Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 238–245

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Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application Ping Liu a, Liangliang Zhao a, Xia Wu a,⇑, Fei Huang a, Minqin Wang b,1, Xiaodan Liu a a Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China b School of Life Sciences, Shandong University, Jinan 250100, PR China

h i g h l i g h t s

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

 The system with AgNPs exhibited

stronger luminescence and higher photostability.  The Qu–AgNPs complex was used as a fluorescence probe for nucleic acids detection.  The Qu–AgNPs system was applied in fluorescence image analysis of protoplasts.

a r t i c l e

i n f o

Article history: Received 26 April 2013 Received in revised form 1 November 2013 Accepted 10 November 2013 Available online 20 November 2013 Keywords: Fluorescence enhancement Quercetin Silver nanoparticles Nucleic acids

a b s t r a c t It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)–Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts. Ó 2013 Elsevier B.V. All rights reserved.

Introduction Silver nanoparticles (AgNPs) have been shown to possess high binding affinity for nucleotides in nucleic acids [1,2]. They have also served as the enhancement reagent in the study of the surface enhanced resonance Raman scattering (SERRS), resonance light scattering (RLS) [3–5] and surface enhanced fluorescence (SEF) [6,7]. The SEF on metal nanoparticles is related to the shape and size of the particle, and the distance between dye molecules and ⇑ Corresponding author. Tel.: +86 531 88365459; fax: +86 531 88564464. 1

E-mail address: [email protected] (X. Wu). Co-author of first.

1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.055

the metal nanoparticles surface. Due to their unique properties and potential applications, many studies have been done on complex probes of AgNPs and dyes [3,8]. Farca˘u and Astilean [9] demonstrated a 28-fold emission enhancement of Rose Bengal fluorophore when it was placed about 1 nm above silver halfshells. Xu’s lab [10] reported the use of fluorescence probes hybridized by Aptamer/Oligomer-A/Cy3-modified AgNPs and Aptamer/ Oligomer-B/Cy3-modified AgNPs for the ultrasensitive detection of immunoglobulin E (IgE). They also exhibited a new fluorescence aptasensor based on the SEF effect of silver nanoparticles for the detection of adenosine [11]. In our previous study in which AgNPs–Curcumin (CU)-Cetyltrimethylammonium Bromide (CTAB) was used for the detection of nucleic acids. It showed that AgNPs

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could serve as a fluorescence enhancement reagent for the CU– CTAB nucleic acids system [12]. Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone, Qu) is a ubiquitous plant flavonoid found in many herbs and fruits (Fig. 1). The interactions between Qu and biomacromolecule [13,14] are commonly studied through fluorescence spectra of Qu complexes. Combining with metal ions [15] or biomacromolecule, the Qu complexes could exhibit higher fluorescence intensity than Qu alone. Our previous study found that DNA could enhance the fluorescence intensity of Qu [16]. And a sensitive determination method has been established. Here, we describe the effective enhancement effect of AgNPs on the fluorescence of the Qu-nucleic acid system. The complex system containing AgNPs coupled with Qu was used in the determination of nucleic acids in solution and in an agarose gel. We also used the complex system to probe and observe the fluorescence microimage of Arabidopsis thaliana (A. thaliana) protoplasts. In contrast to the system without AgNPs, the fluorescence of the AgNPs–fsDNA–Qu system showed an obvious synergistic enhancement effect. In addition, the interaction mechanism of the system of AgNPs–fsDNA–Qu was studied using multiple techniques, including fluorescence spectrometry, fluorescence polarization (FP), UV–vis spectrometry, circular dichroism (CD) and transmission electronic microscopy (TEM), etc. Experimental Apparatus Fluorescence and fluorescence polarization measurements were performed with a LS-55 spectrofluorimeter (Perkin Elmer, USA) in a 1 cm quartz cuvette. All the absorption spectra were measured using a U-4100 spectrophotometer (Hitachi, Japan). The TEM images were taken on a JEOL JEM-1400 transmission electron microscope. All circular dichroism spectra were performed on a J-810S circular dichroism spectrometer (JASCO, Japan). All pH measurements were made with a Delta 320-S acidity meter (Mettler Toledo, Shanghai). Agarose gel electrophoresis was performed in a horizontal gel electrophoresis apparatus (DYY-6C, Beijing Liuyi instrument factory, China). The kDNA separation pattern was analyzed with a Syngene fluorescence gel imaging system (Syngene, USA). The fluorescence images were obtained using a fluorescence microscope with a CCD camera (Nikon ECLIPSE TE2000-U).

