Inorganica Chimica Acta 393 (2012) 142–153

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Inorganica Chimica Acta

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Review

Gold nanoparticles for diagnostic sensing and therapy

Feng Lu a,b, Tennyson L. Doane a, Jun-Jie Zhu b,⇑, Clemens Burda a,⇑a Center for Chemical Dynamics and Nanomaterials Research, Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USAb State Key Lab. of Analytical Chemistry for Life Science, 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:Available online 26 June 2012

Metals in Medicine Special Issue

Keywords:Gold nanoparticlesSensorBiosensorDrug deliveryTherapy

Feng Lu obtained his B.S.doctorate under the directwith Dr. Clemens Burda. H

Tennyson Doane obtainpursuing his doctorate unspectroscopy, transport, a

0020-1693/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ica.2012.05.038

⇑ Corresponding authors. Tel.: +1 216 368 5313; faE-mail addresses: jjzhu@nju.edu.cn (J.-J. Zhu), burd

a b s t r a c t

Gold nanoparticles (Au NPs) provide a unique platform for biomedical applications. Au NP-based sensorshave been widely employed to detect many different chemicals and disease-related biomolecules. Recentresearch on drug delivery and therapy with Au NPs has also indicated attractive and promising prospectsfor future applications. In this invited review we will provide an overview of the use of Au NPs for diag-nostic sensing and therapy applications.

� 2012 Elsevier B.V. All rights reserved.

in the Department for Intensive Instruction of Kuang Yaming Honors School from Nanjing University. Now he is pursuing hision of Professor Jun-jie Zhu at Nanjing University, and currently he is a visiting student at Case Western Reserve Universityis research interests include magnetic nanomaterials, bionanomaterials and multi-functional nanocomposites.

ed his B.S. from Eastern Nazarene College under the guidance of Dr. Lowell Hall and Dr. Timothy Wooster. Currently, he isder the direction of Dr. Clemens Burda at Case Western Reserve University. His interests include bionanomaterials, ultrafastnd surface properties of nanomaterials. He resides in Cleveland, OH with his wife Jennie who is an oncology nurse.

ll rights reserved.

x: +1 216 368 3006 (C. Burda), tel./fax: +86 25 8359 7204 (J.-J. Zhu).a@case.edu (C. Burda).

Jun-Jie Zhu is Professor of Chemistry at Nanjing University, China. He was born in Shanghai, China, in 1960 and received his B.S. (1984) and Ph.D.(1993) degrees from the Department of Chemistry, Nanjing University, under the supervision of Professor Hong Gao. In 1993, he began his academiccareer at Nanjing University. Between 1998 and 1999, he was a postdoctoral fellow with Professor Aharon Gedanken in BarIlan University, Israel. Hiscurrent research interests are in the fields of materials and biological analytical chemistry, mainly focused on the study of biological analyticalchemistry including bioelectrochemistry, nanoelectrochemistry, and the fabrication of biosensors and nanosensors. The syntheses of nanoparticlesusing sonochemistry, sonoelectrochemistry, and microwave chemistry method are also currently being investigated.

Clemens Burda obtained his Ph.D. from the University of Basel, Switzerland. He was a postdoctoral fellow at the Georgia Institute of Technology withProf. El-Sayed. Currently he holds faculty positions in Chemistry and Materials Science and Engineering at Case Western Reserve University, inCleveland, Ohio. His research focus is on nanomaterials chemistry and laser spectroscopy. He is an author of several book chapters, and leadinvestigator of over a hundred publications in the field of nanoscience. He is a founder and co-director of the Center for Chemical Dynamics at CaseWestern Reserve University.

F. Lu et al. / Inorganica Chimica Acta 393 (2012) 142–153 143

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432. Gold nanoparticle-based sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

2.1. Electrochemical and electrochemiluminescence sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1442.2. Colorimetric sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462.3. Fluorescent sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1482.4. Other sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

3. Gold nanoparticle-based drug delivery and therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

3.1. Gold nanoparticles as drug delivery vehicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1493.2. Gold nanoparticles for improved photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503.3. Gold nanoparticles for enhanced photothermal therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

4. Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

1. Introduction

Nanosized materials have been a source of particular interestfor scientists over the course of the past two decades [1]. Goldnanoparticles (Au NPs), with their distinct physical and chemicalproperties [2], are one of the most widely investigated, whichcan be easily seen from the explosion of Au NPs-related publica-tions these years. To date, a large number of methods has alreadybeen developed to synthesize gold nanoparticles [3–5]. Amongthem, there are two classic ways which are widely employed.The first classic method was developed by Turkevich et al. in1951 [6]. In this system, Au NPs were prepared by reducing HAuCl4

with sodium citrate in boiling water. The size can be tuned form 16to 150 nm via changing the gold-to-citrate ratio [7]. These water-soluble and negatively charged Au NPs are often used to assemblenano-composites [8,9] and nano-interfaces [10] via electrostaticinteractions. The second popular method was developed by Brustet al. in 1994 [11]. AuCl4

� was reduced by sodium borohydridein the presence of alkane thiols in a two phase (water–toluene)system. These Au NPs are quite stable when capped with alkanethiols and can be thoroughly dried and redispersed in organicsolvents. The most attractive part is that, unlike citrate-Au NPs,

the alkane thiol stabilized Au NPs can be further functionalizedby ligand exchange directly without any aggregation. Since thesepioneering works, the synthesis of Au NPs has made great progresswith the rapid development of nanoscience. In addition tospherical particles, many other Au nanostructures can be achievedwith tunable size, such as nanorods [12,13], nanoshells [14], nano-cages [15] and nanocubes [16]. Today, a greater emphasis is placedon protectors and functional ligands because surface functionalityis considered to be crucial for potential applications [5,17].

