View Article Online View Journal
Analyst Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: G. Jie, Y. Qin, Q. Meng and J. Wang, Analyst, 2014, DOI: 10.1039/C4AN01465K.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/analyst
Page 1 of 4
Analyst View Article Online
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
206x127mm (150 x 150 DPI)
Analyst Accepted Manuscript
DOI: 10.1039/C4AN01465K
Analyst
Page 2 of 4
CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
View Article Online
ARTICLE TYPE
Autocatalytic Amplified Detection of DNA Based on CdSe Quantum Dots/Folic Acid Electrochemiluminescence Energy Transfer System Guifen Jie*, Yingqiang Qin, Qingmin Meng, Jialin Wang* 5
10
15
20
25
30
35
40
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X First published on the web Xth XXXXXXXXX 200X DOI: 10.1039/b000000x
In this work, electrochemiluminescence energy transfer (ECL-ET) from CdSe quantum dots (QDs) to folic acid was firstly reported, and applied to amplified detection of DNA by using a novel DNAzyme-mediated autocatalytic system. The development of DNA biosensors capable of amplified detection of DNA is being greatly motivated by its potential applications in molecular diagnostics, forensic investigations, genetics therapy, and biomedical development,1 and numerous electrical,2 optical,3 or microgravimetric4 amplified DNA sensors have been reported. Different catalysts, such as enzymes,5 catalytic nucleic acids (DNAzymes),6 or metal nanoparticles (NPs),7 were used for the amplified detection of DNA. Amplified DNA detection was accomplished by an autocatalytic and catabolic DNAzyme-mediated process.8 The easy synthetic preparation of DNAzymes and the reduced nonspecific adsorption of DNAzymes make catalytic nucleic acids attractive reporter units. The use of DNA-based machines has recently been suggested as a means to stimulate the autonomous replication of a catalytic reporter as the result of DNA sensing.9 A further approach for amplifying the DNA detection has included the use of the Zn2+-dependent ligation DNAzyme and the isothermal autonomous synthesis of the ligation DNAzyme units, as a result of the DNA recognition event.10 Electrochemiluminescence (ECL) as a promising tool has attracted much attention in sensing and detecting trace amounts of samples due to its high sensitivity, wide dynamic concentration response range, as well as its potential and spatial controlment.11−12 Recent research demonstrated that QDs are promising ECL tags for selective determination of macromolecules in real samples.13 The marriage of energy transfer with electrochemiluminescence has produced a new technology named electrochemiluminescence energy transfer (ECL-ET), which can realize effective and sensitive detection of biomolecules.14 To obtain optimal ECL-ET efficiency, perfect energy overlapped donor/acceptor pair is of great importance. Notably, the tunable emission wavelength and broad absorption
55
60
65
70
75
80
spectra of QDs render them ideal active donor or acceptor for ECL-ET. 14 Considerable works have been done in NCs ECL resonance energy transfer (RET). Xu and coworkers first demonstrated that the ECL from manganese-doped CdS NCs could be quenched by proximal Au NPs, such ECL energy transfer platform allowed DNA detection.14 Subsequently, QDs-based ECL energy transfer assays for DNA detection,15 cytosensing,16 aptamer,17 and immunoassay have been further explored.18 However, most of the ECL-ET assays used gold nanoparticles as the quencher, which has limits in biolabeling due to the steric hindrance to the electrode. Folic acid as a micromolecule was reported to be acted as an effective quencher for fluorescence.19 Furthermore, DNAzymes as enzyme mimics have attracted considerable attention in ECL techniques due to their high catalytic activity and good stability.20 So far, the QDs-based ECL-ET assays using folic acid as quencher combined with cyclic amplification technique has not been reported. Herenin, we present a novel strategy on DNAzyme-mediated autocatalytic amplified detection of DNA based on electrochemiluminescence energy transfer (ECL-ET) from CdSe quantum dots to folic acid. Characterization of the CdSe QDs. Figure 1A showed the transmission electron microscopy (TEM) image of the CdSe QDs, the nanoparticles possessed uniform morphology and size, and the average diameter was about 5~6 nm. Figure 1B presents the photoluminescence (PL) spectra of the CdSe QDs, the PL emission peak of the QDs was at 650 nm, and the PL intensity was high, indicating that the CdSe QDs possess good luminescent property and has promising optical applications.
