Analytica Chimica Acta 848 (2014) 67–73
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Ultrasensitive determination of DNA sequences by flow injection chemiluminescence using silver ions as labels Lichun Zheng, Xiuhui Liu *, Min Zhou, Yongjun Ma, Guofan Wu, Xiaoquan Lu ** Key Laboratory of Bioelectrochemistry & Enviromental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, No. 967 Anning East Road, Lanzhou, Gansu 730070, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A novel approach for ultrasensitive FI–CL detection of DNA sequences was developed. A detection probe labeled with Ag+ was synthesized for the first time. The method possesses high sensitivity and selectivity with a detection limit of 3.3 pM.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2014 Received in revised form 17 July 2014 Accepted 22 July 2014 Available online 24 July 2014
We presented a new strategy for ultrasensitive detection of DNA sequences based on the novel detection probe which was labeled with Ag+ using metallothionein (MT) as a bridge. The assay relied on a sandwich-type DNA hybridization in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on Fe3O4@Au composite magnetic nanoparticles (MNPs), and then the Ag+-modified detection probes were used to monitor the presence of the specific DNA targets. After being anchored on the hybrids, Ag+ was released down through acidic treatment and sensitively determined by a coupling flow injection–chemiluminescent reaction system (Ag+–Mn2+–K2S2O8–H3PO4– luminol) (FI–CL). The experiment results showed that the CL intensities increased linearly with the concentrations of DNA targets in the range from 10 to 500 pmol L1 with a detection limit of 3.3 pmol L1. The high sensitivity in this work may be ascribed to the high molar ratio of Ag+–MT, the sensitive determination of Ag+ by the coupling FI–CL reaction system and the perfect magnetic separation based on Fe3O4@Au composite MNPs. Moreover, the proposed strategy exhibited excellent selectivity against the mismatched DNA sequences and could be applied to real samples analysis. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Nucleic acid biosensors Flow injection–chemiluminescent detection Metallothioneins Silver ions Magnetic nanoparticles
1. Introduction The detection of DNA sequences has received a huge interest in the fields of clinical diagnosis, genetics therapy, and a variety of biomedical studies recently [1,2]. Thus, there has been an
* Corresponding author. Tel.: +86 931 7972366; fax: +86 931 7971323. ** Corresponding author. Tel.: +86 931 7971276; fax: +86 931 7971276. E-mail addresses: [email protected], [email protected] (X. Liu), [email protected] (X. Lu). http://dx.doi.org/10.1016/j.aca.2014.07.033 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
increasing demand to develop a simple and sensitive method in order to detect the specific oligonucleotide sequence. As well known, various techniques had been developed for detection of DNA hybridization, and their sensitivities depended mainly on the specific activity of the labels which linked to the oligonucleotide probes [3–5]. Dyes, enzymes, and quantum dots (QDs) are the most popular labels for DNA analysis. Dyes such as cyanine 5 (Cy5), carboxyfluorescein (FAM), and tetramethyl-6-carboxy-rhodamine (TAMRA) are the classical fluorophores which have been widely used in DNA analysis. But the quantitative measurements remain a challenge because of low fluorescence intensities and
68
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
susceptibility to photobleaching [6,7]. Other detection techniques which employed enzymes to generate spectrophotometric [8], electrochemical [9,10], or chemiluminescent [11] signals had achieved good linearity and high sensitivity. However, they were also hampered because the activity of the enzyme was destroyed easily by its environment conditions (temperature, pH value, etc.). In recent years, QDs have been used as labels for the detection of DNA due to theirs unique florescent [12,13] and chemiluminescent [14] properties. In addition, the labels, QDs, can be dissolved in acidic solution to produce a large number of corresponding metal ions which will yield well-resolved highly sensitive stripping voltammetric signals for the targets [15]. But the preparation of QDs is often a time-consuming work and requires harsh conditions. More important, it is difficult to control their sizes. In previous work [16], we determined three different DNA targets simultaneously based on the usage of different encoding metal ions as tags, in which the novel detection probes, ssDNA/MT conjugates, covered with different metal ions (Zn2+, Cd2+, and Pb2+) were synthesized. Then the encoding metal ions were used to differentiate the signals of three kinds of virus DNA. The proposed method was proved to be simple and sensitive. Previously, other researchers found that MT is able to bind more monovalent metal ions (Cu+, Ag+, etc.) than bivalent metal ions (Zn2+, Cd2+, Pb2+, etc.) [17–20]. Therefore, in this paper, we proposed a strategy to prepare a new detection probe labeled with Ag+, and detected it by the coupling CL reaction (Ag+–Mn2+–K2S2O8–H3PO4–luminol) [21] equipping with the FI–CL system. Meanwhile, as-prepared Fe3O4@Au composite magnetic nanoparticles (MNPs) were used as substrate to immobile the capture probes. Therefore, it is believed that the proposed method possesses great potential for ultrasensitive detection of DNA hybridization. 2. Experimental 2.1. Materials The Zn7–MT from rabbit liver was purchased from Botai Biotech (Dalian, China). Succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate (SMCC) came from Pierce. 1-Hexanethiol (MCH) was acquired from Aladdin (Shanghai, China). HAuCl4, FeCl36H2O, FeCl24H2O, tetramethylammonium hydroxide (TMOH), dimethyl sulfoxide (DMSO), AgNO3, NaOH, Tris(hydroxymethyl)aminomethane (Tris), NaCl, NaH2PO4 and Na2HPO4 were all purchased from Beijing Chemical Reagents Co. (Beijing, China). Other chemicals employed were all of analytical grade, and Milli-Q water was used in all experiments. All oligonucletides were provided by Sangon (Shanghai, China). The capture probes were thiolated with a (CH2)3 spacer at the 30 ends and the detection probes were modified with amino groups with a (CH2)6 spacer at the 5C ends. Their base sequences were: Human enterovirus-related oligonucletides: Capture probe: 50 -CAGACACTGTTGGTA-(CH2)6-SH-30 Detection probe: 50 -H2N-(CH2)6-GAATAGCGTCAGAAT-30 Complementary target (t1): 50 -TACCAACAGTGTCTGATTCTGACGCTATTC-30 One-base mismatched target (t2): 50 -TACCGACAGTGTCTGATTCTGACGCTATTC-30 Four-base mismatched target (t3): 50 -TACCGGAACTGTCTGATTCTGACGCTATTC-30 Noncomplementary target (t4): 50 -GCCGCGTGTTTTAGCACATAGCGATGTCAC-30 2.2. Apparatus A Jasco spectropolarimeter (model J-715) connected to a computer was used for CD measurements. The UV–vis absorption
spectra were recorded on a UV-1102 UV–vis spectrophotometer. The transmission electron microscope (TEM) and energy dispersive X-ray (EDX) images were acquired from a FEI TF20 field emission transmission electron microscope. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at the SE 400 vertical unit (GE Healthcare). The zeta potential of the samples was determined on a Nano-ZS Zetasizer ZEN3600 dynamic light scattering instrument. The magnetization was measured by a 7304 vibrating sample magnetometer. The composition and crystal line structure of the samples were analyzed on a Philips X’pert multipurpose X-ray diffraction system. The CL signals were measured by an IFFL-D analyzer. 2.3. Procedures 2.3.1. Metal ion binding reactions The lyophilized Zn7–MT was firstly dissolved in 0.02 mol L1 Tris– HClO4 buffer at pH 7.4. Metal binding experiments were carried out by sequentially adding molar ratio aliquots of Ag+ (using AgNO3) and allowed to incubate for 30 min at room temperature. All manipulations involving the metal ions and protein solutions were performed in a nitrogen atmosphere. Circular dichroism (CD) spectroscopy and ultraviolet–visible (UV–vis) absorption spectroscopy were used to monitor the binding reaction of Ag+ to the protein. The products (Ag–MT) were purified by a centricon-3 centrifugal concentrator to remove any unbound Ag+. 2.3.2. Conjugation of Ag–MT to NH2–ssDNA SMCC, the heterobifunctional crosslinker, was firstly dissolved in DMSO, and then diluted with water immediately prior to use to a final concentration of SMCC of 5%. To prevent the hydrolytic degradation of the NHS ester, the conjugation was usually performed at pH 7.3. Phosphate-buffered saline (PBS = 0.1 mol L1 sodium phosphate, 0.15 mol L1 sodium chloride, pH 7.4) and the amine- and sulfhydryl-free buffer were used as conjugation buffer. A 40-fold molar of crosslinker over the amount of MT was used to react simultaneously with MT and excess NH2–ssDNA for 1 h at room temperature. The unreacted SMCC and NH2–ssDNA were removed from the reaction product by ultrafiltration. The conjugation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the polyacrylamide gel was 18%. The electrophoresis started with an initial voltage of 30 V, then maintained at this voltage until the sample had completely entered the stacking gel. Finally, the voltage applied 80 V for 3.5 h. After electrophoresis, the gel was treated with 0.1% AgNO3, and then developed with 1.5% NaOH and 0.4% formaldehyde. 2.3.3. Preparation of Fe3O4@Au composite MNPs The Fe3O4 MNPs were prepared by coprecipitation according to a method mentioned in the previous reports [22,23]. Briefly, 5.4 g of FeCl36H2O and 2.0 g of FeCl24H2O were dissolved in 10 mL of 0.4 mol L1 HCl solution under vigorous stirring. The coprecipitation of Fe3O4 MNPs was carried out in a three-neck round-bottom flask and protected under a pure N2 atmosphere. The above mixture solution was added dropwise to 125 mL of 0.5 mol L1 NaOH, which was preheated to 80 C before the coprecipitation reaction. The reaction was vigorously stirred for about 30 min, resulting in a pale yellow color solution which finally turned into dark black. The black precipitate was collected by sedimentation with the help of an external magnetic field and washed gradually with 0.1 mol L1 HNO3 and 0.01 mol L1 HNO3. To obtain oxidized Fe3O4 MNPs, the particles were then dissolved in 0.01 mol L1 HNO3 and heated at 90 C with stirring until the color of solution became brown. The oxidized Fe3O4 MNPs were suspended in 50 mL of 0.1 mol L1 TMOH at pH 11 after washing with water.