Compared to other nanomaterials, Au NPs are chemically stable,non-toxic and easy to functionalize. DNA [18–20], enzymes [9,21,22], antibodies [10,23] and some functional polymers [24,25] canbe easily conjugated with Au NPs without affecting their activitiesin most cases [26]. Different functionalized Au NPs can be designedand assembled as required. Thus, Au NPs provide a promising plat-form for various types of applications in bio-nanotechnology. Insensing and diagnosis, biomolecule conjugated Au NPs providenew assays with increased sensitivity and selectivity. With highsurface area, Au NPs work efficiently as drug delivery vehicles,and these drugs can be delivered to specific areas if targeting li-gands are utilized [27]. Moreover, some Au nanostructures haveinherent photothermal properties which can be used for direct ther-

Fig. 1. Schematic of the different strategies used for the integration of Au NPs into DNA sensing systems: (A) dissolving of Au NPs using a HBr/Br2 mixture followed by Au(III)ion detection; (B) direct detection of Au NPs anchored onto the surface of the genosensor; (C) conductometric detection, (D) enhancement with silver or gold followed bydetection; (E) Au NPs as carriers of other Au NPs; (F) Au NPs as carriers of other electroactive labels. Reprinted with permission from Ref. [54].

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apy [28]. With these advantages in diagnostic sensing and therapy,Au NPs have been established as a valuable tool within the realm oflife science. In this review we will cover a selection of recent devel-oped Au NP-based sensors and provide an overview of Au NPs indrug delivery systems and therapeutic applications.

2. Gold nanoparticle-based sensors

The design of a sensor mainly focuses on two aspects: sensitivityand selectivity, both of which must be maximized in the develop-ment of an efficient sensor. Despite significant progress, however,establishing how to sensitively detect an analyte in a complexenvironment remains challenging. With an increased ability toaccurately detect viruses, cancer cells, and other biomolecules inblood samples, many diseases could potentially be diagnosed earlyor even prevented. Therefore, developing new analytical methods isboth necessary and urgent. As a kind of novel material, nanoparti-cles show new strategies to enhance signal response and may helpdeveloping novel sensors for biomedical applications. Au NPs havebeen unique among them, providing a functional platform to helpwith complex biomedical problems [26].

It is well known that sensors should contain at least two basicelements: detectable signal and the change of signal by the analyte.Due to the versatility of Au NPs, various signals can be utilized tobuild up sensors which utilize electrochemical response [21,29–31], photoluminescence [32–35], UV–vis absorption [36], andRaman scattering [37]. Electrochemistry, photoluminescence andcolorimetry are the most widely used techniques in sensing dueto their good sensitivity, relatively low cost, and common detectionequipment. The use of metallic Au NPs provides both excellent con-ductivity and surface plasmon resonance (SPR) absorbance due toits small size, allowing for facile electrochemical and colometricexperiments. The interactions between some biomolecules (as inantibody-antigen pairs, biotin and avidin, complementary DNAchains, etc.) are always very effective and specific [10,35,38], evenin vivo. Therefore, after the combination of features of biomoleculesand the properties of Au NPs, great selectivity and sensitivity ofdetection can be obtained.

2.1. Electrochemical and electrochemiluminescence sensors

With the features of high stability, good biocompatibility, excel-lent conductivity and large surface to volume ratio [5], Au NPs pro-vide a great interface for biological recognition and electronictransduction which makes them become a common part in bio-electrochemistry [39]. They can be either assembled on the surfaceof an electrode as a special substrate or assembled with electroac-tive species to be a nanoprobe. To date, Au NPs are widely appliedin electrochemical sensors, biosensors and electro-catalysis.

Direct electron transfer (DET) between electrode and redox pro-teins has become an important field for both biologists and electro-chemists after its initial discovery in 1977 by Eddowes et al. [40]and Yeh et al. [41]. Biosensors based on DET are called third gener-ation biosensors [42]. These mediator-free biosensors can reduceinterfering reactions and make the assaying highly selective [43].However, electron transfer between proteins and electrodes israther slow. This is due to the fact that (a) the electroactive centeris deeply embedded in the protein and (b) the protein adsorbed onthe electrode may have an unfavorable orientation which could in-crease the electron transduction distance [44,45]. Therefore, AuNPs are employed as a substrate to immobilize these proteins orenzymes to provide a suitable microenvironment on the electrodeand lend more freedom of orientation to enhance their DET behav-ior [5]. In addition to improving the orientation of substrates onelectrode surfaces, Au NPs also can conduct electrons from the pro-tein and transport them to the supporting electrode quite effi-ciently [46]. Dong and co-workers developed a novel horseradishperoxidase (HRP) biosensor by assembling Au NPs onto a thiol-rich3-dimensional sol–gel network supported by a gold electrode [21].First, a 3-dimensional silica gel was assembled by immersing thegold electrode in a hydrolyzed (3-mercaptopropyl)-trimethoxysi-lane sol–gel, and then Au NPs were absorbed into the networkvia covalent Au–S bonding to the matrix. Once the nano-frame-work was established, HPR was immobilized on the surface of AuNPs. The prepared biosensor exhibited high sensitivity, long-termstability and fast amperometric responses (2.5 s) to H2O2. The lin-ear detection range was from 5.0 lM to 10.0 mM with a detectionlimit of 2.0 lM. Later, Wang’s group achieved quasi-reversible and

Fig. 2. (A) Secondary structure of the DNAzyme. (B) Cleavage of substrate strand by the enzyme strand in the presence of Pb2+. (C) Schematics of the colorimetric Pb2+ sensordesign. (D) Schematics of the new optimized colorimetric sensor design. Reprinted with permission from Ref. [77].