85
*Corresponding author: Key Laboratory of Sensor Analysis of Tumor
45
50
Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China. E-mail: guifenjie @126.com; Email:[email protected] † Electronic Supplementary Information (ESI) available: Experimental details, UV-vis, TEM and the optimization and selectivity are included in the supporting information.and additional figures. See DOI: 10.1039/b000000x/
This journal is © The Royal Society of Chemistry [year]
Figure 1. Representative TEM image (A), and PL spectra (B) of the CdSe QDs,
90
ECL behavior of the CdSe quantum dots. The ECL– potential curve and cyclic voltammogram (CV) of the CdSe quantum dots were shown in Figure 2. A cathodic peak at –1.05 V is observed in the CV response, corresponding to the reduction of S2O82−. There is an ECL peak at –1.40 V in the ECL–potential curve, which results from the reaction of the CdSe QDs with
Journal Name, [year], [vol], 00–00 | 1
Analyst Accepted Manuscript
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
DOI: 10.1039/C4AN01465K www.rsc.org/xxxxxx | XXXXXXXX
Page 3 of 4
Analyst View Article Online
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
S2O82-. The possible ECL mechanisms are as follows. The QDs are reduced to nanocrystalline species (QD-•), while the S2O82- is reduced to strong oxidant SO4-•. Then SO4-• reacts with QD-• to form the excited state (QD*) that could emit light. 30 5
QD+e-→QD-• S2O82-+e-→SO42-+SO4-• QD-•+SO4-•→QD*+SO42QD*→QD+hν
(1) (2) (3) (4) 35
10
40
15
Figure 2（A）ECL–potential curve and cyclic voltammogram（B）of the CdSe QDs on the electrode.
20
Mechanism for DNAzyme-mediated amplified detection of DNA based on ECL quenching of CdSe QDs by folic acid. The amplified ECL detection of DNA based on Mg2+-dependent DNAzyme autocatalytic system was successfully designed.
45
50
55
Scheme 1 shows the fabrication principle of the strategy. CdSe QDs were firstly assembled on the electrode by using PAMAMCNT nanocomposites, showing high ECL signal. The singlestranded hairpin loop of sequence 4 contains an adenine ribonucleotide and acts as the substrate for the Mg2+-dependent DNAzyme. The stem region of hairpin DNA4 contains the sequence of the target analyte DNA 1, and the quencher folic acid are tethered to the stem end. After the hairpin substrate with the quencher was linked to the CdSe QDs on the electrode, the QDs ECL was effectively quenched. Upon interaction of analyte 1 with unit 3, the hairpin structure is opened, then units 2 and 3 were assembled into the active DNAzyme structure. The synergistic binding of the substrate 4 to the DNAzyme structure results in the cleavage of hairpin substrate in the presence of Mg2+ ions, and the fragment with the quencher was released, then the QDs ECL was enhanced. As the quencher unit includes the base sequence of the analyte, which leads to the enhanced formation of the DNAzyme structure and the increased cleavage of unit 4, with the concomitant generation of higher ECL signal for analyte sensing by autocatalytic amplified technique. Preparation and characterization of the modified electrode for ECL detection. Fig. 3 shows the field-emission scanning electron microscopy (FE-SEM) images of the modified electrodes. The negatively charged multi-walled carbon nanotubes (CNTs) were firstly assembled on the electrode by using PAMAM dendrimers, then the CdSe QDs were covalently conjugated to the dendrimer NCs. It could be found that a thick film of PAMAM-CNTs nanocomposites were homogeneously formed on the electrode (Fig. 3A). By comparison, the well– dispersed CdSe QDs spread over the electrode more uniformly due to the smaller size of QDs (Fig. 3B).
60
65
70
75
80
25
Figure 1 Schematic representation for the strategy of amplified ECL detection of DNA based on Mg2+-dependent DNAzyme autocatalytic system.
2 | Journal Name, [year], [vol], 00–00
85
Figure 3 Representative SEM images of (A) PAMAM-CNTs; (B) PAMAM- CNTs-QDs on the surface of electrode.
