jie Zhu ∗

Key Lab. of Analytical Chemistry for Life Science (MOE), School of Chemistry and Chemical Engineering, Nanjing University, Hankou Road 22, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:Received 14 March 2009Received in revised form 3 May 2009Accepted 7 May 2009Available online 14 May 2009

Keywords:Fluorescence resonance energy transferNear infraredQuantum dotsAu nanorods

a b s t r a c t

A novel sensing system based on the near infrared (NIR) fluorescence resonance energy transfer (FRET)between Mn:CdTe quantum dots (Qdots) and Au nanorods (AuNRs) was established for the detection ofhuman IgG. The NIR-emitting Qdots linked with goat anti-human IgG (Mn:CdTe-Ab1) and AuNRs linkedwith rabbit anti-human IgG (AuNRs-Ab2) acted as fluorescence donors and acceptors, respectively. FREToccurred by human IgG with the specific antigen–antibody interaction. And human IgG was detectedbased on the modulation in FRET efficiency. The calibration graph was linear over the range of 0.05–2.5 �Mof human IgG under optimal conditions. The proposed sensing system can decrease the interference ofbiomolecules in NIR region and increase FRET efficiency in optimizing the spectral overlap of AuNRs withMn:CdTe Qdots. This method has great potential for multiplex assay with different donor–acceptor pairs.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Fluorescence resonance energy transfer (FRET) is a powerfultechnique for characterizing and probing short distance-dependentinteractions between the donor and the acceptor (Lakowicz, 1999;Clapp et al., 2006). Recently, luminescent semiconductor nanocrys-tals or quantum dots (Qdots) are proved to be effective FRET donorsdue to their broad excitation spectra and tunable, narrow and sym-metric photoemission. These unique characteristics could enhanceFRET as an effective technique for the application in molecularbinding event studies, protein conformation change, and biologicalassays (Clapp et al., 2006; Roda et al., 2009; Sapsford et al., 2006;Algar and Ulrich, 2008; Li et al., 2008).

In most of the Qdots-based FRET approaches, organic dyes areusually used as the acceptors (Medintz et al., 2003; Shi et al.,2007; Chong et al., 2007). However, the dyes commonly sufferfrom photo-bleaching problem. In recent years, several researcheshave demonstrated that Au nanoparticles (AuNPs) are promisingcandidates as the acceptors for FRET system, for their capacityof inducing strong fluorescence quenching and without photo-bleaching (Wargnier et al., 2004; Oh et al., 2005; Pons et al., 2007;Shan et al., 2009). However, the absorption spectra of AuNPs withdifferent sizes cannot be easily resolved because the surface plas-mon resonance peaks broaden with the increase of diameter atlonger wavelength (above 650 nm). This disadvantage limits AuNPsapplication in FRET for multiple assays.

∗ Corresponding author. Tel.: +86 25 83594976; fax: +86 25 83594976.E-mail address: jjzhu@nju.edu.cn (J.-J. Zhu).

Compared with AuNPs or organic dyes, Au nanorods (AuNRs)have attracted much attention in biomedical applications due totheir attractive features such as two peaks assignable to the trans-verse and longitudinal surface plasmon resonance bands, highextinction coefficient and broad absorption range that can be tunedby changing their aspect ratio (Link et al., 1999; Thomas et al.,2004; Gole and Murphy, 2005; Huang et al., 2006; Li et al., 2009).AuNRs are good quenchers for Qdots fluorescence (Nikoobakht etal., 2002). Moreover, the strong characteristic absorption bands inthe visible to near infrared (NIR) region make AuNRs suitable accep-tor for FRET system especially in NIR region (650–1100 nm).

Nowadays, biosensing in NIR region has attracted consider-able attention because of the low absorption coefficient for mostbiomolecules (Klostranec and Chan, 2006; Weissleder, 2001; Huanget al., 2006). Although many Qdots-based FRET systems have beenreported and widely used, most of them were in visible region. Themain challenge of moving Qdots-based FRET system from visible toNIR window is to find a good matching donor–acceptor pair. AuNRswith tunable absorption peaks in NIR region could offer excellentoverlap with Qdots emission for multiplexed biological detection.However, to the best of our knowledge, there was no report aboutFRET sensing in NIR region with Qdots–AuNRs bioconjugates.

Herein, we demonstrate a near infrared sensing system for thedetection of human IgG based on the FRET between Mn:CdTeQdots and AuNRs. The NIR-emitting Qdots linked with goat anti-human IgG (Mn:CdTe-Ab1) acted as fluorescence donors. TheNIR-absorption AuNRs linked with rabbit anti-human IgG (AuNRs-Ab2) acted as acceptors. Then FRET occurred by the conjugationbetween Mn:CdTe-Ab1 and AuNRs-Ab2 in the presence of humanIgG. The calibration graph for human IgG detection is linear over the

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Biosensors and Bioelectronics 24 (2009) 3352–3358

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Enhanced electrochemiluminescence of CdSe quantum dots composited withCNTs and PDDA for sensitive immunoassay

Guifen Jie, Lingling Li, Chao Chen, Jie Xuan, Jun-Jie Zhu ∗

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:Received 10 March 2009Received in revised form 23 April 2009Accepted 24 April 2009Available online 3 May 2009