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Fig. 3. (A) Aggregation of oligonucleotide functionalized Au NPs with a DNA target. (B) Color change of the solution from red to blue after aggregation of Au NPs. (C)Monitoring of the aggregation process. Reprinted with permission from Ref. [74].

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direct electrochemistry of Cytochrome c in a similar gel-NPelectrode assembly [47]. Zhu’s group fabricated a series of AuNPs contained in hybrid materials, such as Au NPs-CaCO3 [48],Au NPs-C@SiO2 [49] and Au NPs-polystyrene@polyaniline [50], tofacilitate the direct electron transfer behavior. Layer-by-layer(LBL) methods based on electrostatic interaction are another wayto enhance direct electron transfer. Chen and co-workers assem-

bled films of toluidine (TB) and Au NPs on a gold electrode bythe LBL technique [51]. After HRP was immobilized in {TB/Au}n

films, the electrode showed a rapid and sensitive response toH2O2 with a detection limit of 70 nM. Recently, graphene wasintroduced into this system as well [52]. Huang et al. modified aglassy carbon electrode with a Au NP-graphene nanocompositefilm [53] to promote the electron transfer between catalase and

146 F. Lu et al. / Inorganica Chimica Acta 393 (2012) 142–153

the underlying electrode. These sensors exhibited high sensitivityto H2O2 with a detection limit of 50 nM.

Au NPs have also been demonstrated to be effective geno-sensorsand immunosensors. In most cases, sensing of specific gene se-quences is based on a hybridization event between the target DNAand Au NPs functionalized with the complementary DNA chain. AuNP-based electrochemical sensing of DNA has been reviewed byMerkoci and co-workers [54]. Fig. 1 shows their schematic of the sixmost important strategies to detect DNA with Au NPs, consisting of:Au(III) ion detection after dissolving Au NPs with acid, direct detec-tion of Au NPs anchored on the electrode surface, conductometricdetection with silver, detection followed by silver-on-gold enhance-ment, Au NPs as carriers for other Au NPs and electroactive labels.

Signal amplification by silver accumulation on Au NPs is a verysuccessful technique in electrochemical sensors [55]. Wang andcoworkers reported a novel silver-enhanced electrochemical strip-ping sensor for DNA with 83-fold enhancement of the signal re-sponse [56]. As shown in Fig. 1D, silver was precipitated on AuNPs after DNA hybridization, then dissolved in HNO3 andsubsequently measured by anodic stripping voltammetry with adetection limit in the femtomolar range. A sandwich type electronicDNA sensor with silver enhancement of conductivity was reportedby Mirkin’s group [57]. The sensor employed a mircoelectrode arraywith 20 lm gaps within which short oligonucleotides were immo-bilized. The target DNA and oligonucleotides functionalized Au NPswere then subsequently hybridized to the short oligonucleotides,and subequently silver was deposited onto the Au NPs to enhanceconductivity (correspond to Fig. 1C). By measuring the resistancevalues across the electrode gaps, a sensitivity of 500 femtomolarwas achieved with a point mutation selectivity factor of about100,000:1 [57].

Alternatively, instead of the hybridization between DNA chains,immunosensors are based on the hybridization between antibodiesand antigens. Some antigens can be the biomarkers for cancer orother diseases [58–60], so the development of ultrasensitive elec-trochemical immunosensors will play an important role in clinicaldiagnosis. Immunosensors for antigens can be designed with sim-ilar strategies as genosensors shown in Fig. 1 [9,61–64]. Since anantigen can be recognized by multiple different antibodies at thesame time [65], sandwich type structures (antibody1-antigen–antibody2) are most widely employed in immunosensors. Typi-cally, antibody-1 is immobilized on the electrode and antibody-2is conjugated with Au NPs. Using this sandwich type structure,Zhu et al. achieved an amplified immunosensor with a Au NP/C hy-brid material with a detection limit of 5.6 pg mL�1 [9].

Au NPs also play a special role in electrochemiluminescence(ECL) sensors. ECL signals of both molecules RuðbpyÞ2þ3 [66], lumi-nol [67], etc.) and nanocrystals (CdS [68], CdSe [69], etc.) can beamplified by Au NPs. Zhang and co-workers found that after apply-ing a dense monolayer of silica coated Au NPs on the surface ofCdSe-CdS quantum dots, the ECL signal was about 17 times higherthan with pure quantum dots alone [70]. Recently, the ECL behav-ior of fluorescent gold nanoclusters (quantum-sized Au NPs) wasreported by both Chen’s group [71] and Zhu’s group [72]. Interest-ingly, the ECL signal of gold nanoclusters also can be amplified byAu NPs. These examples illustrate how Au NPs can play an impor-tant role in electrochemistry-based sensors for a variety of chemi-cals, biomolecules and cells, with high detection sensitivity andselectivity. For a more detailed description of the synthesis andelectrochemical sensor applications of Au NPs, please refer to theexcellent review by Guo and Wang [5].

2.2. Colorimetric sensors

One of the most prominent features of Au NPs is their visible-light absorbance, a result of surface plasmon resonance (SPR) in-

duced by nanoscale dimensions which is not present in its atomicor bulk form. This unique optical property is generated by the col-lective oscillation of the conduction electrons at the nanoparticlesurface induced by an interacting electromagnetic field [73]. It isworth noting that the SPR band can be influenced not only by size,shape, temperature, and solvent but also by the distance betweenthe particles. When Au NPs are close to each other or to a differentkind of nanoparticle, the SPR band will significantly red-shift andthe color of the solution color may change from red to blue[74,75]. This phenomenon provides an opportunity for simple col-orimetric assays with Au NPs.

