Ratiometric fluorescence biosensor based on CdTe quantum and carbon dots for double strand DNA detection

Sensors and Actuators B 244 (2017) 585–590

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Ratiometric fluorescence biosensor based on CdTe quantum and carbon dots for double strand DNA detection Si-Si Liang, Liang Qi, Rui-Ling Zhang, Meng Jin, Zhi-Qi Zhang ∗ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China

a r t i c l e

i n f o

Article history: Received 18 September 2016 Received in revised form 14 December 2016 Accepted 5 January 2017 Available online 6 January 2017 Keywords: Ratiometric fluorescence biosensor HIV dsDNA Quantum dots Mitoxantrone

a b s t r a c t A ratiometric fluorescence biosensor was developed for detection of double strand DNA (dsDNA). The sensor consists of water-soluble fluorescent carbon dots (CDs) and 3-mercapropionic acid-coated cadmium telluride (CdTe) quantum dots exhibiting emissions peaks at 435 and 599 nm, respectively, under single-wavelength excitation (360 nm). CdTe QDs fluorescence was quenched by mitoxantrone via electron transfer and was restored in the presence of dsDNA, while the fluorescence intensity of CDs remained almost constant, providing a ratiometric means of dsDNA detection. The relative fluorescence intensity ration is directly proportional to the concentration of dsDNA between 0 and 50 nM, and the detection limit is 1.0 nM. Meanwhile, common organic compounds including amino acids, nucleotides, bovine serum albumin, single strand DNA and RNA had not significant interference in the detection mode. The novel nanosensor is simple, rapid, and convenient since it does not require modification or separation procedures, and was applied to the detection of HIV dsDNA in synthetic samples and human serum samples with satisfactory results. © 2017 Elsevier B.V. All rights reserved.

1. Introduction DNA is central to biochemical processes including gene expression, gene transcription, mutagenesis, and carcinogenesis [1–5]. Nucleic acid probes have been designed as diagnostic and therapeutic tools that are based on small molecule-DNA interactions such as intercalation, groove binding, and electrostatic interaction [6–10]. Various organic dyes have been incorporated into DNA probes, including cyanine dyes, indoles, and imidazoles. DNA can be detected by spectrophotometry [11,12], fluorescence [13–17], surface-enhanced Raman scattering [18,19], and electrochemical approaches [20–22]. Fluorescence-based methods are widely used owing to their high sensitivity, reproducibility, accuracy, rapid response, and low cost. Most fluorescence sensors target specific DNA sequences [23–26]. In fact, DNA is rarely present in the single-stranded form (ssDNA), either naturally or after PCR amplification. Therefore, methods that detect native or double strand DNA (dsDNA) are a more robust and flexible diagnostic tool [27]. For instance, a family

∗ Corresponding author. E-mail address: [email protected] (Z.-Q. Zhang). http://dx.doi.org/10.1016/j.snb.2017.01.032 0925-4005/© 2017 Elsevier B.V. All rights reserved.

of ruthenium complexes such as Ru(phen)3 2+ and its derivatives has been developed as fluorescent DNA probes [28,29]. One such probe is an ionic conjugate of water-soluble thioglycolic acid-capped CdTe quantum dots (QDs) and Ru(bpy)2 (dppx)2+ for dual-color detection of dsDNA [30]. Another dsDNA biosensor is based on photoinduced electron transfer from glutathione-capped CdTe QDs to a praseodymium(III)-rutin complex [31]. Moreover, a (CdTe QDs–safranine T)-based fluorescence “off/on” biosensor was developed for herring sperm DNA detection [17]. Various metal-organic frameworks have been employed to detect human immunodeficiency virus (HIV) 1 dsDNA [32,33]. However, these unidimensional fluorescence signal methods are easily influenced by external conditions or manual operation. Photoluminescent nanoparticles have unique optical and electronic properties owing to quantum-size confinement effectsincluding high fluorescence quantum yield, size- and compositiontunable emission, and resistance to photobleaching [34–37] that provide important advantages over organic dye and lanthanide probes. In this study, we developed a QD-based dual-emission ratiometric fluorescence sensor for DNA detection that consists of water-soluble red-emitting 3-mercaptopropionic acid-capped CdTe QDs and biocompatible green-emitting carbon dots (CDs) (Scheme 1). This sensor differs from traditional ratiometric sensors

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Scheme 1. Schematic diagram of the CdTe–CDs–MTX system to detect dsDNA.

