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

Trends in Analytical Chemistry 124 (2020) 115786

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Recent advances in the bioanalytical and biomedical applications of DNA-templated silver nanoclusters Jiaqi Xu a, Xuanmeng Zhu a, Xi Zhou a, Farjana Yeasmin Khusbu b, **, Changbei Ma a, * a b

School of Life Sciences, Central South University, Changsha, 410013, China Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 410013, China

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 27 December 2019

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. © 2019 Elsevier B.V. All rights reserved.

Keywords: Silver nanocluster Biosensing Bioimaging Biomedicine Amplification

1. 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 Fermiwavelength 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 [3e11]. 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

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (F.Y. Khusbu), [email protected]. cn (C. Ma). https://doi.org/10.1016/j.trac.2019.115786 0165-9936/© 2019 Elsevier B.V. All rights reserved.

fluorescence and application strategies of DNA-templated silver nanoclusters (DNA-AgNCs) [13,14]. Compared with another emerging DNA-templated copper nanoclusters (DNA-CuNCs), DNAAgNCs 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 [19e21]. Classified by the type of DNA template, ssDNA [22], dsDNA [23], triple-stranded

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(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 clusternucleation 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 DNAAgNCs 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 AueAg/CueAg 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. 1.1. Synthesis and fluorescence mechanism 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 Agþ and DNA bases is formed, thus nucleation of neutral clusters occurs. The binding between Agþ 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 eOH 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 AgeAg 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 AgeAg chain and the affinity of Agþ for template DNA control the color of emission [14]. The excitation wavelength (lex) 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 (lmax) shifts from 257 to 267 nm. When Agþ is reduced, the lmax 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 lmax shifts to 262 nm. Following the addition of BH4
the lmax 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 €rster resonance energy the ET process provided by AgNCs is Fo 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]. 2. Biosensing applications Biosensing including detection of DNA, RNA, protein, enzyme activity, small molecules and ions are the basic application field of silver nanoclusters. They eliminate tedious steps in the traditional methods, while guaranteeing sensitivity and specificity in the identification tactics. 2.1. DNA detection Low-cost, portable, versatile and highly sensitive detection of DNA is of vital importance in pharmacology, disease identification, precision medicine, and other biomedical fields. The unique advantages of DNA-templated AgNCs arise from their alterable emission wavelength. The purple to near-infrared spectral region is obtained by adjusting the base sequence and structure of the DNA template. For example, a single base mismatch on the template can influence the fluorescence emission of AgNCs to a great extent, which facilitates a simple direct detection of single nucleotide polymorphisms (SNPs) [48]. These properties offer adequate flexibility in detection process. While specific binding of nucleic acid and application of advanced sensing methods ensure the specificity and sensitivity, DNA-templated AgNCs play a crucial part in DNA structural analysis [49], detection of sequence-specific DNA, SNP,

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Table 1 DNA-templated AgNCs based biosensor for DNA detection. Target

Method

Linear range

LOD

Sample

REF

p53 gene HBB gene HIV DNA HIV-1 DNA DNA HIV DNA

MB NCB exoIII-assisted recycling amplification exoIII-assisted recycling amplification CHA, NCB HCR

250e2500 nM 0e1000 nM 0e100 nM 0e100 nM 0.5e15 nM 10e200 nM

3.57 nM 1.2 nM 35 pM 0.29 nM 20 pM 1.18 nM

[50] [57] [53] [54] [34] [56]

Werner Syndrome-relevant gene

RCA

0.02e80 nM

8.5 pM

H1N1 and H5N1 H1N1 gene

MB ThT

0e2 mM 10e400 nM

25 nM 10 nM

fetal calf serum human serum human serum cells lysates; human serum / human serum human serum / human serum

disease-related DNA and pathogen DNA. The main features of each detection method are listed in Table 1. SNPs are common single nucleotide genetic variations serving as biomarkers for diseaserelated genes. For instance, mutations in tumor suppressor gene BRCA1/2 involve many SNPs in coding or noncoding sequences, which is a common cause of hereditary breast cancer. Several techniques based on DNA-AgNCs for the detection of deletion or single-base mismatch DNA in BRCA1 gene were successfully built [20,27]. It is not surprising that most DNA detection described here demonstrated a procedure of SNP detection [18,22,27], which further proved application advantages of DNA-AgNCs. For the detection of sequence-specific DNA, utilizing DNA-AgNCs based MB for a turn-on analysis is effective [50]. In this arrangement, a blocking sequence and a sequence for the formation of AgNCs

[58] [22] [62]

constructed the stem part of a hairpin loop. The fluorescence of DNA-AgNCs was quenched by the blocking sequence, until a specific DNA hybridized with the loop and opened the hairpin loop. A late-model label-free method using the intergrowth of the emitter pair was discovered recently (Fig. 1A) [51]. By placing two dark emitters at the termini of DNA with a spacer of decent length in between, the emission intensity was significantly increased. AuNPs were creatively applied here as “nanoquencher” to quench the fluorescence of the emitter pair via surface plasmon enhanced energy transfer (SPEET) process. The specificity was achieved through different inducing properties of ssDNA and dsDNA toward AuNP aggregation. In the presence of target DNA, AuNP aggregation happened and weakened the SPEET process. Thus a red-to-blue color change of AuNPs and a related fluorescence could be

Fig. 1. Schematic illustration of DNA detection: (A) Intergrowth of the emitter pair [51]. (B) Hybridization chain reaction (HCR) light up AgNCs strategy [56]. (C) Catalyzed hairpin assembly (CHA) light up AgNCs strategy [34]. (D) A ratiometric catalyzed-assembly (RCA) amplified NCB system [58]. (E) Quartz crystal microbalance (QCM) based biosensor [60]. (F) A sandwich-type structures formed by DNA-AuNPs [18].

