Biosensors and Bioelectronics 58 (2014) 17–21
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Fluorescence detection of Pb2 þ based on the DNA sequence functionalized CdS quantum dots Siyu Liu, Weidan Na, Shu Pang, Xingguang Su n Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 December 2013 Received in revised form 5 February 2014 Accepted 7 February 2014 Available online 18 February 2014
In this paper, we have developed a simple and rapid method for the detection of Pb2 þ based on the DNA sequence capped CdS quantum dots (QDs). We utilized the designed guanine (G)-rich DNA sequence (PS2.M) as a coating reagent to synthesize the DNA-capped CdS QDs. The designed G-rich DNA sequence PS2.M can bind with hemin to form G-quadruplex/hemin complex with K þ , accompanied by the fluorescence quenching of CdS QDs via the photoinduced electron transfer. Pb2 þ can induce conformational changes in the G-quadruplex/hemin complex to release the hemin molecules, so the quenched fluorescence of CdS QDs could be recovered. Therefore, the new fluorescent analysis system could be applied for the detection of Pb2 þ based on the label-free DNA sequence capped CdS QDs. & 2014 Elsevier B.V. All rights reserved.
Keywords: CdS quantum dots Fluorescence Quadruplex Hemin Pb2 þ
1. Introduction Pb2 þ , as a toxic metal ion, has severe effects on the human health and the environment. Even very low levels of Pb2 þ can cause neurological, cardiovascular, and develop-mental disorders in human beings (Needleman, 2004; Winder et al., 1982). Therefore, it is very important to develop a sensitive, simple and selective method to detect Pb2 þ in biological and environmental samples. Compared to the traditional methods to detect metal ions, such as chemiluminescence (Coale et al., 1992), atomic absorption (Zamaow et al., 1998), inductively coupled plasma (ICP) atomic emission spectroscopy (Davis et al., 2007), the strategy based on the fluorescence changes respond to metal ions, is being used in research widely, owing to its operational simplicity, high sensitivity and real-time detection. But most of the developed fluorescence detections for Pb2 þ easily suffered the interference from other heavy metal ions. In recent years, with the developments of the investigation on the interactions between Pb2 þ and DNA, several different types of DNA molecules have been designed for the selective determination of Pb2 þ (Elbaz et al., 2008; Shimron et al., 2010). Several methods, such as colorimetric(Liu and Lu, 2003), electrochemical (Shen et al., 2008) and electrochemiluminescence (Zhu et al., 2009) assay, have been successfully applied to detect Pb2 þ ions. However, complicated operation, high cost, expensive and special
n
Corresponding author. Tel.: þ 86 431 85168352. E-mail address: [email protected] (X. Su).
http://dx.doi.org/10.1016/j.bios.2014.02.013 0956-5663 & 2014 Elsevier B.V. All rights reserved.
substance or long operation time existed in these methods. Therefore, to develop a simple, facile and rapid method for the selective detection of Pb2 þ has attracted great concern. DNAzyme can serve as sensors in detection of metal ions through their induced changes of DNA configuration that can effectively affect activity of the DNAzymes (Kim et al., 2007; Wei et al., 2008). Recently, a frequently studied DNAzyme is horseradish peroxidise (HRP)-mimicking DNAzyme, that is usually a complex of guanine-quadruplex (G-quadruplex) and hemin. This G-quadruplex/hemin complex stabilized by cation like K þ can catalyze the oxidation of many organic molecules (e.g., luminol) (Sen and Gilbert, 1988; Li et al., 2009a). Comparing with K þ , Pb2 þ has a higher efficiency to stabilize the G-quadruplex, which would make K þ -stabilized G-quadruplex/hemin complex undergo a conformational change, accompanied by a decrease in the catalytic activity (Li et al., 2009b, 2010; Zhu et al., 2011). This class of DNAzyme has been utilized in amplified biosensing for Pb2 þ (Li et al., 2009b, 2010, 2011; Chen et al.,2013). Li et al. (2011) developed a sensitive and selective amperometric sensing platform for Pb2 þ based on a Pb2 þ -induced G-rich DNA conformational switch from a random-coil to G-quadruplex with crystal violet . Fluorescent detection of Pb2 þ has been demonstrated in a simple and sensitive method based on the Pb2 þ induced conformational change of DNA sequence (Liu et al., 2009; Guo et al., 2012; Li et al., 2013; Zhan et al., 2013). However, the high cost of labeling DNA sequence and some specific fluorescence substrates limits its further application. Recently, a series of research work on one pot synthesized aptamer-functionalized QDs has been reported (Zhang
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et al., 2012, 2013a, 2013b; Choi et al., 2006; Joe et al., 2005; Ma et al., 2010; Bigham and Coffer, 2000; Choi et al., 2006). Zhang et al., offered a strategy to synthesize phosphorothioate modified DNAfunctionalized Zn2 þ doped CdTe QDs through a facile one-pot hydrothermal route and DNA was directly attached to the surface of QDs (Zhang et al., 2013a, 2013b). Zhang et al. (2012) showed the preparation of DNA–CdTe QDs by a one-pot method with glutathione and a specific sequence DNA as a co-ligands to stabilize the QDs. These one-pot synthesized aptamer-functionalized QDs have been developed as a new class of fluorophores that could be applied for the detection of various biologically important analytes based on different signal-transducing mechanisms. In this paper, we utilized a convenient and inexpensive approach to synthesize label-free DNA sequence PS2.M capped CdS QDs (G-CdS QDs) whose fluorescence was effectively quenched by hemin in the presence of K þ . Pb2 þ can induce the K þ stabilized G-quadruplexes/ hemin complex to undergo a conformation transition that would release the hemin cofactor. And in this case, the quenched fluorescence would be recovered again. This change was based on the special interactions between Pb2 þ and DNA sequence PS2.M. Therefore, the analysis system could be utilized to selectively detect Pb2 þ .
