Biosensors and Bioelectronics 56 (2014) 51–57
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Sensitive single-color fluorescence “off–on” switch system for dsDNA detection based on quantum dots-ruthenium assembling dyads Rui Zhang a,1, Dongxu Zhao a,1, Hui-Guo Ding b, Yan-Xiang Huang b, Hai-Zheng Zhong c, Hai-Yan Xie a,n a
School of Life Science, Beijing Institute of Technology, Beijing 100081, China Beijing YouAn Hospital, Capital Medical University, China c School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 1 October 2013 Received in revised form 13 December 2013 Accepted 18 December 2013 Available online 3 January 2014
Due to the high importance of detecting DNA with both fast speed and high sensitivity, we proposed a new dsDNA detection method relying on a novel single-color fluorescence “off–on” switch system. Water-soluble glutathione capped CdTe QDs (emission at 605 nm) was prepared for taking advantage of the readily tunable emission property of QDs. Initially, QDs was completely quenched by the Ru (phen)2(dppz)2 þ , as the spontaneous formation of QDs-Ru assembling dyads. Then, in the case of the addition of dsDNA, the Ru(phen)2(dppz)2 þ was removed away from the CdTe QDs, producing free CdTe QDs and the Ru-dsDNA complex. Both of them could be excited at the same wavelength and emit overlaid fluorescence. This single-color fluorescence “off–on” signal was sensitive to the concentration of dsDNA. Native dsDNA with the concentration of 10 pg/mL could be detected when 0.5 nM CdTe QDs was used, and ssDNA, RNA or BSA had no interference on it. With this system, the dsDNA samples of hepatitis B virus (HBV) patients were tested. The results were in good agreement with those detected by fluorescence quantitative PCR (P 40.05), and for those samples with very low DNA concentrations, this system could provide more accurate results, demonstrating the possible clinical applicability of this “off– on” switch system. For this system, chemical conjugation or labeling of probes is not required, and unmodified native DNA targets could be detected in less than half an hour. Therefore, a simple, fast, sensitive, low cost, highly selective and practically applicable detection system for dsDNA has been described. & 2014 Elsevier B.V. All rights reserved.
Keywords: dsDNA detection Fluorescence Off–on switch Quantum dots Single-color
1. Introduction The unique optical and electronic properties of quantum dots (QDs) have attracted extensive application studies in many fields, especially in biomedicine sensing and biomedicine imaging (Sapsford et al., 2006; Freeman and Willner, 2012). Among which, those based on fluorescence resonance energy transfer (FRET) or the photoinduced electron/charge transfer between the QDsmodified probes and the dye-labeled targets have been intensively investigated (Jiang et al., 2009; Rogach et al., 2009; Peng et al., 2007; Algar and Krull, 2010; Algar et al., 2011; Algar et al., 2012; Biju et al., 2012). For these studies, suitable fluorescence donor and acceptor design is the most important work. A complete “turn-off” switch system is highly preferred. However the detection based on “turn-off” mode is often restricted due to the low signal/background ratio. It also often provides false positive results because of
n
Corresponding author. Tel.: þ 86 10 68915940; fax: þ86 10 68915956. E-mail address: [email protected] (H.-Y. Xie). 1 Contributed equally.
0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.059
the nonspecific adsorption or interference from interferents and other quenchers. Then significant progress is made to respond to this challenge by developing the “off–on” switch systems (Huang et al., 2013; Ghosh et al., 2013; You et al., 2013; Du et al., 2012; Yao et al., 2012; Yan et al., 2012; McMahon and Gunnlaugsson, 2012; Ren and Xu, 2009). In this type of systems, QDs are first allowed to interact with the quencher, which is modified with the ligand for binding with the analyte, so as to turn off the strong fluorescence signal of QDs. Based on the specific interaction between the ligand and the analyte, addition of the analyte can remove the quencher away from QDs, leading to the recovery of QDs fluorescence. These systems are more reliable and preferable, because the turn-on mechanism can greatly reduce false positive results and the strong fluorescence of QDs can provide very high signal/background ratio. So “off–on” switch systems have drawn intensive attention for investigation and been successfully used in different biomedical labeling and detection (Zhang et al., 2013; Zheng et al., 2012; Kieger et al., 2011; Zhang et al., 2009b; Zhao et al., 2009b). It has been found that the charges of the QDs could be efficiently transferred to the Ru(II) complexes when they were combined by electrostatic adsorption, which leads to the quenching of the QDs0
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emission (Sykore et al., 2006; Sandros et al., 2005; Huang et al., 2008). On the other hand, Ru(II) complexes are typical molecular ‘Light Switches’ of nucleic acids. They can bind double-stranded DNA (dsDNA) with strong affinity, producing very stable fluorescence, while free Ru(II) complexes have no fluorescence in aqueous solution (Friedman et al., 1990; Wang et al., 2005; Lim et al., 2009). Based on these properties, a dual-color fluorescence switch system for dsDNA detection was reported (Zhao et al., 2009a), in which the recognition of dsDNA by Ru(bpy)2(dppx)2 þ could be detected via both the restoration of the green fluorescence of QDs and the emergence of red fluorescence emission signal of the Ru (bpy)2(dppx)2 þ intercalated DNA complex (Ru-dsDNA). This method is simple, but the two signals are not sensitive enough to the concentration of dsDNA, so its detection limit is rather high up to 5 ng/mL and the linear range is also narrow over 1.0 10 8– 2.0 10 7 M 1. Surely, it is necessary to develop such a method for biosensor application in practice. In this paper, we reported a novel single-color fluorescence “off–on” switch system for sensitive dsDNA detection. It was composed of glutathione capped CdTe QDs and the [Ru (phen)2(dppz)]2 þ , where phen ¼1,10-phenanthroline and dppz ¼dipyrido[3,2-a:20 , 30 -c] phenazine. The CdTe QDs with emission at 605 nm was prepared at first. Then it was completely quenched by the [Ru(phen)2(dppz)]2 þ by forming QDs-Ru assembling dyads. With the addition of dsDNA, [Ru(phen)2(dppz)]2 þ was removed away from CdTe QDs, producing free CdTe QDs and Ru-dsDNA complex. Both of them could be simultaneously excited and would emit fluorescence with the maximum intensity at about 605 nm (Scheme 1). Such combined and single-color fluorescence could greatly improve the sensitivity of the system, which could specifically and selectively detect native dsDNA at a level of 10 pg/mL when 0.5 nM CdTe QDs were used. It could detect the hepatitis B virus (HBV) DNA with a very low detection limit of only 5 IU/mL, or even 2 IU/mL and excellent linear relationship from 5 IU/mL to 1 103 IU/mL. For 51 HBV positive clinical samples, the detection results using this system were well consistent with those using the commercial kit (for complementary DNA samples, by t test P 40.05). For the samples at very low DNA concentrations more accurate results could be obtained using this detection system. Therefore, we would provide a facile, fast, quantitative, and label-free method for dsDNA detection, which is very likely to be suitable for practical application.
2. Experimental 2.1. Materials and reagents All chemicals used were of at least analytical grade. All solutions were prepared using deionized water as solvent.
