DNA Functional Gold and Silver Nanomaterials for Bioanalysis - ACS

Chapter 12

Downloaded by UNIV OF ARIZONA on November 27, 2012 | http://pubs.acs.org Publication Date (Web): November 26, 2012 | doi: 10.1021/bk-2012-1113.ch012

DNA Functional Gold and Silver Nanomaterials for Bioanalysis Wei-Yu Chen, Yen-Chun Shiang, Chi-Lin Li, Arun Prakash Periasamy, and Huan-Tsung Chang* Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan *E-mail: [email protected]

Deoxyribonucleic acids (DNA) exhibit many predominant capabilities such as specific binding affinities, catalytic activities, and/or chemical stability. On the other hand, gold and silver nanomaterials (NMs) possess unique sizeand shape-dependence optical properties, large surface area, biocompatibility, and high stability. These properties have enabled the extensive use of DNA with gold or silver NMs in optical biosensors. DNA-conjugated gold nanoparticles have become most popular optical probes for various targets, with advantages of high sensitivity and selectivity. Fluorescent DNA-templated silver nanoclusters (DNA–Ag NCs) have features of molecule-like optical properties, easy preparation, and good biocompatibility. In this chapter, we highlight the synthesis of water-soluble DNA-functionalized gold and silver NMs, and their optical properties and applications in bioanalysis and cell imaging.

Introduction Nanomaterials (NMs) have sizes in the range of 1–100 nm, which have become one of the most fascinating materials in the past ten years (1–3). They possess strong size- and shape-dependence chemical and physical properties, which are quite different from those of their corresponding bulk materials, mainly because of quantum effect (4). The past decade have witnessed progressive advances in the synthesis, characterization, and application of a variety of NMs, © 2012 American Chemical Society In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


including gold nanoparticles (Au NPs) (5), gold nanodots (Au NDs) (6), quantum dots (QDs) such as CdSe and CdTe (7, 8), magnetic nanoparticles (MNPs) (9, 10), titanium dioxide nanoparticles (TiO2 NPs) (11), silica nanoparticles (Si NPs) (12), carbon quantum dots (C–dots) (13, 14), graphene (15, 16), metal nanoclusters (NCs) (17), and so on. The as-synthesized NMs have been widely employed in many fields because of their unique size- and shape-dependence optical (e.g., surface plasmon resonance (SPR), surface enhanced Raman scattering (SERS), and fluorescence), electronic, magnetic, and catalytic properties, which make them ideal candidates as signaling elements for being sensitive biosensors (18–20). In general, most NMs are prepared through bottom-up or top-down approaches; “bottom-up” approaches involve the self-assembly of small sized structures into larger structures and “top-down” approaches are production of nanoscale structures from large materials (21). Although some of the NMs such as Au NMs have shown their strong interactions with analytes possessing thiol and amino residues, most of them do not provide high selectivity for specific analytes from complicated biological samples (22). In order to prepare functional Au NMs having high affinity towards analytes of interest, recognition elements such as small organic ligands, peptides, proteins, and deoxyribonucleic acid (DNA) have been conjugated to their surfaces through simple adsorption and covalent bonding (23–25). Relative to proteins, DNA is much more stable. More interestingly, some DNA molecules have been found selective for analytes, depending on their sequence and conformation (26, 27). These particular DNA molecules are short sequences of single-stranded DNA (ssDNA), which are called aptamers. Aptamers are commonly identified in vitro from vast combinatorial libraries through a process known as systematic evolution of ligands by exponential enrichments (SELEX) (28–30). Some aptamers even possess enzyme activity, including ribozymes and deoxyribozymes (DNAzymes) that are often coordinated to metal ions such as Mg2+ or Pb2+ as cofactors (31, 32). DNAzymes with the assistance of metal ions providing activity enhancement over 100-fold have been found, revealing their great potential in sensing of metal ions (33, 34). Relative to antibodies, aptamers are more stable and easier to synthesize, and are available at a lower cost, but have fewer applications because not many aptamers are available. Aptamers can be synthesized automatically and easily modified with a mercapto or amino group in the 5′- or 3′-end of the ssDNA. The thiol-aptamers are assembled onto the surface of Au NMs through Au–S bonding (35, 36). Amino group modified aptamers can be easily assembled onto the surface of bare or carboxyl groups modified Au NMs (36). When compared to free aptamers, aptamers that are conjugated with NMs provide the advantages of greater resistance to nuclease digestion and higher affinity toward targets (37, 38). Equilibrium dissociation constants (Kd) of aptamers with their targets such as metal ions, small organic molecules, peptides, proteins, nucleic acids, carbohydrates, or even whole cells are usually in the range of picomolar (pM) to micromolar (µM), close to those of antibodies for antigens (39). Aptamer conjugated NMs are also renowned for biomedical applications (40, 41). For example, TBAs–hTBA29 (a 29-base sequence providing TBA29 functionality, a T3 linker, and a 15-base sequence for hybridization) and hTBA15 (a 15-base sequence providing TBA15 functionality, a T3 linker, and a 15-base 288 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