Colorimetric biosensors by using DNAzyme-directed assemblyof Au NPs have been fabricated by Liu and Lu [76–79], providinga highly sensitive and selective strategy to detect lead ions in leadcontaining paints. They chose DNA functionalized Au NPs as thecolorimetric reporter group in conjunction with a DNAzyme thatwas highly specific for Pb2+ for analyte recognition. The DNAzymesystem contains an enzyme strand (17E) and a substrate strand(17DS) as showed in Fig. 2A. In the presence of Pb2+, the enzymestrand catalyzes hydrolytic cleavage of the substrate strand(Fig. 2B). The design of this sensor with DNAzyme is shown inFig. 2C, where 13-nm-diameter Au NPs were capped with a specialsequence of DNA which can hybridize to the substrate. This causesAu NP aggregation and leads to a color change from red to blue. Inthe presence of Pb2+, however, the enzyme strand can catalyzehydrolytic cleavage of the substrate strand (SubAu), so that theaggregation is prevented and red color remains. This biosensor iscapable to detect Pb2+ between 100 nM and 4 lM with excellentselectivity, as other divalent metals tested could not induce a colorchange. Later, they optimized this biosensor by using ‘‘tail-to-tail’’nanoparticle alignment (Fig. 2D) and a larger (42 nm diameter)nanoparticle size [77]. The newly designed sensor can detect Pb2+

at ambient temperatures within 10 min. The authors further foundthat positive ions such as Cu2+ and UO2þ

2 can also be detected witha similar DNAzyme technique [80,81].

Besides heavy metal ions, Liu and Lu demonstrated a generaldesign of colorimetric biosensors with specific aptamers andDNA modified Au NPs to detect analytes, including adenosineand cocaine [82]. Au NPs were linked to an aptamer sequence con-taining DNA to form aggregates which disassembles when the ana-lyte was added. Adenosine could be detected in a range from 0.3 to2 mM, and cocaine was quantified in concentrations of 50–500 lM.Recently, Kim et al. reported a novel Au NP-based colorimetric apt-amer based sensor for highly sensitive and specific detection ofoxytetracycline (OTC). OTC could be readily discriminated fromthe other tetracyclines and the detection limit was lowered to25 nM [83]. Based on the catalytic reaction of acetylcholine ester-ase and the assembly of lipoic acid modified Au NPs, a label freesensor for organophosphate contained nerve agents and pesticideswas developed by Xie and co-workers with a detection limit in thepM range [84]. Lu et al. synthesized 1-(2-mercaptoethyl)-1,3,5-triazinane-2,4,6-trione (MTT) capped Au NPs by ligand-exchangeand employed them as a melamine assay in raw milk. In the pres-ence of melamine, MTT-stabilized Au NPs aggregated due to thehydrogen-bonding recognition between melamine and MTT. Thismethod provides a low concentration (2.5 ppb) detection observa-ble by the eye within 1 min [85].

Bio-analytes such as proteins, oligonucleotides, and cells are di-rectly related to certain diseases. Therefore, the assays of theseanalytes play an important role in diagnostics and are directly re-lated to human health. Early in 1997, Mirkin and co-workers dem-onstrated the ability of DNA functionalized Au NPs to establishcolorimetric sensors for specific oligonucleotides based on Au NPself assembly [74]. As shown in Fig. 3A, Au NPs were modified withtwo different mercaptoalkyl oligonucleotides, which are comple-mentary to both ends of the target oligonucleotide. Thus, in the

Fig. 4. Schematic illustration of FRET-based glucose sensor. Reprinted with permission from Ref. [98].

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Fig. 5. Schematics of Au NP-based sensor and its operation principle. Au, gold nanoparticle; F, fluorophore; S, sulfur atom. Reprinted with permission from Ref.[91].

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F. Lu et al. / Inorganica Chimica Acta 393 (2012) 142–153 147

presence of a target oligonucleotide, the mercaptoalkyl oligonucle-otides on the Au NPs start to hybridize with the target leading tothe diagnostic Au NP aggregation. Due to the aggregation of theAu NPs, the color of the solution changes from red to blue(Fig. 3B). Further investigation about the influence of size of theAu NPs showed that larger particles with reduced DNA surface cov-erage can lead to higher sensitivity and a larger detection range[86]. Recently, Zu et al. reported a new method to detect target oli-gonucleotides under extremely low salt concentrations which canprevent the formation of secondary structures in nucleic acid tar-gets and make the target sequences fully accessible. Au NPs herewere functionalized with morpholino oligonucleotides which al-lows the salt-independent binding of the Au NP probes to the tar-get oligonucleotides [87]. The sharp melting transitions of theassembly provide a discrimination of single-base imperfection,such as substitution, deletion and insertion [87]. In addition toDNA detection, proteins can also be analyzed with Au NP-basedcolorimetric sensors as well. Chang and co-workers demonstrated

a Au NP-based label free detection of thrombin in blood plasmadown to picomolar concentrations [88]. Fibrillar fibrin-Au NP con-jugate aggregation can be induced by the addition of thrombin intoa solution mixed with Au NPs and excess fibrinogen. The concen-tration of thrombin can be identified by simply measuring theabsorbance of the supernatants. This sensor is quite sensitive witha linear detection range of 0.1–10 pM, and was also demonstratedto be selective to other proteins and proteases. By using a newmechanism known as ‘‘hairpin aptamer sticky-end pairing-inducedAu NPs assembly’’, Wu et al. developed a colorimetric assay meth-od for proteins as well [89]. With immunoglobulin E (IgE) as ananalyte model, a wide semi-quantitative detection range of 1 pMto 10 nM can be achieved which is visible to the eye. In addition,the colorimetric detection of cancer cells has been reported byLiu et al. using an aptamer-nanoparticle strip biosensor [90]. Underoptimal conditions, this novel strip biosensor provided a lowdetection limit, with a 4000 Ramos cell sensitive to the eye andan 800 Ramos cell sensitive to a strip reader [90].

ig. 7. Structure of the Au NP carrier (covalent approach) and schematicf intracellular GSH-mediated surface monolayer exchange reaction whichleases the payload. Reprinted with permission from Ref.20].