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and diluting to 100 mL with anhydrous ethanol. A stock solution of silver nanoparticles (5.0  103 mol L1 calculated by the concentration of the silver ion added) was synthesized by an electrochemical method as described in reference [17]. 1.0 g PVP k30 was dissolved in mixed solution of 1.0  102 mol L1 of AgNO3 (25.0 mL) and 1 mol L1 of KNO3 (5.0 mL), then diluting it to 50 mL with 0.22 lm-filtered ultra pure water (18.25 MX cm). The mixed solution was electrolyzed with stirring at 800 r/min for 12 min. The solution color changed gradually from colorless to yellow. The plasma resonance absorption peak of AgNPs is at 402 nm. All the solutions used were stored in a refrigerator at 0–4 °C. A formic–NaOH buffer solution was prepared with 0.2 mol L1 formic acid solution adjusted to pH 4.40 with 0.2 mol L1 NaOH solution. Unless otherwise noted, all reagents and solvents were analytical grade and ultra pure water was used throughout. Procedure Fluorescence spectra To a 10-mL colorimetric tube, solutions were added in the following order: 0.3 mL of 0.2 mol L1 formic–NaOH buffer solution (pH = 4.40), an appropriate amount of nucleic acids, 0.3 mL of 5.0  105 mol L1 AgNPs and 0.15 mL of 1.0  104 mol L1 Qu. The mixture was diluted to 5 mL with water and allowed to stand for 50 min. The excitation and emission slits were both 10 nm and the scan speed was 500 nm min1. The fluorescence intensity at 490 nm with an excitation wavelength of 440 nm was recorded. The enhanced fluorescence intensity of the AgNPs–fsDNA–Qu system was calculated as DIf ¼ If  I0f where If and I0f represent the intensity of the system with and without nucleic acids. Fluorescence polarization Fluorescence polarization were recorded on a Perkin–Elmer LS55 spectrofluorometer, both excitation and emission slits set at 10 nm. Solution used in the measurements of fluorescence polarization was prepared in the same way as described in fluorescence spectra measurements. The solutions were excited at 440 nm and the fluorescence was monitored at 490 nm through a pair of polariser filters. The Fluorescence polarization parameter P is defined as:

P¼

I==  I? I== þ I?

where I// and I\, are the fluorescence components parallel to and orthogonal to the polarization of the exciting light.

Chemicals Stock solutions of nucleic acids (1.0  104 g mL1) were prepared by dissolving commercial fish sperm DNA (fsDNA) (Sigma), salmon serum DNA (smDNA) (Chemical Co. USA) or yeast RNA (yRNA) (Beijing Baitai Co., China) in 0.05 mol L1 sodium chloride solution, respectively. A stock solution of Qu (1.0  103 mol L1) was made by dissolving 0.0302 g of Qu in absolute ethyl alcohol

Fig. 1. The chemical structure of quercetin.

Agarose gel electrophoresis protocol Prior to gel casting, 0.6 g dried low temperature agarose gel was dissolved in 60 mL TAE buffer (1  pH 8.3 tris-acetate-EDTA buffer) by being heated in a microwave for 2–4 min and was then poured into a 8 cm  8 cm glass mold, which was fitted with a well-forming comb, and the processing procedures were performed according to Sambrook and Russel [18]. The conventional kDNA/ HindIII + EcoR I digest molecular weight markers (kDNA marker) were used as DNA samples in the experiment. A total of 20 lL of DNA mixture containing 6 lL kDNA marker samples (1 lL, 0.5 lg kDNA marker + 1 lL of 6  DNA Loading Dye + 4 lL of deionized water) and 14 lL Qu or Qu–AgNPs solution (Qu: 7.5  105 mol L1; AgNPs: 7.5  105 mol L1; formic–NaOH: 3.6  102 mol L1, pH = 4.40) were loaded in each sample well. Then 1  TAE electrophoresis buffer was added into a horizontal electrophoresis apparatus until the buffer just covered the agarose gel. Electrophoresis was performed at 70 V for 30–60 min at room temperature, depending on the desired separation. After electrophoresis, the gel was cut into two pieces, one was immersed into

