Fluorescent Gold Nanoclusters: Recent Advances in Sensing and

Review pubs.acs.org/ac

Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging Li-Yi Chen, Chia-Wei Wang, Zhiqin Yuan, and Huan-Tsung Chang*

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Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan mercaptopropionic acid.7 The second category is from reduction of Au3+ in the presence of a ligand or template (protein) such as bovine serum albumin (BSA).8 The optical properties of biocompatible Au NCs are dependent on their size, surface ligand or template, and the surrounding medium, and thus they can be studied to develop sensitive and selective sensing and imaging systems for the detection of various analytes. The growing popularity of Au NCs in analytical applications has been realized in these few years. Several excellent review papers dealing with Au NCs from the viewpoint of analytical chemistry have been reported to highlight their potential for the analysis of environmental and biological samples.1,9 This review focuses on recent advances in Au NCs based sensing and imaging systems between 2012 and 2014. Current challenges and future prospects of Au NCs for fundamental studies and analytical applications will be provided.

CONTENTS

Synthesis Chemical Reduction Photoreduction Chemical Etching Optical Properties Size Effect Ligand Effect Structure and Charge Effects Sensing Heavy Metal Ions Inorganic Anions Small Biomolecules Proteins Imaging In Vitro In Vivo Conclusions and Outlooks Author Information Corresponding Author Notes Biographies Acknowledgments References

216 217 218 218 218 218 218 219 219 219 220 221 222 223 223 225 226 227 227 227 227 227 227

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SYNTHESIS Important parameters for controlling the size, structure, oxidation state, and surface properties of Au NCs and thus their optical properties include the species and concentration of chemicals (ligands) or templates, the concentration of Au3+, the species and concentration of reducing agents, pH of the solution, as well as reaction temperature and time. To prepare stable and highly fluorescent Au NCs, chemicals, peptides, polymers, and proteins that act as capping agents are required. Figure 1 displays various Au NCs that emit fluorescence in different spectral regions that can be prepared by selecting

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luorescent gold nanoclusters (Au NCs) or nanodots (NDs) with sizes smaller than 3 nm are a specific type of gold nanomaterials. In this review, Au NCs are used to represent fluorescent Au nanomaterials with sizes smaller than 3 nm. Unlike the most popular and well-known spherical, large gold nanoparticles, Au NCs do not exhibit surface plasmon resonance (SPR) absorption in the visible region but have fluorescence in the visible to near-infrared (NIR) region. With advantages of long lifetime, large Stokes shift, and biocompatibility, Au NCs have become interesting sensing and imaging materials.1−4 Although Au NCs prepared from Au3+ in the presence of small thiol compounds such as 2-phenylethanethiol (PhCH2CH2SH) have been reported over the past decade,5 their use for bioapplications have not been well recognized, mainly because of their low quantum yield (usually less than 1%), poor water dispersibility, photo and chemical instability, and difficulty for conjugation. In the past decade, many strategies for the preparation of stable, water dispersible, highly fluorescent, and biocompatible Au NCs have been reported.6−8 There are two major categories elucidating the recent advanced techniques for the preparation of Au NCs. The first category is through etching of larger sizes of nonfluorescent gold nanoparticles (Au NPs) by thiol compounds such as © 2014 American Chemical Society

Figure 1. Schematic illustration of synthetic strategies for fluorescent Au NCs and effect of ligands on their fluorescence. Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2015 Published: October 2, 2014 216

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Figure 2. Optical spectra for two represented Au NCs. (A) Curves a and b are for the UV−vis absorption spectra of RNase A and RNase A−Au NCs, respectively, and curve c is for the fluorescence spectrum of RNase A−Au NCs. Inset: fluorescent photographs for RNase A (blue) and RNase A−Au NCs (red). Reprinted with permission from Kong, Y.; Chen, J.; Gao, F.; Brydson, R.; Johnson, B.; Heath, G.; Zhang, Y.; Wu, L.; Zhou, D. Nanoscale 2013, 5, 1009−1017 (ref 27). Copyright 2013 Royal Society of Chemistry. (B) The fluorescence spectra and photographs of Au NCs prepared by using Al2O3 NPs as supports. Reprinted with permission from Chen, P.-C.; Yeh, T.-Y.; Ou, C.-M.; Shih, C.-C.; Chang, H.-T. Nanoscale 2013, 5, 4691−4695 (ref 37). Copyright 2013 Royal Society of Chemistry.

or carboxylic groups of PAMAM, PAMAM-stabilized Au NCs were prepared through a chemical reduction using NaBH4 as a reducing agent.18,19 By varying the molar ratio of PAMAM to Au ions from 1:1 to 1:15, different sizes of Au NCs, including Au5, Au8, Au13, Au23, and Au31, were prepared. The PAMAMstabilized Au NCs emit at wavelengths from the UV to near infrared (NIR) region, with QYs ranging from 10% to 70%. Biomolecules such as peptides and proteins have been proven as structure-defined scaffolds to induce the nucleation and growth of Au NCs. BSA is the most commonly used protein for the preparation of Au NCs.8 The cysteine and histidine residues in BSA can coordinate with Au3+ ions, while the tyrosine residues can reduce Au3+ ions to form Au NCs that are stabilized by BSA. To obtain strong reducing strength of the tyrosine residues, preparation of Au NCs in the presence of a protein is conducted in solution at a pH value >10.0 (above the pKa of tyrosine). To have strong reducing strength and capping capability, a protein at a high concentration (usually 0.62 mM) is required. Many other proteins such as lysozyme,20−22 transferrin-family proteins, 23,24 horseradish peroxidase (HRP),25 DNase I,26 and ribonuclease A (RNase A)27 have been used to prepare Au NCs, with QYs ranging from 4.3− 12.0%. For example, Figure 2A shows that RNase A−Au NCs fluoresce strongly at the NIR region (emission wavelength, 682 nm) when excited at 365 nm, with a QY of ∼12.0% and a Stokes shift of ∼210 nm.27 Usually, there are 20−25 Au atoms per protein template. By varying the molar ratio of protein/ Au3+, different sizes of Au NCs with various emission colors and QYs can be prepared. In addition, preparation of proteinstabilized Au NCs can be optimized by controlling solution pH and ionic strength and reaction temperature. Solution pH is important in controlling the reducing strength, protein conformation, and capping capability of the protein, and thus various sizes of Au NCs can be prepared at various pH values. Porcine pepsin with strong pH-dependent conformational states presents a good example for the preparation of various sizes of Au NCs.28 Pepsin-stabilized Au NCs with different sizes of Au5 (Au8), Au13, and Au25 were prepared at pH values of 9.0, 1.0, and 12.0, respectively.29 They have blue-, green-, and red colors of emission, respectively. Reaction temperature can be used to control the reaction rate and protein conformation and thus the size of Au NCs. Heating-assisted and microwaveassisted approaches have been applied to accelerate the