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
Au-shell coating was performed by reduction of Au3+ on the surface of Fe3O4 MNPs using a modification of previous procedure [24]. Firstly, 5 mL of N(CH3)4-stabilized, oxidized Fe3O4 MNPs solution were added to 50 mL of 0.01 mol L1 sodium citrate and stirred for 30 min to exchange absorbed OH with citrate ions to make the magnetic-core solution. Next, the solution was ultrasonicated for 15 min and then heated to boiling with vigorous stirring. Then, 1% HAuCl4 (2 mL) was added as soon as the reaction solution reached the boiling point. The mixture was boiled for 15 min and stirred for an additional 15 min while cooling. The obtained Fe3O4@Au composite MNPs were magnetically separated from the Au NPs formed during the process. 2.3.4. Hybridization procedures 30 mL of Fe3O4@Au composite MNPs (8 mg mL1) were firstly transferred into a 1.5 mL centrifuge vial, and then washed twice with buffer A (0.01 mol L1 PBS, 0.2 mol L1 NaCl, pH 7.4) by magnetic separation and resuspended in 200 mL of buffer A containing 10 mmol L1 of thiolated capture probes by shaking. The solution was incubated overnight at room temperature with gentle shaking. The unbound DNA sequences were washed away with the same buffer, supernatant removal. Subsequently, the capture probes-attached composite MNPs were resuspended in buffer A containing 0.1 mmol L1 MCH solution and incubated for 2 h. Then, the capture probes/MCH-attached composite MNPs were washed three times with water and resuspended in 50 mL of water before use. The sandwich assay involved a dual hybridization event. Corresponding amounts of the targets were added into 100 mL of buffer B (0.01 mol L1 PBS, 0.15 mol L1 NaCl, 0.1% Tween 20, pH 7.4) containing 5 mL of the capture probes/MCHattached composite MNPs solution. After hybridization for 2 h at 37 C, the resulting composite MNPs were separated magnetically from the solution and washed twice with buffer B. And then the composite MNPs were resuspended in 100 mL of buffer B containing 5 mL of the as-prepared detection probes and incubated for 2 h at 37 C. The obtained composite MNPs were washed three times with water. Afterward, release of the Ag+ from MT molecules was happened in 100 mL of 1 mol L1 HCl for 10 min. At last, the FI–CL detection of Ag+ was performed according to the standard procedures below. 2.3.5. Standard procedures for FI–CL detection The pH value of the acid solution containing Ag+ was adjusted to be neutral by adding 5 mol L1 NaOH, and the resultant mixture was then transferred into a 5 mL beaker containing 1 mL of 2% (m/ v) K2S2O8, 200 mL of 6 mmol L1 MnSO4, and 200 mL of 1:1 (v/v)
69
H3PO4 and immediately incubated in a 85 C water bath for 8 min; thus, the resultant KMnO4 was produced. The reaction was stopped with flowing cold water, and the solution was diluted to 10 mL. The signals of luminol–KMnO4 CL system were measured by an FI–CL analyzer. 3. Results and discussion 3.1. The fabrication of the biosensor and the detection process The principle of the protocol presented in this work is shown in Fig. 1. At first, the Fe3O4@Au composite MNPs with core–shell structure were synthesized by reduction of Au3+ in the presence of Fe3O4 NPs (A). Secondly, the thiolated capture probes were immobilized on the composite MNPs through the Au S bands (B). Then, the novel detection probes which consist of ssDNA and MT molecules covered with Ag+ were prepared and hybridized with the targets in a sandwich mode (C and D). After hybridization, the Ag+ was released from MT in the acid solution (E). Finally, the released Ag+ was sensitively determined by the coupling CL reaction (Ag+–Mn2+–K2S2O8–H3PO4–luminol) equipped with the FI–CL system (F and G). This coupling CL reaction was based on the strong catalytic effect of Ag+ on the reaction of Mn2+–K2S2O8– H3PO4 [25]. The catalytically produced KMnO4 could further oxidize luminol in an alkaline medium, which would generate a strong CL signal, and the CL intensities are proportional to the amount of DNA targets. The originality of this design is that MT is used as a bridge to combine ssDNA and Ag+. MT is a small metalloprotein characterized by its high cysteine content (approximately 30% of its amino acid sequence), absence of disulfide bonds, and lack of aromatic amino acids, particularly its high metal affinity [26]. Previous research found that 20 cysteine thiolates of each MT are capable of binding to 18 Ag+ [27,28]. 3.2. Characterization of the detection probes 3.2.1. Metal ion binding reactions CD spectra were used to characterize the formation of a series of complexes between Ag+ and the cysteine thiolate groups in zinc MT. As shown in Fig. 2 (curve a), one can see that there was only one positive band near 243 nm which was characteristic of Zn7–MT [28] before Ag+ was added. After titration with Ag+, the 243 nm positive band gradually diminished in intensity while a negative band near 265 nm and a positive band near 293 nm gradually intensified as shown in curve b–d, indicating that the Zn2+ in MT was displaced by Ag+ to form the new species. As we know, the MT
Fig. 1. Schematic representation of the protocol: (A) formation of the Fe3O4@Au composite MNPs; (B) modification with the thiolated probes; (C) hybridization with the targets; (D) hybridization with the detection probes; (E) release of Ag+; (F and G) the coupling CL reaction equipped with the FI–CL system.
