Functional gold nanoparticles for sensing applications

2.1 Small molecule ligand-functionalized AuNPs

A wide variety of ligands have been incorporated into the surface layer of conventional LSPR-active nanoparticles (typically >5 nm), allowing them to be used in sensing of biomolecules and metal ions.

Meanwhile, the synthesis and functionalization of ultrasmall AuNCs still need some efforts. Many research groups are exploring different reducing agents to synthesize monodispersed AuNCs. In fact, some small molecules (such as glutathione [26], captopril [27]) can also be used to direct the synthesis of AuNCs. For example, Perrault and Chan [28] recently demonstrated the use of hydroquinone as a reducing agent and successfully produced AuNCs. Jin’s group produced glutathione (SG)-protected water-soluble AuNCs [Au₂₅(SG)₁₈] and conclude that Au₂₅(SG)₁₈ clusters should adopt the two-shell structure using mass spectrometry and optical spectroscopy analyses. Moreover, this work recommended that all Au₂₅(SR)₁₈ clusters capped by different types of thiolate ligands investigated in this work (R=G, CH₂CH₂Ph, C₆H₁₃, C₁₂H₂₃) should adopt a common two-shell structure, which benefits future studies of this unique Au₂₅ cluster material. The two-shell structure of Au₂₅(SR)₁₈ clusters not being influenced by thiolate types indicates the particular structural stability of the cluster. The special chiral optical signals from the Au₂₅(SG)₁₈ clusters are demonstrated to be imparted by the chiral glutathione ligands, rather than by the chirality of the Au₂₅ core. Because of the unsatisfactory stability of Au₂₅(SG)₁₈ in solution under thermal conditions, the same group further prepared thermally stable, water-soluble captopril (Capt)-protected Au₂₅(Capt)₁₈ nanoclusters. Both Au₂₅(Capt)₁₈ and Au₂₅(SG)₁₈ nanoclusters show fluorescence centered at ~700 nm and have intriguing chiroptical features. The high thermal stability and distinct optical properties of Au₂₅(Capt)₁₈ render this material quite promising for biological applications.

Very recently, Guo et al. [29] reported a simple, one-pot method for the synthesis of water-soluble, red-emitting, highly fluorescent AuNCs using 11-mercaptoundecanoic acid (11-MUA) as the protecting agent. It was found that the fluorescent AuNCs with a quantum yield of ~6.92% and a large Stokes shift could selectively detect Cu²⁺ in aqueous solution, with a detection limit of about 87 nm. The mechanism of the fluorescence quench of 11-MUA-AuNPs by Cu²⁺ was due to the coordination of Cu²⁺ with the carboxyl group of 11-MUA, which might block the charge transfer from the complex of S-Au to AuNPs. The carboxylic group is a poor donor and Cu²⁺ is a poor acceptor. Knoppe et al. [30] prepared novel camphor-10-thiolate protected AuNCs (Au₂₅(CamS)₁₈), which are characterized by circular dichroism and mass spectrometry. The crude reaction mixture was size-separated using gel permeation chromatography. Au₂₅(CamS)₁₈ shows clear chiroptical properties. In a similar approach, 1-phenylethylthiol was used as a protecting agent and yields a polydisperse mixture of clusters. These results indicated that size distribution was found to be ligand dependent. Other interesting chiral molecule-protected AuNPs have been reported recently using N-isobutyryl-l-cysteine or N-isobutyryl-d-cysteine as the stabilizer [31].

2.2 Polymer- or dendrimer-functionalized AuNPs

The polymer-assisted synthesis of AuNPs has received considerable attention because of the small concentrations of homopolymers and block copolymers capable of stabilizing nanoparticles effectively by steric stabilization. For example, Brust’s group [32] reported a one-step method that led to near-monodispersed AuNPs in the presence of a water-soluble alkyl thioether end-functionalized poly(methacrylic acid) stabilizer. The particle size could be controlled by the molar ratio of Au to the capping ligand, and the particles were readily obtained in both aqueous and nonaqueous solutions. Liao and Hafner [33] replaced the stabilizing surfactant bilayer surrounding the Au nanorods using thiol-terminated poly(ethylene glycol) (PEG). The amphiphilic characteristics of PEG ensures that particles coated with PEG have a high degree of biocompatibility and high affinity toward protein. The use of PEG to modify the surface of AuNPs strongly increases the efficiency of cellular uptake compared with the unmodified AuNPs [34]. In addition to the above novel polymer ligands, some other functional polymers such as polyampholyte [35], linear polymers [36], amphiphilic polymers [37], polyetherimide [38, 39] have been designed and used as good stabilizers for obtaining robust AuNPs probes for detection and biological applications. Gaikwad and Rout [38] group reported in situ generation of silver nanoparticles (AgNPs) in an amorphous polyetherimide film by thermal reduction and solvent-induced chemical reduction has shown fine control on their size and distribution. The polyetherimide’s Ag nanocomposite coating shows poorer barrier protection to the steal in the presence of sodium chloride and humid atmosphere than the polyetherimide alone. Duan and Niew [39] used a ligand-induced etching process in which hyperbranched and multivalent coordinating polyetherimide polymers react with preformed Au nanocrystals to form atomic Au clusters. The Au clusters are soluble in water and are highly fluorescent upon UV light excitation. Mass spectrometry data indicate that the light-emitting species is Au₈.