suitable capping ligands. Approaches based on chemical reduction and photoreduction have been applied to prepare Au NCs from Au3+ in the presence of capping agents in the absence/presence of reducing agents. Some proteins can act as reducing and capping agents, and thus no additional reducing agent is needed. Alternatively, etching of larger Au NPs (usually 2−4 nm) by thiol compounds in alkaline solution is useful for the preparation of Au NCs. Chemical Reduction. Stable Au NCs are commonly prepared through the reduction of Au3+ then Au+ to Au in the presence of reducing and capping agents. Thiol compounds are often used as a capping agent mainly because they can form strong Au−S bonding with Au atoms/ions. Common reducing agents used to prepare Au NCs in the presence of thiol compounds are sodium borohydride (NaBH4) and tetrakis(hydroxymethyl)phosphonium chloride (THPC). Stable glutathione-stabilized Au NCs (GSH−Au NCs) have been prepared from Au3+ in the presence of glutathione (GSH) using NaBH4 as a reducing agent.10 After purification and separation, various sizes of GSH−Au NCs that emit at the emission wavelengths from the visible to NIR region with the quantum yields (QYs) of 12.0), 11-MUA provides strong etching ability to etch the surface Au atoms and provide strong coordination ability to form stable 11-MUA−Au complexes on the surface of each Au core to stabilize it, yielding fluorescent 11-MUA capped Au NCs (11-MUA−Au NCs) with a QY of 3.1%.7 One feature of this approach is that the size of Au NCs and thus their optical properties can be controlled by using different thiol compounds. Alkanethiol-bound Au NCs prepared by using different chain lengths of alkanethiols as ligands have the emission wavelengths within the range of 501− 613 nm, with QYs ranging from 0.0062 to 3.1%. Furthermore, the emission wavelength of 11-MUA−Au NCs can be tuned from 525 to 456 nm by controlling the molar ratio of Ag+/Au3+ from 0 to 1.6.36 The blue shift is related to the changes in size and the content of Ag atoms or Ag+ ions in the as-prepared Au/ Ag NCs. Various sizes of Au NCs that emit different colors have also been prepared by using Al2O3 NPs as supports in the presence of HAuCl4 and 6-mercaptohexanol (Figure 2B).37 Penicillamine (PA) adsorbed on the surfaces of Al2O3 reduces Au3+ ions to Au+ ions that are further reduced to form small Au NPs by THPC. The as-formed Au NPs are further etched by 6mercaptohexanol under irradiation of blue light-emitting diode (LED). Stronger fluorescent Au NCs can be prepared much more rapidly under LED irradiation than in the day light (4 h vs 3 days). These fluorescent Au NCs exhibit a number of attractive optical properties: tunable fluorescent wavelength, long lifetime (>200 ns), and large Stokes shift (>100 nm). These properties provide the feasibility for sensing proteins in biological samples and metal ions in environmental samples.7,37−41 GSH-stabilized Au NCs (Au25SG18) can be further etched with octanethiol, leading to the formation of brightly redemitting Au23SG18.42 Didodecyldimethylammonium bromide

stabilized Au NPs can be etched with bidentates such as dihydrolipoic acid to prepare highly red-fluorescent Au NCs.43 Nonfluorescent Au NCs can be used to prepare highly fluorescent Au NCs with a QY of 5.4% when using GSH as an etching agent.44 In addition, polymer-stabilized NCs can also be used to prepare Au NCs through a ligand-induced etching process. For instance, polyethylenimine containing multivalent imine groups was used to replace the original capping agent dodecylamine and to etch the Au NPs, leading to the formation of Au8 NCs with a emission of 445 nm and a QY of 10−20%.45

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OPTICAL PROPERTIES Size Effect. Au NCs consist of several to hundreds of Au atoms with sizes commensurate to the Fermi wavelength of electrons.46 In this size regime, the strong quantum confinement of free electrons leads to the discrete electronic states and thus Au NCs exhibit molecule-like properties. Au NCs show dramatically different optical and chemical properties when compared to those of Au NPs with sizes greater than 3 nm.47,48 Au NCs do not possess apparent surface plasmon resonance (SPR) absorption but exhibit fluorescence at the wavelengths ranging from the visible to NIR region.49 Many studies have revealed that the optical properties of Au NCs highly depend on their sizes, structures, oxidation states, and surface ligands as well as environmental parameters such as temperature, pH, and ionic strength. Although the detail mechanism for the fluorescence of Au NCs is not completely understood yet, a basic model has been proposed.50 The free-electron theory is of particular importance to understand the fundamental optical properties of Au NCs, which works quite well in most aspects. The free electrons on the nanoparticle surface give rise to the polarization in an electronic field and the electron number determines the sizedependent plasmonic optics.51 However, when the free electron number decreases to a critical value where the nanoparticle size approaches the Fermi wavelength, the continuous band breaks up into discrete energy levels.52 The numeric size of the energy level spacing (Eδ) is a determining factor for the fluorescence of Au NCs. When using the thermal energy (ET that is equal to kBT, where kB is the Boltzmann constant) as a criterion, Au NCs can emit only when Eδ is much larger than ET. A mobile electron hole can appear and current can flow when Eδ is much smaller than or comparable to ET. Like organic fluorophores, Au NCs exhibit stronger fluorescence at low temperature than that of high temperature, mainly because the ET is smaller at lower temperature. The relationship among the Eδ value of Au NCs and Au atom number (N) and the Fermi energy (Ef) is simply represented in eq 1:46 Eδ = Ef /N1/3

(1)

When the fluorescence of Au NCs is solely attributed to the metal centered electron transition, the fluorescence of Au NCs undergoes a red shift upon increasing their size and no emission in the visible region can be observed when N is larger than 30. Such a size dependent emission prediction works for some cases such as PAMAM-stabilized Au NCs that do not possess fluorescence when N reaches 55 (1.2 nm).19 However, later studies showed that some thiolate ligands stabilized Au NCs (∼2 nm) can emit strong fluorescence in the green or red region, suggesting other emission mechanisms may exist.7,53 Ligand Effect. Through the charge transfer from S atom to the Au center (LMCT) or LMMCT when considering the Au− 218

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Au aurophilic interaction, some Au+ complexes containing thiolate ligands can fluoresce. For instance, Au+−alkylthiolate complexes show a maximum emission at ∼610 nm, which is similar to those thiolate ligands stabilized large Au NCs, suggesting that LMCT/LMMCT is also dedicated to the appearance of fluorescence in Au NCs.54 The strong covalent Au−S bonds largely affect the electronic structure of Au NCs and result in producing complicated molecular orbitals, making it difficult to predict the emission of thiolated ligand stabilized Au NCs. Compared to metal centered electron transition induced fluorescence, LMCT/LMMCT induced emissions always show a longer fluorescence lifetime, partly because LMCT/LMMCT can affect the excited state relaxation dynamics.55 It is believed that the fluorescence of Au NCs arises from the metal centered free-electron transition (NC core) and LMCT/LMMCT. For example, Au25(SC6H13)18 that is composed of a centered icosahedral Au13 core and an exterior shell made of six S−Au−S−Au−S staples exhibit NIR fluorescence (LMCT/LMMCT) and green fluorescence (metal centered free-electron transition) when excited at 514 and 400 nm, respectively.50,56 Since the electronic flow in LMCT/LMMCT is from S to Au/Au−Au, the electron donating capability of surface ligands plays an important role in determining the QY of Au NCs. Using Au25 with different capping agents as a model, the ligand dependent fluorescence is evident. 5 5 The QYs of [Au25(SC2H4Ph)18]−, [Au25(SC12H25)18]−, and [Au25(SC6H13)18]− are ∼1 × 10−4, 5 × 10−5, 2 × 10−5, respectively. Their fluorescence intensity decreases in the order of [Au 2 5 (SC 2 H 4 Ph) 1 8 ] − > [Au 2 5 (SC 1 2 H 2 5 ) 1 8 ] − > [Au25(SC6H13)18]−, which is consistent with the order of electron donating capability of the thiolated ligands. In other words, increasing electron donating capability of sulfur atom could enhance the fluorescence of Au NCs. It has been reported that partial displacement of GSH with a long chain peptide containing many nitrogen and oxygen atoms results in an enhancement (∼1.8 times) in fluorescence. This suggests that except the electron donating capability of sulfur atom, the existence of electron rich atoms or groups in surface ligands also enhance the fluorescence of Au NCs. In addition, the surface ligand density on the surface of each NC core also affects their fluorescence, with an evidence of increased fluorescence of 11-MUA−Au NCs upon increasing the concentration of 11-MUA.57 After removal of excess 11MUA, the fluorescence intensity of 11-MUA−Au NCs decreases dramatically as a result of a decrease in 11-MUA density on 11-MUA−Au NCs. The fluorescence intensity can be recovered after introducing new 11-MUA to the solution, which may be due to greater LMCT/LMMCT probability upon increasing thiolate ligand density. In addition, when the surface of each Au NC core is stabilized with dense 11-MUA molecules and 11-MUA/Au complexes, it is more difficult for quenchers such as O2 to access the surface to induce fluorescence quenching. Structure and Charge Effects. In addition to size and ligand, the geometrical structures also contribute to the optical properties of Au NCs. For example, [Au25(PPh3)10(SC2H5)5Cl2](SbF6)2 and Au25(SC2H4Ph)18 are both composed of 25 Au atoms, but they have different optical properties, likely due to their different geometrical structures. The former has a bi-icosahedral structure, while the latter has a core (centered icosahedral Au13)-shell (six S−Au−S−Au−S staples) structure.58 The low energy band of Au25(SC2H4Ph)18

