Talanta 131 (2015) 59–63
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Talanta journal homepage: www.elsevier.com/locate/talanta
Label-free detection of exonuclease III by using dsDNA–templated copper nanoparticles as fluorescent probe Hao Zhang b,a, Zihan Lin a, Xingguang Su a,n a
Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China Key Laboratory for Chemical Cleaner Production Technologies of The Education Department of Jilin Province, Jilin University of Chemical Technology, Jilin 132000, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2014 Received in revised form 17 July 2014 Accepted 21 July 2014 Available online 30 July 2014
30 –50 Exonuclease activities play key roles in maintaining genome stability, so the detection of 30 -50 exonuclease activity is very important for diseases diagnosis and drug development. In this paper, we established a simple, sensitive, low-cost and label-free method to detect the activity of exonuclease III (Exo III) by using double-strand DNA (dsDNA)-templated copper nanoparticles as fluorescent probe. Fluorescent Cu nanoparticles (NPs ) with maximum emission wavelength of 575 nm are formed by using double-strand DNA (dsDNA) as templates. Upon the addition of Exo III, the dsDNA templates would be digested from 30 to 50 , and the formation of fluorescent Cu NPs would be inhibited. Thus, the fluorescence intensity of dsDNA–Cu NPs would decrease. This method exhibits a low detection limit of 0.02 U mL 1 for Exo III. Compared with the previous reports, this method does not need complex DNA sequence design, fluorescence dye label and sophisticated experimental techniques. & 2014 Elsevier B.V. All rights reserved.
Keywords: DNA Copper nanoparticles 30 –50 exonuclease Exonuclease III Fluorescent sensor
1. Introduction Exonucleases are DNA degrading enzymes, which can digest DNA sequences from 30 -termini or 50 -termini. Exonuclease that contains 30 –50 exonuclease activity can remove nucleotides from the 30 -termini at DNA strands. The 30 –50 exonuclease plays important roles in several key cellular and physiological processes, such as repair of DNA double-strand breaks [1], contribution to the fidelity of DNA synthesis [2], promotion of genetic recombination [3] and prevention of genome instability [4]. The lack of 30 –50 exonuclease activity will cause serious diseases. For example, a major 30 –50 exonuclease TREX 1 shows DNA-editing roles in DNA replication or gap filling during DNA repairs [5]. Mutations in TREX 1 have been linked to four distinct diseases, Aicardi–Goutières syndrome, Systemic Lupus Erythematosus, Familial Chilblain Lupus and Retinal Vasculopathy and Cerebral Leukodystrophy [6]. WRN exonuclease is involved in DNA repair, which encodes a 30 –50 exonuclease. WRN exonuclease has been identified in an autosomal recessive genetic disorder Werner syndrome [7]. Therefore, the detection of 30 –50 exonuclease activity is important for diseases diagnosis and drug development.
n
Corresponding author. Tel.: þ 86 431 85168352. E-mail address: [email protected] (X. Su).
http://dx.doi.org/10.1016/j.talanta.2014.07.065 0039-9140/& 2014 Elsevier B.V. All rights reserved.
The traditional methods for detecting 30 –50 exonuclease activity are commonly based on gel-based and/or require the use of radioisotopes labeled DNA [8–10]. However, these methods are costly, unwieldy, time-consuming and necessary security arrangement to control radiographic exposure. Recently, alternative methods for 30 –50 exonuclease activity detection have been developed such as fluorescence methods. For example, Leung et al. developed a label free and G-quadruplex-based fluorescence assay method for the detection of 30 –50 exonuclease activity [11]. Lee et al. reported a new assay platform for DNA exonuclease activity detection based on the preferential binding of single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) to graphene oxide [12]. By using molecular beacons, Wu et al. developed an ultrasensitive and rapid turn-on fluorescence assay for the detection 30 –50 exonuclease activity of exonuclease III [13]. These assay methods showed great advantages over the traditional methods. However, these methods suffered from costly fluorescence dye labeled DNA probes and rigorous design of the DNA sequence. Therefore, the development of a simple, sensitive and low-cost detection method for detecting 30 –50 exonuclease activity is important and would be useful. Recently, ultrasmall fluorescent metal nanoparticles have attracted special research interest in the field of biochemical analysis due to their remarkable optical properties, good biocompatibility and facile surface modification. For example, a simple fluorescence method for cysteine detection has been developed
60
H. Zhang et al. / Talanta 131 (2015) 59–63
