Periodic Fluorescent Silver Clusters Assembled by Rolling Circle

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Periodic Fluorescent Silver Clusters Assembled by Rolling Circle Amplification and Their Sensor Application Tai Ye, Jinyang Chen, Yufei Liu, Xinghu Ji, Guohua Zhou, and Zhike He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/am504035a • Publication Date (Web): 12 Aug 2014 Downloaded from http://pubs.acs.org on August 29, 2014

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ACS Applied Materials & Interfaces

Schematically illustration of the preparation of Ag NCs wires by rolling-circle amplification. 81x40mm (300 x 300 DPI)

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A typical TEM image of the AgNCs (a) and self-assembled Ag NCs (b). 26x10mm (300 x 300 DPI)


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the height-mode AFM image of Ag NCs nanowires 361x270mm (72 x 72 DPI)



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Gel electrophoresis image of Ag NCs nanowires in presence (a) and absence (b) of target. 34x54mm (254 x 254 DPI)


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Fluorescence excitation and emission spectra of Ag NCs before (curve a and b) and after (curve c and d) self-assemble 41x29mm (300 x 300 DPI)



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Confocal microscopy image of the red-emitting Ag NCs nanowires. 63x33mm (300 x 300 DPI)


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Fluorescence profiles of self-assembly Ag NCs nanowires for the specificity analysis with (a) target and phi29 polymerase; (b) target only; (c) phi29 polymerase only. Experimental conditions: target DNA, 0.3 nM; phi29 polymerase, 10 U; Ag NCs, 750 nM; enhance probe, 750 nM. 41x29mm (300 x 300 DPI)



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Bar chart of the fluorescent responses of various DNA sequences. The target DNA and other mismatched strands were all 0.3 nM. 42x29mm (300 x 300 DPI)


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Effects of the amount of Ag NCs 42x29mm (300 x 300 DPI)



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Effects of the amount of phi29 polymerase on the fluorescent intensity of the sensing system 42x29mm (300 x 300 DPI)


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Effects of the time of hybrization on the fluorescent intensity of the sensing system 42x29mm (300 x 300 DPI)



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Effects of the incubation time of the RCA reaction on the fluorescent intensity of the sensing system 42x29mm (300 x 300 DPI)


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Fluorescence intensity of Ag NCs with varieties concentration of the “G-rich helper” sequence. 41x29mm (300 x 300 DPI)



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(A) The fluorescence response to the different concentrations of the target DNA: 6, 18, 30, 60, 120, 180, 300 pM (from bottom to top). (B) The corresponding calibration curve of the Ag NC nanowires strategy for the DNA assay. Inset: shows the linear response at low concentrations of the target DNA. 226x82mm (150 x 150 DPI)


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Periodic Fluorescent Silver Clusters Assembled

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by Rolling Circle Amplification and Their Sensor

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Application

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Tai Ye, Jinyang Chen, Yufei Liu, Xinghu Ji*, Guohua Zhou, and Zhike He*

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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

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College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China.

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KEYWORDS: silver cluster, rolling circle amplification, self-assembly

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ABSTRACT: A simple method for preparing DNA-stabilized Ag nanoclusters (NCs) nanowires

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is presented. To fabricate the Ag NCs nanowires, we use just two unmodified component strands

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and a long enzymatically produced scaffold. These nanowires form at room temperature and

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have periodic sequence units that are available for fluorescence Ag NCs assembled which

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formed three-way junction (TWJ) structure. These Ag NCs nanowires can be clearly visualized

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by confocal microscopy. Furthermore, due to the high efficiency of rolling circle amplify

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reaction in signal amplification, the nanowires exhibit high sensitivity for the specific DNA

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detection with a wide linear range from 6 pM to 300 pM and a low detection limit of 0.84 pM,

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which shows good performance in the complex serum samples. Therefore, these Ag NCs

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nanowires might have great potential in clinical and imaging applications in the future.

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Introduction

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DNA-based assemblies of nanomaterials offer the possibility to fabricate nano-biomaterials

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with precise structures, versatile functions and numerous applications.1-4 Due to DNA’s

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remarkable molecular recognition properties, many strategies, such as tile assembly, scaffolded

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origami and DNA bricks, have been developed to design and produce 1D, 2D, and 3D

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architectures with sophisticated morphologies.5-7 Particularly, since DNA origami technology

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was reported by Rothemund in 2006,8 this bottom-up strategy had widely used in well-define

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construction of nanomaterials

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precise controlled annealing protocols increase the limitation of this strategy.