2. Experiment 2.1. Apparatus The fluorescence spectra were obtained by using a Shimadzu RF-5301 PC spectrofluorophotometer equipped with a xenon lamp using right-angle geometry. UV–vis absorption spectra were obtained by a Varian GBC Cintra 10e UV–vis spectrometer. In both experiments, a 1 cm path-length quartz cuvette was used. FT-IR spectra were recorded using a Bruker IFS66V FT-IR spectrometer equipped with a DGTS detector (32 scans). Dynamic Light Scattering spectra (DLS) were obtained by Zetasizer Nano S90. Transmission electron microscopy (TEM) experiments were performed on a Philips Tecnai F20 TEM operating at 200 KV acceleration voltage. A JASCO Model J-810 spectropolarimeter was utilized to perform the circular dichroism (CD) measurement. 2.2. Reagents All reagents were of at least analytical grade. The water used in all experiments had a resistivity higher than 18 M Ω cm 1. Cadmium(II) chloride (CdCl2), sodium hydroxide (NaOH), sodium sulfide nonahydrate(Na2S 9H2O), trihydroxymethyl aminomethane (Tis), lead nitrate (Pb(NO3)2) and hydrochloric acid were purchased from Shanghai Qingxi Technology Co., Ltd. The purified G-rich DNA sequence (PS2.M: 5'-GTGGGTAGGGCGGGTTGG-3') and hemin (from bovine) were obtained from Sangon Biotechnology Co., Ltd. The 0.1 mol/L Tris–HCl buffered solution (pH 7.8) was used as the medium for detection process. 2.3. The synthesis of DNA sequences PS2.M capped CdS QDs The preparation of G-CdS QDs by a one-step route could be described in the following experiment (Bigham and Coffer, 2000; Choi et al., 2006; Gao and Ma, 2012). For 10 mL synthesis, 40 nmol DNA sequence PS2.M, 4 mL CdCl2 solution (10 mmol/L) and 0.5 mL Tris–HCl buffer solution (pH 7.8, 0.1 mol/L) were added into 10 mL calibrated test tube, and shaken thoroughly for 15 min. After that, 1.2 mL (10 mmol/L Na2S) solution was added into the test tube and diluted to the mark with deionized water followed by thoroughly shaking and equilibrated for 10 min. The fluorescence spectra were recorded from 405 nm to 655 nm with the excitation wavelength of 340 nm. The slit widths of excitation and emission were both 10 nm.
The fluorescence (FL) intensity of the maximum emission peak was recorded. 2.4. Determination of hemin based on the DNA sequences PS2.M capped CdS QDs 500 μL synthesized G-CdS QDs solution, 20 μL KCl solution (2 mmol/L), 200 μL Tris–HCl buffer solution (pH 7.8, 0.1 mol/L) and varying amounts of hemin were successively added into 2 mL calibrated test tube, and then diluted to the mark with deionized water followed by the thoroughly shaking and equilibrated for 1 h. The fluorescence spectra were recorded from 405 nm to 655 nm with the excitation wavelength of 340 nm. The slit widths of excitation and emission were both 10 nm. The fluorescence intensity of the maximum emission peak was used for the quantitative analysis of hemin. 2.5. Determination of Pb2 þ based on the DNA sequences PS2.M capped CdS QDs 500 μL synthesized G–CdS QDs solution, 20 μL KCl solution (2 mmol/L), 200 μL Tris–HCl buffer solution (pH 7.8, 0.1 mol/L), different concentration of Pb2 þ , and 100 μL hemin (50 μmol/L) were successively added into 2 mL calibrated test tube, and then diluted to the mark with deionized water followed by the thoroughly shaking and equilibrated for 1 h. The fluorescence spectra were recorded from 405 nm to 655 nm with the excitation wavelength of 340 nm. The slit widths of excitation and emission were both 10 nm. The fluorescence intensity of the maximum emission peak was used for the quantitative analysis of Pb2 þ concentration.
3. Results and discussion 3.1. Spectra of the DNA sequences PS2.M capped CdS QDs The synthesized process of the DNA sequences PS2.M capped CdS QDs is shown in Scheme 1. The fluorescent CdS QDs could be generated in the presence of Cd2 þ and S2 with a certain amount of DNA sequences PS2.M as stabilizers. The synthesized process of DNA sequences PS2.M functionalized CdS QDs is rapid, simple, low cost, and efficient. We utilized DNA aptamer as capping ligand in the generation of functional CdS QDs that provided a powerful means of rationally controlling CdS QDs properties. The DNA sequence can passivate the surface of QDs, rendering them water-soluble and stable against aggregation, and retain the nucleotides structure that could selectively bind to its target (Zhang et al., 2012, 2013a, 2013b; Choi et al., 2006; Joe et al., 2005; Ma et al., 2010; Bigham and Coffer, 2000). Fig. 1 shows UV–vis absorption (Dash line) and fluorescence (FL) emission spectra (Solid line) of the DNA sequences PS2.M capped CdS QDs in Tris–HCl buffer solution (pH 7.8). As shown the fluorescence emission spectrum demonstrates a well-shaped peak at 540 nm which arises from excitonic emission of CdS QDs (GaraiIbabe et al., 2013a). There is an increase in absorption below 450 nm that is the result of 1Sh–1Se excitonic transition
Scheme 1. Synthesis of DNA sequences PS2.M capped CdS QDs.
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3.2. The effect of pH value and PS2.M concentration on the fluorescence of CdS QDs
Fig. 1. UV–vis absorption (Dash line) and fluorescence emission spectra of DNA sequences PS2.M capped CdS QDs (Solid line) generated in the presence of CdCl2 (4 mmol/L), PS2.M (4 μmol/L) and Na2S (1.2 mmol/L) in 10 mmol/L Tris–HCl buffer solution (pH 7.8).
