Ratiometric Catalyzed-Assembly of NanoCluster Beacons: A

Aug 29, 2017 - *E-mail: [email protected] (L.G.)., *E-mail: [email protected] (F.L.). ... fluorescent signal amplification strategy through the ratiomet...
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Ratiometric Catalyzed-Assembly of NanoCluster Beacons: A Nonenzymatic Approach for Amplified DNA Detection Lei Ge,* Ximei Sun, Qing Hong, and Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, 266109, People’s Republic of China S Supporting Information *

ABSTRACT: In this work, a novel fluorescent transformation phenomenon of oligonucleotide-encapsulated silver nanoclusters (AgNCs) was demonstrated, in which green-emissive AgNCs effectively transformed to red-emissive AgNCs when placed in close proximity to a special DNA fragment (denoted as convertor here). Taking advantage of a catalyzed-hairpin-assembly (CHA) amplification strategy, we rationally and compatibly engineered a simple and sensitive AgNC-based fluorescent signal amplification strategy through the ratiometric catalyzed-assembly (RCA) of green-emissive NanoCluster Beacon (NCB) with a convertor modified DNA hairpin to induce the template transformation circularly. The proposed ratiometric fluorescent biosensing platform based on RCA-amplified NCB (RCA-NCB) emits intense green fluorescence in the absence of target DNA and will undergo consecutively fluorescent signal transformation from green emission to red emission upon exposure to its target DNA. The ratiometric adaptation of the NCB to CHA circuit advances their general usability as biosensing platform with great improvements in detection sensitivity. By measuring the fluorescence intensity ratio of the red emission and green emission, the proposed RCA-NCB platform exhibits sensitive and accurate analytical performance toward Werner Syndrome-relevant gene, the proof-of-concept target in this work. A low detection limit down to the pM level was achieved, which is lower than most of the reported AgNC-based fluorescent DNA biosensors, making the proposed RCA-NCB biosensing strategy appealing in amplifying the ratiometric fluorescent signal for sensitive DNA detection. Moreover, our proposed RCA-NCB platform shows good recovery toward the target DNA in real human serum samples, illustrating their potential promise for clinical and imaging applications in the future. KEYWORDS: catalyzed-hairpin-assembly, DNA detection, fluorescent silver nanoclusters, signal amplification, ratiometric template transformation

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INTRODUCTION Water-soluble oligonucleotide-encapsulated silver nanoclusters (denoted as AgNCs in this work), which contain several to tens of silver atoms with the size comparable to the Fermi wavelength of conduction electron (less than 1 nm for silver), have emerged as a significant new classification of attractive molecular-scale metals and have recently attracted substantial research efforts.1 As promising alternatives to conventional organic dyes and quantum dots, oligonucleotide-encapsulated AgNCs show the advantages of good biocompatibility, low toxicity, easy functionalization, and facile synthesis.2 Due to the application of DNA templates for AgNCs synthesis, the desired emission wavelength can be encoded and regulated throughout the blue-green to near-infrared spectral region by simply modulating the sequence, length, and structure of DNA template without the complicated covalent labeling of different fluorescent dyes/materials.3 Furthermore, the oligonucleotideencapsulated AgNCs exhibit outstanding spectral and photophysical properties in terms of excellent photostability, high photoemission rates, large Stokes shifts, and high luminescence quantum yields.4 More importantly, oligonucleotide-encapsulated AgNCs feature short-lived metastable dark states (30 μs) that can be rapidly optically depopulated, through transient © 2017 American Chemical Society

absorption, to directly and specifically modulate the emissive manifold,5 allowing the unique and specific detection of bright AgNC fluorescence from high background environments. Therefore, not surprisingly, these unique features of the oligonucleotide-encapsulated AgNCs have been not only implemented to develop a variety of optical sensing platforms,6−15 but also render the oligonucleotide-encapsulated AgNCs competent as environmentally friendly and biocompatible fluorophores in cellular visualization16−20 and molecular computing.21−23 During the rapid growth of AgNC-based sensing platforms, adopting a sensitive and compatible signal amplification strategy is a key aspect to acquire high performance biosensing, which can overcome the inherent limitation of 1:1 target-tosignal ratio in conventional AgNC-based sensing platforms. Due to the superiorities of the AgNCs’ DNA ligands, such as exquisite Watson−Crick encoding, predictable and combinatorial structures (e.g., G-quadruplexes), reactivity toward tool enzymes (e.g., nucleases), and base-sequence-guided functional Received: June 23, 2017 Accepted: August 29, 2017 Published: August 29, 2017 32089