70
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
3.2.2. Conjugation of Ag–MT to NH2–ssDNA The conjugation of Ag–MT to NH2–ssDNA was performed as our previous work [16]. Briefly, ssDNA was synthesized first to contain a 30 -aminohexyl linker at the end. Then, the heterobifunctional crosslinking reagent (SMCC) was used as a bridge to combine NH2– ssDNA and Ag–MT. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to assess the conjugation of MT to ssDNA. As shown in Fig. 3, one can see that there are two bands in lane 1 before the crosslinking reaction. However, after the crosslinking reaction, there is only one band in lane 2. Compared to the bands in lane 1, the band in lane 2 showed the lower mobility due to its higher molecular weight after the crosslinking reaction. The result gives the immediate evidence for the production of the (Ag–MT)–ssDNA conjugates (namely the detection probe). 3.3. Characterization of Fe3O4@Au composite MNPs
Fig. 2. CD spectra recorded during the titration of 20 mmol L Zn7–MT solution with the molar ratio aliquots of Ag+ at pH 7.5. (a) 0; (b) 3; (c) 6; (d) 12; (e) 18. 1
protein has the shape of a dumbbell and envelops the metals in two separate cluster-structured domains (a-domain and b-domain) [29–31]. According to the literature reported by Li and Otvos [32], in the initial stages of the titration, the Zn2+ in the b-domain was expected to be displaced selectively and cooperatively by Ag+ to produce the metal-hybrid protein species, (Zn4)a(Ag6)b–MT (curve c). Additional Ag+ would then displace the Zn2+ in the a-domain to produce Ag12–MT (curve d). Therefore, the 243 nm positive band was no longer detectable following addition of 12 equivalents of Ag + (curve d and e), indicating that the Zn2+ in MT were all displaced by Ag+. It is noted that the continued addition of Ag+ caused a progressive increase of 293 nm positive band and reached a maximum at 18 equivalents of Ag+ (curve e). The resulting species was so-called “over-metalated” Ag18–MT [27]. Furthermore, the replacement of Zn2+ in MT by Ag+ induced a large absorption change at 260 nm as shown in Fig. S1, which was attributed to the forming of Ag–MT [20].
TEM and EDX were used to observe the morphological characteristics, intrinsic crystal structures and to assay their composition of the as-synthesized MNPs. As shown in Fig. 4A, after coating of the Fe3O4 MNPs with gold shell, the resulting Fe3O4@Au composite MNPs had an average diameter of about 11 nm and were approximately spherical and uniform in size. An absorption peak at 525 nm in Fig. 4B further confirmed the existence of the Au shell [33]. Compared with Fe3O4 MNPs, a higher negative zeta potential (Fig. S2B) imparted by Fe3O4@Au composite MNPs confirmed an amount of gold coverage on the magnetite surface, which was in agreement with the literature [36]. Fig. 4C illustrates XRD patterns of Fe3O4 MNPs (curve a) and Fe3O4@Au composite MNPs (curve b). Compared with curve a, there are other diffraction peaks at 38.4 , 44.2 , 64.2 and 77.6 in curve b, corresponding to the (111), (2 0 0), (2 2 0) and (3 11) planes of the gold crystal with a cubic phase. And it was found that the diffraction from Fe3O4 became weaker in curve b due to the heavy atom effect of Au [34,35]. The above results suggested that composite particles are Fe3O4/Au hybrid nanostructures and their crystalline structure was face-centered cubic with the dominant crystal planes of (111). The lattice fringe spacings of the Fe3O4 core (3 11 plane) and the Au shell (111 plane) were further observed from the HRTEM images in Fig. S3. The magnetic hysteresis loops were therefore obtained to investigate the magnetism of the MNPs in Fig. 4D. The results also showed that the composite MNPs had a superparamagnetic property with lower coercivity. Upon placement of an external magnet beside the vials, the composite MNPs quickly concentrated on the side of the vial within 1 min, leaving the solution transparent (inset). When the magnet was removed, the composite MNPs were well-redispersed again by shaking or ultrasonic vibration, demonstrating that the magnetic remanence (Mr) of the composite MNPs allowed the particles to be easily redispersed in the absence of a magnetic field. The excellent magnetic response of the composite MNPs to a magnetic field facilitated the separation of such particles for their targeted bioseparation and biodetection application. 3.4. Optimization of the coupling FI–CL reaction system
Fig. 3. The SDS-PAGE image of Ag–MT and ssDNA before (1) and after (2) the crosslinking reaction.
Since the quantitative determination of Ag+ was based on the Ag+–K2S2O8–Mn2+–H3PO4–luminol coupling FI–CL reaction system, several parameters were investigated systematically to establish the optimal conditions for the FI–CL reaction (Figs. S4 and S5). In the catalytic reaction, the optimal amounts of the K2S2O8, Mn2+, and H3PO4 were firstly investigated. Considering the CL intensity, 1 mL of 2% (m/V) K2S2O8, 200 mL of 6 mmol L1 MnSO4, and 200 mL of 1:1 (V/V) H3PO4 were selected in this study.
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
71
Fig. 4. Characterization of the MNPs. (A) TEM image and EDX image (inset) of Fe3O4@Au composite MNPs; (B) UV–vis spectrum of Au NPs (a), Fe3O4 MNPs (b) and Fe3O4@Au composite MNPs (c); (C) XRD patterns of Fe3O4 MNPs (curve a) and Fe3O4@Au composite MNPs (curve b); (D) magnetic hysteresis loops of as-synthesized Fe3O4 MNPs (a) and Fe3O4@Au composite MNPs (b) and photographs of the Fe3O4@Au composite MNPs suspension before and after magnetic separation by an external magnet.
Meanwhile, the optimal incubation temperature and incubation time were 85 C and 8 min, respectively. Since the CL signals originated from the reaction of KMnO4–luminol, 1 mol L1 NaOH and 300 mmol L1 luminol were found to be the optimum for the CL reaction. 3.5. Analytical performance As soon as the novel detection probes, ssDNA–(Ag–MT), had been prepared, the hybridization was performed in a sandwich mode, and then the released tags (Ag+) were determined by the coupling FI–CL reaction system under the optimized experimental conditions. As shown in Fig. 5, the CL intensities were linear to the DNA concentrations in the range of 10–500 pmol L1. The regression equation was shown as Eq. (1) with a correlation coefficient of 0.9937. The limit of detection (LOD) of this method was estimated to be 3.3 pmol L1 at the ratio of signal to noise of 3. Such a LOD is lower than those labeled with Zn2+, Cd2+ and Pb2+ measured by square wave anodic stripping voltammetry in our previous work
[16]. Three repetitive measurements were used for estimating the precision, and the relative standard deviation was about 4.5%. ICL ¼ 3:18 c ðpmol L
1
Þ 13:02
(1)
In addition, the selectivity of the method was investigated by using mismatched targets. Fig. 6 are the resulting column graphics of the CL response for the fully complementary target (t1), onebase mismatched target (t2), four-base mismatched target (t3) and noncomplementary target (t4) at the same concentration of 0.5 nmol L1. The ratios of the CL intensities of the four sequences were 100:30:5:2, respectively. The CL response from four-base mismatched strands was significantly smaller (only 5%) than that of the perfectly complementary targets. The results indicated that the CL assay has a high specificity for DNA hybridization, which is very important for many genetic assays in clinical and forensic applications. Therefore, this novel method may be used for practical application. The stability of the detection probes was estimated from the responses to 0.20 nmol L1 human enterovirus-related
72
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73 +
–K2S2O8–H3PO4–luminol, where the Ag+ were dissolved from DNA probe hybridized with target DNA. The CL intensity demonstrated a wide linear relationship with the concentrations of target DNA ranging from 10 to 500 pmol L1, and the detection limit of 3.3 pmol L1 of DNA were obtained. This biosensor with high sensitivity and selectivity may be ascribed to three reasons as followings: (1) the MT molecules has high metal affinity to Ag+ and each MT is capable of binding to 18 Ag+; (2) Ag+ could be sensitively determined by the coupling FI–CL reaction system (Ag+–Mn2 + –K2S2O8–H3PO4–luminol); (3) the perfect magnetic separation based on the Fe3O4@Au composite MNPs avoided the loss of samples during the analytical process effectively. In addition, the main advantage of this method lies in the simple and time-saving. Therefore, it has great potential utility in the clinical applications for DNA detection. Acknowledgment Fig. 5. CL intensities for the targets in different concentration and the resulting calibration plot (inset).