Keywords:ElectrochemiluminescenceCdSe quantum dotsCdSe QDs–CNTs compositesECL immunosensor

a b s t r a c t

Electrochemiluminescence (ECL) of CdSe quantum dots (QDs) was greatly enhanced by the combinationof carbon nanotubes (CNTs) and poly (diallyldimethylammonium chloride) (PDDA) in the CdSe QDs film,and could successfully be used to develop a sensitive ECL immunosensor for the detection of human IgG(Ag). The novel CdSe QDs–CNTs composites exhibited high ECL intensity, good biocompatibility, and highstability, which held great promise for the fabrication of the ECL biosensors with improved sensitivity.After PDDA as a binding linker was conjugated to the CdSe QDs–CNTs composite film on the electrode,the ECL signal was significantly enhanced. Subsequently, gold nanoparticles (GNPs) assembled onto theCdSe QDs–CNTs/PDDA modified electrode could amplify the ECL signal once again. After antibody (Ab)was immobilized onto the electrode through GNPs, the ECL immunosensor was successfully fabricated.It is for the first time that the unique function of PDDA for enhancing QDs ECL was explored and used todevelop an ECL biosensor. The principle of ECL detection for target Ag is based on the increment of sterichindrance after immunoreaction, which resulted in the decrease of ECL intensity. The Ag concentrationwas determined in the linear range of 0.002–500 ng L−1 with a detection limit of 0.6 pg mL−1. The sensorshowed good fabrication and detection reproducibility, and the assay results were in acceptable agree-ment with the clinical sera tests, showing a promising clinical application. This work opened the newavenues for applying QDs ECL in highly sensitive bioassays.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Semiconductor nanocrystals (NCs, also referred to as quantumdots) have generated great research interest due to their uniqueluminescent properties and relatively low cost (Alivisatos, 1996a,b;Qu and Peng, 2002; Burda et al., 2005) in recent years. Stronglyluminescent semiconductor NCs have found potential applicationsin biological imaging and labeling (Bruchez et al., 1998; Chan andNie, 1998; Han et al., 2001). Luminescent properties are usuallystudied by using photoexcitation (Qu and Peng, 2002), electro-chemiluminescence (ECL) generated by electron injection (Ding etal., 2002), and cathodoluminescence produced by electron impact(Rodriguez-Viejo et al., 1997). ECL as the production of light fromelectrochemically generated reagents has been paid considerableattention due to its versatility, simplified optical setup, low back-ground signal, good temporal and spatial control (Zhang et al.,2008a,b). ECL of II–VI QDs has been extensively studied in bothorganic (Myung et al., 2002, 2003; Bae et al., 2004) and aqueousmedia (Bae et al., 2006; Ding et al., 2006; Wang et al., 2008). Effi-cient and stable ECL emissions can be obtained in aqueous solution

∗ Corresponding author. Tel.: +86 25 83594976; fax: +86 25 83594976.E-mail address: jjzhu@nju.edu.cn (J.-J. Zhu).

by using H2O2 (Zou and Ju, 2004; Ding et al., 2006), S2O82− (Jie

et al., 2007) and O2 (Jiang and Ju, 2007) as coreactants. The QDs-based ECL immunosensors (Jie et al., 2007) and enzyme-based ECLbiosensors for glucose (Jiang and Ju, 2007) have been successfullyfabricated.

However, the reports concerning the detection of biomoleculeswith QDs ECL are relatively scarce, though ECL analysis has manyadvantages over photoluminescence due to the absence of back-ground from unselective photoexcitation. The reasons are partiallythat the ECL of semiconductor NCs is weaker than that of con-ventional luminescent reagents such as luminal or Ru(bpy)3

2+,and that NCs in ECL with a high excited electrochemical poten-tial are unstable. Thus, it is important to select effective methodsto enhance NCs ECL signal for development of ECL biosensors.Thin film technique of semiconductor NCs may provide an effec-tive way for the construction of QDs-based ECL biosensors. Inrecent years, carbon nanotubes (CNTs) have received particularinterest due to their remarkable nanostructure, high electrical con-ductivity, and good chemical stability (Iijima, 1991; Iijima andIchihashi, 1993; Wang et al., 2003). It is reported that carbonnanotubes can be used to enhance the ECL of CdS QDs film byreducing the injection barrier of electrons to the QDs (Ding et al.,2006). ECL of CdSe NCs could be enhanced by combining with car-bon nanotubes—chitosan and 3-aminopropyl-triethoxysilane (Jie

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Biosensors and Bioelectronics 24 (2009) 1074–1082

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Review

Some thoughts on the existence of ion and water channels in highly dense andwell-ordered CH3-terminated alkanethiol self-assembled monolayers on gold

Jianyuan Dai, Zhiguo Li, Jing Jin, Yanqing Shi, Jiongjia Cheng, Jing Kong, Shuping Bi ∗

School of Chemistry and Chemical Engineering, State Key Laboratory of Coordination Chemistry of China & Key Laboratory of MOE for Life Science,Nanjing University, Nanjing 210093, China

a r t i c l e i n f o

Article history:Received 28 April 2008Received in revised form 30 July 2008Accepted 15 August 2008Available online 28 August 2008