As shown in Scheme 1 and Fig. 1, the fluorescent CdS QDs could be generated in the presence of Cd2 þ and S2 with DNA sequences PS2.M as stabilizer. It is generally known that, appropriate pH value and incubation play a key role in the generation process of QDs (Xie et al.,2009; Huang et al., 2007). As shown in Fig. S3A, we systematically investigated the influence of pH value on the generation of the fluorescent PS2.M capped QDs. It can be seen that, the fluorescence emission intensity of PS2.M capped CdS QDs increased with the increase of pH value (from 6.6 to 7.8). When the pH value further increased from 7.8 to 8.6, the fluorescence intensity was almost constant. According to the previous reports on aqueous synthesis of CdS QDs (Garai-Ibabe et al., 2012, 2013a, 2013b), a certain amount of DNA sequences PS2.M was added into distilled water with a fixed precursor ratio of Cd2 þ : S2 of 1:0.3, the optimized DNA sequences PS2.M concentration was investigated in this paper. The fluorescence intensity of the CdS QDs with different DNA sequences PS2.M concentration is shown in Fig. S3B. It can be seen that when the PS2.M concentration reached 0.5 μmol/L, the fluorescence emission peak (around 540 nm) belonging to CdS QDs appeared, and the fluorescence intensity of CdS QDs increased correspondingly with the increasing concentration of PS2.M from 0.5 μmol/L to 4.0 μmol/L. When the PS2. M concentration was higher than 4 mmol/L, the fluorescence intensity of CdS QDs changed slowly. 3.3. The fluorescence quenching of PS2.M capped CdS QDs induced by hemin
Scheme 2. Detection of Pb2 þ based on the DNA sequences PS2.M capped CdS QDs.
characteristic of CdS QDs and a shoulder around 260 nm attributed to DNA sequence PS2.M, indicating the successful binding between DNA and QDs (Matsumoto et al., 1996; Liu et al., 2013). The FT-IR spectra of the CdS crystals without DNA sequences PS2.M as stabilizers and PS2.M-capped CdS QDs were compared to confirm the coordination of the DNA sequences PS2.M on the surface of the CdS QDs. As shown in Fig. S1 curve b, the majority of PS2.M functional groups could be clearly found through the stretching vibrations of CQO (1580 cm 1) and –OH (3410 cm 1), the –NH2 feature (1640 cm 1), the asymmetric stretching vibrations of the PO2 (1280 cm 1), the out of phase symmetrical stretches (1060 cm 1), and the P–O stretches of the main chain (930 cm 1). And these characteristic peaks were not observed in the FT-IR spectra of uncapped CdS (Fig. S1 curve a) that indicated the successful capping of DNA PS2.M on the surface of the CdS QDs. The TEM image and DLS of the DNA PS2.M-capped CdS QDs are shown in Fig. S2A and B.It could be seen that the generated CdS nanoparticles with DNA sequences PS2.M as stabilizers are nearly monodispersed with the diameter from 4 nm to 7 nm. The zeta potential of DNA PS2.M-capped CdS QDs is shown in Fig. S2C. Solution with zeta potential above þ20 mV and below 20 mV is considered stable (Prathna et al., 2011). The zeta potential of PS2. M-capped CdS QDs was 20.88 mV, which indicated that the generated PS2.M-capped CdS QDs could be stable in the solution.
As shown in Scheme 2, in the presence of K þ , the G-rich DNA sequences PS2.M would fold into a G-quadruplex structure by a Hoogsteen hydrogen bond between four G-bases that could effectively bind to hemin molecules to form the G-quadruplex/ hemin complex through external π–π stacking interactions (Sharon et al., 2010; Zhang et al., 2013a, 2013b; Shi et al., 2011). In this study, we investigated the interaction between PS2.M capped CdS QDs and hemin (2.5 μmol/L) by monitoring fluorescence changes. Fig. S4 shows the temporal evolution of fluorescence (FL) intensity of PS2.M capped CdS QDs after the addition of hemin. And it could be seen that the fluorescence intensity gradually decreased and remained nearly constant after 1 h. Such quenching process reached an equilibrium proving the formation of G-quadruplex/hemin complex. The fluorescence quenching was caused by the electron transfer from the PS2.M capped CdS QDs to hemin (Sharon et al., 2010; Zhang et al., 2013a, 2013b). Fig. 2 shows the evolution of fluorescence spectra of G-CdS QDs with increasing hemin concentration. It could be seen that the fluorescence intensity of G-CdS QDs obviously decreased with increasing hemin concentration from 0 to 2.5 μmol/L. Furthermore, Fig. 2 (inset) showed that there was a good linear relationship between the fluorescence quenching ratios F/F0 (F0 and F are the fluoresence intensity of G-CdS QDs without and with hemin ) of G-CdS QDs and the hemin concentration in the range of 0–0.25 μmol/L. The regression equation is F=F 0 ¼ 0:982 0:887½he min ; μmol=L
ð1Þ
The corresponding regression coefficient is 0.990, and the detection limit (S/N ¼3) for hemin was 5 nmol/L. The standard deviation for nine replicate measurements of 0.01 μmol/L hemin was 3.7%. In the concentration range from 0.25 to 2.5 μmol/L, the regression equation could be described as follows: F=F 0 ¼ 0:796 0:122½he min ; μmol=L
ð2Þ
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As shown in Fig. S6A, we investigated the effect of a series of proteins including human serum albumins (HSA), bovine serum albumins (BSA), pepsase (Pep), trypsin (Try), alkaline phosphatese (ALP), urase (Ura) and horseradish peroxidase (HRP) on the fluorescence of PS2.M capped CdS QDs. It could be seen that only hemin can effectively induce the fluorescence quenching of the CdS QDs. The results clearly demonstrate that this DNA sequence functionalized CdS QDs can selectively identify hemin. 3.4. The fluorescence recovery of PS2.M capped CdS QDs-hemin induced by Pb2 þ
Fig. 2. Fluorescence spectra of G-CdS QDs assay system in the presence of 20 μmol/ L K þ and different concentration of hemin (0, 0.01, 0.025, 0.05, 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 μmol/L). Inset: Plot of fluorescence quenching ratios (F/F0) of assay system at fluorescence emission peak versus the concentration of hemin. 10 mmol/ L Tris–HCl buffer solution (pH 7.8) incubation for 60 min. Error bars represent standard deviations from three measurements.
