Biosensors and Bioelectronics 62 (2014) 208–213
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Label-free chemiluminescent aptasensor for platelet-derived growth factor detection based on exonuclease-assisted cascade autocatalytic recycling amplification Sai Bi a,n, Baoyu Luo b, Jiayan Ye b, Zonghua Wang a,n a Laboratory of Fiber Materials and Modern Textiles, the Growing Base for State Key Laboratory, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Qingdao University, Qingdao 266071, PR China b Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2014 Received in revised form 14 June 2014 Accepted 26 June 2014 Available online 2 July 2014
Here an exonuclease III (Exo III)-assisted cascade autocatalytic recycling amplification (Exo-CARA) strategy is proposed for label-free chemiluminescent (CL) detection of platelet-derived growth factor BB (PDGF-BB) by taking advantage of both recognition property of aptamer and cleavage function of Exo III. Functionally, this system consists of a duplex DNA (aptamer–blocker hybrid), two kinds of hairpin structures (MB1 and MB2), and Exo III. Upon recognizing and binding with PDGF-BB, aptamer folds into a close configuration, which initiates the proposed Exo-CARA reaction (Recyclings I-II-III-II). Finally, numerous “caged” G-quadruplex sequences on DNAzyme1 and DNAzyme2 release that intercalate hemin to catalyze the oxidation of luminol by H2O2 to generate an amplified CL signal, achieving excellent specificity and high sensitivity with a detection limit of 6.8 10 13 M PDGF-BB. The proposed strategy has the advantages of simple design, isothermal conditions, homogeneous reaction without separation and washing steps, effective-cost without the need of labeling, and high amplification efficiency, which might be a universal and promising protocol for the detection of a variety of biomolecules whose aptamers undergo similar conformational changes. & 2014 Elsevier B.V. All rights reserved.
Keywords: Analytical method Aptasensor DNAzyme Exponential amplification Label-free detection
1. Introduction The detection of nucleic acids and proteins plays significant role in investigating their functions for the development of molecular diagnostic (Adams et al., 2012). To achieve determination of these biomolecules in complex systems, much effort has been devoted to the development of amplification strategies to improve detection sensitivity and specificity, especially isothermal amplification techniques (Kim and Easley, 2011). In comparison with polymerase chain reaction (PCR) that requires thermal cycling, isothermal amplification has the advantages of easy operation, cost effective, and more tolerant to inhibitory components from a crude sample. So far, various isothermal amplification strategies have been developed for biomolecules detection, such as rolling circle amplification (Zhao et al., 2008), strand-displacement reaction (Krishnan and Simmel, 2011), helicase-dependent n
Corresponding authors. Tel./fax: þ86 0532 85950873. E-mail addresses: [email protected] (S. Bi), [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.bios.2014.06.057 0956-5663/& 2014 Elsevier B.V. All rights reserved.
amplification (Vincent et al., 2004), recombinase polymerase amplification (Rohrman and Richards-Kortum, 2012). Recently, exonuclease III (Exo III) assisted target recycling strategies have been reported for amplified detection of DNA (Liu et al., 2013; Zuo et al., 2010; Bi et al., 2012). Exo III can catalyze the stepwise removal of mononucleotides from 3′-hydroxyl terminus of doublestranded DNA when substrate is a blunt or recessed 3′-terminus, while shows limited activity on single-stranded DNA or duplex DNAs with a protruding 3′ end (Zuo et al., 2010). Thus, as an advantage over nicking endonucleases, Exo III is sequence-independent that does not require a specific recognition site, providing a more versatile platform for biomolecules detection (Freeman et al., 2011; Liu et al., 2012; Xu et al., 2012). Aptamers are single-stranded oligonucleotides that can be isolated in vitro from random sequence libraries through a process termed SELEX (selective evolution of ligands by exponential enrichment) (Famulok et al., 2007). Advantages of aptamers over antibodies include ease synthesis and modification, high stability, unlimited shelf-life, and so on (Li et al., 2010). So far, aptamers have been selected and synthesized for a broad range of analytes,
S. Bi et al. / Biosensors and Bioelectronics 62 (2014) 208–213
including small molecules, metal ions, proteins, and even cells (Tang et al., 2007; Wu et al., 2011). Aptamers can fold into distinct secondary structures and function as receptors for target molecules with high affinity and specificity, which thus have been employed to design variously novel biosensors for signal amplification (i.e., aptasensors) (Iliuk et al., 2011; Nutiu and Li, 2003; Xue et al., 2012, 2010). To achieve rapid, simple and convenient detection of biomarkers, label-free strategies have attracted considerable interest for the development of aptasensors. The main advantage of label-free methods is that they can effectively avoid expensive and tedious labeling procedures, which can retain the highest activity and affinity of the recognition elements (Luo and Davis, 2013; Ma et al., 2013). Particularly, DNA-mediated homogeneous binding assays which perform in solution containing the specimen and all reagents without the need of immobilization, separation or washing steps can minimize the effects of contamination (Sassolas et al., 2011; Zhang et al., 2013). Thus, such homogeneous assays are the most promising techniques for molecular diagnostics and point-of-care applications. Herein, an isothermal, homogeneous, and label-free chemiluminescent (CL) aptasensor has been developed for the detection of platelet-derived growth factor BB (PDGF-BB) that is known to be related to tumor transformation, growth and progression (Heldin and Westermark, 1990) based on Exo III-assisted cascade autocatalytic recycling amplification (Exo-CARA), combing with the induced formation of G-quadruplex for amplified signal transduction. Since this design is simply based on nucleic acid hybridization for 3′-5′ exodeoxyribonuclease activity of Exo III, it can be generally applied to other aptamer-based strategies for label-free detection of various analytes.