from intraband (sp−sp) transition is around 1.8 eV calculated by applying the density function theory, while that in [Au25(PPh3)10(SC2H5)5Cl2](SbF6)2 is originated from the interaction between the two vertex-sharing Au13 icosahedrons. The oxidation state of NC core also affects the optical property of Au NCs. Polyethylenimine-stabilized positively charged Au8 NCs before and after reduction with NaBH4 emit green and blue fluorescence, respectively.45 The shift of absorption peak from ∼350 to 295 nm indicates that the oxidation states of NC core also affects the interaction between NC core and surface ligands. The effect of oxidation state of NC core on the optical properties of thiolate-stabilized Au NCs is mainly because the fluorescence of these Au NCs is largely originated from the charge transfer from S to Au. Oxidation of the NC core can promote the charge transfer efficiency and thus enhances the fluorescence. On the other hand, reducing Au(I) shell to Au(0) can hinder the charge transfer and results in the dramatic decrease of fluorescence. This reduction dependent fluorescence was discovered in 11-MUA-stabilized Au NCs,59 while the oxidation dependent fluorescence of Au NC core was proved when using Au25(SC2H4Ph)18 as a reference model.55

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SENSING Although simple and easy strategies for the preparation of water dispersible and biocompatible Au NCs having QYs greater than 10% have only been realized for few years, the analytical application of Au NCs has gained great attention recently. Having advantages of long lifetime, large Stokes shift, biocompatibility, ease of conjugation, as well as size and ligand dependent fluorescence properties, Au NCs are suitable for developing sensitive and selective sensing systems for various analytes such as trinitrotoluene.60 Heavy Metal Ions. Heavy metal ions such as Hg2+, Cd2+, Pb2+, and Cu2+ bind to various cellular components, such as proteins, enzymes, and nucleic acids, leading to alteration of their biological functions, which may cause serious diseases and death. Because of their high toxicity, the maximum allowed limits (MAL) of Hg2+, Cd2+, Pb2+, and Cu2+ in drinking water set by the environmental protection agency (EPA) of the United States are 0.002 (10), 0.005 (45), 0.015 (72), and 1.3 (21 000) ppm (nM), respectively. It is difficult to detect these ions in complicated biological and environmental samples. Thus, highly sensitive and selective sensing systems are required for their determination. By taking advantage of the 5d10−5d10 interaction between Hg2+ and Au+ that alter electronic structures of Au NCs,31,61 many sensitive and selective sensing systems have been developed for the detection of Hg2+.7,20,21,61 The fluorescence of Au NCs decreases upon increasing the concentration of Hg2+. For example, dihydrolipoic acid capped Au NCs prepared through a microwave-assisted synthetic route were used to detect Hg2+, with a limit of detection (LOD) of 0.5 nM.62 Although this method provides the advantages of simplicity, rapidity, and good sensitivity, its selectivity and sensitivity for Hg2+ are highly dependent on pH and ionic strength of the solution. Therefore, the application in complicated samples is limited. To improve the stability of Au NCs in high-salinity solution, Au NCs were prepared in poly(N-isopropylacrylamide) microgels (Au NC-PNIPAM MGs) (Figure 3).63 The asprepared Au NC-PNIPAM MGs have similar optical properties (excitation/emission wavelengths, 375/520 nm; QY, 3.8%) to that of 11-MUA−Au NCs, with much higher stability against 219

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electron energy of BSA−Au NCs, leading to the blue shift and enhancement of the fluorescence. BSA−Au NCs acting as a reductant to reduce Ag+ has been supported with matrixassisted laser desorption/ionization mass spectrometry and Xray photoelectron spectroscopy data.65 Au NCs have been applied to the detection of Cu2+.66−73 For example, GSH−Au NCs was used to detect Cu2+, based on analyte induced fluorescence quenching, with an LOD of 86 nM.71 Through the complexation between Cu2+ and the carboxyl group of GSH, Au NCs aggregated. Because Hg2+ and Pb2+ also cause fluorescence quenching of GSH−Au NCs, the selectivity is not great. The fluorescence quenching induced by Hg2+ and Pb2+ arises from the metallophilic interactions between Hg2+ and Au+ instead of the complexation with GSH. By adding ethylenediaminetetraacetate (EDTA) as a masking agent, the interference of Pb2+ but not Hg2+ was minimized. The interference from Hg2+ was minimized by the addition of Sn2+.72 A mixture solution of BSA−Au NCs and lysine-stabilized Au NCs has been employed to improve the sensitivity for Cu2+, with an LOD of 0.8 pM.73 Cu2+ coordinates with −COOH and −NH2 of both BSA−Au NCs and lysinestabilized Au NCs, leading to anisotropic growth of the NCs and thereby inducing fluorescence quenching of both Au NCs. A similar strategy based on coordination of Fe and dihydroxyphenylalanine-capped Au NCs has also been applied to the detection of Fe3+, with an LOD of 3.5 μM that is close to the MAL value (∼5.6 μM) in drinking water permitted by the U.S. EPA.74 The o-quinone-containing ligands formed complexes with Fe3+, leading to the aggregation of the Au NCs and thus a decrease in the fluorescence. A fluorescence assay using GSH−Au NCs has been demonstrated for selective determination of Cr3+ and Cr6+ in environmental water samples.75 Compared to Cr3+, Cr6+ has been considered to be 100 times more toxic to cause cancers. Over a pH range from 3.5 to 6.5, Cr3+ quenches the fluorescence of GSH−Au NCs. Through the formation complexes of Cr3+ with GSH, aggregation of GSH−Au NCs occurs, leading to fluorescence quenching. Under acidic conditions, a redox reaction between Cr6+ and GSH occurs, leading to fluorescence quenching of GSH−Au NCs. The quenching efficiency caused by Cr6+ decreases upon increasing the solution pH value. By conducting a stepwise sensing assay using GSH−Au NCs, Cr3+ and Cr6+ in water samples were selectively detected, with LODs of 48 and 9.6 nM, respectively. Cr3+ was detected at pH 6.5 in which Cr6+ nearly showed no fluorescence quenching capability for Au NCs. Because the fluorescence quenching induced by Cr3+ is pH independent, by measuring differential fluorescence intensities obtained at pH 3.5 and 5.0, the concentration of Cr6+ was determined. Dicysteine-capped Au NCs have been employed for the detection of As3+, with an LOD of 53.7 nM that is lower than the MAL (133 nM) of arsenic in drinking water set by the U.S. EPA.76 The addition of As3+ enhances the fluorescence of Au NCs, mainly because the electrons can flow from the electron rich Au NCs to the electron deficient As3+, resulting in an increase in the radiative decay rate of the Au NCs. The average decay time of the fluorescence was shortened from 2.63 to 0.48 ns in the presence of As3+. By simply adding a chelating agent such as succinic acid, As3+ on the surfaces of Au cores can be removed and thus the Au NCs can be reused. Inorganic Anions. Cyanide (CN−) is extremely toxic because it halts the cellular respiration by inhibiting the activity of cytochrome c oxidase in mitochondria. Lysozyme-stabilized

Figure 3. Schematic illustration of PNIPAM MGs incorporated with Au NDs (NCs) for the detection of Hg2+. Reprinted from ref 63. Copyright 2013 American Chemical Society.