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observed. It achieved a detection limit of 2.5 nM and could be applied to discrimination of single-base mismatched targets. The combination of AuNP colloid and DNA-AgNCs produced both visible color change and bright fluorescence emission. This intergrowth of the emitter pair provided new design concept for DNA-templated AgNCs application. For instance, during the detection of DNA secondary structure, a target DNA was employed which contains two cytosine-rich AgNCs template sequences at both ends and a guanine-rich (G-rich) DNA sequence in the middle segment was constructed [52]. Once the G-quadruplex was formed, it drew two darkish DNA-AgNCs close and produced signal. Many amplification strategies have been developed to accomplish more sensitive detection of disease-related DNA biomarkers or genes. Exonuclease III (exo III)'s speciality of catalyzing and digesting blunt or recessed 30 -hydroxyl termini of double-stranded DNA (dsDNA) without recognizing the specific nucleotide sequence has made it a valid tool in DNA-templated AgNCs related detection process. Exo III employed recycling signal amplification was successfully applied to the detection of human immunodeficiency virus type 1 (HIV-1) DNA. Their detection limits are 35 pM and 290 pM, respectively [53,54]. Taking advantage of exo III and MB, a double recycling amplification for human hemochromatosis gene detection was then explored with a detection limit of 120 pM [55]. It is a matter of fact the enzyme-dependent assays are sensitive to reaction conditions, which makes the operation cumbersome. Consequently, more assays were further focused on attractive enzyme-free and label-free techniques. Later, Liu, Li et al. introduced ssDNA-AgNCs as signal transformation strategies in HCR for the first time [33]. A HCR/AgNCs amplification platform utilizing GO as quencher for DNA detection has been built (Fig. 1B) [56]. The AgNCs template was constructed on the hairpin structure of HCR (H1, H2). Target DNA initiated the strand displacement and hybridization event of hairpins and prevented the AgNCs from quenching through the FRET process with GO. A catalyzed hairpin assembly (CHA) amplified NCB system for nucleic acid assay has been developed by Pan, Liang et al., with a DNA detection limit of 20 pM (Fig. 1C) [34,57]. This label-free and enzyme-free isothermal nucleic acid circuits catalyzed cross-opening of two hairpin substrates by an initiator. The CHA reaction system contained the analyte string as the initiator, two hairpins (H1, H2) and one DNAtemplated AgNCs probe. The analyte could open the kinetically impeded hairpin (H1) via a toehold-mediated strand displacement mechanism, which triggered the continuous opening of hairpin (H2). Finally, the generated H1eH2 duplex formed a capturing site for the DNA-templated AgNCs probe. The approaching of the G-rich sequences enhancer and the DNA-templated AgNCs probe constructed a NCB design and produced enhanced fluorescence. Besides, ratiometric detection was also developed by means of a ratiometric catalyzed-assembly (RCA) amplified NCB system (Fig. 1D) [58]. A convertor sequence which served as a special enhancer was rationally designed. Upon hybridization, it could convert the green-emissive ssDNA encapsulated AgNCs to redemissive AgNCs via spatial proximity effect. Then this NCB system was successfully integrated with CHA amplification circuits for highly sensitive ratiometric DNA sensing. Teng et al. found a novel NCB design distinctive from the traditional NCB based on a G-rich sequence enhancer [59]. The efficiency was greatly enhanced when the nucleation sequence and the enhancer were on the opposite sides. Apart from fluorescence properties, emerging new analytical strategies were also adopted to produce stable and reproducible signals. Zhou, Lu et al. employed AgNCs as amplifiers in the quartz crystal microbalance (QCM) biosensor (Fig. 1E) [60]. First, target DNA was captured by the probe DNA-modified gold chip of QCM sensor. Then a silver cluster formed along DNA through

hydroquinone-induced reductive. By this scheme, 87.5 times larger amplified frequency response of QCM sensor was obtained. DNAAgNCs electrochemical sensors using differential pulse voltammetry (DPV) measurements have also been conducted (Fig. 1F) [18]. AuNPs was used as carriers for capturing a mass of C-rich ssDNAAgNCs to synthesize ssDNA-functionalized AuNPs (ssDNA-AuNPs) nanoprobe. Immobilized thiolated capture probe was hybridized with target DNA sequence (tDNA) and ssDNA-AuNPs to form a sandwich-type structure. With the increasing amount of AgNCs, redox reaction of Ag/Agþ increased the absolute reduction peak current of AgNCs. The assay indicated the feasibility of introducing AgNCs as electrochemical signal transducer. Gene expression profiling and molecular diagnostics require simultaneous detection of different nucleic acid to reduce expenditure, save diagnostic time and cut down burdensome procedure. Instead of carrying out the experiments one by one for different nucleic acid, DNA-templated AgNCs provide a ideal platform for multiplex analysis of DNA, thanks to their wide range and adjustability of fluorescence wavelength. Employing two types of ssDNAtemplated AgNCs, with green emission (507 nm) and orange emission (597 nm), multiplexed analysis of Influenza A virus subtype H1N1 gene DNA and subtype H5N1 gene DNA with low detection limit (25 nM) was realized [22]. The target DNA bound with the loop part of label-free/conjugation-free MBs, thus opened the stem-loop structure and exposed the DNA template. By using diverse DNA template and modulating the excitation wavelength, three infectious disease-related genes HIV, H1N1, and H5N1 could be detected in accordance with different emission color simultaneously [22,61]. Combining AgNCs with Thioflavin T (ThT) by a dumbbell shape structure, a multifunctional fluorescent probe for H1N1 and H5N1 virus gene detection was constructed [62]. In the presence of H1N1 virus DNA, the 30 termnius dark AgNCs approached another split DNA-AgNCs to produce a fluorescent nanocluster dimer. While in the presence of H5N1 virus DNA the 50 termnius G-quadruplex was formed and bound with ThT for a specific fluorescent response. DNA-AgNCs could also be applied to detection of epigenetic changes such as DNA methylation of CpG island [23,63], which is closely related with cancer diagnosis. Unmethylated, methylated and even partially methylated DNA in CpG rich sequences were clearly discriminated based on the quenching ability of methyl groups. It is possible that in the presence of methyl group on C5, cytosine no longer interferes with the DNA-AgNCs formation [23]. A biosensor based on photoelectrochemical (PEC) nanosystem has been developed in which the quenching ability of AgNCs against the CdS QDs is employed. In this system of DNA assay, a MB is used that has two segments capable of hybridizing with both target DNA and label DNA. The MB is anchored on the CdS QDs modified electrode and unfolded by target DNA resulting in close interaction between the label of DNAAgNCs and the CdS QDs electrode surface. An ET is generated that triggers the quenching of CdS QDs and provides sufficient reading for DNA bioanalysis. 2.2. RNA detection Versatile DNA-AgNCs based DNA detection techniques were widely exploited to RNA detection too, such as HCR [33,64,65], CHA [34], NCB system [57] and multiple detection [62], as listed in Table 2. MicroRNAs, are a class of single-stranded, endogenous and non-coding RNAs, typically about 19e25 nucleotides in length produced by digesting process catalyzed by Dicer enzyme. There are an estimated 2500 known miRNAs in humans. By negatively regulating the expression of genes at the post-transcriptional level through target mRNA clearance or suppression of the translation processes, thousands of miRNAs play an important part in diseases.