in which one of the fluorescence materials is covalent linked to the surface of silica nanoparticles [38,39], a process that can potentially lead to the loss of fluorescence. Mitoxantrone (MTX) is a synthetic anthraquinone drug with a symmetrical structure comprising a tricyclic planar chromophore and two basic side chains that can intercalate with DNA [40,41]. In this work, MTX quenched the fluorescence of CdTe QDs via an electron transfer mechanism, switching the red fluorescence into an “off” state. On the other hand, MTX intercalated into the double helix structure of DNA and was peeled off of the CdTe QD surface, resulting in the recovery of QD fluorescence at 599 nm. Meanwhile, CDs were insensitive to both MTX and DNA and maintained constant fluorescence intensity, thereby serving as a reference for ratiometric detection of dsDNA. 2. Experimental 2.1. Apparatus Fluorescence measurements were carried out on a LS-55 luminescence spectrometer (Perkin-Elmer, Waltham, MA, USA) in a 400 ␮l quartz cuvette at 37 ◦ C. Ultraviolet–visible light (UV–vis) adsorption spectra were acquired on a TU-1901 spectrophotometer (Puxi Analytic Instrument, Beijing, China). Fourier-transform infrared (FT-IR) spectra ranging from 400 to 4000 cm−1 in KBr were recorded using a Tensor 27 spectrometer (Bruker, Bremen, Germany). Transmission electron microscopy (TEM) was carried out on a Tecnai G2 F20 microscope (FEI, Hillsboro, OR, USA) with an accelerating voltage of 200 kV. 2.2. Reagents Ultrapure water obtained from a Milli-Q water purification system (18.2 M cm, Millipore, Billerica, MA, USA) was used for all experiments. HIV dsDNA (5 -CGA GTT AAG AAG AAA AAA GAT TGA GC-3 /5 -GCT CAA TCT TTT TTC TTC TTA ACT CG-3 ), ssDNA1 (5 -ACC TGG GGG AGT ATT GCG GAG GAA GGT-3 ), ssDNA2 (5 GGT TGG TGT GGT TGG-3 ), and ssDNA3 (5 -GCA GTT TGG AC) were synthesized by Shanghai Sangon Biotechnology Co. (Shanghai, China). RNA (5 -GGC CUG GGC GAG AAG UUU AGG CC-3 ) was synthesized and purified by Takara Biotechnology Co. (Da Lian, China). MTX dihydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA), nucleoside monophosphate, and amino acids were purchased from Shanghai Sangon Biotechnology Co., and 3-mercaptopropionic acid (3-MPA), cadmium chloride (CdCl2 ), tellurium (Te) powder, and sodium

borohydride were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). All reagents were of analytical grade and were used without further purification. 2.3. Methods 2.3.1. Preparation of 3-MPA-cadmium telluride (CdTe) QDs and ammonium citrate CDs Water-soluble 3-MPA-capped CdTe QDs were synthesized as previously described [42]. Briefly, 0.228 g (1 mmol) of CdCl2 ·2.5 H2 O and 1.6 mmol (126 ␮l) 3-MPA was mixed in 40 ml deionized water, and the pH was adjusted to 10.5 by dropwise addition of 2.0 M NaOH solution with stirring. The three-necked flask containing the mixture was heated with a heating jacket under an N2 atmosphere. When the temperature reached 100 ◦ C, 10 ml of the NaHTe solution was added. The mole ratio of Cd:MPA:Te in the reaction was 1:1.5:0.2. Nitrogen was steadily pumped into the mixture with the temperature kept constant to obtain stable, water-compatible 3-MPA-capped CdTe QDs with a fluorescence emission wavelength of 599 nm. Fluorescent CDs were synthesized as previously described [43]. Ammonium citrate (2 g, 8.2 mmol) was dissolved in 25 ml deionized water. The resultant transparent solution was sealed in a 50-ml Teflon-equipped stainless steel autoclave and heated at 160 ◦ C for 6 h. The color of the solution turned from colorless to dark blue. The final solution was diluted and dialyzed against pure water for 2 days. 2.3.2. Analytical procedure To obtain a ratiometric fluorescence sensor with a suitable fluorescence spectrum, a mixture containing CdTe (8 × 10−3 mol L−1 ; 5 ml) and CDs (8.2 × 10−4 mol L−1 ; 1 ml) was prepared. A 10␮l CdTe-CDs solution and different concentrations of MTX were dispersed in a centrifuge tube and diluted to 200 ␮l with phosphate-buffered saline (pH 7.4, 150 mM NaCl), then mixed thoroughly by gentle shaking. The concentration of MTX that was added ranged from 0 to 1000 nM. These above samples were applied to the fluorescent quenching phenomenon investigation. Furthermore, the CdTe–CDs QDs–MTX solution system was used for analyzing fluorescence enhancement of the solution system when introducing dsDNA. The concentration range of dsDNA was from 0 to 175 nM. To obtain the final fluorescence intensity, a two-stage analytical process was carried out with incubation for 10 min at room temperature, followed by recording of fluorescence spectra at an excitation wavelength of 360 nm with excitation and emission band-slits set as 10 nm. Fluorescence spectra were recorded

