Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 17–21

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

Highly selective and sensitive nucleic acid detection basedon polysaccharide-functionalized silver nanoparticles

http://dx.doi.org/10.1016/j.saa.2014.06.0791386-1425/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding authors. Address: The Key Laboratory of Carbohydrate Chemis-try & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122,Jiangsu, China. Tel.: +86 15952819661 (J.-K. Yan). Tel.: +852 34008671 (J.-Y. Wu).

E-mail addresses: jkyan_27@163.com (J.-K. Yan), jian-yong.wu@polyu.edu.hk(J.-Y. Wu).

Jing-Kun Yan a,b,c,⇑, Hai-Le Ma b, Pan-Fu Cai b, Jian-Yong Wu c,⇑a The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122 Jiangsu, Chinab School of Food & Biological Engineering, Jiangsu University, Zhenjiang 212013, Chinac Department of Applied Biology & Chemical Technology, State Key Laboratory of Chinese Medicine and Molecular Pharmacology in Shenzhen,The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

h i g h l i g h t s

� The Oc-AgNPs can be used as a novelsensing platform for nucleic aciddetection.� This sensing system can well

differentiate complementary andmismatch sequences.� The proposed sensing platform can be

developed for practical applications.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 January 2014Received in revised form 27 May 2014Accepted 1 June 2014Available online 21 June 2014

Keywords:PolysaccharideSilver nanoparticlesNucleic acid detectionFluorescent sensing platform

a b s t r a c t

Polysaccharide-functionalized silver nanoparticles (Oc-AgNPs) with a mean diameter of 15 nm were uti-lized as a novel and effective fluorescence-sensing platform for nucleic acid detection. Tests on the oligo-nucleotide sequences associated with the human immunodeficiency virus as a model system showedthat the Oc-AgNPs effectively absorbed and quenched dye-labeled single-stranded DNA through stronghydrogen bonding interactions and slight electrostatic attractive interactions. The proposed system effi-ciently differentiated between complementary and mismatched nucleic acid sequences with high selec-tivity and good reproducibility at room temperature.

� 2014 Elsevier B.V. All rights reserved.

Introduction diagnosis, the occurrence of many diseases, especially cancers,

Nucleic acids are biomolecules vital for gene mutation andexpression, protein synthesis, and heredity. With the developmentof modern molecular biology and its many applications in clinical

has been found closely associated with the abnormal changes innucleic acids [1,2]. Thus, nucleic acid-based detection techniqueshave attracted significant research attention particularly becauseof their potential applications in gene expression profiling and clin-ical disease diagnosis, and treatment [3].

A number of optical and electrochemical methods for nucleicacid detection have been successfully established through hybrid-ization between a target and its complementary probe [4].However, some of these protocols are time-consuming, compli-

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Fig. 1. Fluorescence emission spectra of PHIV (50 nM) under different conditions: (a)PHIV; (b) PHIV + 300 nM T1; (c) PHIV + Oc-AgNPs; and (d) PHIV + Oc-AgNPs + 300 nMT1. Curve e: emission spectrum of Oc-AgNPs sample. Inset: fluorescence intensityratio of the PHIV–Oc-AgNPs complex with F/F0 � 1 (F0 and F are the fluorescenceintensity without and with the presence of T1, respectively) plotted againstlogarithm of the concentration of T1. Excitation was at 480 nm, and the emission

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cated, expensive, and usually require specialized equipment. Withrecent developments in nanotechnology, new, rapid, cost-effective,sensitive, and specific methods have been generated, and appliedin fluorescence-enhanced nucleic acid detection. The homoge-neous fluorescence assays based on fluorescence resonance energytransfer or the quenching mechanism for nucleic acid detectionhave overcome the limitation to the selectivity of a fluorescence-quencher pair in a nanostructure-involved fluorescence assayusing nanoquenchers to quench dyes with different emissionfrequencies [5,6]. A variety of effective nanoquenchers have beendocumented for the detection of nucleic acids such as gold nano-particles [7,8], single-walled carbon nanotubes (SWCNTs) [6],multi-walled carbon nanotubes [9], carbon nanoparticles [10], car-bon nanospheres [11], nano-C60 [12], mesoporous carbon micro-particles [13], graphene oxide [14], polyaniline nanofibers [15],poly(o-phenylenediamine) colloids [16], poly(2,3-diaminonaph-thalene) microspheres [17], poly(p-phenylenediamine) nanobelts[18], tetracyanoquinodime nanoparticles [19], coordination poly-mer colloids and nanobelts [20,21], citrate-capped silver nanopar-ticles (AgNPs) [22], and Ag@poly(m-phenylenediamine) core–shellNPs [23]. Nonetheless, further development is still of significancefor novel nanostructures as effective fluorescence-sensing plat-forms for the detection of nucleic acids.