based on fluorescent silver clusters [14]. Glutathione-bound gold nanoclusters have been used as the fluorescence probe for detection of glutathione S-transferase-tagged proteins [15]. Silver and thioflavin T hybrid nanoclusters with extra bright and photostable emissions showed a new tool for biological imaging [16]. Lately, Mokhir et al. reported that fluorescent copper nanopaticles (Cu NPs) could be formed by using double-strand DNA as templates, whereas single strand DNA templates could not support the formation of Cu NPs [17]. The obtained dsDNA–templated Cu NPs exhibited low-toxicity, excellent fluorescence properties, good water solubility and high biocompatibility. Thus the dsDNA–Cu NPs had been used as fluorescence probes in some biological assays [18–20]. Herein, we designed a simple, sensitive, low cost and label-free strategy to detect the 30 –50 exonuclease activity based on the formation of fluorescent Cu NPs by using dsDNA as templates. The dsDNA was used as both template for Cu NPs formation and substrate of the 30 –50 exonuclease. The exonuclease could digest dsDNA probe to decrease the concentration of dsDNA templates, which would inhibit the formation of fluorescent Cu NPs. Hence, the 30 –50 exonuclease activity could be detected through the changes of the fluorescence intensity of the system. Compared with the previous reported works, this strategy does not need any complex DNA sequence design or fluorescence dye label. Meanwhile, it exhibits high sensitivity and selectivity for detecting the 30 –50 exonuclease activity. 2. Experimental 2.1. Apparatus and instruments Fluorescence measurements were carried out on a Shimadzu RF-5301 PC spectra fluorophotometer. A quartz fluorescence cell with an optical path length of 1.0 cm was used. The fluorescence spectra were obtained with the excitation wavelength of 350 nm. All pH measurements were made with a Starter-2C pH meter (Ohaus Instruments Co. Ltd., Shanghai, China). Transmission electron microscope (TEM) images were obtained with Tecnai G2 F20 S-Twin. 2.2. Reagents DNA oligomers were purchased from Sangon Biotech (Shanghai) Co., Ltd. The sequences of the DNA oligonucleotides were as follows:
1 h at 37 1C, a mixed solution containing 10 mmol L 1 PBS buffer (pH 7.4), sodium ascorbate and 250 mmol L 1 NaCl was introduced. After vibrating for 1 min, 10 μL CuSO4 solutions were added to the mixture solution with the final concentrations of 500 nmol L 1 DNA, 120 μmol L 1 Cu2 þ and 1.5 mmol L 1 sodium ascorbate. The mixed solution was kept for 10 min at room temperature, and then the fluorescent Cu NPs were formed. All experiments were repeated for three times. 2.4. Serum sample assay Human blood samples were collected from local hospital. All the blood samples were obtained through venipuncture and centrifuged at 10,000 rpm for 10 min after stored for 2 h at room temperature. 0.4 mL serum sample was mixed with 0.6 mL acetonitrile. After vigorously shaking for 2 min, the mixture was centrifuged at 10,000 rpm for 10 min. The obtained supernatant was diluted by 10 times with deionized water and the Exo III activity assay was done as described above.
3. Results and discussion 3.1. Sensor design Exonuclease III (Exo III) is well known as a sequenceindependent 30 –50 exonuclease, which has been widely used in biochemical analysis [21,22]. Exo III can catalyze the stepwise removal of mononucleotides from DNA duplexes in the direction from blunt or recessed 30 -termini to 50 -termini, while it is unable to catalyze the removal of bases from single stranded DNA or DNA duplexes with a protruding 30 end [23]. Scheme 1 shows the design of the label-free fluorescent probe for Exo III activity detection. The sensing system contains two completely complementary nucleic acid strands, which are simply designed based on DNA hybridization. The hybridized dsDNA was introduced to act as both templates for Cu NPs formation and substrate of Exo III. In the absence of Exo III, the DNA1 and DNA2 would hybridize to form stable DNA duplexes. Upon the addition of Cu2 þ ions and sodium ascorbate, the formed dsDNA–templated Cu NPs are clustered on dsDNA through the reduction of copper(II) to copper(I) followed by the disproportionation of copper(I) into copper(II) and copper(0) [17]. The formed stable Cu nanoparticles show strong fluorescence. However, in the presence of the Exo III, the stable DNA duplexes will be cleaved into fragments due to the high exodeoxyribonuclease activity
DNA1, 50 -AATAATAAGCTATAATAATT-30 DNA2, 50 -AATTATTATAGCTTATTATT-30 Exonuclease III (Exo III), sodium ascorbate were purchased from Sangon Biotech (Shanghai) Co., Ltd. Glucose, CuSO4 5H2O, MgCl2 6H2O, sodium dehydrogenized phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) were purchased from Aldrich Chemical Co. The water used in all experiments had a resistivity higher than 18 MΩ cm 1. Self-made 10 mmol L 1 PBS buffer (pH 7.4, 10 mmol L 1 NaH2PO4–Na2HPO4) was used as the medium for the detection process. 2.3. Fluorescence assay Typically, in order to prepare dsDNA templates, two ssDNA strands (DNA1, DNA2) were mixed together in 80 μL 10 mmol L 1 PBS buffer (pH 7.4) with 5 mmol L 1 Mg2 þ and reacted for 20 min at 25 1C to form the stable dsDNA. Then, various concentrations of 10 μL Exo III were added to the dsDNA solution. After incubation of
Scheme 1. Schematic illustration of the Exo III detection based on the dsDNA– templated fluorescent Cu NPs..