2. Experimental section 2.1. Chemicals
209
DNAzyme1 and DNAzyme2 to fold into G-quadruplex-hemin complexes. The CL signals were monitored on a Centro LB942 luminometer (Berthold, Germany). Briefly, corresponding samples and 50 mL of luminol (5.0 10 4 M) were firstly added to 96-well plate. And then, 50 mL of H2O2 (5.0 10 4 M) was introduced to each well through automatic injector of the CL equipment. The CL kinetics was recorded for 2.5 min in 0.5 s intervals. The negative high voltage of luminometer was 1200 V. 2.3. Nondenaturing polyacrylamide gel electrophoresis The Exo III-assisted amplification processes were characterized by 12.5% native polyacrylamide gel electrophoresis. The samples were added to 3 mL of loading buffer. Electrophoresis was carried out in 1 Tris–acetate–EDTA (TAE) at 120 V constant voltage for 1 h at room temperature. After staining gels with ethidium bromide (EB, 0.5 μg/mL) for 30 min, the visualization and photography were performed using a digital camera under UV illumination.
3. Results and discussion 3.1. Principle of the proposed Exo-CARA The principle of our proposed Exo-CARA strategy for PDGF-BB detection is shown in Scheme 1. This system consists of an aptamer–blocker hybrid, MB1, MB2, and Exo III. Blocker DNA that 3′ terminus with four bases non-complementary to aptamer plays significantly roles in not only preventing aptamer from uncontrolled folding into an active configuration in the absence of targets, but also resisting the activity of Exo III. In addition, MB1 and MB2 self-hybridize to form stem-loop structures that contain Exo III-resistant 3′ protruding terminus. As a result, the sequences
Olignoculeotides used in the present study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences are listed in Table S1. Exonuclease III (Exo III) was purchased from MBI Fermentas (Canada). Recombinant human platelet-derived growth factor BB (PDGF-BB human) was ordered from Prospec-Tany TechnoGene Ltd. (Israel), which was dissolved in 4 mM HCl containing 0.2% bovine serum albumin (BSA). Hemin, 4-(2-hydroxyethyl)piperazine-1 ethanesulfonic acid sodium salt (HEPES) and luminol were obtained from Aladdin Chemistry Co. Ltd (China). A hemin stock solution (5.0 10 3 M) was prepared in DMSO and stored in the dark at 20 °C. Double-distilled, deionized ultrapure water was used in all experiments. All regents were of analytical grade and used without further purification. 2.2. Exo III-assisted cascade autocatalytic recycling amplification (Exo-CARA) for PDGF-BB detection Firstly, the DNA solutions of aptamer (2.0 10 6 M) and blocker (2.0 10 6 M), MB1 (1.0 10 6 M), MB2 (1.0 10 6 M) were respectively heated to 90 °C for 10 min and allowed to gradually cool to room temperature. Then, a 10 mL of different concentrations of PDGF-BB was incubated with 10 mL of aptamer– blocker solution (1.0 10 6 M) at 37 °C for 15 min to perform the aptamer–target binding reaction. The amplification process was performed by adding 10 mL of MB1 (1.0 10 6 M), 10 mL of MB2 (1.0 10 6 M), 4.5 μL of 10 reaction buffer, 50 units of Exo III, followed by incubating at 25 °C for 30 min. The resulting products were incubated with 50 mL of hemin (1.0 10 8 M) in 25 mM HEPES buffer (pH 7.4, 20 mM KCl, 200 mM NaCl, 1% DMSO (v/v), 0.05% Triton X-100 (w/v)) for 30 min to induce the liberated
Scheme 1. Schematic illustration of the proposed Exo III-assisted cascade autocatalytic recycling amplification (Exo-CARA) strategy (Recyclings I-II-III-II) for label-free CL detection of PDGF-BB. (For interpretation of the references to color in this scheme, the reader is referred to the web version of this article.)