Tellurium powder ( 200 mesh, 99.5%), sodium borohydride (NaBH4, 99%) were purchased from Alfa Aesar (Massachusetts, United States). L-glutathione (GSH, 98%) and urea were purchased from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). CdCl2 2.5H2O, H3BO3, Na2HPO4 12H2O, NaH2PO4 2H2O, HCl, NaOH, KCl, NaHCO3, sodium citrate (C6H5Na3O7), NaCl and other chemical reagents were purchased from the Beijing Chemical Reagent Company (Beijing, China). Calfthymus DNA (ctDNA), yeast ribonucleic acid (RNA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. The different bases of ssDNA were purchased from Sangon Biotech Company Limited. [Ru (phen)2(dppz)]2 þ (phen ¼ 1,10-phenanthroline, dppz ¼dipyrido [3,2-a:20 ,30 -c]phenazine) was supplied by Prof. Zhi-Ke He of Wuhan University, China. HBV magnetic glass particles reagent cassette and HBV lysis reagent cassette were purchased from Roche Molecular Systems Inc. 2.2. Synthesis of CdTe QDs The GSH-capped CdTe quantum dots (QDs) was synthesized according to the scheme reported by Zheng et al. (2007a, 2007b) with a little modification. Briefly, 63.8 mg (0.5 mmol) of tellurium powder and 100 mg of NaBH4 were mixed with 5 mL of nitrogensaturated deionized water. The mixture was stirred for 2 h until it became a light-pink color. This solution was herein referred as the Te precursor. In the second step, 0.2312 g (1 mmol) of CdCl2 2.5H2O, 0.3685 g (1.2 mmol) of GSH, and 100 mL of nitrogensaturated deionized water were loaded into a 250 mL threenecked flask under stirring. The pH was adjusted to be 8–9 by dropwise addition of NaOH solution, and subsequently injected with the NaHTe precursor. The reaction mixture was slowly heated to 100 1C under nitrogen atmosphere, and the growth of CdTe QDs was kept at this temperature for 5.5 h. The obtained CdTe QDs was purified by the addition of 2-propanol and centrifugation. Transmission electron microscopy (TEM) (JEOL JEM-2100 microscope, 200 kV) was used to determine the size and shape of the prepared the CdTe QDs. The concentration of the GSH-capped CdTe QDs was calculated according to Peng and his coworkers (Yu et al., 2003). 2.3. Formation of CdTe–Ru assembling dyads 10 μL of [Ru(phen)2(dppz)]2 þ with different concentrations were respectively added into 90 μL of 0.01 M PBS buffer solution containing 1 μL of CdTe QDs (100 nM). The mixture was kept for 10 min at room temperature to quench the fluorescence of CdTe QDs and form CdTe–Ru assembling dyads. UV–vis absorption spectra data were recorded by a U-3900 spectrophotometer (HITACHI). Fluorescence spectra data were collected with a Fluoromax-4 spectrofluorometer (HORIBA JOBIN YVON) and the excitation wavelength (λex) was 390 nm. The ζ potential of CdTe
Scheme 1. Schematic overview of the single-color fluorescence “off–on” switch system. The CdTe QDs emits fluorescence at 605 nm, which can be completely quenched when it is incubated with the [Ru(phen)2(dppz)]2 þ by forming QDs-Ru assembling dyads. With the addition of dsDNA, [Ru(phen)2(dppz)]2 þ is removed away from CdTe QDs by forming Ru-dsDNA complex, meanwhile, producing free CdTe QDs. Both of them can be simultaneously excited and emit fluorescence with the maximum intensity at about 605 nm.
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QDs and CdTe QDs-Ru assembling dyads in 0.01 M PBS solution at pH 7.4 were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern Instruments). Fluorescence intensity decay curves of CdTe QDs and CdTe QDs-Ru assembling dyads in 0.01 M PBS solution at pH 7.4 were measured on a FELIX32 system (Photon Technology International). All measurements were done in triplicate.
2.4. Fluorescence detection in the presence of targets 10 μL of [Ru(phen)2(dppz)]2 þ (1 μM) was added into 89 μL of 0.01 M PBS buffer solution containing 1 μL of CdTe QDs (100 nM), and the mixture was kept for 10 min to form the QDs-Ru assembling dyads. Then 1 μL of RNA, BSA, ssDNA or dsDNA with different concentrations were added into the QDs-Ru assembling dyads solution and incubated for 15 min at room temperature before fluorescence measurement. After which, for full complementary or part complementary DNA sensing, 1 μL of corresponding ssDNA with different concentrations were added into the solution of QDs-Ru assembling dyads incubated with ssDNA, and then incubated for 30 min at room temperature before fluorescence measurement.
2.5. Calibration curve of HBV DNA 10 μL of [Ru(phen)2(dppz)]2 þ (1 μM) was added into 89 μL of pH 7.4 0.01 M PBS buffer containing 1 μL of CdTe QDs (100 nM), the mixture was kept for 10 min. Then 1 μL of HBV Quantitation Standard (QS) DNA with different concentrations were added into the QDs-Ru assembling dyads solution and incubated for 20 min at room temperature. After which, the fluorescence signals were collected. The calibration curve of HBV QS DNA was based on the change in fluorescence intensity versus the concentration of HBV QS DNA.
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2.6. Detection of HBV DNA clinical samples HBV DNA was extracted from a human serum specimen by using a Hepatitis B Viral DNA Quantitative Fluorescence Diagnostic Kit (Roche Molecular Systems Inc.). Then 1 μL of HBV DNA samples were added into the QDs-Ru assembling dyads solution and incubated for 20 min at room temperature before fluorescence detection. All measurements were done in triplicate. The concentrations of HBV DNA were calculated by fitting the fluorescence intensity to the standard curve of HBV DNA. Statistical analyses were performed using the Paired-samples t test in SPSS (predictive analytics software from IBM) and Po0.05 were considered significant.
3. Results and discussion 3.1. Preparation of CdTe QDs and the construction of the single-color fluorescence “off–on” switch system The CdTe QDs were directly synthesized in water. The glutathione acted as ligands and made the QDs hydrophilic. They were of uniform size distribution and good monodispersivity. According to the statistical report of Nano Measurer software (Nano Measurer 1.2), the particle size was about 3.107 1.0 nm (Fig. S1). The fluorescence was strong with mean (SD) quantum yields of 28.7% (3.1%) (Fig. 1(A)) (Qu and Peng, 2002) and steady at pH 7–11, if the pH values were lower than 7 or higher than 11, the fluorescence intensity of CdTe QDs was very low (Fig. 1(B)). This is maybe because glutathione is easy to shed from the surface of quantum dots which usually happens in such conditions. So it cannot effectively passivate the surface defects of quantum dots, which results in aggregation and subsequent fluorescence intensity decrease of quantum dots. For biological application, the experiments were carried out in pH 7.4 PBS buffer in the following except additional noted. During one month storage at ambient temperature, the fluorescence of the CdTe QDs in general, displayed no significant shift in the
Fig. 1. The optical properties of GSH-capped CdTe QDs and Ru-dsDNA complex. (A) Image of GSH-capped CdTe QDs irradiated under UV light. (B) Fluorescence intensity of CdTe QDs in 0.01 M PBS buffer at different pH. (C) Fluorescence intensity of CdTe QDs in 0.01 M pH 7.4 PBS for various periods. (D) Normalized absorption (a) and (c) and fluorescence emission (b) and (d) spectra of CdTe QDs (a) and (b) and Ru-dsDNA complex (c), and (d).