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Table 2 DNA-templated AgNCs based biosensor for RNA detection. Target

Method

Linear range

LOD

Sample

REF

miRNA-199a miRNA-21 miRNA-141; miRNA-21 miRNA let-7a miRNA-21 miRNA miRNA let-7a AMI-associated miRNAs miRNA-145 miRNA-21

HCR Y-shaped probe G-quadruplex NCB CHA, NCB HCR FRET PET HCR CHA

1.0 fM-0.1 nM 0e1200 nM 1e400 nM; 10e400 nM 0e600 nM 0.75e15 nM 100 pMe0.1 mM 12e300 nM 0.1 nMe8 mM 0.1e1.6 nM 1 aM-104 fM

0.64 fM / 1 nM; 10 nM 22 nM 38 pM 7 pM 6.9 nM 0.06 nM 0.1 nM 0.96 aM

human serum / cell lysates human serum / cell lysates cell lysates cell lysates human serum human serum

[64] [69] [62] [57] [34] [33] [70] [72] [65] [73]

Detection of the dynamically changing levels of miRNAs via DNAAgNCs in response to cellular and environmental signals can greatly benefit disease diagnosis, prognosis and treatment [66]. Dong et al. rationally took advantages of the quenching ability of metal ions Hg2þ to construct an endonuclease Nt.BbvCI-assisted target recycling amplification for miRNA detection [67]. The target sequence, MB probe and assistant probe together formed a Yshape junction structure, which unfolded the haipin MB probe and

released Hg2þ. And then Hg2þ quenched the fluorescence through a d10-d10 metallophilic interaction. In the same research, the detection limit could be as low as 0.6 fM compared with a detection limit of 0.16 nM obtained by simply applying DNA-AgNCs as fluorophore without amplification. By incorporating target-assisted polymerization nicking reaction (TAPNR) and HCR, Yang et al. realized an dual amplification strategy on the sensing electrode (Fig. 2A) [64]. After the target hybridized with a template DNA sequence, DNA

Fig. 2. Schematic illustration of RNA detection: (A) TAPNR and HCR dual amplification strategy [64]. (B) SDA amplification strategy [68]. (C) The locking-to-unlocking system based on fold-back anchored DNA templates [25].

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polymerase and Nt.BbvCI nicking enzyme took turns to play their role and produced massive intermediate sequences. Those sequences were captured on the gold electrode to trigger the HCR process. Hairpin loop H1 and H2 were opened in turns. dsDNA ploymers were thus formed and numerous AgNCs can be generated on the C-rich loop of H1 and H2. Through DPV measurement, the detection limit of target miRNA-199a was 0.64 fM. The significant amplification of the current response could be attributed to the electro-oxidation in the synthetic process of DNA-AgNCs. Strand-displacement amplification (SDA) was also introduced into amplification strategies based on DNA-AgNCs (Fig. 2B) [68]. For example, miR-16-5p and miR-19b-3p were identified as biomarkers to indicate the progression of gastric cancerby constructing hairpin DNA probes with a C-rich sequence as template for AgNCs nucleation and primers with a G-rich sequence for a NCB system. The SDA reaction achieved quick detection of miR-16-5p and miR-19b3p. Upon the presence of miRNAs, the hairpin loop probes were exposed. Then the primers containing G-rich sequence enchancer annealed to the free 30 stem, which triggered SDA reaction and enhanced fluorescence emission at the same time. Under the action of enzymes, target miRNAs were displaced and went back to the next cycling. Two types of hairpin DNA-templated AgNCs were adopted here with red and green emission color for duplex miRNAs Detection. To realize both a strong fluorescence and a high target affinity, a locking-to-unlocking system based on fold-back anchored DNA templates is designed for miRNA detection (Fig. 2C) [25]. Though there are probes made up of a target sensing loop and two C-rich DNA template stem capable of generating strong fluorescence. Utilizing reduced graphene oxide (RGO) as quencher, this Y-shape probe is applied to detection of miRNA in vitro [69]. In the lockingto-unlocking system, a different hairpin loop DNA templates with a target sensing stem connected by a c-rich loop is built. Nevertheless, when the target sensing stem was perfectly complementary, its high thermodynamical stability made it hard to be disrupted by target hybridization with miRNA. The problem can be solved by shorting the opposite strand of target sensing strand, which would lead to a decreased fluorescence in return. After optimizing the length of the opposite strand named a fold back anchor the hairpin loop structure was capable of providing sensitive and selective miRNA detection. Fluorescence resonance energy transfer (FRET) [70], surface plasmon-enhanced energy transfer (SPEET) [71], photoinduced electron transfer (PET) [72] and surface plasmon-enhanced electrochemiluminescence (SPEECL) [73] based methods were also feasible for miRNA detection. For the detection of acute myocardial infarction (AMI)-associated miRNAs group, distance-dependent PET from preformed AgNCS to G-quadruplex/hemin complex was first utilized by Wang et al. [72]. Feng et al. implemented an ultra sensitive detection of miRNA-21 with a detection limit of 0.64 aM, by innovatively utilizing distance dependent SPEECL with the enzyme-free recycling amplification strategy (CHA) [73]. The simplicity, flexibility and sensitivity of DNA-AgNCs based techniques might have an advantage in a certain aspect over widely used conventional methods including RNA blot analysis, microarray, qRT-PCR and sequencing, especially when it comes to identifying abnormal miRNAs in vitro and the spatial distribution of miRNAs in cells [74,75]. The latter will be discussed in the bioimaging section. 2.3. Protein detection Protein is recognized as one of the important biomarkers for cellular events and disease diagnosis. However, due to its low concentration and feature that cannot be amplified, it is hard to

detect protein in many circumstances. In the past years, several fluorescence-based detection methods for protein have been reported. However, these reported methods have some shortcomings such as time-consuming, low sensitivity, poor specificity, requirements of expensive labels and specific antibodies. So there is still challenge in the sensitive detection of protein. In this respect, DNA-AgNCs can offer sensitive and simple outcome. For the weak brightness of DNA-AgNCs, methods based on G-rich sequences [76e79], G-quadruplex [80], and signal amplification [81] were used for enhancement. A label-free fluorescent aptasensor for the detection of human epidermal growth factor receptor-2 (HER2) was developed by Zhang et al. [76]. In the presence of HER2, the highly specific binding of HER2 to HER2-binding aptamer (HApt) caused HApt separating from double stranded DNA templated AgNCs (dsDNA-AgNCs), resulting in the folding of DNA2-AgNCs because of the complementary bases at both ends, which resulted in AgNCs' approach to G-rich sequences, and therefore the enhanced fluorescence intensity. Li et al. first applied DNA-AgMBs in transcription factors (TF) analysis and designed an assay on basis of the switchable fluorescence of AgMBs [79]. In the absence of TFs, under exonuclease III (Exo III) digestion, a double-stranded DNA probe (referred as dsTFs probe) released a single-stranded DNA, which was functioned as a reporter. Then, the reporter continuous consumed the guanine-rich enhancer sequences in AgMBs, triggering downstream Exo III-assisted signal amplification, eventually the fluorescent decreased significantly. On the contrary, in the presence of TFs, the probe was protected by dsTFs from Exo III's digestion and the downstream reaction was blocked, which led to a highly fluorescent state.