DNAzyme-mediated amplified detection of DNA based on ECL quenching of CdSe QDs by folic acid. The strategy feasibility for DNA detection based on ECL quenching of CdSe QDs by folic acid was investigated. As shown in Figure S1, very low background ECL signal was observed on the bare electrode (curve a). When the PAMAM-CNT-CdSe composite QDs were assembled on the electrode, high and stable ECL signal was obtained (curve b), indicating that PAMAM-CNT was an excellent immobilization matrix for CdSe composite QDs. Then folic acid as ECL quencher were close to the QDs on electrode, the ECL signal obviously decreased (curve c), demonstrating that the folic acid efficiently quenched the QDs ECL. In the presence of target analyte together with the mixture containing units 2, 3 and Mg2+ ions, the ECL signal obviously enhanced (curve d), This journal is © The Royal Society of Chemistry [year]
Analyst Accepted Manuscript
DOI: 10.1039/C4AN01465K
Analyst
Page 4 of 4 View Article Online
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
5
10
indicating that the DNAzyme-mediated amplification strategy based on ECL quenching of the QDs could be applied to the detection DNA analyte. Figure 4 shows the ECL signal changes upon analyzing different concentrations of target DNA. In the presence of target DNA by DNAzyme-mediated autocatalytic amplification strategy, the ECL peak signal gradually increased with increasing concentrations of analyte DNA (curve a to f), indicating that the autocatalytic amplification strategy based on ECL quenching of CdSe QDs by folic acid enables the detection of the target DNA.
40
45
50
15
55
60
20
Figure 4. ECL signal changes for detection of different concentrations of target DNA. The concentrations of DNA (nmol·L-1): (a) 0.05; (b) 0.1; (c) 1.0; (d) 10.0; (e) 100.0; (f) 1000.0.
sequences, respectively (Figure S2). The ECL signal change for single-base mismatched sequences was significantly smaller than that of the completely complementary sequences, and the noncomplementary sequences showed no response. The results suggest the autocatalytic amplified strategy has good selectivity for DNA detection. In summary, we provide a novel autocatalytic amplification strategy for detection of DNA based on ECL energy transfer from CdSe QDs to folic acid. Several advantages of this method have been demonstrated: 1) ECL-ET from CdSe QDs to folic acid was firstly reported and applied to amplified detection of DNA, which overcome steric hindrance in traditional quencher such as golds nanoparticles; 2) The DNAzyme-mediated autocatalytic system was combined with QDs ECL for DNA detection, which exhibits higher sensitivity, specificity, and rapid speed; 4) So far, the present work is the first report on the ECL-ET from QDs to folic acid combined with for amplified detection of DNA, which opens a promising new approach to QDs-based ECL bioassays, and has extensive potential in analytical applications. In addition, the ECL-ET system also has the disadvantage of time-consuming in electrode modification, which should stimulate more promising technology in ECL bioassay of QDs. This work was supported by the National Natural Science Foundation of China (No. 21175078), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).
Notes and references 1 1 (a) F. Wang, J. Elbaz, R. Orbach, N. Magen, I. J. m. Willner, Chem. Soc. 2011, 133, 17149; (b) Zhou M, Dong S J. Accounts of Chemical Research, 2011, 44: 1232. 2 W. Tan, K.Wang, T. J. Drake, Curr. Opin. Chem. Biol. 2004, 8, 547. 3 Y. Lu, J. Liu, Curr. Opin. Biotechnol. 2006, 17, 580. 4 F. Lucarelli, S. Tombelli, M. Minunni, G. Marrazza, M. Mascini, Anal. 70 Chim. Acta 2008, 609, 139. 5 G. Liu, Y. Wan, V. Gau, J. Zhang, L. Wang, S. Song, C. Fan, J. Am. Chem. Soc. 2008, 130, 6820. 6 (a) C. H. Lu, F. Wang, I. Willner, J. Am. Chem. Soc. 2012, 134, 10651; (b) F.Wang, J. Elbaz, C. Teller, I. Willner, Angew. Chem. 2011, 123, 75 309. 7 S. J. Kwon, A. J. Bard, J. Am. Chem. Soc. 2012, 134, 10777. 8 F. Wang, J. Elbaz, C. Teller, I. Willner, Angew. Chem., Int. Ed. 2011, 50, 295. 9 W. Xu, X. Xue, T. Li, H. Zeng, X. Liu, Angew. Chem. 2009, 121, 6981 80 10 F. Wang, J. Elbaz, I. Willner, J. Am. Chem. Soc. 2012, 134, 5504. 11 S. F. Liu, X. Zhang, Y. M. Yu, G. Z. Zou, Anal. Chem. 2014, 86, 2784. 12 G. F. Jie, L.Wang, J. X. Yuan, S. S. Zhang, Anal. Chem. 2011, 83, 3873. 85 13 G. Liang, S. Liu, G. Zou, X. Zhang, Anal. Chem. 2012, 84, 10645. 14 M.-S. Wu, H.-W. Shi, L.-J. He, J.-J. Xu, H.-Y. Chen, Anal. Chem. 2012, 84, 4207. 15 H. Zhou, Y.-Y Zhang, J. Liu, J.-J. Xu, H.-Y. Chen, Chem. Commun. 2013, 49, 2246. 90 16 G. F. Jie, J. X. Yuan, Anal. Chem., 2012, 84, 2811. 17 C.-Y. Tian, J.-J. Xu, H.-Y. Chen, Chem. Commun. 2012, 48, 8234. 18 Y. Shan, J.-J. Xu, H.-Y. Chen, Chem. Commun. 2010, 46, 5079. 19 S. Santra, C. Kaittanis, O. J. Santiesteban, J. M. Perez, J. Am. Chem. Soc. 2011, 133,16680. 95 20 H. Zhou, Y. Y. Zhang, J. Liu, J. J. Xu, H. Y. Chen, Chem. Commun. 2013, 49, 2246. 65
Figure 5 The logarithmic calibration curve for DNA detection.