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and nanosensor.12 However, large scale staple strands and

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Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain

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DNA polymerases in which long single stranded (ss) DNA molecules are synthesized on a short

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circular ssDNA template by using a single DNA primer.13 As an effective DNA amplification

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tool, RCA was explored as an important technique for ultrasensitive DNA, RNA, and protein


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detection in diagnostic genomics and proteomics.14-16 Recently, RCA product was demonstrated

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as nano-scaffold for nanostructure construction and nanomaterials assembly.17-20 For example,

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Sleiman and co-workers fabricate DNA nanotube and AuNP assemblies based on a long

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continuous scaffold produced by RCA.21-22 Compare with the long scaffold ssDNA in

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conventional DNA origami (typically 7249 kb M13 bacteriophage genomic DNA), the RCA

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product is characteristic of relatively short periodic sequences which can be easily fold into well-

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define structure.23 In addition, these programmable DNA nanostructures based on RCA products

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were further applied in biomedical applications. Tan and Chen attached the dye-label sequence

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or multidye-modified dUTPs on the replicated product for single molecule detection and

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multiplex fluorescent cellular imaging.24-27

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DNA stabilized fluorescent silver nanoclusters (DNA-Ag NC) have emerged as new powerful

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fluorescence nanomaterials in biology.28-31 Compared with conventional fluorescent materials

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such as quantum dots and organic dyes, silver nanoclusters (Ag NCs) show advantages of easy

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preparation32 and functionalization,33 smaller in size and lower in toxicity.34-35 It is well-known

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that DNA template Ag NCs have sequence dependent fluorescence.36 Some works focus on

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spatially controlling cluster assembly through DNA nanostructure. Yan first employed DNA

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origami structure as nano-scaffold for site-specific synthesis AgNCs by Tollens regeant.37

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O’Neill fabricated AgNCs nanotube using nanotubes assembled from DNA tiles, with hairpin

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appendages on a subset of the tiles serving as the cluster growth sites.38 Willner and co-works

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controlled assembly of Ag NCs through nucleic acid-triggered hybridization chain reaction and

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Y-shape DNA in linear and dendrimer-like structure, respectively.39-40 However, all these

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assembly strategies are based on complex nuclear acid design, and strict annealing process.

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Furthermore, an intrinsic limitation of this type of method is that Ag NC synthesis is an


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integrated step of the detection process when it is involved in analysis applications, and a few

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hours are often needed to generate fluorescence, limiting its practical applications.30 Herein, we

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report an approach to assemble Ag NCs and a “G-rich helper” strand in to nanowires by long

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repetitive DNA strands synthesized from rolling circle amplification. In this approach, Ag NCs

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were pre-synthesized independently. The long enzymatically produced scaffold induces plenty of

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Ag NCs and the “helper” strands in proximity, which brings the end of the “G-rich helper” to

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close proximity to Ag NCs, resulting in a significant fluorescence enhancement.41 Because of the

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tunable fluorescence intensity of Ag NCs, these nanowires exhibit luminescent function in sensor

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application. To the best of our knowledge, the present work is the first constructing Ag NCs

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nanowire as a signal amplification biosensor, based on guanine-rich DNA sequences enhances

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fluorescence.

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EXPERIMENTAL SECTION

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Materials. Silver nitrate (AgNO3) and sodium borohydride (NaBH4) were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). T4 DNA ligase and dNTP were obtained from Takara

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(Dalian, China), while Phi29 DNA polymerase was purchased from New England Biolabs

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((Beijing, China). All oligonucleotide with different sequences were synthesized and HPLC

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purified by Sangon Biotechnology Co., Ltd (Shanghai, China). The sequences of the

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oligonucleotide used in this work are as follows:

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Primer: 5’-GGCGAGACACGGTGCTGCAG-3’

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Padlock: 5’-P-GTGTCTCGCCTTCTTGTTTCCTTTCCTTGA-

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AACTTCTTTCTTTCTTCGCTGCAGCACC-3’


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Nuclear sequence: 5’-TTCTTGTTTCCTTTCCTTGCCCTTAATCCCC-3’

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G-rich helper sequence: 5’-GGGTGGGGTGGGGTGGGGAAACTTCTTTCTTTCTTCG-3’

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MT1: 5’-GGCGAGACAGGGTGCTGCAG-3’