DOI: 10.1021/acsami.7b09034 ACS Appl. Mater. Interfaces 2017, 9, 32089−32096

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measurement of changes in the ratio of the fluorescence intensities at two well-resolved wavelengths, could address this problem. Compared to those fluorescent signal readouts with single emission wavelength, ratiometric fluorescence biosensors56 have achieved improved sensitivity and accuracy for biosensing because the signals of the ratiometric probe are not influenced by probe concentration, illumination intensity, photobleaching, light collection efficiency, and so on, eliminating systematic errors. With these thoughts in mind, a novel AgNC-based fluorescent sensing platform is demonstrated in this work through the compatible and ratiometric integration of CHA amplification circuits with NCBs, which are expected to satisfy unmet challenges in sensitive and accurate biosensing. To realize the ratiometric catalyzed-assembly (RCA) of NCB, we endeavored to explore a novel signal transduction mode for the construction of an RCA-amplifiable NCB (RCA-NCB) platform. In our previous work, we have found that there is a remarkable template transformation phenomenon when a green-emitting AgNCs nucleation sequence (GNuS, 5′-T3TGCCTTTTGGGGACGGATA-3′)57 is connected with a special DNA fragment (5′-CACCGC-T-3′, denoted as convertor in this work), forming a red-emitting AgNCs nucleation sequence (RNuS, 5′-CACCGC-T4-TGCCTTTTGGGGACGGATA-3′).58 On the basis of the above finding, we have constructed a ratiometric NCB based on the hybridization proximity mode.58 Unfortunately, while this reported NCB could provide a sensitive and ratiometric response to its target sequence, the postsynthesis of AgNCs after target hybridization limit their real sample application, and the lack of an alterable overhang sequence as a toehold further hinders their integration into CHA amplification circuits. Thus, taking these motivations into consideration, we wonder whether there is a similar transformation phenomenon when the special DNA fragment is close to the GNuS-encapsulated AgNCs, which will not only provide a new form of ratiometric signal transduction, but also be more competent for CHA amplification circuits. In particular, the ratiometric adaptation of the NCB to CHA circuits may lead to concomitant improvements in the sensitivity, convenience, and programmability of AgNCs-based fluorescent biosensing platforms.