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Nos. 21245004, 21167015, 21365019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.07.033. References
Fig. 6. The column graphics of the CL response of the tags after hybridization with various targets at the same concentration of 0.5 nmol L1. (a) Perfectly matched target (t1), (b) one-base mismatched target (t2), (c) four-base mismatched target (t3) and (d) noncomplementary target (t4).
oligonucletides by using the fresh detection probes and the ones prepared four months ago. It could retain 93.5% of the original value by using the detection probes prepared four months ago. The result showed that the detection probes are good in stability. The simulative real sample analysis containing human enterovirus-related oligonucletides was carried out in 1% human serum. As showed in Fig. S6, we found that the CL intensities were linear to the oligonucletides concentrations in the range of 40–500 pmol L1. The regression equation was shown as Eq. (2) with a correlation coefficient of 0.9979. The limit of detection (LOD) of was estimated to be 13.3 pmol L1 at the ratio of signal to noise of 3. The results indicated that the method has a potential in detecting of human enterovirus-related nucletides directly in biological samples. ICL ¼ 2:90 c ðpmol L
1
Þ 8:57
(2)
4. Conclusions A novel biosensor for ultrasensitive determination of the DNA sequences was developed by detecting the CL intensity of Ag+–Mn2
[1] R.C. dos Santos, C. Kranz, J. Kowalik, H. Yamanaka, B. Mizaikoff, M. Josowicz, Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tipintegrated electrodes, Anal. Chem. 80 (2007) 237–245. [2] K. Klepárník, P. Bo9 cek, DNA diagnostics by capillary electrophoresis, Chem. Rev. 107 (2007) 5279–5317. [3] J.E.N. Dolatabadi, O. Mashinchian, B. Ayoubi, A.A. Jamali, A. Mobed, D. Losic, Y. Omidi, M.D.L. Guardia, Optical and electrochemical DNA nanobiosensors, Trends Anal. Chem. 30 (2011) 459–472. [4] H. Ju, H. Zhao, Electrochemical biosensors for DNA analysis, Front. Biosci. 10 (2005) 37–46. [5] T.G. Drummond, M.G. Hill, J.K. Barton, Electrochemical DNA sensors, Nat. Biotechnol. 21 (2003) 1192–1199. [6] S. Weiss, Fluorescence spectroscopy of single biomolecules, Science 283 (1999) 1676–1683. [7] X. Fang, W. Tan, Imaging single fluorescent molecules at the interface of an optical fiber probe by evanescent wave excitation, Anal. Chem. 71 (1999) 3101– 3105. [8] Y. Zhang, Z. Tang, J. Wang, H. Wu, A. Maham, Y. Lin, Hairpin DNA switch for ultrasensitive spectrophotometric detection of DNA hybridization based on gold nanoparticles and enzyme signal amplification, Anal. Chem. 82 (2010) 6440–6446. [9] Ó.A. Loaiza, E. Jubete, E. Ochoteco, G. Cabañero, H. Grande, J. Rodríguez, Gold coated ferric oxide nanoparticles based disposable magnetic genosensors for the detection of DNA hybridization processes, Biosens. Bioelectron. 26 (2010) 2194–2200. [10] G. Liu, Y. Wan, V. Gau, J. Zhang, L. Wang, S. Song, C. Fan, An enzyme-based EDNA sensor for sequence-specific detection of femtomolar DNA targets, J. Am. Chem. Soc. 130 (2008) 6820–6825. [11] E.S. Mansfield, J.M. Worley, S.E. Mckenzie, S. Surrey, E. Rappaport, P. Fortina, Nucleic acid detection using non-radioactive labelling methods, Mol. Cell. Probe. 9 (1995) 145–156. [12] D. Cui, B. Pan, H. Zhang, F. Gao, R. Wu, J. Wang, R. He, T. Asahi, Self-assembly of quantum dots and carbon nanotubes for ultrasensitive DNA and antigen detection, Anal. Chem. 80 (2008) 7996–8001. [13] M. Han, X. Gao, J.Z. Su, S. Nie, Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules, Nat. Biotechnol. 19 (2001) 631–635. [14] X. Freeman, Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer—substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots, J. Am. Chem. Soc. 133 (11) (2011) 597–11604. [15] J. Wang, G. Liu, A. Merkoçi, Electrochemical coding technology for simultaneous detection of multiple DNA targets, J. Am. Chem. Soc. 125 (2003) 3214– 3215. [16] L. Zheng, X. Li, P. Liu, G. Wu, X. Lu, X. Liu, Simultaneous detection of multiple DNA targets based on encoding metal ions, Biosens. Bioelectron. 52 (2014) 354–359.
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73 [17] Y. Fu, M. Xu, X. Li, M. Du, J. Wang, F. Zhou, Voltammetric studies of cadmiumand zinc-containing metallothioneins at nafion-coated mercury thin film electrodes, Electroanalysis 20 (2008) 888–893. [18] Ò. Palacios, À. Leiva-Presa, S. Atrian, R. Lobinski, A study of the Pb(II) binding to recombinant mouse Zn7–metallothionein 1 and its domains by ESI TOF MS, Talanta 72 (2007) 480–488. [19] D.R. Winge, Copper coordination in metallothionein, Method Enzymol. 205 (1991) 458–469. [20] R. Bofill, Ó. Palacios, M. Capdevila, N. Cols, R. González-Duarte, S. Atrian, P. González-Duarte, A new insight into the Ag+ and Cu+ binding sites in the metallothionein b domain, J. Inorg. Biochem. 73 (1999) 57–64. [21] Y. Jia, Z. Wang, Z. Zhao, Z. Zhang, Determination of trace silver(I) with combined chemiluminescence reaction system, J. Anal. Sci. 4 (1996) 308– 310. [22] M. Mikhaylova, D.K. Kim, C.C. Berry, A. Zagorodni, M. Toprak, A.S.G. Curtis, M. Muhammed, BSA immobilization on amine-functionalized superparamagnetic iron oxide nanoparticles, Chem. Mater. 16 (2004) 2344–2354. _ du, I.H. [23] U. Tamer, Y. Gündog Boyacı, K. Pekmez, Synthesis of magnetic core– shell Fe3O4–Au nanoparticle for biomolecule immobilization and detection, J. Nanopart. Res. 12 (2010) 1187–1196. [24] T.T. Hien Pham, C. Cao, S.J. Sim, Application of citrate-stabilized gold-coated ferric oxide composite nanoparticles for biological separations, J. Magn. Magn. Mater. 320 (2008) 2049–2055. [25] G. Zheng, J. Wu, Z. Chen, Q. Liu, Determination of trace-silver(I) by combined chemiluminescence reaction system, Chinese J. Anal. Chem. 23 (1995) 401– 403. [26] M.J. Stillman, Metallothioneins, Coord. Chem. Rev. 144 (1995) 461–511.