Some research works have proved that Pb2 þ -stabilized Gquadruplexes have shorter M–O and O–O bonds than K þ -stabilized G-quadruplexes (Kotch et al., 2000), which indicates that Pb2 þ has a higher efficiency for stabilizing G-quadruplexes than K þ . Therefore, Pb2 þ could competitively bind to K þ -stabilized G-quadruplexes/hemin complex, resulting in a conformation transition that would release the hemin cofactor (Li et al., 2009; Li et al., 2010; Guo et al., 2012). In this work, we utilized CD measurements to study the conformational change before and after adding Pb2 þ . As shown in Fig. S6B, in the absence of Pb2 þ , the K þ -stabilized PS2.M had a positive band around 295 nm in the CD spectrum. Upon the addition of Pb2 þ , a positive peak appeared at 310 nm, that is the typical characteristic of Pb2 þ -stabilized antiparallel G-quadruplexes (Li et al.,2009; Li et al., 2010). At the same time, we also studied the influence of Pb2 þ concentration on the fluorescence of G-CdS QDs quenched by 2.5 μmol/L hemin. From Fig. 3, it can be seen that the fluorescence of G-CdS QDs quenched by hemin increased with the increase of Pb2 þ concentration from 0 to 2.0 μmol/L. As shown in Fig. 3 (inset), there is a good linear relationship between the fluorescence intensity ratio F/F0 (F is the fluorescence intensity of the G-CdS QDs quenched by hemin in the presence of Pb2 þ and F0 is the fluorescence intensity of the original G-CdS QDs) and the Pb2 þ concentration in the range from 0.02 to 1.0 μmol/L. The regression equation is F=F 0 ¼ 0:502 þ 0:365½Pb
2þ
; μmol=L
ð3Þ
The corresponding regression coefficient is 0.999, and the detection limit for Pb2 þ was 10 nmol/L. The standard deviation for nine replicates measurements of 0.05 μmol/L Pb2 þ is 3.2%. Fig. 3. Fluorescence spectra of G-CdS QDs assay system in the presence of 20 μmol/ L K þ , 2.5 μmol/L hemin and different concentration of Pb2 þ (0, 0.02, 0.05, 0.10, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 μmol/L). Inset: Plot of fluorescence intensity ratio (F/F0) of assay system at fluorescence emission peak versus the concentration of Pb2 þ . 10 mmol/L Tris–HCl buffer solution (pH 7.8) incubation for 60 min. Error bars represent standard deviations from three measurements.
The corresponding regression coefficient is 0.997. It is well known that hemin molecule is negatively charged with an isoelectric point (pI) of 7.0 (Wang et al., 2010) and hence the electrostatic interaction between hemin and the DNA sequence in our assay system (pH 7.8) is very weak. As mentioned in the previous reports (Sharon et al., 2010; Zhang et al., 2012), nonspecific binding of hemin to the nucleic acid-functionalized QDs can only lead to minute quenching of the fluorescence of the QDs. As shown in Fig. S5, in the absence of K þ , the fluoresecence intensity of G-CdS QDs almost remained the same with increasing hemin concentration from 0 to 2.5 μmol/L. The fluorescence quenching of the QDs originated from the specific organization of the G-quadruplex/hemin structure on the QDs, and a Gquadruplex structure with π-stacked G-quartets and an ion channel in its center would favor DNA sequence as the medium for electron transfer to a great extent (Sharon et al., 2010; Zhang et al., 2012). Therefore, G-CdS QDs-K þ assay system could be also utilized to selectively detect hemin.
3.5. Selectivity assay for Pb2 þ based on the PS2.M capped CdS QDs The selectivity of our DNA functionalized CdS QDs for the assay of Pb2 þ was investigated, a number of common metal ions such as Hg2 þ , Ag þ , Cu2 þ , Zn2 þ , Ni2 þ , Mn2 þ , Ca2 þ , Ba2 þ , Mg2 þ , Na þ are adopted in place of Pb2 þ . As shown in Fig. 4A, although the concentration of other metal ion was higher than that of Pb2 þ , only Pb2 þ could cause an obvious fluorescence recovery of the GCdS QDs quenched by hemin. We further tested this assay system with 2 μmol/L Pb2 þ in the coexistence of other metal ion. As shown in Fig. 4B, the response of this assay system to Pb2 þ was almost unaffected by the coexistence of other tested metal ion. The results suggest that our assay is selective for Pb2 þ detection. Compared with the previous reports about fluorescence probe for Pb2 þ (Li et al., 2011, 2013; Chen et al.,2013; Liu et al.,2009; Guo et al.,2012; Zhan et al., 2013), our proposed method for the assay of Pb2 þ is simple, low-cost and high selectivity and has a similar or superior detection limit and dynamic range. 3.6. Analytical applications To test the applicability of the proposed methods, it was applied to determine the Pb2 þ ions in tap water and spring water samples. The water samples were filtered several times through
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Fig. 4. Selectivity study. (A) The fluorescence intensity of the assay system containing DNA sequence PS2.M capped CdS QDs and 2.5 mmol/L hemin in the presence of individual metal ions (2 mmol/L Pb2 þ , 5 mmol/L other metal ions) and (B) The fluorescence intensity of the assay system containing DNA sequence PS2.M capped CdS QDs and 2.5 mmol/L hemin in the presence of 2 mmol/L Pb2 þ ( first bar) and at the coexistence of 2 mmol/L Pb2 þ with 5 mmol/L other metal ions, respectively. Error bars represent standard deviations from three measurements.
qualitative filter paper. The pH was adjusted to 7.0 using NaOH solution before analysis. The results showed that Pb2 þ was not detected in these real samples, so the samples were spiked with standard Pb2 þ solution. The averages of three replicate determination results are shown in Table S1. The accuracy of the proposed method was evaluated by determining the average recovery of Pb2 þ in real samples. From Table S1, it can be seen that the RSD was lower than 4.2%, and the average recoveries of Pb2 þ in the real samples was in the range of 90–108%.
4. Conclusion In summary, we have synthesized the DNA sequence PS2.M functionalized CdS QDs and utilized it as a turn-on fluorescent Pb2 þ probe based on Pb2 þ -induced conformation transition of PS2. M G-quadruplexes/hemin complex. This assay is simple and has low-cost, and the DNA functionalized CdS QDs probe exhibits good selectivity for Pb2 þ over other metal ions.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21075050, 21275063) and the Science and Technology Development project of Jilin province, China (no. 20110334).