Dendrimers are also a good type of stabilizer for nanoparticles because they can provide multiple metal-binding functionalities that are different from conventional linear polymers. Using dendrimers as protecting agents for the synthesis of AuNPs have the following advantages [40]: (1) the dendrimer templates themselves are of fairly uniform composition and structure, and therefore, they yield well-defined AuNPs; (2) the dendrimer arms are very effective in preventing nanoparticle aggregation; (3) the terminal groups on the dendrimer periphery can be modified for purposes of nanoparticle solubility in different media; (4) the AuNPs protected by dendrimers are typically very small in size (e.g., 1–5 nm), which will participate in various catalytic reactions. For early examples, Garcia et al. [41] and Esumi and Torigoe [42] studied the formation of AuNPs within poly(amidoamine) (PAMAM) dendrimers carrying sugar, amine, methyl ester, and alkyl groups on their periphery. However, relatively large particles were formed both in the interior and on the dendrimer exterior. Later, Crooks et al. [43] reported highly monodispersed, 1- to 2-nm-diameter AuNCs using OH-containing PAMAM dendrimers as agents. In addition to PAMAM dendrimers, other dendrimers such as poly(propyleneimine) dendrimers [44], oligothia, and dendron [45, 46] have recently been extended as novel ligands for the synthesis of AuNPs. Water-soluble Au₈ nanoclusters have also been prepared by Zheng et al. [47] within hydroxy-terminated second- and fourth-generation (G2-OH and G4-OH) dendrimers. AuNCs fluoresce at various wavelengths that are tunable with the size as well as the type of ligands on the AuNCs surface.

2.3 Biomacromolecule-functionalized AuNPs

There are two methods to modify the surface of AuNPs with biomolecules, that is, through covalent and noncovalent interactions. Noncovalent interactions can provide interesting assemblies of AuNPs with biomolecules by simple physical methods such as hydrophobic-hydrophobic interaction and charge-pairing [48]. To efficiently enhance the specificity and sensitivity of the detection signal, it is necessary to seek suitable methods for the functionalization of NPs with biomolecules as recognition and signal-triggering elements. Thus, it is highly desirable to synthesize aqueous stabilized AuNPs using the covalent methods with biomacromolecules [such as oligonucleotides (ODN), DNA, and protein]. Among them, sulfur-containing ligands are predominant as the most effective functional group for AuNPs due to strong Au-thiol (Au-S) bonding, leading to highly stable AuNPs. In one of the early examples of bionanotechnology, Mirkin et al. [49] and Alivisatos et al. [50] independently reported the creation of ODN-AuNP conjugates through Au-S bond formation. Later, Rosi et al. [51] prepared antisense ODN-modified AuNPs with different numbers of ODN strands per particle. Antisense particle A prepared from a tetrathiol-modified antisense ODN have a lower quantity of ODN strands on the surface of AuNPs than antisense particles B modified with a monothiol species because tetrathiol with a higher binding affinity occupies a larger surface area of the AuNPs. Demers et al. [52] determined the number of thiol-derivatized single-stranded ODNs bound to AuNPs and their extent of hybridization with complementary ODNs in solution using a fluorescence-based method. The method presented here has an important application for optimizing the sensitivity of AuNP-based ODN detection methods. The protein- and DNA-protected AuNPs or AuNCs will be discussed next.

3.1 Detecting heavy metal ions

The development of precise and sensitive metal ion sensors in a solution has always been a major research thrust in the analytical application of AuNPs [53, 54]. The reason is simple – we need a sensor system that we can use to provide on-site and real-time monitoring of metal ions (e.g., Hg²⁺, Cd²⁺, Pb²⁺, Cu²⁺) in applications such as industrial wastewater monitoring, environmental biology, and clinical toxicology [55, 56]. Here, we review various designs of metal ion sensors based on AuNPs and their use for sensitive and selective detection and quantification of metal ions.

3.1.1 Colorimetric sensors using AuNPs

Colorimetric sensors using AuNPs have been widely explored and have important applications in the sensitive detection of metal ions [57–66]. The AuNP-based colorimetric assay does not use organic co-solvents, enzymatic reactions, light-sensitive dye molecules, or sophisticated instrumentation, thereby overcoming some of the limitations of conventional methods. A simple colorimetric sensor is desired because the cost can be minimized and they can be made for portable real-time detection platforms. Chai et al. [57] demonstrated a sensitive colorimetric detection method for sensing Pb²⁺ ions using glutathione (GSH)-functionalized AuNPs (GS-AuNPs). The Pb²⁺ ions were detected by colorimetric response of AuNPs due to the aggregation of GS-AuNPs in the presence of Pb²⁺. The GS-AuNPs showed excellent selectivity for Pb²⁺, and its detection limit was 100 nm. The colorimetric sensor can be used for on-site and real-time detection of Pb²⁺. Liu and Lu [58] reported a colorimetric Pb²⁺ sensor based on the assembly of AuNPs by a Pb²⁺-dependent DNAzyme. However, heating and cooling processes could give rise to color change in the sensor for reporting the target. Liu and Lu [59] further improved the sensor for the fast detection of Pb²⁺ at room temperature, rather than relying on the heating/cooling processes. This improvement is due to the alignment of the nanoparticles in a “tail-to-tail” manner, rather than “head-to-tail”. Moreover, AuNPs size is the key factor that allows a fast color change. Liu and coworkers [60–62] have developed a fast and simple colorimetric sensor for on-site and real-time heavy metal cation detection based on a DNAzyme modification of AuNPs. The sensor has a detection limit of 3 nm for Pb²⁺, which is much lower than the US Environmental Protection Agency limit for Pb²⁺ in drinking water.

Liu and colleagues [63] and Lee and Mirkin [64] have also developed a highly sensitive and selective Hg²⁺ detection assay based on the Hg²⁺-mediated formation of T-Hg²⁺-T base pairing. This is based on Hg²⁺-induced thymine-thymine (T-T) mismatches in DNA-modified AuNPs to form particle aggregates at room temperature with a concomitant colorimetric response. The concentration of Hg²⁺ can be determined by the change of the solution color at a given temperature or the melting temperature (T_m) of the DNA-AuNP aggregates. Significantly, this method can, in principle, be used to detect other metal ions by substituting the thymidine in the study with synthetic artificial bases that can selectively bind to other metal ions. Lee et al. [65] developed a chip-based scanometric method for the detection of Hg²⁺. This approach uses the cooperative binding and catalytic properties of DNA-functionalized AuNPs and the selective binding of a T-T mismatch for Hg²⁺. The sensitivity of this assay is estimated to be 10 nm Hg²⁺, and the assay showed an excellent selectivity for Hg²⁺ among many metal ions.