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emission maximum or rapid descent in strength (Fig. 1(C)). These superior properties made them reliable in practical application. The readily tunable optical property of QDs made it feasible to prepare QDs with anticipated emission wavelength. In order to construct the single-color fluorescence “off–on” switch system, the maximum emission wavelength of the CdTe QDs was tuned to be 605 nm, which was almost overlaid with that of the dsDNA bonded [Ru(phen)2(dppz)]2 þ (Ru-dsDNA). It is worth noting that both the CdTe QDs and Ru-dsDNA had significant UV–vis absorption over a wavelength range of 350–500 nm (Fig. 1(D)). Thus, they can be simultaneously excited, emitting the fluorescence of the same wavelength. Ru complex has been proved to be an excellent quencher of QDs fluorescence (Yeh et al., 2010). It was found that when the CdTe QDs were mixed with [Ru(phen)2(dppz)]2 þ , the fluorescence of the QDs gradually decreased with increasing [Ru (phen)2(dppz)]2 þ concentration (Fig. 2(A)). Temporal fluorescence intensity change of the CdTe QDs (1 nM) in the presence of 100 nM [Ru(phen)2(dppz)]2 þ was also monitored for 20 min. Minimum and stable quenched fluorescence intensity was observed after a 10 min interaction (Fig. 2B). When the concentration of the QDs was 1 nM, 100 nM [Ru(phen)2(dppz)]2 þ was needed to almost completely quench the fluorescence (fluorescence quenching efficiency: 96.1%). And when the concentration of the CdTe QDs increased to 10 nM or decreased to 0.5 nM, 1 μM or 50 nM [Ru (phen)2(dppz)]2 þ was needed, respectively (Fig. S2). Therefore, the ratio of [Ru(phen)2(dppz)]2 þ to QDs was kept to be 100:1. In aqueous solution, water-soluble GSH-capped CdTe QDs were negatively charged. As a result, after the addition of [Ru (phen)2(dppz)]2 þ , the positively charged [Ru(phen)2(dppz)]2 þ formed an ionic conjugate (QDs-Ru assembling dyads) with QDs due to electrostatic attraction. The ζ potential of the CdTe QDs gradually increased with increasing [Ru(phen)2(dppz)]2 þ and reached almost to a constant value after the molar ratio of [Ru (phen)2(dppz)]2 þ to QDs attained to 100:1 (Table S1). These results suggested that about 100 molecules of [Ru(phen)2(dppz)]2 þ were
electrostatically adsorbed and assembled around the surface of 1 quantum dot to quench the fluorescence. Since there was no overlap between the fluorescence emission of CdTe QDs and the absorbance of [Ru(phen)2(dppz)]2 þ (Fig. 1(D)), fluorescence resonance energy transfer is excluded. To explore the possible mechanism of the [Ru(phen)2(dppz)]2 þ quenching CdTe QDs fluorescence, their fluorescence intensity decay lifetime was collected. Mean fluorescent decay lifetime of QDs and the QDs-Ru assembling dyads was 25.7 and 25.5 ns (Fig. S3A), respectively. Meanwhile, the UV–vis spectra of the CdTe QDs gradually changed following the addition of the [Ru (phen)2(dppz)]2 þ (Fig. S3B). Both these results indicated that static quenching and ground state complex formation may be the quenching mechanism (Zhang et al., 2009a). Therefore; the quenching behavior was investigated by using the conventional static quenching equation lg ðF 0 –FÞ=F ¼ lg K A þn lg C Q where F0 and F were the fluorescence of QDs before and after the addition of [Ru(phen)2(dppz)]2 þ with different concentrations, respectively; CQ was the concentration of the added [Ru (phen)2(dppz)]2 þ , KA was the ground state complex binding constant, and n is the binding sites. As shown in Fig. 2(C), the linear range was 1–60 nM, KA was 4.3 107 M 1, and n was about 1.1, indicating [Ru(phen)2(dppz)]2 þ was an excellent quencher to GSH-capped CdTe QDs fluorescence. 3.2. Specific interaction between [Ru(phen)2(dppz)]2 þ and dsDNA Besides acting as excellent quencher to QDs, [Ru (phen)2(dppz)]2 þ was also known for its specific and strong interaction with dsDNA by intercalating into the minor grooves and major grooves in double-helixs structure of dsDNA. So, in principle, the addition of dsDNA to the solution of QDs-Ru assembling dyads should remove the [Ru(phen)2(dppz)]2 þ away from QDs. As it could be seen (Fig. 3(A)), for the solution of QDs-Ru
Fig. 2. [Ru(phen)2(dppz)]2 þ complex quenching the fluorescence of CdTe QDs. (A) Fluorescence emission spectra of CdTe QDs (1 nM) in 0.01 M PBS (pH 7.4, λex ¼ 390 nm) mixed with increasing amount of [Ru(phen)2(dppz)]2 þ . From (a) to (m), the concentrations of [Ru(phen)2(dppz)]2 þ were 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM, respectively. (B) Temporal fluorescence intensity change of 1 nM CdTe QDs in the presence of 100 nM [Ru(phen)2(dppz)]2 þ (pH 7.4, λex ¼ 390 nm). (C) Static quenching plot of CdTe QDs quenched by [Ru(phen)2(dppz)]2 þ in 0.01 M PBS (pH 7.4).
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assembling dyads, the single fluorescence emission with maximum peak at 605 nm gradually increased with the increasing amount of added dsDNA. On the contrary, direct interaction between CdTe QDs and dsDNA caused some decrease of the QDs fluorescence (Fig. S4). This may be due to the effect of dsDNA on the surface of CdTe QDs or the glutathione ligands. There was no obvious fluorescence increase when ssDNA, RNA or BSA was added to the solution of QDs-Ru assembling dyads. However, when the ssDNA and its corresponding complementary ssDNA were successively introduced into the solution of QDs-Ru assembling dyads, the fluorescence restoration emerged (Fig. S5). Moreover the signal gradually increased with the increasing in the complementary degree of the two ssDNA (Fig. S6). So it was reasonable to conclude that dsDNA could specifically interact with [Ru (phen)2(dppz)]2 þ , resulting the removal of the [Ru (phen)2(dppz)]2 þ from the surface of the QDs and the formation of the Ru-dsDNA conjugates. As a result, the QDs fluorescence recovered and the signal of Ru-dsDNA appeared concomitantly. It was interesting to note that the response sensitivity and capacity of this system to the dsDNA was dependent only on the total amount of the dsDNA instead of the base composition or chain length of the dsDNA (Table S2 and Fig. S7). This could be explained by the mechanism of [Ru(phen)2(dppz)]2 þ interacting with dsDNA. [Ru(phen)2(dppz)]2 þ intercalates into the minor grooves and major grooves in double-helix structure of dsDNA. This interaction was based on the double helix structure of the dsDNA instead of others, such as the sequence, base composition, etc. Therefore, the intensity of the fluorescence depends on the number of dsDNA
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molecules that can destroy the non-fluorescent QDs-Ru assembling dyads to form the luminescent Ru-dsDNA and then to release free CdTe QDs. Such performance made this “off–on” switch system very competent in the clinical detection application for those diseases causing DNA damage or the change of dsDNA concentration in blood. 3.3. Performance evaluation and optimization of the “off–on” switch system When PBS was used, the emission of the CdTe QDs could be restored 80.4% of its original value if sufficient dsDNA was added (Fig. 4(A)). If borate buffer solution (BBS) was used, the recovery rate increased to 85% (Fig. S8). Similarly, different anions or cations such as Cl , HPO42 , H2PO4 , citric acid, HCO3 , BO33 , OH and urea, or cations, such as Na þ , K þ , Mg2 þ , Ca2 þ , Zn2 þ and Fe3 þ , had no effect on the detection ability though some ions had a little influence on the fluorescence intensity (Fig. S9), while both the detection limit and linear detection range did not vary obviously (results did not show). So PBS was used as solution except additional noted. The recovered luminescence was stable. It decreased only 7% within 6 h (Fig. 4(B)). As shown in Fig. 3(B), according to the relationship between the increased fluorescence intensity and the concentrations of added dsDNA (a signal-tonoise ratio of 3 is considered as the detection limit), when 1 nM CdTe QDs was used, the detection limit for dsDNA was 0.05 ng/mL and the detection range was 0.05 ng/mL to 1 μg/mL. The linear detection range was 0.5–100 ng/mL. The detection limit could be
Fig. 3. Single-color fluorescence “off–on” system for dsDNA detection. (A) Fluorescence emission spectra of QDs-Ru assembling dyads at different concentrations of dsDNA. From (a) to (o), the concentrations of dsDNA were 0, 0.05, 0.5, 5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, 1000 ng/mL, respectively. (B) F–F0 (increased fluorescence intensity) vs the concentrations of dsDNA (inset: linear relationship between the increased fluorescence intensity and the concentrations of dsDNA). The QDs-Ru assembling dyads was formed by incubating 1 nM CdTe QDs with 100 nM [Ru(phen)2(dppz)]2 þ in 0.01 M PBS (pH 7.4, λex ¼ 390 nm).