features (e.g., DNAzymes), DNA recycling amplification strategies have shown maximal suitability in the development of various sensitive AgNC-based sensing platforms,24−32 most of which, however, employed tool enzymes as biocatalysts. Although sensitive, these enzyme-based strategies encounter the disadvantages of being expensive and need special reaction conditions due to the irreversible denaturation of enzymes. Therefore, great attention has been focused on signal amplification without the use of enzymes, e.g., by employing dynamic DNA self-assembly technology, which has provided an attractive signal-amplification approach for AgNC-based biochemical analytical applications. For instance, based on the hybridization chain reaction (HCR), Willner’s group demonstrated the triggered autonomous, isothermal self-assembly of AgNCs-functionalized DNA nanowires with controlled luminescence properties;33 and Chen et al. have successfully employed the mimic oxidase catalytic character of AgNCs to construct an amplified electrochemical aptasensor.34 Moreover, based on the DNAzyme-catalyzed cleavage of DNA, Zhang and co-workers developed an AgNC-based catalytic and molecular beacon for sensitive fluorescent analysis of DNAzyme cofactors.35 Nevertheless, to the best of our knowledge, the diversity of reported enzyme-free DNA recycling amplification strategies employed in AgNC-based sensing platforms is very limited and almost exclusively based on HCR or DNAzyme catalysis. Because of its excellent programmability, modularity, scalability, and robustness, the nonenzymatic catalyzed-hairpinassembly (CHA) amplification strategies36 have been proven particularly useful to both transduce and amplify the detection signals of DNA and other analytes. Moreover, CHA is operated in an autonomous and reconfigurable manner and requires only the design of base-pairing between DNA strands, leading to immense improvements not only in the sensitivity of sensing platform but also in the convenience of signal amplification.37−41 Although CHA has been incorporated into various detection modalities to facilitate signal generation and amplification,42−44 to date, there is no report on the CHAamplified AgNCs-based fluorescent sensing platform. Another key component involved in the design and construction of sensitive AgNC-based sensing platforms is to adopt a compatible fluorescent signal readout strategy/probe. Among the numerous AgNC-based fluorescent probes, “lightup” nucleic acid probe, one of the most successful AgNC fluorescent signal readout probes termed as NanoCluster Beacon (NCB),45−47 has strongly attracted interest from the research community for both basic and applied studies. For conventional NCB design, a guanine-rich oligonucleotide sequence and a yellow-emissive AgNC nucleated by cytosinerich oligonucleotide sequence48 are usually employed. When the AgNCs with weak yellow emission were placed in close proximity to the guanine-rich oligonucleotide sequences through hybridization, they were transformed into bright redemitting AgNCs. NCBs exhibit unique properties such as being label-free and having a great enhancement ratio.49 Together with the excellent properties of oligonucleotide-stabilized AgNCs, NCBs have been widely used in the construction of sensing platforms for various molecules,50−52 and have become a powerful tool for bioimaging.53 Despite the aforementioned advantages, however, most of the reported AgNC-based sensing platforms employed single-emission turn-on/off signal readout strategies, which are limited by sensing interferences originating from environmental and/or experimental conditions. Ratiometric fluorescent signal readout strategies,54,55 which allow the

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EXPERIMENTAL SECTION

Chemicals and Materials. All the HPLC-purified oligonucleotides with different sequences were custom-synthesized and freezedried by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The sequences of the oligonucleotide used in this work are summarized in the Supporting Information, SI. All the synthetic oligonucleotides were received as powders and centrifuged so that they would reside at the bottom of the containers. The powder was then dissolved with 20 mM sodium acetate solution (NaAc, Sigma-Aldrich, pH 7.5) to give stock solutions of 100 μM. Silver nitrate (AgNO3, 99.99%, analytical grade) and sodium borohydride (NaBH4, 99.99%, analytical grade) were obtained from Sigma-Aldrich Chemical Co (St. Louis, MO, U.S.A.) and directly used without additional purification. All solutions were prepared with ultrapure water obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, U.S.A.) with an electrical resistance of 18.2 MΩ· cm. Nondenaturing polyacrylamide gel electrophoresis was conducted on a Mini PROTEAN Tetra Cell (BIO-RAD, America) in 1 × trisborate-EDTA (TBE) buffer at 110 V for 40 min. The gel was stained with 1 × SYBR Green I solution, the images of which were acquired on the Gel Doc XR+ Imaging System (BIO-RAD, America). Synthesis of AgNCs. First, all the oligonucleotide hairpins were separately heated at 88 °C for 10 min to dissociate any intermolecular 32090