2.4. Enzyme activity assay Assessment of enzyme activity is of vital importance due to their regulatory roles in maintaining cellular homeostasis, and it can be disrupted by disease. Genetic or non-genetic factors can be responsible for the aberrant activity of enzymes; therefore, several bioassays have been developed to quantify enzyme activity. In order to solve the shortcomings of traditional detection methods, strategy based on DNA-AgNCs has been designed to detect and measure the activity of several enzymes (Table 3) [82e94].

2.4.1. Uracil-DNA glycosylase (UDG) Uracil-DNA glycosylase (UDG) was one of the enzymes which involved in base excision repair (BER) function as the protector of life via sustaining the integrity of gene, because diverse elements such as external environment and endogenous biological metabolism processes triggered DNA damage, which may result in genetic diseases. Several research developments confirmed that abnormal activity of UDG may induce the base excision repair process to be malfunctioned to a certain extent and may be attached to many diseases. Zhang et al. reported a UDG assay platform by combining the two-tailed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and T7 RNA polymerase, and it can produce a large number of microRNA sequences, and entropy-driven reaction (Fig. 3A) [82]. In addition, the UDG activity can transfer to DNAAgNC fluorescence by using a well-designed AuNP probe. Two significant advances are included in the well-designed assay platform: First, two-tailed RT-qPCR-aided recycling amplification strategy can reduce the background and reduce the complicated operation. Second, the label-free AgNC-DNA fluorescence intensity is quenched by AuNPs, which minimize the fluctuation of background more easily.

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Table 3 DNA-templated AgNCs based biosensor for protein and enzyme detection. Target

Method

Linear range

LOD

Sample

Ref

HER2 PSA PSA PTK7 NF-kB p50 UDG T4 PNK Dam MTase ALP Chymotrypsin DNase I ADA

G-rich sequences G-rich sequences PET C-rich sequences exoIII-assisted signal amplification RT-qPCR G-rich sequence C-loop G-rich sequence GO/FRET GO Aptamer

8.5e225 fM 2e150 ng/mL 0e100 pM 30 pMe2 nM 30 pMe1.5 nM 0.001e0.01 U/mL 0.01e12.5 U/mL 0.4e20 U/mL 1e800 U/L 0e50.0 ng/mL 0e10 U/ml 0e200 mM

0.0904 fM 1.14 ng/mL 10 pM 12 pM 10 pM 0.1 mU/mL 0.01 U/mL 0.1 U/mL 1 U/L 3 ng/mL 0.10 U/ml 2.7 mM

human serum human serum / HeLa cell DLD-1 cell HeLa cell PC-3 cell human serum human serum human serum bovine urine human serum

[76] [78] [80] [81] [79] [82] [84] [87] [89] [91] [92] [93]

Fig. 3. Schematic illustration of protein detection: (A) The two-tailed assay platform detection strategy [82]. (B) AuNPs/ERGO hybrids and hybridization chain reactions (HCR) amplification strategy [86]. (C) The kissing complexes-induced aptasensor detection strategy [93]. (D) The target-assisted isothermal exponential amplification strategy [94].

2.4.2. Telomerase As a ribonucleoprotein reverse transcriptase, Telomerase can maintain the length and activity of telomeres, keeping chromosome terminal from degradation and maintaining chromosome stability. However, cells are able to proliferate indefinitely under the condition of cellular telomerase overexpression, which would make a normal cell a potential cancer cell. Therefore, telomerase is considered as a cancer marker and developing a sensitive and reliable sensing platform for detecting telomerase activity is of great value. To this end, Peng et al. reported a study on the fluorescence enhancement of DNA-AgNCs induced by metal ions [83]. The enhancement is greatly dependent on the primary sequence and secondary structure of the DNA strands. Thus, strategy based on a label-free AgNCs-based MB was designed to detect the activity of telomerase. Nonfluorescent MB-AgNCs in phosphate buffer emit dramatic red fluorescence when the hairpin structure of MB was opened. Telomerase primer could be elongated by telomerase, which resulted in unfolding of MB in a strand-displacement reaction. On the basis of the different brightnesses of AgNCs produced by the two DNA templates, telomerase activity could be detected. The MB-AgNCs sensing platform provided a simple and low-cost method to detect telomerase activity and

showed great potential in the construction of cost-effective probes for biomolecular detection. 2.4.3. T4 polynucleotide kinase As a kind of DNA repair enzyme, T4 polynucleotide kinase (T4 PNK) can catalyze the transfer of phosphate group of ATP or other nucleoside triphosphates to the 50 -OH terminal of single strand DNA, double strand DNA or RNA. It has also been confirmed that the aberrant T4 PNK activity can result in the disorder of phosphorylation level in nucleic acids, which are associated with many kinds of human diseases including Rothmudthomson syndrome, Werner syndrome, and Bloom's syndrome. Li et al. reported a turn-off fluorometric method on the basis of the use of DNA-templated AgNCs [84]. DNA probes with terminal 50 hydroxyl groups were used as substrates for DNA phosphatases. If subsequently treated with Lambda exonuclease (l exo) and T4 PNK, the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated, which leads to a sharp decrease of fluorescence caused by the proximity of the G-rich region and the C-rich region in the AgNCs. This makes it possible to determine the activity of T4 PNK by fluorescence kinetics.

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Fig. 4. Schematic illustration of biothiols detection: (A) Quenching effect of biothiols by energy transfer from DNA-AgNCs to thiol group [97]. (B)The enhancement of ECL intensity of triplex DNA by biothiols [24]. (C) NCB system for an ultra sensitive Cys detection [98]. (D) Ratiometric detection of biothiols [99].