Keywords:CH3-terminated alkanethiolSelf-assembled monolayerGold electrodePermeationChannel

a b s t r a c t

Through extensive and systematic reviewing of literatures, this paper presents some thoughts on theexistence of ion and water channels in highly dense and well-ordered CH3-terminated alkanethiol self-assembled monolayers (CnSH-SAMs) on gold: (1) based on the combinational effect of “the van der Waalsinteraction between the neighbor hydrocarbon chains”, “the framework size of CnSH molecule” and “thesurface lattice structure of CnSH-SAMs on gold”, a simple ideal model of the close-packed CnSH-SAMsis proposed for the first time. The channel with an appropriate diameter of about 3 Å (∼3 Å) existingin SAMs on Au is deduced, which is found large enough for ions and water molecules to permeate; (2)through summarizing the literature reports for various experiments (e.g. scan microscopy techniquesand electrochemical methods, etc.), the existence of the ion and water channels (∼3 Å) in close-packedCnSH-SAMs is verified; (3) furthermore, the effect of the ions and water molecules permeation on thestudies of the SAMs’ electron tunneling process is discussed. This simple ideal model of the close-packedCnSH-SAMs established by us may clarify the controversies about the permeation mechanism of ions andwater molecules in CnSH-SAMs.

© 2008 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10742. Establishment of the simple ideal model for the CnSH-SAMs on gold—the existence of ion and water channels (∼3 Å) in SAMs . . . . . . . . . . . . . . 10753. Experimental evidences for the existence of ion and water channels (∼3 Å) in close-packed CnSH-SAMs on gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078

3.1. Proved by the STM and AFM images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10783.2. Proved by the vapor deposition of metal atoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10783.3. Proved by the underpotential deposition (upd) of metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10793.4. Proved by the variation of differential capacitance Cd with v (CV) and the variation of phase angle ˚ with f (EIS) in inert electrolyte

without any redox species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10794. The effect of ions and water molecules permeation on the studies of the SAMs’ electron tunneling process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080

4.1. The basic characteristics of alkanethiol SAMs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10804.2. The effect of ions and water molecules permeation on the studies of the SAMs’ electron tunneling process . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080

5. Conclusions and outlooks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081

1. Introduction

SAMs are the novel organic super-thin films developed in thepast two decades that are able to deeper understand the interface

∗ Corresponding author. Tel.: +86 25 86205840; fax: +86 25 83317761.E-mail address: bisp@nju.edu.cn (S. Bi).

phenomenon and the relationship between structure and propertyat the atomic and molecular level (Calhoun et al., 2008; Crivillers etal., 2008; Feng et al., 2008; Mullen and Rabe, 2008). SAMs derivedfrom the adsorption of alkanethiol on gold have been extensivelystudied as a typical interface structure due to their ease of prepara-tion and stability (Lee et al., 2005; Love et al., 2005; Song et al., 2007;Tao and Bernasek, 2007; Wang et al., 2007). Although alkanethiolSAMs are almost the simplest self-assembly system and their basic

0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2008.08.034

Biosensors and Bioelectronics 24 (2008) 35–40

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

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A chemiluminescent immunosensor based on antibody immobilized carboxylicresin beads coupled with micro-bubble accelerated immunoreaction for fastflow-injection immunoassay

a a b,∗ a, H a,∗

), Dep

columred bap thform

lex. Te-en

ed. Thcolu

ient officienoaffit, and

Zhanjun Yang , Zhifeng Fu , Feng Yan , Hong Liua Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of Chinab Jiangsu Institute of Cancer Prevention and Cure, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:Received 25 November 2007Received in revised form 18 February 2008Accepted 11 March 2008Available online 18 March 2008

Keywords:Flow-injection analysisCarboxylic resinChemiluminescenceImmunoassayMicro-bubble�-Fetoprotein

a b s t r a c t

A novel immunoaffinityimmunoassay was prepacould fast recognize and trantigen, which was firstlya sandwich immunocompreaction to produce enzymimmunoassay was proposeration of immunoaffinitywith a correlation coefficintra- and inter-assay coeage stability of the immunflexible, sensitive, low-cos

1. Introduction

Flow-injection immunoassay (FIIA) is one of the most usefuldetection techniques for improving cumbersome, time-consuming,and labor-intensive traditional immunoassays (Pollema et al., 1992;Gunaratna et al., 1993). It has been widely applied to many fieldsincluding environmental monitoring, food safety, pharmaceuticalanalysis, and clinical diagnosis (Fu et al., 2007, 2008; Wu et al.,2007) due to its small sample-cost, reduced sample handling step,acceptable reusability, good reproducibility, less time consuming,and easy automation for high sample throughout. This techniquecan be performed in both homogeneous and heterogeneous sys-tems. The latter usually uses a solid support to immobilize eitherthe antibody or antigen, thus permits the separation of free portionfrom the bound immunocomplex. The most commonly used sup-ports include bead materials, such as silica, agarose, sepharose andpolystyrene, and membranes (Liu et al., 1991; Morais et al., 1999).They have been extensively used for preparation of immunoaffin-ity columns or immunosensors for development of flow-injectionimmunoassay system. This work uses for the first time carboxylicresin beads to prepare a novel immunoaffinity column. This mate-

∗ Corresponding authors. Tel.: +86 25 83593593; fax: +86 25 83593593.E-mail addresses: yanfeng2007@sohu.com (F. Yan), hxju@nju.edu.cn (H. Ju).