H. Zhang et al. / Talanta 131 (2015) 59–63
61
Fig. 1. Fluorescence spectra of obtained Cu NPs under different conditions. (a) DNA1 strand þDNA2 strand, (b) DNA1 strandþDNA2 strandþ Exo III, (c) DNA1 strand and (d) DNA2 strand. Conditions: 500 nmol L 1 DNA1, 500 nmol L 1 DNA2, 3 U mL 1 Exo III, 1.5 mmol L 1 sodium ascorbate, 120 μmol L 1 Cu2 þ ; PBS 10 mmol L 1, pH 7.4.
of Exo III on DNA duplexes. Hence, the introduction of Cu2 þ ions and ascorbate cannot form the fluorescent Cu NPs due to the lack of the dsDNA templates. Accordingly, low fluorescence intensity is observed. Thus, the Exo III activity can be detected via the changes of the fluorescence intensity in the system. To demonstrate the feasibility of our new strategy for detecting 30 –50 exonuclease activity, fluorescence emission spectra of the sensing system under different conditions were recorded. As shown in Fig. 1, the dsDNA successfully worked as templates for the formation of fluorescent Cu NPs and high fluorescence intensity could be observed (Fig. 1curves a). However, when Exo III was added into the system, the dsDNA would be digested into fragments from the 30 -termini. For the lack of dsDNA templates, the formation of Cu NPs would not happen, resulting in low fluorescence intensity (Fig. 1curves b). In addition, by using either DNA1 strand (Fig. 1, curves c) or DNA2 strand (Fig. 1, curves d), no fluorescent signal could be detected, which proved that the ssDNA would not be used as effective templates for the formation of Cu NPs. In other words, in the absence of dsDNA or only in the presence of ssDNA templates, fluorescent Cu NPs would not formed. The results indicated that our proposed strategy could be used to detect Exo III activity. Since it has been reported that whether Cu NPs are emissive or not is dependent on their sizes [24], the TEM images of the CuNPs before and after the addition of Exo III have been investigated. As shown in Fig. 2, the addition of Exo III will inhibit the formation of the dispersive Cu NPs, which indicates that the DNA duplexes will be cleaved into fragments due to the addition of Exo III, and the introduction of Cu2 þ ions and ascorbate cannot form the fluorescent Cu NPs due to the lack of the dsDNA templates. 3.2. Optimization of sensor To obtain the optimal experimental conditions for Exo III activity assay, we investigated the effects of double-stranded DNA concentrations, the concentrations of Cu2 þ and sodium ascorbate, and the reaction time on the fluorescence intensity of dsDNA–Cu NPs. Because dsDNA acted as the templates for the formation of fluorescent Cu NPs, we firstly studied the effect of dsDNA concentrations on the fluorescence intensity of Cu NPs. As shown in Fig. S1, the fluorescence intensity of dsDNA–Cu NPs increased with
Fig. 2. The TEM images of the CuNPs before (A) and after (B) the addition of 5 U mL 1 Exo III.
the increase of dsDNA concentration, and there was a good linear correlation between the fluorescence intensity and dsDNA concentrations in the range of 100 nmol L 1 to 1000 nmol L 1 (Fig. S1 inset). It indicated that the formation of Cu NPs specifically depended on the concentration of dsDNA with the other conditions fixed. In the further experiments, we used a dsDNA concentration of 500 nM, because the corresponding fluorescence intensity could satisfy the detection requirements. As the source of the formed fluorescent Cu NPs, Cu2 þ was an important factor in influencing the fluorescence intensity of the probe. To form Cu NPs effectively, a certain high Cu2 þ ion/dsDNA ratio was necessary. The effect of the concentrations of Cu2 þ was investigated in this paper. As shown in Fig. S2A, the fluorescence intensity of the Cu NPs sharply increased with the increase of Cu2 þ ion concentration. At low Cu2 þ ion concentration, Cu2 þ ions preferred to bind to the backbone phosphate negative groups through nonspecific electrostatic attraction [25]. When Cu2 þ ion concentration was further increased, with much higher affinity than the phosphate negative groups, the Cu2 þ ion began to interact with DNA bases [26], and could be reduced by ascorbate to form stable fluorescent Cu nanoparticles. Hence, the enhancement of fluorescence intensity was observed. When the concentration of Cu2 þ ion was higher than 120 μmol L 1, the fluorescence intensity decreased gradually. It might be due to the decreasing of the dsDNA templates with the destruction of the DNA double helix by the generated hydroxyl radicals at the higher
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Cu2 þ concentration [27]. Hence, the 120 μmol L 1 of Cu2 þ concentration was used in the further experiments. Considering high concentrated Cu2 þ /ascorbate mixture would degrade the dsDNA templates via the produced hydroxyl radicals, the concentration of sodium ascorbate was also optimized. It could be seen in Fig. S2B, the fluorescence intensity increased remarkably with the increasing of ascorbate concentrations, indicating that the ascorbate efficiently acted as a reducing agent and stabilizing ligand in the formation of Cu NPs [28]. When the concentrations of ascorbate exceeded 1.5 mmol L 1, the fluorescence intensity was decreased. The reason of high ascorbate concentrations influencing the formation of the Cu NPs would be similar with that of high concentration of Cu2 þ ion. So, we chose 1.5 mmol L 1 of sodium ascorbate in further experiment. Finally, the effect of reaction time was studied. Without the addition of Exo III, the reaction time was recorded after starting the reduction reaction between Cu2 þ ions and ascorbate in the presence of dsDNA. As shown in Fig. S3A, the fluorescence intensity of dsDNA–Cu NPs increased with the increase of reaction time and achieved a plateau within 10 min, which indicated that the formation of Cu NPs by the reduction of Cu2 þ with ascorbate was nearly complete in 10 min. So the reduction time of 10 min was used in the following experiments. For using the dsDNA–Cu NPs as fluorescent probe to detect the Exo III activity, the effect of incubation time was investigated with the addition of 5 U mL 1 Exo III. It could be seen in Fig. S3B, the fluorescence intensity of dsDNA–Cu NPs decreased quickly and reached the plateau after 30 min. It had been reported that the DNA digestion proceeded faster with higher concentration of Exo III [12]. That is to say, at the lower concentration of Exo III, more time was required for the complete digestion of dsDNA. Considering the complete digestion of dsDNA by Exo III, we used 60 min of incubation time in this study.