2.4.4. DNA methyltransferase DNA methyltransferases (MTases) assist DNA methylation, which is a vital long-term gene silencing mechanism and involved in gene regulation. Many studies have reported that abnormal expression of DNA MTase is closely related to various diseases and bacterial infections, thus DNA MTase shows potential in drug screening and anticancer treatment, especially in early diagnosis of tumors. The aberrant DNA MTase activity could be regarded as markers for early diagnosis of cancer and other diseases. A tripleamplified biosensor was designed for the purpose of ultrasensitive methyltransferase (Dam MTase) activity detection and inhibitor screening on basis of in-situ synthesized silver nanoclusters (AgNCs)which is considered as signal probes to provide amplified current signal coupling with gold nanoparticles/electrochemical reduced graphene oxide (AuNPs/ERGO) hybrids and HCR amplification strategy (Fig. 3B) [86]. For biosensor preparation, the AuNPs/ ERGO hybrids were firstly generated on the electrode surface, then the dsDNA structures which comprised the specific recognition sequences (50 -G-A-T-C-30 ) of the restrictive endonuclease DpnI and the Dam MTase were formed on the electrode. In the presence of Dam MTase, partial dsDNA structures could be methylated on the electrode which could be digested by DpnI and could not undergo HCR process. The unmethylated dsDNA structures underwent HCR process and further hybridized with DNA1 and DNA2 to form dsDNA superstructures, which was an effective template for AgNCs in-situ synthesis. The signal output for Dam MTase detection was the oxidation peak current. Due to its high sensitivity, this biosensor had potential in clinical diagnosis and inhibitor screening. In addition, Kermani et al. used a cytosine-rich DNA loop (C-loop) as a signal indicator for the detection of M.SssI DNA MTase [87], and Liu et al. designed a hairpin-shaped DNA probe with 50 -Crich/G-rich-30 tails for Dam MTase detection [88]. 2.4.5. Alkaline phosphatase Alkaline phosphatase (ALP) is a type of enzyme that is able to catalyze the removal of phosphate groups from various phosphorylated substrates, such as small molecules, nucleic acids and proteins. Studies have shown that ALP plays an important role in many physiological processes including cell metabolism and signal transduction. Since the level of serum ALP is closely related to

numerous human diseases, it is commonly used as a biomarker for clinical disease diagnosis. Ma et al. have presented a method for the detection of ALP activity based on the design of a G-rich DNA lightup DNA-AgNCs probe and exonuclease cleavage reaction [89]. In addition, Ma et al. have portrayed a new copper-mediated on-off switch for detecting ALP based on DNA-AgNCs [90]. 2.4.6. Adenosine deaminase The analysis of adenosine using the kissing complexes-induced aptasensor is depicted schematically in (Fig. 3C) [93]. Zhang et al. reported on the integration of AgNCs and riboswitches for the development of a kissing complexes-induced aptasensor (KCIA). They apply the tunable riboswitches properties of this strategy to demonstrate the multiplexes analysis of adenosine and adenosine deaminase (ADA). A new strategy for the determination of proteins using an exponential amplification reaction coupled with fluorescent DNA-scaffolded silver nanoclusters is reported (Fig. 3D) [94]. 2.5. Small molecules detection DNA-AgNCs could also be applied in detection of small molecules including biomolecules and synthetic molecules [95e109]. Generally, small molecules can affect the probe itself, and the detection mechanism of small molecules includes two paths. (1) Target molecules directly affect the properties of AgNCs. (2) Target molecules alter the structure of DNA template to indirectly modulate the properties of AgNCs, which are often accomplished by constructing a aptamer-functionalized fluorescent AgNCs [85,86]. Biothiols including cysteine (Cys) and glutathione (GSH) were detected in the first path. They directly quenched the fluorescence intensity of DNA-AgNCs and changed their microenvironment (Fig. 4A) [97]. Since increasing concentration of Cys and GSH induced the formation of AgeS bond, energy transfer from DNAAgNCs to thiol group happened and the secondary structure of the DNA template was changed. Further investigation suggested that larger nanostructures were formed and the average lifetime of DNA-AgNCs was decreased. Wu et al. investigated the stable electrochemiluminescence (ECL) property of triplex DNA-AgNCs and applied it for detection of L -cysteine (Cys) (Fig. 4B) [24]. Recently, Li

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et al. further introduced NCB system for an ultrasensitive Cys detection (Fig. 4C) [98]. They directly linked a core sequence for AgNCs nucleation to a G-rich aptameric sequence for enhancement. The strategy showed an excellent detection limit of 0.05 nM with a linear range of 0.1e100 nM. Ratiometric detection of biothiols was also realized utilizing the unique quenching property of metal ion Hg2þ (Fig. 4D) [99]. DNA-AgNCs with two fluorescence emission peaks (red and green) were synthesized. Hg2þ as a modulator could quench the red fluorescence emission and increase the green fluorescence emission simultaneously. Cys and GSH, however, had an opposite effect. Based on this, Ired/Igreen was measured and a highly sensitive and selective method was developed. For the first path, nanoclusters sometimes were rationally designed to fit the need of detection [100]. For instance, DNA-templated Au/AgNCs were synthesized for ascorbic acid (AA) detection on account that the reducibility of AA could induce the aggregation of DNA-Au/Ag NC, while having little effect on DNA-AgNCs. ATP detection was based on the second path. As a common crucial small molecule for organism, ATP can induce the change of the secondary structure of DNA, which might be an important reason for an induced fluorescence change of DNA-AgNCs. Recently, Xu et al. found a meaningful phenomenon when using the BT3T3 and BT3T3R DNA which could bind with ATP for the synthesis of ssDNA-AgNCs or emitter pair AgNCs [101]. A dumbbell-shaped DNA template serving for the multiple preparations of AgNCs and CuNPs was designed [102]. Exo I and Exo III were used to degrade the dumbbell-shaped DNA. While in the presence of ATP, their activities were inhibited. 2.6. Detection of ions Detection of related ions is very important in clinical analysis and environmental monitoring. By introducing quencher Hg2þ into DNA-templated AgNCs [110], a turn-off strategy for selective detection of Hg2þ was developed with a detection limit of 4.5 nM [111]. However, fluorescence quenching of other emission wavelength by Hg2þ was also found and applied. A pH and adenosine triphosphate (ATP) concentration-dependent DNA-AgNCs were applied to Hg2þ detection [112]. Recently, a dual weaken exo IIIassisted recycling amplification turn-off method for Hg2þ detection was implemented by Ma et al. [113]. Positively charged AuNPs and Hg2þ were introduced successively as quencher; a DNA duplex was formed through T-Hg2þ-T coordination chemistry. Then exo III digested the duplex and released AuNPs to set on the cycle. Although Hg2þ can only provide a negative readout signal, this dual weaken design realized a low detection limit of 2.3 pM. DNA-AgNCs’ property of adjustable emission wavelength depending on structure was cleverly applied to Agþ detection [114]. Lee et al. found that Agþ might induce a dimeric structure of Cyt 12AgNCs, which would result in a fluorescence change from red to green. Dual color detection was also applied to Pb2þ detection [115]. By combining the Pb2þ-dependent DNAzyme with alterable emission wavelength DNA-AgNCs, Wang et al. established a sensitive platform for metal ion detection. Another accurate Pb2þ detection technique was fabricated based on ssDNA templated AgNCs emitter pair [116]. With AgNCs template at the two termini and a specific Pb2þ aptamer segment placed in the middle, a canonical emitter pair for turn-on detection was constructed. Pb2þ induced the aptamer to form a G-quadruplex structure which could draw two darkish DNA/AgNCs near, resulting in a fluorescence light-up. In general, highly sensitive DNA-AgNCs based ion detection methods are much more inexpensive, simple and convenient than the standard method inductively coupled plasma mass spectrometry (ICP-MS). Meanwhile, the good biocompatibility of DNA-AgNCs make them environmentally friendly.