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2.3. Fluorescent sensors

With the ability to quench molecular excited states, Au NPsfunction as effective photoluminescence (PL) quenchers in design-ing fluorescence-based sensors [91]. When the fluorophore is nearthe surface of the Au NP, fluorescence can be efficiently quenchedby energy transfer mechanisms. Typically, the mechanisms are di-vided into Förster resonance energy transfer (FRET) and nanometalsurface energy transfer (NSET). Further research shows that smallAu NPs (1.4 and 3 nm) have negligible localized surface plasmonresonance absorption and quench fluorescence mainly via a NSETmechanism, while larger Au NPs (15 and 80 nm) utilize a FRETmechanism [92,93]. The critical parameter for a fluorescent sensorusing Au NPs is the change in distance between the fluorophoreand Au NP, which can be accomplished by the biological recogni-tion event between the biomolecule modified Au NP and a givenfluorophore probe.

Chen et al. developed a turn-on FRET assay for Hg2+ with Au NPsand Rhodamine 6G [94]. In the presence of Hg2+, Rhodamine 6Gcould be separated from the surface of the Au NPs and the fluores-cence emission from the dye was recovered. The sensor showed agood linear range over 0.5–35.5 nM. An NSET-based Hg2+ sensorhas been established by Wu and co-workers by using DNA-conju-gated quantum dots (QDs) and Au NPs. DNA on the surface of QDsand Au NPs start to hybridize in the presence of Hg2+ due to the for-mation of Hg2+-complexes. As a result, the photoluminescence ofthe QDs was quenched by NSET providing a turn-off fluorescenceassay method [95]. QDs were first introduced into Au NP-basedFRET sensors in 2005 by Kim and co-workers to detect avidin[32]. After that, many assay methods have been developed withFRET between QDs and Au NPs to detect different analytes, Pb2+

[96], 2,4,6-trinitrotoluene [97], glucose [98], and type IV collage-nase [99]. In the same year, Li et al. employed upconversion-lumi-nescent nanoparticles into Au NP-based FRET sensors for thedetection of trace amounts of avidin [35]. These upconversionnanoparticles were excited with IR radiation and greatly reducedthe fluorescence from the biological samples (background),improving both the sensitivity and detection accuracy. Fig. 4 showsa scheme detailing how to fabricate a Au NP-based fluorescent sen-sor. Au NPs were modified with thiolated b-cyclodextrins (b-SH-CDs) and QDs were conjugated with concanavalin A (ConA). Afterthe specific combination of ConA and b-SH-CDs, fluorescence wasquenched by energy transfer between the QDs and Au NPs, whichwas recovered in the presence of glucose due to competition withb-SH-CDs for the binding sites of ConA [98].

The change of oligonucleotide conformation upon hybridizationevents provides a sensitive and selective fluorescent analyticalmethod to detect target specific oligonucleotide strands. Tyagiand Kramer developed a novel approach to recognize specificDNA strands in solution with hairpin shaped single strand DNAprobes (molecular beacons) based on fluorescence resonance en-ergy transfer in 1996 [100]. The probes possessed a stem-and-loopstructure, where the loop is complementary to the target and the

Fig. 6. Schematic of the design of Au NP-drug nanoconjugates. Adapted withpermission from Ref. [117].

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stem is formed by two complementary arm sequences on eitherside of the probe sequence. These two complementary arm se-quences can be functionalized with both a fluorescent moietyand a fluorescent quenching moiety on either side. In the presenceof target DNA, the loop part of the probe hybridizes with the targetforcing the arm sequences apart so that the fluorophore movesaway from the quencher and the fluorescence is recovered. Later,Dubertret and co-workers applied this technology for DNA detec-tion with the help of Au NPs as the designated quencher [33]. Byemploying Au NPs instead of the organic quencher (DABCYL),100-fold sensitivity was achieved and the ability to detect singleDNA mismatches was enhanced 8-fold. After this breakthrough,Maxwell et al. developed a new class of nanobiosensors witharch-like oligonucleotide molecules attached on the surface of AuNPs [91], similar to the principles employed in the molecular bea-con sensors shown in Fig. 5. Oligonucleotides labeled with a thiolgroup and a fluorophore were immobilized on Au NPs to form anarch-like structure. The fluorescence was significantly quenchedin this structure due to the short distance between the fluorophoreand quencher. Upon target binding, the constrained conformationopened, and the fluorophore diffused away from the quencher,leading to a fluorescence turn-on detection of specific DNA targets.Mirkin’s group developed another kind of nanoprobe, known as‘‘nano-flares’’, to detect mRNA and adenosine triphosphate (ATP)in living cells [101,102]. The nano-flare consisted of a Au NP corefunctionalized with oligonucleotides which were hybridized withshort dye-terminated fluorescent reporter sequences. In the pres-ence of analyte, the probe bound selectively to the target and thefluorescent reporters was released from the nanoparticle likeflares. These nano-flares show great advantages for intracellulardetection without any requirements for microinjection or auxiliarytransfection reagents, high resistance to enzymatic degradationand low toxicity [101].

2.4. Other sensors

In addition to these commonly used techniques, there are vari-ous other ways to fabricate sensors with Au NPs, including surfaceenhanced Raman scattering (SERS) [37,104], surface plasmon reso-nance scattering [23,104,105], bio-barcode assays [38,106] andchemiluminescence (CL) [107,108]. Ray and co-workers utilizedcysteine modified Au NPs as an efficient SERS probe to detect traceamount of trinitrotoluene (TNT). They demonstrated that TNT

Fig. 8. Targeting effect of EGF-Au NP-Pc 4 conjugates in brain tumor-bearing mice via fluorescence imaging. The white circle indicates the tumor position in the brain.Adapted with permission from Ref. [27].