25
30
35
Figure 5 displays the relationship between the ECL signal changes (∆I) and the concentrations of analyte DNA (0.05 nmol·L-1 to 1000 nmol·L-1). The ∆I was logarithmically related to the analyte concentrations in the range from 0.05 nmol·L-1 to 1000 nmol·L-1 (R = 0.995) with a detection limit of 0.05nmol·L-1 at 3σ, which is comparable to the previously reported detection of DNA. A series of five duplicate measurements of 1.0 nmol·L-1 were used for estimating the precision, and the relative standard deviation (RSD) was 6.8%, showing good reproducibility of the ECL method. The selectivity of the autocatalytic amplified detection of DNA based on ECL quenching of CdSe QDs was investigated by using unit 3 to hybridize with completely complementary tDNA, single-base mismatched DNA and noncomplementary DNA This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3
Analyst Accepted Manuscript
DOI: 10.1039/C4AN01465K
Analyst Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: G. Jie, Y. Qin, Q. Meng and J. Wang, Analyst, 2014, DOI: 10.1039/C4AN01465K.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/analyst
Page 1 of 4
Analyst View Article Online
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
206x127mm (150 x 150 DPI)
Analyst Accepted Manuscript
DOI: 10.1039/C4AN01465K
Analyst
Page 2 of 4
CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
View Article Online
ARTICLE TYPE
Autocatalytic Amplified Detection of DNA Based on CdSe Quantum Dots/Folic Acid Electrochemiluminescence Energy Transfer System Guifen Jie*, Yingqiang Qin, Qingmin Meng, Jialin Wang* 5
10
15
20
25
30
35
40
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X First published on the web Xth XXXXXXXXX 200X DOI: 10.1039/b000000x
In this work, electrochemiluminescence energy transfer (ECL-ET) from CdSe quantum dots (QDs) to folic acid was firstly reported, and applied to amplified detection of DNA by using a novel DNAzyme-mediated autocatalytic system. The development of DNA biosensors capable of amplified detection of DNA is being greatly motivated by its potential applications in molecular diagnostics, forensic investigations, genetics therapy, and biomedical development,1 and numerous electrical,2 optical,3 or microgravimetric4 amplified DNA sensors have been reported. Different catalysts, such as enzymes,5 catalytic nucleic acids (DNAzymes),6 or metal nanoparticles (NPs),7 were used for the amplified detection of DNA. Amplified DNA detection was accomplished by an autocatalytic and catabolic DNAzyme-mediated process.8 The easy synthetic preparation of DNAzymes and the reduced nonspecific adsorption of DNAzymes make catalytic nucleic acids attractive reporter units. The use of DNA-based machines has recently been suggested as a means to stimulate the autonomous replication of a catalytic reporter as the result of DNA sensing.9 A further approach for amplifying the DNA detection has included the use of the Zn2+-dependent ligation DNAzyme and the isothermal autonomous synthesis of the ligation DNAzyme units, as a result of the DNA recognition event.10 Electrochemiluminescence (ECL) as a promising tool has attracted much attention in sensing and detecting trace amounts of samples due to its high sensitivity, wide dynamic concentration response range, as well as its potential and spatial controlment.11−12 Recent research demonstrated that QDs are promising ECL tags for selective determination of macromolecules in real samples.13 The marriage of energy transfer with electrochemiluminescence has produced a new technology named electrochemiluminescence energy transfer (ECL-ET), which can realize effective and sensitive detection of biomolecules.14 To obtain optimal ECL-ET efficiency, perfect energy overlapped donor/acceptor pair is of great importance. Notably, the tunable emission wavelength and broad absorption