Fig. 4. The recovery rate of CdTe QDs and stability of recovered luminescence. Fluorescence emission spectra of 1 nM CdTe QDs (a), QDs-Ru assembling dyads formed by incubating 1 nM CdTe QDs with 100 nM [Ru(phen)2(dppz)]2 þ (b) and QDs-Ru assembling dyads incubated with 1 μg/mL dsDNA (c) in 0.01 M pH 7.4 PBS, λex ¼340 nm (there is negligible direct fluorescence emission from Ru-dsDNA upon excitation at 340 nm). (B) Fluorescence intensity of QDs-Ru assembling dyads incubated with 1 μg/mL dsDNA in 0.01 M pH 7.4 PBS for various periods, λex ¼390 nm.
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further improved simply by decreasing the amount of the CdTe QDs used in the QDs-Ru assembling dyads formation. For example, the detection limit decreased to 10 pg/mL when 0.5 nM CdTe QDs was used, this was 1/500 of reported results (Zhao et al., 2009a). The wide response range of dsDNA concentration and the sensitive detection limit were due to the single-color fluorescence characteristic of our “off–on” switch system. First of all, it made the fluorescence signal sensitive to the relative increase of the dsDNA concentration. Second, in this system the excessive quencher had little influence, which usually hampered the response sensitivity of subsequent dsDNA detection in other systems. On the contrary, the quencher, [Ru(phen)2(dppz)]2 þ could also emit fluorescence upon the addition of dsDNA. So a large quantity of quencher could be used so as to completely turn off the fluorescent CdTe QDs. The precision of this dsDNA detection system was presented by the intra-assay variability, which was the variability of the same sample repeatedly assayed several times. Here, dsDNA solutions with different concentrations were respectively detected three times with the same batch of QDs-Ru assembling dyads, and the intra-assay variability (Table S3) was calculated by the mean of the coefficient of variation (CV% ¼SD/mean) of the parallel results. The intra-assay variability of 0.9% proved that the precision and the reproducibility of this detection system were very good. 3.4. Application to detection of clinical samples To demonstrate the possibility and feasibility of the single-color fluorescence “off–on” switch system for clinical application, the DNA of hepatitis B virus (HBV), which is one among the typical dsDNA viruses, was detected. Excitingly, the QDs-Ru assembling dyads formed by using 1 nM CdTe QDs could detect HBV DNA with a concentration lower than 5 IU /mL (Fig. S10A), even as low as 2 IU/mL (IU: International Unit, 1 IU HBV DNA E 5.82 copies HBV DNA), when the concentration of CdTe QDs was adjusted to be 0.5 nM (Fig. S11). But the detection limit of commercial detection kit carried out by real time fluorescence quantitative PCR was 10 IU/mL, and the reliable results were available only if DNA concentrations were higher than 100 IU/mL. Moreover, the fluorescence enhancement of the assembling dyads was found to be well proportional to the concentration of HBV DNA from 5 IU/mL to 1000 IU/mL (Fig. S10B). Combined with dilution, it was very easy to detect HBV DNA in a very wide concentration range. A double-blind trial of 56 samples of HBV patients randomly assigned was performed in our study, among which, 51 clinical samples were detected to be dsDNA positive using both the “offon” switch system and the fluorescence quantitative PCR (Table S4). Statistical analysis was performed using the Paired-samples t test in SPSS (predictive analytics software from IBM). The sig. (2-tailed) ¼0.48 (P¼ 0.48 40.05), indicating the results detected by both methods were in good agreement. Furthermore, for 40 in total 51 DNA positive samples, the differences between two groups of test results were less than 1%. In addition, there were another 5 samples with very low HBV DNA concentrations which were generally determined to be negative by the fluorescence quantitative PCR (o 100 IU/mL), but our method could provide more exact results. 3 of them were proved to be HBV DNA positive and the DNA concentrations were 25.5 IU/mL, 15.1 IU/mL and 7.3 IU/mL (Table 1). All these results demonstrated the possible clinical applicability of this “off–on” switch system. Contrary to the fluorescence quantitative PCR assays which required expensive commercial reagents and sophisticated instruments for routine detection, this method was much faster and simpler. The preparation of the QDs-Ru assembling dyads does not concern any chemical conjugation, and native DNA targets can be directly detected without modification. The time consumption is less than half an hour. The cost is low.
Table 1 The detection results of the two methods for HBV DNA clinical samples with low concentrations. Samples number
Method 1a (IU/mL)
Method 2b (IU/mL)
1 2 3 4 5
o100 o100 o100 o100 o100
24.5 15.1 7.3 o 1.0 o 1.0
a b
Fluorescence quantitative PCR. Single-color fluorescence “off–on” switch system.
4. Conclusions In summary, to our knowledge, for the first time, by constructing the single-color fluorescence “off–on” switch system, a simple, sensitive, quantitative, label-free and fast detection method for dsDNA has been developed. By our method, dsDNA of 0.05 ng/mL to 1 μg/mL can be sensitively detected with a detection limit of 10 pg/mL. The satisfactory detection of HBV DNA clinical samples validated the promising prospects of this method for clinic application. Moreover, the response sensitivity and detection capacity for dsDNA of this system depend on the total amount of the dsDNA instead of its base composition or the chain length. Therefore, it could be popularized to detection of other diseases causing DNA damage.
Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, No. 2011CB933600), the National Natural Science Foundation of China (No. 21372028) and the 863 Program (No. 2013AA032204).
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Rogach, A.L., Klar, T.A., Lupton, J.M., Meijerink, A., Feldmann, J., 2009. J. Mater. Chem. 19, 1208–1221. Sandros, M.G., Gao, D., Benson, D.E., 2005. J. Am. Chem. Soc. 127, 12198–12199. Sapsford, K.E., Pons, T., Medintz, I.L., Mattoussi, H., 2006. Sensors 6, 925–953. Sykore, M., Petruska, M.A., Alstrum-Acevedo, J., Bezel, I., Meyer, T.J., Klimov, V.I., 2006. J. Am. Chem. Soc. 128, 9984–9985. Wang, J., Jiang, Y.X., Zhou, C.S., Fang, X.H., 2005. Anal. Chem. 77, 3542–3546. Yan, F.Y., Cao, D.L., Yang, N., Yu, Q.H., Wang, M., Chen, L., 2012. Sensor. Actuat. B-Chem 162, 313–320. Yao, J.L., Gao, X., Sun, W.L., Fan, X.Z., Shi, S., Yao, T.M., 2012. Inorg. Chem. 51, 12591–12593. Yeh, H.Y., Yates, M.V., Mulchandania, A., Chen, W., 2010. Chem. Commun. 46, 3914–3916. You, J., Hu, H.Z., Zhou, J.P., Zhang, L.N., Zhang, Y.P., Kondo, T., 2013. Langmuir 29, 5085–5092.