Fig. 9. Fluorescence image of a tumor-bearing mouse 1 min after the injectionof Au NP-Pc 4 conjugates. Reprinted with permission from Ref.[24].

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could be detected down to pM levels with excellent selectivity overother nitro compounds and heavy metals [103]. Tian et al. reporteda shell coated-Au NPs based enhanced Raman spectroscopy ap-proach, where the Raman signal could be amplified when the AuNPs were coated with ultrathin silica or alumina shells. With thisnovel technique, mannoproteins at the wall of a yeast cell and pes-ticide residues on citrus fruits could be monitored by Raman spec-tra [36]. Ren’s group investigated so called ‘‘photon burst’’ behaviorafter laser beam excitation, which was due to both plasmon reso-nance scattering and the Brownian motion of Au NPs. With thistechnique, they developed an ultra-sensitive and highly selectiveassay platform for the detection of cancer biomarkers and DNA.The detection limit was about 1 fM for DNA and several hundredfM for several cancer biomarkers [105]. Mirkin and co-workersconstructed a bio-barcode-based DNA detection method withDNA conjugated Au NPs and magnetic microparticles with PCR-likesensitivity [38]. Using the catalytic activity of Au NPs and a lumi-nol-H2O2 based chemiluminescence system, Li and Qi developeda sensitive, label free, aptamer-based biosensor for thrombin witha detection limit of about 26 fM [107]. In addition, Lu et al. com-bined the optical properties of Au NPs with HPLC techniques tofabricate a novel post-column detection method for HPLC assaysof homocysteine [109].

3. Gold nanoparticle-based drug delivery and therapy

3.1. Gold nanoparticles as drug delivery vehicles

The ability to transport drugs to a diseased target tissue is cru-cial in medicine because targeted drug delivery is able to improvethe therapeutic efficacy (increased drug accumulation in target tis-sue) and minimize side effects (lowered drug accumulation inhealthy tissue) [110]. The unique chemical and physical propertiesof Au NPs provide promise for drug delivery applications. First, AuNPs are stable and non-toxic, so they do not degrade during thetransport and are safe to normal tissue. Second, functional or targetgroups can be easily attached to the surface of an Au NP using thiolbased covalent bonding approaches. Finally, the photo-physicalproperties can be utilized to remotely trigger the release of drugsfrom the NP surface [111]. Research into functionalized Au NPsas drug delivery vehicles has been heavily explored [112]. Herewe will focus on the aspects of gold nanoparticle-based drug deliv-ery, drug loading, targeted transport and controlled release.

Drugs can be loaded onto Au NPs by either non-covalent orcovalent approaches [113]. Non-covalent approaches are designedto load drugs without modification so that the therapeutic efficacyof the drug will be completely retained, while covalent conjugationrequires chemical modification of the drug to link it with Au NPswhich may cause unknown side effects or reduced therapeutic effi-cacy [114,115]. Conversely, covalent approaches provide the‘‘nanodrug’’ with satisfactory stability, and allow for a more selec-tive drug release from the particle [116].

Several examples of non-covalent drug delivery have been re-ported [26]. The Burda group functionalized Au NPs with polyeth-ylene glycol (PEG) to provide an amphiphilic environment for the

non-covalent adsorption of silicon phthalocyanine 4 (Pc 4, a hydro-phobic photodynamic drug) [24,117]. The design of the drug deliv-ery vehicle and the structure of Pc 4 are shown in Fig. 6. Thesewater-soluble nanoconjugates have long circulation lifetimes inthe blood and can deliver hydrophobic drugs efficiently and deeplyinto the tumor by passive accumulation [118]. Rotello et al. utilizeda ‘‘hydrophobic pocket’’ in the monolayer of Au NP to entraphydrophobic dyes or drugs non-covalently [113]. In both of theseexamples, the non-covalently loaded hydrophobic drugs usuallydiffuse when the nanoparticles are in a lipophilic environment(for example, cell membranes). Interestingly, Rotello’s group alsodemonstrated that drugs or DNA covalently loaded on Au NPscan be released by glutathione (GSH) through molecular exchange[119,120]. Fig. 7 shows the design of the Au NP carrier with cova-lently linked payload and the release mechanism. GSH with itsthiol group was able to trigger the release of fluorescein isothiocy-anate (FITC) or doxorubicin through the ligand exchange reaction.As the intracellular level of GSH is much higher than extracellular,the Au NP carrier provides efficient intracellular release which isdemonstrated by in vitro experiments [120].

Au NPs with drug payload can be delivered to a given target bypassive accumulation as well as active targeting [121]. Defectivevascular architecture and poor lymphatic drainage in solid tumorsallow for an enhanced permeability and retention (EPR) effect forNPs to accumulate passively in cancerous tissues. In contrast, thetargeting of NPs can be improved using active targeting by modify-ing the surface of Au NPs with targeting moieties such as peptidesand proteins, which can recognize specific biomarkers in targetcells. Due to the overexpression of biomarkers in certain diseasessuch as cancer, targeted Au NPs can be accumulated in a targetedarea selectively over other tissue. For example, Cheng et al. usedan epidermal growth factor (EGF) peptide conjugated to Au NPsto deliver non-covalently adsorbed drug molecules into brain tu-mors with improved efficiency and selectivity (Fig. 8). A signifi-

Fig. 10. (A) Photograph of a mouse with a tumor during photothermal therapy.Thermographic images of nanocage injected (B–E) and saline injected (F–I)tumor bearing mice at different times (from top to bottom: 1, 3, 5, and 10 min).(J) Average tumor temperature as a function of exposure time. All scalebars are 1 cm. Reprinted with permission from Ref.[144].