Wang et al. [66] recently demonstrated that Mg²⁺ could be detected based on EcoRI-modified AuNPs (Figure 1A). In this assay, a specifically designed double-stranded DNA (dsDNA) that contains an EcoRI recognition site and complementary sticky ends was used. The reason that this assay is able to discriminate Mg²⁺ from other metal ions is that the EcoRI could cleave the dsDNA in the presence of Mg²⁺ as a factor (Figure 1B).

Figure 1

EcoRI-modified AuNPs for colorimetric detection of Mg²⁺.

(A) Schematic design of the method. (B) Colorimetric response of the detection system in the presence of different metal ions. Adapted from Ref. [66].

3.1.2 Fluorescent AuNCs as sensors for heavy metal ions

AuNCs (often <2 nm) differ from the above plasmonic nanoparticles (typically >2 nm) in many aspects. Owing to the ultrasmall size, nanoclusters exhibit discrete electronic structure and distinctive properties such as enhanced photoluminescence [6]. The luminescence-based detection methods are often more sensitive than the LSPR response-based detection schemes because the former involves much lower background. The properties of AuNCs are highly sensitive to the number of Au atoms in the nanocluster. Recently, an interesting structure-property relationship between AuNC size and its emission wavelength was reported, that is, the blue fluorescence shifts with decreasing number of Au atoms in the cluster. The fluorescent Au clusters could be synthesized in the solution using several methods: (1) chemical reduction of Au³⁺ ions in the presence of thiol ligands [67–69]; (2) core etching of AuNPs into smaller clusters by etchants such as polymers [70, 71]; (3) template-assisted synthesis inside the cavities of dendrimers [72] and proteins [73].

Bovine serum albumin (BSA), the most abundant plasma protein widely used in applications such as sensing, self-assembly, and imaging [74], was chosen as the model protein for the synthesis of AuNCs. It was recently used to stabilize Au particles with size ranging from nanometers to micrometers. The protein-directed synthesis was adapted to produce human apo-transferrin-stabilized AuNCs with strong emission in the near-IR region for cell imaging and targeting [75]. As described previously, reducing agents are avoided for protein-directed syntheses to improve biocompatibility and retain native protein structure. Xie et al. [76] first developed a simple, “green” method to synthesize highly fluorescent [quantum yield (QY) ~6%] AuNCs using a protein-templated method, which was subsequently adopted by many research groups. The AuNCs consist of 25 Au atoms (Au₂₅) and emit intense red fluorescence (emission 640 nm) (Figure 2). The surface of the cluster is stabilized by a small amount of Au⁺ (about 17%), which should have strong and specific interactions with Hg²⁺. Xie et al. [77] further investigated the sensing mechanism. It was based on the high-affinity metallophilic Hg²⁺-Au⁺ interactions, which effectively quenched the fluorescence of AuNCs (Figure 3). This one-step method is simple, fast, highly selective, and ultrasensitive for Hg²⁺ over other metal ions. It can detect Hg²⁺ ions at concentrations as low as 0.5 nm. Moreover, it can be easily developed as a paper test strip to facilitate routine Hg²⁺ monitoring. Hu et al. [78] observed that the interaction of BSA-protected AuNCs consist of Au₂₅ with Hg²⁺. In this work, ethylenediaminetetraacetate (EDTA) was used in competition with BSA-AuNCs for Hg²⁺. It is noted that the quenched fluorescence of BSA-AuNCs by Hg²⁺ was not recovered when adding to EDTA, which confirmed that the complex formation occurred between BSA and Hg²⁺ by S-Hg bonds and a photoinduced electron transfer process was the primary mechanism for fluorescence quenching.

Figure 2

(A) Photographs of BSA (1) powder and (2) aqueous solution and BSA-AuNCs (3) aqueous solution and (4) powder under (top) visible and (bottom) UV light. (B) Optical absorption (dash lines) and photoemission (solid lines, λ_ex=470 nm) spectra of aqueous solution of BSA (blue) and BSA-AuNCs (red). The inset shows the photoexcitation spectrum of BSA-AuNCs. From Ref. [76].

Figure 3

(A) Schematic of Hg²⁺ sensing based on the fluorescence quenching of AuNCs resulting from high-affinity metallophilic Hg²⁺-Au⁺ bonds. (B) Photoemission spectra (λ_ex=470 nm) and (inset) photographs under UV light (354 nm) of AuNCs (20 mm) in the (1) absence and (2) presence of Hg²⁺ ions (50 mm). From Ref. [77].

Liu et al. [79] prepared BSA-stabilized AuNCs according to the method described by Xie et al. [76]. The BSA-stabilized AuNCs have a high selectivity toward CN^- due to the unique Elsner reaction between cyanide (CN^-) and the Au atoms of AuNCs, which is based on the CN^- etching-induced fluorescence quenching of AuNCs. The relevant mechanism is shown in Figure 4. With this sensor, the lowest concentration to quantify toxic anions CN^- could be down to 200 nm, which is approximately 14 times lower than the maximum level (2.7×10^-6 m) of cyanide in drinking water permitted by the World Health Organization. Furthermore, experimental results show that this fluorescent sensor exhibits excellent recoveries (over 93%). This AuNC-based fluorescent sensor could find applications in highly sensitive and selective detection of CN^- in food, soil, water, and biological samples.

Figure 4

Schematic representation of the AuNC-based sensor for CN^-. From Ref. [79].