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Yu, W.W., Qu, L.H., Guo, W.Z., Peng, X.G., 2003. Chem. Mater. 15, 2854–2860. Zhang, M., Lv, Q.L., Yue, N.N., Wang, H.Y., 2009a. Spectrochim. Acta Part A 72, 572–576. Zhang, Y., Li, Y., Yan, X.P., 2009b. Anal. Chem. 81, 5001–5007. Zhang, Y., Liu, J.M., Yan, X.P., 2013. Anal. Chem. 85, 228–234. Zhao, D., Chan, W.H., He, Z.K., Qiu, T., 2009a. Anal. Chem. 81, 3537–3543. Zhao, Y., Zhang, X.B., Han, Z.X., Qiao, L., Li, C.Y., Jian, L.X., Shen, G.L., Yu, R.Q., 2009b. Anal. Chem. 81, 7022–7030. Zheng, J., Jiao, A.L., Yang, R.H., Li, H.M., Li, J.S., Shi, M.L., Ma, C., Jiang, Y., Deng, L., Tan, W.H., 2012. J. Am. Chem. Soc. 134, 19957–19960. Zheng, Y., Gao, S., Ying, J.Y., 2007a. Adv. Mater. 19, 376–380. Zheng, Y., Yang, Z., Ying, J.Y., 2007b. Adv. Mater. 19, 1475–1479.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Sensitive single-color fluorescence “off–on” switch system for dsDNA detection based on quantum dots-ruthenium assembling dyads Rui Zhang a,1, Dongxu Zhao a,1, Hui-Guo Ding b, Yan-Xiang Huang b, Hai-Zheng Zhong c, Hai-Yan Xie a,n a
School of Life Science, Beijing Institute of Technology, Beijing 100081, China Beijing YouAn Hospital, Capital Medical University, China c School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 1 October 2013 Received in revised form 13 December 2013 Accepted 18 December 2013 Available online 3 January 2014
Due to the high importance of detecting DNA with both fast speed and high sensitivity, we proposed a new dsDNA detection method relying on a novel single-color fluorescence “off–on” switch system. Water-soluble glutathione capped CdTe QDs (emission at 605 nm) was prepared for taking advantage of the readily tunable emission property of QDs. Initially, QDs was completely quenched by the Ru (phen)2(dppz)2 þ , as the spontaneous formation of QDs-Ru assembling dyads. Then, in the case of the addition of dsDNA, the Ru(phen)2(dppz)2 þ was removed away from the CdTe QDs, producing free CdTe QDs and the Ru-dsDNA complex. Both of them could be excited at the same wavelength and emit overlaid fluorescence. This single-color fluorescence “off–on” signal was sensitive to the concentration of dsDNA. Native dsDNA with the concentration of 10 pg/mL could be detected when 0.5 nM CdTe QDs was used, and ssDNA, RNA or BSA had no interference on it. With this system, the dsDNA samples of hepatitis B virus (HBV) patients were tested. The results were in good agreement with those detected by fluorescence quantitative PCR (P 40.05), and for those samples with very low DNA concentrations, this system could provide more accurate results, demonstrating the possible clinical applicability of this “off– on” switch system. For this system, chemical conjugation or labeling of probes is not required, and unmodified native DNA targets could be detected in less than half an hour. Therefore, a simple, fast, sensitive, low cost, highly selective and practically applicable detection system for dsDNA has been described. & 2014 Elsevier B.V. All rights reserved.
Keywords: dsDNA detection Fluorescence Off–on switch Quantum dots Single-color
1. Introduction The unique optical and electronic properties of quantum dots (QDs) have attracted extensive application studies in many fields, especially in biomedicine sensing and biomedicine imaging (Sapsford et al., 2006; Freeman and Willner, 2012). Among which, those based on fluorescence resonance energy transfer (FRET) or the photoinduced electron/charge transfer between the QDsmodified probes and the dye-labeled targets have been intensively investigated (Jiang et al., 2009; Rogach et al., 2009; Peng et al., 2007; Algar and Krull, 2010; Algar et al., 2011; Algar et al., 2012; Biju et al., 2012). For these studies, suitable fluorescence donor and acceptor design is the most important work. A complete “turn-off” switch system is highly preferred. However the detection based on “turn-off” mode is often restricted due to the low signal/background ratio. It also often provides false positive results because of
n
Corresponding author. Tel.: þ 86 10 68915940; fax: þ86 10 68915956. E-mail address: [email protected] (H.-Y. Xie). 1 Contributed equally.
0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.059
the nonspecific adsorption or interference from interferents and other quenchers. Then significant progress is made to respond to this challenge by developing the “off–on” switch systems (Huang et al., 2013; Ghosh et al., 2013; You et al., 2013; Du et al., 2012; Yao et al., 2012; Yan et al., 2012; McMahon and Gunnlaugsson, 2012; Ren and Xu, 2009). In this type of systems, QDs are first allowed to interact with the quencher, which is modified with the ligand for binding with the analyte, so as to turn off the strong fluorescence signal of QDs. Based on the specific interaction between the ligand and the analyte, addition of the analyte can remove the quencher away from QDs, leading to the recovery of QDs fluorescence. These systems are more reliable and preferable, because the turn-on mechanism can greatly reduce false positive results and the strong fluorescence of QDs can provide very high signal/background ratio. So “off–on” switch systems have drawn intensive attention for investigation and been successfully used in different biomedical labeling and detection (Zhang et al., 2013; Zheng et al., 2012; Kieger et al., 2011; Zhang et al., 2009b; Zhao et al., 2009b). It has been found that the charges of the QDs could be efficiently transferred to the Ru(II) complexes when they were combined by electrostatic adsorption, which leads to the quenching of the QDs0
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emission (Sykore et al., 2006; Sandros et al., 2005; Huang et al., 2008). On the other hand, Ru(II) complexes are typical molecular ‘Light Switches’ of nucleic acids. They can bind double-stranded DNA (dsDNA) with strong affinity, producing very stable fluorescence, while free Ru(II) complexes have no fluorescence in aqueous solution (Friedman et al., 1990; Wang et al., 2005; Lim et al., 2009). Based on these properties, a dual-color fluorescence switch system for dsDNA detection was reported (Zhao et al., 2009a), in which the recognition of dsDNA by Ru(bpy)2(dppx)2 þ could be detected via both the restoration of the green fluorescence of QDs and the emergence of red fluorescence emission signal of the Ru (bpy)2(dppx)2 þ intercalated DNA complex (Ru-dsDNA). This method is simple, but the two signals are not sensitive enough to the concentration of dsDNA, so its detection limit is rather high up to 5 ng/mL and the linear range is also narrow over 1.0 10 8– 2.0 10 7 M 1. Surely, it is necessary to develop such a method for biosensor application in practice. In this paper, we reported a novel single-color fluorescence “off–on” switch system for sensitive dsDNA detection. It was composed of glutathione capped CdTe QDs and the [Ru (phen)2(dppz)]2 þ , where phen ¼1,10-phenanthroline and dppz ¼dipyrido[3,2-a:20 , 30 -c] phenazine. The CdTe QDs with emission at 605 nm was prepared at first. Then it was completely quenched by the [Ru(phen)2(dppz)]2 þ by forming QDs-Ru assembling dyads. With the addition of dsDNA, [Ru(phen)2(dppz)]2 þ was removed away from CdTe QDs, producing free CdTe QDs and Ru-dsDNA complex. Both of them could be simultaneously excited and would emit fluorescence with the maximum intensity at about 605 nm (Scheme 1). Such combined and single-color fluorescence could greatly improve the sensitivity of the system, which could specifically and selectively detect native dsDNA at a level of 10 pg/mL when 0.5 nM CdTe QDs were used. It could detect the hepatitis B virus (HBV) DNA with a very low detection limit of only 5 IU/mL, or even 2 IU/mL and excellent linear relationship from 5 IU/mL to 1 103 IU/mL. For 51 HBV positive clinical samples, the detection results using this system were well consistent with those using the commercial kit (for complementary DNA samples, by t test P 40.05). For the samples at very low DNA concentrations more accurate results could be obtained using this detection system. Therefore, we would provide a facile, fast, quantitative, and label-free method for dsDNA detection, which is very likely to be suitable for practical application.