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of horseradish peroxidase (HRP)-mimicking DNAzyme are caged in the duplex structures of the stems of MB1 and MB2, respectively. In the presence of PDGF-BB, aptamer recognizes and binds with PDGF-BB to form a consensus secondary structure motif of threeway helix junction with a conserved single-stranded loop at the branch point, resulting in the Exo III-catalyzed digestion of aptamer from 3′ end. This reaction recycles the PDGF-BB, which is then free to bind another aptamer to trigger a new cleavage process (Recycling I). At the same time, blocker is also autonomously released, which acts as a secondary target to activate the Recycling II by recognizing and hybridizing with the protruding 3′ end of MB1 to form a blunt 3′ terminus. As a result, Exo III hydrolytically digests the 3′ end of MB1, liberating blocker for another Recycling II and yielding DNAzyme1 that includes HRPmimicking DNAzyme sequence (blue) and target recognition sequence (green) to hybridize with the protruding 3′ end of MB2 probe to activate Recycling III with the assistance of Exo III. During Recycling III, DNAzyme2 containing the sequence of HRP-mimicking DNAzyme (blue) and the same sequence of blocker (red) liberates, which in turn triggers Recycling II and repeats the above cyclic process to complish the Exo-CARA reaction (Scheme 1A). Finally, the caged HRP-mimicking DNAzyme sequences in DNAzyme1 and DNAzyme2 can be exponentially produced, which assemble into HRP-mimicking DNAzymes in the presence of hemin that catalyze the oxidation of luminol by H2O2 to generate an amplified CL signal (Scheme 1B). 3.2. Feasibility of the proposed strategy The reaction pathways of the assay were confirmed by polyacrylamide gel electrophoresis (PAGE) (Fig. 1). As shown in lanes 1–3, the aptamer–blocker hybrid, MB1 and MB2 respectively displayed only one band, which can stably coexist in the absence of PDGF-BB and Exo III (lane 7). In the presence of PDGF-BB, a slower and a faster migration bands appeared in lane 4, revealing the binding complex of aptamer and PDGF-BB and separation of blocker from aptamer. However, the brightness of released blocker DNA with only 15 bases in lane 4 was relatively weak, which could be explained as EB stains single-stranded DNA much less efficiently than double-stranded DNA, so that relative brightness between different bands should not be compared (Zhang et al., 2007). Moreover, aptamer–blocker was treated with Exo III in the absence (lane 5) or presence of PDGF-BB (lane 6). From lane 5, in the absence of PDGF-BB, no reaction occurred even in the presence of Exo III. Upon the addition of PDGF-BB, besides the migration band of blocker, a small molecular weight product that corresponded to the cleaved aptamer was observed in lane 6. This phenomenon can also be seen in lane 8 that the mixture of aptamer–blocker, MB1, and MB2 was treated with both Exo III and PDGF-BB. Thus, the proposed Exo-CARA reaction was carried
out as design which was triggered by target and executed by Exo III. 3.3. Optimization of the Exo-CARA assay for PDGF-BB detection 3.3.1. Influence of aptamer–blocker hybrid The original aptamer sequence of PDGF-BB used in this assay is 35 bases with an open secondary structure in the absence of PDGF-BB, which occurs conformational change to form a close conformation of a three-way helix junction when binding to PDGF-BB that brings 3′ and 5′ ends within a stem (Green et al., 1996). However, it has been reported that a significant fraction of original PDGF-BB aptamer will be in close conformation even in the absence of target protein, leading to non-specific folding and producing a high background (Zhao et al., 2011). To overcome this problem, a blocker DNA with four bases (T4) noncomplementary to the aptamer at its 3′ end was introduced. The results of Fig. 2A demonstrate that the blocker DNA can significantly prevent the non-specific background and further improve the sensitivity of the amplification assay. Additionally, it should be noted that the length of the stem of aptamer has influence on not only the formation of the folded structure upon addition of PDGF-BB to promote the replacement of blocker DNA, but also the efficiency of the Exo IIIassisted cleavage process that is strongly depends on the ability of the 3′ end of the aptamer to hybridize with its outer sequences and the stability of the formed stem. According to previous investigations, the aptamer probe with 6 bp stem was selected in this assay due to its good performance in facilitating replacement of blocker DNA and activating the following Exo-CARA process in the presence of PDGF-BB, but remaining inactive in the absence of target (Zhang and Zhang, 2012; Cai et al., 2011). 3.3.2. Influence of Exo III amounts The influence of the amount of Exo III used in the Exo-CARA system was investigated by detecting 1.0 10 10 M PDGF-BB in the presence of 0, 25 units, 50 units, 75 units and 100 units Exo III, respectively. From the results of Fig. 2B, the CL intensities increased with increasing the Exo III amounts. The maximum change of the CL intensity between PDGF-BB and the negative control without PDGF-BB (I I0) was obtained when the amount of Exo III was 50 units, after which the I I0 leveled off. Therefore, 50 units Exo III were chosen as the optimum condition for PDGF-BB detection in the following study. 3.3.3. Influence of reaction time Reaction time is another important factor of the proposed ExoCARA system. As shown in Fig. 2C, for the detection of PDGF-BB at the concentration of 1.0 10 10 M, with the increase of reaction time, the CL intensities increased which reached the maximum after 30 min. As control, the blank sample (0 M PDGF-BB) at each time was treated in the same way as Exo-CARA. From the results, no obvious CL change was observed for the negative control without PDGF-BB. Therefore, the reaction time of 30 min was adopted for the proposed Exo-CARA system. 3.4. Sensitivity for PDGF-BB detection
Fig. 1. Polyacrylamide gel electrophoresis (PAGE) of the reaction pathways. Lane 1: aptamer–blocker; lane 2: MB1; lane 3: MB2; lane 4: aptamer–blockerþ PDGF-BB; lane 5: aptamer–blockerþ Exo III; lane 6: aptamer–blockerþ PDGF-BB þ Exo III; lane 7: aptamer–blockerþMB1 þMB2; lane 8: aptamer–blockerþ MB1 þ MB2 þ PDGFBB þ Exo III. M represents 500-bp DNA ladder.
To demonstrate the sensitivity of the proposed Exo-CARA assay, PDGF-BB with different concentrations were detected by CL measurement under the optimum conditions. As shown in Fig. 3A, the CL intensity increased when the concentration of PDGF-BB was in the range from 1.0 10 12 to 1.0 10 8 M, indicating that the copy number of liberated DNAzymes was highly dependent on and directly proportional to the concentration of target protein. Fig. 3B shows the linear calibration curve corresponding to the relative CL peak intensities (I I0) versus
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Fig. 2. Effects of (A) the blocker DNA, (B) the amount of Exo-III, and (C) the reaction time on the Exo-CARA system. The CL intensities were obtained by detecting DNAzymes produced through the Exo-CARA reaction in the presence (I) and in the absence (negative control, I0) of 1.0 10 10 M PDGF-BB.