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Qu, and the other was immersed into Qu–AgNPs solution. After 5 min, the gel was placed on a UV light box. The images of the fluorescence of the Qu-kDNA marker and Qu–AgNPs-kDNA marker (the time of exposure was 430 ms) were analyzed with a Syngene gel imaging system.

Fluorescence microimage Calli of A. thaliana was induced from seedlings on MS medium supplemented with 1 mg/L 2, 4-D (MS1 medium) and 0.7% (w/v) agar. Suspension cells were induced from the friable and embryogenic calli cultured on liquid MS1 medium on a gyratory shaker (200 r/min) and subcultured weekly [19]. Protoplasts were isolated from approximately 2-month-old cell suspension cultures in an enzyme solution (1.6% (w/v) Cellulase Onozuka RS10 (Yakult Biochemicals, Tokyo, Japan), 0.3% (w/v) Macerozyme R10 (Yakult Biochemicals), 7% (w/v) mannitol and 0.12% (w/v) CaCl22H2O (pH = 5.5)) [20]. After being incubated on the shaker (30 r/min) for 2–3 h at 25 °C, the protoplast–enzyme mixture was filtered through a 50 lm mesh nylon sieve and washed for 3 times with washing solution (7% (w/v) mannitol and 0.12% (w/v) CaCl22H2O) by centrifugation at 500 r/min for 3 min [19]. One little drop (about 6–8 lL) of the above protoplasts were softly transferred onto the slides using micropipettes. The slides were stained with 20 lL Qu or Qu–AgNPs solution (Qu: 7.5  105 mol L1; AgNPs: 7.5  105 mol L1; formic–NaOH: 3.6  102 mol L1, pH = 4.40) for 20–30 min at 25 °C. The images were observed under a fluorescence microscope with a CCD camera.

Results and discussion Fluorescence spectra Fluorescence spectra of the systems of Qu, nucleic acids and AgNPs are shown in Fig. 2. The fluorescence of Qu (curve 2) is weak. Its excitation and emission peaks are at 440 nm and 515 nm, respectively. AgNPs can increase slightly the fluorescence intensity of Qu system. The peak positions of excitation and emission spectra remain nearly unchanged (curve 3). Whereas the fluorescence intensity of Qu increases 5-fold after adding fsDNA to the system (curve 4), and the emission peak shows a blue shift to 490 nm. That means that when Qu combines to DNA, the conjugate rigid plane structure of Qu is formed, resulting in an increase of the fluorescence intensity [16]. After adding AgNPs to the Qu–fsDNA(yRNA, smDNA) system, the fluorescence intensity of Qu–fsDNA(yRNA, smDNA) increases dramatically, without significant change in the maximum emission wavelength (curves 7, 6 and 5 vs curve 4). These results indicate that there are synergistic interactions among the nucleic acids, Qu and AgNPs.

Effects of pH and choice of buffer solution The effects of pH on the fluorescence intensity of the systems were tested (see Supplementary Fig. S1). A large fluorescence intensity enhancement is observed over the range from pH 4.0 to 5.5, with a maximum DIf obtained at pH 4.40. The influence of different buffers on the fluorescence intensity of this system was also tested at pH (4.40 ± 0.05). The relative DIf (%) for formic acid– NaOH, Britton–Robinson (BR), citric acid–sodium citrate, HMTA– HCl and NaAc–HAc were 100, 60.8, 2.7, 32.1 and 9.8, respectively. So formic acid–NaOH was chosen for the remaining experiments. Experimental result has been proved that the optimum concentration of formic–NaOH buffer was 1.2  102 mol L1.