2. Experimental 2.1. Materials and reagents All chemicals used were of at least analytical grade. All solutions were prepared using deionized water as solvent.
Tellurium powder ( 200 mesh, 99.5%), sodium borohydride (NaBH4, 99%) were purchased from Alfa Aesar (Massachusetts, United States). L-glutathione (GSH, 98%) and urea were purchased from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). CdCl2 2.5H2O, H3BO3, Na2HPO4 12H2O, NaH2PO4 2H2O, HCl, NaOH, KCl, NaHCO3, sodium citrate (C6H5Na3O7), NaCl and other chemical reagents were purchased from the Beijing Chemical Reagent Company (Beijing, China). Calfthymus DNA (ctDNA), yeast ribonucleic acid (RNA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. The different bases of ssDNA were purchased from Sangon Biotech Company Limited. [Ru (phen)2(dppz)]2 þ (phen ¼ 1,10-phenanthroline, dppz ¼dipyrido [3,2-a:20 ,30 -c]phenazine) was supplied by Prof. Zhi-Ke He of Wuhan University, China. HBV magnetic glass particles reagent cassette and HBV lysis reagent cassette were purchased from Roche Molecular Systems Inc. 2.2. Synthesis of CdTe QDs The GSH-capped CdTe quantum dots (QDs) was synthesized according to the scheme reported by Zheng et al. (2007a, 2007b) with a little modification. Briefly, 63.8 mg (0.5 mmol) of tellurium powder and 100 mg of NaBH4 were mixed with 5 mL of nitrogensaturated deionized water. The mixture was stirred for 2 h until it became a light-pink color. This solution was herein referred as the Te precursor. In the second step, 0.2312 g (1 mmol) of CdCl2 2.5H2O, 0.3685 g (1.2 mmol) of GSH, and 100 mL of nitrogensaturated deionized water were loaded into a 250 mL threenecked flask under stirring. The pH was adjusted to be 8–9 by dropwise addition of NaOH solution, and subsequently injected with the NaHTe precursor. The reaction mixture was slowly heated to 100 1C under nitrogen atmosphere, and the growth of CdTe QDs was kept at this temperature for 5.5 h. The obtained CdTe QDs was purified by the addition of 2-propanol and centrifugation. Transmission electron microscopy (TEM) (JEOL JEM-2100 microscope, 200 kV) was used to determine the size and shape of the prepared the CdTe QDs. The concentration of the GSH-capped CdTe QDs was calculated according to Peng and his coworkers (Yu et al., 2003). 2.3. Formation of CdTe–Ru assembling dyads 10 μL of [Ru(phen)2(dppz)]2 þ with different concentrations were respectively added into 90 μL of 0.01 M PBS buffer solution containing 1 μL of CdTe QDs (100 nM). The mixture was kept for 10 min at room temperature to quench the fluorescence of CdTe QDs and form CdTe–Ru assembling dyads. UV–vis absorption spectra data were recorded by a U-3900 spectrophotometer (HITACHI). Fluorescence spectra data were collected with a Fluoromax-4 spectrofluorometer (HORIBA JOBIN YVON) and the excitation wavelength (λex) was 390 nm. The ζ potential of CdTe
Scheme 1. Schematic overview of the single-color fluorescence “off–on” switch system. The CdTe QDs emits fluorescence at 605 nm, which can be completely quenched when it is incubated with the [Ru(phen)2(dppz)]2 þ by forming QDs-Ru assembling dyads. With the addition of dsDNA, [Ru(phen)2(dppz)]2 þ is removed away from CdTe QDs by forming Ru-dsDNA complex, meanwhile, producing free CdTe QDs. Both of them can be simultaneously excited and emit fluorescence with the maximum intensity at about 605 nm.
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QDs and CdTe QDs-Ru assembling dyads in 0.01 M PBS solution at pH 7.4 were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern Instruments). Fluorescence intensity decay curves of CdTe QDs and CdTe QDs-Ru assembling dyads in 0.01 M PBS solution at pH 7.4 were measured on a FELIX32 system (Photon Technology International). All measurements were done in triplicate.
2.4. Fluorescence detection in the presence of targets 10 μL of [Ru(phen)2(dppz)]2 þ (1 μM) was added into 89 μL of 0.01 M PBS buffer solution containing 1 μL of CdTe QDs (100 nM), and the mixture was kept for 10 min to form the QDs-Ru assembling dyads. Then 1 μL of RNA, BSA, ssDNA or dsDNA with different concentrations were added into the QDs-Ru assembling dyads solution and incubated for 15 min at room temperature before fluorescence measurement. After which, for full complementary or part complementary DNA sensing, 1 μL of corresponding ssDNA with different concentrations were added into the solution of QDs-Ru assembling dyads incubated with ssDNA, and then incubated for 30 min at room temperature before fluorescence measurement.
2.5. Calibration curve of HBV DNA 10 μL of [Ru(phen)2(dppz)]2 þ (1 μM) was added into 89 μL of pH 7.4 0.01 M PBS buffer containing 1 μL of CdTe QDs (100 nM), the mixture was kept for 10 min. Then 1 μL of HBV Quantitation Standard (QS) DNA with different concentrations were added into the QDs-Ru assembling dyads solution and incubated for 20 min at room temperature. After which, the fluorescence signals were collected. The calibration curve of HBV QS DNA was based on the change in fluorescence intensity versus the concentration of HBV QS DNA.
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2.6. Detection of HBV DNA clinical samples HBV DNA was extracted from a human serum specimen by using a Hepatitis B Viral DNA Quantitative Fluorescence Diagnostic Kit (Roche Molecular Systems Inc.). Then 1 μL of HBV DNA samples were added into the QDs-Ru assembling dyads solution and incubated for 20 min at room temperature before fluorescence detection. All measurements were done in triplicate. The concentrations of HBV DNA were calculated by fitting the fluorescence intensity to the standard curve of HBV DNA. Statistical analyses were performed using the Paired-samples t test in SPSS (predictive analytics software from IBM) and Po0.05 were considered significant.
3. Results and discussion 3.1. Preparation of CdTe QDs and the construction of the single-color fluorescence “off–on” switch system The CdTe QDs were directly synthesized in water. The glutathione acted as ligands and made the QDs hydrophilic. They were of uniform size distribution and good monodispersivity. According to the statistical report of Nano Measurer software (Nano Measurer 1.2), the particle size was about 3.107 1.0 nm (Fig. S1). The fluorescence was strong with mean (SD) quantum yields of 28.7% (3.1%) (Fig. 1(A)) (Qu and Peng, 2002) and steady at pH 7–11, if the pH values were lower than 7 or higher than 11, the fluorescence intensity of CdTe QDs was very low (Fig. 1(B)). This is maybe because glutathione is easy to shed from the surface of quantum dots which usually happens in such conditions. So it cannot effectively passivate the surface defects of quantum dots, which results in aggregation and subsequent fluorescence intensity decrease of quantum dots. For biological application, the experiments were carried out in pH 7.4 PBS buffer in the following except additional noted. During one month storage at ambient temperature, the fluorescence of the CdTe QDs in general, displayed no significant shift in the
Fig. 1. The optical properties of GSH-capped CdTe QDs and Ru-dsDNA complex. (A) Image of GSH-capped CdTe QDs irradiated under UV light. (B) Fluorescence intensity of CdTe QDs in 0.01 M PBS buffer at different pH. (C) Fluorescence intensity of CdTe QDs in 0.01 M pH 7.4 PBS for various periods. (D) Normalized absorption (a) and (c) and fluorescence emission (b) and (d) spectra of CdTe QDs (a) and (b) and Ru-dsDNA complex (c), and (d).