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from 380 to 700 nm, and fluorescence intensities of CDs at 435 nm and CdTe at 599 nm were used for quantitative analysis of dsDNA. Unless otherwise specified, all spectra were obtained at least in triplicate. 3. Results and discussion 3.1. Characterization of CdTe QDs and CDs The FT-IR spectra of CdTe (Fig. S1A) [44] and CDs showed surface functional groups; the abundance of hydroxyl and carboxyl groups on the surface of the latter indicated excellent water solubility (Fig. S2A) [43]. The TEM analysis of aqueous 3-MPA-capped CdTe QDs revealed monodispersed particles with near-uniform size and a diameter of about 5 nm (Fig. S1B), whereas CDs were approximately spherical and showed good size distribution and excellent monodispersity, with an average size of around 2 nm (Fig. S2B). 3.2. Effect of MTX and dsDNA on the fluorescence spectra of CdTe−CDs system The effect of different agents on CdTe–CDs was examined by comparing the fluorescence spectra of CdTe–CDs alone, CdTe–CD–dsDNA, CdTe–CD–MTX, and CdTe–CD–MTX–dsDNA under the same experimental conditions (Fig. 1). Direct interaction of CdTe–CDs with dsDNA did not alter the fluorescence signal. CdTe fluorescence was decreased in the presence of MTX, but addition of dsDNA restored the signal at 599 nm, indicating that CdTe–CDs are suitable for ratiometric fluorescence detection of dsDNA. 3.3. Effect of pH We also investigated the effect of pH on CdTe−CDs and found that fluorescence intensity of CDs slightly increased very slightly with the increase of pH, it’s was not sensitive to pH. While CdTe had weak fluorescence under acidic conditions (Fig. 2A). To establish the optimal conditions for CdTe–CDs with MTX and dsDNA, the pH was varied between 6.0 and 12.0 (20 mM phosphate buffer with 150 mM NaCl) and the fluorescence intensity ratio–defined as I599 /I435 –was determined (Fig. 2B, C). I1 and I2 represented the fluorescence intensity ratio of CdTe−CDs in the presence of MTX only or both MTX and dsDNA, respectively. Under slightly alkaline conditions (pH optimum of 7.4), I2 –I1 was higher. We therefore used phosphate buffer at pH 7.4 for subsequent dsDNA detection. We also determined that the fluorescence intensity of CdTe–CDs

Fig. 1. The fluorescence spectra of CdTe–CDs (10 ␮l) in different system. (a) CdTe–CDs; (b) CdTe–CDs + dsDNA (175 nM); (c) CdTe–CDs + MTX (1000 nM); (d) CdTe–CDs + MTX (1000 nM) + dsDNA (25 nM).

(I599 /I435 ) remained stable over 5 h at pH 7.4 (Fig. S3). In fact, even after being stored for one months, there was no significant change of fluorescence intensity of CdTe–CDs system. 3.4. Quenching effect of MTX on CdTe−CDs Addition of MTX to CdTe–CDs solution reduced the fluorescence intensity of CdTe–especially the emission peak at 599 nm–while that of CDs remained constant. It was found that 1000 nM MTX presented a quenching effect of ∼85%. I599 /I435 was used for linear fit analysis at different MTX concentrations (Fig. 3). The quenching constant (Ksv ) was calculated with the following equation [45]: F0 /F = (f a Ksv )−1 [Q]−1 + f a −1 ,

(1)

where F is the difference in I599 /I435 measured in the absence (F0 ) and presence (F) of MTX at concentration [Q], fa is the fraction of maximum accessible fluorescence, and Ksv is the effective quenching constant for accessible fluorophores. The dependence of F0 /F on the reciprocal of MTX concentration ([Q]−1 ) was linear, with a slope of (fa Ksv )−1 and a y intercept of fa −1 . The constant Ksv −1 is the quotient of the ordinate fa −1 and the slope (fa Ksv )−1 . A curvefitting analysis for Eq. (1) suggested that the fluorescence of CdTe QDs could be quenched by MTX with a quenching constant (Ksv ) of 1.167 × 103 Lmol −1 (Fig. S4).