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3. Bioimaging applications Biomedical imaging as an efficient tool to study the location and size of tumor, track the biodistribution of theranostic agents and diagnosis in the early age has been considered an important way to improve cancer treatment survival. With the development of confocal laser scanning microscopy, fluorescence-based detection method has been well developed. Owing to the strong fluorescence, facile synthesis, versatile procedure and adjustable structure, the properties and strategies of DNA-AgNCs used in biosensing were organically integrated for bioimage in complex circumstances. Thus, DNA-templated AgNCs have become a versatile agent for cell surface biomarker imaging, intracellular nucleic acid imaging and specific type cell imaging. Even magnetic resonance imaging can be modified with the help of DNA-AgNCs [117,118].

3.1. Cell surface biomarker imaging Biomarkers on specific individual cell types are useful imaging and diagnostic indicators. For example, folate receptors (FRs), which are over-expressed in many human cancerous cells are common-used antigens or biomarkers. Cancer cell imaging could be achieved by imaging FRs on cell surface. A wash-free quantification and imaging of FR was conducted via terminal protection assay strategy [119]. Folate-linked DNA probe with a small molecule ligand folate at 30 end and DNA template at 50 terminal was constructed for FR detection and imaging. Exo-1 was used to obtain a wash-free procedure by degrading the free probe and quench the fluorescence. Upon the specific recognition and binding between folate-linked DNA probe with cell surface FR, terminal protection worked to protect the surface-bound probes from exo-1 reaction and realized fluorescence imaging. Another folate-conjugation bioimaging methods have been exploited via single-walled carbon nanotubes (SWCNTs) utilized FRET system [120]. Likewise with the assistance of exo-1 and terminal protection mechanism, the binding between folate with FR served as a switch to control the FRET process from DNA-AgNCs to SWCNTs. DNA-templated AgNCs designed as DNA sequences conjugated with donor molecules were also used for other biomarkers imaging such as mannose receptors which were over-expressed in certain cancer cells [121]. Besides, aptamer-biomarker recognition and immune combination were also feasible strategies. With the application of aptamer-functionalized AgNCs, bioimage using multicolor emissions corresponding to different cell surface biomarkers was developed [122]. AS1411 aptamer, as a common-used, FDAapproved and antiproliferative G-rich DNA sequence was frequently used. The aptamer could not only specifically bind with a shuttle protein, nucleolin, which is expressed in active breast cancer cell surfaces, but also act as an G-rich enhancer for fluorescence emission of the AgNCs. AS1411 aptamer formed G-quadruplex provided guanine bases to bring in high brightness of DNAAgNCs [26]. Based on AS1141 aptamer-nucleolin recognition and antigen-antibody binding, fluorescence imaging and electrochemical techniques were integrated by Cao et al. [123]. AS1141 aptamer was functionalized on ITO electrode to selectively capture nucleolin over-expressed MCF-7 cancer cells. Then the prearranged (anti-MUC 1)-(DNA-AgNCs) conjugate could bind to the mucin protein, which is a transmembrane glycoprotein on breast cancer cell surfaces. The sandwich structure formed on this design provided a dual signal probe for both imaging and quantitative detection of cancer cells. MUC1 aptamer-functionalized AgNCs have also been constructed by Li et al. for MCF-7 breast cancer cells imaging and specific recognition [124].

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3.2. Internalization and stability enhancement for bioimaging in the cell To some extent, biomarkers imaging is equivalent to corresponding specific type cell imaging. However, certain cell imaging as well as intracellular nucleic acid imaging further involve a internalization process of DNA-AgNCs. Though the entry of the DNA-AgNCs probes into the cell automatically via cell endocytosis might work [125]. Internalization assisted with nucleic acid aptamer recognition is the most commonly used. Moreover, cationic polyelectrolyte [126], MnO2 nanosheets [118] and so on are also advanced feasible tools for improving the cell permeability of DNA-AgNCs. Unlike using microinjection or cationic liposomes, these methods take advantages of DNA-nanoclusters and are more convenient. For example, aforementioned AS1141 aptamernucleolin recognition could efficiently induce internalization mainly via nucleolin-mediated endocytosis. Thanks to the great properties of AS1141 aptamer, it was universally utilized in cell image [127,128]. Another cell-specific DNA aptamer, Sgc8c, which could bind human protein tyrosine kinase-7 (PTK 7) and be internalized efficiently was also widely used [129,130]. Utilizing Sgc8c aptamer for specific internalization, a tumor growth marker mRNA TK1 was imaged (Fig. 5A) [129]. Sgc8c aptamer was linked with a poly-cytosine sequence and complementary DNA (cDNA) to form a hairpin structure probe. Upon entering the cell the probe hybridizes with the target mRNA, and an enhanced fluorescence signal as well as spectrum-shift of DNA-AgNCs are observed. The multiplexed color emissions corresponding to target mRNA make a 2channel efficient bioimage possible. Thus, signal to background ratio could be greatly increased for monitoring of relatively low expression nucleic acid in living cells. A novel siRNA delivery and tracing technique was developed with the Sgc8c aptamer involved hairpin structure probe (Fig. 5B) [130]. Streptavidin was functioned as a bridge between the target siRNA and Sgc8c aptamerfunctionalized AgNCs through biotin-streptavidin interactions. Both the siRNA and aptamer were labeled with small molecule biotin, and biotin-streptavidin interactions resulted in the formation of a AgNCs-streptavidin-siRNA complex. The complex could be specifically internalized into cancer cells and produce fluorescence signals. Through cell staining and microscopic image, intracellular AgNCs were found mainly accumulated in the nuclei. According to the results, a possible RNAi pathway was brought up. miRNA detection is a wide application field for DNA-AgNCs. DNA-AgNCs based fluorescence in situ hybridization (FISH) detection for miRNA was also capable with the help of high-resolution imaging (Fig. 5C) [75]. Nonspecific internalization was analyzed under prepared buffer circumstance. A hairpin structure capture probe with target mRNA hybridization sequence, DNA-AgNCs hybridization sequence and G-rich fluorescence enhancement sequence was delivered into cells. In the presence of target mRNA, the probe is unfolded and facilitated the fluorescence production of DNA-AgNCs. Moreover, to enhance the thermal stability of the hybridized double strands, sequences trans with the C-rich sequences arranged in a cis-trans direction from the 50 to 30 ends were used. With this setting three different mRNA miR-16-5p, miR-19b3p and miR-101-3p were detected in gastric cancer cells. This FISH strategy also found that they were mainly located in the nucleus. Though turn-on fluorescence signal is regarded as an important demand for bioimage, miRNA Let-7i, which is associated with early diagnosis of traumatic brain injury (TBI), is also imaged with a switched off method [125]. In the presence of Let-7i, hairpin structure responsible for both AgNCs nucleation and target recognition was unfolded, thus led to the quenching of fluorescence (Fig. 5D). As for intracellular signal amplification, Zhu et al. reported an intracellular DNA double strand breaks (DSBs) imaging for the