Under the optimal conditions, the formed dsDNA–Cu NPs was used as fluorescent probe for Exo III detection. As shown in Fig. 3, when a series of different concentrations (0.05 U mL 1 to 5 U mL 1) of Exo III were introduced to the dsDNA–Cu NPs system,
the fluorescence intensity of the formed dsDNA-CuNPs gradually decreased with the increasing concentrations of Exo III. Fig. 3 inset described the relationship between the Exo III concentration and the fluorescence intensity of dsDNA–Cu NPs at the maximum emission wavelength. A linear correlation existed between the fluorescence intensity and the concentration of Exo III in the range of 0.05 U mL 1 to 2 U mL 1. The regression equation was F¼ 251.72[Exo III]þ641.04 with a correlation coefficient of 0.999. The limit of detection (LOD) for Exo III was 0.02 U mL 1. The LOD was calculated by the equation LOD¼(3σ/s), where σ was the standard deviation of nine blank signals and s was the slope of the calibration curve. This LOD was much lower than that of the reported G-quadruplex-based label-free fluorescence method [11] and comparable to the labeled fluorescent method [13]. The relative standard deviation (RSD) was 2.2% for the determination of 1 U mL 1 Exo III (n¼6), which indicated a good reproducibility. To demonstrate the selectivity of the present method, we investigated the effect of a series of 15 U mL 1 nonspecific enzymes on the fluorescence intensity of Cu NPs, including lysozyme, Exo I, EcoRI and Hind III. Exo I belongs to a similar exonuclease family to Exo III, which could catalyze the removal of mononucleotides on single-stranded DNA from the 30 to 50 direction. EcoRI and Hind III belong to the restriction endonuclease, which could cleave the DNA within a defined sequence. Lysozyme belongs to neither exonuclease nor endonuclease. As shown in Fig. 4, under the same conditions for Exo III detection, only Exo III caused an obvious fluorescence decrease. These results indicated that the proposed method has good selectivity for Exo III detection. We further studied the practical application of the proposed method by detecting Exo III activity in human serum samples. The results are listed in Table 1. Results show that the concentrations of Exo III in human serum can be detected with RSD lower than 3.2% and the average recoveries of Exo III in the human serum samples were in the range of 96.8–101%. We use UV–vis method [29] as a contrast method to detect Exo III in the human serum samples. It can be seen that the results obtained by the proposed method were in good agreement with those provided by UV–vis method. The results demonstrate that our proposed lable-free fluorescence sensing platform is feasible for detecting Exo III activity in human serum sample.
Fig. 3. Fluorescence spectra for the determination of Exo III at different concentrations: (a) 0 U mL 1, (b) 0.05 U mL 1, (c) 0.1 U mL 1, (d) 0.25 U mL 1, (e) 0.5 U mL 1, (f) 0.75 U mL 1, (g) 1 U mL 1, (h) 2 U mL 1, (i) 3 U mL 1, (j) 5 U mL 1. Inset: the linear relationship between the fluorescence intensity and the Exo III concentrations. Conditions: 500 nmol L 1 dsDNA; 1.5 mmol L 1 sodium ascorbate; 120 μmol L 1 Cu2 þ ; PBS 10 mmol L 1, pH 7.4.
Fig. 4. Selectivity of dsDNA–Cu NPs sensing system for Exo III detection. The concentrations of all the interferents are fixed on 15 U mL 1 and the concentrations of ExoIII is 3 U mL 1. Conditions: 500 nmol L 1 dsDNA; 1.5 mmol L 1 sodium ascorbate; 120 μmol L 1 Cu2 þ ; PBS 10 mmol L 1, pH 7.4.
3.3. Exo III detection
H. Zhang et al. / Talanta 131 (2015) 59–63
Reference
Table 1 Detection of Exo III activity in human serum sample. Samples UV–vis method Found (μmol L 1)
Our label-free fluorescence sensing platform Found added Found Recovery (μmol L 1) (μmol L 1) (μmol L 1) (%)
RSD (%, n¼ 3)
1
0.49
0.51
2
0.98
0.99
2.6 1.0 3.2 1.3
0.25 0.5 0.25 0.5
0.74 1.02 1.20 1.47
63
97.4 101 96.8 98.7
4. Conclusion In this paper, we established a simple, sensitive, low-cost and label-free method to detect Exo III. The design of dsDNA sequence is simply based on DNA hybridization, which could be digested by Exo III. For the dsDNA acting as templates for Cu NPs formation, the digestion of dsDNA would cause the decrease of the formed Cu NPs, resulting in the decreased fluorescence intensity. Hence, the concentration of Exo III can be detected through the changes of the fluorescence intensity in the sensor system. Compared with the previous reported works, this strategy does not need any complex DNA sequence design, fluorescence dye label, and sophisticated experimental techniques. At the same time, this method exhibits high sensitivity and selectivity for the detection of Exo III. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21075050, No. 21275063) and the science and technology development project of Jilin province, China (No. 20110334). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.07.065.