salt. In the presence of 500 mM NaCl, a 9% increase in the fluorescence intensity of the Au NC-PNIPAM MGs was observed, while a 55% fluorescence quenching was found in the 11-MUA−Au NCs. The change in the microstructure of the gel and local environmental condition induced by the salt are responsible for the increased fluorescence intensity of the Au NC-PNIPAM MGs. 11-MUA−Au NCs became unstable and eventually aggregated upon increasing the NaCl concentration, leading to fluorescence quenching. Owing to the electrostatic repulsion with the MGs, the ions cannot access to the surfaces of Au NCs easily. The Au NC-PNIPAM MGs provided LODs for Hg2+ of 1.9 and 1.7 nM in the presence and absence of 500 mM NaCl, respectively. This probe was successfully applied to the determination of the concentration of Hg2+ in representative fish samples, revealing its excellent practicability for monitoring of Hg2+ levels in complicated biological samples. Au NCs on electrospun poly(vinyl alcohol) nanofibers allowed detection of Hg2+ by the naked eye down to 250 nM.64 A uniform distribution of Au NCs on the nanofibers significantly enhances the purity and homogeneity of the color and fluorescence intensity, which is important for the sensing of Hg2+. In the presence of Hg2+, the fluorescence of Au NCs decreased because of the formation of metallophilic bonds between Hg2+ and Au+. When using a confocal laser scanning microscope (excitation/emission wavelengths, 488/610 nm), this approach provided an LOD of 5.0 nM. This approach has the advantages of high stability, self-standing ability, naked-eye detection, selectivity, reproducibility, and easy handling. A sensitive and selective assay has been developed for Hg2+ and Ag+ using Al2O3 supported Au NCs, based on the analyte induced fluorescence quenching.37 Having a similar electron configuration to Hg2+, Ag+ also interacts with Au+ through the d10−d10 interaction, leading to analyte-induced fluorescence quenching. By adding excess amounts of PA as a masking agent, this assay is sensitive (LOD, 1.5 nM) and selective toward Ag+ over Hg2+, mainly because PA forms more stable complexes with Hg2+ (log K1 = 16.15) than with Ag+ (log K1 = 12.42). Protein-capped Au NCs have also been applied to the detection of Hg2+ and Ag+. BSA−Au NCs have been used as a label-free probe for Hg2+.61 The fluorescence of BSA−Au NCs decreases in the presence of Hg2+ due to Hg2+−Au+ interactions. This assay provided an LOD of 0.5 nM, with linearity over the concentration range of 1.0−20 nM. BSA−Au NCs have also been used for the detection of Ag+, with an LOD of 0.1 μM.32 When reacting with Ag+, the emission wavelength of BSA−Au NCs shifted from 604 to 567 nm and the fluorescence intensity enhanced when excited at 350 nm. Meanwhile, the absorption of BSA−Au NCs at 435 nm increased while that at 515 nm decreased. This is mainly because of the change in the surface 220

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Au NCs have been utilized for the selective detection of CN−, with an LOD of 190 nM.77 The sensing mechanism is based on the oxidation of Au NCs in the presence of O2 by CN− to form stable [Au(CN)2]− complexes. This assay is extremely selective, mainly because of high stability of Au NCs. Like most of the CN− etching based sensing systems that is slow and requires a high concentration of CN−, the sensitivity and assay speed of this assay are limited. Au NCs based macroporous sensing films have been developed for naked-eye detection of CN− based on the gold cyanidation process.78 The macroporous structure of the film considerably enhances their interaction sites and favors efficient diffusion of the analyte, leading to sensitive and rapid detection of CN−. During the soaking process, these macropores are filled with water, which facilitates instant capture and concentration of the volatilized CN−, resulting in a signal amplification effect. The practical use of this system was further validated for the detection of CN− in red wine, juice, coffee, and real-time monitoring of CN− release in cassava processing. Sulfide (S2−) is well-known as an environmental pollutant but also an important gaseous signal transmitter. The toxicity of S2−, which is similar to CN−, is attributed to the formation of the complexes with iron in the mitochondrial cytochrome enzymes, thus preventing cellular respiration. BSA−Au NCs have been used for the sensitive and selective detection of S2−, with an LOD of 29 nM.79 The sensing mechanism is based on the reaction of S2− with BSA−Au NCs to form Au2S precipitate, leading to the degradation of the structure of BSA−Au NCs. As a result, the fluorescence decreased upon increasing in the concentration of S2−. The assay was further applied toward the detection of S2− in river water samples, showing great potential in real sample analysis. Alternatively, a turn-on fluorescence assay using 1-(10-mercaptodecyl)-5methylpyrimidine-2,4-dione and 11-MUA comodified Au NCs has been developed for the detection of S2−, with an LOD of 0.5 μM.80 Owing to the poor water solubility of 1-(10mercaptodecyl)-5-methylpyrimidine-2,4-dione, aggregation of the Au NCs occurs within 2 days, leading to decreased fluorescence. Adding S2− to the Au NC aggregates, adsorption of S2− onto the surface of Au NCs leads to increases in the surface charge. As a result of increased static repulsion, the aggregation disassembles and their fluorescence recovers. Nitrite (NO2−), which presents at trace levels in nature but high concentrations in wastewater, reacts with secondary amines to form carcinogenic nitrosamines under acidic conditions. BSA−Au NCs have been employed for the sensitive and selective detection of NO2−, with an LOD of 1.0 nM.81 Interaction of NO2− with BSA−Au NCs leads to the formation of aggregates and thus fluorescence quenching. The X-ray photoelectron spectroscopy data revealed that the binding energy for the Au 4f7/2 electrons in BSA−Au NCs in the presence of nitrite is higher than that in the absence of nitrite, showing that NO2− induced an increase in the oxidation state of the Au atoms in the Au NCs.82 Similarly, myristic acid and 11-mercaptoundecanol cocapped Au NCs have been used to detect NO2− down to 40 nM.83 Small Biomolecules. A sensing system using glucose oxidase-functionalized Au NCs has been developed for the detection of glucose, with an LOD of 0.7 μM.84 Fluorescence quenching of the Au NCs takes place in the presence of glucose, as a result of oxidation of the Au core to form Au+ by the enzymatic product of H2O2 and aggregation of the NCs. Successful determination of the concentrations of glucose in human urine and serum samples revealed that the simple,

selective, and sensitive assay holds great potential for clinical applications. NIR fluorescent trypsin stabilized Au NCs (try− Au NCs) and cysteamine-capped Au NPs (cyst−Au NPs) have been used for the detection of heparin via surface plasmon enhanced energy transfer (Figure 4).85 The efficiency of surface

Figure 4. Schematic illustration for selective detection of heparin through surface plasmon enhanced energy transfer between try−Au NCs and cyst−Au NPs. Reprinted from ref 85. Copyright 2013 American Chemical Society.

energy transfer between Au NPs and fluorophores is enhanced due to the unique SPR property of Au NPs, leading to fluorescence quenching. The negatively charged try−Au NCs and the positively charged cyst−Au NPs get closer through their electrostatic interaction. In addition, a large overlap of the SPR absorption spectrum of cyst−Au NPs (absorption wavelength 524 nm) and the emission spectrum (excitation and emission wavelengths, 520/690 nm) of the try−Au NCs is also responsible for great energy transfer efficiency. In the presence of negatively charged heparin, the interaction between the cyst−Au NPs and the try−Au NCs becomes weaker as a result of heparin induced aggregation of the cyst−Au NPs. As a result, the fluorescence of the try−Au NCs increases upon increasing the concentration of heparin, allowing the detection of heparin down to 0.05 μg mL−1. GSH−Au NCs and protein stabilized Au NCs both have been used for the detection of phosphate-containing biomolecules such as adenosine-5′-triphosphate (ATP) in cell lysate and human plasma samples.86,87 The fluorescence of GSH−Au NCs is quenched upon their interaction with Fe3+, mainly because of the electron transfer between GSH−Au NCs and Fe3+. Once the Fe3+ is released from the GSH−Au NCs through its strong complexation with phosphate-containing molecules, the fluorescence is recovered. Because the formation constant of Fe3+ with multiphosphates is higher than that with monophosphates, this assay is slightly more sensitive for pyrophosphate than for ATP, with LODs of 43 and 28 μM, respectively. Similarly, chicken egg white protein stabilized Au NCs have been used for the turn-on detection of ATP and pyrophosphate, with LODs of 19 and 5 μM, respectively.87 A sensitive assay using urease-stabilized Au NCs has been developed for the detection of urea in blood samples.88 Upon increasing the concentration of urea, the fluorescence of the Au NCs decreases, allowing detection of urea down to 1.0 mM. Urease catalyzes the conversion of urea into NH3 and CO2, leading to generation of NH4+. Through the interaction of NH4+ with the negative surface charge of the Au NCs, aggregation of Au NCs and consequent fluorescence quenching take place. Deficiency of folic acid has been linked to elevated levels of serum homocysteine, which implicates an independent risk factor for coronary artery disease and stroke. On the basis of analyte induced fluorescence quenching, a simple, rapid method using BSA−Au NCs has been demonstrated for the 221