The convergence of biotechnology and nanotechnology has ledto the development of hybrid nanomaterials that incorporate thehighly selective catalytic and recognition properties of biomole-cules, such as proteins, enzymes, DNA and polysaccharides, withthe unique electronic, photonic, and catalytic features of nanopar-ticles (NPs) [24]. Because of the unique recognition, transport, andcatalytic properties of biomolecules, biomolecule-functionalizedNPs can provide electronic or optical transduction of biologicalphenomena in the development of novel biosensors [25,26]. In arecent study, we prepared polysaccharide-functionalized AgNPsusing 4-acetamido-2,2,6,6-tetramethypiperidine-1-oxyl radical(4-acetamido-TEMPO)-oxidized curdlan (Oc) as both reducingand stabilizing agents for the green synthesis of AgNPs from silvernitrate. The polysaccharide-capped AgNPs (Oc-AgNPs) were well-dispersed and spherical in shape with a mean diameter of 15 nmin aqueous solution [27]. To the best of our knowledge, the useof Oc-AgNPs as a fluorescence-sensing platform for nucleic aciddetection has not yet been reported.

In this study, therefore, we evaluated the feasibility of utilizingOc-AgNPs as a novel and effective fluorescence-sensing platformfor nucleic acid detection using an oligonucleotide sequence asso-ciated with the human immunodeficiency virus (HIV) as a modelsystem.

Experimental

Materials and chemicals

The Oc-AgNPs were prepared by the method as reported previ-ously, and the molecular weight of Oc was about 1.2 � 105 Da bysize-exclusion chromatography with multi-angle laser-light scat-tering analysis [27]. All chemically synthesized oligonucleotideswere obtained from Shanghai Sangon Biotechnology Co. Ltd.(Shanghai, China). DNA concentrations were determined by mea-surement of absorbance at 260 nm. All chemicals and solventswere of laboratory grade and used without further purification.Deionized water was used in all of the experiments. Oligonucleo-tide sequences used are listed as follows (mismatch underlined).

(1) FAM dye-labeled ssDNA (PHIV): 50-FAM-AGT CAG TGT GGAAAA TCT CTA GC-30.

(2) Complementary target to PHIV (T1): 50-GCT AGA GAT TTT CCACAC TGA CT-30.

(3) Single-base mismatched target to PHIV (T2): 50-GCT AGA GATTGT CCA CAC TGA CT-30.

(4) Two-base mismatched target to PHIV (T3): 50-GCT AGA GATTGT ACA CAC TGA CT-30.

(5) Noncomplementary target to PHIV (T4): 50-TTT TTT TTT TTTTTT TTT TTT TT-30.

Instrumentation and conditions

Fluorescence emission spectra were recorded on a Cary Eclipsespectrofluorometer (Agilent, USA). Zeta potential measurementwas performed on a Malvern Zetasizer Nano (3000SHA, MalvernInstruments Ltd., UK) at 25 �C. The volume of each sample usedfor fluorescence measurement was 900 lL in 50 mM Tris–HCl buf-fer containing 5 mM MgCl2 (pH 7.4), and the concentration of Oc-AgNPs stock solution was 900 nM. All experiments were carriedout at room temperature (about 25 �C) unless specified otherwise.

Results and discussion

Oc-AgNPs for nucleic acid detection

In this study, stable and well-dispersed AgNPs were preparedby a facile and simple green method based on oxidized curdlan(Oc) was used as a new fluorescent sensing platform for nucleicacid detection [27]. Fig. 1 shows the fluorescence emission spectraof PHIV, the FAM-labeled probe, under different conditions. Thepresence of the fluorescein-based dye induced PHIV to exhibitstrong fluorescence emission at �520 nm in the absence of Oc-AgNPs in the PHIV buffer (curve a). However, in the presence ofOc-AgNPs, the fluorescence emission was quenched by about 65%(curve c), indicating the strong absorption of single-strandedDNA (ssDNA) by Oc-AgNPs. In contrast, the PHIV–Oc-AgNP complexexhibited significant fluorescence enhancement upon incubationwith the complementary target T1 over a period of 30 min, leadingto a fluorescence recovery of about 85% (curve d).