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emission maximum or rapid descent in strength (Fig. 1(C)). These superior properties made them reliable in practical application. The readily tunable optical property of QDs made it feasible to prepare QDs with anticipated emission wavelength. In order to construct the single-color fluorescence “off–on” switch system, the maximum emission wavelength of the CdTe QDs was tuned to be 605 nm, which was almost overlaid with that of the dsDNA bonded [Ru(phen)2(dppz)]2 þ (Ru-dsDNA). It is worth noting that both the CdTe QDs and Ru-dsDNA had significant UV–vis absorption over a wavelength range of 350–500 nm (Fig. 1(D)). Thus, they can be simultaneously excited, emitting the fluorescence of the same wavelength. Ru complex has been proved to be an excellent quencher of QDs fluorescence (Yeh et al., 2010). It was found that when the CdTe QDs were mixed with [Ru(phen)2(dppz)]2 þ , the fluorescence of the QDs gradually decreased with increasing [Ru (phen)2(dppz)]2 þ concentration (Fig. 2(A)). Temporal fluorescence intensity change of the CdTe QDs (1 nM) in the presence of 100 nM [Ru(phen)2(dppz)]2 þ was also monitored for 20 min. Minimum and stable quenched fluorescence intensity was observed after a 10 min interaction (Fig. 2B). When the concentration of the QDs was 1 nM, 100 nM [Ru(phen)2(dppz)]2 þ was needed to almost completely quench the fluorescence (fluorescence quenching efficiency: 96.1%). And when the concentration of the CdTe QDs increased to 10 nM or decreased to 0.5 nM, 1 μM or 50 nM [Ru (phen)2(dppz)]2 þ was needed, respectively (Fig. S2). Therefore, the ratio of [Ru(phen)2(dppz)]2 þ to QDs was kept to be 100:1. In aqueous solution, water-soluble GSH-capped CdTe QDs were negatively charged. As a result, after the addition of [Ru (phen)2(dppz)]2 þ , the positively charged [Ru(phen)2(dppz)]2 þ formed an ionic conjugate (QDs-Ru assembling dyads) with QDs due to electrostatic attraction. The ζ potential of the CdTe QDs gradually increased with increasing [Ru(phen)2(dppz)]2 þ and reached almost to a constant value after the molar ratio of [Ru (phen)2(dppz)]2 þ to QDs attained to 100:1 (Table S1). These results suggested that about 100 molecules of [Ru(phen)2(dppz)]2 þ were
electrostatically adsorbed and assembled around the surface of 1 quantum dot to quench the fluorescence. Since there was no overlap between the fluorescence emission of CdTe QDs and the absorbance of [Ru(phen)2(dppz)]2 þ (Fig. 1(D)), fluorescence resonance energy transfer is excluded. To explore the possible mechanism of the [Ru(phen)2(dppz)]2 þ quenching CdTe QDs fluorescence, their fluorescence intensity decay lifetime was collected. Mean fluorescent decay lifetime of QDs and the QDs-Ru assembling dyads was 25.7 and 25.5 ns (Fig. S3A), respectively. Meanwhile, the UV–vis spectra of the CdTe QDs gradually changed following the addition of the [Ru (phen)2(dppz)]2 þ (Fig. S3B). Both these results indicated that static quenching and ground state complex formation may be the quenching mechanism (Zhang et al., 2009a). Therefore; the quenching behavior was investigated by using the conventional static quenching equation lg ðF 0 –FÞ=F ¼ lg K A þn lg C Q where F0 and F were the fluorescence of QDs before and after the addition of [Ru(phen)2(dppz)]2 þ with different concentrations, respectively; CQ was the concentration of the added [Ru (phen)2(dppz)]2 þ , KA was the ground state complex binding constant, and n is the binding sites. As shown in Fig. 2(C), the linear range was 1–60 nM, KA was 4.3 107 M 1, and n was about 1.1, indicating [Ru(phen)2(dppz)]2 þ was an excellent quencher to GSH-capped CdTe QDs fluorescence. 3.2. Specific interaction between [Ru(phen)2(dppz)]2 þ and dsDNA Besides acting as excellent quencher to QDs, [Ru (phen)2(dppz)]2 þ was also known for its specific and strong interaction with dsDNA by intercalating into the minor grooves and major grooves in double-helixs structure of dsDNA. So, in principle, the addition of dsDNA to the solution of QDs-Ru assembling dyads should remove the [Ru(phen)2(dppz)]2 þ away from QDs. As it could be seen (Fig. 3(A)), for the solution of QDs-Ru
Fig. 2. [Ru(phen)2(dppz)]2 þ complex quenching the fluorescence of CdTe QDs. (A) Fluorescence emission spectra of CdTe QDs (1 nM) in 0.01 M PBS (pH 7.4, λex ¼ 390 nm) mixed with increasing amount of [Ru(phen)2(dppz)]2 þ . From (a) to (m), the concentrations of [Ru(phen)2(dppz)]2 þ were 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM, respectively. (B) Temporal fluorescence intensity change of 1 nM CdTe QDs in the presence of 100 nM [Ru(phen)2(dppz)]2 þ (pH 7.4, λex ¼ 390 nm). (C) Static quenching plot of CdTe QDs quenched by [Ru(phen)2(dppz)]2 þ in 0.01 M PBS (pH 7.4).