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cantly enhanced drug accumulation was achieved in brain tumorsby employing the tumor-specific peptide [27].

Once the drug-Au NP conjugate reached its target, the release ofthe drug loaded from the NP can be triggered by changes in envi-ronment, such as pH, polarity, intracellular biomolecules, or ac-tively via light irradiation [112]. As described earlier [26,27],non-covalently adsorbed hydrophobic drugs diffuse from the AuNP into the cell when they come across a hydrophobic environ-ment (such as a cell membrane) without uptake of the NP carrier.Alternatively, NP drug carriers can take advantage of the low pHlevels in endosomes/lysosomes (pH 5–6) [122] and the tumor mi-cro-environment (pH 6.2–6.7) [123] through pH responsive linkersto release drugs in specific areas [124,125]. Wang et al. tethereddoxorubicin with PEG on Au NPs through an acid-labile hydrazone(Hyd) linker, and found that the synthesized DOX-Hyd@Au NPsachieved enhanced drug accumulation in MCF-7/ADR cancer cells[124]. Controlled release can also be governed by intracellularbiomolecules via exchange reaction or enzymatic degradation.Alexander et al. have confirmed the capacity of drug release fromDNA-capped AuNPs by target DNA [126]. In addition, externalstimuli (for example, light) can be used to induce drug release aswell. Rotello et al. demonstrated a UV light-controlled release pro-cess of caged anticancer drugs with a photocleavable linkage togold nanoparticles [127]. An et al. used Au NPs as a switch forUV light induced drug release from thermosensitive Au NPs-liposome composites [128]. Burdick and co-workers used near-infrared (NIR) light in vivo to trigger the release of drug frompolymer-gold nanorod (Au NR) composites [129]. Due to the localheating of the Au nanorod upon irradiation, the temperature of thepolymer composite will be higher than its glass transition temper-ature (Tg) under NIR exposure resulting in partial polymer meltingand controlled release is achieved. The authors also demonstrateda triggered and stepwise release of doxorubicin via multiple cyclesof NIR irradiation.

3.2. Gold nanoparticles for improved photodynamic therapy

In addition to Au NPs serving as unique vehicles to deliver drugsand DNA into cells for drug treatment and gene therapy [121],respectively, Au NPs can also be an integral part of therapeutic ac-tion. Generally, the role of the Au NP in therapy is primarilythrough photodynamic therapy (PDT) or photothermal therapy(PTT) [130]. PDT as a photo-induced noninvasive method to dam-age target cells showed great clinical potential and has been anattractive area of research within the last two decades. The PDTtreatment method is performed with light-sensitizing agents,termed photosensitizers, which can generate reactive oxygen spe-cies (ROS) to induce cell apoptosis and necrosis [118]. Unfortu-nately, photosensitizers with excellent PDT performance(phthalocyanines and porphyrins) are often hydrophobic so thata transport carrier, such as a PEGylated Au NP, is required to makethem water soluble for medical use [131]. In addition, PDT drugson Au NPs can be protected from recognition and clearance duringthe transport.

Russell and co-workers utilized thiol-functionalized phthalocy-anine (Pc) instead of alkane thiols to prepare Pc stabilized goldnanoparticles with the modified Burst method. These Pc-Au NPconjugates became soluble in polar and protic solvents (e.g. etha-nol) in which free Pc can’t be dissolved. Surprisingly, fluorescenceof Pc was not quenched by gold nanoparticles due to the long al-kane chain in Pc and singlet oxygen (1O2, excited-state singlet oxy-gen) quantum yield increased ca. 50% after immobilization the ongold nanoparticles [132]. Later, experiments both in vitro andin vivo were performed to demonstrate the efficiency and mecha-nism of PDT with this Pc-Au NP conjugates [131,133]. After irradi-ation of Au NP incubated HeLa cells, significant cell mortality was

achieved demonstrating the great PDT efficacy of Pc-Au NP conju-gates. In vivo PDT of melanoma with nano-conjugates intrave-

F. Lu et al. / Inorganica Chimica Acta 393 (2012) 142–153 151

nously injected was investigated in C57 mice. In the event that thetumor could not be completely cured, the growth of the tumor wasslowed by PDT treatment with Pc-Au NP conjugates, and TEM stud-ies suggested the mechanism of PDT treatment for tumor is viadestruction of the vasculature. All data showed that the employ-ment of Au NPs greatly improved the PDT efficacy in vivo but thepersistence of these nanoparticles in liver and spleen remained aserious problem to be solved for clinic applications. Recently, anti-bodies and PEG were introduced into Pc-Au NP conjugates toachieve targeting capabilities and improve stability in physiologi-cal environments [134]. Pc and PEG were immobilized on the sur-face of gold nanoparticles through thiol linkers, while antibodieswere attached to the terminal end of the PEG chains via amidebonds. After anti-HER2 antibodies were conjugated to Au NPs, agood selectivity in PDT treatment for SK-BR-3 cells was achieveddue to the overexpressed HER2 receptor.