Muhammed et al. [80] reported the synthesis of AuNC with an Au₃₈ core using BSA as the etching agent (shown in Figure 5). Luminescence of the novel AuNC is exploited as a “turn-off” sensor for Cu²⁺ ions and a “turn-on”sensor for glutathione detection. Metal-enhanced luminescence (MEL) of AuNCs in the presence of AgNPs has been demonstrated, and a 9-fold maximum enhancement was observed [80]. This is the first report of the observation of MEL from AuNCs. The stability of the clusters in a wide pH range, and their luminescence, solvent stability, and minimum change in the secondary structure of BSA make the AuNC-BSA system very interesting for diverse applications in ion sensing and cell imaging.

Figure 5

(Left) Photoluminescence profiles of BSA (traces i and ii) and BSA protected gold quantum clusters (AuQC@BSA) (traces iii and iv). (Right) photographs of AuQC@BSA solution under white light (A) and UV light (B), confocal luminescence image of the QC (C), and photographs of QC powder under irradiation by white light (D) and UV light (E). From Ref. [80].

Protein-mediated synthesis proves to be quite flexible. Subtle changes to the reaction can yield AuNCs with different emission properties. Inspired by this discovery, there have been many research works on protein-stabilized fluorescent AuNCs involving proteins such as lysozyme (Lyz) [81], lactoferrin (Lf) [81, 82], and pepsin [83, 84].

A new kind of highly fluorescent AuNCs has been synthesized through a protein-directed process in basic aqueous solution using Lyz as the reducing and stabilizing agent [81]. Lyz is a protein of choice for experimentation with bio-nanosystems because of its commercial availability and antibacterial properties. The Lyz-stabilized fluorescent Au clusters have an average size of 1 nm, emission at ~657 nm, and with a quantum yield of 5.6%. The fluorescence could be specifically quenched by Hg²⁺, and thus, the clusters can be used as a sensor for sensitive and selective Hg²⁺ detection. A detection limit of 10 nm has been demonstrated [81] compared with the 0.5-nm limit of detection (LOD) reported by Xie et al. [77]. Similar results were also reported by Lin and Tseng [82], who used Lyz as the reducing and stabilizing agent to synthesize fluorescent AuNCs (denoted as Au-631). The Au-631 clusters were capable of sensing Hg²⁺ and CH₃Hg⁺ through the interaction between Hg²⁺ (or CH₃Hg⁺) and Au⁺ on the Au surface; the LODs for Hg²⁺ and CH₃Hg⁺ were 3 pm and 4 nm, respectively. Au-631 not only provided the first example for detecting CH₃Hg⁺ but also offered a ~330-fold improvement in the detection of Hg²⁺ compared with the BSA-stabilized AuNCs.

Recently, Deng’s group [85] synthesized fluorescent AuNC and AgNC on an eggshell membrane (as shown in Figure 6), which contained the Lyz protein rich in cysteine units. The eggshell membrane is a solid-state platform and offers several advantages, including its unique near-IR emission and being cost-effective and “green”, which allows for many potential applications such as heterogeneous catalysis, surface-enhanced Raman scattering interface, fluorescent patterning, chemical sensing paper, fluorescent surface patterning, and anticounterfeiting.

Figure 6

Schematic representation of an eggshell membrane (ESM)-based multimodal platform for the synthesis of fluorescent AuNC and AgNC. From Ref. [85].

Lf is a multifunctional protein in the transferrin family, which are all iron-binding glycoproteins. Owing to the composition of Lf (34 cysteine residues and 22 tyrosine residues) [86], it could be a candidate for making biocompatible and functionalized AuNCs and can be used for some interesting applications. Xavier et al. [87] synthesized the functional AuNCs and found the capability of the clusters for sensing Cu²⁺ selectively at concentration levels of parts per million. Further studies showed that Förster resonance energy transfer (FRET) occurred between the protein and the cluster (Figure 7).

Figure 7

Schematic of the occurrence of FRET between Lf (NLf) and cluster. Inset shows the photographs of (i) NLf and (ii) Lactoferrin protected gold quantum clusters (AuQC@NLf) (from left to right) taken in visible light (above) and in UV light (below). From Ref. [87].

From the above, it can be found that most of the different protein-stabilized AuNCs have red to near-IR fluorescence originating from AuNCs with an average size of 25 or 38 Au atoms. If smaller AuNCs can be synthesized, they would exhibit blue fluorescence. There is an interesting structure-property relationship between AuNC size and their emission wavelength, i.e., the blue fluorescence shifts with decreasing number of Au atoms in the cluster. Until now, there have been reports that AuNCs preferentially contain a magic number of Au atoms (e.g., 2, 8, 11, 13, 18, 22, 25, 28, 39, or 55), as these are the most stable arrangements of Au atoms [88, 89]. The number of Au atoms in AuNCs drastically influences the emission wavelength. For example, Au₈ NCs give a maximum emission at 450 nm, Au₁₀ NCs at ~490 nm, and Au₁₃ at ~510 nm [47, 83, 90]. The emission wavelength of the red-emitting papain-AuNCs was close to that of BSA-Au₂₅ NCs (λ_em=640 nm) [76] and pepsin-Au₂₅ NCs (λ_em=670 nm) [83].

Recently, an impressive study has been carried out on the pH-dependent synthesis of pepsin-mediated AuNCs that emit blue, green, and red fluorescence from Au₅ (Au₈), Au₁₃, and Au₂₅, respectively, as shown in Figure 8 [83]. Pepsin is a gastric aspartic proteinase that plays an integral role in the digestive process of vertebrates. The red-emitting Au₂₅ NCs were stabilized by pepsin in an alkaline solution. Strongly acidic conditions produced Au₁₃ NCs stabilized by autolytic peptide strands from pepsin with a green fluorescence. To reduce the core size to blue-emitting Au₈ and Au₅, a dramatic jump to higher pH effectively etched the Au₁₃ core to the much smaller size. The pepsin-mediated Au₂₅ NCs were also found to be useful as fluorescent sensors for the detection of Pb²⁺ ions by fluorescence enhancement and the detection of Hg²⁺ ions by fluorescence quenching.