Fig. 2. (A) Effect of pH values on the fluorescence intensities of CdTe–CDs system. (B) Effect of pH values on the I599 /I435 ratio of CdTe–CDs in the CdTe-CDs alone, CdTe–CDs–MTX system and CdTe–CDs–MTX–dsDNA system. CeTe–CDs: 10 ␮l; MTX: 1000 nM; DNA: 25 nM. (C) The difference at I599 /I435 ratio of CdTe−CDs system in the presence of MTX (I1 ) and in the presence of MTX, dsDNA (I2 ). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Fluorescence spectra of CdTe–CDs at the addition of MTX (0, 125, 250, 375, 500, 750, 875, 1000 nM) in phosphate buffer solution (pH 7.4, 150 mM NaCl). Insert: linear relationship between the fluorescence intensity ratio of I599 /I435 and MTX concentration in the range of 0–1000 nM. CdTe–CDs: 10 ␮l.

The quenching of QD photoluminescence by quinones is attributed to excited-state electron transfer from the former to the latter [46]. As an anthraquinone derivative that is a known electron acceptor, MTX can be adsorbed onto the QD surface, resulting in the quenching of QD photoluminescence emission via electron transfer. The fluorescence spectra of CdTe–CDs and UV–vis absorption spectra of MTX were measured, MTX showed clear absorption at around 610 and 670 nm, which overlapped with the red-emitting CdTe in the CdTe–CDs spectrum (Fig. S5). Thus, fluorescence quenching was attributed to the inner-filter effect of fluorescence. 3.5. Ratiometric fluorescence detection of dsDNA The fluorescence intensity of CdTe–CDs at 599 nm was restored by adding increasing amounts of dsDNA to the system (0–175 nM) (Fig. 4A). This was due to the strong and specific binding of dsDNA to MTX, which displaced the latter from the CdTe QDs surface. Moreover, since both CdTe QDs and dsDNA are negatively charged, electrical repulsion caused the intercalated DNA to move further away from the CdTe QDs. The fluorescence intensity ratio of CdTe and CDs showed a linear response to dsDNA concentration in the range of 0–50 nM; the calibration curve was expressed as I599 /I435 = 0.03443 + 0.5716 [dsDNA] (Fig. 4B). Ratiometric fluorescence detection by CdTe−CDs yielded a

Fig. 5. The fluorescence responses of the CdTe–CDs–MTX system to different interferences. (nucleoside monophosphate 10 ␮M, RNA 50 nM, BSA 10 ␮M and amino acid 10 ␮M).

linear fit with a correlation coefficient of 0.9980. The detection limit was estimated as 1.0 nM according to the definition of three times the deviation of the blank signal. Compared to single-fluorescence recovery of CdTe QDs and the corresponding linear curve (LOD: 3.038 nM, R2 = 0.9389) (Fig. S6), it was evident that the ratiometric fluorescence method (Fig. 4) was more sensitive and reliable for dsDNA detection. The performance characteristics of the biosensor as compared to other methods of dsDNA detection are shown in Table 1. 3.6. Effect of interfering substances The high selectivity for an analyte of interest in a complex background of competing species is a challenge in biosensor development. To investigate the selectivity of biosensor, a variety of biologically relevant chemical substances such as nucleoside monophosphates, amino acids, BSA, RNA, and ssDNA were introduced to the system to explore the interference effect. It can be seen from Fig. 5, the signal of fluorescence intensity ratio at I599 /I435 for addition of large molecule BSA and smaller ones such as nucleoside monophosphates and amino acids was obviously lower than that of dsDNA, and almost same as the blank. RNA, which lacks the double helix structure, could not specifically combine with MTX and had a little effect. Likewise, ssDNA had a little effect on the emission spectra of the sensor (Fig. S7), and did not interfere with the detec-

Fig. 4. (A) The fluorescence spectra of CdTe–CDs–MTX system in the presence of different concentrations of dsDNA (0, 5, 12.5, 25, 37.5, 50, 75, 100, 125, 150, 175 nM). (B) The linear relationship between the fluorescence intensity ratio of I599 /I435 and dsDNA concentration in the range of (0–50) nM.

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Table 1 Comparisons of the performance characteristics of developed biosensor with other methods for the determination of dsDNA. Methods

Materials

Detection limit

Refs.