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ACS Applied Materials & Interfaces interaction and slowly cooled to room temperature to ensure the formation of the desired secondary structures. For AgNC synthesis, 20 μL of 188 μM freshly prepared AgNO3 was added into 10 μL of 30 μM AgNCs-modified hairpin DNA solution followed by vigorous shaking of the mixture for 30 s. After the incubation in the dark at 4 °C for 30 min, this obtained DNA-silver solution was reduced quickly by 20 μL of 94 μM freshly prepared NaBH4 solution, followed by vigorous shaking of the solution for another 30 s. The reaction was then diluted to a final volume of 100 μL with 50 μL NaAc solution and held in the dark at 4 °C for 1.0 h prior to use. Analytical Procedure. The typical analytical procedure for this RCA-NCB platform could be briefly described as follows: First, 20 μL of DNA target sequence at different concentrations was mixed with 10 μL of 6.0 μM recognition-toehold probe (RTP), 10 μL of 6.0 μM recognition-branch-migration probe (RBMP), 10 μL of 6.0 μM convertor-modified hairpin DNA, and 10 μL of 6.0 μM AgNCsmodified hairpin DNA followed by swirling for 10 s. Then, the reaction mixture was allowed to react at room temperature for 100 min. For all the fluorescence excitation and emission spectra scans for the above-mentioned experiment, the resultant reaction mixture was diluted with 60 μL of NaAc solution at room temperature and was then transferred into a 1 cm path length quartz cuvette before the fluorescent measurements, which were performed with an F-4600 fluorescence spectrophotometer (Hitachi Ltd., Japan). For emission scans, the emission spectra of red-emitting AgNCs were recorded from 625 to 800 nm with an excitation wavelength of 610 nm. Emission spectra of green-emitting AgNCs were recorded over the wavelength ranges of 525 to 800 nm upon excitation at 500 nm. For the fluorescence contour maps, the excitation wavelengths were scanned from 430 to 700 nm, and the emission intensities were collected over the wavelength ranges of 500 to 800 nm. Both emission and excitation were scanned using 10 nm increment steps.

Figure 1. (A) 2D fluorescence contour map of T*-GNuS-encapsulated AgNCs; (B−H) time-dependent 2D fluorescence contour map changes upon subjecting the T*-GNuS-encapsulated AgNCs to the Conv-T sequence for a time-interval of (B) 5 min; (C) 10 min; (D) 15 min; (E) 20 min; (F) 25 min; (G) 30 min; and (H) 35 min.

GNuS show no difference in fluorescence profiles compared with that held within GNuS only (Figure S1A). The timedependent fluorescence features of the T*-GNuS-encapsulated AgNCs upon interaction with Conv-T are shown in Figure 1B− H in the form of contour maps. After the addition of Conv-T into a solution containing T*-GNuS-encapsulated AgNCs, as shown in Scheme 1, the binding of two complementary tails brings the convertor sequence and GNuS-encapsulated AgNCs into close proximity. As a result, significant fluorescence increase of red emission (Figure 1B−H) was observed whereas the green emission of the solution decreased remarkably, and both of them leveled off to a constant value after ca. 25 min (Figure 1F), suggesting that the Conv-T sequence could effectively trigger the transformation of green-emissive GNuSencapsulated AgNCs to red-emissive AgNCs with the aid of complementary tails. The transmission electron microscopy (TEM, Hitachi HT7700, Hitachi, Japan) observations revealed that an average size of green-emissive AgNCs with good crystallinity is about 2.18 nm (Figure S2A), which is slightly smaller than that of red-emissive AgNCs (ca. 2.53 nm, Figure S2B). These results identify the transformation between two different AgNCs. To further demonstrate that the AgNC transformations are due to the spatial proximity between the convertor sequence and the GNuS-encapsulated AgNCs, we then implemented several control experiments in this work. First, Conv-T was split into two portions (Figure S3), i.e., the convertor sequence (Conv) and the complementary tail sequence (T). For the second control experiment, as shown in Figure S4, we appended a noncomplementary tail of adenine−thymine-rich sequence to the 3′-end of convertor (denoted as Conv-N). Finally, the stability of the greenemissive AgNCs encapsulated in GNuS (Figure S1) and T*GNuS (Figure S5), respectively, was investigated to exclude its self-transformations. Compared to Conv-T, no fluorescence change was observed when T*-GNuS-encapsulated AgNCs were subjected to the Conv-N sequence (Figure S4) or the mixture of convertor sequence and tail sequence (Figure S3) for a time interval of 140 min. Considering the good stability of the as-prepared green-emissive AgNCs (Figure S1 and Figure S5), the results of these control experiments evidently confirm that the close proximity through hybridization is the key to AgNC transformation. With this hybridization-induced AgNC transformation strategy in hand, the question becomes whether it allows the compatible and ratiometric integration with CHA amplification

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RESULTS AND DISCUSSION The principle of this novel AgNC transformation phenomenon is illustrated in Scheme 1. As is well-known, homopolymer Scheme 1. Hybridization-Induced AgNCs Transformation Strategya

a Squares and arrows drawn on DNA strands represent 5′ termini and 3′ termini, respectively.