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Fluorescence detection of Pb2 þ based on the DNA sequence functionalized CdS quantum dots Siyu Liu, Weidan Na, Shu Pang, Xingguang Su n Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 December 2013 Received in revised form 5 February 2014 Accepted 7 February 2014 Available online 18 February 2014
In this paper, we have developed a simple and rapid method for the detection of Pb2 þ based on the DNA sequence capped CdS quantum dots (QDs). We utilized the designed guanine (G)-rich DNA sequence (PS2.M) as a coating reagent to synthesize the DNA-capped CdS QDs. The designed G-rich DNA sequence PS2.M can bind with hemin to form G-quadruplex/hemin complex with K þ , accompanied by the fluorescence quenching of CdS QDs via the photoinduced electron transfer. Pb2 þ can induce conformational changes in the G-quadruplex/hemin complex to release the hemin molecules, so the quenched fluorescence of CdS QDs could be recovered. Therefore, the new fluorescent analysis system could be applied for the detection of Pb2 þ based on the label-free DNA sequence capped CdS QDs. & 2014 Elsevier B.V. All rights reserved.
Keywords: CdS quantum dots Fluorescence Quadruplex Hemin Pb2 þ
1. Introduction Pb2 þ , as a toxic metal ion, has severe effects on the human health and the environment. Even very low levels of Pb2 þ can cause neurological, cardiovascular, and develop-mental disorders in human beings (Needleman, 2004; Winder et al., 1982). Therefore, it is very important to develop a sensitive, simple and selective method to detect Pb2 þ in biological and environmental samples. Compared to the traditional methods to detect metal ions, such as chemiluminescence (Coale et al., 1992), atomic absorption (Zamaow et al., 1998), inductively coupled plasma (ICP) atomic emission spectroscopy (Davis et al., 2007), the strategy based on the fluorescence changes respond to metal ions, is being used in research widely, owing to its operational simplicity, high sensitivity and real-time detection. But most of the developed fluorescence detections for Pb2 þ easily suffered the interference from other heavy metal ions. In recent years, with the developments of the investigation on the interactions between Pb2 þ and DNA, several different types of DNA molecules have been designed for the selective determination of Pb2 þ (Elbaz et al., 2008; Shimron et al., 2010). Several methods, such as colorimetric(Liu and Lu, 2003), electrochemical (Shen et al., 2008) and electrochemiluminescence (Zhu et al., 2009) assay, have been successfully applied to detect Pb2 þ ions. However, complicated operation, high cost, expensive and special
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Corresponding author. Tel.: þ 86 431 85168352. E-mail address: [email protected] (X. Su).
http://dx.doi.org/10.1016/j.bios.2014.02.013 0956-5663 & 2014 Elsevier B.V. All rights reserved.
substance or long operation time existed in these methods. Therefore, to develop a simple, facile and rapid method for the selective detection of Pb2 þ has attracted great concern. DNAzyme can serve as sensors in detection of metal ions through their induced changes of DNA configuration that can effectively affect activity of the DNAzymes (Kim et al., 2007; Wei et al., 2008). Recently, a frequently studied DNAzyme is horseradish peroxidise (HRP)-mimicking DNAzyme, that is usually a complex of guanine-quadruplex (G-quadruplex) and hemin. This G-quadruplex/hemin complex stabilized by cation like K þ can catalyze the oxidation of many organic molecules (e.g., luminol) (Sen and Gilbert, 1988; Li et al., 2009a). Comparing with K þ , Pb2 þ has a higher efficiency to stabilize the G-quadruplex, which would make K þ -stabilized G-quadruplex/hemin complex undergo a conformational change, accompanied by a decrease in the catalytic activity (Li et al., 2009b, 2010; Zhu et al., 2011). This class of DNAzyme has been utilized in amplified biosensing for Pb2 þ (Li et al., 2009b, 2010, 2011; Chen et al.,2013). Li et al. (2011) developed a sensitive and selective amperometric sensing platform for Pb2 þ based on a Pb2 þ -induced G-rich DNA conformational switch from a random-coil to G-quadruplex with crystal violet . Fluorescent detection of Pb2 þ has been demonstrated in a simple and sensitive method based on the Pb2 þ induced conformational change of DNA sequence (Liu et al., 2009; Guo et al., 2012; Li et al., 2013; Zhan et al., 2013). However, the high cost of labeling DNA sequence and some specific fluorescence substrates limits its further application. Recently, a series of research work on one pot synthesized aptamer-functionalized QDs has been reported (Zhang
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et al., 2012, 2013a, 2013b; Choi et al., 2006; Joe et al., 2005; Ma et al., 2010; Bigham and Coffer, 2000; Choi et al., 2006). Zhang et al., offered a strategy to synthesize phosphorothioate modified DNAfunctionalized Zn2 þ doped CdTe QDs through a facile one-pot hydrothermal route and DNA was directly attached to the surface of QDs (Zhang et al., 2013a, 2013b). Zhang et al. (2012) showed the preparation of DNA–CdTe QDs by a one-pot method with glutathione and a specific sequence DNA as a co-ligands to stabilize the QDs. These one-pot synthesized aptamer-functionalized QDs have been developed as a new class of fluorophores that could be applied for the detection of various biologically important analytes based on different signal-transducing mechanisms. In this paper, we utilized a convenient and inexpensive approach to synthesize label-free DNA sequence PS2.M capped CdS QDs (G-CdS QDs) whose fluorescence was effectively quenched by hemin in the presence of K þ . Pb2 þ can induce the K þ stabilized G-quadruplexes/ hemin complex to undergo a conformation transition that would release the hemin cofactor. And in this case, the quenched fluorescence would be recovered again. This change was based on the special interactions between Pb2 þ and DNA sequence PS2.M. Therefore, the analysis system could be utilized to selectively detect Pb2 þ .