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assembling dyads, the single fluorescence emission with maximum peak at 605 nm gradually increased with the increasing amount of added dsDNA. On the contrary, direct interaction between CdTe QDs and dsDNA caused some decrease of the QDs fluorescence (Fig. S4). This may be due to the effect of dsDNA on the surface of CdTe QDs or the glutathione ligands. There was no obvious fluorescence increase when ssDNA, RNA or BSA was added to the solution of QDs-Ru assembling dyads. However, when the ssDNA and its corresponding complementary ssDNA were successively introduced into the solution of QDs-Ru assembling dyads, the fluorescence restoration emerged (Fig. S5). Moreover the signal gradually increased with the increasing in the complementary degree of the two ssDNA (Fig. S6). So it was reasonable to conclude that dsDNA could specifically interact with [Ru (phen)2(dppz)]2 þ , resulting the removal of the [Ru (phen)2(dppz)]2 þ from the surface of the QDs and the formation of the Ru-dsDNA conjugates. As a result, the QDs fluorescence recovered and the signal of Ru-dsDNA appeared concomitantly. It was interesting to note that the response sensitivity and capacity of this system to the dsDNA was dependent only on the total amount of the dsDNA instead of the base composition or chain length of the dsDNA (Table S2 and Fig. S7). This could be explained by the mechanism of [Ru(phen)2(dppz)]2 þ interacting with dsDNA. [Ru(phen)2(dppz)]2 þ intercalates into the minor grooves and major grooves in double-helix structure of dsDNA. This interaction was based on the double helix structure of the dsDNA instead of others, such as the sequence, base composition, etc. Therefore, the intensity of the fluorescence depends on the number of dsDNA
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molecules that can destroy the non-fluorescent QDs-Ru assembling dyads to form the luminescent Ru-dsDNA and then to release free CdTe QDs. Such performance made this “off–on” switch system very competent in the clinical detection application for those diseases causing DNA damage or the change of dsDNA concentration in blood. 3.3. Performance evaluation and optimization of the “off–on” switch system When PBS was used, the emission of the CdTe QDs could be restored 80.4% of its original value if sufficient dsDNA was added (Fig. 4(A)). If borate buffer solution (BBS) was used, the recovery rate increased to 85% (Fig. S8). Similarly, different anions or cations such as Cl , HPO42 , H2PO4 , citric acid, HCO3 , BO33 , OH and urea, or cations, such as Na þ , K þ , Mg2 þ , Ca2 þ , Zn2 þ and Fe3 þ , had no effect on the detection ability though some ions had a little influence on the fluorescence intensity (Fig. S9), while both the detection limit and linear detection range did not vary obviously (results did not show). So PBS was used as solution except additional noted. The recovered luminescence was stable. It decreased only 7% within 6 h (Fig. 4(B)). As shown in Fig. 3(B), according to the relationship between the increased fluorescence intensity and the concentrations of added dsDNA (a signal-tonoise ratio of 3 is considered as the detection limit), when 1 nM CdTe QDs was used, the detection limit for dsDNA was 0.05 ng/mL and the detection range was 0.05 ng/mL to 1 μg/mL. The linear detection range was 0.5–100 ng/mL. The detection limit could be
Fig. 3. Single-color fluorescence “off–on” system for dsDNA detection. (A) Fluorescence emission spectra of QDs-Ru assembling dyads at different concentrations of dsDNA. From (a) to (o), the concentrations of dsDNA were 0, 0.05, 0.5, 5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, 1000 ng/mL, respectively. (B) F–F0 (increased fluorescence intensity) vs the concentrations of dsDNA (inset: linear relationship between the increased fluorescence intensity and the concentrations of dsDNA). The QDs-Ru assembling dyads was formed by incubating 1 nM CdTe QDs with 100 nM [Ru(phen)2(dppz)]2 þ in 0.01 M PBS (pH 7.4, λex ¼ 390 nm).
Fig. 4. The recovery rate of CdTe QDs and stability of recovered luminescence. Fluorescence emission spectra of 1 nM CdTe QDs (a), QDs-Ru assembling dyads formed by incubating 1 nM CdTe QDs with 100 nM [Ru(phen)2(dppz)]2 þ (b) and QDs-Ru assembling dyads incubated with 1 μg/mL dsDNA (c) in 0.01 M pH 7.4 PBS, λex ¼340 nm (there is negligible direct fluorescence emission from Ru-dsDNA upon excitation at 340 nm). (B) Fluorescence intensity of QDs-Ru assembling dyads incubated with 1 μg/mL dsDNA in 0.01 M pH 7.4 PBS for various periods, λex ¼390 nm.
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further improved simply by decreasing the amount of the CdTe QDs used in the QDs-Ru assembling dyads formation. For example, the detection limit decreased to 10 pg/mL when 0.5 nM CdTe QDs was used, this was 1/500 of reported results (Zhao et al., 2009a). The wide response range of dsDNA concentration and the sensitive detection limit were due to the single-color fluorescence characteristic of our “off–on” switch system. First of all, it made the fluorescence signal sensitive to the relative increase of the dsDNA concentration. Second, in this system the excessive quencher had little influence, which usually hampered the response sensitivity of subsequent dsDNA detection in other systems. On the contrary, the quencher, [Ru(phen)2(dppz)]2 þ could also emit fluorescence upon the addition of dsDNA. So a large quantity of quencher could be used so as to completely turn off the fluorescent CdTe QDs. The precision of this dsDNA detection system was presented by the intra-assay variability, which was the variability of the same sample repeatedly assayed several times. Here, dsDNA solutions with different concentrations were respectively detected three times with the same batch of QDs-Ru assembling dyads, and the intra-assay variability (Table S3) was calculated by the mean of the coefficient of variation (CV% ¼SD/mean) of the parallel results. The intra-assay variability of 0.9% proved that the precision and the reproducibility of this detection system were very good. 3.4. Application to detection of clinical samples To demonstrate the possibility and feasibility of the single-color fluorescence “off–on” switch system for clinical application, the DNA of hepatitis B virus (HBV), which is one among the typical dsDNA viruses, was detected. Excitingly, the QDs-Ru assembling dyads formed by using 1 nM CdTe QDs could detect HBV DNA with a concentration lower than 5 IU /mL (Fig. S10A), even as low as 2 IU/mL (IU: International Unit, 1 IU HBV DNA E 5.82 copies HBV DNA), when the concentration of CdTe QDs was adjusted to be 0.5 nM (Fig. S11). But the detection limit of commercial detection kit carried out by real time fluorescence quantitative PCR was 10 IU/mL, and the reliable results were available only if DNA concentrations were higher than 100 IU/mL. Moreover, the fluorescence enhancement of the assembling dyads was found to be well proportional to the concentration of HBV DNA from 5 IU/mL to 1000 IU/mL (Fig. S10B). Combined with dilution, it was very easy to detect HBV DNA in a very wide concentration range. A double-blind trial of 56 samples of HBV patients randomly assigned was performed in our study, among which, 51 clinical samples were detected to be dsDNA positive using both the “offon” switch system and the fluorescence quantitative PCR (Table S4). Statistical analysis was performed using the Paired-samples t test in SPSS (predictive analytics software from IBM). The sig. (2-tailed) ¼0.48 (P¼ 0.48 40.05), indicating the results detected by both methods were in good agreement. Furthermore, for 40 in total 51 DNA positive samples, the differences between two groups of test results were less than 1%. In addition, there were another 5 samples with very low HBV DNA concentrations which were generally determined to be negative by the fluorescence quantitative PCR (o 100 IU/mL), but our method could provide more exact results. 3 of them were proved to be HBV DNA positive and the DNA concentrations were 25.5 IU/mL, 15.1 IU/mL and 7.3 IU/mL (Table 1). All these results demonstrated the possible clinical applicability of this “off–on” switch system. Contrary to the fluorescence quantitative PCR assays which required expensive commercial reagents and sophisticated instruments for routine detection, this method was much faster and simpler. The preparation of the QDs-Ru assembling dyads does not concern any chemical conjugation, and native DNA targets can be directly detected without modification. The time consumption is less than half an hour. The cost is low.
Table 1 The detection results of the two methods for HBV DNA clinical samples with low concentrations. Samples number
Method 1a (IU/mL)
Method 2b (IU/mL)
1 2 3 4 5
o100 o100 o100 o100 o100
24.5 15.1 7.3 o 1.0 o 1.0
a b
Fluorescence quantitative PCR. Single-color fluorescence “off–on” switch system.
4. Conclusions In summary, to our knowledge, for the first time, by constructing the single-color fluorescence “off–on” switch system, a simple, sensitive, quantitative, label-free and fast detection method for dsDNA has been developed. By our method, dsDNA of 0.05 ng/mL to 1 μg/mL can be sensitively detected with a detection limit of 10 pg/mL. The satisfactory detection of HBV DNA clinical samples validated the promising prospects of this method for clinic application. Moreover, the response sensitivity and detection capacity for dsDNA of this system depend on the total amount of the dsDNA instead of its base composition or the chain length. Therefore, it could be popularized to detection of other diseases causing DNA damage.
Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, No. 2011CB933600), the National Natural Science Foundation of China (No. 21372028) and the 863 Program (No. 2013AA032204).
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