73
[27] Ò. Palacios, K. Polec-Pawlak, R. Lobinski, M. Capdevila, P. González-Duarte, Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies, J. Biol. Inorg. Chem. 8 (2003) 831–842. [28] A.J. Zelazowski, M.J. Stillman, Silver binding to rabbit liver zinc metallothionein and zinc a and b fragments. Formation of silver metallothionein with Ag(I): protein ratios of 6, 12, and 18 observed using circular dichroism spectroscopy, Inorg. Chem. 31 (1992) 3363–3370. [29] E.H. Fischer, E.W. Davie, Recent excitement regarding metallothionein, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 3333–3334. [30] D.E.K. Sutherland, M.J. Stillman, The magic numbers of metallothionein, Metallomics 3 (2011) 444–463. [31] T.T. Ngu, M.J. Stillman, Metal-binding mechanisms in metallothioneins, Dalton Trans. 28 (2009) 5425–5433. [32] H. Li, J.D. Otvos, HPLC characterization of Ag+ and Cu+ metal exchange reactions with Zn- and Cd–metallothioneins, Biochemistry 35 (1996) 13937–13945. [33] Y. Bai, J. Li, J. Xu, H. Chen, Ultrasensitive electrochemical detection of DNA hybridization using Au/Fe3O4 magnetic composites combined with silver enhancement, Analyst 135 (2010) 1672–1679. [34] Z. Cui, L. Jiang, W. Song, Y. Guo, High-yield gas–liquid interfacial synthesis of highly dispersed Fe3O4 nanocrystals and their application in lithium-ion batteries, Chem. Mater. 21 (2009) 1162–1166. [35] X. Zhou, W. Xu, Y. Wang, Q. Kuang, Y. Shi, L. Zhong, Q. Zhang, Fabrication of cluster/shell Fe3O4/Au nanoparticles and application in protein detection via a SERS method, J. Phys. Chem. C 114 (2010) 19607–19613. [36] M. Arsianti, M. Lim, S.N. Lou, L.Y. Goon, C.P. Marquis, R. Amal, Bi-functional gold-coated magnetite composites with improved biocompatibility, J. Colloid Interface Sci. 354 (2011) 536–545.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Ultrasensitive determination of DNA sequences by flow injection chemiluminescence using silver ions as labels Lichun Zheng, Xiuhui Liu *, Min Zhou, Yongjun Ma, Guofan Wu, Xiaoquan Lu ** Key Laboratory of Bioelectrochemistry & Enviromental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, No. 967 Anning East Road, Lanzhou, Gansu 730070, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A novel approach for ultrasensitive FI–CL detection of DNA sequences was developed. A detection probe labeled with Ag+ was synthesized for the first time. The method possesses high sensitivity and selectivity with a detection limit of 3.3 pM.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2014 Received in revised form 17 July 2014 Accepted 22 July 2014 Available online 24 July 2014
We presented a new strategy for ultrasensitive detection of DNA sequences based on the novel detection probe which was labeled with Ag+ using metallothionein (MT) as a bridge. The assay relied on a sandwich-type DNA hybridization in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on Fe3O4@Au composite magnetic nanoparticles (MNPs), and then the Ag+-modified detection probes were used to monitor the presence of the specific DNA targets. After being anchored on the hybrids, Ag+ was released down through acidic treatment and sensitively determined by a coupling flow injection–chemiluminescent reaction system (Ag+–Mn2+–K2S2O8–H3PO4– luminol) (FI–CL). The experiment results showed that the CL intensities increased linearly with the concentrations of DNA targets in the range from 10 to 500 pmol L1 with a detection limit of 3.3 pmol L1. The high sensitivity in this work may be ascribed to the high molar ratio of Ag+–MT, the sensitive determination of Ag+ by the coupling FI–CL reaction system and the perfect magnetic separation based on Fe3O4@Au composite MNPs. Moreover, the proposed strategy exhibited excellent selectivity against the mismatched DNA sequences and could be applied to real samples analysis. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Nucleic acid biosensors Flow injection–chemiluminescent detection Metallothioneins Silver ions Magnetic nanoparticles
1. Introduction The detection of DNA sequences has received a huge interest in the fields of clinical diagnosis, genetics therapy, and a variety of biomedical studies recently [1,2]. Thus, there has been an
* Corresponding author. Tel.: +86 931 7972366; fax: +86 931 7971323. ** Corresponding author. Tel.: +86 931 7971276; fax: +86 931 7971276. E-mail addresses: [email protected], [email protected] (X. Liu), [email protected] (X. Lu). http://dx.doi.org/10.1016/j.aca.2014.07.033 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
increasing demand to develop a simple and sensitive method in order to detect the specific oligonucleotide sequence. As well known, various techniques had been developed for detection of DNA hybridization, and their sensitivities depended mainly on the specific activity of the labels which linked to the oligonucleotide probes [3–5]. Dyes, enzymes, and quantum dots (QDs) are the most popular labels for DNA analysis. Dyes such as cyanine 5 (Cy5), carboxyfluorescein (FAM), and tetramethyl-6-carboxy-rhodamine (TAMRA) are the classical fluorophores which have been widely used in DNA analysis. But the quantitative measurements remain a challenge because of low fluorescence intensities and
68
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
susceptibility to photobleaching [6,7]. Other detection techniques which employed enzymes to generate spectrophotometric [8], electrochemical [9,10], or chemiluminescent [11] signals had achieved good linearity and high sensitivity. However, they were also hampered because the activity of the enzyme was destroyed easily by its environment conditions (temperature, pH value, etc.). In recent years, QDs have been used as labels for the detection of DNA due to theirs unique florescent [12,13] and chemiluminescent [14] properties. In addition, the labels, QDs, can be dissolved in acidic solution to produce a large number of corresponding metal ions which will yield well-resolved highly sensitive stripping voltammetric signals for the targets [15]. But the preparation of QDs is often a time-consuming work and requires harsh conditions. More important, it is difficult to control their sizes. In previous work [16], we determined three different DNA targets simultaneously based on the usage of different encoding metal ions as tags, in which the novel detection probes, ssDNA/MT conjugates, covered with different metal ions (Zn2+, Cd2+, and Pb2+) were synthesized. Then the encoding metal ions were used to differentiate the signals of three kinds of virus DNA. The proposed method was proved to be simple and sensitive. Previously, other researchers found that MT is able to bind more monovalent metal ions (Cu+, Ag+, etc.) than bivalent metal ions (Zn2+, Cd2+, Pb2+, etc.) [17–20]. Therefore, in this paper, we proposed a strategy to prepare a new detection probe labeled with Ag+, and detected it by the coupling CL reaction (Ag+–Mn2+–K2S2O8–H3PO4–luminol) [21] equipping with the FI–CL system. Meanwhile, as-prepared Fe3O4@Au composite magnetic nanoparticles (MNPs) were used as substrate to immobile the capture probes. Therefore, it is believed that the proposed method possesses great potential for ultrasensitive detection of DNA hybridization. 2. Experimental 2.1. Materials The Zn7–MT from rabbit liver was purchased from Botai Biotech (Dalian, China). Succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate (SMCC) came from Pierce. 1-Hexanethiol (MCH) was acquired from Aladdin (Shanghai, China). HAuCl4, FeCl36H2O, FeCl24H2O, tetramethylammonium hydroxide (TMOH), dimethyl sulfoxide (DMSO), AgNO3, NaOH, Tris(hydroxymethyl)aminomethane (Tris), NaCl, NaH2PO4 and Na2HPO4 were all purchased from Beijing Chemical Reagents Co. (Beijing, China). Other chemicals employed were all of analytical grade, and Milli-Q water was used in all experiments. All oligonucletides were provided by Sangon (Shanghai, China). The capture probes were thiolated with a (CH2)3 spacer at the 30 ends and the detection probes were modified with amino groups with a (CH2)6 spacer at the 5C ends. Their base sequences were: Human enterovirus-related oligonucletides: Capture probe: 50 -CAGACACTGTTGGTA-(CH2)6-SH-30 Detection probe: 50 -H2N-(CH2)6-GAATAGCGTCAGAAT-30 Complementary target (t1): 50 -TACCAACAGTGTCTGATTCTGACGCTATTC-30 One-base mismatched target (t2): 50 -TACCGACAGTGTCTGATTCTGACGCTATTC-30 Four-base mismatched target (t3): 50 -TACCGGAACTGTCTGATTCTGACGCTATTC-30 Noncomplementary target (t4): 50 -GCCGCGTGTTTTAGCACATAGCGATGTCAC-30 2.2. Apparatus A Jasco spectropolarimeter (model J-715) connected to a computer was used for CD measurements. The UV–vis absorption
spectra were recorded on a UV-1102 UV–vis spectrophotometer. The transmission electron microscope (TEM) and energy dispersive X-ray (EDX) images were acquired from a FEI TF20 field emission transmission electron microscope. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at the SE 400 vertical unit (GE Healthcare). The zeta potential of the samples was determined on a Nano-ZS Zetasizer ZEN3600 dynamic light scattering instrument. The magnetization was measured by a 7304 vibrating sample magnetometer. The composition and crystal line structure of the samples were analyzed on a Philips X’pert multipurpose X-ray diffraction system. The CL signals were measured by an IFFL-D analyzer. 2.3. Procedures 2.3.1. Metal ion binding reactions The lyophilized Zn7–MT was firstly dissolved in 0.02 mol L1 Tris– HClO4 buffer at pH 7.4. Metal binding experiments were carried out by sequentially adding molar ratio aliquots of Ag+ (using AgNO3) and allowed to incubate for 30 min at room temperature. All manipulations involving the metal ions and protein solutions were performed in a nitrogen atmosphere. Circular dichroism (CD) spectroscopy and ultraviolet–visible (UV–vis) absorption spectroscopy were used to monitor the binding reaction of Ag+ to the protein. The products (Ag–MT) were purified by a centricon-3 centrifugal concentrator to remove any unbound Ag+. 2.3.2. Conjugation of Ag–MT to NH2–ssDNA SMCC, the heterobifunctional crosslinker, was firstly dissolved in DMSO, and then diluted with water immediately prior to use to a final concentration of SMCC of 5%. To prevent the hydrolytic degradation of the NHS ester, the conjugation was usually performed at pH 7.3. Phosphate-buffered saline (PBS = 0.1 mol L1 sodium phosphate, 0.15 mol L1 sodium chloride, pH 7.4) and the amine- and sulfhydryl-free buffer were used as conjugation buffer. A 40-fold molar of crosslinker over the amount of MT was used to react simultaneously with MT and excess NH2–ssDNA for 1 h at room temperature. The unreacted SMCC and NH2–ssDNA were removed from the reaction product by ultrafiltration. The conjugation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the polyacrylamide gel was 18%. The electrophoresis started with an initial voltage of 30 V, then maintained at this voltage until the sample had completely entered the stacking gel. Finally, the voltage applied 80 V for 3.5 h. After electrophoresis, the gel was treated with 0.1% AgNO3, and then developed with 1.5% NaOH and 0.4% formaldehyde. 2.3.3. Preparation of Fe3O4@Au composite MNPs The Fe3O4 MNPs were prepared by coprecipitation according to a method mentioned in the previous reports [22,23]. Briefly, 5.4 g of FeCl36H2O and 2.0 g of FeCl24H2O were dissolved in 10 mL of 0.4 mol L1 HCl solution under vigorous stirring. The coprecipitation of Fe3O4 MNPs was carried out in a three-neck round-bottom flask and protected under a pure N2 atmosphere. The above mixture solution was added dropwise to 125 mL of 0.5 mol L1 NaOH, which was preheated to 80 C before the coprecipitation reaction. The reaction was vigorously stirred for about 30 min, resulting in a pale yellow color solution which finally turned into dark black. The black precipitate was collected by sedimentation with the help of an external magnetic field and washed gradually with 0.1 mol L1 HNO3 and 0.01 mol L1 HNO3. To obtain oxidized Fe3O4 MNPs, the particles were then dissolved in 0.01 mol L1 HNO3 and heated at 90 C with stirring until the color of solution became brown. The oxidized Fe3O4 MNPs were suspended in 50 mL of 0.1 mol L1 TMOH at pH 11 after washing with water.