55
60
65
70
75
80
spectra of QDs render them ideal active donor or acceptor for ECL-ET. 14 Considerable works have been done in NCs ECL resonance energy transfer (RET). Xu and coworkers first demonstrated that the ECL from manganese-doped CdS NCs could be quenched by proximal Au NPs, such ECL energy transfer platform allowed DNA detection.14 Subsequently, QDs-based ECL energy transfer assays for DNA detection,15 cytosensing,16 aptamer,17 and immunoassay have been further explored.18 However, most of the ECL-ET assays used gold nanoparticles as the quencher, which has limits in biolabeling due to the steric hindrance to the electrode. Folic acid as a micromolecule was reported to be acted as an effective quencher for fluorescence.19 Furthermore, DNAzymes as enzyme mimics have attracted considerable attention in ECL techniques due to their high catalytic activity and good stability.20 So far, the QDs-based ECL-ET assays using folic acid as quencher combined with cyclic amplification technique has not been reported. Herenin, we present a novel strategy on DNAzyme-mediated autocatalytic amplified detection of DNA based on electrochemiluminescence energy transfer (ECL-ET) from CdSe quantum dots to folic acid. Characterization of the CdSe QDs. Figure 1A showed the transmission electron microscopy (TEM) image of the CdSe QDs, the nanoparticles possessed uniform morphology and size, and the average diameter was about 5~6 nm. Figure 1B presents the photoluminescence (PL) spectra of the CdSe QDs, the PL emission peak of the QDs was at 650 nm, and the PL intensity was high, indicating that the CdSe QDs possess good luminescent property and has promising optical applications.
85
*Corresponding author: Key Laboratory of Sensor Analysis of Tumor
45
50
Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China. E-mail: guifenjie @126.com; Email:[email protected] † Electronic Supplementary Information (ESI) available: Experimental details, UV-vis, TEM and the optimization and selectivity are included in the supporting information.and additional figures. See DOI: 10.1039/b000000x/
This journal is © The Royal Society of Chemistry [year]
Figure 1. Representative TEM image (A), and PL spectra (B) of the CdSe QDs,
90
ECL behavior of the CdSe quantum dots. The ECL– potential curve and cyclic voltammogram (CV) of the CdSe quantum dots were shown in Figure 2. A cathodic peak at –1.05 V is observed in the CV response, corresponding to the reduction of S2O82−. There is an ECL peak at –1.40 V in the ECL–potential curve, which results from the reaction of the CdSe QDs with
Journal Name, [year], [vol], 00–00 | 1
Analyst Accepted Manuscript
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
DOI: 10.1039/C4AN01465K www.rsc.org/xxxxxx | XXXXXXXX
Page 3 of 4
Analyst View Article Online
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
S2O82-. The possible ECL mechanisms are as follows. The QDs are reduced to nanocrystalline species (QD-•), while the S2O82- is reduced to strong oxidant SO4-•. Then SO4-• reacts with QD-• to form the excited state (QD*) that could emit light. 30 5
QD+e-→QD-• S2O82-+e-→SO42-+SO4-• QD-•+SO4-•→QD*+SO42QD*→QD+hν
(1) (2) (3) (4) 35
10
40
15
Figure 2（A）ECL–potential curve and cyclic voltammogram（B）of the CdSe QDs on the electrode.
20
Mechanism for DNAzyme-mediated amplified detection of DNA based on ECL quenching of CdSe QDs by folic acid. The amplified ECL detection of DNA based on Mg2+-dependent DNAzyme autocatalytic system was successfully designed.