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MT2: 5’-GGCGTGACAGGGTGCTGCAG-3’

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MT3: 5’-GGCGTGACAGGGTGCTCCAG-3’

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Apparatus. Fluorescence spectra were obtained with a RF-5301PC spectrophotometer

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(Shimadzu, Japan) equipped with a 150 W xenon lamp (Ushio Inc, Japan). Gels were imaged by

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a ChemiDoc XRD system (Bio-Rad, USA). Morphology observation of Ag NCs and self-

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assembled Ag NCs in PBS buffer (pH 7.0, 20 mM) was carried out on TEM machine (JEM-2100

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microscope) at an acceleration voltage of 200 kV. The morphology of Ag NCs nanowires was

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characterized with a Veeco Multimode atomic force microscope (Veeco, USA). The

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fluorescence was analyzed by confocal laser scanning microscopy (Andor RevolutionXD, United

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Kingdom) equipped with a 543 nm argon laser for elf-assembled Ag NCs.

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Preparation of Ag nanocluster. DNA stabilized Ag nanoclusters were synthesized according

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to literature procedure with some modifications.41-42 Briefly, 15 µL 100 µM nuclear sequence

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was mixed with 73 µL 20 mM phosphate (pH 7.0) buffer and then 6 µL 1.5 mM AgNO3 solution

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was added to provide a Ag+ -to- DNA molar ratio of 6:1. After cooling at 4 ºC for 15 min, this

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mixture was reduced quickly by 6 µL 1.5 mM NaBH4, followed by vigorous shaking for 1 min.

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The reaction was kept in the dark at 4 ºC for 6 hours before used.


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Ligation and RCA reaction. For the ligation reactions with T4 DNA ligase, the reaction

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mixture consisted of ligation buffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 1 mM ATP), 350

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U of T4 DNA ligase, 0.5 µM padlock probe, and an appropriate amount of target DNA in a

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reaction volume of 50 µL. Before the ligase was added, the reaction mixture was heated at 90 ºC

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for 10 min. After the reaction mixture had been cooled to room temperature, the ligase was

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added and the reaction mixture was incubated at 37 ºC for 2h. The product of the ligation

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reaction was added to the 100 µL RCA reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10

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mM MgCl2, 10 mM (NH4)2SO4, 200 µM of dNTP, and 10 U of phi29 DNA polymerase. The

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RCA reactions were performed at 37 ºC for 4 h and terminated by incubation at 65 ºC for 10 min.

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Fluorescence Detection. The product of the ligation reaction and 750 nM of Ag NCs and G-

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rich helper sequence were combined in a 2.5 mL centrifuge tube and diluted to 300 µL with 20

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mM phosphate buffer (pH 7.0, 200 mM NaNO3). After incubation for 2h at room temperature,

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the fluorescence spectra were measured. The excitation wavelength was 560 nm, and the

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fluorescence emission intensity was measured at 611 nm.

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RESULTS AND DISCUSSION

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Scheme 1 schematically illustrates the RCA process and its scaffolding for periodical Ag NCs

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assemblies. The system consists of nano-scaffold preparation and assembling process. For

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nanoscaffold preparation, the padlock probe was firstly ligated and circularized with the primer

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as the template in the presence of T4 DNA ligase. Subsequently, the primer initiates RCA

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reaction by the high-displacement activity of the phi29 DNA polymerase for producing long

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enzymatically produced scaffold DNA. In the process of assembly, the Ag NCs and G-rich

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helper sequences are bind to the periodic sequence of the scaffold DNA. Because of the


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fluorescence enhancement of silver nanoclusters (Ag NCs) in proximity to guanine-rich DNA

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sequence, the bright Ag NCs nanowires are form.

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Scheme 1. Schematically illustration of the preparation of Ag NCs wires by rolling-circle

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amplification.

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As shown in Figure 1A, Ag NCs of 2 nm diameter were prepared by the classical NaBH4

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reduction route.36 In the presence of the long enzymatically backbone (Scheme 1), plenty of

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monodisperse Ag NCs are stacking on the DNA nano-wires forming self-assembled Ag NCs

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(Figure 1B). The morphology of the Ag NCs nanowires was investigated by atomic force

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microscopy (AFM). As shown in Figure 1C, micrometer-long wires are observed with the

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height of 1.4 nm, resulting from the height of the duplex structure of the DNA in TWJ structure.