Figure 8

Schematic illustration of the pH-dependent synthesis of pepsin-mediated AuNCs with blue-, green-, and red-fluorescent emission. From Ref. [83].

Glutathione (SG) was also used as a stabilizing biomolecule, producing red emission similar to BSA- and Lyz-AuNCs, but with an emission wavelength further red-shifted. The red shift indicates a larger size of the nanoclusters (~2 nm). Glutathione-protected AuNCs were highly sensitive to the essential biological metal ions Cu²⁺ based on aggregation-induced fluorescence quenching [91]. Wu et al. [92] reported a novel type of Ag⁺ sensor using well-defined AuNCs Au₂₅(SG)₁₈. Their experimental results demonstrated that Au₂₅(SG)₁₈ can be a potential Ag⁺ nanosensor with a detection limit of approximately 200 nm based on the fluorescence enhancement and good selectivity against other 20 types of metal cations. Furthermore, three factors were found to be primarily responsible for the unique fluorescence enhancement in Au₂₅(SG)₁₈ when sensing Ag⁺: the oxidation state change of Au₂₅(SG)₁₈, the interaction of Ag⁰ with Au₂₅, and the interaction of Ag⁺ with Au₂₅(SG)₁₈. This work indicates another potential application of AuNCs and offers new strategies for NC-based chemical sensing and also reveals a new way to influence NCs chemistry for potential applications. Moreover, Wu et al. [93] investigated the potential use of Au₂₅(SG)₁₈ as a fluorescent iodide sensor. The detection limit of 400 nm and excellent selectivity among 12 types of anion (F^-, Cl^-, Br^-, I^-, NO₃^-, ClO₄^-, HCO₃^-, IO₃^-, SO₄^2-, SO₃^2-, CH₃COO^-, and C₆H₅O₇^3-) make Au₂₅(SG)₁₈ a good candidate for iodide sensing. The sensing mechanism is affinity-induced ratiometric and enhanced fluorescence, which has rarely been reported previously and may provide an alternative strategy for devising nanocluster-based sensors.

Pradeep’s group [94] reported a FRET between the metal core and the ligand in Au₂₅(SG)₁₈ using dansyl chromophores attached to the cluster core through glutathione linkers. The dansyl chromophore functionalization of the cluster has been carried out by two different routes. Efficient energy transfer from the dansyl donor to the Au₂₅ core is manifested by way of the reduced lifetime of the excited state of the former and drastic quenching of its fluorescence. The donor-acceptor separation observed in the two synthetic routes reflects the asymmetry in the ligand binding on the cluster core, which is in agreement with the recent theoretical and experimental results on the structure of Au₂₅.

All the reports on the use of Au nanoparticles and nanoclusters have suggested that Au-based sensors are able to sense trace amounts of heavy metal ions. These reported sensors have a great potential for real-time monitoring of concentration of heavy metal ions in the industries, which use a high quantity of heavy metals in catalytic processes.

3.2 Sensing of glucose

The detection of glucose has been widely used as a clinical indicator of diabetes and recently gained more attention in the analytical biochemistry fields [95–98]. To date, electrochemical methods are considered to be useful for sensing glucose because a higher-sensitivity detection can be achieved. Most of the electrochemical methods are based on the use of the enzyme glucose oxidase (GOx), which selectively catalyzes the oxidation of β-d-glucose to glucolactone and reduction of oxygen to hydrogen peroxide. The electrochemical biosensing of glucose is based on two mechanisms: one is the redox reaction of mediators that are attached onto the electrode surface along with the enzyme and the other mechanism is the electrochemical oxidation of enzymatically generated H₂O₂. The flow of electrons released (or required) in this redox reaction can be measured as electrical current. AuNPs serve as excellent biocompatible surfaces for the immobilization of enzymes and proteins because the interaction between amino and cysteine groups of proteins with AuNPs is as strong as that of the commonly used thiols. Thus, amino acids and proteins may be directly immobilized on AuNPs without any modification [99, 100]. Much of the research on biosensors involving AuNPs has been devoted to enzyme electrodes [101, 102]. Among the various strategies followed, a useful and simple way consists of the direct deposition of NPs onto the electrode surface. The introduction of AuNPs has several advantages. First, an electrode covered with a layer of NPs has a much higher surface roughness and thus larger surface area, which leads to higher currents. Second, because of the small curvature of small Au particles, the contact of the Au particle with the enzyme can be more “intimate”, i.e., located in close to the reactive center, which can facilitate electron transport [9, 103]. For example, a glucose biosensor was prepared by the covalent attachment of GOx to an AuNP monolayer-modified Au electrode. Chen’s group [104] demonstrated a novel biocomposite made of chitosan hydrogel, GOx, and AuNPs by a direct and facile electrochemical deposition method under enzyme-friendly conditions for glucose biosensor. The biocomposite provided a shelter for the enzyme to retain its bioactivity even under harsh conditions, and the decorated AuNPs in the biocomposite offered excellent affinity to enzyme. The biosensor exhibited a rapid response (within 7 s) and a linear calibration range from 5.0 μm to 2.4 mm, with a detection limit of 2.7 μm for the detection of glucose. Fan’s group [105] recently coupled AuNPs with horseradish peroxidase (HRP) to assemble catalytic nanoconjugates (HRP-AuNPs) for indirect glucose detection. The results showed that a proper mixing ratio of HRP/AuNPs can significantly improve the catalytic activity for the reaction of glucose, an effect arising from increased spatial coupling between enzymes (Figure 9). This glucose sensor has a wide dynamic range (0.4–80 mm). The LOD was ~0.4 mm (note: normal blood glucose level in humans is about 4–6 mm). The conjugated nanostructure is extremely easy to prepare and thus holds good potential in developing simple, robust, and cost-effective biosensors.

Figure 9

Schematic showing HRP-AuNPs catalyzing the glucose involved cascade reaction [105].