detection and monitoring of cell apoptosis [131]. DNA-AgNCs were synthesized and hybridized with a BHQ-labeled DNA sequence to quench the fluorescence (Fig. 5E(a)). Then terminal deoxynucleotidyl transferase (TdT) mediated the DNA polymerization by catalyzing the addition of poly-dA to the 30 -OH ends of the DSBs in the nucleus of apoptotic cells (Fig. 5E(b)). Finally, quantities of DNAAgNCs pobes hybridized with the poly-dA chains and the toehold strand displacement contributed to both quencher-labeled sequence removing and signal amplification (Fig. 5E(c)). 4. Therapeutic application Imaging-guided cancer therapy is a promising theranostic platform that integrates diagnosis and treatment. Photothermal therapy (PTT) is emerging noninvasive tumor therapy technique with deep tissue penetration ability and weak side effects [118,132]. Silver nanoparticles have added new dimension and are successfully applied to PTT in vivo [133]. Recently, the remarkable photothermal properties of DNA-AgNCs were first reported by Wu et al. [132]. They could efficiently convert optical energy from specific wavelength light into local heat, thus kill cancer cells. Tumor targeting was a indispensable part in PTT. Cell surface glycans were selected as target via a special Dibenzocyclooctyne (DBCO) -functionalized DNA. Aberrant glycans could be modified on many types of cancer cell surface through glycosylation. By using azido-sugars (Ac4ManNAz, Ac4galNAz) as label on the target protein, two hairpin structures of DNA-AgNCs (H1 and H2) for HCR and a DBCOfunctionalized DNA (H3) were assembled for in situ imaging of glycans. The azide group of azido-sugars can covalently couple with H3 via click reaction, and then H3 as a initiator triggers the HCR process on cell surface. High fluorescence responses were obtained as expected. While performing the detraction and imaging of cell surface glycans, laser irradiation at 808 nm at a power density of 1 W cm
2 for 10 min was used for PTT both in vitro and in vivo. Temperature change in tumor was analyzed by means of IR thermal camera. And the in vivo photothermal images manifested a rapidly upward tumor temperature of 54.3 C only within the tumor area. DNA-AgNCs with HCR process showed well-performed functions in cancer cell killing and tumor growth suppression. Nucleic acid delivery provides a promising paradigm for treatment of various diseases. siRNA plays an important role in RNA interference (RNAi) by collaborating with the RNA-induced silencing complex (RISCs). A novel siRNA target delivery and tracing technique developed with a aptamer-functionalized AgNCsstreptavidin- siRNA complex was mentioned above [130]. The complex could be specifically internalized into cancer cells, then the RNAi pathway which down-regulates the expression of a specific target gene most likely worked to transport itself from cytoplasm into nucleus. The probe was proved to efficiently decrease the expression of target mRNA and protein. And intracellular image worked well to elucidate the function of siRNA. Combining target delivery, intracellular image, pathway tracking and mechanism research, DNA-AgNCs applied in this field might make significant contributions to precision medicine. Drug screening is another important field which can greatly induce the development of therapy. A nonspecific simple way is to do drugs efficacy test through the highly sensitive biosensing function of DNA-AgNCs. By monitoring the concentration of cytochrome c (Cyt c), a metalloprotein related with cell apoptosis, natural drug screening was conducted [134]. Several methods have been explored to detect and discover anticancer drugs. Homoadenine binding molecules, for example, are a type of small molecules that promote the formation of an anti-parallel non-WatsonCrick homo-adenine DNA duplex. Adenine-rich nucleic acid sequences are a crucial part of the chromatin and influence mRNA

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Fig. 5. Schematic illustration of intracellular nucleic acid imaging: (A) A 2-channel intracellular mRNA imaging [129]. (B) A novel siRNA delivery and tracing technique based on AgNCs-streptavidin-siRNA complex [130]. (C) DNA-AgNCs based fluorescence in situ hybridization (FISH) detection for miRNA [75]. (D) A switched off method for miRNA monitoring [125]. (E) Terminal deoxynucleotidyl transferase (TdT) mediated DNA polymerization for intracellular DNA double strand breaks (DSBs) imaging [131].