Addition of the complementary target T1 to PHIV in the absenceof Oc-AgNPs showed a slight influence on the fluorescence

was monitored at 520 nm. All measurements were done in Tris–HCl buffer in thepresence of 5.0 mM Mg2+ (pH 7.4).

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Fig. 2. Fluorescence intensity histograms of PHIV–Oc-AgNPs complex and PHIV–Oc-AgNPs complex + T1 using 0, 50, 100, 150, 200, 250, and 300 lL of Oc-AgNPs samplein this system ([PHIV] = 50 nM; [T1] = 300 nM). Excitation was at 480 nm, and theemission was monitored at 520 nm. All measurements were done in Tris–HCl bufferin the presence of 5.0 mM Mg2+ (pH 7.4).

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Fig. 4. (a) Fluorescence quenching of PHIV (50 nM) by Oc-AgNPs and (b) fluores-cence recovery of PHIV–Oc-AgNPs by T1 (300 nM) as a function of incubation time.Excitation was at 480 nm, and the emission was monitored at 520 nm. Allmeasurements were done in Tris–HCl buffer in the presence of 5.0 mM Mg2+ (pH7.4).

J.-K. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 17–21 19

intensity (curve b). The Oc-AgNPs showed no fluorescence emis-sion at 520 nm (curve e) and therefore had no contribution tothe fluorescence intensity detected of each sample. Fig. 1 insetshows the fluorescence intensity changes (F/F0 � 1) of the PHIV–Oc-AgNP complex in the presence of varied T1 concentrations,where F0 and F are FAM fluorescence intensities at 520 nm in theabsence and the presence of T1, respectively. In the DNA concentra-tion range of 2.5–300 nM, a dramatic increase of FAM fluorescenceintensity was observed, confirming that the detection methodusing the Oc-AgNPs–DNA assembly was sensitive and effectivefor investigating biomolecular interactions. This result is consistentwith our previous report on the interaction of Oc-AgNPs withbovine serum albumin [28]. The limit of detection (LOD) is calcu-lated by following equation [29]:

LOD ¼ kDSb=q1 ð1Þ

Here, KD = 3, Sb was the standard deviation of blank sample, q1 wasthe slope of the calibration line, respectively. In this study, a goodlinear relationship (the linear equation is y = 0.23x + 0.103, correla-tion coefficient R2 = 0.990) was obtained in the range of 2.5–25 nMfor the concentration of T1, the standard deviation of logarithm ofmaximum optical intensity of blank sample is 0.02 (n = 7), that is

Fig. 3. A schematic diagram (not to scale) to illustrate the fluorescence-en

meaning log (LOD) = 3 � 0.02/0.23 = 0.26, Therefore, the LOD ofthe assay is calculated to be 1.8 nM (3r).

The zeta potential of the Oc-AgNPs in Tris–HCl buffer containing5.0 mM Mg2+ (pH 7.4) was measured to be +2.08 mV. Because ofthe presence of Mg2+ (5 mM) in buffer solution, the negativelycharged property of Oc-AgNPs was shielded. Therefore, thereshould be slight electrostatic attractive interactions between Oc-AgNPs and negatively charged backbone of ssDNA. Furthermore,carboxylic curdlan bearing the b-1,3-polyglucuronic acid structureprepared from 4-acetamido-TEMPO-mediated oxidation haspotential for producing gene carriers, bio-nanomaterials and otherchiral nanowires through hydrogen bonding interactions, hydro-phobic interactions, and electrostatic attraction [30]. The strongaffinity of as-synthesized Oc-AgNPs toward ssDNA was most likelyvia strong inter-molecular hydrogen bonding interactions withtheir hydroxyl and carboxylate groups. These functional groupscould be contributed to the absorption of ssDNA on the Oc-AgNPsurfaces. In addition, Oc-AgNPs which is different from thereported nanoquenchers is not a p-rich structure [7–21,23], andthus there should not be p–p stacking interactions between ssDNAbases and Oc-AgNPs.

hanced nucleic acid detection using Oc-AgNPs as a sensing platform.