Fig. 3. (A) CL kinetic curves obtained by the Exo-CARA strategy in response to different concentrations of PDGF-BB: 0, 1.0 10 12, 1.0 10 11, 1.0 10 10, 1.0 10 9, 1.0 10 8 M. (B) Corresponding calibration curve of relative CL peak intensities (I I0) versus the logarithm of PDGF-BB concentrations (M). Error bars represent the standard deviation of three repeated measurements.
different concentrations of PDGF-BB in logarithmic scales. The regression equation was expressed as ΔI ¼6220.24log Cþ 76422.61 with a correlation coefficient (R) of 0.9949, where ΔI and C represented the relative CL intensity (I I0) and the concentration of PDGF-BB (M), respectively. The limit of detection (LOD) was calculated to be 6.8 10 13 M through evaluating the average
response of blank plus three times the standard deviation, which was comparable with other most sensitive aptamer-based methods for PDGF-BB detection (Table S1) and 10-fold lower than that obtained by using only aptamer–blocker and MB1 to perform Recycling I and Recycling II (see control experiment in Supporting information). The relative standard deviation (RSD) obtained by
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Fig. 4. The detection specificity of the proposed Exo-CARA assay. Target protein (PDGF-BB) at the concentration of 1.0 10 10 M and three non-target proteins (thrombin, lysozyme, and BSA) at the concentrations of 1.0 10 7 M were used to investigate the relative CL intensity (I I0) responding to a particular protein. All the experiments were carried out under the optimum conditions. Error bars represent the standard deviation of three repeated measurements.
1.0 10 10 M PDGF-BB was 5.8% for five measurements, indicating a desirable reproducibility of the present biosensing system for protein assay.
obtained by ELISA assay as a reference method that is the most standard method currently for PDGF-BB detection. It has been reported that the dilution of serum samples by 10-fold or more have little matrix effects on protein detection (Zhao et al., 2009). Thus, the blood samples used in this assay were diluted to 100-fold prior to analysis, which were analyzed as described in Section 2.2. In addition, the interferences of oxidizable species in the diluted blood plasma sample, such as ascorbic acid (AA) and uric acid (UA), can be ignored for CL detection of PDGF-BB (see Supporting information for detail). As shown in Table 1, the PDGF-BB concentrations obtained by the present method were in good agreement with those determined by ELISA with the maximum relative deviation of 9.5% and a correlation coefficient of 0.9799, indicating our detection method compared favorably with ELISA for PDGF-BB detection in human blood plasma samples. Moreover, it has been reported that ELISA assay for PDGF-BB detection covering 4 orders of magnitude from 10 pM to 10 nM with an LOD at picomolar level. Thus, the developed aptasensor offered a wider linear range (5 orders of magnitude from 1.0 pM to 10 nM) and a lower LOD (1 order of magnitude). In addition, it should be noted that in comparison with ELISA our method was much simpler (homogeneous reaction and aptamer recognition), cost-effective (label-free), rapid ( 1 h), signal highly amplified and sensitive enough to be used for PDGF-BB quantitation in clinical samples (0.4–0.7 nM in human serum and 0.008–0.04 nM in human plasma) (Fang et al., 2001).
3.5. Detection specificity The detection specificity is another essential factor for an aptamer-based assay. The relative CL changes induced by three nontarget proteins (thrombin, lysozyme, and BSA) were investigated. Relative CL intensity was defined as the difference in the CL intensity between a certain sample with protein and the negative control without protein. As shown in Fig. 4, in comparison with the value upon PDGF-BB, the relative CL intensities corresponding to the nontarget proteins were extremely low even though their concentrations are 103-fold higher than that of target species. Therefore, the CL signal was generated by the specific interaction between the aptamer and its target, indicating the excellent specificity of the present aptasensor for PDGF-BB detection. In addition, the blocker DNA was designed that hybridized to aptamer with 11 bases, resulting in a melting point of 47.9 °C when the concentrations of aptamer–blocker and Mg2 þ were 1.0 mM and 12.5 mM, respectively. Thus, the aptamer–blocker hybrid was stable at room temperature and physiological pH, which could significantly limit nonspecific release of blocker DNA and further inhibit false positive detection signals. 3.6. Real sample assay To further demonstrate the applicability of this assay in clinical diagnosis, five human blood plasma specimens were measured by the proposed aptasensor, which were compared with the results Table 1 The detection of PDGF-BB in human blood plasma samples by the proposed ExoCARA aptasensor and ELISA method.
4. Conclusion In conclusion, a novel Exo-CARA-based CL aptasensor has been developed for PDGF-BB detection. In comparison with other aptamerbased methods for PDGF-BB analysis, this assay has the distinct advantages of simplicity (homogeneous reaction), rapidity (within 1 h), signal great amplification (cascade autocatalytic recycling amplification and DNAzymes as reporters to catalyze signal), high sensitivity (an LOD of 6.8 10 13 M), and excellent specificity (aptamer recognition). In addition, the proposed Exo-CARA strategy has been successfully applied in human blood plasma analysis, promising as a convenient point-of-care diagnostics. Moreover, this strategy can be versatilely extended to the detection of other target proteins, such as thrombin and lysozyme, based on analyte-induced self-assembly of the aptamer subunits and Exo III as an amplifying catalyst.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21375056 and 21105052), the Program for New Century Excellent Talents in University of Ministry of Education of People's Republic of China (NCET-12-1024), and the University Doctoral New Teacher Foundation of the Ministry of Education (20113719120001).
Appendix A. Supplementary information
PDGF-BB concentration (nM)a Sample
Proposed Exo-CARA aptasensor
ELISA
Relative deviation (%)
1 2 3 4 5
0.74 0.94 0.45 0.51 0.46
0.79 0.90 0.48 0.54 0.42
6.3 4.4 6.2 5.6 9.5
a
Each value is the average of three measurements.