Other than covalently linked drug-gold nanoparticle conjugates,this group has developed a non-covalent approach to deliver PDTdrug via PEGylated Au NPs (Fig. 6) and studied its PDT efficacy,delivery mechanism and pharmacokinetics systematically[24,27,117,118]. After Pc 4 was absorbed on Au NPs, the singletoxygen quantum yield decreased compared to free Pc 4 but theauthors found that this did not influence its PDT performance be-cause in vivo the Pc 4 is released from the particle in hydrophobicenvironments. Nearly complete mortality of HeLa cells could beachieved by subsequent irradiation with light at the intensity of1 J/cm2 after the drugs were released. In vivo experiments furtherindicated the capability of Au NPs to deliver PDT drugs, as fluores-cence monitoring of the drug revealed significant accumulation ofPc 4 in tumors as fast as 1 min post-injection (Fig. 9) [24]. In addi-tion, targeted delivery and specific uptake were accomplished byEGF targeting peptide functionalized Au NPs (Fig. 8) [27]. All thesestudies demonstrated the capability of Au NPs in PDT and providedoverall a valid approach for hydrophobic drug delivery.

Instead of simply delivering PDT drugs, recent research by theHwang group suggested that singlet oxygen can be formed by22 nm gold nanoparticles directly without organic photosensitiz-ers [135]. Although the singlet oxygen quantum yield of Au NPs(3.7%) is not nearly as appreciable as conventional dyes, the excel-lent stability and extraordinary high molar extinction coefficientscan easily compensate this shortcoming. The authors conclude thatthis unprecedented observation provides an opportunity for AuNPs to serve as a new generation of PDT reagents for cancertreatment.

3.3. Gold nanoparticles for enhanced photothermal therapy

Photothermal therapy (PTT) is a technique to damage cells bylocalized heat or hyperthermia generated by Au NPs after irradia-tion with light [130]. Gold nanostructures with a strong SPRabsorption can rapidly increase in temperature if irradiated withresonant light and thus have become a favorite energy converterfor PTT treatment [130]. Spherical Au NP based PTT was first dem-onstrated by Lin et al. in 2003 by using Au NPs with short laserpulses to generate highly localized cell damage in vitro [136]. Theyfound only �500 nanoparticles are required to inflict lethality to-wards each cell under their experiment conditions. In addition,El-sayed and co-workers provided fundamental research to opti-mize Au NP-based PTT, including calculations of heat generationefficiency with different particle size and shape [137] and studiedthe intensity of laser powers and threshold temperature needed toinduce cell death [138].

In order to achieve PTT in vivo, the absorption peak of the goldnanoparticles should be tuned to the biological transmission (ortherapeutic) window in the NIR range to avoid physiological fluidsand tissues absorption. As recently stated by El-Sayed and cowork-

ers [139], gold nanorods, nanoshells and nanocage are three mostfavored and widely employed nanostructure as PTT agents due totheir tunable absorption in NIR window. The same authors havedemonstrated the feasibility of NIR laser induced cellular hyper-thermia by gold nanorods in vivo. Both direct and intravenousadministration of PEGylated gold nanorods were performed andfound to be effective in inhibiting the growth of tumor in mice over13 days with NIR PTT [140]. Au nanoshells (gold coated silica NPs)were presented in 2003 by Halas and West as viable PTT agents.They demonstrated the excellent PTT capability of gold nanoshellsunder NIR light illumination both in vitro and in vivo [141]. Afterthe tumor had accumulated gold nanoshells, the target was ex-posed to a NIR laser (4 W/cm2) for 6 min, resulting in an averagetemperature increment of about 33 �C and subsequently inducedcellular damage. Tissues after PTT treatment underwent coagula-tion, with cell shrinkage and loss of nuclear staining indicating irre-versible thermal damage. In 2007, Xia and co-workers reported theuse of antibody conjugated Au nanocages to kill SK-BR-3 breast can-cer cells with NIR PTT treatment in vitro [142,143]. Later, they fur-ther studied the PTT performance of gold nanocages in vivo [144].Gold nanocages were found to accumulate in tumors by passive tar-geting, and after sufficient accumulation could be heated to over 50centigrade in one minute of treatment. In comparison, no obviouschanges were observed in the saline injected control (Fig. 10). Fur-thermore, metabolic activity of the tumor decreased by 70% after24 h treatment which strongly confirmed the NIR PTT capabilityof gold nanocages. In addition to these more common agents, hol-low gold nanospheres, nanocubes and other gold nanostructuresalso showed their ability in PTT treatment [145,146].

Recently, some groups have attempted to combine photother-mal therapy with photodynamic therapy by using PDT drugs con-jugated gold nanostructures [147–150]. Choi et al. immobilized anegatively charged photosensitizer Al(III) phthalocyanine chloridetetrasulfonic acid (AlPcS4) on PEGylated gold nanorods via electro-static interaction to prepare AuNR-AlPcS4 complex with satisfac-tory stability. Triggered release of AlPcS4 by heat generated fromgold nanorods with NIR light irradiation was observed. The thera-peutic efficacy was improved by this dual-modality photodynamicand photothermal therapy although PDT alone also showed effec-tive anticancer therapeutic effects in vivo [148]. Similar resultswere also observed by using indocyanine green conjugated AuNPs and Au NRs [149].

4. Summary and outlook

The versatility of Au nanoparticles has allowed researchers towidely investigate them for implementation in both diagnosticbiomedical sensing and therapy. The ease of surface engineeringtogether with sensitive responses to the surrounding environmentprovides Au NP-based sensors with superior sensitivity and selec-tivity. It is expected that the development of these biosensors willbe invaluable in the diagnosis of human diseases. Recent researchon Au NP-based therapy also shows great promise for new meth-odologies of treating difficult diseases. Here, we have shown thatAu NPs are excellent drug delivery vectors, and can play a criticalrole in both photodynamic therapy and photothermal therapy.Due to the many favorable inherent physical properties of AuNPs for biological applications, it is expected that they will remaina critical building block for sensors and therapeutics for years tocome.

Acknowledgement

The authors would like to appreciate the support of NationalNatural Science Foundation of China (NSFC 21020102038).

152 F. Lu et al. / Inorganica Chimica Acta 393 (2012) 142–153

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