strands of T and A bases have no obvious influence on the formation of AgNCs.59 Therefore, to address the question of whether green-emissive AgNCs held in GNuS can change their emission wavelength when brought together with the convertor sequence, as shown in Scheme 1 and Table S1, we rationally extend the convertor and GNuS, respectively, with a 20 nt complementary tail of adenine−thymine-rich sequence from the 3′-end of convertor (denoted as Conv-T) and the 5′-end of GNuS (denoted as T*-GNuS). The 2D fluorescence contour map of T*-GNuS-encapsulated AgNCs (Figure 1A) exhibits a maximum excitation and emission wavelength of 500 and 565 nm, respectively. As expected, the synthesized AgNCs on T*32091

DOI: 10.1021/acsami.7b09034 ACS Appl. Mater. Interfaces 2017, 9, 32089−32096

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ACS Applied Materials & Interfaces Scheme 2. Ratiometric Catalyzed-Assembly of NanoCluster Beacona

a

For clarification and simplicity, the sequences of DNA molecules are grouped into encoded domains, each of which represents a short fragment of DNA sequence. And complementary domains were denoted with an asterisk after the same letter.

circuits, which are always designed to bring two DNA-hairpin structures together through DNA hybridizations. Scheme 2 depicts the configuration of the proposed RCA-NCB platform for multiplexed detection of nucleic acid targets. As a proof-ofprinciple, we designed a RCA-NCB platform for the detection of Werner Syndrome (WS)-relevant gene target.60 As shown in Scheme 2, a typical CHA configuration usually contains a pair of DNA hairpin structures (HP1 and HP2). In order to combine the AgNC transformation with the CHA circuit to form the RCA-NCB platform, in which products of the CHA reaction should initiate the AgNC transformations, as shown in Scheme 2, we appended the GNuS sequence onto the 3′-end of HP1 (denoted as GNuS-HP1) and attached the convertor sequence onto the 5′-end of HP2 (denoted as Conv-HP2). Thus, both the convertor sequence and the GNuS sequence would be exposed on the same end of the final CHA product. After the annealing process, GNuS-HP1 and Conv-HP2 formed the hairpin structure. GNuS-HP1 was then employed as template to synthesize green-emissive fluorescence AgNCs (AgNCs∥GNuS-HP1) in the presence of Ag+ through the reduction by NaBH4. Comparing the fluorescent profiles of the AgNCs encapsulated in GNuS-HP1 (Figures 2A and S6) with that in GNuS (Figure S1), the similar fluorescence peak with good stability reveals that the HP1 hairpin structure did not obviously affect the fluorescent features of GNuS-stabilized AgNCs. In this work, we employed a target-driven DNA association strategy to initiate the CHA reaction in this RCA-NCB platform, which contained two recognition probes, i.e., recognition-toehold probe (RTP) and recognition-branchmigration probe (RBMP). As shown in Scheme 2, the toehold domain (domain-a) and branch-migration domains (domain-b and domain-c) lie on RTP and RBMP, respectively, and were connected to the recognition domains (domain-x* and domainy*). In the absence of a nucleic acid target, as shown in Scheme 2, the spontaneous hybridization between RTP, RBMP, AgNCs∥GNuS-HP1, and Conv-HP2 is kinetically hindered by rationally and carefully designing their sequences, avoiding unintended secondary structures and misfolding. Figure 2

Figure 2. (A) 2D fluorescence contour map of AgNCs∥GNuS-HP1; (B−H) time-dependent 2D fluorescence contour map changes observed upon subjecting the AgNCs∥GNuS-HP1 to the mixture of RTP, RBMP, and Conv-HP2 for a time-interval of (B) 20 min; (C) 40 min; (D) 60 min; (E) 80 min; (F) 100 min; (G) 120 min; and (H) 140 min.