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.057.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Label-free chemiluminescent aptasensor for platelet-derived growth factor detection based on exonuclease-assisted cascade autocatalytic recycling amplification Sai Bi a,n, Baoyu Luo b, Jiayan Ye b, Zonghua Wang a,n a Laboratory of Fiber Materials and Modern Textiles, the Growing Base for State Key Laboratory, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Qingdao University, Qingdao 266071, PR China b Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2014 Received in revised form 14 June 2014 Accepted 26 June 2014 Available online 2 July 2014
Here an exonuclease III (Exo III)-assisted cascade autocatalytic recycling amplification (Exo-CARA) strategy is proposed for label-free chemiluminescent (CL) detection of platelet-derived growth factor BB (PDGF-BB) by taking advantage of both recognition property of aptamer and cleavage function of Exo III. Functionally, this system consists of a duplex DNA (aptamer–blocker hybrid), two kinds of hairpin structures (MB1 and MB2), and Exo III. Upon recognizing and binding with PDGF-BB, aptamer folds into a close configuration, which initiates the proposed Exo-CARA reaction (Recyclings I-II-III-II). Finally, numerous “caged” G-quadruplex sequences on DNAzyme1 and DNAzyme2 release that intercalate hemin to catalyze the oxidation of luminol by H2O2 to generate an amplified CL signal, achieving excellent specificity and high sensitivity with a detection limit of 6.8 10 13 M PDGF-BB. The proposed strategy has the advantages of simple design, isothermal conditions, homogeneous reaction without separation and washing steps, effective-cost without the need of labeling, and high amplification efficiency, which might be a universal and promising protocol for the detection of a variety of biomolecules whose aptamers undergo similar conformational changes. & 2014 Elsevier B.V. All rights reserved.
Keywords: Analytical method Aptasensor DNAzyme Exponential amplification Label-free detection
1. Introduction The detection of nucleic acids and proteins plays significant role in investigating their functions for the development of molecular diagnostic (Adams et al., 2012). To achieve determination of these biomolecules in complex systems, much effort has been devoted to the development of amplification strategies to improve detection sensitivity and specificity, especially isothermal amplification techniques (Kim and Easley, 2011). In comparison with polymerase chain reaction (PCR) that requires thermal cycling, isothermal amplification has the advantages of easy operation, cost effective, and more tolerant to inhibitory components from a crude sample. So far, various isothermal amplification strategies have been developed for biomolecules detection, such as rolling circle amplification (Zhao et al., 2008), strand-displacement reaction (Krishnan and Simmel, 2011), helicase-dependent n
Corresponding authors. Tel./fax: þ86 0532 85950873. E-mail addresses: [email protected] (S. Bi), [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.bios.2014.06.057 0956-5663/& 2014 Elsevier B.V. All rights reserved.
amplification (Vincent et al., 2004), recombinase polymerase amplification (Rohrman and Richards-Kortum, 2012). Recently, exonuclease III (Exo III) assisted target recycling strategies have been reported for amplified detection of DNA (Liu et al., 2013; Zuo et al., 2010; Bi et al., 2012). Exo III can catalyze the stepwise removal of mononucleotides from 3′-hydroxyl terminus of doublestranded DNA when substrate is a blunt or recessed 3′-terminus, while shows limited activity on single-stranded DNA or duplex DNAs with a protruding 3′ end (Zuo et al., 2010). Thus, as an advantage over nicking endonucleases, Exo III is sequence-independent that does not require a specific recognition site, providing a more versatile platform for biomolecules detection (Freeman et al., 2011; Liu et al., 2012; Xu et al., 2012). Aptamers are single-stranded oligonucleotides that can be isolated in vitro from random sequence libraries through a process termed SELEX (selective evolution of ligands by exponential enrichment) (Famulok et al., 2007). Advantages of aptamers over antibodies include ease synthesis and modification, high stability, unlimited shelf-life, and so on (Li et al., 2010). So far, aptamers have been selected and synthesized for a broad range of analytes,
S. Bi et al. / Biosensors and Bioelectronics 62 (2014) 208–213
including small molecules, metal ions, proteins, and even cells (Tang et al., 2007; Wu et al., 2011). Aptamers can fold into distinct secondary structures and function as receptors for target molecules with high affinity and specificity, which thus have been employed to design variously novel biosensors for signal amplification (i.e., aptasensors) (Iliuk et al., 2011; Nutiu and Li, 2003; Xue et al., 2012, 2010). To achieve rapid, simple and convenient detection of biomarkers, label-free strategies have attracted considerable interest for the development of aptasensors. The main advantage of label-free methods is that they can effectively avoid expensive and tedious labeling procedures, which can retain the highest activity and affinity of the recognition elements (Luo and Davis, 2013; Ma et al., 2013). Particularly, DNA-mediated homogeneous binding assays which perform in solution containing the specimen and all reagents without the need of immobilization, separation or washing steps can minimize the effects of contamination (Sassolas et al., 2011; Zhang et al., 2013). Thus, such homogeneous assays are the most promising techniques for molecular diagnostics and point-of-care applications. Herein, an isothermal, homogeneous, and label-free chemiluminescent (CL) aptasensor has been developed for the detection of platelet-derived growth factor BB (PDGF-BB) that is known to be related to tumor transformation, growth and progression (Heldin and Westermark, 1990) based on Exo III-assisted cascade autocatalytic recycling amplification (Exo-CARA), combing with the induced formation of G-quadruplex for amplified signal transduction. Since this design is simply based on nucleic acid hybridization for 3′-5′ exodeoxyribonuclease activity of Exo III, it can be generally applied to other aptamer-based strategies for label-free detection of various analytes.