sequence for hybridization) conjugated with Au NPs that are modified with captured DNA provide enzymatic inhibition of thrombin (42). Among the NMs, Au NPs are most commonly used for the preparation of aptamers functionalized optical sensors, mainly because of their strong SPR absorption with extremely high absorption (extinction) coefficients (108–1010 M-1 cm-1) in the visible region, along with simple preparation, high stability, and biocompatibility. In addition, the surface chemistry of Au NPs is versatile, allowing conjugation with various functional elements (e.g., organic acids, aminothiols, amphiphilic polymers, antibodies, nucleic acids, and proteins) through strong Au–S or Au–N covalent bonding (Figure 1A) or through physical adsorption (Figure 1B) (24, 25). Loss of their recognition to analytes due to a change in conformation and desorption sometimes occur when the aptamers are adsorbed on the surfaces of Au NMs. In order to provide high affinity, covalent bonding of aptamers to Au NMs that had been modified with thiol compounds having a suitable spacer and functional groups is suggested (Figure 1C) (43). However, it is not an easy task and loss of hydrophilicity of Au NMs occurs sometimes. Integrating nanotechnology with functional nucleic acids have opened up new strategies for biomolecules detection (44–47). Most popular sensing mechanism is based on hybridization of DNA bound to the Au NPs with complementary ssDNAs to form double helix strands, leading to crosslinking aggregation and thus the shift in SPR peaks from 520 nm to 650 nm (48, 49). In addition, “non-crosslinking” assays are applied for the detection of analytes based on their induced changes in the surface charge density of Au NPs. Relative to citrate-reduced Au NPs, ssDNA-modified Au NPs are more stable and remain red in color in solution containing NaCl at the concentration above 0.5 M (50). When the ssDNA on Au NPs surface is hybridized with its complementary DNA sequence, aggregation occur through the formation of double helix as a result of reduces in their surface negatively charged density (50). A decrease in surfaces negatively charged density and a change in the aptamer conformation (from random coil to secondary) also occurs in the aptamer conjugated Au NPs (Apt–Au NPs) in the presence of metal ions. Fan and co-workers demonstrated that potassium binding aptamer-modified Au NPs aggregated immediately upon the addition of K+, through K+-induced formation of four-stranded tetraplex (G-quartet) structure (51). In addition to colorimetric assays, Au NPs have been widely used in different kinds of analytical techniques, including fluorescence (52, 53), SERS (54, 55), SPR (56, 57) and mass spectrometry (MS) (58–60). In contrast to Au NPs that absorb light, metal nanoclusters (NCs) that consist of a few atoms present a new type of fluorescent NMs for sensing of analytes of interest (41, 61–65). These metal NCs have molecule-like properties and no longer exhibit plasmonic properties (17, 66, 67). As their size is comparable to the de Broglie wavelength at the Fermi level, NCs display dramatically different optical and chemical properties from NPs with diameters greater than 2 nm (17, 66, 67). The energy levels tend to become closer upon increasing the number of atoms in NCs, resulting in size-dependent optical properties (17, 66, 67). In addition, the electronic close shell and odd-even effects play increasing roles in determining the dissociation energies of NCs; for example the dissociation energies of Ag NCs 289 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


increase from 1 to 3 eV, when the number of Ag atoms increases from 2 to 25 (68–70). Among various metallic NCs, Au and Ag NCs are the most popular, mainly because of their fluorescence properties, biocompatibility, large Stokes shift, and high emission rates (17, 41, 61, 65). Au and Ag NCs that are prepared in the presence of ligands, including phosphine, thiolates, polymers, proteins, and DNA possess fluorescence at different wavelengths with significantly different quantum yields (17, 61, 62, 65, 67). Proteins such as bovine serum albumin (BSA) are commonly used as templates for the preparation of water-soluble, stable, and biocompatible Au NCs (71–75). On the other hand, DNA as templates are used for the preparation of Ag NCs, mainly because mismatched cytosine–cytosine pairs in DNA duplexes are stabilized by Ag+ ions (76–79). In the past decade, we have witnessed numerous DNA-functionalized Au and Ag NMs based sensing systems, including absorption, fluorescence, SERS, and amperometry. This chapter covers the synthesis and optical properties of DNA functional Au NPs and Ag NCs and their applications based on absorption and fluorescence detection modes.