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Figure 1. A typical TEM image of the Ag NCs (A) and self-assembled Ag NCs (B), and the

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height-mode AFM image of Ag NCs nanowires (C). Inset: Cross-section analysis of the resulting

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Ag NCs nanowires.

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The successful preparation of Ag NCs wires by RCA was also confirmed by gel

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electrophoresis. In the gel, a strong fluorescence band corresponding to DNA with length in

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excess of 10000 bp of the marker was observed (Figure 2A, lane a), which would lead to chains

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of DNA in many micrometers in length. In absence of target DNA, there was no RCA product

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observed (Figure 2A, lane b). The successful assemble of Ag NCs onto the scaffold DNA

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resulting remarkable change of fluorescence spectrum. As shown in Figure 2B, the unassembled

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Ag NCs show negligible excitation and emission spectrum (curve a and b). In presence of

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scaffold DNA, mono-dispersed Ag NCs were tethered in proximity of guanine-rich DNA

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sequences, and the significant enhance of fluorescent spectrum was observed (curve c and d),

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which were consistent with the previous reports.42-44 The resulting DNA-stabilized Ag NCs

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nanowires were further imaged by confocal fluorescence microscopy. Figure 2C depicts a

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confocal microscopy image of the resulting red-emitting Ag NCs nanowires. Compared with

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previous report about Ag NCs assembly, there is no need careful sequence design involved in

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this method and the room temperature hybridization strategy reduced the error hybridization in

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tedious annealing process. Meanwhile, the synthesis of Ag NCs is independent of the DNA

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assembly, which circumvents the interference of unstable NaBH4 during the Ag NC synthesis.


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Figure 2. (A) Gel electrophoresis image of Ag NCs nanowires in presence (a) and absence (b) of

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target. (B) Fluorescence excitation and emission spectra of Ag NCs before (curve a and b) and

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after (curve c and d) self-assemble. (C) Confocal microscopy image of the red-emitting Ag NCs

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nanowires. Scale bar is 10 µm.

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Due to the highly effective of RCA reaction in signal amplification, we combined the Ag NCs

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assembling and RCA reaction for the specific DNA detection. The primer of 20 mer

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oligonucleotide sequence was chose as a target DNA. The feasibility of the design is presented in

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Figure 3. In the presence of the target DNA, the product of the RCA reaction as a nanoscaffold,

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puts the two tailored sequence fragments in proximity. As a result, the fluorescence of Ag NCs

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enhances greatly. The resulting Ag NCs featured an emission band centred at 611 nm when

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excited at 560 nm (Fig. 3 a). In contrast, almost no fluorescence at 611 nm was observed (Fig. 3

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b, c) without the target DNA and phi29 DNA polymerase, respectively, as there was no long

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tandem repeated sequence produced by RCA reaction. Thus, the Ag NCs and enhancer probes

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could not be gathered in proximity, and no fluorescence enhancement was observed. Although

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many papers have reported some DNA detection methods based on fluorescent Ag NCs,45-47 the

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sensitivity of Ag NCs nanosensor might be limited if each target DNA generates only one

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fluorescent AgNC. Meanwhile, few reports combine the signal amplify and Ag NCs, higher

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background signal lower the detection limit.48-49 In addition, target-triggered isothermal


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amplification strategy can achieve ultrahigh sensitivity,48, 50-51 the unstable NaBH4 limit the

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reproducible of the assay. Herein, we combine the RCA product and Ag NCs assembled for

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constructing Ag NCs nanowires and also show good performance in sensing amplification,

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which circumvents the disadvantage of the mentioned above.

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Figure 3.Fluorescence profiles of self-assembly Ag NCs nanowires for the specificity analysis

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with (a) target and phi29 polymerase; (b) target only; (c) phi29 polymerase only. Experimental

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conditions: target DNA, 0.3 nM; phi29 polymerase, 10 U; Ag NCs, 750 nM; enhance probe, 750

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nM.

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In this strategy, RCA was a crucial step, which mediated the generation and amplification of

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the fluorescence signal. In order to achieve the system’s best sensing performance, several

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experimental parameters were investigated (Figure S1). The amount of phi29 DNA polymerase

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and RCA reaction time were set at 10 U and 4 hours, respectively. We found that the

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concentration of Ag NCs and the self-assemble equilibrium between the RCA product and Ag

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NCs were two key factors in this strategy. As shown in Figure S1C, the fluorescence intensity

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increased with the increase of Ag NCs concentration. When the concentration of Ag NCs

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reached 750 nM, the maximum fluorescence intensity was achieved. Thereafter, the fluorescence

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response exhibited a gradual decrease with a further increase in the concentration of Ag NCs. As


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a result, 750 nM of Ag NCs was selected for further investigation. We also studied the non-

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specificity of fluorescence enhancement of Ag NCs due to the high concentration of “G-rich

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helper” DNA in reaction system (Figure S2). In absence of target DNA, with increasing the

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concentration of the G-rich helper” DNA, the background signal showed neglected enhancement.