20 J.-K. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 17–21

Effect of the amount of Oc-AgNPs

The fluorescence intensity and subsequent fluorescence recov-ery of the proposed sensing system were significantly affected bythe amount of Oc–AgNPs in the system. As shown in Fig. 2, thefluorescence quenching efficiency of the system increased withincreasing amount of Oc-AgNPs. In contrast, the fluorescencerecovery efficiency decreased with increasing Oc-AgNP amount.Increasing the amount of Oc-AgNPs could increase the adsorptionof dye-labeled ssDNA and improve the quenching efficiency. How-ever, excessive Oc-AgNPs in the system could absorb the targetmolecules and prevent their hybridizing with the absorbed ssDNA,which led to decreases in both hybridization efficiency and recov-ery efficiency. The optimal amount of Oc–AgNPs was about 200 lL.

Process of nucleic acid detection and kinetic behaviors

Fig. 3 is a postulated scheme for the fluorescence-enhancednucleic acid detection process using Oc-AgNPs as a sensing plat-form. Two steps were involved in the detection of nucleic acids:(1) Oc-AgNP adsorption of the dye-labeled ssDNA because ofstrong hydrogen bonding interactions and slight electrostaticattractive interactions, which leads to fluorescence quenching ofFAM due to their close approximation [31] and (2) hybridizationof FAM-ssDNA with T1 via strong Watson–Crick hydrogen bondinginteractions in the presence of the complementary target T1 to pro-duce a rigid dsDNA that detaches from the Oc-AgNPs, resulting influorescence recovery.

The kinetic behaviors of PHIV and the Oc-AgNPs, as well as thoseof the PHIV–Oc-AgNP complex with T1, were investigated by col-lecting their time-dependent fluorescence emission spectra. Asshown in Fig. 4, in the absence of the complementary target and

Table 1Comparison of the analytical performance of different nanostructures toward nucleic acid

Platform Structure Particle size (nm) Zeta potential(mV)

Interaction fo

Oc-AgNPs Sphericalcolloids

15 2.08 Hydrogen bon

SWNTs Nanotube A few nanometersto tens ofnanometers

p–p stacking

MWCNTs Nanotubes 20–35 �29.6 p–p stackingCNPs Spherical

colloids25–40 �7.19 p–p stacking

CNSs Carbonnanosphere

60–140 �0.16 p–p stacking

Nano-C60 Sphere 5–25 �13.7 p–p stackingMC Mesoporous

structureA length of 400 nmand a width of200 nm

�35.8 p–p stacking

PANI Nanofibers 33.8 p–p stacking,electrostatic a

POPD Sphericalcolloids

450–550 �12.7 p–p stacking

PDAN Sphericalmicroparticles

600–950 4.5 p–p stacking

PNs Nanobelts Several hundred-nanometers

1.06 p–p stacking

TNs Nanoparticles 100–500 �5.36 p–p stackingCPCs Spherical

colloids�350 42.1 p–p stacking,

electrostatic aCPNBs Nanobelts 100–150 �1.59 p–p stackingAgNPs Spherical

colloids50–100

APCSNPs Core–shell 3.5 p–p stacking

in the presence of Oc-AgNPs, the fluorescence intensity of PHIV

(curve a) rapidly decreased within the first 2 min and graduallyreached equilibrium over a period of 30 min. This finding suggeststhat ssDNA adsorption occurs much faster on the Oc-AgNPs thanon SWCNTs (65 min) [6] but similar on citrate-capped AgNPs(30 min) [22] and MC (30 min) [13]. In contrast, in the presenceof the complementary target, the fluorescence intensity of thePHIV–Oc-AgNP complex (curve b) exhibited a rapid increase withinthe first 5 min, followed by a slow increase over a period of 30 min.The best fluorescence response was obtained after a 30 min incu-bation period. All these observations indicated that the fluores-cence quenching and the subsequent recovery process in ourpresent system were rapid. As a biomolecule-functionalized metalNP, the Oc-AgNP has significance differences from other chemi-cally-synthesized nanostructures in terms of structure, morphol-ogy as well as physicochemical properties, and in turn thedifferent absorption mode and kinetics of Oc-AgNPs from the othernanostructures. Table 1 summarizes the properties and the analyt-ical performance of the Oc-AgNPs and those nanostructures in theliterature toward nucleic acid detection.