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Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Label-free detection of exonuclease III by using dsDNA–templated copper nanoparticles as fluorescent probe Hao Zhang b,a, Zihan Lin a, Xingguang Su a,n a
Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China Key Laboratory for Chemical Cleaner Production Technologies of The Education Department of Jilin Province, Jilin University of Chemical Technology, Jilin 132000, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2014 Received in revised form 17 July 2014 Accepted 21 July 2014 Available online 30 July 2014
30 –50 Exonuclease activities play key roles in maintaining genome stability, so the detection of 30 -50 exonuclease activity is very important for diseases diagnosis and drug development. In this paper, we established a simple, sensitive, low-cost and label-free method to detect the activity of exonuclease III (Exo III) by using double-strand DNA (dsDNA)-templated copper nanoparticles as fluorescent probe. Fluorescent Cu nanoparticles (NPs ) with maximum emission wavelength of 575 nm are formed by using double-strand DNA (dsDNA) as templates. Upon the addition of Exo III, the dsDNA templates would be digested from 30 to 50 , and the formation of fluorescent Cu NPs would be inhibited. Thus, the fluorescence intensity of dsDNA–Cu NPs would decrease. This method exhibits a low detection limit of 0.02 U mL 1 for Exo III. Compared with the previous reports, this method does not need complex DNA sequence design, fluorescence dye label and sophisticated experimental techniques. & 2014 Elsevier B.V. All rights reserved.
Keywords: DNA Copper nanoparticles 30 –50 exonuclease Exonuclease III Fluorescent sensor
1. Introduction Exonucleases are DNA degrading enzymes, which can digest DNA sequences from 30 -termini or 50 -termini. Exonuclease that contains 30 –50 exonuclease activity can remove nucleotides from the 30 -termini at DNA strands. The 30 –50 exonuclease plays important roles in several key cellular and physiological processes, such as repair of DNA double-strand breaks [1], contribution to the fidelity of DNA synthesis [2], promotion of genetic recombination [3] and prevention of genome instability [4]. The lack of 30 –50 exonuclease activity will cause serious diseases. For example, a major 30 –50 exonuclease TREX 1 shows DNA-editing roles in DNA replication or gap filling during DNA repairs [5]. Mutations in TREX 1 have been linked to four distinct diseases, Aicardi–Goutières syndrome, Systemic Lupus Erythematosus, Familial Chilblain Lupus and Retinal Vasculopathy and Cerebral Leukodystrophy [6]. WRN exonuclease is involved in DNA repair, which encodes a 30 –50 exonuclease. WRN exonuclease has been identified in an autosomal recessive genetic disorder Werner syndrome [7]. Therefore, the detection of 30 –50 exonuclease activity is important for diseases diagnosis and drug development.
n
Corresponding author. Tel.: þ 86 431 85168352. E-mail address: [email protected] (X. Su).
http://dx.doi.org/10.1016/j.talanta.2014.07.065 0039-9140/& 2014 Elsevier B.V. All rights reserved.
The traditional methods for detecting 30 –50 exonuclease activity are commonly based on gel-based and/or require the use of radioisotopes labeled DNA [8–10]. However, these methods are costly, unwieldy, time-consuming and necessary security arrangement to control radiographic exposure. Recently, alternative methods for 30 –50 exonuclease activity detection have been developed such as fluorescence methods. For example, Leung et al. developed a label free and G-quadruplex-based fluorescence assay method for the detection of 30 –50 exonuclease activity [11]. Lee et al. reported a new assay platform for DNA exonuclease activity detection based on the preferential binding of single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) to graphene oxide [12]. By using molecular beacons, Wu et al. developed an ultrasensitive and rapid turn-on fluorescence assay for the detection 30 –50 exonuclease activity of exonuclease III [13]. These assay methods showed great advantages over the traditional methods. However, these methods suffered from costly fluorescence dye labeled DNA probes and rigorous design of the DNA sequence. Therefore, the development of a simple, sensitive and low-cost detection method for detecting 30 –50 exonuclease activity is important and would be useful. Recently, ultrasmall fluorescent metal nanoparticles have attracted special research interest in the field of biochemical analysis due to their remarkable optical properties, good biocompatibility and facile surface modification. For example, a simple fluorescence method for cysteine detection has been developed
60