Fig. 2. Fluorescence spectra of the Qu–fsDNA–AgNPs system. (a) excitation spectra (kem = 490 nm); (b) emission spectra (kex = 440 nm). Curves: (1) fsDNA–AgNPs; (2) Qu; (3) Qu–AgNPs; (4) Qu–fsDNA; (5) AgNPs–smDNA-Qu; (6) AgNPs–yRNA-Qu; (7) AgNPs–fsDNA–Qu. Conditions: Qu: 3.0  106 mol L1; fsDNA, smDNA, yRNA: 1.0  106 g mL1; AgNPs: 3.0  106 mol L1; and formic–NaOH buffer solution: 0.012 mol L1 (pH = 4.40).

Effect of molar ratio of Qu to AgNPs Using a fixed AgNPs concentration of 3.0  106 mol L1, the effect of the molar ratio of Qu to AgNPs on the DIf was tested(see Supplementary Fig. S2). The results showed that when the molar ratio of Qu to AgNPs was less than 1:1, the DIf increased sharply with the increasing molar ratio of Qu to AgNPs and then reached a maximum and remained roughly constant in the range of 1:1 to 3:1. When the molar ratio of Qu:AgNPs exceeded 3:1, the DIf decreased. So a 1:1 M ratio of Qu:AgNPs was chosen for further research.

Fluorescence signal stability over time The effect of the order of reagent addition on the fluorescence intensity of the system was tested. Adding formic–NaOH buffer solution, nucleic acids, AgNPs and Qu in this sequence gave the strongest fluorescence enhancement. Under the optimum conditions, after all reagents had been added, the fluorescence intensity reached a maximum after 50 min and remained stable for over 4 h, whereas the fluorescence intensity of the Qu–fsDNA system only

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remained stable for 2 h, meaning that the fluorescence intensity of AgNPs–fsDNA–Qu was more stable than that of Qu–fsDNA.

and molecular biology. The DNA sample was isolated from seedlings of A. thaliana by using the CTAB method [21]. The concentration of DNA was 5.20  104 g mL1, as determined by a Biophotometer (Eppendorf Co). The sample was diluted 1000 times and the DNA determined by our proposed method. The mean value of five measurements was 5.17  104 g mL1 and the relative standard deviation was 2.1% (n = 5). Hence, the proposed method was suitable for the determination of trace amount of nucleic acids in this sample.

Effect of foreign substances The interference of foreign substances such as amino acids and metal ions on the system was tested and the results are shown in Table 1. All the amino acids and metal ions tested except Al3+ had little effect on the system fluorescence, therefore would not interfere with the detection of fsDNA within ± 5% relative error.

Agarose gel electrophoresis analysis Preparation of the calibration curves and determination of detection limits

Fig. 3 shows the electrophoresis pattern of kDNA molecular weight markers. The profile pattern of the 564 bp kDNA fragment clearly appeared immediately after the agarose gel was immersed into a solution of Qu–AgNPs, which meant that almost all kDNA band patterns stained clearly by Qu–AgNPs. But only after the agarose gel was immersed into Qu solution for five minutes, could the profile pattern of kDNA fragments larger than 1904 bp be discerned(Fig. 3a). So the fluorescence intensity of the kDNA marker stained by Qu was lower than that by the Qu–AgNPs systems. The results suggest that the AgNPs improved the detection rate of kDNA markers by agarose gel electrophoresis analysis, and that

Under the optimum conditions, nucleic acids could remarkably enhance the fluorescence intensity of the system of Qu–AgNPs. A linear relationship was obtained between DIf and the concentration of nucleic acids (see Supplementary Fig. S3). All the parameters are presented in Table 2. The detection limits (S/N = 3) of fsDNA, smDNA and yRNA were 4.2  109 g mL1, 8.8  109 g mL1 and 6.6  109 g mL1 respectively, according to the 3Sb/S criterion, where S is the slope for the range of the linearity used and Sb is the standard deviation of the blank (n = 11). Therefore the AgNPs–Qu probe could detect many nucleic acids, and we applied it for analysis of an actual DNA sample and for DNA agarose gel electrophoresis analysis. Analytical applications Analysis of an actual biological sample An A. thaliana DNA sample was tested by the standard addition method. Because of its small genome and the completion of its whole genome sequencing, A. thaliana is used as a model species and offers important advantages for basic research in genetics

Table 1 Interference from foreign substances. Foreign substances

Concentration coexisting (105 mol L-1)