Huang et al. [106] described a new method for a highly sensitive determination of hydrogen peroxide, glucose, and uric acid based on FRET using AuNPs as energy acceptors (Figure 10). The efficient FRET was been demonstrated between tyramide-labeled tetramethyl rhodamine (TMR, as energy donors) and HRP-conjugated AuNPs (as energy acceptors), which attributed to the formation of TMR-labeled HRP-AuNPs or TMR-labeled BSA-AuNPs in the presence of H₂O₂. The conjugation between the tyramide-labeled TMR and protein-conjugated AuNPs in the presence of H₂O₂ initiated the tyramide reaction and in turn served as a bridge to make the distance between the donor and the acceptor short enough for FRET to occur. The results suggest that the FRET system was simple and applicable for the detection of H₂O₂ with a low detection limit. The method was successfully applied for the determination of glucose by coupling with GOx- and uricase-mediated reaction. The established methods are at least one order of magnitude more sensitive than the commercially available methods.

Figure 10

Schematic illustration for the FRET process between tyramide-labeled TMR (donor) and HRP-AuNPs (acceptor). From Ref. [106].

A comparison of the analytical performance of different GOx biosensor designs based on several SAM-modified electrodes shows that a configuration involving colloidal Au bound to cysteamine monolayers self-assembled on an Au disk electrode exhibited a high sensitivity and a long biosensor lifetime in comparison with other GOx biosensors [107]. Figure 11 shows the different strategies for GOx biosensors using tailored AuNP-modified electrodes.

Figure 11

Schemes of different GOx biosensors constructed by means of different tailored AuNP-modified electrode surfaces.

(A) GOx/colloidal Au-cysteamine-AuE; (B) GOx/colloidal Au-cysteamine/cysteamine-AuE; (C) GOx/cysteamine-electrodeposited AuNP-GCE or GOx/3-mercaptopropionic acid (MPA)-electrodeposited AuNP-GCE. Adapted from Ref. [107].

Kwon et al. [108] fabricated high-performance glucose biosensors based on enzyme precipitate coating in AuNP-conjugated single-walled carbon nanotube (SWCNT) network films. AuNPs were decorated in the SWCNT network films using spontaneous reduction of Au salt. The SWCNTs behaved as electron donors and the Au ions (Au³⁺) accepted electrons from SWCNTs, forming nanoparticles on the surface of SWCNTs, which increased the electron transfer rate. Recently, Zargoosha et al. [109] reported a novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H₂O₂ by the effective immobilization of GOx/CNTs/AuNPs in nafion film on graphite support. CNTs and AuNPs offer excellent catalytic activity toward H₂O₂ generation in the enzymatic reaction between GOx and glucose, which would enable the sensitive determination of glucose. Under the optimum condition, the linear response range of glucose was found to be 2.25×10^-6 to 1.75×10^-4 m, and the detection limit was 1.00×10^-6 m. The CL biosensor exhibited good storage stability, i.e., 80% of its initial response was retained after 10 days of storage at pH 7.0. Because the CL system is free of uric acid interference and the sensitivity of the CL permits glucose analysis of sample sizes as small as 10 μl, the method may be applied in both routine clinical analysis and research.

An ultrasensitive electrochemical biosensor for glucose determination was reported based on CdTe-CdS core-shell quantum dot as ultrafast electron transfer relay between graphene-Au nanocomposite and AuNPs [110]. The biosensor showed high sensitivity (5762.8 nA/nm/cm), low detection limit (S/N=3) (3×10^-12 m), fast response time (0.045 s), wide calibration range (from 1×10^-11 m to 1×10^-8 m), and good long-term stability (26 weeks). This high sensitivity could be ascribed to the improvement of the conductivity between graphene nanosheets due to introduction of AuNPs as well as to the ultrafast charge transfer from CdTe-CdS quantum dot to graphene nanosheets and AuNPs due to the unique electrochemical properties of the quantum dot and good biocompatibility of AuNPs for GOx (Figure 12). The as-prepared biosensor provides the best sensitivity among all the biosensors based on graphene or its composites for the detection of glucose up to now. This study also opens up a new challenge and approach to explore the electrochemical features of graphene or its nanocomposites for the potential utilizations.

Figure 12

(A) Procedure for fabrication of GOx/AuNP/CdTe-CdS/G-AuNP/GE. (B) Electrocatalytic oxidation of glucose at the GOx/AuNP/CdTe-CdS/G-AuNP/GE. From Ref. [110].

3.3 Protein analysis

The use of AuNPs for protein analysis/detection is also a very interesting research field. In the past decade, AuNPs/protein conjugates have found increasing applications as bioanalytical, diagnostic, and/or immunohistochemical probes. AuNP-based colorimetric assays for the detection of proteins or protein-protein interactions have been documented elsewhere.

Liu and Huo [111] reported AuNPs with 12-nm diameter as probes for the determination of proteins by resonance Rayleigh scattering (RRS) techniques. In a weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged AuNPs to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), BSA, and ovalbumin (Ova) can bind AuNPs to form larger-volume products (the diameter of the AuNP-HAS conjugates is ~23 nm) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, remarkable enhancement of the RRS, and the appearance of the RRS spectra. At the same time, the second-order scattering (SOS) and frequency-double scattering (FDS) intensities are also enhanced. The binding products of AuNPs with different proteins have similar spectral characteristics, and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 nm for FDS. The scattering enhancement is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity, and the detection limit is 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova. The methods have good selectivity. Thus, the RRS method for the determination of trace proteins using an AuNPs probe has been developed. Because AuNP probes do not need to be modified chemically in advance, the method is very simple and fast.