0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2008.03.007

uangxian Juartment of Chemistry, Nanjing University, Nanjing 210093, PR China

n used as an immunosensor for flow-injection chemiluminescent (CL)y immobilizing antibody on carboxylic resin beads. The immunosensore immunocomplex of horseradish peroxidase (HRP)-labeled antibody anded with a micro-bubble accelerated pre-incubation process, to producehe HRP introduced in the immunoaffinity column could catalyze the CLhanced emission. With �-fetoprotein (AFP) as a mode, a flow-injection CLe whole assay for one sample, including the pre-incubation and the regen-

mn, could be performed within 16 min. The linear range was 1.0–80 ng/mlf 0.998 and a detection limit of 0.1 ng/ml at a signal/noise ratio of 3. Thets of variation at 20 ng/ml AFP were 1.2% and 8.5%, respectively. The stor-

nity column and the accuracy for sample detection were acceptable. Thisrapid method is valuable for clinical immunoassay.

© 2008 Elsevier B.V. All rights reserved.

rial is low-cost and robust with excellent rigidity and mechanicalproperty, and can immobilize much more biomolecules than usualmembrane materials due to its large surface-to-volume ratio. Thehigh immobilization capability can provide the immunoaffinity col-

umn with good sensitivity for flow-injection chemiluminescent(CL) immunoassay.

In FIIA, the incubation time is a bottleneck to the improve-ment of assay speed. This process is usually controlled by masstransport of immunoreagents and kinetics of immunoreaction. Thelatter can be accelerated by a heating equipment. However, itincreases the cost and complexity of manipulation. Furthermorethe increased temperature is limited due to the denaturalizationof proteins at high temperature. Thus antibody–antigen binding isgenerally limited by mass transport rather than by binding kinet-ics (i.e., recognition rate) (Stenberg et al., 1986; Nygren et al.,1987; Myszka et al., 1997), and accelerating the mass transportof immunoreagents has attracted considerable interest for short-ening the incubation time of immunoreaction and improving thesample throughput of immunoassay. Various approaches have beenused to accelerate the mass transport of the antigen and antibody,such as electric-field-driven method (Ewalt et al., 2001), magneticstirring (Tanaka and Imagawa, 2005), magnetic field combinedwith superparamagnetic labels (Driskell et al., 2007), low-powermicrowave radiation (Previte et al., 2006), and capture substraterotation (Luxton et al., 2004). They also need complex or espe-

Biosensors and Bioelectronics 23 (2008) 1896–1899

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

Short communication

Electrochemiluminescence of CdSe quantum dots for immunosensingof human prealbumin

Guifen Jie, Haiping Huang, Xiaolian Sun, Jun-Jie Zhu ∗

Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), School of Chemistry and Chemical Engineering,Nanjing University, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:Received 23 November 2007Received in revised form 28 February 2008Accepted 28 February 2008Available online 18 March 2008

Keywords:ElectrochemiluminescenceCdSe quantum dotsECL immunosensor

a b s t r a c t

We describe a non-labeled electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots(QDs) for the detection of human prealbumin (PAB, antigen). The immunosensor was fabricated by layerby layer coupled with nanoparticle-amplification techniques. After two gold nanoparticle layers wereself-assembled onto the gold electrode surface through cysteamine, anti-PAB (antibody) were conjugatedwith –COOH groups of both the CdSe QDs and cysteine, which were linked to the gold nanoparticle-modified electrode. The principle of ECL detection was that the immunocomplex inhibited the ECL reactionbetween CdSe QDs and K2S2O8, which resulted in the decrease of ECL intensity. On the one hand, theimmunocomplex increased the steric hindrance. On the other hand, the immunocomplex maybe inhibitthe transfer of K2S2O8 to the surface of the CdSe QD-electrode. The PAB concentration was determined inthe range of 5.0 × 10−10 to 1.0 × 10−6 g mL−1, and the detection limit was 1.0 × 10−11 g mL−1. The developedCdSe QD-based ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol forprotein detection, which could be applied in more bioanalytical systems.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Highly luminescent semiconductor nanocrystals (NCs), oftenreferred to as quantum dots (QDs), have received tremendousattention for their possible luminescent applications in aqueoussolution (Bruchez et al., 1998; Chan and Nie, 1998). With size-tunable narrow emission spectra and broad excitation spectra, theyhave been widely used as multicolored photoluminescent probes,biological luminescent labels (Gill et al., 2006; Zhelev et al., 2006;Jin et al., 2005; Charbonniere et al., 2006) and bioimagings (Medintzet al., 2005; Larson et al., 2003). Since the breakthrough works onthe use of QDs in 1998 (Bruchez et al., 1998; Chan and Nie, 1998),multifarious water-soluble QDs have been synthesized to show dif-ferent optical properties, biocompatibilities, and cellular toxicities(Wang et al., 2006; Jin et al., 2006; Hoshino et al., 2004; Duanand Nie, 2007; Choi et al., 2006). Some bioinorganic conjugatesmade with CdSe/ZnS core-shell NCs and antibodies have showntheir potential applications in fluoroimmunoassays (Goldman etal., 2002a,b, 2004; Mattoussi et al., 2000). Luminescent propertiesof semiconductor nanocrystals are usually investigated by photolu-minescence (PL) (Qu and Peng, 2002), electrochemiluminescence(ECL) (Bae et al., 2004; Zou and Ju, 2004; Miao et al., 2005), cathodo-