Change of DIf (%)

Na+, CO2 3 K+, Cl Mg2+, Cl Zn2+, Ac Na+, Cl Al3+, Cl  NHþ 4 , Cl Ca2+, Cl

13

4.9

5 13 11 80 0.9 3.2 12.5 4.0

4.9 4.8 5.7 4.6 5.0 3.8 4.2 5.3

3.8 1.3 2.4 2.5 16.4 4.4

5.4 4.6 5.5 3.9 4.9 4.1

Mn2+, SO2 4 L-HB CMP TMP AMP L-Asp L-Phe

Fig. 3. Agarose gel electrophoresis analysis. (a) Electrophoresis pattern of Lambda DNA size marker (cut with EcoR I + Hind III) stained with Qu for 5 min; (b) Electrophoresis pattern of Lambda DNA size marker (cut with EcoR I + Hind III) stained with Qu–AgNPs for 5 min. Conditions: Qu: 7.5  105 mol L1; AgNPs: 7.5  105 mol L1; and formic–NaOH: 3.6  102 mol L1, pH = 4.40.

Conditions: Qu: 3.0  106 mol L1; fsDNA: 1.0  106 g mL1; AgNPs: 3.0  106 mol L1; and formic–NaOH buffer solution: 0.012 mol L1 (pH = 4.40).

Table 2 Analytical parameter of this method. Nucleic acids fsDNA yRNA smDNA

Linear range (g mL1) 9

6

8.0  10 –2.0  10 5.0  108–5.0  106 2.0  108–8.0  107

Regression equation (g mL1) 8

DIf = 3.18 + 1.92  10 C DIf = 8.33 + 1.42  108 C DIf = 0.38 + 1.28  108 C

Detection limit (g mL1)

Correlation coefficient

4.2  109 6.6  109 8.8  109

0.999 0.993 0.997

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the Qu–AgNPs system could be regarded as a DNA indicator, which may be able to replace toxic substances such as ethidium bromide (EB) in the future. Fluorescence image analysis of protoplasts of A. thaliana Fig. 4 shows the optical and fluorescence microscopy images of A. thaliana protoplasts. The fluorescence intensity of the A. thaliana protoplasts increased remarkably in the presence of Qu–AgNPs, meaning the contents of the plant cell are more sensitive to the Qu–AgNPs than Qu. We deduce that the Qu–AgNPs complex system combines with nucleic acids and other substances in the A. thaliana cells. Clearly, the surface fluorescence enhancement effect of AgNPs plays a key role in the system. More detailed investigations on the fluorescence enhancement mechanism in this system are in progress. Interaction mechanism of the system of Qu–AgNPs–fsDNA The interaction between Qu, AgNPs and fsDNA The UV–vis absorption spectra of the system are shown in Fig. 5. Qu exhibits absorption bands with peaks at 368 nm (band I) and 253 nm (band II), respectively. The band I at 368 nm was related to the n–p* transitions, which corresponds to the cinnamoyl system consisting of ring B-ring C. Whereas the band II at 253 nm was related to the p–p* chromophoric transitions, which corresponds to benzoyl system consisting of ring A (see Fig. 1) [22,23]. Upon adding fsDNA and AgNPs–fsDNA to the Qu system, the absorption intensity of band II showed only overlay of the absorption of the each components. Comparing to the Qu, the absorption