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hydrophobic phospholipid membrane, the resulting fluorescent 11-MUA−Au NCs−liposome (11-MUA−Au NCs/Lip) hybrids have been utilized for the selective detection of phospholipase C (PLC) (Figure 5).99 PLC catalyzes the

determination of the concentration of folic acid, with an LOD of 41 nM.89 Upon increasing the concentration of folic acid, the fluorescence of BSA−Au NCs decreases, while the absorbance at 350 nm increases. The result suggests that the quenching is attributed to the interaction between FA and BSA and changes in the local environment around the Au NCs. Cu2+-modulated BSA−Au NCs have been used for the detection of dopamine that is an important neurotransmitter.90 Through strong interaction of Cu2+ with dopamine, Cu2+ induced fluorescence quenching of BSA−Au NCs is suppressed. Having high sensitivity (LOD, 0.1 μM) and selectivity, this assay is practical for the detection of dopamine in serum samples. By taking advantage of specific binding (formation constant 5.5 × 105 L mol−1) between human serum albumin (HSA) and bilirubin, HSA-stabilized Au NCs (HSA−Au NCs) have been used for the detection of bilirubin based on analyte induced fluorescence quenching.91 This approach provided an LOD of 248 nM for bilirubin. The fluorescence response toward bilirubin is constant over a wide pH (6.0−9.0) and temperature (25−50 °C) range, showing its practicality for the analysis of biological samples. Proteins. Proteins play an important role in many biological processes. Functionalization of Au NCs with recognition molecules such as antibody, aptamer, and carbohydrate has been successfully developed to improve their selectivity toward proteins.38−41,92 A multifunctional boron nitride (BN) sheet decorated with Au NCs as fluorescent or an electrochemical labels has been employed for the detection of interleukin-6.93 Fluorescent Au NCs were decorated in poly(diallyl-dimethylammonium) chloride modified BN (PDDA-BN) sheets by an electrostatic layer-by-layer assembly. The PDDA-BN−Au NCs was then conjugated with an antibody (Ab2) to form PDDABN−Au NCs−Ab2 bioconjugates. Using the bioconjugates, fluorescent and electrochemical sandwich bioaffinity assays were conducted for the detection of interleukin-6 with LODs of 0.03 ng mL−1 and 1.3 pg mL−1, respectively. By adapting a similar strategy, porous calcium carbonate (CaCO3) spheres were used for loading Au NCs to fabricate CaCO3/Au NCs hybrid spheres through electrostatic interaction.94 Functionalization of CaCO3/Au NCs hybrid spheres with HRP/antibody conjugates (CaCO3/Au NCs/HRP−Ab2) were further employed to construct a dual fluorescent and electrochemical system for the detection of cancer biomarker neuron-specific enolase. In this system, Ab1 immobilized multiwalled carbon nanotubes provide a surface for enolase binding, while CaCO3/ Au NCs/HRP−Ab2 bioconjugates act as a capture for further detection. The fluorescence and electrochemical signals can be achieved by releasing Au NCs and HRP-catalyzed oxidation of o-phenylenediamine from the captured CaCO3/Au NCs/ HRP−Ab2, respectively. The assay provided LODs for enolase of 2.0 and 0.10 pg mL−1, separately based on the fluorescence and electrochemical signals against the enolase concentration. On the basis of the fact that the fluorescent intensity of Au NCs depend on the surface ligand and environment, BSA−Au NCs have been applied for the detection of proteases and acetylcholinesterase.95−98 Protease catalyzes the hydrolysis of the protein shell, leading to removal of BSA from BSA−Au NCs. As a result, Au core has a greater chance of exposure to quenchers such as oxygen molecules (O2), leading to a decrease in fluorescence. This approach allowed the detection of proteinase K down to ∼1 ng mL−1.98 More recently, by etching Au NPs (∼3 nm) with 11-MUA molecules in

Figure 5. Cartoon illustration of the operation of the 11-MUA−Au NC/Lip probe for the detection of PLC through control of O2induced fluorescence quenching by the 11-MUA−Au NC/Lip probe. Reprinted from ref 99. Copyright 2013 American Chemical Society.

hydrolysis of phosphatidylcholine, leading to the formation of diacylglycerol and phosphocholine products and decomposition of liposome. The as-formed diacylglycerol further interacts with 11-MUA−Au NCs via hydrophobic interactions and inhibits the O2-mediated quenching. As a result, the fluorescent intensity of 11-MUA−Au NCs/Lip is proportional to the concentration of PLC, allowing detection of PLC down to 0.21 nM. This assay is extremely selective toward PLC over other tested proteins, enzymes, and phospholipase. Having the advantage of high sensitivity and selectivity, this assay is practical for the determination of PLC concentration in breast cancer cells (MCF-7 and MDA-MB-231 cell lines) and for the study of the interaction of PLC with its inhibitor such as D609. The activity of pyrophosphatase has been determined using 11MUA−Au NCs based on Cu2+ induced fluorescence quenching.100 The fluorescence quenching is suppressed in the presence of pyrophosphate that interacts with Cu2+ strongly. The pyrophosphate-Cu2+ complex becomes unstable in the presence of pyrophosphatase that specifically catalyzes the hydrolysis of PPi. As a result, the fluorescence of 11-MUA−Au NCs was quenched by the released Cu2+. This approach allowed determination of PPase activity over the range from 1 to 20 mU, with an LOD less than 1 mU. Peptide-templated Au NCs have been used to develop a realtime and label-free sensing approach for the detection of posttranslational modification enzymes and peptidase, including histone deacetylase 1, protein kinase A, and elastase, with LODs at the subpicomolar level.101,102 Enzymatic modification of the peptides causes significant fluorescence quenching of the Au NCs, mainly because the chemical modifications destroy the protective peptide coating on Au NCs. As a result, O2-mediated quenching and oxidation of the fluorescent Au NCs take place 222