∗ Corresponding author. Tel.: +86 25 83594976; fax: +86 25 83317761.E-mail address: jjzhu@nju.edu.cn (J.-J. Zhu).

luminescence, and chemiluminescence (CL) (Wang et al., 2005).Among them, ECL is a useful technique for both fundamental studyand analytical applications (Bae et al., 2004; Ding et al., 2002;Myung et al., 2002, 2003). In recent years, Bard and co-workers(Ding et al., 2002; Myung et al., 2002, 2004; Bae et al., 2004)have reported ECL of semiconductor nanocrystals in organic sol-vent. Subsequently, ECL of CdS (Miao et al., 2005; Ding et al., 2006)and CdSe NCs in aqueous solution (Zou and Ju, 2004; Jiang and Ju,2007a,b; Liu et al., 2007) have also been observed and applied toH2O2 or glucose sensing. However, so far no immunosensor usingECL of QDs has been reported. Therefore, the development of ECLimmunosensor fabricated with CdSe QDs is of great significance forbiological analysis.

Prealbumin (PAB), a stable circulating glycoprotein synthesizedin the liver, is a reliable index of liver function. Blood serum PABdecreased in varieties of hepatitis, cirrhosis and hepatocarcinoma(Guo, 2006). So the detection of PAB was worthwhile for early diag-nosing serious hepatitis and evaluating clinical results.

This work developed a promising CdSe QD-based ECLimmunosensor for prealbumin detection via self-assembly and goldnanoparticle-amplification techniques. The CdSe QDs and anti-prealbumin were directly immobilized onto the gold nanoparticle-modified electrode. The specific immunoreaction of prealbuminwith anti-prealbumin resulted in the decrease of ECL inten-sity which could be used to detect the prealbumin. This ECLimmunosensor is rapid, specific, and ultrasensitive for bioassays. In

0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2008.02.028

Available online at www.sciencedirect.com

Biosensors and Bioelectronics 23 (2008) 1666–1673

Horseradish peroxidase-functionalized gold nanoparticle labelfor amplified immunoanalysis based on gold nanoparticles/

carbon nanotubes hybrids modified biosensor

Rongjing Cui, Haiping Huang, Zhengzhi Yin, Di Gao, Jun-Jie Zhu ∗Key Laboratory of Analytical Chemistry for Life Science (MOE), School of Chemistry and Chemical Engineering,

Nanjing University, Nanjing 210093, People’s Republic of China

Received 18 October 2007; received in revised form 2 January 2008; accepted 29 January 2008Available online 13 February 2008

Abstract

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybridsplatform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a modelprotein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix andmade the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRPlabels and secondary antibodies (Ab2) linked to GNPs at high HRP/Ab2 molar ratio. The approach provided a linear response range between 0.125and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and couldbe used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.© 2008 Elsevier B.V. All rights reserved.

Keywords: Electrochemical biosensor; Amplified immunoassay; Hybrids

1. Introduction

Achieving high sensitivity is a major goal in immunoas-say, such as the monitoring of disease markers and infectiousagents or biothreat agents. The achievement of high sensitivityrequires innovative approaches that couple different amplifica-tion platforms and amplification processes (Wang, 2005). Byincorporation with nanoparticles (NPs), the enormous signalenhancement associated with the use of NPs amplifying labelsand with the formation of NPs/biomolecule conjugations pro-vides the basis for ultrasensitive electrical detection of tracebiomolecule (Cui et al., 2007; Daniel and Astruc, 2004; Katzand Willner, 2004). It has been reported that the NPs-basedamplification platforms and amplification processes dramati-cally enhanced the intensity of the electrochemical signal andlead to ultrasensitive bioassays (Hansen et al., 2006; Das etal., 2006; Yu et al., 2006). For example, Wang et al. havedeveloped a highly sensitive bioelectronic protocol for detect-

∗ Corresponding author. Fax: +86 25 83594976.E-mail address: jjzhu@nju.edu.cn (J.-J. Zhu).

ing proteins based on CNTs as carriers for numerous enzymetags (Wang et al., 2003). Further sensitivity enhancement wasachieved by a layer-by-layer (LbL) assembly of multilayerenzymes films on the CNTs template (Munge et al., 2005). Thecoupling of CNTs carriers and protein multilayer architectureswere shown to maximize the ratio of enzyme tags per bindingevent and hence to offer a remarkably high amplification fac-tor. Among the various NPs, gold nanoparticles (GNPs) havebeen recognized as a versatile and efficient label for the conju-gation of biomolecules due to their rapid and simple chemicalsynthesis, a narrow size distribution, and efficient coating bythiols or other bioligands (Katz et al., 2004; Merkocui et al.,2005; Pumera et al., 2005; Zhang et al., 2002; Schneider etal., 2000a, 2000b). Recently, a novel double-codified nanola-bel based on GNPs modified with horseradish peroxidase (HRP)conjugated antibody for enhanced immunoanalysis was reported(Ambrosi et al., 2007). Yet, amplified transduction of biologi-cal recognition events remains a major challenge to electricalbioassays.