2. Experimental section 2.1. Chemicals
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DNAzyme1 and DNAzyme2 to fold into G-quadruplex-hemin complexes. The CL signals were monitored on a Centro LB942 luminometer (Berthold, Germany). Briefly, corresponding samples and 50 mL of luminol (5.0 10 4 M) were firstly added to 96-well plate. And then, 50 mL of H2O2 (5.0 10 4 M) was introduced to each well through automatic injector of the CL equipment. The CL kinetics was recorded for 2.5 min in 0.5 s intervals. The negative high voltage of luminometer was 1200 V. 2.3. Nondenaturing polyacrylamide gel electrophoresis The Exo III-assisted amplification processes were characterized by 12.5% native polyacrylamide gel electrophoresis. The samples were added to 3 mL of loading buffer. Electrophoresis was carried out in 1 Tris–acetate–EDTA (TAE) at 120 V constant voltage for 1 h at room temperature. After staining gels with ethidium bromide (EB, 0.5 μg/mL) for 30 min, the visualization and photography were performed using a digital camera under UV illumination.
3. Results and discussion 3.1. Principle of the proposed Exo-CARA The principle of our proposed Exo-CARA strategy for PDGF-BB detection is shown in Scheme 1. This system consists of an aptamer–blocker hybrid, MB1, MB2, and Exo III. Blocker DNA that 3′ terminus with four bases non-complementary to aptamer plays significantly roles in not only preventing aptamer from uncontrolled folding into an active configuration in the absence of targets, but also resisting the activity of Exo III. In addition, MB1 and MB2 self-hybridize to form stem-loop structures that contain Exo III-resistant 3′ protruding terminus. As a result, the sequences
Olignoculeotides used in the present study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences are listed in Table S1. Exonuclease III (Exo III) was purchased from MBI Fermentas (Canada). Recombinant human platelet-derived growth factor BB (PDGF-BB human) was ordered from Prospec-Tany TechnoGene Ltd. (Israel), which was dissolved in 4 mM HCl containing 0.2% bovine serum albumin (BSA). Hemin, 4-(2-hydroxyethyl)piperazine-1 ethanesulfonic acid sodium salt (HEPES) and luminol were obtained from Aladdin Chemistry Co. Ltd (China). A hemin stock solution (5.0 10 3 M) was prepared in DMSO and stored in the dark at 20 °C. Double-distilled, deionized ultrapure water was used in all experiments. All regents were of analytical grade and used without further purification. 2.2. Exo III-assisted cascade autocatalytic recycling amplification (Exo-CARA) for PDGF-BB detection Firstly, the DNA solutions of aptamer (2.0 10 6 M) and blocker (2.0 10 6 M), MB1 (1.0 10 6 M), MB2 (1.0 10 6 M) were respectively heated to 90 °C for 10 min and allowed to gradually cool to room temperature. Then, a 10 mL of different concentrations of PDGF-BB was incubated with 10 mL of aptamer– blocker solution (1.0 10 6 M) at 37 °C for 15 min to perform the aptamer–target binding reaction. The amplification process was performed by adding 10 mL of MB1 (1.0 10 6 M), 10 mL of MB2 (1.0 10 6 M), 4.5 μL of 10 reaction buffer, 50 units of Exo III, followed by incubating at 25 °C for 30 min. The resulting products were incubated with 50 mL of hemin (1.0 10 8 M) in 25 mM HEPES buffer (pH 7.4, 20 mM KCl, 200 mM NaCl, 1% DMSO (v/v), 0.05% Triton X-100 (w/v)) for 30 min to induce the liberated
Scheme 1. Schematic illustration of the proposed Exo III-assisted cascade autocatalytic recycling amplification (Exo-CARA) strategy (Recyclings I-II-III-II) for label-free CL detection of PDGF-BB. (For interpretation of the references to color in this scheme, the reader is referred to the web version of this article.)