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based on fluorescent silver clusters [14]. Glutathione-bound gold nanoclusters have been used as the fluorescence probe for detection of glutathione S-transferase-tagged proteins [15]. Silver and thioflavin T hybrid nanoclusters with extra bright and photostable emissions showed a new tool for biological imaging [16]. Lately, Mokhir et al. reported that fluorescent copper nanopaticles (Cu NPs) could be formed by using double-strand DNA as templates, whereas single strand DNA templates could not support the formation of Cu NPs [17]. The obtained dsDNA–templated Cu NPs exhibited low-toxicity, excellent fluorescence properties, good water solubility and high biocompatibility. Thus the dsDNA–Cu NPs had been used as fluorescence probes in some biological assays [18–20]. Herein, we designed a simple, sensitive, low cost and label-free strategy to detect the 30 –50 exonuclease activity based on the formation of fluorescent Cu NPs by using dsDNA as templates. The dsDNA was used as both template for Cu NPs formation and substrate of the 30 –50 exonuclease. The exonuclease could digest dsDNA probe to decrease the concentration of dsDNA templates, which would inhibit the formation of fluorescent Cu NPs. Hence, the 30 –50 exonuclease activity could be detected through the changes of the fluorescence intensity of the system. Compared with the previous reported works, this strategy does not need any complex DNA sequence design or fluorescence dye label. Meanwhile, it exhibits high sensitivity and selectivity for detecting the 30 –50 exonuclease activity. 2. Experimental 2.1. Apparatus and instruments Fluorescence measurements were carried out on a Shimadzu RF-5301 PC spectra fluorophotometer. A quartz fluorescence cell with an optical path length of 1.0 cm was used. The fluorescence spectra were obtained with the excitation wavelength of 350 nm. All pH measurements were made with a Starter-2C pH meter (Ohaus Instruments Co. Ltd., Shanghai, China). Transmission electron microscope (TEM) images were obtained with Tecnai G2 F20 S-Twin. 2.2. Reagents DNA oligomers were purchased from Sangon Biotech (Shanghai) Co., Ltd. The sequences of the DNA oligonucleotides were as follows:
1 h at 37 1C, a mixed solution containing 10 mmol L 1 PBS buffer (pH 7.4), sodium ascorbate and 250 mmol L 1 NaCl was introduced. After vibrating for 1 min, 10 μL CuSO4 solutions were added to the mixture solution with the final concentrations of 500 nmol L 1 DNA, 120 μmol L 1 Cu2 þ and 1.5 mmol L 1 sodium ascorbate. The mixed solution was kept for 10 min at room temperature, and then the fluorescent Cu NPs were formed. All experiments were repeated for three times. 2.4. Serum sample assay Human blood samples were collected from local hospital. All the blood samples were obtained through venipuncture and centrifuged at 10,000 rpm for 10 min after stored for 2 h at room temperature. 0.4 mL serum sample was mixed with 0.6 mL acetonitrile. After vigorously shaking for 2 min, the mixture was centrifuged at 10,000 rpm for 10 min. The obtained supernatant was diluted by 10 times with deionized water and the Exo III activity assay was done as described above.
3. Results and discussion 3.1. Sensor design Exonuclease III (Exo III) is well known as a sequenceindependent 30 –50 exonuclease, which has been widely used in biochemical analysis [21,22]. Exo III can catalyze the stepwise removal of mononucleotides from DNA duplexes in the direction from blunt or recessed 30 -termini to 50 -termini, while it is unable to catalyze the removal of bases from single stranded DNA or DNA duplexes with a protruding 30 end [23]. Scheme 1 shows the design of the label-free fluorescent probe for Exo III activity detection. The sensing system contains two completely complementary nucleic acid strands, which are simply designed based on DNA hybridization. The hybridized dsDNA was introduced to act as both templates for Cu NPs formation and substrate of Exo III. In the absence of Exo III, the DNA1 and DNA2 would hybridize to form stable DNA duplexes. Upon the addition of Cu2 þ ions and sodium ascorbate, the formed dsDNA–templated Cu NPs are clustered on dsDNA through the reduction of copper(II) to copper(I) followed by the disproportionation of copper(I) into copper(II) and copper(0) [17]. The formed stable Cu nanoparticles show strong fluorescence. However, in the presence of the Exo III, the stable DNA duplexes will be cleaved into fragments due to the high exodeoxyribonuclease activity
DNA1, 50 -AATAATAAGCTATAATAATT-30 DNA2, 50 -AATTATTATAGCTTATTATT-30 Exonuclease III (Exo III), sodium ascorbate were purchased from Sangon Biotech (Shanghai) Co., Ltd. Glucose, CuSO4 5H2O, MgCl2 6H2O, sodium dehydrogenized phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) were purchased from Aldrich Chemical Co. The water used in all experiments had a resistivity higher than 18 MΩ cm 1. Self-made 10 mmol L 1 PBS buffer (pH 7.4, 10 mmol L 1 NaH2PO4–Na2HPO4) was used as the medium for the detection process. 2.3. Fluorescence assay Typically, in order to prepare dsDNA templates, two ssDNA strands (DNA1, DNA2) were mixed together in 80 μL 10 mmol L 1 PBS buffer (pH 7.4) with 5 mmol L 1 Mg2 þ and reacted for 20 min at 25 1C to form the stable dsDNA. Then, various concentrations of 10 μL Exo III were added to the dsDNA solution. After incubation of
Scheme 1. Schematic illustration of the Exo III detection based on the dsDNA– templated fluorescent Cu NPs..