Another long-standing goal in electrochemical immunosen-sors is stability and activity of the immobilized biocomponentson solid support. Taking account of the advantages of GNPs

0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2008.01.034

Biosensors and Bioelectronics 24 (2008) 35–40

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

A chemiluminescent immunosensor based on antibody immobilized carboxylicresin beads coupled with micro-bubble accelerated immunoreaction for fastflow-injection immunoassay

a a b,∗ a, H a,∗

), Dep

columred bap thform

lex. Te-en

ed. Thcolu

ient officienoaffit, and

Zhanjun Yang , Zhifeng Fu , Feng Yan , Hong Liua Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of Chinab Jiangsu Institute of Cancer Prevention and Cure, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:Received 25 November 2007Received in revised form 18 February 2008Accepted 11 March 2008Available online 18 March 2008

Keywords:Flow-injection analysisCarboxylic resinChemiluminescenceImmunoassayMicro-bubble�-Fetoprotein

a b s t r a c t

A novel immunoaffinityimmunoassay was prepacould fast recognize and trantigen, which was firstlya sandwich immunocompreaction to produce enzymimmunoassay was proposeration of immunoaffinitywith a correlation coefficintra- and inter-assay coeage stability of the immunflexible, sensitive, low-cos

1. Introduction

Flow-injection immunoassay (FIIA) is one of the most usefuldetection techniques for improving cumbersome, time-consuming,and labor-intensive traditional immunoassays (Pollema et al., 1992;Gunaratna et al., 1993). It has been widely applied to many fieldsincluding environmental monitoring, food safety, pharmaceuticalanalysis, and clinical diagnosis (Fu et al., 2007, 2008; Wu et al.,2007) due to its small sample-cost, reduced sample handling step,acceptable reusability, good reproducibility, less time consuming,and easy automation for high sample throughout. This techniquecan be performed in both homogeneous and heterogeneous sys-tems. The latter usually uses a solid support to immobilize eitherthe antibody or antigen, thus permits the separation of free portionfrom the bound immunocomplex. The most commonly used sup-ports include bead materials, such as silica, agarose, sepharose andpolystyrene, and membranes (Liu et al., 1991; Morais et al., 1999).They have been extensively used for preparation of immunoaffin-ity columns or immunosensors for development of flow-injectionimmunoassay system. This work uses for the first time carboxylicresin beads to prepare a novel immunoaffinity column. This mate-

∗ Corresponding authors. Tel.: +86 25 83593593; fax: +86 25 83593593.E-mail addresses: yanfeng2007@sohu.com (F. Yan), hxju@nju.edu.cn (H. Ju).

0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2008.03.007

uangxian Juartment of Chemistry, Nanjing University, Nanjing 210093, PR China

n used as an immunosensor for flow-injection chemiluminescent (CL)y immobilizing antibody on carboxylic resin beads. The immunosensore immunocomplex of horseradish peroxidase (HRP)-labeled antibody anded with a micro-bubble accelerated pre-incubation process, to producehe HRP introduced in the immunoaffinity column could catalyze the CLhanced emission. With �-fetoprotein (AFP) as a mode, a flow-injection CLe whole assay for one sample, including the pre-incubation and the regen-

mn, could be performed within 16 min. The linear range was 1.0–80 ng/mlf 0.998 and a detection limit of 0.1 ng/ml at a signal/noise ratio of 3. Thets of variation at 20 ng/ml AFP were 1.2% and 8.5%, respectively. The stor-

nity column and the accuracy for sample detection were acceptable. Thisrapid method is valuable for clinical immunoassay.

© 2008 Elsevier B.V. All rights reserved.

rial is low-cost and robust with excellent rigidity and mechanicalproperty, and can immobilize much more biomolecules than usualmembrane materials due to its large surface-to-volume ratio. Thehigh immobilization capability can provide the immunoaffinity col-

umn with good sensitivity for flow-injection chemiluminescent(CL) immunoassay.

In FIIA, the incubation time is a bottleneck to the improve-ment of assay speed. This process is usually controlled by masstransport of immunoreagents and kinetics of immunoreaction. Thelatter can be accelerated by a heating equipment. However, itincreases the cost and complexity of manipulation. Furthermorethe increased temperature is limited due to the denaturalizationof proteins at high temperature. Thus antibody–antigen binding isgenerally limited by mass transport rather than by binding kinet-ics (i.e., recognition rate) (Stenberg et al., 1986; Nygren et al.,1987; Myszka et al., 1997), and accelerating the mass transportof immunoreagents has attracted considerable interest for short-ening the incubation time of immunoreaction and improving thesample throughput of immunoassay. Various approaches have beenused to accelerate the mass transport of the antigen and antibody,such as electric-field-driven method (Ewalt et al., 2001), magneticstirring (Tanaka and Imagawa, 2005), magnetic field combinedwith superparamagnetic labels (Driskell et al., 2007), low-powermicrowave radiation (Previte et al., 2006), and capture substraterotation (Luxton et al., 2004). They also need complex or espe-

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Available online at www.sciencedirect.com

Biosensors and Bioelectronics 23 (2007) 682–687

Direct electrochemistry of lactate dehydrogenaseimmobilized on silica sol–gel modified

gold electrode and its application

Junwei Di a,b, Jiongjia Cheng a, Quan Xu a, Huie Zheng a, Jingyue Zhuang a,Yongbo Sun a, Keyu Wang c, Xiangyin Mo c, Shuping Bi a,∗

a School of Chemistry & Chemical Engineering, State Key Laboratory of Coordination Chemistry of China & KayLaboratory of MOE for Life Science, Nanjing University, Nanjing 210093, China

b Department of Chemistry, Suzhou University, Suzhou 215006, Chinac Materials Science Key Laboratory, Nanjing Normal