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
Au-shell coating was performed by reduction of Au3+ on the surface of Fe3O4 MNPs using a modification of previous procedure [24]. Firstly, 5 mL of N(CH3)4-stabilized, oxidized Fe3O4 MNPs solution were added to 50 mL of 0.01 mol L1 sodium citrate and stirred for 30 min to exchange absorbed OH with citrate ions to make the magnetic-core solution. Next, the solution was ultrasonicated for 15 min and then heated to boiling with vigorous stirring. Then, 1% HAuCl4 (2 mL) was added as soon as the reaction solution reached the boiling point. The mixture was boiled for 15 min and stirred for an additional 15 min while cooling. The obtained Fe3O4@Au composite MNPs were magnetically separated from the Au NPs formed during the process. 2.3.4. Hybridization procedures 30 mL of Fe3O4@Au composite MNPs (8 mg mL1) were firstly transferred into a 1.5 mL centrifuge vial, and then washed twice with buffer A (0.01 mol L1 PBS, 0.2 mol L1 NaCl, pH 7.4) by magnetic separation and resuspended in 200 mL of buffer A containing 10 mmol L1 of thiolated capture probes by shaking. The solution was incubated overnight at room temperature with gentle shaking. The unbound DNA sequences were washed away with the same buffer, supernatant removal. Subsequently, the capture probes-attached composite MNPs were resuspended in buffer A containing 0.1 mmol L1 MCH solution and incubated for 2 h. Then, the capture probes/MCH-attached composite MNPs were washed three times with water and resuspended in 50 mL of water before use. The sandwich assay involved a dual hybridization event. Corresponding amounts of the targets were added into 100 mL of buffer B (0.01 mol L1 PBS, 0.15 mol L1 NaCl, 0.1% Tween 20, pH 7.4) containing 5 mL of the capture probes/MCHattached composite MNPs solution. After hybridization for 2 h at 37 C, the resulting composite MNPs were separated magnetically from the solution and washed twice with buffer B. And then the composite MNPs were resuspended in 100 mL of buffer B containing 5 mL of the as-prepared detection probes and incubated for 2 h at 37 C. The obtained composite MNPs were washed three times with water. Afterward, release of the Ag+ from MT molecules was happened in 100 mL of 1 mol L1 HCl for 10 min. At last, the FI–CL detection of Ag+ was performed according to the standard procedures below. 2.3.5. Standard procedures for FI–CL detection The pH value of the acid solution containing Ag+ was adjusted to be neutral by adding 5 mol L1 NaOH, and the resultant mixture was then transferred into a 5 mL beaker containing 1 mL of 2% (m/ v) K2S2O8, 200 mL of 6 mmol L1 MnSO4, and 200 mL of 1:1 (v/v)
69
H3PO4 and immediately incubated in a 85 C water bath for 8 min; thus, the resultant KMnO4 was produced. The reaction was stopped with flowing cold water, and the solution was diluted to 10 mL. The signals of luminol–KMnO4 CL system were measured by an FI–CL analyzer. 3. Results and discussion 3.1. The fabrication of the biosensor and the detection process The principle of the protocol presented in this work is shown in Fig. 1. At first, the Fe3O4@Au composite MNPs with core–shell structure were synthesized by reduction of Au3+ in the presence of Fe3O4 NPs (A). Secondly, the thiolated capture probes were immobilized on the composite MNPs through the Au S bands (B). Then, the novel detection probes which consist of ssDNA and MT molecules covered with Ag+ were prepared and hybridized with the targets in a sandwich mode (C and D). After hybridization, the Ag+ was released from MT in the acid solution (E). Finally, the released Ag+ was sensitively determined by the coupling CL reaction (Ag+–Mn2+–K2S2O8–H3PO4–luminol) equipped with the FI–CL system (F and G). This coupling CL reaction was based on the strong catalytic effect of Ag+ on the reaction of Mn2+–K2S2O8– H3PO4 [25]. The catalytically produced KMnO4 could further oxidize luminol in an alkaline medium, which would generate a strong CL signal, and the CL intensities are proportional to the amount of DNA targets. The originality of this design is that MT is used as a bridge to combine ssDNA and Ag+. MT is a small metalloprotein characterized by its high cysteine content (approximately 30% of its amino acid sequence), absence of disulfide bonds, and lack of aromatic amino acids, particularly its high metal affinity [26]. Previous research found that 20 cysteine thiolates of each MT are capable of binding to 18 Ag+ [27,28]. 3.2. Characterization of the detection probes 3.2.1. Metal ion binding reactions CD spectra were used to characterize the formation of a series of complexes between Ag+ and the cysteine thiolate groups in zinc MT. As shown in Fig. 2 (curve a), one can see that there was only one positive band near 243 nm which was characteristic of Zn7–MT [28] before Ag+ was added. After titration with Ag+, the 243 nm positive band gradually diminished in intensity while a negative band near 265 nm and a positive band near 293 nm gradually intensified as shown in curve b–d, indicating that the Zn2+ in MT was displaced by Ag+ to form the new species. As we know, the MT
Fig. 1. Schematic representation of the protocol: (A) formation of the Fe3O4@Au composite MNPs; (B) modification with the thiolated probes; (C) hybridization with the targets; (D) hybridization with the detection probes; (E) release of Ag+; (F and G) the coupling CL reaction equipped with the FI–CL system.