displays the fluorescent features of the mixture without nucleic acid target as a function of time. As expected, only a slight conversion of green-emission to red-emission was observed over 140 min incubation. These results implied that the coexistence of RTP, RBMP, AgNCs∥GNuS-HP1, and ConvHP2 results in a low background signal leakage in the absence of target. Upon the introduction of the nucleic acid target, as shown in Scheme 2, RTP and RBMP could corecognize the nucleic acid target to produce an RTP-target-RBMP structure, which brought the toehold (domain-a) and branch-migration domains (domain-b and domain-c) into close proximity to hybridize with the domain-a*,b*,c* of AgNCs∥GNuS-HP1. Domain-a of the RTP-target-RBMP structure hybridizes to domain-a* on AgNCs∥GNuS-HP1 as a toehold and, then, opens the stem part of AgNCs∥GNuS-HP1 through the toehold-initiated branch migration reaction. At the same time, the conformational change of the AgNCs∥GNuS-HP1 thus results in the exposure of domain-c. This newly released domain-c can then bind to the 3′-end overhang (domain-c* as toehold) of Conv32092

DOI: 10.1021/acsami.7b09034 ACS Appl. Mater. Interfaces 2017, 9, 32089−32096

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Figure 3. (A−H) Time-dependent 2D fluorescence contour map changes observed upon treatment of the RCA-NCB platform with 80 nM WS target sequence for a time-interval of (B) 20 min; (C) 40 min; (D) 60 min; (E) 80 min; (F) 100 min; (G) 120 min; and (H) 140 min.

HP2 to initiate a secondary toehold-mediated branch migration reaction, which ultimately form the energetically favored AgNCs∥GNuS-HP1@Conv-HP2 complex. During the secondary branch migration, the RTP-target-RBMP structure dissociates simultaneously from the AgNCs∥GNuS-HP1@ Conv-HP2 complex and is available to further catalyze the next round of dynamic assembly between AgNCs∥GNuS-HP1 and Conv-HP2. After CHA reaction, as shown in Scheme 2, the convertor sequence on Conv-HP2 is colocalized with the GNuS-stabilized AgNCs on GNuS-HP1 at the same end of the GNuS-HP1@Conv-HP2 duplex and, thus, converts numerous green-emissive AgNCs to red-emissive AgNCs, causing dramatically enhanced fluorescent change of the DNA-Ag solution for highly sensitive detection of target DNA. Figure 3 illustrates the time-dependent fluorescent contour map change of the CHA reaction mixture upon treatment with a fixed concentration of the target DNA. As the reaction time increases, the two fluorescent emission peaks of the CHA reaction mixture show opposite changes, with the green emission decreasing and the red one intensifying greatly, and then they level-off after ca. 100 min. To further ensure the feasibility of our RCA-NCB platform, it is critical to confirm the compatibly designed CHA reaction by native polyacrylamide gel electrophoresis (PAGE) experiments. Figure 4 displays the PAGE analysis of the formation of the RTP-target-RBMP structure (Figure 4A) and the catalyzedassembly of the AgNCs∥GNuS-HP1 and Conv-HP2 (Figure 4B). Lanes a, b, and c in both parts A and B of Figure 4 were loaded with target WS, RTP, and RBMP, respectively. Lanes h and i in Figure 4B were loaded with AgNCs∥GNuS-HP1 and Conv-HP2, respectively. Benefiting from the rational design of the DNA sequences, as illustrated by parts A and B of Figure 4, all the aforementioned electrophoresis lines exhibit a single and narrow electrophoresis band, respectively, indicating that no obvious secondary structure is observed in each carefully designed DNA structure. As negative control, compared with the aforementioned electrophoresis lines, no obvious change of band position and brightness was observed in the mixture of RTP and RBMP (lane-d in Figure 4A) as well as the mixture of AgNCs∥GNuS-HP1 and Conv-HP2 (lane-j in Figure 4B). Similarly, lane-k in Figure 4B shows the products of CHA reaction in the absence of target WS. Only four obvious electrophoresis bands were clearly observed in lane-k with the similar brightness and mobility to that in lanes-b, -c, -h, and -i,