peak of AgNPs–Qu was red shifted from 253 nm to 264 nm. Whereas a similar change in absorption band I was exhibited in the systems of AgNPs–Qu and fsDNA–Qu. The original peak at 368 nm weakened and a new peak emerged at 438 nm. We concluded that the structure changed of the rings B–C of Qu more significantly than that of ring A when Qu interacted with fsDNA and AgNPs. From the fluorescence and UV–vis spectra (Figs. 2 and 5), it can be seen that the absorbance at 438 nm of the AgNPs–Qu system is the biggest among them, but its fluorescence intensity increases slightly. Which may be related to the energy dissipation in AgNPs, resulting from Qu directly combing to AgNPs surface [24]. Whereas after adding nucleic acid to the Qu system, the absorption peak at 368 nm decreased meanwhile the absorbance at 438 nm increased and the fluorescence intensity increased 5 times. Evidently fsDNA could change the structure of Qu and they form a new complex. And comparing with the absorption spectrum of fsDNA–Qu, the absorbance at 368 nm of fsDNA– Qu with AgNPs is lower meanwhile its absorbance at 438 nm is higher. But their absorption intensities are between that of fsDNA–Qu and AgNPs–Qu (Fig. 5). And the fluorescence intensity enhancement of fsDNA–Qu in the presence of AgNPs has doubled (Fig. 2). It indicates that AgNPs can further improve the fluorescence intensity of fsDNA–Qu. That is the plasmon effect of AgNPs helps to enhance the fluorescence intensity of the Qu-nucleic acids system. In addition, AgNPs solution prepared have an absorption peak at 402 nm (see Fig. 5 inset 1), which is corresponding to their plasmon resonance absorption. The inset 2 in Fig. 5 is the absorption spectra of AgNPs–Qu (vs Qu) and AgNPs–fsDNA–Qu (vs Qu–fsDNA). From Fig. 5 inset 2, it can be seen that the maximum absorption peaks of AgNPs both undergo a bathochromic shift to about 430 nm. They display that the size of AgNPs increase in the presence of Qu or Qu–fsDNA. Which is confirmed by the TEM

Fig. 4. Microscope images of agglutinate A. thaliana protoplasts with Qu or Qu–AgNPs. (A) optical microscope image with Qu; (B) fluorescence microscope image with Qu; (C) optical microscope image with Qu–AgNPs; and (D) fluorescence microscope image with Qu–AgNPs. Bar = 10 lm; Conditions: Qu: 7.5  105 mol L1; AgNPs: 7.5  105 mol L1; formic–NaOH: 3.6  102 mol L1, pH = 4.40.

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To further study the interaction between AgNPs–Qu and fsDNA, the fluorescence polarization values (P) of the system versus the concentration of fsDNA were tested (see Supplementary Fig. S4). As we know, when chromophore intercalates into the DNA helix, its rotational motion is restricted and the fluorescence polarization of the bound chromophore should be increased [25]. Herein, the increase of the concentration of fsDNA was negatively correlated to the P values of the system, meaning that the AgNPs–Qu or Qu combined with fsDNA in non-intercalation binding mode [26]. The P values of Qu–AgNPs and Qu alone were 0.468 and 0.320, respectively. The increase of P in the Qu–AgNPs system should be attributed to Qu conglutination with AgNPs and the rotation slowdown of Qu molecules. And the variation tendency of the P value of the AgNPs–Qu–fsDNA system was similar to the Qu–fsDNA system. Therefore it is likely that Qu is connected directly with fsDNA in the AgNPs–Qu–fsDNA system. Based on the UV–vis and fluorescence polarization spectra, we conclude that in the AgNPs–fsDNA–Qu system, fsDNA acts as a bridge between Qu and AgNPs which assures a proper distance of Qu from AgNPs, resulting in the fluorescence enhancement of the system. A DNA denaturalization experiment was performed to test the effect on the fluorescence response of the system. Two samples containing only fsDNA were heated to different temperatures: one was kept at room temperature, and the other was denatured by heating to 100 °C for 5 min, then freezing in an ice-bath for 10 min and thawing at room temperature. Hence, the second sample was denatured such that the strands separate completely into a single-stranded structure compared to the native structure of DNA in the first sample. The fluorescence spectra (see Supplementary Fig. S5) showed that the DNA samples could both enhance the fluorescence of Qu–AgNPs but the enhancement was greater for the native DNA. Thereby it was considered that the intact helix within the double strands actively binds Qu–AgNPs, whereas the single stranded DNA also had obvious enhancement function, which showed that there was an interaction between single-strand fsDNA and Qu–AgNPs. The present results suggest there are two main interaction modes between Qu–AgNPs and fsDNA, the groove binding and electrostatic force, and the latter was dominant.