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Fig. 1. Fluorescence spectra of obtained Cu NPs under different conditions. (a) DNA1 strand þDNA2 strand, (b) DNA1 strandþDNA2 strandþ Exo III, (c) DNA1 strand and (d) DNA2 strand. Conditions: 500 nmol L 1 DNA1, 500 nmol L 1 DNA2, 3 U mL 1 Exo III, 1.5 mmol L 1 sodium ascorbate, 120 μmol L 1 Cu2 þ ; PBS 10 mmol L 1, pH 7.4.
of Exo III on DNA duplexes. Hence, the introduction of Cu2 þ ions and ascorbate cannot form the fluorescent Cu NPs due to the lack of the dsDNA templates. Accordingly, low fluorescence intensity is observed. Thus, the Exo III activity can be detected via the changes of the fluorescence intensity in the system. To demonstrate the feasibility of our new strategy for detecting 30 –50 exonuclease activity, fluorescence emission spectra of the sensing system under different conditions were recorded. As shown in Fig. 1, the dsDNA successfully worked as templates for the formation of fluorescent Cu NPs and high fluorescence intensity could be observed (Fig. 1curves a). However, when Exo III was added into the system, the dsDNA would be digested into fragments from the 30 -termini. For the lack of dsDNA templates, the formation of Cu NPs would not happen, resulting in low fluorescence intensity (Fig. 1curves b). In addition, by using either DNA1 strand (Fig. 1, curves c) or DNA2 strand (Fig. 1, curves d), no fluorescent signal could be detected, which proved that the ssDNA would not be used as effective templates for the formation of Cu NPs. In other words, in the absence of dsDNA or only in the presence of ssDNA templates, fluorescent Cu NPs would not formed. The results indicated that our proposed strategy could be used to detect Exo III activity. Since it has been reported that whether Cu NPs are emissive or not is dependent on their sizes [24], the TEM images of the CuNPs before and after the addition of Exo III have been investigated. As shown in Fig. 2, the addition of Exo III will inhibit the formation of the dispersive Cu NPs, which indicates that the DNA duplexes will be cleaved into fragments due to the addition of Exo III, and the introduction of Cu2 þ ions and ascorbate cannot form the fluorescent Cu NPs due to the lack of the dsDNA templates. 3.2. Optimization of sensor To obtain the optimal experimental conditions for Exo III activity assay, we investigated the effects of double-stranded DNA concentrations, the concentrations of Cu2 þ and sodium ascorbate, and the reaction time on the fluorescence intensity of dsDNA–Cu NPs. Because dsDNA acted as the templates for the formation of fluorescent Cu NPs, we firstly studied the effect of dsDNA concentrations on the fluorescence intensity of Cu NPs. As shown in Fig. S1, the fluorescence intensity of dsDNA–Cu NPs increased with
Fig. 2. The TEM images of the CuNPs before (A) and after (B) the addition of 5 U mL 1 Exo III.
the increase of dsDNA concentration, and there was a good linear correlation between the fluorescence intensity and dsDNA concentrations in the range of 100 nmol L 1 to 1000 nmol L 1 (Fig. S1 inset). It indicated that the formation of Cu NPs specifically depended on the concentration of dsDNA with the other conditions fixed. In the further experiments, we used a dsDNA concentration of 500 nM, because the corresponding fluorescence intensity could satisfy the detection requirements. As the source of the formed fluorescent Cu NPs, Cu2 þ was an important factor in influencing the fluorescence intensity of the probe. To form Cu NPs effectively, a certain high Cu2 þ ion/dsDNA ratio was necessary. The effect of the concentrations of Cu2 þ was investigated in this paper. As shown in Fig. S2A, the fluorescence intensity of the Cu NPs sharply increased with the increase of Cu2 þ ion concentration. At low Cu2 þ ion concentration, Cu2 þ ions preferred to bind to the backbone phosphate negative groups through nonspecific electrostatic attraction [25]. When Cu2 þ ion concentration was further increased, with much higher affinity than the phosphate negative groups, the Cu2 þ ion began to interact with DNA bases [26], and could be reduced by ascorbate to form stable fluorescent Cu nanoparticles. Hence, the enhancement of fluorescence intensity was observed. When the concentration of Cu2 þ ion was higher than 120 μmol L 1, the fluorescence intensity decreased gradually. It might be due to the decreasing of the dsDNA templates with the destruction of the DNA double helix by the generated hydroxyl radicals at the higher
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Cu2 þ concentration [27]. Hence, the 120 μmol L 1 of Cu2 þ concentration was used in the further experiments. Considering high concentrated Cu2 þ /ascorbate mixture would degrade the dsDNA templates via the produced hydroxyl radicals, the concentration of sodium ascorbate was also optimized. It could be seen in Fig. S2B, the fluorescence intensity increased remarkably with the increasing of ascorbate concentrations, indicating that the ascorbate efficiently acted as a reducing agent and stabilizing ligand in the formation of Cu NPs [28]. When the concentrations of ascorbate exceeded 1.5 mmol L 1, the fluorescence intensity was decreased. The reason of high ascorbate concentrations influencing the formation of the Cu NPs would be similar with that of high concentration of Cu2 þ ion. So, we chose 1.5 mmol L 1 of sodium ascorbate in further experiment. Finally, the effect of reaction time was studied. Without the addition of Exo III, the reaction time was recorded after starting the reduction reaction between Cu2 þ ions and ascorbate in the presence of dsDNA. As shown in Fig. S3A, the fluorescence intensity of dsDNA–Cu NPs increased with the increase of reaction time and achieved a plateau within 10 min, which indicated that the formation of Cu NPs by the reduction of Cu2 þ with ascorbate was nearly complete in 10 min. So the reduction time of 10 min was used in the following experiments. For using the dsDNA–Cu NPs as fluorescent probe to detect the Exo III activity, the effect of incubation time was investigated with the addition of 5 U mL 1 Exo III. It could be seen in Fig. S3B, the fluorescence intensity of dsDNA–Cu NPs decreased quickly and reached the plateau after 30 min. It had been reported that the DNA digestion proceeded faster with higher concentration of Exo III [12]. That is to say, at the lower concentration of Exo III, more time was required for the complete digestion of dsDNA. Considering the complete digestion of dsDNA by Exo III, we used 60 min of incubation time in this study.