70
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
3.2.2. Conjugation of Ag–MT to NH2–ssDNA The conjugation of Ag–MT to NH2–ssDNA was performed as our previous work [16]. Briefly, ssDNA was synthesized first to contain a 30 -aminohexyl linker at the end. Then, the heterobifunctional crosslinking reagent (SMCC) was used as a bridge to combine NH2– ssDNA and Ag–MT. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to assess the conjugation of MT to ssDNA. As shown in Fig. 3, one can see that there are two bands in lane 1 before the crosslinking reaction. However, after the crosslinking reaction, there is only one band in lane 2. Compared to the bands in lane 1, the band in lane 2 showed the lower mobility due to its higher molecular weight after the crosslinking reaction. The result gives the immediate evidence for the production of the (Ag–MT)–ssDNA conjugates (namely the detection probe). 3.3. Characterization of Fe3O4@Au composite MNPs
Fig. 2. CD spectra recorded during the titration of 20 mmol L Zn7–MT solution with the molar ratio aliquots of Ag+ at pH 7.5. (a) 0; (b) 3; (c) 6; (d) 12; (e) 18. 1
protein has the shape of a dumbbell and envelops the metals in two separate cluster-structured domains (a-domain and b-domain) [29–31]. According to the literature reported by Li and Otvos [32], in the initial stages of the titration, the Zn2+ in the b-domain was expected to be displaced selectively and cooperatively by Ag+ to produce the metal-hybrid protein species, (Zn4)a(Ag6)b–MT (curve c). Additional Ag+ would then displace the Zn2+ in the a-domain to produce Ag12–MT (curve d). Therefore, the 243 nm positive band was no longer detectable following addition of 12 equivalents of Ag + (curve d and e), indicating that the Zn2+ in MT were all displaced by Ag+. It is noted that the continued addition of Ag+ caused a progressive increase of 293 nm positive band and reached a maximum at 18 equivalents of Ag+ (curve e). The resulting species was so-called “over-metalated” Ag18–MT [27]. Furthermore, the replacement of Zn2+ in MT by Ag+ induced a large absorption change at 260 nm as shown in Fig. S1, which was attributed to the forming of Ag–MT [20].
TEM and EDX were used to observe the morphological characteristics, intrinsic crystal structures and to assay their composition of the as-synthesized MNPs. As shown in Fig. 4A, after coating of the Fe3O4 MNPs with gold shell, the resulting Fe3O4@Au composite MNPs had an average diameter of about 11 nm and were approximately spherical and uniform in size. An absorption peak at 525 nm in Fig. 4B further confirmed the existence of the Au shell [33]. Compared with Fe3O4 MNPs, a higher negative zeta potential (Fig. S2B) imparted by Fe3O4@Au composite MNPs confirmed an amount of gold coverage on the magnetite surface, which was in agreement with the literature [36]. Fig. 4C illustrates XRD patterns of Fe3O4 MNPs (curve a) and Fe3O4@Au composite MNPs (curve b). Compared with curve a, there are other diffraction peaks at 38.4 , 44.2 , 64.2 and 77.6 in curve b, corresponding to the (111), (2 0 0), (2 2 0) and (3 11) planes of the gold crystal with a cubic phase. And it was found that the diffraction from Fe3O4 became weaker in curve b due to the heavy atom effect of Au [34,35]. The above results suggested that composite particles are Fe3O4/Au hybrid nanostructures and their crystalline structure was face-centered cubic with the dominant crystal planes of (111). The lattice fringe spacings of the Fe3O4 core (3 11 plane) and the Au shell (111 plane) were further observed from the HRTEM images in Fig. S3. The magnetic hysteresis loops were therefore obtained to investigate the magnetism of the MNPs in Fig. 4D. The results also showed that the composite MNPs had a superparamagnetic property with lower coercivity. Upon placement of an external magnet beside the vials, the composite MNPs quickly concentrated on the side of the vial within 1 min, leaving the solution transparent (inset). When the magnet was removed, the composite MNPs were well-redispersed again by shaking or ultrasonic vibration, demonstrating that the magnetic remanence (Mr) of the composite MNPs allowed the particles to be easily redispersed in the absence of a magnetic field. The excellent magnetic response of the composite MNPs to a magnetic field facilitated the separation of such particles for their targeted bioseparation and biodetection application. 3.4. Optimization of the coupling FI–CL reaction system
Fig. 3. The SDS-PAGE image of Ag–MT and ssDNA before (1) and after (2) the crosslinking reaction.
Since the quantitative determination of Ag+ was based on the Ag+–K2S2O8–Mn2+–H3PO4–luminol coupling FI–CL reaction system, several parameters were investigated systematically to establish the optimal conditions for the FI–CL reaction (Figs. S4 and S5). In the catalytic reaction, the optimal amounts of the K2S2O8, Mn2+, and H3PO4 were firstly investigated. Considering the CL intensity, 1 mL of 2% (m/V) K2S2O8, 200 mL of 6 mmol L1 MnSO4, and 200 mL of 1:1 (V/V) H3PO4 were selected in this study.
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73
71
Fig. 4. Characterization of the MNPs. (A) TEM image and EDX image (inset) of Fe3O4@Au composite MNPs; (B) UV–vis spectrum of Au NPs (a), Fe3O4 MNPs (b) and Fe3O4@Au composite MNPs (c); (C) XRD patterns of Fe3O4 MNPs (curve a) and Fe3O4@Au composite MNPs (curve b); (D) magnetic hysteresis loops of as-synthesized Fe3O4 MNPs (a) and Fe3O4@Au composite MNPs (b) and photographs of the Fe3O4@Au composite MNPs suspension before and after magnetic separation by an external magnet.
Meanwhile, the optimal incubation temperature and incubation time were 85 C and 8 min, respectively. Since the CL signals originated from the reaction of KMnO4–luminol, 1 mol L1 NaOH and 300 mmol L1 luminol were found to be the optimum for the CL reaction. 3.5. Analytical performance As soon as the novel detection probes, ssDNA–(Ag–MT), had been prepared, the hybridization was performed in a sandwich mode, and then the released tags (Ag+) were determined by the coupling FI–CL reaction system under the optimized experimental conditions. As shown in Fig. 5, the CL intensities were linear to the DNA concentrations in the range of 10–500 pmol L1. The regression equation was shown as Eq. (1) with a correlation coefficient of 0.9937. The limit of detection (LOD) of this method was estimated to be 3.3 pmol L1 at the ratio of signal to noise of 3. Such a LOD is lower than those labeled with Zn2+, Cd2+ and Pb2+ measured by square wave anodic stripping voltammetry in our previous work
[16]. Three repetitive measurements were used for estimating the precision, and the relative standard deviation was about 4.5%. ICL ¼ 3:18 c ðpmol L
1
Þ 13:02
(1)
In addition, the selectivity of the method was investigated by using mismatched targets. Fig. 6 are the resulting column graphics of the CL response for the fully complementary target (t1), onebase mismatched target (t2), four-base mismatched target (t3) and noncomplementary target (t4) at the same concentration of 0.5 nmol L1. The ratios of the CL intensities of the four sequences were 100:30:5:2, respectively. The CL response from four-base mismatched strands was significantly smaller (only 5%) than that of the perfectly complementary targets. The results indicated that the CL assay has a high specificity for DNA hybridization, which is very important for many genetic assays in clinical and forensic applications. Therefore, this novel method may be used for practical application. The stability of the detection probes was estimated from the responses to 0.20 nmol L1 human enterovirus-related
72
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73 +
–K2S2O8–H3PO4–luminol, where the Ag+ were dissolved from DNA probe hybridized with target DNA. The CL intensity demonstrated a wide linear relationship with the concentrations of target DNA ranging from 10 to 500 pmol L1, and the detection limit of 3.3 pmol L1 of DNA were obtained. This biosensor with high sensitivity and selectivity may be ascribed to three reasons as followings: (1) the MT molecules has high metal affinity to Ag+ and each MT is capable of binding to 18 Ag+; (2) Ag+ could be sensitively determined by the coupling FI–CL reaction system (Ag+–Mn2 + –K2S2O8–H3PO4–luminol); (3) the perfect magnetic separation based on the Fe3O4@Au composite MNPs avoided the loss of samples during the analytical process effectively. In addition, the main advantage of this method lies in the simple and time-saving. Therefore, it has great potential utility in the clinical applications for DNA detection. Acknowledgment Fig. 5. CL intensities for the targets in different concentration and the resulting calibration plot (inset).