Ratiometric fluorescence Fluorescence Fluorescence Light scattering Light scattering Fluorescence Ratiometric fluorescence

Gly-CDs and EB TGA-CdTe/CdS QDs GSH-CdTe Acridine red CdTe/CdS QDs GSH-CdTe QDs CdTe and CDs

0.47 ␮M 0.41 (␮g/ml) 0.0262 (␮g/ml) 0.100 (␮g/ml) 0.146 (␮g/ml) 0.0108 (␮g/ml) 1.0 nM/0.016 (␮g/ml)

[47] [48] [49] [50] [51] [52] This work

Table 2 Determination results of dsDNA in synthetic samples. Sample

Compositiona

Added (nM)

Found (nM)

RSD (n = 5, %)

Recovery (n = 5, %)

1 2 3

AMP, Lysine, Proline, Alanine, RNA CMP, Histidine, Phenylalanine, Tyrosine TMP, Arginine, Glutamine, BSA

12.5 25 30

12.63 24.59 29.94

1.2 1.7 3.9

101.0 98.37 99.80

a The concentration: AMP, CMP, TMP and BSA 500 nM, Lysine, Proline, Alanine, Histidine, Phenylalanine, Tyrosine, Arginine, and Glutamine 500 nM, and RNA 25 nM, respectively.

tion of dsDNA. These data demonstrate that developed biosensor have high selectivity toward to dsDNA. 3.7. Analysis of HIV dsDNA samples The applicability of the biosensor for HIV dsDNA detection was evaluated using three synthetic samples containing nucleoside monophosphate, amino acids, RNA, and BSA (Table 2). The biosensor showed good reproducibility with the relative standard deviations between 1.2% and 3.9%, and desirable recoveries ranged from 98.37% to 101.0%. In order to further evaluate the feasibility in real samples, the analysis of HIV dsDNA in fresh serum samples was carried out using the standard addition method. The human serum samples were collected from healthy volunteers, which were centrifuged at 13000 rpm for 3 min and were diluted 100-fold with PBS before detection. As could be seen from Table 3, the relative standard deviations were less than 5.0%, and the recoveries were satisfactory. These results revealed that the biosensor developed in this study can be successfully applied to real samples. 4. Conclusion A water-soluble CdTe–CDs biosensor was developed for the detection of HIV dsDNA in biological samples. In this system, MTX acted as a fluorescence “off/on” switch for red-emitting CdTe QDs through electron transfer. No fluorescence resonance energy transfer occurred between CDs and CdTe QDs. CdTe fluorescence increased as a result of intercalation of MTX and dsDNA, while blue-emitting CDs were insensitive to MTX, allowing ratiometric dsDNA detection. This biosensor has advantages over other sensing methods because it is simple in design and offers a convenient “mix-and-detect” protocol for rapid dsDNA detection. Moreover, the CdTe and CDs did not require chemical modification after their preparation, making it cost-effective. The CdTe-CDs ratiometric fluorescence sensor eliminated background interference and reduced the fluctuation of detection conditions with built-in calibration of two emission peaks, ensuring more reliable results compared to Table 3 Determination results of dsDNA in human serum samples. Sample

Added (nM)

Found (nM)

RSD (n = 5, %)

Recovery (n = 5, %)