Differentiation of complementary and mismatched sequences

As shown in Fig. 5, the fluorescence intensity of the PHIV–Oc-AgNP complex with the different targets decreased as the numberof mismatched bases increased. The F/F0 values obtained uponaddition of 300 nM T2 and T3 to the PHIV–Oc-AgNP complex wereabout 88% and 67% respectively of the values obtained upon theaddition of 300 nM T1 to the same complex. The mismatched targetexhibited lower hybridization ability and fluorescence recoveryefficiency with the adsorbed dye-labeled ssDNA probe than withthe complementary target. A slight change in the fluorescence

detection.

rce Fluorescencequenching/recovery

Quenchingtime (min)

Recoverytime (min)

Detectionlimit (nM)

Ref

ding 65%/85% 30 30 1.8 Thiswork

90%/70% 65 65 5 [6]

78.5%/78.2% 25 65 33 [9]82%/80% 25 40 [10]

96.5%/57.4% 25 30 0.1 [11]

74%/88% 10 25 0.025 [12]61.1%/66.1% 30 30 2.5 [13]

ttraction92.5%/94.4% 10 10 2.5 [15]

63%/56% 1 40 3 [16]

69.5%/71.2% 10 40 10 [17]

92%/55% 30 60 30 [18]

54%/72% 40 40 1.5 [19]

ttraction66%/90% 45 65 5 [20]

66%/73% 40 40 5 [21]48%/88% 30 15 5 [22]

83%/87% 30 50 0.25 [23]

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Fig. 5. Fluorescence emission spectra of PHIV (50 nM) at different conditions: (a)PHIV–Oc-AgNPs complex; (b) PHIV–Oc-AgNPs complex + 300 nM T1; (c) PHIV–Oc-AgNPs complex + 300 nM T2; (d) PHIV–Oc-AgNPs complex + 300 nM T3; (e) PHIV–Oc-AgNPs complex + 300 nM T4. Inset: fluorescence intensity histograms with errorbar. Excitation was at 480 nm, and the emission was monitored at 520 nm. Allmeasurements were done in Tris–HCl buffer in the presence of 5.0 mM Mg2+ (pH7.4).

J.-K. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 17–21 21

intensity was observed when 300 nM T4 was added to the PHIV–Oc-AgNP complex, suggesting that the observed fluorescenceenhancement in the present system was attributed to base pairingbetween the probe and its target. The inset in Fig. 5 shows the cor-responding fluorescence intensity histograms. The histograms sug-gest the good reproducibility of the present system. The resultsindicate that the proposed nucleic acid detection platform was ableto differentiate effectively between complementary andmismatched nucleic acid sequences with high sensitivity and goodreproducibility. Furthermore, the relative standard deviations(RSDs) for the measurements of PHIV–Oc-AgNP complex with300 nM different targets were found to be 1.4–3.5% (n = 7), wassmaller than 10%, indicating that the proposed method had goodreproducibility and stability. This demonstration revealed thatthe method could be used for the detection of nucleic acid. Thesensing system obtained in this work thus has a great potentialfor practical and useful mismatch detection in future applications.

Conclusions

The Oc-AgNPs synthesized using a green method were well-dispersed and quite stable in aqueous solution. The Oc-AgNPs weresuccessfully applied as an effective sensing platform for fluores-cence-enhanced nucleic acid detection. This sensing system candistinguish complementary and mismatch nucleic acid sequences

with high selectivity and good reproducibility. It is a promisingfluorescence-enhanced detection method that is also sensitiveand selective toward target molecules.

Acknowledgements

This work was supported financially by the Open Foundation ofKey Laboratory of Carbohydrate Chemistry & Biotechnology Minis-try of Education (KLCCB-KF201202), the China’s Postdoctoral Sci-ence Fund Projects (2013M531292), the Priority Ace-demicProgram Development (PAPD) of Jiangsu Higher Education Institu-tions and the Guangdong Education University-Industry Coopera-tion Project (2008B090500002).

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