2. Experiment 2.1. Apparatus The fluorescence spectra were obtained by using a Shimadzu RF-5301 PC spectrofluorophotometer equipped with a xenon lamp using right-angle geometry. UV–vis absorption spectra were obtained by a Varian GBC Cintra 10e UV–vis spectrometer. In both experiments, a 1 cm path-length quartz cuvette was used. FT-IR spectra were recorded using a Bruker IFS66V FT-IR spectrometer equipped with a DGTS detector (32 scans). Dynamic Light Scattering spectra (DLS) were obtained by Zetasizer Nano S90. Transmission electron microscopy (TEM) experiments were performed on a Philips Tecnai F20 TEM operating at 200 KV acceleration voltage. A JASCO Model J-810 spectropolarimeter was utilized to perform the circular dichroism (CD) measurement. 2.2. Reagents All reagents were of at least analytical grade. The water used in all experiments had a resistivity higher than 18 M Ω cm 1. Cadmium(II) chloride (CdCl2), sodium hydroxide (NaOH), sodium sulfide nonahydrate(Na2S 9H2O), trihydroxymethyl aminomethane (Tis), lead nitrate (Pb(NO3)2) and hydrochloric acid were purchased from Shanghai Qingxi Technology Co., Ltd. The purified G-rich DNA sequence (PS2.M: 5'-GTGGGTAGGGCGGGTTGG-3') and hemin (from bovine) were obtained from Sangon Biotechnology Co., Ltd. The 0.1 mol/L Tris–HCl buffered solution (pH 7.8) was used as the medium for detection process. 2.3. The synthesis of DNA sequences PS2.M capped CdS QDs The preparation of G-CdS QDs by a one-step route could be described in the following experiment (Bigham and Coffer, 2000; Choi et al., 2006; Gao and Ma, 2012). For 10 mL synthesis, 40 nmol DNA sequence PS2.M, 4 mL CdCl2 solution (10 mmol/L) and 0.5 mL Tris–HCl buffer solution (pH 7.8, 0.1 mol/L) were added into 10 mL calibrated test tube, and shaken thoroughly for 15 min. After that, 1.2 mL (10 mmol/L Na2S) solution was added into the test tube and diluted to the mark with deionized water followed by thoroughly shaking and equilibrated for 10 min. The fluorescence spectra were recorded from 405 nm to 655 nm with the excitation wavelength of 340 nm. The slit widths of excitation and emission were both 10 nm.
The fluorescence (FL) intensity of the maximum emission peak was recorded. 2.4. Determination of hemin based on the DNA sequences PS2.M capped CdS QDs 500 μL synthesized G-CdS QDs solution, 20 μL KCl solution (2 mmol/L), 200 μL Tris–HCl buffer solution (pH 7.8, 0.1 mol/L) and varying amounts of hemin were successively added into 2 mL calibrated test tube, and then diluted to the mark with deionized water followed by the thoroughly shaking and equilibrated for 1 h. The fluorescence spectra were recorded from 405 nm to 655 nm with the excitation wavelength of 340 nm. The slit widths of excitation and emission were both 10 nm. The fluorescence intensity of the maximum emission peak was used for the quantitative analysis of hemin. 2.5. Determination of Pb2 þ based on the DNA sequences PS2.M capped CdS QDs 500 μL synthesized G–CdS QDs solution, 20 μL KCl solution (2 mmol/L), 200 μL Tris–HCl buffer solution (pH 7.8, 0.1 mol/L), different concentration of Pb2 þ , and 100 μL hemin (50 μmol/L) were successively added into 2 mL calibrated test tube, and then diluted to the mark with deionized water followed by the thoroughly shaking and equilibrated for 1 h. The fluorescence spectra were recorded from 405 nm to 655 nm with the excitation wavelength of 340 nm. The slit widths of excitation and emission were both 10 nm. The fluorescence intensity of the maximum emission peak was used for the quantitative analysis of Pb2 þ concentration.
3. Results and discussion 3.1. Spectra of the DNA sequences PS2.M capped CdS QDs The synthesized process of the DNA sequences PS2.M capped CdS QDs is shown in Scheme 1. The fluorescent CdS QDs could be generated in the presence of Cd2 þ and S2 with a certain amount of DNA sequences PS2.M as stabilizers. The synthesized process of DNA sequences PS2.M functionalized CdS QDs is rapid, simple, low cost, and efficient. We utilized DNA aptamer as capping ligand in the generation of functional CdS QDs that provided a powerful means of rationally controlling CdS QDs properties. The DNA sequence can passivate the surface of QDs, rendering them water-soluble and stable against aggregation, and retain the nucleotides structure that could selectively bind to its target (Zhang et al., 2012, 2013a, 2013b; Choi et al., 2006; Joe et al., 2005; Ma et al., 2010; Bigham and Coffer, 2000). Fig. 1 shows UV–vis absorption (Dash line) and fluorescence (FL) emission spectra (Solid line) of the DNA sequences PS2.M capped CdS QDs in Tris–HCl buffer solution (pH 7.8). As shown the fluorescence emission spectrum demonstrates a well-shaped peak at 540 nm which arises from excitonic emission of CdS QDs (GaraiIbabe et al., 2013a). There is an increase in absorption below 450 nm that is the result of 1Sh–1Se excitonic transition
Scheme 1. Synthesis of DNA sequences PS2.M capped CdS QDs.
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3.2. The effect of pH value and PS2.M concentration on the fluorescence of CdS QDs
Fig. 1. UV–vis absorption (Dash line) and fluorescence emission spectra of DNA sequences PS2.M capped CdS QDs (Solid line) generated in the presence of CdCl2 (4 mmol/L), PS2.M (4 μmol/L) and Na2S (1.2 mmol/L) in 10 mmol/L Tris–HCl buffer solution (pH 7.8).