Serum 1 Serum 2 Serum 3

12.5 25 45

13.76 25.97 44.52

3.9 2.9 3.6

110.0 103.9 98.88

single-emission signal sensors. Taken together, these findings indicate that CdTe–CDs can be used for the rapid and specific detection of dsDNA with minimal interference from common biomolecules present in the sample. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21275098) and Natural Science Grand Research Program of Shaanxi Province (No. 2013SZS08-Z01). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.01.032. References [1] S. Sreelatha, P.R. Padma, M. Umadevi, Protective effects of coriandrum sativum extracts on carbon tetrachloride-induced hepatotoxicity in rats, Food Chem. Toxicol. 47 (2009) 702–708. [2] D. Gholamreza, E.N.D. Jafar, J. Abolghasem, A.Z. Karim, M.A. Seyed, K. Soheila, Spectroscopic studies on the interaction of quercetin-terbium (III) complex with calf thymus DNA, DNA Cell Biol. 30 (2011) 195–201. [3] G. Michels, W. Watjen, P. Niering, B. Steffan, Q.H. Tran Thi, Y. Chovolou, A. Kampkotter, A. Bast, P. Proksch, R. Kahl, Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells, Toxicology 206 (2005) 337–348. [4] M.A. Grodick, N.B. Muren, J.K. Barton, DNA charge transport within the cell, Biochemistry 54 (2015) 962–973. [5] N.B. Muren, J.K. Barton, Electrochemical assay for the signal-on detection of human dna methyltransferase activity, J. Am. Chem. Soc. 135 (2013) 16632–16664. [6] S.N. Liu, Y.Q. Tu, W. Li, P. Wu, H. Zhang, C.X. Cai, Assay methods of DNA methylation and their applications in cancer diagnosis and therapy, Chin. J. Anal. Chem. 139 (2011) 1451–1458. [7] P.G. Sammes, G. Yahioglu, 1,10-Phenanthroline: a versatile ligand, Chem. Soc. Rev. 23 (1994) 327–334. [8] C.V. Kumar, E.H. Asunction, DNA binding studies and site selective fluorescence sensitization of an anthryl probe, J. Am. Chem. Soc. 115 (1993) 8547–8553. [9] S.Y. Bi, C.Y. Qiao, D.Q. Song, Y. Tian, D.J. Gao, Y. Sun, H.Q. Zhang, Study of interactions of flavonoids with DNA using acridine orange as a fluorescence probe, Sens. Actuators B 119 (2006) 199–208. [10] J.P. Yuan, W.W. Guo, X.R. Yang, E.K. Wang, Anticancer drug-dna interactions measured using a photoinduced electron-transfer mechanism based on luminescent quantum dots, Anal. Chem. 81 (2009) 362–368. [11] Y.K. Jung, H.G. Park, Colorimetric detection of clinical DNA samples using an intercalator-conjugated polydiacetylene sensor, Biosens. Bioelectron. 72 (2015) 127–132. [12] M.I. Kim, K.S. Parka, H.G. Park, Ultrafast colorimetric detection of nucleic acidsbased on the inhibition of the oxidase activity of cerium oxide nanoparticles, Chem. Commun. 50 (2014) 9577–9580.