University, Nanjing 210097, China

Received 8 April 2007; received in revised form 6 July 2007; accepted 3 August 2007Available online 10 August 2007

bstract

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol–gel film on gold electrode was investigated, and an obviousathodic peak at about −200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversiblelectrode process involving a one electron transfer reaction with the charge-transfer coefficient (α) of 0.79 and the apparent heterogeneous electronransfer rate constant (Ks) of 3.2 s−1. The activated voltammetric response and decreased charge-transfer resistance of Ru(NH3)6

2+/3+ on the LDH-odified electrode provided further evidence. The surface morphologies of silica sol–gel and the LDH embedded in silica sol–gel film were

haracterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated.he calibration range of lactic acid was from 2.0 × 10−6 to 3.0 × 10−5 mol L−1 and the detection limit was 8.0 × 10−7 mol L−1 at a signal-to-noise

atio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimentalesults of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modifiedlectrode can be applied as a biomarker to study the pollution effect in the environment.

2007 Elsevier B.V. All rights reserved.

actic a

2Idvoepm

eywords: Lactate dehydrogenase (LDH); Direct electrochemistry; Sol–gel; L

. Introduction

It is well-known that enzyme plays a considerably impor-ant role in the biological systems. Moreover, the direct electronransfer between enzyme and electrode can provide a good

odel close to the original biological redox process for mecha-istic and thermodynamic studies on the direct electrochemistryf enzyme. In application, the achievements of this field have

een used to develop the third-generation biosensors and havettracted more and more attentions because of their simplicitynd selectivity without using redox mediators (Patolsky et al.,

∗ Corresponding author. Tel.: +86 25 86205840; fax: +86 25 83317761.E-mail address: bisp@nju.edu.cn (S. Bi).

(tep

nt

956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2007.08.002

cid; Resorcinol

004; Willner, 2002; Willner and Katz, 2000; Xiao et al., 2003).t was also demonstrated that the effective ways to obtain theirect electron transfer of enzymes were to incorporate them intoarious films modified on the electrode surfaces with the additionf different promoters (Di et al., 2006). For example, the directlectrochemistry was obtained by immobilizing cytochrome c inoly aniline nano-networks (Zhang et al., 2006) or incorporatingicroperoxidase onto carbon nanotubes modified Pt electrode

Wang et al., 2005). In our previous works, the direct electronransfer of catalase immobilized on silica sol–gel modified goldlectrode (Di et al., 2006) and adding Au nanoparticles as a

romoter (Di et al., 2005) were also reported.

Lactate dehydrogenase (LDH) (EC 1.1.1.27) is a nicoti-amide adenine dinucleotide NAD(H)-dependent oxidoreduc-ase that catalyzes the conversion from pyruvic acid to lactic

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Biosensors and Bioelectronics 22 (2006) 649–655

Nanocrystalline diamond modified gold electrodefor glucose biosensing

Wei Zhao, Jing-Juan Xu ∗, Qing-Qing Qiu, Hong-Yuan ChenThe Key Lab of Analytical Chemistry for Life Science (MOE), Department of Chemistry,

Nanjing University, Nanjing 210093, PR China

Received 23 November 2005; received in revised form 19 January 2006; accepted 30 January 2006Available online 23 March 2006

bstract

Boron-doped diamond has drawn much attention in electrochemical sensors. However there are few reports on non-doped diamond because of itseak conductivity. Here, we reported a glucose biosensor based on electrochemical pretreatment of non-doped nanocrystalline diamond (N-NCD)odified gold electrode for the selective detection of glucose. N-NCD was coated on gold electrode and glucose oxidase (GOx) was immobilized

nto the surfaces of N-NCD by forming amide linkages between enzyme amine residues and carboxylic acid groups on N-NCD. The anodicretreatment of N-NCD modified electrode not only promoted the electron transfer rate in the N-NCD thin film, but also resulted in a dramaticmprovement in the reduction of the dissolved oxygen. This performance could be used to detect glucose at negative potential through monitoring

he current change of oxygen reduction. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence ofommon interferents, such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA). A wide linear calibration range from 10 �M to 15 mMnd a low detection limit of 5 �M were achieved for the detection of glucose.

2006 Elsevier B.V. All rights reserved.

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eywords: Non-doped nanocrystalline diamond; Electrochemical pretreatment

. Introduction

Due to the various electron orbit characteristics and thenisotropy of carbon element, carbon materials contain char-cteristics of nearly all the matters on the earth, thus attractingntense attention of scientists and engineers. A large degreef carbon materials have been employed for the developmentf biosensors. Glassy carbon (GC), carbon paste, graphitend carbon fibres have been well established for many yearss electrode materials (Cosnier et al., 1999; Kulys, 1999;orozova et al., 2000; Netchiporouk et al., 1995). Theseaterials allow for the easy enzyme immobilization and

ossess reproducible electrochemical behavior. Recently, novelarbon materials, such as carbon fullerenes, carbon nanotubes

CNT) and diamond lead new directions in the design ofiosensors (Nednoor et al., 2004; Poh et al., 2004). They offerots of possibilities for the biosensor construction, for example,

∗ Corresponding author. Fax: +86 25 83594862.E-mail address: xujj@nju.edu.cn (J.-J. Xu).