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of horseradish peroxidase (HRP)-mimicking DNAzyme are caged in the duplex structures of the stems of MB1 and MB2, respectively. In the presence of PDGF-BB, aptamer recognizes and binds with PDGF-BB to form a consensus secondary structure motif of threeway helix junction with a conserved single-stranded loop at the branch point, resulting in the Exo III-catalyzed digestion of aptamer from 3′ end. This reaction recycles the PDGF-BB, which is then free to bind another aptamer to trigger a new cleavage process (Recycling I). At the same time, blocker is also autonomously released, which acts as a secondary target to activate the Recycling II by recognizing and hybridizing with the protruding 3′ end of MB1 to form a blunt 3′ terminus. As a result, Exo III hydrolytically digests the 3′ end of MB1, liberating blocker for another Recycling II and yielding DNAzyme1 that includes HRPmimicking DNAzyme sequence (blue) and target recognition sequence (green) to hybridize with the protruding 3′ end of MB2 probe to activate Recycling III with the assistance of Exo III. During Recycling III, DNAzyme2 containing the sequence of HRP-mimicking DNAzyme (blue) and the same sequence of blocker (red) liberates, which in turn triggers Recycling II and repeats the above cyclic process to complish the Exo-CARA reaction (Scheme 1A). Finally, the caged HRP-mimicking DNAzyme sequences in DNAzyme1 and DNAzyme2 can be exponentially produced, which assemble into HRP-mimicking DNAzymes in the presence of hemin that catalyze the oxidation of luminol by H2O2 to generate an amplified CL signal (Scheme 1B). 3.2. Feasibility of the proposed strategy The reaction pathways of the assay were confirmed by polyacrylamide gel electrophoresis (PAGE) (Fig. 1). As shown in lanes 1–3, the aptamer–blocker hybrid, MB1 and MB2 respectively displayed only one band, which can stably coexist in the absence of PDGF-BB and Exo III (lane 7). In the presence of PDGF-BB, a slower and a faster migration bands appeared in lane 4, revealing the binding complex of aptamer and PDGF-BB and separation of blocker from aptamer. However, the brightness of released blocker DNA with only 15 bases in lane 4 was relatively weak, which could be explained as EB stains single-stranded DNA much less efficiently than double-stranded DNA, so that relative brightness between different bands should not be compared (Zhang et al., 2007). Moreover, aptamer–blocker was treated with Exo III in the absence (lane 5) or presence of PDGF-BB (lane 6). From lane 5, in the absence of PDGF-BB, no reaction occurred even in the presence of Exo III. Upon the addition of PDGF-BB, besides the migration band of blocker, a small molecular weight product that corresponded to the cleaved aptamer was observed in lane 6. This phenomenon can also be seen in lane 8 that the mixture of aptamer–blocker, MB1, and MB2 was treated with both Exo III and PDGF-BB. Thus, the proposed Exo-CARA reaction was carried
out as design which was triggered by target and executed by Exo III. 3.3. Optimization of the Exo-CARA assay for PDGF-BB detection 3.3.1. Influence of aptamer–blocker hybrid The original aptamer sequence of PDGF-BB used in this assay is 35 bases with an open secondary structure in the absence of PDGF-BB, which occurs conformational change to form a close conformation of a three-way helix junction when binding to PDGF-BB that brings 3′ and 5′ ends within a stem (Green et al., 1996). However, it has been reported that a significant fraction of original PDGF-BB aptamer will be in close conformation even in the absence of target protein, leading to non-specific folding and producing a high background (Zhao et al., 2011). To overcome this problem, a blocker DNA with four bases (T4) noncomplementary to the aptamer at its 3′ end was introduced. The results of Fig. 2A demonstrate that the blocker DNA can significantly prevent the non-specific background and further improve the sensitivity of the amplification assay. Additionally, it should be noted that the length of the stem of aptamer has influence on not only the formation of the folded structure upon addition of PDGF-BB to promote the replacement of blocker DNA, but also the efficiency of the Exo IIIassisted cleavage process that is strongly depends on the ability of the 3′ end of the aptamer to hybridize with its outer sequences and the stability of the formed stem. According to previous investigations, the aptamer probe with 6 bp stem was selected in this assay due to its good performance in facilitating replacement of blocker DNA and activating the following Exo-CARA process in the presence of PDGF-BB, but remaining inactive in the absence of target (Zhang and Zhang, 2012; Cai et al., 2011). 3.3.2. Influence of Exo III amounts The influence of the amount of Exo III used in the Exo-CARA system was investigated by detecting 1.0 10 10 M PDGF-BB in the presence of 0, 25 units, 50 units, 75 units and 100 units Exo III, respectively. From the results of Fig. 2B, the CL intensities increased with increasing the Exo III amounts. The maximum change of the CL intensity between PDGF-BB and the negative control without PDGF-BB (I I0) was obtained when the amount of Exo III was 50 units, after which the I I0 leveled off. Therefore, 50 units Exo III were chosen as the optimum condition for PDGF-BB detection in the following study. 3.3.3. Influence of reaction time Reaction time is another important factor of the proposed ExoCARA system. As shown in Fig. 2C, for the detection of PDGF-BB at the concentration of 1.0 10 10 M, with the increase of reaction time, the CL intensities increased which reached the maximum after 30 min. As control, the blank sample (0 M PDGF-BB) at each time was treated in the same way as Exo-CARA. From the results, no obvious CL change was observed for the negative control without PDGF-BB. Therefore, the reaction time of 30 min was adopted for the proposed Exo-CARA system. 3.4. Sensitivity for PDGF-BB detection
Fig. 1. Polyacrylamide gel electrophoresis (PAGE) of the reaction pathways. Lane 1: aptamer–blocker; lane 2: MB1; lane 3: MB2; lane 4: aptamer–blockerþ PDGF-BB; lane 5: aptamer–blockerþ Exo III; lane 6: aptamer–blockerþ PDGF-BB þ Exo III; lane 7: aptamer–blockerþMB1 þMB2; lane 8: aptamer–blockerþ MB1 þ MB2 þ PDGFBB þ Exo III. M represents 500-bp DNA ladder.
To demonstrate the sensitivity of the proposed Exo-CARA assay, PDGF-BB with different concentrations were detected by CL measurement under the optimum conditions. As shown in Fig. 3A, the CL intensity increased when the concentration of PDGF-BB was in the range from 1.0 10 12 to 1.0 10 8 M, indicating that the copy number of liberated DNAzymes was highly dependent on and directly proportional to the concentration of target protein. Fig. 3B shows the linear calibration curve corresponding to the relative CL peak intensities (I I0) versus
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Fig. 2. Effects of (A) the blocker DNA, (B) the amount of Exo-III, and (C) the reaction time on the Exo-CARA system. The CL intensities were obtained by detecting DNAzymes produced through the Exo-CARA reaction in the presence (I) and in the absence (negative control, I0) of 1.0 10 10 M PDGF-BB.