As shown in Scheme 1 and Fig. 1, the fluorescent CdS QDs could be generated in the presence of Cd2 þ and S2 with DNA sequences PS2.M as stabilizer. It is generally known that, appropriate pH value and incubation play a key role in the generation process of QDs (Xie et al.,2009; Huang et al., 2007). As shown in Fig. S3A, we systematically investigated the influence of pH value on the generation of the fluorescent PS2.M capped QDs. It can be seen that, the fluorescence emission intensity of PS2.M capped CdS QDs increased with the increase of pH value (from 6.6 to 7.8). When the pH value further increased from 7.8 to 8.6, the fluorescence intensity was almost constant. According to the previous reports on aqueous synthesis of CdS QDs (Garai-Ibabe et al., 2012, 2013a, 2013b), a certain amount of DNA sequences PS2.M was added into distilled water with a fixed precursor ratio of Cd2 þ : S2 of 1:0.3, the optimized DNA sequences PS2.M concentration was investigated in this paper. The fluorescence intensity of the CdS QDs with different DNA sequences PS2.M concentration is shown in Fig. S3B. It can be seen that when the PS2.M concentration reached 0.5 μmol/L, the fluorescence emission peak (around 540 nm) belonging to CdS QDs appeared, and the fluorescence intensity of CdS QDs increased correspondingly with the increasing concentration of PS2.M from 0.5 μmol/L to 4.0 μmol/L. When the PS2. M concentration was higher than 4 mmol/L, the fluorescence intensity of CdS QDs changed slowly. 3.3. The fluorescence quenching of PS2.M capped CdS QDs induced by hemin
Scheme 2. Detection of Pb2 þ based on the DNA sequences PS2.M capped CdS QDs.
characteristic of CdS QDs and a shoulder around 260 nm attributed to DNA sequence PS2.M, indicating the successful binding between DNA and QDs (Matsumoto et al., 1996; Liu et al., 2013). The FT-IR spectra of the CdS crystals without DNA sequences PS2.M as stabilizers and PS2.M-capped CdS QDs were compared to confirm the coordination of the DNA sequences PS2.M on the surface of the CdS QDs. As shown in Fig. S1 curve b, the majority of PS2.M functional groups could be clearly found through the stretching vibrations of CQO (1580 cm 1) and –OH (3410 cm 1), the –NH2 feature (1640 cm 1), the asymmetric stretching vibrations of the PO2 (1280 cm 1), the out of phase symmetrical stretches (1060 cm 1), and the P–O stretches of the main chain (930 cm 1). And these characteristic peaks were not observed in the FT-IR spectra of uncapped CdS (Fig. S1 curve a) that indicated the successful capping of DNA PS2.M on the surface of the CdS QDs. The TEM image and DLS of the DNA PS2.M-capped CdS QDs are shown in Fig. S2A and B.It could be seen that the generated CdS nanoparticles with DNA sequences PS2.M as stabilizers are nearly monodispersed with the diameter from 4 nm to 7 nm. The zeta potential of DNA PS2.M-capped CdS QDs is shown in Fig. S2C. Solution with zeta potential above þ20 mV and below 20 mV is considered stable (Prathna et al., 2011). The zeta potential of PS2. M-capped CdS QDs was 20.88 mV, which indicated that the generated PS2.M-capped CdS QDs could be stable in the solution.
As shown in Scheme 2, in the presence of K þ , the G-rich DNA sequences PS2.M would fold into a G-quadruplex structure by a Hoogsteen hydrogen bond between four G-bases that could effectively bind to hemin molecules to form the G-quadruplex/ hemin complex through external π–π stacking interactions (Sharon et al., 2010; Zhang et al., 2013a, 2013b; Shi et al., 2011). In this study, we investigated the interaction between PS2.M capped CdS QDs and hemin (2.5 μmol/L) by monitoring fluorescence changes. Fig. S4 shows the temporal evolution of fluorescence (FL) intensity of PS2.M capped CdS QDs after the addition of hemin. And it could be seen that the fluorescence intensity gradually decreased and remained nearly constant after 1 h. Such quenching process reached an equilibrium proving the formation of G-quadruplex/hemin complex. The fluorescence quenching was caused by the electron transfer from the PS2.M capped CdS QDs to hemin (Sharon et al., 2010; Zhang et al., 2013a, 2013b). Fig. 2 shows the evolution of fluorescence spectra of G-CdS QDs with increasing hemin concentration. It could be seen that the fluorescence intensity of G-CdS QDs obviously decreased with increasing hemin concentration from 0 to 2.5 μmol/L. Furthermore, Fig. 2 (inset) showed that there was a good linear relationship between the fluorescence quenching ratios F/F0 (F0 and F are the fluoresence intensity of G-CdS QDs without and with hemin ) of G-CdS QDs and the hemin concentration in the range of 0–0.25 μmol/L. The regression equation is F=F 0 ¼ 0:982 0:887½he min ; μmol=L
ð1Þ
The corresponding regression coefficient is 0.990, and the detection limit (S/N ¼3) for hemin was 5 nmol/L. The standard deviation for nine replicate measurements of 0.01 μmol/L hemin was 3.7%. In the concentration range from 0.25 to 2.5 μmol/L, the regression equation could be described as follows: F=F 0 ¼ 0:796 0:122½he min ; μmol=L
ð2Þ
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As shown in Fig. S6A, we investigated the effect of a series of proteins including human serum albumins (HSA), bovine serum albumins (BSA), pepsase (Pep), trypsin (Try), alkaline phosphatese (ALP), urase (Ura) and horseradish peroxidase (HRP) on the fluorescence of PS2.M capped CdS QDs. It could be seen that only hemin can effectively induce the fluorescence quenching of the CdS QDs. The results clearly demonstrate that this DNA sequence functionalized CdS QDs can selectively identify hemin. 3.4. The fluorescence recovery of PS2.M capped CdS QDs-hemin induced by Pb2 þ
Fig. 2. Fluorescence spectra of G-CdS QDs assay system in the presence of 20 μmol/ L K þ and different concentration of hemin (0, 0.01, 0.025, 0.05, 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 μmol/L). Inset: Plot of fluorescence quenching ratios (F/F0) of assay system at fluorescence emission peak versus the concentration of hemin. 10 mmol/ L Tris–HCl buffer solution (pH 7.8) incubation for 60 min. Error bars represent standard deviations from three measurements.