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956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2006.01.026

ose oxidase; Biosensor

lectron mediation, nano-biosensor fabrication and enzymentrapment.

Diamond-based materials, because of their unique prop-rties such as biocompatibility, chemical inertness, chemicaltability, wide potential window and optical transparency prop-rties, which are superior to the traditional carbon materials,.g. GC and highly oriented pyrolytic graphite (HOPG), havettracted increasing attention in biosensor and biochip appli-ations (Haymond et al., 2002; Lora Huang and Chang, 2004;eng et al., 2005). The functionalized surfaces of diamond are

xcellent for the self-assembly of proteins. Hamers and cowork-rs employed nanocrystalline diamond thin films for DNAybridization via photochemical modification of diamond sur-aces, and showed that both as semiconductors, nanocrystallineiamond has superior properties over the crystalline silicon forhe DNA hybridization (Yang et al., 2002; Strother et al., 2002,003). Proteins such as cytochrome c, antibodies and enzymes

lso have been immobilized on diamond films for biosensorpplications (Lora Huang and Chang, 2004; Huang et al., 2004;u et al., 2004). Since for electrochemical applications, the dia-ond thin film must possess a good conductivity, most work

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Biosensors and Bioelectronics 22 (2006) 768–773

Short communication

Direct electrochemistry of horseradish peroxidase based on biocompatiblecarboxymethyl chitosan–gold nanoparticle nanocomposite

Qin Xu a, Chun Mao b, Ni-Na Liu a, Jun-Jie Zhu a,∗, Jian Sheng b

a School of Chemistry and Chemical Engineering, Laboratory of Life Analytical Chemistry, Nanjing University, Nanjing 210093, PR Chinab Research Center of Surface and Interface Chemical and Engineering Technology, Nanjing University, Nanjing 210093, PR China

Received 7 November 2005; received in revised form 8 January 2006; accepted 8 February 2006Available online 5 April 2006

bstract

The nanocomposite composed of carboxymethyl chitosan (CMCS) and gold nanoparticles was successfully prepared by a novel and in situ process.t was characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectrophotometer (FTIR). The nanocompositeas hydrophilic even in neutral solutions, stable and inherited the properties of the AuNPs and CMCS, which make it biocompatible for enzymes

mmobilization. HRP, as a model enzyme, was immobilized on the silica sol–gel matrix containing the nanocomposite to construct a novel H2O2

iosensor. The direct electron transfer of HRP was achieved and investigated. The biosensor exhibited a fast amperometric response (5 s), a goodinear response over a wide range of concentrations from 5.0 × 10−6 to 1.4 × 10−3 M, and a low detection limit of 4.01 × 10−7 M. The apparent

ichaelis–Menten constant (KappM ) for the biosensor was 5.7 × 10−4 M. Good stability and sensitivity were assessed for the biosensor.

2006 Elsevier B.V. All rights reserved.

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eywords: Nanocomposite; Biocompatibility; Direct electrochemistry

. Introduction

Direct electron transfer between an electrode and a redoxnzyme is very important for the fundamental studies and theonstruction of biosensors. However, because of its unfavorablerientation on the electrode surface or the adsorption of impu-ities to make it denature, the enzyme often exhibits sluggishlectron transfer at conventional electrodes. Some efforts haveeen made to enhance its electron transfer. In some cases, media-ors have been used to facilitate the electron transfers of enzymesXu et al., 2003), but the low molecular weight soluble medi-tor can leach out from the electrode and be lost into the bulkolution. This may lead to a significant signal loss and affect thetability of the biosensor. The other means is the immobilizationf the enzymes on different substrates (Xiao et al., 2000; Jia etl., 2002; Dai et al., 2004; Ferapontova, 2004; Liu et al., 2005).

Gold nanoparticles (AuNPs) have attracted much more atten-ion in recent years especially in biological and chemicalesearches because of their biocompatibility (Daniel and Astruc,

∗ Corresponding author. Tel.: +86 25 83594976; fax: +86 25 83594976.E-mail address: jjzhu@netra.nju.edu.cn (J.-J. Zhu).

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956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2006.02.010

004). They can provide an environment that is similar to theature for enzyme immobilization (Crumbliss et al., 1992, 1993;rown et al., 1996). On the other hand, the nanoparticles canct as tiny conduction centers to facilitate electron transferFerapontova et al., 2001; Shipway et al., 2000; Bharathi et al.,001). In most of the cases, the AuNPs were immobilized on thelectrode by assembling (Lei et al., 2003) or physically encap-ulating into porous sol–gel network (Bharathi and Lev, 1998).owever, Natan (Musick et al., 1997) claimed that the step-by-

tep assembly process was time-consuming and the possibilityf contaminating gold colloid due to improper rinsing stages. Onhe other hand, the nanoparticles encapsulated were always inoose association or physical contact with the electrode and theyould show some leakage because of their nano-sized dimen-

ions.It is well-known that the polymer–nanoparticles composite

ossess the interesting electrical, optical and magnetic propertiesuperior to those of the parent polymer and nanoparticles (Grocet al., 1998; Huynh et al., 1999; Guo et al., 2000). Metal nanopar-

icles dispersed in polymeric matrices display the increasedtability, improved processability, recyclability and solubility invariety of solvents (Shenhar et al., 2005). These nanocompos-

tes can be used for the selective detection of chemical vapors