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more easily. The fluorescence of Au NCs decreases upon increasing concentration of the enzymes, allowing quantitation of the enzymes. Adsorption of proteins onto Au NCs surfaces has been demonstrated to significantly enhance the fluorescence of Au NCs, depending on the nature of proteins such as size and shape.103 The enhancement of fluorescent intensity follows the order of protein size: apotransferrin (80 kDa) > human serum albumin (66 kDa) > lysozyme (14 kDa). In contrast to the protein induced fluorescence enhancement, cytochrome c exhibits fluorescence quenching of Au NCs via electron transfer. Fluorescence quenching of 11-MUA−Au NCs induced by heme-containing proteins, including hemoglobin, cytochrome c, and myoglobin, has been demonstrated through redox reactions between the 11-MUA−Au NCs and the Fe(II) atoms of hemin units.104 The Stern−Volmer quenching constants for hemin, cytochrome c, hemoglobin, and myoglobin reveal that the decreasing order of their quenching efficiency is in good agreement with the increasing order of their sizes and decreasing order of their reduction potentials. The assay is selective to heme-containing proteins over other common proteins such as BSA found in blood. Having an LOD of 0.5 nM for hemoglobin in biological buffer, 11-MUA−Au NCs is practical for the diagnosis of diseases associated with changes in the levels of hemoglobin. BSA−Au NCs have been used as a fluorescent probe for the detection of separated human serum proteins in gel (Figure 6),

allowed detection of some low abundance proteins such as zincalpha-2-glycoprotein (ZAG), vitamin D-binding protein precursor, and complement component C3. In addition, this method was able to differentiate protein profiles of the serum samples from patients with liver disease and from healthy people. Alternatively, an array-based sensing strategy has been applied for protein discrimination.106 The fluorescence intensity of protein-stabilized Au NCs can be enhanced by depositing on plasmonic substrates, which is an approximately 20-fold enhancement of fluorescence emission. The plasmonenhanced fluorescence results from resonant coupling of fluorescence emission with localized surface plasmon on metallic nanostructures. Protein-stabilized Au NCs were directly added on silver substrates, leading to increased fluorescence intensity when excited at 405 nm. Because the target proteins interact with protein-stabilized Au NCs and thus influence the enhanced fluorescence process, distinct fluorescent image patterns were obtained using a laser scanning confocal microscope. Red-emitting human serum albumin stabilized Au NCs have been proven to provide unique affinity toward pathogenic bacteria including Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), mainly because the peptide motif of human serum albumin stabilized Au NCs specifically interacts with the cell wall of the two bacterial strains.107 The high-affinity and sensitive assay was applied for the rapid screening of S. aureus and MRSA at the concentrations down to ∼106 cells mL−1 by the naked eye. However, the assay is not able to distinguish S. aureus from MRSA.

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IMAGING Among various types of Au NCs, protein-stabilized Au NCs are especially suitable for cell imaging and therapy,108 mainly because of their unique functionality, ease in conjugation, biocompatibility, large Stoke shift, long lifetime, as well as photo and chemical stability. Advanced techniques in imaging have shown that multifunctional Au NCs/composites are practical for in vivo imaging and efficient therapy of various diseases. To make Au NCs as ideal candidates for imaging, some important issues such as their affinity toward specific cells or organs, stability during delivery, and cell penetration have to be considered carefully. In Vitro. In recent years, many efforts have been dedicated to modify Au NCs with recognition molecules to enhance their performance in tumor diagnosis and therapy. Recognition elements can be conjugated to protein-stabilized Au NCs through their reactions with thiol, amino, and carboxyl residues on proteins. For instance, cancer cell recognized molecules (e.g., folic acid and herceptin) have been intensely used to functionalize BSA−Au NCs by using the conventional 1-ethyl3-(3-(dimethylamino)propyl)carbodiimide (EDC) coupling chemistry.109−111 Cells labeled with these functionalized BSA−Au NCs can be clearly imaged with the NIR-emitting fluorescence (emission wavelengths 700−800 nm) when excited at the wavelengths over 480−550 nm, with minimum interference from autofluorescence of biological matrixes. Many proteins have been used to prepare protein-stabilized Au NCs that maintain the biological function of the protein template, and thus they can be used directly to target specific cells without conducting any postreactions. For example, human transferrin-stabilized Au NCs and insulin-stabilized Au NCs can directly target transferrin receptor overexpressed cells (e.g.,

Figure 6. Detection of proteins in human serum after native polyacrylamide gel electrophoresis: (A) BSA−Au NC-based fluorescence imaging, (B) fluorescence image with oxygen low-temperature plasma pretreated Au NCs, (C) silver stain, and (D) CBB-R250 stain. Reprinted with permission from Zhang, J.; Sajid, M.; Na, N.; Huang, L.; He, D.; Ouyang, J. Biosens. Bioelectron. 2012, 35, 313−318 (ref 105). Copyright 2012 Elsevier.

with a sensitivity of 7−14 times higher than that of traditional staining detection methods (e.g., Coomassie Brillant Blue R250 stain and silver stain).105 The BSA−Au NCs were treated with oxygen type low-temperature plasma (LTP) and then incubated with on-gel proteins for subsequent fluorescence imaging. The oxygen LTP treatment has the potentials to improve the complexation of BSA−Au NCs and proteins in gels through van der Waals force and hydrogen-bonding interaction in an acidic environment (pH 3.3), which may effectively enhance the Au NCs-based fluorescence imaging detection without additional steps (e.g., destaining, immobilizing). Having a greater sensitivity, labeling with BSA−Au NCs 223

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positions revealed that these brightly fluorescent PA−Au NCs were inside the cells as well as attached to the outside of the plasma membrane. Large aggregates of most of the PA−Au NCs were found inside the cells, revealing that a specific endocytosis pathway is likely involved in the internalization process. Several water dispersible Au NCs such as insulinstabilized Au NCs and GSH−Au NCs have been also applied for two-photon cellular imaging.33,119 GSH−Au NCs with high two-photon absorption cross section (∼189 740 GM) and exceptional photostability were utilized for both one- and twophoton excitation cell imaging on SH-SY5Y human neuroblastoma cells.119 Similar to one-photon excitation results, the two-photon cell imaging clearly showed the particles inside the cells and no autofluorescence from the cells. In addition, the cells were imaged with different z-position scans under twophoto excitation, confirming that the GSH−Au NCs were internalized inside the cells. Having long lifetime (usually >100 ns) than that (few nanoseconds) of common biomolecules, Au NCs is ideal for fluorescence lifetime imaging, mainly because interference from most intrinsic fluorescence can be removed.120 For example, dihydrolipoic acid-protected Au NCs with bright NIR emission and long fluorescence lifetime (500−800 ns) have been applied to monitoring of the live HeLa cells internalization process.121 The fluorescence lifetime images clearly display the distributions of Au NCs in both of the intracellular and membrane regions. Furthermore, fluorescence lifetime images not only reveal the uptake of Au NCs but also provide information on changes in the local environment of cells. For instance, lipoic acid-capped Au NCs have been used as versatile nanothermometry devices for cell imaging by taking advantage of the temperature-dependent fluorescence lifetime and emission intensity, which change considerably over the physiological temperature range (15−45 °C).122 By applying a pixel-by-pixel basis to fit the fluorescence decay of each pixel with a triexponential function, lifetime maps of the Au NCs in HeLa cells were recorded by the intensity-weighted average lifetime, showing a shorter fluorescence lifetime upon increasing temperature. Fluorescent Au NCs have been used in live cell imaging, allowing real-time investigation of the changes in the intracellular environment.53,123−128 For example, cysteamine conjugated GSH−Au NCs have been employed to show pHdependent membrane adsorption of the Au NCs within a biological pH range (5.3−7.4).53 The fluorescence intensity of the cells increased dramatically upon decreasing the extracellular pH values to pH 6.0 and further to 5.3, implying stronger adsorption of the Au NCs to the cell membrane in a slightly acidic environment. The predominant driving force arises from electrostatic attraction between cell membrane and cysteamine ligand. Very recently, dual-emission Au NCs (BSA− Ce/Au NCs) have been used for monitoring of local pH values inside cells.126 The BSA−Ce/Au NCs are stable at various ionic strengths and under photoirradiation for up to 6 h. At the same excitation wavelength (325 nm), the NCs emit at 410 and 650 nm with intensities that are pH dependent and independent, respectively. The fluorescence emission band at 410 nm is assigned to BSA−Ce complexes, while that at 650 nm is due to the NCs. The pH-dependent change in the fluorescent intensity is reversible and related to the stability of BSA−Ce complexes. Upon increasing pH value, the fluorescent intensity at 410 nm increases. On the other hand, the fluorescence intensity at 650 nm is pH independent. By taking advantage of the pH