Figure 1. Functionalization of Au NPs through (A) covalent, (B) non-covalent (physical adsorption), and (C) covalent conjugation with a suitable spacer. Figure C is reprinted with permission from reference (43). Copyright 2006 American Chemical Society. 290 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

DNA functional Au NPs (DNA–Au NPs) Preparation and Optical Properties of DNA Functional Au NPs


Preparation Au NPs are usually prepared from Au3+ ions (HAuCl4) with reducing agents such as citrate (80, 81). It is well known that the strength and concentration of a reducing agent are important factors affecting the seeding and growth rates of Au NPs (80, 81). A strong and/or high concentration of reducing agent is used for the preparation of small sizes of Au NPs. For example, NaBH4 and citrate are separately used to prepare Au NPs having sizes smaller and larger than 5 nm, respectively. By controlling citrate concentration, different sizes of Au NPs are prepared. For example, 32- and 56-nm Au NPs are easily prepared by adding 1% trisodium citrate solutions (0.5 and 0.3 mL, respectively) rapidly to 0.01% HAuCl4 solutions (50 mL) under reflux, in which the mixtures react for 8 min. Although citrate ions stabilize the Au NPs, they are readily replaced by thiol derivated DNA through Au–S bonding for further applications. Generally, preparation of DNA–Au NPs is carried out by simply mixing citrate-Au NPs and thiol-modified oligonucleotides in aqueous solution. Relative to citrate-Au NPs, DNA–Au NPs are more stable in solution containing high salt and/or at high temperature. The stability and hybridization efficiency of DNA–Au NPs are highly dependent on the surface density of DNA. At low surface density, DNA tend to form flat structures on the surfaces of Au NPs, leading to low stability and weak recognition ability. Salt aging of DNA–Au NPs in 100 mM NaCl overnight is effective to prepare stable DNA–Au NPs having greater surface density of DNA (49). However steric effects occur when the density of the DNA on the Au NPs surfaces is too high, leading to losses in the sensitivity and reproducibility. In one of our previous studies, we found that the surface density of DNA molecules on Au NPs affect the sensitivity for Hg2+ ions significantly (82). Thus, it is important to select suitable concentrations of DNA and Au NPs to prepare DNA–Au NPs. In order to minimize surface effect and thus to provide high hybridization efficiency, DNA containing a spacer sequence (e.g., 15 bases of thymine) and a recognition sequence is recommended (83). The main advantages of preparation of DNA–Au NPs through covalent bonding include easy control of the density of DNA and stable DNA molecules on the surfaces of Au NPs. Although Au NPs can be simply conjugated with DNA through electrostatic attractions, they are not as stable as covalently bonded DNA–Au NPs in high-ionic-strength solution. In addition, control of the conformation of DNA on the Au NPs surface is difficult.

Optical Properties The optical properties of Au NPs are mainly dominated by SPR, involving the collective oscillation of electrons at their surfaces, in resonance with the incident electromagnetic radiation (84, 85). The SPR absorption wavelengths of Au NPs are dependent on their size, shape, and refractive index; therefore, any changes in surface structure, aggregation, or medium’s compositions may induce 291 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


colorimetric changes in their dispersions. The SPR band observed at 520–530 nm for the Au NPs reveal the sizes ranging from 5–20 nm in diameter (5). Moreover, the SPR band is red shifted upon increasing the size of Au NPs. Unlike spherical Au NPs, Au nanorods and Au–Ag nanorods exhibit two SPR bands; for example, Au–Ag nanorods have a transverse band at 508–532 nm and longitudinal band at 634–743 nm, depending on their aspect ratio (length/width) (86–88). On the contrary to large Au NPs, small Au NPs (

DNA Functional Gold and Silver Nanomaterials for Bioanalysis - ACS

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