Figure 4. Native polyacrylamide gel electrophoresis confirmation of (Gel board A) the RTP-target-RBMP structure and (Gel board B) the catalyzed-assembly of the AgNCs∥GNuS-HP1 and Conv-HP2. Lane-a, WS; lane-b, RTP; lane-c, RBMP; lane-d, RTP+RBMP; lane-e, WS +RTP; lane-f, WS+RBMP; lane-g, WS+RTP+RBMP; lane-h, AgNCs∥GNuS-HP1; lane-i, Conv-HP2; lane-j, AgNCs∥GNuSHP1+Conv-HP2; lane-k, RTP+RBMP+AgNCs∥GNuS-HP1+ConvHP2; lane-l, WS+RTP+RBMP+AgNCs∥GNuS-HP1+Conv-HP2.

respectively. These results implied that the designed DNA structures assembled scarcely in the absence of the target WS. In the presence of target WS, as shown in lane-e and lane-f of Figure 4A, both of the RTP and RBMP could hybridize with the target sequence, respectively. When the target WS was subjected to the mixture of RTP and RBMP (lane-g), a new electrophoresis band was observed at the shorter electrophoresis distance, confirming the formation of the RTP-targetRBMP structure. Once the RTP-target-RBMP structure was introduced into the mixture of AgNCs∥GNuS-HP1 and ConvHP2, the electrophoresis band corresponding to AgNCs∥GNuS-HP1 and Conv-HP2 disappears (lane-l) due to the consumption of the two DNA hairpins in CHA recycling circuit. At the same time, a new electrophoresis band corresponding to AgNCs∥GNuS-HP1@Conv-HP2 complex appears at the minimum electrophoresis distance in lane-l, revealing that the CHA reactions were successfully initiated as expected. Furthermore, after the CHA reaction, the recovery of the electrophoresis band corresponding to RTP-target-RBMP structure (lane-l) confirmed that RTP-target-RBMP structure served as a catalyst to circularly catalyze the CHA reaction. Thus, these results validated the enzyme-free and isothermal assembly of AgNCs∥GNuS-HP1 and Conv-HP2. The aforementioned ratiometric fluorescent response of the RCA-NCB platform prompted us to investigate its target 32093

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none of the tested single-base mismatched target gave R670/565 higher than that produced by 50 pM target and was very close to that of the blank test. These results revealed that this RCANCB platform was able to discriminate perfectly matched and single-base mismatched DNA targets, indicating that the specificity of the proposed promising platform toward the target DNA was acceptable. After proving the sensitivity and specificity of our RCA-NCB sensing platform, the application of which was further evaluated through the spike-in experiments to determine the recovery of target DNA in human serum (purchased from Shanghai LincBio Science Co., Ltd.) samples under the aforementioned conditions. Upon the stability investigation of AgNCs (Figures S7 and S8) in NaAc solutions containing different amounts of human serum or Cl−, 1% diluted human serum samples were chosen as the detection condition for practical application. Three different concentrations of WS at 0.5, 5.0, and 50 nM were separately added into the diluted human serum and then the as-prepared samples were assayed using the proposed RCANCB sensing platform. The measured results are listed in Table 1. The data obtained show clearly that the recovery values for

responsive capability for amplified WS detection. Thus, the analytical performance of the RCA-NCB platform was examined by challenging the system with different target concentrations. Figure 5A shows the time-dependent lumines-

Figure 5. (A) Time-dependent fluorescence spectra of the redemitting (top) and green-emitting (down) AgNCs upon challenging the RCA-NCB platform with 0.02 nM (▲), 1.5 nM (●), and 80 nM (■) target DNA. (B) Fluorescent emissions of reaction mixtures containing 1.0 μM RTP, 1.0 μM RBMP, 1.0 μM AgNCs∥GNuS-HP1, 1.0 μM Conv-HP2, and varying concentrations of WS (from a to j: 0, 0.02, 0.05, 0.15, 0.5, 1.5, 5.0, 12, 30, and 80 nM). (C) The linear relationship of the logarithmic value of R670/565 versus the logarithm of WS concentration. (D) Selectivity of the propose RCA-NCB platform.