A549 cells) and insulin receptor overexpressed cells (e.g., C2C12 cells), respectively.23,33 It is worth to note that human apotransferrin (iron-free form) stabilized Au NCs (apoTf−Au NCs) showed no significant quenching effect of the fluorescent intensity after iron loading. Hence, cell labeled with iron loaded apoTf−Au NCs clearly showed a strong membrane association after colocalization of the cell membrane and the Au NCs, mainly because the transferrin receptor expressed on the cell surface has high affinity toward iron loaded transferrin.23 On the other hand, the intense red fluorescence of insulin-stabilized Au NCs overlapped with that of the fully differentiated C2C12 myoblasts in the cytoplasm instead of the undifferentiated C2C12 myoblasts, indicating that the insulin-stabilized Au NCs bind to the insulin receptor overexpressed C2C12 cells. Like insulin, the insulin-stabilized Au NCs differentiated C2C12 myoblasts and then entered the cell through receptor-mediated endocytosis.33 A density tunable dendrimeric array has been developed for in situ tracing of sialic acid density by using GSH−Au NCs functionalized with 3-aminophenylboronic acid (APBA−Au NCs) as signal reporter probes.112 Selectivity of this assay is based on the high affinity of boronic acid with glycans on the surface of cells (e.g., BGC-823 cells). When cells were added to the APBA−Au NCs conjugated dendrimer-modified slide, the fluorescent APBA−Au NCs probes were specifically bound to the cells through a covalent linkage between APBA and sialic acid. As a result, the cells were released from the slide, leading to a decrease in fluorescence intensity of the probe on the slide. The amount of unloaded probe from the slide depends on the dendrimer density, cell number and the expression extent of sialic acids on the cell surface. In addition to estimation of the surface density of sialic acid on the surface of the cells, this sensing system allows monitoring of subtle changes of glycan density in response to drugs such as sialidase. Au NCs are suitable for tracking subcellular localization.113−115 A bifunctional peptide, called CCYTAT, containing two functional domains for Au NCs synthesis and for cell nucleus targeting was used to prepare CCYTAT−Au NCs.114 The TAT is a classic nucleus localization signal sequence, allowing localization of the red emitting CCYTAT−Au NCs in the cell nuclei that can be clearly observed by using a confocal microscope. To enhance cell penetration and localization of Au NCs in mitochondria, a mitochondria-targeted fluorescent probe was prepared.115 Triphenylphosphonium (TPP) cation and chitosan-coated Au NCs (Au NCs@CS) were used to prepare Au NCs@CS−TPP (probe). TPP is a delocalized lipophilic cation, which can pass through phospholipid bilayers easily and accumulates into highly negatively charged mitochondria of living cells. Like use of a standard mitochondria-targeted fluorescent probe (Mito Tracker), the blue emitting Au NCs@CS−TPP is specifically accumulated in the mitochondria of a cell. High two-photon absorption cross sections at 800 nm116 makes water dispersible Au NCs as promising probes for twophoton cellular imaging. Notably, two-photon excitation imaging technique provides high penetration depth and highspatial resolution in living tissue imaging, with minimization of tissue scattering and autofluorescence background in multiphoton microscopy.117 PA−Au NCs have been used to observe the live imaging of human cervical cells (HeLa) by using a twophoton excitation confocal microscope under the excitation of an 810 nm laser.118 3D reconstruction images of cells pretreated with the PA−Au NCs collected at different z224

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dependent and independent properties at the two emission wavelengths, BSA−Ce/Au NCs holds great potential as a dual emission probe for ratiometric determination of pH values. The cell images shown in Figure 7 display the local pH values inside

Figure 7. Confocal images of HeLa cells under different pH values: (A) blue, (B) red, and (C) merged images. Reprinted with permission from Chen, Y.-N.; Chen, P.-C.; Wang, C.-W.; Lin, Y.-S.; Ou, C.-M.; Ho, L.-C.; Chang, H.-T. Chem. Commun. 2014, 50, 8571−8574 (ref 126). Copyright 2014 Royal Society of Chemistry.

cells treated with BSA−Ce/Au NCs, the blue cell images (corresponding to the fluorescence at 410 nm) are weaker upon decreasing pH values from 8.5 to 5.0, while the red fluorescence images (at 650 nm) are almost the same. A dualemission fluorescent nanocomplex of Au NCs-decorated silica particles has been demonstrated as a novel nanosensor for live cell imaging of highly reactive oxygen species (ROS).127 The GSH−Au NCs is sensitive and selective to ROS such as hydroxyl radical (•OH), hypochlorite (ClO−), and peroxynitrite (ONOO−). When the Au NCs-decorated silica particles are excited at a single wavelength (405 nm), they emit two different emission colors; one is from the Au NCs that is responsive to ROS, and the other is from the dye-encapsulated silica particles that acts as an internal reference (Figure 8A). Live cell imaging studies revealed that the probe allows rapid imaging of ROS signaling with high contrast in cells, which provides great potential for real-time monitoring of intracellular ROS signaling events (Figure 8B). GSH−Au NCs have also been applied to monitoring of intracellular antioxidants such as ascorbic acid that can protect the fluorescence of Au NCs against quenching by ROS.128 In Vivo. Compared to the often used blue and green fluorescent Au NCs for biological labeling, water-dispersible Au NCs with NIR emission have higher penetration capability and thus they can be ideal for live animal imaging.129−133 BSA−Au NCs have been used for in vivo imaging.130 The whole-body imaging observed in real-time after intravenous injection of BSA−Au NCs solution into nude mice reveals that the BSA− Au NCs were retained in the circulation at 5 h post injection and the fluorescence decreases noticeably within 24 h. The study also found that the uptake of BSA−Au NCs by the reticuloendothelial system (e.g., liver and spleen) is relatively low in comparison with other nanomaterials, partly due to their ultrasmall hydrodynamic size (∼2.7 nm). GSH−Au NCs have been applied to in vivo imaging, including biodistribution, renal

Figure 8. (A) Schematic illustration of hROS detection using a dualemission fluorescent nanocomplex of Au NCs-decorated silica particles. (B) Confocal fluorescence microscopy images of HeLa cells treated with (a) no ROS, (b) H2O2, (c) HClO, and (d) SIN-1, after incubating with DEFN for 1 h at 37 °C. Reprinted from ref 127. Copyright 2013 American Chemical Society.

clearance, pharmacokinetics, and tumor accumulation.131,132 To understand the renal clearance profile of Au NCs, fluorescent GSH−Au NCs have been used for in vivo fluorescence and Xray computed tomography (CT) imaging to real-time visualization of urinary excretion of these Au NCs within 24 h after intravenous injection into a mice body.131 The study found that GSH−Au NCs can be efficiently cleared out of the body with evidence of only 3.7% of the GSH−Au NCs accumulated in the liver; over 50% of the GSH−Au NCs were found in urine. The minimization of liver and spleen accumulation of the GSH−Au NCs can be ascribed to their very small particle sizes and low affinities to serum proteins. Moreover, the renal-clearable GSH−Au NCs have also been demonstrated with a much longer tumor retention time and faster normal tissue clearance.132 These advantages allow GSH−Au NCs to detect a tumor rapidly without severe accumulation in the reticuloendothelial system organs, making them very promising for cancer diagnosis and therapy. Au NCs-based fluorescence turn-on probes have been developed for in vitro and in vivo bioimaging.133 The fluorescence of transferrin-stabilized Au NCs conjugated with graphene oxide (GO) is very weak, mainly because GO is a very efficient quencher. Through interaction of transferrin with its receptor, the fluorescence increased as a result of the release of transferrin-stabilized Au NCs from GO surface. It is noted that the adsorption of transferrin-stabilized Au NCs onto the GO 225