Some research works have proved that Pb2 þ -stabilized Gquadruplexes have shorter M–O and O–O bonds than K þ -stabilized G-quadruplexes (Kotch et al., 2000), which indicates that Pb2 þ has a higher efficiency for stabilizing G-quadruplexes than K þ . Therefore, Pb2 þ could competitively bind to K þ -stabilized G-quadruplexes/hemin complex, resulting in a conformation transition that would release the hemin cofactor (Li et al., 2009; Li et al., 2010; Guo et al., 2012). In this work, we utilized CD measurements to study the conformational change before and after adding Pb2 þ . As shown in Fig. S6B, in the absence of Pb2 þ , the K þ -stabilized PS2.M had a positive band around 295 nm in the CD spectrum. Upon the addition of Pb2 þ , a positive peak appeared at 310 nm, that is the typical characteristic of Pb2 þ -stabilized antiparallel G-quadruplexes (Li et al.,2009; Li et al., 2010). At the same time, we also studied the influence of Pb2 þ concentration on the fluorescence of G-CdS QDs quenched by 2.5 μmol/L hemin. From Fig. 3, it can be seen that the fluorescence of G-CdS QDs quenched by hemin increased with the increase of Pb2 þ concentration from 0 to 2.0 μmol/L. As shown in Fig. 3 (inset), there is a good linear relationship between the fluorescence intensity ratio F/F0 (F is the fluorescence intensity of the G-CdS QDs quenched by hemin in the presence of Pb2 þ and F0 is the fluorescence intensity of the original G-CdS QDs) and the Pb2 þ concentration in the range from 0.02 to 1.0 μmol/L. The regression equation is F=F 0 ¼ 0:502 þ 0:365½Pb
2þ
; μmol=L
ð3Þ
The corresponding regression coefficient is 0.999, and the detection limit for Pb2 þ was 10 nmol/L. The standard deviation for nine replicates measurements of 0.05 μmol/L Pb2 þ is 3.2%. Fig. 3. Fluorescence spectra of G-CdS QDs assay system in the presence of 20 μmol/ L K þ , 2.5 μmol/L hemin and different concentration of Pb2 þ (0, 0.02, 0.05, 0.10, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 μmol/L). Inset: Plot of fluorescence intensity ratio (F/F0) of assay system at fluorescence emission peak versus the concentration of Pb2 þ . 10 mmol/L Tris–HCl buffer solution (pH 7.8) incubation for 60 min. Error bars represent standard deviations from three measurements.
The corresponding regression coefficient is 0.997. It is well known that hemin molecule is negatively charged with an isoelectric point (pI) of 7.0 (Wang et al., 2010) and hence the electrostatic interaction between hemin and the DNA sequence in our assay system (pH 7.8) is very weak. As mentioned in the previous reports (Sharon et al., 2010; Zhang et al., 2012), nonspecific binding of hemin to the nucleic acid-functionalized QDs can only lead to minute quenching of the fluorescence of the QDs. As shown in Fig. S5, in the absence of K þ , the fluoresecence intensity of G-CdS QDs almost remained the same with increasing hemin concentration from 0 to 2.5 μmol/L. The fluorescence quenching of the QDs originated from the specific organization of the G-quadruplex/hemin structure on the QDs, and a Gquadruplex structure with π-stacked G-quartets and an ion channel in its center would favor DNA sequence as the medium for electron transfer to a great extent (Sharon et al., 2010; Zhang et al., 2012). Therefore, G-CdS QDs-K þ assay system could be also utilized to selectively detect hemin.
3.5. Selectivity assay for Pb2 þ based on the PS2.M capped CdS QDs The selectivity of our DNA functionalized CdS QDs for the assay of Pb2 þ was investigated, a number of common metal ions such as Hg2 þ , Ag þ , Cu2 þ , Zn2 þ , Ni2 þ , Mn2 þ , Ca2 þ , Ba2 þ , Mg2 þ , Na þ are adopted in place of Pb2 þ . As shown in Fig. 4A, although the concentration of other metal ion was higher than that of Pb2 þ , only Pb2 þ could cause an obvious fluorescence recovery of the GCdS QDs quenched by hemin. We further tested this assay system with 2 μmol/L Pb2 þ in the coexistence of other metal ion. As shown in Fig. 4B, the response of this assay system to Pb2 þ was almost unaffected by the coexistence of other tested metal ion. The results suggest that our assay is selective for Pb2 þ detection. Compared with the previous reports about fluorescence probe for Pb2 þ (Li et al., 2011, 2013; Chen et al.,2013; Liu et al.,2009; Guo et al.,2012; Zhan et al., 2013), our proposed method for the assay of Pb2 þ is simple, low-cost and high selectivity and has a similar or superior detection limit and dynamic range. 3.6. Analytical applications To test the applicability of the proposed methods, it was applied to determine the Pb2 þ ions in tap water and spring water samples. The water samples were filtered several times through
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Fig. 4. Selectivity study. (A) The fluorescence intensity of the assay system containing DNA sequence PS2.M capped CdS QDs and 2.5 mmol/L hemin in the presence of individual metal ions (2 mmol/L Pb2 þ , 5 mmol/L other metal ions) and (B) The fluorescence intensity of the assay system containing DNA sequence PS2.M capped CdS QDs and 2.5 mmol/L hemin in the presence of 2 mmol/L Pb2 þ ( first bar) and at the coexistence of 2 mmol/L Pb2 þ with 5 mmol/L other metal ions, respectively. Error bars represent standard deviations from three measurements.
qualitative filter paper. The pH was adjusted to 7.0 using NaOH solution before analysis. The results showed that Pb2 þ was not detected in these real samples, so the samples were spiked with standard Pb2 þ solution. The averages of three replicate determination results are shown in Table S1. The accuracy of the proposed method was evaluated by determining the average recovery of Pb2 þ in real samples. From Table S1, it can be seen that the RSD was lower than 4.2%, and the average recoveries of Pb2 þ in the real samples was in the range of 90–108%.
4. Conclusion In summary, we have synthesized the DNA sequence PS2.M functionalized CdS QDs and utilized it as a turn-on fluorescent Pb2 þ probe based on Pb2 þ -induced conformation transition of PS2. M G-quadruplexes/hemin complex. This assay is simple and has low-cost, and the DNA functionalized CdS QDs probe exhibits good selectivity for Pb2 þ over other metal ions.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21075050, 21275063) and the Science and Technology Development project of Jilin province, China (no. 20110334).
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