590

S.-S. Liang et al. / Sensors and Actuators B 244 (2017) 585–590

[13] E. Sharon, R. Freeman, I. Willner, CdSe/ZnS quantum dots-G-quadruplex/hemin hybrids as optical DNA sensors and aptasensors, Anal. Chem. 82 (2010) 7073–7077. [14] R. Freeman, X.Q. Liu, I. Willner, Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA metal ions, and aptamer substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots, J. Am. Chem. Soc. 133 (2011) 11597–11604. [15] X.Q. Liu, F. Wang, R. Aizen, O. Yehezkeli, I Willner, Graphene oxide/nucleic-acid-stabilized silver nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic DNAs, J. Am. Chem. Soc. 35 (2013) 11832–11839. [16] X.W. He, N. Ma, A general strategy for label-free sensitive DNA detection based on quantum dot doping, Anal. Chem. 86 (2014) 3676–3681. [17] L.L. Wang, S.P. Liu, C.X. Hao, X.L. Zhang, C.Q. Wang, Y.Q. He, A dual-fluorescence biosensor assembled by quantum dots and phenazinium dyes: a comparative study for DNA detection, Sens. Actuators B 229 (2016) 145–154. [18] B. Duan, J.J. Zhou, Z. Fang, C.X. Wang, X.J. Wang, H. Hemond, M. Chan-Park, H.W. Duan, surface enhanced raman scattering by graphenenanosheet-gapped plasmonic nanoparticle arrays for multiplexed DNA detection, Nanoscale 7 (2015) 12606–12613. [19] T.W. Lin, H.Y. Wu, T.T. Tasi, Y.H. Laia, H.H. Shen, Surface-enhanced raman spectroscopy for DNA detection by the self-assembly of Ag nanoparticles onto Ag nanoparticle-graphene oxide nanocomposites, Phys. Chem. Chem. Phys. 17 (2015) 18443–18448. [20] R.Y. Li, L. Liu, H.X. Bei, Z.j. Li, Nitrogen-doped multiple graphene aerogel/gold nanostar as the electrochemical sensing platform for ultrasensitive detection of circulating free DNA in human serum, Biosens. Bioelectron. 79 (2016) 457–466. [21] X.Y. Lin, Y.N. Ni, S. Kokot, An electrochemical DNA-sensor developed with the use of methylene blue as a redox indicator for the detection of DNA damage induced by endocrine-disrupting compounds, Anal. Chim. Acta 867 (2015) 29–37. [22] H.P. Peng, Y. Hua, P. Liu, Y.N. Deng, P. Wang, W. Chen, A.L. Liu, Y.Z. Chen, X.H. Lin, Label-free electrochemical DNA biosensor for rapid detection of mutidrug resistance gene based on Au nanoparticles/toluidine blue-graphene oxide nanocomposites, Sens. Actuators B 207 (2015) 269–276. [23] X.Q. Liu, F.A. Wang, R. Aizen, O. Yehezkeli, I. Willner, Graphene oxide/nucleic-acid-stabilized silver nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic DNAs, J. Am. Chem. Soc. 135 (2013) 11832–11839. [24] W.B. Qiang, X. Wang, W. Li, X. Chen, H. Li, D.K. Xu, A fluorescent biosensing platform based on the polydopamine nanospheres intergrating with exonuclease III-assisted target recycling amplification, Biosens. Bioelectron. 71 (2015) 143–149. [25] J. Chao, Z.H. Li, J. Li, H.Z. Pengb, S. Su, Q. Li, C.F. Zhu, X.L. Zuo, S.P. Song, L.H. Wang, L.H. Wang, Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays, Biosens. Bioelectron. 81 (2016) 92–96. [26] Q.S. Guo, Z.X. Bai, Y.Q. Liu, Q.J. Sun, A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms, Biosens. Bioelectron. 77 (2016) 107–110. [27] I. Ghosh, C.I. Stains, A.T. Ooi, D.J. Segal, Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics, Mol. Biosyst. 2 (2006) 551–560. [28] J.K. Barton, Metals and DNA: molecular left-handed complements, Science 233 (1986) 727–734. [29] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, Binding modes and base specificity of Tris (phenanthroline) ruthenium (II) enantiomers with nucleic acids: tuning the stereoselectivity, J. Am. Chem. Soc. 108 (1986) 2081–2088. [30] D. Zhao, W.H. Chan, Z.K. He, T. Qiu, Quantum dot-ruthenium complex dyads: recognition of double-strand DNA through dual-color fluorescence detection, Anal. Chem. 81 (2009) 3537–3543. [31] Z.Q. Liu, S.P. Liu, X.D. Wang, P.P. Li, Y.Q. He, A novel quantum dots-based off-on fluorescent biosensor for highly selective and sensitive detection of double-strand DNA, Sens. Actuators B 176 (2013) 1147–1153. [32] S.P. Yang, S.R. Chen, S.W. Liu, X.Y. Tang, L. Qin, G.H. Qiu, J.X. Chen, W.H. Chen, Platforms formed from a three-dimensional Cu-based zwitterionic metal-organic framework and probe ss-dna: selective fluorescent biosensors for human immunodeficiency virus 1 ds-DNA and sudan virus RNA sequences, Anal. Chem. 87 (2015) 12206–12214. [33] H.Q. Zhao, G.H. Qiu, Z. Liang, M.M. Li, B. Sun, L. Qin, S.P. Yang, W.H. Chen, J.X. Chen, A zinc(II)-based two-dimensional MOF for sensitive and selective sensing of HIV-1 ds-DNA sequences, Anal. Chim. Acta 922 (2016) 55–63. [34] X. Michalet, F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S.T. Weiss, Quantum dots for live cells in vivo imaging and diagnostics, Science 307 (2005) 538–544. [35] A. Barati, M. Shamsipur, H. Abdollahi, Hemoglobin detection using carbon dots as a fluorescence probe, Biosens. Bioelectron. 71 (2015) 470–475.