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Nos. 21245004, 21167015, 21365019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.07.033. References
Fig. 6. The column graphics of the CL response of the tags after hybridization with various targets at the same concentration of 0.5 nmol L1. (a) Perfectly matched target (t1), (b) one-base mismatched target (t2), (c) four-base mismatched target (t3) and (d) noncomplementary target (t4).
oligonucletides by using the fresh detection probes and the ones prepared four months ago. It could retain 93.5% of the original value by using the detection probes prepared four months ago. The result showed that the detection probes are good in stability. The simulative real sample analysis containing human enterovirus-related oligonucletides was carried out in 1% human serum. As showed in Fig. S6, we found that the CL intensities were linear to the oligonucletides concentrations in the range of 40–500 pmol L1. The regression equation was shown as Eq. (2) with a correlation coefficient of 0.9979. The limit of detection (LOD) of was estimated to be 13.3 pmol L1 at the ratio of signal to noise of 3. The results indicated that the method has a potential in detecting of human enterovirus-related nucletides directly in biological samples. ICL ¼ 2:90 c ðpmol L
1
Þ 8:57
(2)
4. Conclusions A novel biosensor for ultrasensitive determination of the DNA sequences was developed by detecting the CL intensity of Ag+–Mn2
[1] R.C. dos Santos, C. Kranz, J. Kowalik, H. Yamanaka, B. Mizaikoff, M. Josowicz, Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tipintegrated electrodes, Anal. Chem. 80 (2007) 237–245. [2] K. Klepárník, P. Bo9 cek, DNA diagnostics by capillary electrophoresis, Chem. Rev. 107 (2007) 5279–5317. [3] J.E.N. Dolatabadi, O. Mashinchian, B. Ayoubi, A.A. Jamali, A. Mobed, D. Losic, Y. Omidi, M.D.L. Guardia, Optical and electrochemical DNA nanobiosensors, Trends Anal. Chem. 30 (2011) 459–472. [4] H. Ju, H. Zhao, Electrochemical biosensors for DNA analysis, Front. Biosci. 10 (2005) 37–46. [5] T.G. Drummond, M.G. Hill, J.K. Barton, Electrochemical DNA sensors, Nat. Biotechnol. 21 (2003) 1192–1199. [6] S. Weiss, Fluorescence spectroscopy of single biomolecules, Science 283 (1999) 1676–1683. [7] X. Fang, W. Tan, Imaging single fluorescent molecules at the interface of an optical fiber probe by evanescent wave excitation, Anal. Chem. 71 (1999) 3101– 3105. [8] Y. Zhang, Z. Tang, J. Wang, H. Wu, A. Maham, Y. Lin, Hairpin DNA switch for ultrasensitive spectrophotometric detection of DNA hybridization based on gold nanoparticles and enzyme signal amplification, Anal. Chem. 82 (2010) 6440–6446. [9] Ó.A. Loaiza, E. Jubete, E. Ochoteco, G. Cabañero, H. Grande, J. Rodríguez, Gold coated ferric oxide nanoparticles based disposable magnetic genosensors for the detection of DNA hybridization processes, Biosens. Bioelectron. 26 (2010) 2194–2200. [10] G. Liu, Y. Wan, V. Gau, J. Zhang, L. Wang, S. Song, C. Fan, An enzyme-based EDNA sensor for sequence-specific detection of femtomolar DNA targets, J. Am. Chem. Soc. 130 (2008) 6820–6825. [11] E.S. Mansfield, J.M. Worley, S.E. Mckenzie, S. Surrey, E. Rappaport, P. Fortina, Nucleic acid detection using non-radioactive labelling methods, Mol. Cell. Probe. 9 (1995) 145–156. [12] D. Cui, B. Pan, H. Zhang, F. Gao, R. Wu, J. Wang, R. He, T. Asahi, Self-assembly of quantum dots and carbon nanotubes for ultrasensitive DNA and antigen detection, Anal. Chem. 80 (2008) 7996–8001. [13] M. Han, X. Gao, J.Z. Su, S. Nie, Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules, Nat. Biotechnol. 19 (2001) 631–635. [14] X. Freeman, Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer—substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots, J. Am. Chem. Soc. 133 (11) (2011) 597–11604. [15] J. Wang, G. Liu, A. Merkoçi, Electrochemical coding technology for simultaneous detection of multiple DNA targets, J. Am. Chem. Soc. 125 (2003) 3214– 3215. [16] L. Zheng, X. Li, P. Liu, G. Wu, X. Lu, X. Liu, Simultaneous detection of multiple DNA targets based on encoding metal ions, Biosens. Bioelectron. 52 (2014) 354–359.
L. Zheng et al. / Analytica Chimica Acta 848 (2014) 67–73 [17] Y. Fu, M. Xu, X. Li, M. Du, J. Wang, F. Zhou, Voltammetric studies of cadmiumand zinc-containing metallothioneins at nafion-coated mercury thin film electrodes, Electroanalysis 20 (2008) 888–893. [18] Ò. Palacios, À. Leiva-Presa, S. Atrian, R. Lobinski, A study of the Pb(II) binding to recombinant mouse Zn7–metallothionein 1 and its domains by ESI TOF MS, Talanta 72 (2007) 480–488. [19] D.R. Winge, Copper coordination in metallothionein, Method Enzymol. 205 (1991) 458–469. [20] R. Bofill, Ó. Palacios, M. Capdevila, N. Cols, R. González-Duarte, S. Atrian, P. González-Duarte, A new insight into the Ag+ and Cu+ binding sites in the metallothionein b domain, J. Inorg. Biochem. 73 (1999) 57–64. [21] Y. Jia, Z. Wang, Z. Zhao, Z. Zhang, Determination of trace silver(I) with combined chemiluminescence reaction system, J. Anal. Sci. 4 (1996) 308– 310. [22] M. Mikhaylova, D.K. Kim, C.C. Berry, A. Zagorodni, M. Toprak, A.S.G. Curtis, M. Muhammed, BSA immobilization on amine-functionalized superparamagnetic iron oxide nanoparticles, Chem. Mater. 16 (2004) 2344–2354. _ du, I.H. [23] U. Tamer, Y. Gündog Boyacı, K. Pekmez, Synthesis of magnetic core– shell Fe3O4–Au nanoparticle for biomolecule immobilization and detection, J. Nanopart. Res. 12 (2010) 1187–1196. [24] T.T. Hien Pham, C. Cao, S.J. Sim, Application of citrate-stabilized gold-coated ferric oxide composite nanoparticles for biological separations, J. Magn. Magn. Mater. 320 (2008) 2049–2055. [25] G. Zheng, J. Wu, Z. Chen, Q. Liu, Determination of trace-silver(I) by combined chemiluminescence reaction system, Chinese J. Anal. Chem. 23 (1995) 401– 403. [26] M.J. Stillman, Metallothioneins, Coord. Chem. Rev. 144 (1995) 461–511.
73
[27] Ò. Palacios, K. Polec-Pawlak, R. Lobinski, M. Capdevila, P. González-Duarte, Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies, J. Biol. Inorg. Chem. 8 (2003) 831–842. [28] A.J. Zelazowski, M.J. Stillman, Silver binding to rabbit liver zinc metallothionein and zinc a and b fragments. Formation of silver metallothionein with Ag(I): protein ratios of 6, 12, and 18 observed using circular dichroism spectroscopy, Inorg. Chem. 31 (1992) 3363–3370. [29] E.H. Fischer, E.W. Davie, Recent excitement regarding metallothionein, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 3333–3334. [30] D.E.K. Sutherland, M.J. Stillman, The magic numbers of metallothionein, Metallomics 3 (2011) 444–463. [31] T.T. Ngu, M.J. Stillman, Metal-binding mechanisms in metallothioneins, Dalton Trans. 28 (2009) 5425–5433. [32] H. Li, J.D. Otvos, HPLC characterization of Ag+ and Cu+ metal exchange reactions with Zn- and Cd–metallothioneins, Biochemistry 35 (1996) 13937–13945. [33] Y. Bai, J. Li, J. Xu, H. Chen, Ultrasensitive electrochemical detection of DNA hybridization using Au/Fe3O4 magnetic composites combined with silver enhancement, Analyst 135 (2010) 1672–1679. [34] Z. Cui, L. Jiang, W. Song, Y. Guo, High-yield gas–liquid interfacial synthesis of highly dispersed Fe3O4 nanocrystals and their application in lithium-ion batteries, Chem. Mater. 21 (2009) 1162–1166. [35] X. Zhou, W. Xu, Y. Wang, Q. Kuang, Y. Shi, L. Zhong, Q. Zhang, Fabrication of cluster/shell Fe3O4/Au nanoparticles and application in protein detection via a SERS method, J. Phys. Chem. C 114 (2010) 19607–19613. [36] M. Arsianti, M. Lim, S.N. Lou, L.Y. Goon, C.P. Marquis, R. Amal, Bi-functional gold-coated magnetite composites with improved biocompatibility, J. Colloid Interface Sci. 354 (2011) 536–545.