45
50
55
Scheme 1 shows the fabrication principle of the strategy. CdSe QDs were firstly assembled on the electrode by using PAMAMCNT nanocomposites, showing high ECL signal. The singlestranded hairpin loop of sequence 4 contains an adenine ribonucleotide and acts as the substrate for the Mg2+-dependent DNAzyme. The stem region of hairpin DNA4 contains the sequence of the target analyte DNA 1, and the quencher folic acid are tethered to the stem end. After the hairpin substrate with the quencher was linked to the CdSe QDs on the electrode, the QDs ECL was effectively quenched. Upon interaction of analyte 1 with unit 3, the hairpin structure is opened, then units 2 and 3 were assembled into the active DNAzyme structure. The synergistic binding of the substrate 4 to the DNAzyme structure results in the cleavage of hairpin substrate in the presence of Mg2+ ions, and the fragment with the quencher was released, then the QDs ECL was enhanced. As the quencher unit includes the base sequence of the analyte, which leads to the enhanced formation of the DNAzyme structure and the increased cleavage of unit 4, with the concomitant generation of higher ECL signal for analyte sensing by autocatalytic amplified technique. Preparation and characterization of the modified electrode for ECL detection. Fig. 3 shows the field-emission scanning electron microscopy (FE-SEM) images of the modified electrodes. The negatively charged multi-walled carbon nanotubes (CNTs) were firstly assembled on the electrode by using PAMAM dendrimers, then the CdSe QDs were covalently conjugated to the dendrimer NCs. It could be found that a thick film of PAMAM-CNTs nanocomposites were homogeneously formed on the electrode (Fig. 3A). By comparison, the well– dispersed CdSe QDs spread over the electrode more uniformly due to the smaller size of QDs (Fig. 3B).
60
65
70
75
80
25
Figure 1 Schematic representation for the strategy of amplified ECL detection of DNA based on Mg2+-dependent DNAzyme autocatalytic system.
2 | Journal Name, [year], [vol], 00–00
85
Figure 3 Representative SEM images of (A) PAMAM-CNTs; (B) PAMAM- CNTs-QDs on the surface of electrode.
DNAzyme-mediated amplified detection of DNA based on ECL quenching of CdSe QDs by folic acid. The strategy feasibility for DNA detection based on ECL quenching of CdSe QDs by folic acid was investigated. As shown in Figure S1, very low background ECL signal was observed on the bare electrode (curve a). When the PAMAM-CNT-CdSe composite QDs were assembled on the electrode, high and stable ECL signal was obtained (curve b), indicating that PAMAM-CNT was an excellent immobilization matrix for CdSe composite QDs. Then folic acid as ECL quencher were close to the QDs on electrode, the ECL signal obviously decreased (curve c), demonstrating that the folic acid efficiently quenched the QDs ECL. In the presence of target analyte together with the mixture containing units 2, 3 and Mg2+ ions, the ECL signal obviously enhanced (curve d), This journal is © The Royal Society of Chemistry [year]
Analyst Accepted Manuscript
DOI: 10.1039/C4AN01465K
Analyst
Page 4 of 4 View Article Online
Published on 29 August 2014. Downloaded by Nipissing University on 14/10/2014 10:47:09.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
5
10
indicating that the DNAzyme-mediated amplification strategy based on ECL quenching of the QDs could be applied to the detection DNA analyte. Figure 4 shows the ECL signal changes upon analyzing different concentrations of target DNA. In the presence of target DNA by DNAzyme-mediated autocatalytic amplification strategy, the ECL peak signal gradually increased with increasing concentrations of analyte DNA (curve a to f), indicating that the autocatalytic amplification strategy based on ECL quenching of CdSe QDs by folic acid enables the detection of the target DNA.
40
45
50
15
55
60
20
Figure 4. ECL signal changes for detection of different concentrations of target DNA. The concentrations of DNA (nmol·L-1): (a) 0.05; (b) 0.1; (c) 1.0; (d) 10.0; (e) 100.0; (f) 1000.0.
sequences, respectively (Figure S2). The ECL signal change for single-base mismatched sequences was significantly smaller than that of the completely complementary sequences, and the noncomplementary sequences showed no response. The results suggest the autocatalytic amplified strategy has good selectivity for DNA detection. In summary, we provide a novel autocatalytic amplification strategy for detection of DNA based on ECL energy transfer from CdSe QDs to folic acid. Several advantages of this method have been demonstrated: 1) ECL-ET from CdSe QDs to folic acid was firstly reported and applied to amplified detection of DNA, which overcome steric hindrance in traditional quencher such as golds nanoparticles; 2) The DNAzyme-mediated autocatalytic system was combined with QDs ECL for DNA detection, which exhibits higher sensitivity, specificity, and rapid speed; 4) So far, the present work is the first report on the ECL-ET from QDs to folic acid combined with for amplified detection of DNA, which opens a promising new approach to QDs-based ECL bioassays, and has extensive potential in analytical applications. In addition, the ECL-ET system also has the disadvantage of time-consuming in electrode modification, which should stimulate more promising technology in ECL bioassay of QDs. This work was supported by the National Natural Science Foundation of China (No. 21175078), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).