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metabolism. DNA-AgNCs based NCB system induced by targets was first used to screen a homo-adenine binding molecule Coralyne which have antileukemic activity [135]. While promoting the formation of homo-adenine DNA duplex, target Coralyne induced the proximity of DNA-AgNCs and the enhancer sequence, thus produced bright red fluorescence. This design offers a novel strategy for screening of other homo-adenine binding molecules as well. Since that a large percentage of chemotherapeutic anticancer drugs act via interacting with cancer cell DNA, using DNA extracted from the cancer cell as the templated DNA of the DNA/AgNCs could realize specific binding between DNA-AgNCs and target drugs. Iridium (III) compounds (IrC) were detected and analyzed through fluorescence and resonance light scattering (RLS) measurement [136]. Two kinds of IrC (IrC1 and IrC2) were inserted into the human liver cancer SMMC-7721 DNA duplex. They resulted in a decreased fluorescence intensity and an increased RLS intensity of SMMC-7721 DNA templated AgNCs. Further Cell toxicity assay and cell apoptosis assay were conducted to investigate the anticancer properties of IrC. Finally, IrC1 was proved to be a promising cancer drug. This work provides a method for screening of a particular kind of anticancer drugs, which interact with previously known DNA. 5. Conclusion and perspective In this review, we summarized the recent advancement of DNAAgNCs in the biomedical application including biosensing, bioimaging and therapy. As a new type of functional nanomaterial, the application advantages of DNA-AgNCs have been expounded in a lot of researches. Here we conclude them into the following points. (1) Ultra small size: With sizes approach the Fermi wavelength of electrons, discrete energy of AgNCs is the fundamental source of their extraordinary optical, electrical and chemical properties different from nanoparticles. (2) Solubility: Soluble DNA-AgNCs facilitate the basic biosensing application, as well as detection in real samples. (3) Strong and stable fluorescence: Fluorescence properties of AgNCs are the most frequently used. Thanks to them, novel designed probes can be constructed prevailing over other fluorophores. (4) Adjustable emission: DNA-AgNCs with multiple color make multifunctional and simultaneous monitoring possible. Their emission wavelength has a close relationship with the template DNA, solvent environment and so on. (5) Facile synthesis: A convenient synthesis of various DNA-AgNCs can be easily achieved. The construction of a related system usually does not require sophisticated operations. (6) Biocompatibity: DNA-AgNCs can be efficiently introduced into biological systems. Despite that a micromolar concentration of AS1411 aptamer-functionalized AgNCs could induce cancer cell death [128], most cytotoxicity tests indicate that DNA-AgNCs with a concentration less than 1 mM post almost no effect on cell viability [122,123,127,129]. Although this novel nanomaterial has made great progress with the application of improved strategies, it still faces questions and challenges. Ever since they were invented, structures and fundamental understanding remain a major problem. A precise formation and stabilization process of DNA-AgNCs are still uncertain. What's more, considering the adjustable properties, establishment of structure-property correlations may be able to promote its development to the greatest extent. By synthesizing DNA template according to a pre-existing “color code” [137], DNA-AgNCs with functions perfectly corresponding to demanding can be obtained. Integrated with other materials, better features may show up as a newly developed structure. DNA-AgNCs fixed in Au nanoparticles showed electrochemical properties [18]. DNA-templated AueAg/ CueAg bimetallic nanoclusters also hold unique features [35,36]. Combined application could bring broader prospects which are not available for a single material.

DNA-AgNCs still have a long way to go for clinical applications, but they open up a novel path for precision medicine. Aptamer functionalized DNA-AgNCs can specifically recognize cell surface biomarks and be internalized into endochylema or nucleus. They image certain pathological cells with manually set colors. Nucleic acid including siRNA, mRNA and gene fragment have the potential to be delivered into cells via DNA-AgNCs constructed platform. Using DNA template which can hybridize with nucleic acids in cells, gene expression process may be modified and monitored. For those applications, the photostability and biostability of DNA-AgNCs need to be further improved. Improved stability makes it possible to use less concentration DNA-AgNCs. And only when the dosage is decreased, cytotoxicity can be substantially reduced. Measures for other fluorophores which are generally taken to achieve high level of photostability also can be applied for this potential class of fluorophores. Improving surface encapsulation and using photostabilizing agents as well as better passivation may boost the photostability and biostability properties. Future studies may direct to finding potential stabilizer as well as nanoclusters with more customized properties (e.g., flexibility, ultra-fluorescence feature and more selectivity). With further progress in mechanism and application, we believe that DNA-AgNCs’ unprecedented unique properties will finally benefit medical progress. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 21205142), State Key Laboratory of Chemo/ Biosensing and Chemometrics, Hunan University (2017006), The Research Innovation Program for Graduates of Central South University (2018zzts384, 2019zzts453). References [1] R. Jin, C. Zeng, M. Zhou, Y. Chen, Atomically precise colloidal metal nanoclusters and nanoparticles: fundamentals and opportunities, Chem. Rev. 116 (2016) 10346e10413. [2] I. Chakraborty, T. Pradeep, Atomically precise clusters of noble metals: emerging link between atoms and nanoparticles, Chem. Rev. 117 (2017) 8208e8271. [3] Y. Chen, M.L. Phipps, J.H. Werner, S. Chakraborty, J.S. Martinez, DNA templated metal nanoclusters: from emergent properties to unique applications, Acc. Chem. Res. 51 (2018) 2756e2763. [4] Y. Yu, B.Y.L. Mok, X.J. Loh, Y.N. Tan, Rational design of biomolecular templates for synthesizing multifunctional noble metal nanoclusters toward personalized theranostic applications, Adv. Healthc. Mater. 5 (2016) 1844e1859. [5] J.M. Obliosca, C. Liu, H.-C. Yeh, Fluorescent silver nanoclusters as DNA probes, Nanoscale 5 (2013) 8443e8461. [6] Y. Tao, M. Li, J. Ren, X. Qu, Metal nanoclusters: novel probes for diagnostic and therapeutic applications, Chem. Soc. Rev. 44 (2015) 8636e8663. [7] A. Tlahuice-Flores, R.L. Whetten, M. Jose-Yacaman, Ligand effects on the structure and the electronic optical properties of anionic Au-25(SR)(18) clusters, J. Phys. Chem. C 117 (2013) 20867e20875. [8] Y. Bao, H.-C. Yeh, C. Zhong, S.A. Ivanov, J.K. Sharma, M.L. Neidig, D.M. Vu, A.P. Shreve, R.B. Dyer, J.H. Wernerothers, formation and stabilization of fluorescent gold nanoclusters using small molecules, J. Phys. Chem. C 114 (2010) 15879e15882. [9] J. Zheng, R.M. Dickson, Individual water-soluble dendrimer-encapsulated silver nanodot fluorescence, J. Am. Chem. Soc. 124 (2002) 13982e13983. [10] X. Zhang, J.-Y. Wang, Y.-Z. Huang, M. Yang, Z.-N. Chen, Silver(i) nanoclusters of carbazole-1,8-bis(acetylide): from visible to near-infrared emission, Chem. Commun. 55 (2019) 6281e6284. [11] A. Pandya, A.N. Lad, S.P. Singh, R. Shanker, DNA assembled metal nanoclusters: synthesis to novel applications, RSC Adv. 6 (2016) 113095e113114. [12] J.T. Petty, J. Zheng, N.V. Hud, R.M. Dickson, DNA-templated Ag nanocluster formation, J. Am. Chem. Soc. 126 (2004) 5207e5212. [13] B. Han, E. Wang, DNA-templated fluorescent silver nanoclusters, Anal. Bioanal. Chem. 402 (2011) 129e138. [14] S.Y. New, S.T. Lee, X.D. Su, DNA-templated silver nanoclusters: structural correlation and fluorescence modulation, Nanoscale 8 (2016) 17729e17746. [15] Q. Cao, J. Li, E. Wang, Recent advances in the synthesis and application of copper nanomaterials based on various DNA scaffolds, Biosens. Bioelectron. 132 (2019) 333e342.

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