With its strong light scattering, AuNPs could be an excellent light-scattering enhancer using light-scattering techniques. Huo’s group [112] and Dai’s group [113] demonstrated the proof of concept of light-scattering detections using AuNPs for light-scattering assay of both proteins and DNA targets as illustrated in Figure 13. Using a noncompetitive assay format, a sandwich-type antibody-antigen binding or DNA hybridization was transduced into AuNPs aggregation formation, which was detected by dynamic light scattering (DLS) and subsequently correlated to the target analyte concentration. Due to the extremely strong light-scattering intensity of AuNPs around their SPR band region, this assay can potentially be highly sensitive for biomolecular detection and analysis. It is a single-step, washing-free, and amplification-free process; therefore, it is very easy to conduct and may also be automated. In addition, the assay involves the use of very small volumes of samples and AuNP probes (total of 10 μl or less), which can lead to substantial reduction in cost. High throughput 96- or 384-microtiter plate readers are becoming a standard feature of current DLS instruments, which means the new assay developed in the research can be easily adapted for a high-throughput biomolecular analysis.

Figure 13

Illustration of a one-step homogeneous biomolecular assay using AuNP probes as lightscattering enhancers coupled with dynamic light-scattering detection. From Ref. [112].

The highly specific molecular recognition properties of aptamers have been combined with the unique optical properties of AuNPs for the development of a dry-reagent strip biosensor that enables qualitative (visual-based)/quantitative detection of proteins within minutes. A model system comprising thrombin as an analyte and a pair of aptamer probes is used to demonstrate the proof of concept on the conventional lateral flow test strip by Xu et al. [114] (Figure 14). The assay avoids the multiple incubation and washing steps performed in most current aptamer-based protein analyses. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a portable “strip reader”. The response of the biosensor is linear over the range of 5–100 nm of thrombin, with a detection limit of 2.5 nm (S/N=3). By comparing the analytical performance of the aptamer-based strip biosensor with the antibody-based strip sensor, we can demonstrate that aptamers are equivalent or superior to antibodies in terms of specificity and sensitivity. The sensor has been used successfully for the detection of thrombin in human plasma samples. It shows great promise for use of aptamer-functionalized AuNP probes in dry reagent strip biosensors for point-of-care or in-field detection of proteins.

Figure 14

Schematic illustration of the configuration and measurement principle of the aptamer-based strip biosensor.

(A) Configuration of the biosensor. (B) The principle of visual detection in the presence and absence of thrombin. (C) Quantitative detection with a portable strip reader. From Ref. [114].

Wang et al. [115] developed protein biosensors using DNA-modified AuNPs by combining AuNPs (as the fluorescence quencher) and aptamer (as the probe). Three strategies, Ap-Ad-AuNP, Ap-Im-AuNP, and Ap-Hy-AuNP, were tested. Among the three strategies, the Ap-Ad-AuNP method was the simplest, but its protein detection was not satisfactory. Both the Ap-Im-AuNP and the Ap-Hy-AuNP strategies were more complicated in the preparation of probes, but they offered better performance in protein detection. The results showed that the Ap-Im-AuNP strategy had the highest affinity constant and, subsequently, the most sensitive detection limit. Therefore, under suitable experimental conditions, the Ap-Im-AuNP method was the best for protein detection.

Min et al. [116] developed for the first time a highly sensitive electrochemical lectin protein biosensor using carbohydrate-stabilized AuNPs and Ag-enhancement technique (Figure 15). Mannose-stabilized AuNPs on the sandwich-type complex of the concanavalin A (Con A) serve as the nucleation sites for the Ag-enhancement process and thus greatly amplify the electrochemical signals, which are dependent on Con A concentration. Based on this detection method, the present lectin protein biosensor exhibited a wide linear dynamic range of almost three orders of magnitude for Con A concentration from 0.084 to 50.0 g/ml, with a remarkable detection limit of 0.070 g/ml, which is much lower compared with those obtained with the reported microgravimetric and colorimetric detection methods. The present biosensing method is simple, rapid, sensitive, and inexpensive. Thus, it provides a versatile tool for the analysis of clinically important lectin proteins containing several binding sites.

Figure 15

Schematic illustration of the electrochemical lectin biosensor based on mannose-stabilized AuNPs and Ag enhancement. From Ref. [116].

The combination of optical and electrochemical properties of AuNPs with different detection techniques has been demonstrated in protein determination. Ambrosi et al. [117] synthesized a novel double-codified nanolabel based on an AuNP modified with an anti-human immunoglobulin G (IgG) peroxidase (HRP)-conjugated antibody that allows enhanced spectrophotometric and electrochemical detection of antigen human IgG (HIgG) as a model protein. The detection limit of this novel double-codified nanoparticle-based assay is much lower than those typically achieved by ELISA tests. Using the same detection principle, Zhu’s group also developed AuNPs/CNT hybrid platforms with HRP-functionalized AuNPs labels for a sensitive detection of HIgG as a model protein [118].

3.4 DNA determination

AuNPs are the most widely used nanoparticles for DNA detection for several reasons: simple synthetic procedures are available for obtaining AuNPs with well-controlled diameters, shapes, and optical properties; AuNPs possess extremely high extinction coefficients (e.g., 2.7×10⁸ m^-1 cm^-1 at ~520 nm for 13-nm spherical AuNPs), and thus, slight aggregation may result in an intense color change; the large surface area of nanoparticles allows hundreds of capture probe DNAs to be loaded.

