Recent advances in the bioanalytical and biomedical applications of DNA-templated silver nanoclusters

Highlights

• DNA-AgNCs have displayed advantages including ultra-small size, solubility, stable fluorescence emission.

• The applications of DNA-AgNCs to biosensing were summarized.

• The applications of DNA-AgNCs in bioimaging and therapy were summarized.

• Potential opportunities and challenges regarding this field is discussed.

Abstract

Silver nanoclusters stabilized with DNA scaffolds, with excellent physical and chemical properties, have emerged as a versatile tool in biomedical systems. Silver nanoclusters as a late-model nanomaterial with a small size less than 2 nm shows good stability and strong fluorescence. By using DNA with diverse sequence and structures, soluble DNA-templated silver nanoclusters are facilely prepared. They possess tunable fluorescence emission and are suitable with multifunctional designs. Extensive efforts have been put into the application potential of this biocompatible material for biosensing, bioimaging and therapy. Here we are committed to outline the recent advances of DNA-templated silver nanoclusters in biomedical application. The future prospect and challenges are then discussed considering rising progress. Though there is still a long way for illuminating the mechanism behind traits and conducting further clinical trials, we believe DNA-templated silver nanoclusters’ unprecedented unique properties will finally benefit medical progress.

Introduction

Metal nanoclusters (NCs) composed of several to a few hundred metal atoms have attracted a great deal of attention. Different from metal nanoparticles (>2 nm), the small size of metal nanoclusters (sub-nanometer to ~2 nm) which approaches the Fermi-wavelength of electrons to endow them with discrete energy levels and special molecule-like properties [1,2]. Being regarded as a bridge between molecular materials and nanoparticles, metal nanoclusters such as silver (Ag), gold (Au) and copper (Cu) nanoclusters have been extensively applied in biosensing, bioimaging, delivery, immunostimulatory agents and antibacterial agents by utilizing their unique features including biocompatibility, high stability, tunable fluorescence, facile synthesis and large strokes shift [[3], [4], [5], [6], [7], [8], [9], [10], [11]].

Silver nanoclusters (AgNCs) offer a dynamic role as ultrabright fluorescent. DNA-templated metal nanocluster was first synthesized with silver by Dickson's group [12]. Since then a tremendous amount of progress has been made on the synthesis, principle of fluorescence and application strategies of DNA-templated silver nanoclusters (DNA-AgNCs) [13,14]. Compared with another emerging DNA-templated copper nanoclusters (DNA-CuNCs), DNA-AgNCs show a wider range and more adjustable fluorescence wavelength. Moreover, the photostability and biocompatibility of DNA-AgNCs make them more applicable to biological systems [15]. By using a rationally designed DNA sequence, significant fluorescence of DNA-AgNCs can be retained for one year [16], which is much longer than the fluorescence time DNA-CuNCs can maintain. Also, AgNCs are brighter the AuNCs and their lower raw material price and richer earth stock give them advantages over AuNCs [17]. AuNCs provide longer emission wavelength and are mostly used as an energy acceptor, whereas AgNCs are utilized as an energy donor. However, AgNCs provide high quantum yield thereby show much admissibility. DNA-AgNCs as considerably stable fluorophore have better fluorescence properties than organic dyes and lower toxicity, smaller size than quantum dots [5]. They efficiently avoid the sophisticated and time-consuming labeling process and further extend the application scale to electrochemical sensors [18]. The derived nucleic acid conjugated products are gained easily that makes the applications much more feasible. Wide practical application of DNA-AgNCs should firstly be attributed to their tunable spectral region from purple to near infrared [[19], [20], [21]]. Classified by the type of DNA template, ssDNA [22], dsDNA [23], triple-stranded (ts) DNA [24], hairpin loop [25], G-quadruplex [26] and i-motif [27] DNA display various fluorescence emission wavelength and intensity [28]. Among them, ssDNA and hairpin loop are the most frequently used type based on their flexible structures and easy construction. A novel DNA-AgNCs synthesized with coiled DNA sequences exhibited yellow and red fluorescence emissions, which greatly facilitate the application of DNA-AgNCs as molecular beacons (MBs) [29]. A nanocluster beacon (NCB) system was first established by Yeh et al. It was found that the red fluorescence of DNA-AgNCs could be enhanced up to 500-fold with a signal/background ratio of 175 when placed in proximity to guanine-rich DNA sequences [30]. Besides, multicolor NCB system was also reported as a complementary palette. Both the alteration of the cluster-nucleation sequence and linker sequence could result in a spectrum-shift [31,32]. Utilizing DNA-AgNCs based NCB system, biomedical analysis for low concentration molecules become much more feasible.