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surface is through π−π stacking and hydrophobic patches. Practicality of this assay was validated by the fluorescence turnon imaging of cancer cells and small animals. Since transferrin exhibits high specificity and affinity toward transferrin receptor, the restoration of fluorescent intensity of transferrin-stabilized Au NCs/GO was observed in the presence of transferrin receptor. In addition, this approach allowed for imaging of HeLa cells and HeLa tumor-bearing mice. Because each imaging technique has its own unique advantages along with intrinsic limitations, multimodal imaging based on the integration of two or more imaging techniques can provide complementary information and allow reliable and accurate diagnosis of diseases.134 For example, to strengthen the integration of both fluorescence and single photon emission CT imaging techniques, radioactive 198Au was used to synthesize radioactive NIR-emitting GSH−Au NCs.135 The bright NIR-emitting fluorescence and the radioactive gamma emission render these Au NCs with potential applications as dual-modality imaging probes. Au NCs-based nanoprobes with NIR fluorescence, magnetic resonance imaging (MRI) contrast, and 1O2-sensitized have been demonstrated and applied in intracellular bioimaging.136 Through the conjugation of NIRemitting BSA−Au NCs with epidermal growth factor functionalized Fe3O4, the as-prepared multimodal nanocomposites can be specifically delivered into cells via endocytosis, which can be visualized with the combination of NIR fluorescence and MRI for live cell imaging. Because the NIR-emitting Au NCs are also responsible to sense dissolved oxygen and produce 1O2, a third imaging modality is easily realized by using a singlet oxygen sensor green dye. Similarly, gadolinium(III) functionalized BSA−Au NCs have been synthesized as fluorescence/MRI/ CT-based multimodal imaging probes for in vivo tumor-bearing mice.137 The fluorescence, CT, and T1-weighted MRI signal derived from gadolinium(III) functionalized BSA−Au NCs were all very distinguishable in the tumor region from other tissues, indicating a significant accumulation of the probe in tumor tissues through the enhanced permeation and retention effect and a reduced clearance from the tumor (Figure 9). The development of multifunctional agents for cancer therapy has been focused on the combination of cancer targeting, therapy, and diagnosis.138 Because fluorescent Au NCs exhibit a great deal of advantage for being a contrast agent in in vitro and in vivo imaging, the conjugation of anticancer drug with Au NCs has been developed for cancer cell imaging and therapy.139−143 Such multifunctional nanomaterials are called theranostic nanocomposites. A pH-responsive polymeric nanocarrier fabricated with fluorescent Au NCs, recognition molecules, and anticancer drugs provided the efficacy of selective targeting, in situ imaging, and anticancer therapy.143 The GSH-stabilized Au NCs and the targeting ligand (folic acid) were tethered to the amphiphilic copolymer poly(DBAMco-NAS-co-HEMA) to form nanocomposite. Then, the nanocomposite was self-assembled with the hydrophobic drug (paclitaxel) to form a core−satellites nanocomposite. Through the degradation of the pH-labile linkages in the polymer, the encapsulated drugs were released in mildly acidic endosomal/ lysosomal compartments. Both in vitro and in vivo results revealed that the GSH-stabilized Au NCs provide positioning information and the drug has a therapeutic action at the folate overexpressing cancerous cells, showing this theranositc nanocomposite is useful for the early detection and therapy of cancerous cells. By utilizing a similar strategy, a core−shell structured multifunctional nanocarrier was developed by using

Figure 9. In vivo (A) fluorescence, (B) MRI, and (C) CT imaging of tumor bearing mice after the tail injection of BSA−Au NCs based multimodal imaging probe. Reprinted with permission from Hu, D.H.; Sheng, Z.-H.; Zhang, P.-F.; Yang, D.-Z.; Liu, S.-H.; Gong, P.; Gao, D.-Y.; Fang, S.-T.; Ma, Y.-F.; Cai, L.-T. Nanoscale 2013, 5, 1624−1628 (ref 137). Copyright 2013 Royal Society of Chemistry.

Au NCs as core and folate-conjugated amphiphilic block copolymer as a shell.144 The Au NCs copolymers not only have great potential as tumor-targeted drug delivery but also have an assistant role in the treatment of cancer by introducing hydrophobic drugs.

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CONCLUSIONS AND OUTLOOKS Many simple strategies have been developed for the preparation of biocompatible Au NCs from Au3+ in the presence of small thiol compounds, peptide, or proteins. The nature and concentration of the ligand, reaction temperature and time, solution pH, and ionic strength all play important roles in determining the formation of Au NCs and thus their chemical and optical properties. These Au NCs have large Stokes shift and long lifetime and hence many of them have become useful materials for developing sensitive sensing and imaging systems. However, their QY values are usually less than 20% and their affinity toward a specific target is not great. To further improve their sensitivity and selectivity, strategies for the preparation of Au NCs having high QY and high affinity are still needed. 226

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Strategies for the preparation of stable Au based bimetal NCs have been shown effective for enhanced QY of Au NCs.145,146 Using several metal ions for the preparation of NCs having different optical properties is a trend for the preparation of ratiometric probes for sensing of pH, free radicals, and important biomarkers. Since ligand has an important role in determining the optical properties of Au NCs, use of more than two different types of ligands such as cationic and anionic thiol ligands to prepare Au NCs is possible to prepare more stable and highly fluorescent Au NCs. Many protein-stabilized Au NCs with other types of nanomaterials have been used to prepare functional nanocomposites that provide multifunctionality such as targeting, sensing, and therapy. However, it requires a great amount of proteins to prepare stable and fluorescent Au NCs, limiting the use of expensive proteins for the preparation of Au NCs. Thus, strategies for the preparation of Au NCs in the presence of small amounts of expensive proteins are required. Using mixtures of BSA with an expensive protein or DNA may be useful to prepare functional Au NCs. Since solution pH, ionic strength, and reaction temperature all have great effects on the protein conformation, these factors must be optimized. Most of the sensing approaches using Au NCs are based on analyte induced fluorescence enhancement or quenching. Although time-resolved fluorescence has been popular for many years, its use combined with Au NCs has not been well recognized. Having long lifetimes, Au NC based time-resolved fluorescence techniques shall soon become more popular. Use of Au NPs as a quencher and Au NCs as a donor, a fluorescence resonance energy transfer based approach was demonstrated for the sensitive and selective detection of platelet-derived growth factors few years ago.147 Advances in the preparation of various functional nanomaterials and Au NCs shall benefit for the development of more sensitive fluorescence energy transfer based techniques. It is our strong belief that more and more sensitive and selective sensing and imaging techniques using Au based NCs will soon become golden standards for clinical applications.

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2013 with Dr. Yan He and Dr. Edward S. Yeung. His research interests focus on synthesis, formation mechanism investigation, and analytical application of metal nanoclusters. Huan-Tsung Chang is currently a Professor of the Department of Chemistry, National Taiwan University. He is a fellow of the Royal Society of Chemistry. He obtained his Ph.D. from the Department of Chemistry, Iowa State University in 1994 with Dr. Edward S. Yeung. His current research interests include nanotechnology, green chemistry, biosensors, and mass spectrometry.

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ACKNOWLEDGMENTS

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REFERENCES

We are grateful for the support from the Ministry of Science and Technology of Taiwan (Contract Number NSC101-2113M-002-002-MY3). Z. Yuan is grateful to the Ministry of Science and Technology of Taiwan for a postdoctoral fellowship under Contract NSC 103-2811-M-002-169.

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AUTHOR INFORMATION

Corresponding Author

* Phone/fax: 011-886-2-33661171. E-mail: [email protected]. tw. Notes

The authors declare no competing financial interest. Biographies Li-Yi Chen received her M.S. degree from the National Chiao Tung University, College of Biological Science and Technology in 2009. She is currently a Ph.D. student at the Department of Chemistry, National Taiwan University, under the supervision of Prof. Huan-Tsung Chang. Her research interests focus on developing hybrid gold nanomaterials for bioapplications. Chia-Wei Wang is currently a Ph.D. student in Prof. Huan-Tsung Chang’s group at National Taiwan University. He received his B.S. degree from the department of chemistry at National Taiwan University in 2012. His research interests focus on synthesis of functional nanoparticles for developing highly sensitive optical systems. Zhiqin Yuan is currently a postdoctoral fellow at the Department of Chemistry, National Taiwan University. He obtained his Ph.D. from College of Chemistry and Chemical Engineering, Hunan University, in 227

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