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On the other hand, the time of forming three-way junction influenced the signal to noise of this

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approaching. The fluorescence intensity increased with increasing the time of self-assemble in

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the range 0~120 min and reached the plateau at 120 min. Thus, 120 min of hybridization time

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was selected for this study.

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Figure 4 shows representative fluorescence responses with different concentrations of target

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DNA under the optimized experimental conditions. A linear dependence between the

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fluorescence intensity and the concentration of the target DNA was obtained in the range from 6

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pM to 120 pM with a limit of detection (LOD) as low as 0.84 pM (3σ).

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Figure 4. (A) The fluorescence response to the different concentrations of the target DNA: 6, 18,

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30, 60, 120, 180, 300 pM (from bottom to top). (B) The corresponding calibration curve of the

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Ag NC nanowires strategy for the DNA assay. Inset: shows the linear response at low

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concentrations of the target DNA.

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To evaluate the selectivity of this method, four DNA sequences, including target, the single-

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base mismatched target (MT1), two-base mismatched target (MT2) and the three-base

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mismatched target (MT3) at the same concentration (0.3 nM) were selected, and the result were

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shown in Figure 5. Taking advantage of padlock probe in highly specific single nucleotide

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polymorphism detection,52 the Ag NCs nanowires exhibit good selectivity to mismatch target.

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The fluorescence intensity of the target DNA is approximately 2-fold higher than that in response

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to MT1, about 3-fold higher to MT2, and about 4-fold higher to MT3. All these observations

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indicate that the present biosensor can easily differentiate between perfect matched and

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mismatched targets, and therefore, provides a great opportunity for single-nucleotide

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polymorphism analysis.

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Figure 5. Relative fluorescence response from different inputs of (a) target DNA, (b) one-base

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mismatched sequence (MT1), (c) two-base mismatched sequence (MT2), and (d) three-base

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mismatched sequence (MT3). The target DNA and other mismatched strands were all 0.3 nM.

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To demonstrate the feasibility of the practical application of this sensor, three concentrations

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of target (9, 16 and 23 pM) were spiked into diluted human serum. Due to the serum with high

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concentration shows a remarkable emission peak under experimental condition which influences

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the Ag NCs fluorescence. Thus, 1% serum was chose as the detection condition of practical


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application. The recovery values were in the range of 94.5-102.5% (Table S1), indicating that the

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sensor is capable of analysing real biological samples with a high serum percentage by diluting

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the real sample.

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CONCLUSIONS

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In conclusion, we have developed a facile method to prepare Ag NC nanowires. They are made

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from two tailored single strand that will come together only in the presence of an enzymatically

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produced backbone, via addressable binding regions. Furthermore, the tuneable fluorescence

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intensity of these nanowires can be used as nanosensor for specific DNA detection with RCA

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reaction. This novel approach has shown some unique features. First, the assay does not involve

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any chemical modification. Second, due to the G rich sequence enhance fluorescence of Ag NCs,

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the assay does not require troublesome separation procedures. In view of these advantages, this

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fluorescent DNA nanomaterial has great potential in the DNA diagnostics and clinical analysis.

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ASSOCIATED CONTENT

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Supporting Information

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The effects of Effects of the incubation time of the RCA reaction, the amount of phi29

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polymerase, the amount of Ag NCs and the time of hybrization on the fluorescent intensity of

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the sensing system; Recovery of the target DNA in spiked serum samples. This material is

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available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author


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*E-mail: [email protected]. (Z.H.).

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Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval

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to the ﬁnal version of the manuscript.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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This work was financially supported by the National Key Scientific Program Nanoscience and

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Nanotechnology (2011CB933600), the Suzhou Nanotechnology Special Project (ZXG2013028),

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the National Natural Science Foundation of China (21275109 and 21205089).

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REFERENCES

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SYNOPSIS for TOC only

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Periodic Fluorescent Silver Clusters Assembled by Rolling Circle

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