Notes and references 1 1 (a) F. Wang, J. Elbaz, R. Orbach, N. Magen, I. J. m. Willner, Chem. Soc. 2011, 133, 17149; (b) Zhou M, Dong S J. Accounts of Chemical Research, 2011, 44: 1232. 2 W. Tan, K.Wang, T. J. Drake, Curr. Opin. Chem. Biol. 2004, 8, 547. 3 Y. Lu, J. Liu, Curr. Opin. Biotechnol. 2006, 17, 580. 4 F. Lucarelli, S. Tombelli, M. Minunni, G. Marrazza, M. Mascini, Anal. 70 Chim. Acta 2008, 609, 139. 5 G. Liu, Y. Wan, V. Gau, J. Zhang, L. Wang, S. Song, C. Fan, J. Am. Chem. Soc. 2008, 130, 6820. 6 (a) C. H. Lu, F. Wang, I. Willner, J. Am. Chem. Soc. 2012, 134, 10651; (b) F.Wang, J. Elbaz, C. Teller, I. Willner, Angew. Chem. 2011, 123, 75 309. 7 S. J. Kwon, A. J. Bard, J. Am. Chem. Soc. 2012, 134, 10777. 8 F. Wang, J. Elbaz, C. Teller, I. Willner, Angew. Chem., Int. Ed. 2011, 50, 295. 9 W. Xu, X. Xue, T. Li, H. Zeng, X. Liu, Angew. Chem. 2009, 121, 6981 80 10 F. Wang, J. Elbaz, I. Willner, J. Am. Chem. Soc. 2012, 134, 5504. 11 S. F. Liu, X. Zhang, Y. M. Yu, G. Z. Zou, Anal. Chem. 2014, 86, 2784. 12 G. F. Jie, L.Wang, J. X. Yuan, S. S. Zhang, Anal. Chem. 2011, 83, 3873. 85 13 G. Liang, S. Liu, G. Zou, X. Zhang, Anal. Chem. 2012, 84, 10645. 14 M.-S. Wu, H.-W. Shi, L.-J. He, J.-J. Xu, H.-Y. Chen, Anal. Chem. 2012, 84, 4207. 15 H. Zhou, Y.-Y Zhang, J. Liu, J.-J. Xu, H.-Y. Chen, Chem. Commun. 2013, 49, 2246. 90 16 G. F. Jie, J. X. Yuan, Anal. Chem., 2012, 84, 2811. 17 C.-Y. Tian, J.-J. Xu, H.-Y. Chen, Chem. Commun. 2012, 48, 8234. 18 Y. Shan, J.-J. Xu, H.-Y. Chen, Chem. Commun. 2010, 46, 5079. 19 S. Santra, C. Kaittanis, O. J. Santiesteban, J. M. Perez, J. Am. Chem. Soc. 2011, 133,16680. 95 20 H. Zhou, Y. Y. Zhang, J. Liu, J. J. Xu, H. Y. Chen, Chem. Commun. 2013, 49, 2246. 65
Figure 5 The logarithmic calibration curve for DNA detection.
25
30
35
Figure 5 displays the relationship between the ECL signal changes (∆I) and the concentrations of analyte DNA (0.05 nmol·L-1 to 1000 nmol·L-1). The ∆I was logarithmically related to the analyte concentrations in the range from 0.05 nmol·L-1 to 1000 nmol·L-1 (R = 0.995) with a detection limit of 0.05nmol·L-1 at 3σ, which is comparable to the previously reported detection of DNA. A series of five duplicate measurements of 1.0 nmol·L-1 were used for estimating the precision, and the relative standard deviation (RSD) was 6.8%, showing good reproducibility of the ECL method. The selectivity of the autocatalytic amplified detection of DNA based on ECL quenching of CdSe QDs was investigated by using unit 3 to hybridize with completely complementary tDNA, single-base mismatched DNA and noncomplementary DNA This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3
Analyst Accepted Manuscript
DOI: 10.1039/C4AN01465K