The aggregation of AuNPs leads to a new absorption band at longer wavelengths. The first reports by Mirkin et al. [49] focused on the detection of single-stranded DNA (ssDNA) by using two types of AuNPs, both coated with a complementary strand to a portion of the target ssDNA. Upon hybridization, the Au colloid changed its color from red to blue. The milestone discovery by Rosi and Mirkin [119] was that the red-to-blue color change from AuNP aggregation pointed out the possibility of using AuNPs as a DNA detection agent. Later, extensive investigations by Mirkin and coworkers were carried out to detect DNA [120–124]. In these experiments, two populations of Au nanospheres are functionalized with two noncomplementary strands of ODNs. When a solution mixture is exposed to target DNA that is complementary to both sequences of surface-bound DNA, the target DNA will bind to both nanoparticle probes via DNA hybridization and induce nanoparticle aggregation (Figure 16). Aggregation-induced SPR red shift, as visualized by the solution color change from red to blue, offers the capability to detect low concentrations of DNA using simple UV-vis spectroscopy. The signal for hybridization was governed by the optical properties of the nanoparticles, which depended in part on their spacing within the aggregate. Although the detection system was not optimized, it was able to detect 10 fm of target DNA. After the milestone work from Mirkin’s group, DNA-functionalized AuNPs have become popular sensing materials for DNA detection. ODN-functionalized AuNPs also exhibit sharper melting transitions and higher melting temperatures than the corresponding fluorophore-labeled ODN [125, 126], which made ODN-AuNPs particularly attractive and such ODN-AuNPs have become a widely applicable DNA probe. The ODN functionalization of AuNPs is usually achieved through covalent binding or electrostatic interactions [127]. Simple colorimetric hybridization assays that exploit the color change caused by nanoparticle aggregation can be designed and thiol-linked ODN-modified AuNPs represent inexpensive and easy ways to synthesize nanoparticles that can be used for colorimetric detection of DNA targets [128].

Figure 16

Schematic illustration of DNA detection based on hybridization-induced AuNP aggregation.

The cross-link aggregation of ODN-functionalized AuNPs in the presence of complementary target DNA results in a change of solution color from red to blue [119].

Gao et al. [129] recently developed a novel approach for DNA detection using label-free citrate-protected AuNPs coupled with DLS. Most previous work on DNA detection involving the use of AuNP requires the conjugation of an ssDNA probe attached to AuNPs. The assay developed by Gao et al. is based on the different binding affinity of ssDNA and dsDNA to citrate-protected AuNPs. ssDNA can bind or adsorb to AuNPs through Au-N bonding between AuNPs and the base moiety on DNA to form a protective layer on the AuNP surface. Citrate-protected AuNPs tend to aggregate at a salt concentration higher than 10 mm due to the disruption of the electrical double layer. When the citrate-AuNPs are protected by an ssDNA layer, the AuNPs are well stabilized and remain monodispersed in high-salt-content solution. In contrast, when an ssDNA probe is hybridized with a target DNA to form a dsDNA, the dsDNA loses its binding affinity to citrate-AuNPs. Therefore, citrate-AuNPs are not protected by dsDNA molecules against salt, and thus, aggregation occurs. The detection limit can reach the 10-fm range. A recent side-by-side comparative study conducted by Pylaev et al. [130] on DNA sequence detection also confirmed that the dynamic light-scattering detection is significantly more sensitive than the colorimetric assay.

The interparticle aggregation of functionalized AuNPs, caused by probe-target hybridization, can occur through cross-linking and non-cross-linking events. Jian and Huang [131] reported a colorimetric detection of DNA sequences using a non-cross-linking approach. They found that the spatial distance between AuNP and thrombin (i.e., a serine protease involved in a multitude of biological processes) has a strong influence on the catalytic activity of thrombin. This effect can be harnessed to develop complementary colorimetric techniques for DNA assays. As shown in Figure 17, DNA-modified AuNPs are mixed with thrombin and a thrombin-binding aptamer. Upon addition of fibrinogen-modified AuNPs, thrombin is able to cleave fibrinogen into many fragments, resulting in the formation of particle aggregates. However, in the presence of a complementary target DNA, thrombin and AuNPs form a sandwich structure with the target DNA. Owing to the steric hindrance and the electrostatic charge of the particles, the biological activity of thrombin is substantially suppressed.

Figure 17

Schematic representation of colorimetric DNA detection based on enzymatic activity modulation. Adapted from Ref. [131].

Other detection techniques have also been applied in the AuNP-based DNA assay for improving detection sensitivity and selectivity [112, 132]. It has been further reported that the DNA detection can be done by directly monitoring the light-scattering change of the assay solution. According to Mie or Rayleigh scattering theory, the scattered light intensity increases with increased particle size. For Rayleigh scattering, the scattering light intensity is proportional to the sixth power of the radius of the particle. When AuNPs form clusters in solution because of target analyte binding, the scattered light intensity could increase dramatically. This is the reason why light scattering-based detection techniques are generally far more sensitive than light absorption-based assays. Du et al. [133] demonstrated the detection of HIgG and DNA using resonance light-scattering technique of an AuNP-modified structure-switching aptamer. Dong et al. [134] reported a significant sensitivity enhancement in the electrical detection of DNA hybridization in SWCNT networks (SNFETs) via the introduction of reporter DNA-AuNP conjugates in the hybridization step. With detection limits in the femtomolar range, SNFET-based biosensors and immunosensors may be adapted to the detection of a variety of biomarkers for applications ranging from femtomolar diagnostics to in vitro diagnostics.

Liu et al. [135] calculated the interaction of water-soluble Au₅₅(Ph₂PC₆H₄SO₃H)₁₂C_l6 (~1.4 nm) with natural DNA (Figure 18). The molecular modeling calculations showed that interaction exists between bare Au₅₅ clusters and the major grooves of B-DNA in aqueous media under the experimental conditions, which behave differently from that metal nanoparticles organize along the DNA double strands by electrostatic interactions. Although B-DNA could be transformed into A-DNA under the ultrahigh vacuum conditions in the electron microscope. The ultrahigh vacuum conditions lead to the loss of water from B-DNA, resulting in shrinking and reduction of the dehydrated major groove height from ca. 1.4 to 0.7 nm [136]. One might expect that the shrinking process is avoided due to the presence of the 1.4-nm Au spheres or, alternatively, the elimination of the clusters might occur. Instead, the 1.4-nm spheres are degraded to 0.7-nm clusters (corresponding to Au₁₃) that form stable complexes with the water-free A-DNA. Interstrand interactions lead to the wire-like structure.

Figure 18

(Left) Modeling of the interaction of bare Au₅₅ clusters with the major grooves of B-DNA. (Right) Molecular modeling of Au₁₃ clusters in A-DNA. From Ref. [135].