On account of their superior fluorescence characteristics, a growing number of strategies have been conducted with DNA-AgNCs for biomedical application. Hybridization chain reaction (HCR), catalyzed hairpin assembly (CHA) and functional enzymes were used for signal amplification [33,34]. Au Nanoparticles assisted ssDNA-AgNCs as well as DNA-templated Au–Ag/Cu–Ag bimetallic nanoclusters were built for electrochemical probes and ions detection [18,35,36]. In this review, we will summarize the recent advancement of DNA-AgNCs in the biomedical application including biosensing, bioimaging and therapy, which provide an insight into the future prospects of this fascinating nanomaterial.

The emission of DNA-AgNCs ranges from violet to near-infrared (NIR) region. Basically, the outcome depends on the experimental conditions and DNA ligands. The pH, temperature, buffer, or salt, as well as sequences and length of DNA and presence of metal ions, influence the fluorescence emission [37]. As proper adjustment of experimental conditions is delicate, preferences towards DNA template modifications are observed. Structurally, various ligands have been utilized to increase the stability of metal NCs and to protect them from oxidation and aggregation. For example, biopolymers, synthetic polymers, dendrimers, thiol-compounds, and inorganic matrices have been used [38].

Currently, the preparation of DNA-AgNCs involves two methods: direct preparation and the cluster-shuttle approach utilizing a template DNA, a Ag salt and a strong reductant. AgNO₃ and NaBH₄ are most used Ag salt and reductant, respectively. In the first approach, phosphate or acetate buffer is used with an optimal pH [39]. In the case of cluster-shuttle method, the salt is first mixed with 3-(2-aminoethylamino)propyl-trimethoxy silane (APTMOS) in methanol and later poly acrylic acid (PAA). Although direct preparation is much more favorable than the cluster-shuttle approach, the later one is sensitive in terms of pH response and increases the number of clusters proportionately (micro-to milli-litres).

In the mixture of Ag salt and template DNA, a strong interaction with affinities more than 10⁵ M⁻¹ between Ag⁺ and DNA bases is formed, thus nucleation of neutral clusters occurs. The binding between Ag⁺ and DNA bases is assumed to occurr in the position of N7 of purines and N3 of pyrimidines. It is demonstrated that the affinity of cytosine and guanine is much stronger than adenine and thymine hence the first two are preferred to incorporate in the design of DNA templates [40]. The properties of sugar-phosphate backbone are also significant for the synthesis of DNA-AgNCs. A backbone with high flexibility provides simple folding of the template. This can be achieved by introducing DNA-RNA chimera scaffolds as an extra –OH group of ribose sugar intensifies dipolar and steric interactions [41]. This particular feature secures the high fluorescent property of Ag-NC emitter. Additionally, a backbone containing phosphorothioate (PS) also influences the fluorescence emission of AgNCs. Next, a number of Ag-DNA complexes form more condensed silver adducts followed by the agglomeration of Ag-DNA clusters in which multiple Ag–Ag bonds are produced [42].

The optical characteristics are governed by the shape of clusters that are supported by the DNA template. It is postulated that the rod-shaped cluster core, the bending angle of Ag–Ag chain and the affinity of Ag⁺ for template DNA control the color of emission [14]. The excitation wavelength (λ_ex) can be measured by using a short path length and applying low concentration of DNA. Using UV ranging from 260 to 270 nm, the DNA-AgNCs can be excited and imaged with a UV transilluminator. Interestingly, the quantum yields and fluorescence flow can be enhanced by molecular crowding [43].

Dickson's group showed the response of electronic transition: for the 12-base oligonucleotides, after the binding of Ag to base, the absorption maximum (λ_max) shifts from 257 to 267 nm. When Ag⁺ is reduced, the λ_max reaches 256 nm followed by enhanced molar absorptivity, which is explained by the formation of overlapping electronic bands for small Ag clusters. So, Ag clusters gradually grow resulting in absorption in visible range, and the λ_max shifts to 262 nm. Following the addition of BH⁴⁻ the λ_max reaches 426 nm after 9 min. The absorbance decreases gradually in 12 h and displays an absorption range between 424 and 520 nm. The observed fluorescence for the DNA-AgNCs lies at approximately 630 nm upon excitation between 240 and 300 nm having the intensity maximized at 260 nm excitation. It is postulated that the emission is governed via energy transfer (ET). The AgNCs follow similar photoluminescence theory of AuNCs; however the energy accepting capability of AgNCs is ill-defined. A study has demonstrated that the ET process provided by AgNCs is Förster resonance energy transfer (FRET)-based [44]. It is showed that AgNCs can be functionalized with different photophysical properties and act as energy acceptors [45]. Thus, two energy-donors have been proposed based on FRET exploiting the off-on or ratiometric fluorescence signaling [46]. Furthermore, compartmentalization of donor and acceptor has been shown to alter the FRET process by suppressing the signal [47].