Fig. 3. (A) CL kinetic curves obtained by the Exo-CARA strategy in response to different concentrations of PDGF-BB: 0, 1.0 10 12, 1.0 10 11, 1.0 10 10, 1.0 10 9, 1.0 10 8 M. (B) Corresponding calibration curve of relative CL peak intensities (I I0) versus the logarithm of PDGF-BB concentrations (M). Error bars represent the standard deviation of three repeated measurements.
different concentrations of PDGF-BB in logarithmic scales. The regression equation was expressed as ΔI ¼6220.24log Cþ 76422.61 with a correlation coefficient (R) of 0.9949, where ΔI and C represented the relative CL intensity (I I0) and the concentration of PDGF-BB (M), respectively. The limit of detection (LOD) was calculated to be 6.8 10 13 M through evaluating the average
response of blank plus three times the standard deviation, which was comparable with other most sensitive aptamer-based methods for PDGF-BB detection (Table S1) and 10-fold lower than that obtained by using only aptamer–blocker and MB1 to perform Recycling I and Recycling II (see control experiment in Supporting information). The relative standard deviation (RSD) obtained by
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Fig. 4. The detection specificity of the proposed Exo-CARA assay. Target protein (PDGF-BB) at the concentration of 1.0 10 10 M and three non-target proteins (thrombin, lysozyme, and BSA) at the concentrations of 1.0 10 7 M were used to investigate the relative CL intensity (I I0) responding to a particular protein. All the experiments were carried out under the optimum conditions. Error bars represent the standard deviation of three repeated measurements.
1.0 10 10 M PDGF-BB was 5.8% for five measurements, indicating a desirable reproducibility of the present biosensing system for protein assay.
obtained by ELISA assay as a reference method that is the most standard method currently for PDGF-BB detection. It has been reported that the dilution of serum samples by 10-fold or more have little matrix effects on protein detection (Zhao et al., 2009). Thus, the blood samples used in this assay were diluted to 100-fold prior to analysis, which were analyzed as described in Section 2.2. In addition, the interferences of oxidizable species in the diluted blood plasma sample, such as ascorbic acid (AA) and uric acid (UA), can be ignored for CL detection of PDGF-BB (see Supporting information for detail). As shown in Table 1, the PDGF-BB concentrations obtained by the present method were in good agreement with those determined by ELISA with the maximum relative deviation of 9.5% and a correlation coefficient of 0.9799, indicating our detection method compared favorably with ELISA for PDGF-BB detection in human blood plasma samples. Moreover, it has been reported that ELISA assay for PDGF-BB detection covering 4 orders of magnitude from 10 pM to 10 nM with an LOD at picomolar level. Thus, the developed aptasensor offered a wider linear range (5 orders of magnitude from 1.0 pM to 10 nM) and a lower LOD (1 order of magnitude). In addition, it should be noted that in comparison with ELISA our method was much simpler (homogeneous reaction and aptamer recognition), cost-effective (label-free), rapid ( 1 h), signal highly amplified and sensitive enough to be used for PDGF-BB quantitation in clinical samples (0.4–0.7 nM in human serum and 0.008–0.04 nM in human plasma) (Fang et al., 2001).
3.5. Detection specificity The detection specificity is another essential factor for an aptamer-based assay. The relative CL changes induced by three nontarget proteins (thrombin, lysozyme, and BSA) were investigated. Relative CL intensity was defined as the difference in the CL intensity between a certain sample with protein and the negative control without protein. As shown in Fig. 4, in comparison with the value upon PDGF-BB, the relative CL intensities corresponding to the nontarget proteins were extremely low even though their concentrations are 103-fold higher than that of target species. Therefore, the CL signal was generated by the specific interaction between the aptamer and its target, indicating the excellent specificity of the present aptasensor for PDGF-BB detection. In addition, the blocker DNA was designed that hybridized to aptamer with 11 bases, resulting in a melting point of 47.9 °C when the concentrations of aptamer–blocker and Mg2 þ were 1.0 mM and 12.5 mM, respectively. Thus, the aptamer–blocker hybrid was stable at room temperature and physiological pH, which could significantly limit nonspecific release of blocker DNA and further inhibit false positive detection signals. 3.6. Real sample assay To further demonstrate the applicability of this assay in clinical diagnosis, five human blood plasma specimens were measured by the proposed aptasensor, which were compared with the results Table 1 The detection of PDGF-BB in human blood plasma samples by the proposed ExoCARA aptasensor and ELISA method.
4. Conclusion In conclusion, a novel Exo-CARA-based CL aptasensor has been developed for PDGF-BB detection. In comparison with other aptamerbased methods for PDGF-BB analysis, this assay has the distinct advantages of simplicity (homogeneous reaction), rapidity (within 1 h), signal great amplification (cascade autocatalytic recycling amplification and DNAzymes as reporters to catalyze signal), high sensitivity (an LOD of 6.8 10 13 M), and excellent specificity (aptamer recognition). In addition, the proposed Exo-CARA strategy has been successfully applied in human blood plasma analysis, promising as a convenient point-of-care diagnostics. Moreover, this strategy can be versatilely extended to the detection of other target proteins, such as thrombin and lysozyme, based on analyte-induced self-assembly of the aptamer subunits and Exo III as an amplifying catalyst.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21375056 and 21105052), the Program for New Century Excellent Talents in University of Ministry of Education of People's Republic of China (NCET-12-1024), and the University Doctoral New Teacher Foundation of the Ministry of Education (20113719120001).
Appendix A. Supplementary information
PDGF-BB concentration (nM)a Sample
Proposed Exo-CARA aptasensor
ELISA
Relative deviation (%)
1 2 3 4 5
0.74 0.94 0.45 0.51 0.46
0.79 0.90 0.48 0.54 0.42
6.3 4.4 6.2 5.6 9.5
a
Each value is the average of three measurements.
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.057.
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