[36] Y.Q. Dong, R.X. Wang, G.L. Li, C.Q. Chen, Y.W. Chi, G.N. Chen, Polyamine-functionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions, Anal. Chem. 84 (2012) 6220–6224. [37] B.F. Shi, Y.B. Su, L.L. Zhang, R.j. Liu, M.J. Huang, S.L. Zhao, Nitrogen-rich functional groups carbon nanoparticles based fluorescent pH sensor with broad-range responding for environmental and live cells applications, Biosens. Bioelectron. 82 (2016) 233–239. [38] S.S. Liu, J. Zhao, K. Zhang, L. Yang, M.T. Sun, H. Yu, Y.H. Yan, Y.J. Zhang, L.J. Wu, S.H. Wang, Dual-emissive fluorescence measurements of hydroxyl radicals using a coumarin-activated silica nanohybrid probe, Analyst 141 (2016) 2296–2302. [39] Q. Mu, Y. Li, H. Xu, Y.F. Ma, W.H. Zhu, X.H. Zhong, Quantum dots-based ratiometric fluorescence probe for mercuric ions in biological fluids, Talanta 119 (2014) 564–571. [40] B.S. Parker, S.M. Cutts, C. Cullinane, D.R. Phillips, Formaldehyde activation of mitoxantrone yields CpG and CpA specific DNA adducts, Nucl. Acids Res. 28 (2000) 982–990. [41] B.S. Parker, T. Buley, B.J. Evison, S.M. Cutts, G.M. Neumann, M.N. Iskander, D.R. Phillips, A molecular understanding of mitoxantrone-DNA adduct formation, J. Biol. Chem. 279 (2004) 18814–18823. [42] Z.Y. Gu, L. Zou, Z. Fang, W.H. Zhu, X.H. Zhong, One-pot synthesis of highly luminescent CdTe/CdS core/shell nanocrystals in aqueous phase, Nanotechnology 19 (2008) 135604–135611. [43] Z. Yang, M.H. Xu, Y. Liu, F.J. He, F. Gao, Y.J. Su, H. Wei, Y.F. Zhang, Nitrogen-doped carbon-rich, highly photoluminescent carbon dots from ammonium citrate, Nanoscale 6 (2014) 1890–1895. [44] Y.T. Huang, Y.W. Lan, Q.L. Yi, H.L. Huang, Y.L. Wang, J.P. Lu, Microwave-assisted synthesis of CdTe quantum dots using 3-mercaptopropionic acid as both a reducing agent and a stabilizer, Chem. Res. Chin. Univ. 32 (2016) 16–19. [45] Y.J. Hu, Y. Liu, X.H. Xiao, Investigation of the interaction between berberine and human serum albumin, Biomacromolecules 10 (2009) 517–521. [46] C. Burda, T.C. Green, S. Link, M.A. El-Sayed, Electron shuttling across the interface of cdse nanoparticles monitored by femtosecond laser spectroscopy, J. Phys. Chem. B 103 (1999) 1783–1788. [47] S. Huang, L. Wang, F.W. Zhu, W. Su, J.R. Sheng, C.H. Huang, Q. Xiao, A ratiometric nanosensor based on fluorescent carbon dots for label-free and highly selective recognition of DNA, RSC Adv. 5 (2015) 44587–44597. [48] P.P. Li, S.P. Liu, S.G. Yan, X.Q. Fan, Y.Q. He, A sensitive sensor for anthraquinone anticancer drugs and hsDNA based on CdTe/CdS quantum dots fluorescence reversible control, Colloid Surf. A 392 (2011) 7–15. [49] Z.Q. Liu, S.P. Liu, X.D. Wang, P.P. Li, Y.Q. He, A novel quantum dots-based off/on fluorescent biosensor for highly selective and sensitive detection of double-strand DNA, Sens. Actuators B 176 (2013) 1147–1153. [50] M. Wang, J.H. Yang, X. Wu, F. Huang, Study of the interaction of nucleic acids with acridine red and CTMAB by a resonance light scattering technique and determination of nucleic acids at nanogram levels, Anal. Chim. Acta 422 (2000) 151–158. [51] W.J. Liang, Z.Q. Liu, S.P. Liu, J.D. Yang, Y.Q. He, A novel surface modification strategy of CdTe/CdS QDs and its application for sensitive detection of ct-DNA, Sens. Actuators B 196 (2014) 336–344. [52] L.L. Wang, S.P. Liu, C.X. Hao, X.L. Zhang, C.Q. Wang, Y.Q. He, A dual-fluorescence biosensor assembled by quantum dots and phenazinium dyes: a comparative study for DNA detection, Sens. Actuators B 229 (2016) 145–154.

Biographies Si-Si Liang is a Master candidate in the School of Chemistry and Chemical Engineering, Shaanxi Normal University. Research interests: molecular spectroscopy analysis, synthesis of nanophase materials and the development of biosensors. Liang Qi is a Doctor candidate in the School of Chemistry and Chemical Engineering, Shaanxi Normal University. Research interest: ligand-RNA interactions. Rui-Ling Zhang is a master candidate in the School of chemistry and chemical engineering, Shaanxi Normal University. Research interests: enzyme activity assay based on fluorescent sensors. Meng Jin is a master candidate in the School of chemistry and chemical engineering, Shaanxi Normal University. Research interest: analytical chemistry. Zhi-Qi Zhang is a professor in the School of chemistry and chemical engineering, Shaanxi Normal University. He received his doctor degree (analytical chemistry) from School of chemistry & materials science, Northwest University in 2004. His current research interests include bioanalytical chemistry, pharmaceutical analysis, and new materials for separation and enrichment.