Under the optimal conditions, the formed dsDNA–Cu NPs was used as fluorescent probe for Exo III detection. As shown in Fig. 3, when a series of different concentrations (0.05 U mL 1 to 5 U mL 1) of Exo III were introduced to the dsDNA–Cu NPs system,
the fluorescence intensity of the formed dsDNA-CuNPs gradually decreased with the increasing concentrations of Exo III. Fig. 3 inset described the relationship between the Exo III concentration and the fluorescence intensity of dsDNA–Cu NPs at the maximum emission wavelength. A linear correlation existed between the fluorescence intensity and the concentration of Exo III in the range of 0.05 U mL 1 to 2 U mL 1. The regression equation was F¼ 251.72[Exo III]þ641.04 with a correlation coefficient of 0.999. The limit of detection (LOD) for Exo III was 0.02 U mL 1. The LOD was calculated by the equation LOD¼(3σ/s), where σ was the standard deviation of nine blank signals and s was the slope of the calibration curve. This LOD was much lower than that of the reported G-quadruplex-based label-free fluorescence method [11] and comparable to the labeled fluorescent method [13]. The relative standard deviation (RSD) was 2.2% for the determination of 1 U mL 1 Exo III (n¼6), which indicated a good reproducibility. To demonstrate the selectivity of the present method, we investigated the effect of a series of 15 U mL 1 nonspecific enzymes on the fluorescence intensity of Cu NPs, including lysozyme, Exo I, EcoRI and Hind III. Exo I belongs to a similar exonuclease family to Exo III, which could catalyze the removal of mononucleotides on single-stranded DNA from the 30 to 50 direction. EcoRI and Hind III belong to the restriction endonuclease, which could cleave the DNA within a defined sequence. Lysozyme belongs to neither exonuclease nor endonuclease. As shown in Fig. 4, under the same conditions for Exo III detection, only Exo III caused an obvious fluorescence decrease. These results indicated that the proposed method has good selectivity for Exo III detection. We further studied the practical application of the proposed method by detecting Exo III activity in human serum samples. The results are listed in Table 1. Results show that the concentrations of Exo III in human serum can be detected with RSD lower than 3.2% and the average recoveries of Exo III in the human serum samples were in the range of 96.8–101%. We use UV–vis method [29] as a contrast method to detect Exo III in the human serum samples. It can be seen that the results obtained by the proposed method were in good agreement with those provided by UV–vis method. The results demonstrate that our proposed lable-free fluorescence sensing platform is feasible for detecting Exo III activity in human serum sample.
Fig. 3. Fluorescence spectra for the determination of Exo III at different concentrations: (a) 0 U mL 1, (b) 0.05 U mL 1, (c) 0.1 U mL 1, (d) 0.25 U mL 1, (e) 0.5 U mL 1, (f) 0.75 U mL 1, (g) 1 U mL 1, (h) 2 U mL 1, (i) 3 U mL 1, (j) 5 U mL 1. Inset: the linear relationship between the fluorescence intensity and the Exo III concentrations. Conditions: 500 nmol L 1 dsDNA; 1.5 mmol L 1 sodium ascorbate; 120 μmol L 1 Cu2 þ ; PBS 10 mmol L 1, pH 7.4.
Fig. 4. Selectivity of dsDNA–Cu NPs sensing system for Exo III detection. The concentrations of all the interferents are fixed on 15 U mL 1 and the concentrations of ExoIII is 3 U mL 1. Conditions: 500 nmol L 1 dsDNA; 1.5 mmol L 1 sodium ascorbate; 120 μmol L 1 Cu2 þ ; PBS 10 mmol L 1, pH 7.4.
3.3. Exo III detection
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Reference
Table 1 Detection of Exo III activity in human serum sample. Samples UV–vis method Found (μmol L 1)
Our label-free fluorescence sensing platform Found added Found Recovery (μmol L 1) (μmol L 1) (μmol L 1) (%)
RSD (%, n¼ 3)
1
0.49
0.51
2
0.98
0.99
2.6 1.0 3.2 1.3
0.25 0.5 0.25 0.5
0.74 1.02 1.20 1.47
63
97.4 101 96.8 98.7
4. Conclusion In this paper, we established a simple, sensitive, low-cost and label-free method to detect Exo III. The design of dsDNA sequence is simply based on DNA hybridization, which could be digested by Exo III. For the dsDNA acting as templates for Cu NPs formation, the digestion of dsDNA would cause the decrease of the formed Cu NPs, resulting in the decreased fluorescence intensity. Hence, the concentration of Exo III can be detected through the changes of the fluorescence intensity in the sensor system. Compared with the previous reported works, this strategy does not need any complex DNA sequence design, fluorescence dye label, and sophisticated experimental techniques. At the same time, this method exhibits high sensitivity and selectivity for the detection of Exo III. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21075050, No. 21275063) and the science and technology development project of Jilin province, China (No. 20110334). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.07.065.
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