Fig. 5. The UV–vis absorption spectra of the system. Fig. 5 and Fig. 5 inset 2: Conditions: Qu: 8.0  106 mol L1; AgNPs: 7.0  106 mol L1; fsDNA: 5.0  106 g mL1; formic–NaOH buffer solution: 0.012 mol L1 (pH = 4.40) Fig. 5 inset 1: Conditions: AgNPs: 5.0  103 mol L1.

images (see Section 5.3). From the above, we infer that there is interaction among Qu, AgNPs and nucleic acids. In this AgNPs– fsDNA–Qu system, the nucleic acids provide a suitable distance between Qu and AgNPs and make the surface plasmon resonance effect of AgNPs more evident, resulting in fluorescence enhancement of the fsDNA–Qu system.

Fig. 6. The CD spectra of the system. (1) fsDNA; (2) AgNPs–fsDNA; (3) Qu–fsDNA; (4) AgNPs–fsDNA–Qu. Conditions: Qu: 3.0  106 mol L1; fsDNA: 1.0  106 g mL1; AgNPs: 3.0  106 mol L1; and formic–NaOH buffer solution: 0.012 mol L1 (pH = 4.40).

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Fig. 7. TEM images of the system. (a) AgNPs; (b) AgNPs–fsDNA; (c) AgNPs–Qu; (d) AgNPs–fsDNA–Qu. Conditions: AgNPs: 3.0  106 mol L1; fsDNA: 1.0  106 g mL1; Qu: 3.0  106 mol L1; and formic–NaOH buffer solution: 0.012 mol L1 (pH = 4.40).

Conformation change of fsDNA The circular dichroism spectra in the UV range can be used to monitor the conformational transition of DNA. To explain the interaction between fsDNA and the Qu–AgNPs system, the CD spectra of the system were taken (Fig. 6). It is known that a positive Cotton effect at 275 nm corresponds to base stacking, and a negative Cotton effect at 245 nm corresponds to helicity of nucleic acid [27]. Upon adding Qu or AgNPs to fsDNA, the ellipticity at 245 nm and 275 nm both decrease and the minimum ellipticity is also red shifted. Upon adding Qu–AgNPs to the fsDNA system, the ellipticity at 245 nm decreases greatly, but it increases correspondingly at 275 nm and is even higher than that of native fsDNA. Therefore, the synergistic action between Qu and AgNPs can lead to more obvious conformational transitions of fsDNA, resulting in more effective interaction among Qu, AgNPs and fsDNA.

The aggregates of Qu–fsDNA–AgNPs TEM images of the system are shown in Fig. 7. The AgNPs are spherical in shape and well dispersed with a diameter of about 15 nm (Fig. 7a). In the presence of fsDNA (Fig. 7b), regular wire-like aggregates of 30 nm in average size are developed. In the system of Qu and AgNPs, larger aggregates appear with about 70 nm diameters (Fig. 7c). In the AgNPs–fsDNA–Qu system, the size of the aggregates is about 40 nm (Fig. 7d). In these systems, the diameters and shapes of the aggregates are different, which confirms that the AgNPs play an important role in the fluorescence enhancement

of the AgNPs–fsDNA–Qu system because of the synergy of between Qu and fsDNA with AgNPs. Conclusions A new method for the determination of nucleic acids was established based on the fluorescence enhancement effect of Qu–fsDNA in the presence of AgNPs. The detection limits (S/N = 3) for the nucleic acids were reduced to the ng mL1 level. This complex system of Qu with AgNPs was used for the determination of nucleic acids in actual samples, and for the analysis of nucleic acids after agarose gel electrophoresis and in fluorescence microscopy of A. thaliana protoplasts. Satisfactory results were obtained. Investigation into the interaction mechanisms of the Qu–fsDNA–AgNPs system indicated that both groove binding and electrostatic interactions exist between Qu–AgNPs and fsDNA. The nucleic acid acts as a bridge between AgNPs and Qu, providing a suitable distance between them that leads to the fluorescence enhancement of the system. The system with AgNPs exhibits stronger fluorescence efficiency and higher photostability. Acknowledgements This work was supported by the Natural Science Foundation of Shandong Province, China (Grant No. ZR2013BM025). We gratefully express deep appreciation to Dr. Houyi Ma (Shandong University) for his help in preparation of AgNPs. We also thank Dr. Pamela Holt for editing the manuscript.

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