Table 1. Recoveries of RCA-NCB Platform for WS Spiked Human Serum Samples

cence changes of the proposed RCA-NCB platform upon analyzing different concentration of WS target sequence. Obviously, the generation rate of AgNCs∥GNuS-HP1@ConvHP2 complex is directly proportional to the concentration of target WS. Figure 5B depicts the fluorescence spectra of the proposed RCA-NCB platform upon analyzing different concentrations of WS target sequence for a fixed response time of 100 min. As shown in Figure 5B, the emission intensity of RCA-NCB at 565 nm decreased gradually whereas the emission at 670 nm intensified continuously with elevated concentration of target WS. In this work, the fluorescent intensity ratio between red emission and green emission (R670/565) was calculated as the output signal for this RCA-NCB platform. As seen in Figure 5C, the logarithmic (lg) value of R670/565 exhibited an excellent linearity toward the logarithm of target WS concentration in the dynamic range of 0.02 to 80 nM, which provided the basis for quantitative detection of target WS. The resulting linear regression equation was lgR670/565 = 0.46792 × lg[cWS(nM)] + 0.01213 with a linear correlation coefficient of 0.9952 (n = 6) and a detection limit of 8.5 pM at 3σ, demonstrating the potential of RCA-NCB platform for sensitive target DNA detection. To the best of our knowledge, the detection limit of the proposed RCA-NCB platform is lower than most of the reported AgNCs-based fluorescence biosensors. The detailed comparison of the proposed RCA-NCB platform with others was listed in Table S3. The specificity of the developed RCA-NCB platform was then evaluated by substituting the WS target sequence with one-base mismatched targets, each of which was tested at relatively high concentration under the same experimental conditions. It was found that, as summarized in Figure 5D,

samples

spiked (nM)

measured (nM)

recovery (%)

sample-1 sample-2 sample-3

0.5 5.0 50

0.516 ± 0.048 4.79 ± 0.29 51.3 ± 1.84

103.2 95.8 102.6

the spiked WS are in the range from 95.8 to 103.2%, indicating that our proposed RCA-NCB sensing platform for DNA detection possesses an acceptable recovery and has a potential to be applied in real biological samples.

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CONCLUSIONS In summary, on the basis of the remarkable fluorescence transformation upon convertor proximity, together with the reconfigurable CHA reaction, we have developed a sensitive and selective ratiometric AgNC-based fluorescent biosensing platform through the ratiometric catalyzed-assembly of ConvHP2 with green-emissive NCB, i.e., AgNCs∥GNuS-HP1. In the presence of target DNA, AgNCs∥GNuS-HP1 is transformed to a new AgNCs∥GNuS-HP1@Conv-HP2 complex with strong red emission (λmax = 670 nm) but otherwise remains green (λmax = 565 nm). Gel electrophoresis results confirmed that the designed CHA circuit can work well. By integrating the signal amplification of CHA reaction with the accuracy of ratiometric strategy, the proposed AgNCs-based fluorescent biosensing platform was achieved with a low detection limit and a wide linear range, and was successfully applied to detection of target WS in human serum. This achievement may not only enable the use of RCA strategy as an efficient signal amplifier for NCBs, but also suggest a novel paradigm for a AgNC-based fluorescent biosensing platform in which target recognition, ratiometric signal transduction, and amplification can be compatibly integrated via modular programming and design of the sensing probes. Furthermore, the development of the RCA transduction will offer new opportunities for ratiometric signal amplification circuit design and can be extended to other signal amplification strategies, such as HCR. 32094

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b09034. Sequences of oligonucleotides used in this work, TEM images of AgNCs, 2D fluorescence contour plots of control experiments as described in the text, and comparison of the present study with other fluorescence biosensors (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.G.). *E-mail: [email protected] (F.L.). ORCID

Feng Li: 0000-0002-3894-6139 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (31501570 and 21575074), Natural Science Foundation of Shandong Province, China (ZR2014BQ011), Basic Research Program of Qingdao (16-5-155-jch), Research Foundation for Distinguished Scholars of Qingdao Agricultural University (663-1115003 and 6631113311), and The Special Foundation for Distinguished Taishan Scholars of Shandong Province (ts201511052).

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ACS Applied Materials & Interfaces

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